EXERCISE AND ADIPOSE TISSUE GLUT4
By
Marcelo Alejandro Flores Opazo, BPT, MPhil
August 2017
The Department of Physiology The University of Melbourne Melbourne, Victoria 3010 AUSTRALIA
TABLE OF CONTENTS
DECLARATION...... VII
ACKNOWLEDGEMENTS ...... VIII
ABSTRACT ...... IX
ABBREVIATIONS ...... XI
LIST OF FIGURES AND TABLES ...... XV
CHAPTER ONE: REVIEW OF LITERATURE ...... 1
1.1 INTRODUCTION...... 1
1.2 GLUT4 PROTEIN STRUCTURE ...... 3
1.3 INSULIN AND CONTRACTION-STIMULATED GLUT4 TRANSLOCATION 6
1.4 GLUT4 EXPRESSION IN SKELETAL MUSCLE AND ADIPOSE TISSUE ...... 8
1.4.1 Domain 1 ...... 9
1.4.2 Adipose tissue-specific element (ASE) ...... 10
1.4.3 Myocyte enhancer factors ...... 10
1.4.4 GLUT4 expression is regulated by GEF – MEF2A/MEF2D interaction...... 11
1.4.5 The role of HDACs in GLUT4 expression ...... 12
1.4.6 Potential involvement of histone acetyltransferases ...... 15
1.5 ADIPOSE TISSUE GLUT4 EXPRESSION ...... 16
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1.5.1 SREBP-1c...... 16
1.5.2 LXRs ...... 17
1.5.3 KLF15 ...... 19
1.5.4 C/EBPα ...... 19
1.5.5 Peroxisome proliferator-activated receptor-γ ...... 22
1.5.6 Free-fatty acids ...... 23
1.5.7 FOXO1 ...... 24
1.6 FACTORS INFLUENCING ADIPOSE TISSUE GLUT4 EXPRESSION ...... 25
1.6.1 The importance of adipose tissue GLUT4 ...... 27
1.6.2 Adipose tissue GLUT4 over-expression and insulin action ...... 28
1.6.3 Nutritional factors influencing adipose tissue GLUT4 expression ...... 30
1.6.4 Insulin resistance, type 2 diabetes mellitus, diet-induced obesity and adipose tissue
GLUT4 ...... 33
1.6.5 Exercise and adipose tissue GLUT4 ...... 39
1.7 SUMMARY ...... 43
1.8 AIM AND OBJECTIVES ...... 44
2 CHAPTER TWO: DIET AND EXERCISE EFFECTS ON ADIPOSE TISSUE
GLUT4 ...... 45
2.1 INTRODUCTION...... 45
2.2 METHODS: ...... 47
2.2.1 Adipose tissue samples: ...... 47
2.2.2 Adipose tissue homogenisation: ...... 48
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2.2.3 Immunoblotting: ...... 49
2.2.4 Primary antibodies: ...... 50
2.2.5 Statistical analysis: ...... 50
2.3 RESULTS: ...... 51
2.3.1 Effect of HFD on epididymal adipose tissue GLUT4: ...... 51
2.3.2 Effect of HFD on CREB/ATF3 pathway: ...... 52
2.3.3 Effect of HFD on TLR4/NF-κβ pathway: ...... 52
2.3.4 Effect of 4 weeks of exercise-training on adipose tissue GLUT4 protein in mice fed
on chow and HFD diets: ...... 52
2.3.5 Effect of muscle-specific HSP72-Tg over-expression on adipose tissue GLUT4: ... 53
2.4 DISCUSSION ...... 59
2.5 CONCLUSION ...... 66
3 CHAPTER THREE: SHORT-TERM EXERCISE TRAINING AND ADIPOSE
TISSUE GLUT4 IN HUMANS ...... 68
3.1 INTRODUCTION...... 68
3.2 METHODS ...... 70
3.2.1 Subjects: ...... 70
3.2.2 Peak pulmonary oxygen consumption (VO2 peak): ...... 70
3.2.3 Anthropometric measurements: ...... 71
3.2.4 Exercise training:...... 71
3.2.5 Muscle and adipose tissue biopsies: ...... 72
3.2.6 Adipose tissue homogenization:...... 72
III
3.2.7 Skeletal muscle homogenization: ...... 72
3.2.8 Immunoblotting: ...... 73
3.2.9 Adipose tissue histological sections: ...... 74
3.2.10 Adipocyte size: ...... 74
3.2.11 Statistical analysis: ...... 75
3.3 RESULTS ...... 76
3.3.1 Characteristics of the subjects: ...... 76
3.3.2 Short-term training adaptations in skeletal muscle: ...... 76
3.3.3 Short-term training adaptations in adipose tissue: ...... 76
3.4 DISCUSSION ...... 85
3.5 CONCLUSION ...... 90
4 CHAPTER FOUR: EXERCISE SERUM EFFECTS ON GLUT4 IN HUMAN
PRIMARY ADIPOCYTES ...... 92
4.1 INTRODUCTION...... 92
4.2 METHODS: ...... 94
4.2.1 Subjects: ...... 94
4.2.2 Blood sampling and serum extraction: ...... 94
4.2.3 Preliminary experiments: ...... 95
4.2.4 Adipose tissue biopsy: ...... 95
4.2.5 SVF-derived adipocyte proliferation and differentiation: ...... 95
4.2.6 Dose response to ECS: ...... 96
4.2.7 Treatment with ECS, IL-6, FGF21 and isoproterenol: ...... 96
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4.2.8 Gene expression in human primary adipocytes: ...... 97
4.2.9 cDNA synthesis and RT-qPCR: ...... 97
4.2.10 Immunoblotting: ...... 98
4.2.11 Glucose uptake: ...... 98
4.2.12 Oil-red-O staining (ORO): ...... 99
4.2.13 Statistical analysis: ...... 99
4.3 RESULTS ...... 101
4.3.1 Time-response effect of exercise serum on GLUT4 mRNA expression in human
primary adipocytes: ...... 101
4.3.2 Effect of ECS on GLUT4 expression levels in human primary adipocytes: ...... 101
4.3.3 Effect of exercise serum on adipogenic markers: ...... 101
4.3.4 Effect of exercise serum on insulin-stimulated glucose uptake: ...... 102
4.3.5 Effects of Isoproterenol, IL-6 and FGF21 on GLUT4: ...... 102
4.3.6 Effects of Isoproterenol, IL-6 and FGF21 on adipogenic markers: ...... 102
4.4 DISCUSSION ...... 113
4.5 CONCLUSION ...... 118
5 CHAPTER FIVE: SUMMARY AND FINAL CONCLUSIONS ...... 120
REFERENCES ...... 130
6 CHAPTER 6: SUPPLEMENTARY INFORMATION...... 175
6.1 Characterization of GLUT4 and adipogenic genes expression in 3T3-L1 cells during adipogenic differentiation...... 175
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6.2 Gene expression of adipogenic markers in 3T3-L1 cells during differentiation: 176
6.3 Differentiation of human SVF-derived pre-adipocytes and exposure to conditioned serum:...... 178
6.4 Lipogenic gene expression in human primary adipocytes ...... 178
6.5 SUPPLEMENTARY RESULTS ...... 180
6.5.1 Expression of adipogenic markers in 3T3-L1 adipocytes during differentiation: .. 180
6.5.2 Expression of glucose transporters expression in 3T3-L1 adipocytes during
differentiation: ...... 180
6.5.3 Dose-response effect of exercise serum on GLUT4 protein in human primary
adipocytes: ...... 181
6.5.4 Dose-dependent effect of exercise serum on lipogenic markers: ...... 181
6.6 GLUT4 expression and adipogenic profile in 3T3-L1 adipocytes ...... 189
VI
DECLARATION
This is to certify that:
The thesis entitled Exercise and Adipose Tissue GLUT4 has been submitted in total fulfilment of the degree of Doctor of Philosophy and comprises only my original, previously unpublished work in the Department of Physiology, Faculty of Medicine, Dentistry and
Health Sciences, The University of Melbourne. All the experimental work was performed by the author, with the exception of muscle and adipose tissue biopsies which were performed by Dr Andrew Garnham, and paraffin processing, slicing and staining of human adipose tissue histological sections which were performed by the Histology service of the Faculty of
Medicine, Dentistry and Health Sciences.
Due acknowledgement has been made in the text to all other material used.
The thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices.
Marcelo Alejandro Flores Opazo August 2017
Department of Physiology Faculty of Medicine, Dentistry and Health Sciences The University of Melbourne Parkville, Victoria, 3010 AUSTRALIA
VII
ACKNOWLEDGEMENTS
I owe my deepest gratitude to Prof. Mark Hargreaves whose encouragement, support and supervision throughout my candidature enabled me to acquire rigorous scientific thinking and to build a strong foundation towards my professional development as a physiotherapist and as a researcher in the field of exercise sciences. Despite the numerous difficulties and hard times, his words and guidance helped me in keeping the inspiration alive.
I would also like to thanks those who made this thesis possible:
To Dr. Darren Henstridge, from the Cellular and Molecular Metabolism lab at Baker Institute for his supervision during my first year on my PhD, and the opportunity to develop skills and laboratory techniques.
To Prof. Sean McGee and Dr. Shona Morrison from the Metabolic Research Unit at Deakin University, for their supervision during my second year on my PhD and the opportunity to learn cell culture techniques.
To Prof. Matthew Watt from the Lipid Metabolism lab at Monash University, for his supervision during my third year of my PhD at his lab, and all the experience and technical support with adipocyte biology and in vitro experiments.
To my colleagues Arthe Raajendiran, for teaching and patiently helping me with human primary adipocytes and in vitro experiments, and Thomas Tsiloulis for helping me with the in vitro experiments and data analysis.
To Assoc. Prof. Robyn Murphy for the opportunity to collaborate, for her support during the last stage of my PhD, and help with western blotting.
To Prof. Assam El-Osta, Prof. Mark Febbraio, and Dr. Martin Witham for the opportunity to collaborate.
To the subjects who participated in the studies.
Lastly, to my beloved wife Daniela and my children Martin, Laura and Rafael for the inspiration, love and unconditional support.
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ABSTRACT
GLUT4 is the major insulin-sensitive glucose transporter expressed predominantly in skeletal and cardiac muscles, and adipose tissue where it mediates insulin-stimulated glucose transport. Notwithstanding the major role of skeletal muscle in insulin–mediated glucose disposal, adipose tissue glucose uptake and the expression of GLUT4 have been shown to be of importance for the systemic regulation of insulin sensitivity and glucose homeostasis.
Alterations in intracellular glucose flux owing to defective GLUT4 in adipocytes have been associated with impaired glucose tolerance and hyperinsulinemia. Interestingly, downregulation of adipose tissue GLUT4 expression is a common defect described in insulin resistant states including obesity, metabolic syndrome, and diabetes, whereas muscle GLUT4 expression remains unaltered. Exercise training increases adipose tissue GLUT4 expression in rodents, and our lab has previously shown that 4 weeks of exercise training normalizes the expression of adipose tissue GLUT4 in patients with T2DM. However, the mechanisms involved in exercise-induced up-regulation of AT-GLUT4 are still unknown. The broad aim of the present research project was to examine the effect of exercise on adipose tissue
GLUT4 expression. With this purpose, three studies were conducted, using mice, human primary adipocytes and healthy volunteers, in an attempt to gain a better understanding of: 1) how a high fat diet affects the expression of adipose tissue GLUT4, 2) how exercise may be beneficial to prevent this effect; and 3) how this effect may occur. Results obtained in this thesis showed that adipose tissue GLUT4 protein is rapidly reduced by a high fat diet, suggesting this could be an early defect contributing to diet-induced insulin resistance.
Concurrent four weeks of exercise training with high fat diet increased GLUT4 protein in high fat-fed mice, however, no training effect was observed in chow-fed mice. This finding may imply a longer exposure is needed in chow-fed animals to induce an exercise training
IX effect on adipose tissue GLUT4. In healthy humans, ten days of short-term exercise training did not affect adipose tissue GLUT4 levels in subcutaneous adipose tissue, a result that supports the suggested longer exposure to the exercise stimulus in normal conditions in mice.
Conversely, serum from exercised healthy volunteers increased GLUT4 protein and mRNA in human primary adipocytes in vitro, suggesting that circulating factor(s) may mediate exercise effects on adipose tissue GLUT4 expression. The identification of such circulating factors released during exercise, potentially mediating the increase in adipose tissue GLUT4, would require a broad spectrum of experimental approaches aiming to explore tissue(s) of origin, mechanisms of production and secretion (e.g. cleavage and release of mature proteins or vesicle-associated transport), delivery to target tissue, and signal transduction of the candidate factor(s).
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ABBREVIATIONS
AICAR 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside
ACC acyl-coA-carboxylase
AMP adenosine monophosphate
ATP adenosine triphosphate
AMPK AMP-activated protein kinase
ASE adipose tissue-specific binding element
ATGL adipose tissue-triglyceride lipase
BMI body mass index
CAMKII calcium2+/calmodulin-dependent kinase II
CREB cAMP-dependent response element protein
ChREBP carbohydrate response element binding protein
CPT-1 carnitine-palmitoyl-transferase
C/EBP CCAAT/Enhancer-binding protein cAMP cyclic-AMP
COX cyclooxygenase
DG diacylglycerol
ECS exercise-conditioned serum
EGFP enhanced green fluorescent protein
ER endoplasmic reticulum
ERK 1/2 extracellular-signal-regulated kinase 1 and 2
ET exercise training
FA fatty acids
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FAS fatty acid synthase
FATP1 fatty acid transporter protein 1
FGF21 fibroblast growth factor 21
FOXO forkhead box
GCN5 general control of amino-acid synthesis
GDP guanidine diphosphate
GEF GLUT4 enhancer factor
GLUT glucose transporter
GPAT glycerol-3-phosphate acyltransferase
GSV GLUT4 storage vesicles
GTP guanidine triphosphate
HAT histone acetyltransferases
HDAC histone deacetylases
HFD high fat diet
HIIT high intensity interval training
HSP heat shock protein
InsR insulin receptor
IR insulin resistance
IRE insulin response element
IRS1 insulin receptor substrate 1
KLF15 krüppel-like factor 15
LPL lipoprotein lipase
LXRs liver X receptors
MAPK mitogen activated protein kinase
MEF2 myocyte enhancer factor 2
XII min minutes mRNA messenger ribonucleic acid mTORC1 mammalian target of rapamycin complex 1
NAD nicotinamide adenine dinucleotide
NADPH nicotinamide adenine dinucleotide phosphate
NF-1 nuclear factor I
NF-Κb nuclear factor kappa light chain enhancer of activated B cells
NMNT nicotinamide N-methyltransferase
PDK-1 phosphoinositide dependent protein kinase-1
PDK4 pyruvate dehydrogenase 4
PGC-1α peroxisome proliferator-activated receptor-γ coactivator-1alpha
PGC-1β peroxisome proliferator-activated receptor-γ coactivator-1beta
PGE2 prostaglandin E2
PGJ2 prostaglandin J2
PI3K phosphatidylinositol-3-kinase
PIP2 phosphatidylinositol-4,5 phosphate
PIP3 phosphatidylinositol-3,4,5 phosphate
PKA protein kinase A
PKC protein kinase C
PL phospholipids
PPARγ peroxisome proliferator-activated receptor-gamma
PPO peak power output
Prep1 pbx-regulating protein-1
RBP4 retinol-binding protein 4
RXR retinol X receptor
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SCAT subcutaneous adipose tissue
SET7 su(var)3-9, enhancer-of-zeste, trithorax domain-containing
lysine methyltransferase 7
SLC2A solute carrier family 2A
SREBP-1c sterol response element binding protein-1c
STZ streptozotocin
SVF stromal vascular fraction
TG triglycerides
TGFβ transforming-growth factor beta
TGN trans-golgi network
TLR toll-like receptor
TNF-α tumor necrosis factor-alpha
TUG tether containing UBX domain for GLUT4
VAT visceral adipose tissue
ZDF zucker diabetic fatty rats
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LIST OF FIGURES AND TABLES
FIGURE 1.1 Regulatory mechanisms of GLUT4 expression in the adipose tissue...... 42
FIGURE-2.1: Time-course effect of HFD in adipose tissue GLUT4 protein...... 54
FIGURE-2.2: Time-course effect of HFD in CREB, p-CREB (ser133) and ATF3 protein .... 55
FIGURE-2.3: Time-course effect of HFD in the activation of TLR4/NF-κβ ...... 56
FIGURE-2.4: Effect of High fat diet and exercise-training on adipose GLUT4 protein...... 57
FIGURE-2.5: Effect of muscle-specific HSP72-Tg on adipose tissue GLUT4 protein...... 58
TABLE 3.1: Characteristics of the subjects...... 77
FIGURE 3.1: Effect of short-term training on skeletal muscle GLUT4 protein...... 78
FIGURE 3.2: Effect of short-term training on skeletal muscle COX-IV protein...... 79
FIGURE 3.3: Effect of short-term training on adipose tissue GLUT4 and GLUT1 proteins . 80
FIGURE 3.4: Effect of short-term training on adipose tissue MEF2A and HDAC5 proteins. 81
FIGURE 3.5: Effect of short-term training on adipose tissue COX-IV and FABP4 proteins. 82
FIGURE 3.6: Effect of short-term training on adipose tissue OXPHOS proteins...... 83
FIGURE 3.7 Effect of short-term training on adipocyte cell size...... 84
TABLE 4.1: Characteristics of the subjects ...... 103
TABLE 4.2: Human primary adipocytes differentiation protocol ...... 104
TABLE 4.3: Adipogenic genes TaqMan probes...... 104
FIGURE 4.1: Time course of exercise serum effects on GLUT4 mRNA in human primary adipocytes ...... 105
FIGURE 4.2: Effect of ECS on GLUT4 protein in human primary adipocytes...... 106
FIGURE 4.3: Effect of exercise serum on GLUT4 mRNA expression in human primary adipocytes...... 107
FIGURE 4.4: Effect of exercise serum on adipogenic marker mRNA expression in human primary adipocytes...... 108
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FIGURE 4.5: Effect of exercise serum on adipogenic marker protein content in human primary adipocytes...... 109
FIGURE 4.6: Effect of exercise serum on insulin-stimulated glucose uptake in human primary adipocytes...... 110
FIGURE 4.7: Effect of isoproterenol, IL-6 and FGF-21 treatments on GLUT4 mRNA expression...... 111
FIGURE 4.8: Effect of isoproterenol, IL-6, FGF-21, and 10% FBS treatments on adipogenic marker mRNA expression...... 112
SUPPLEMENTARY TABLE 6.1: Primer sequence for genes of interest in 3T3-L1 ...... 182
SUPPLEMENTARY TABLE 6.2: Lipogenic genes, primers sequence ...... 182
SUPPLEMENTARY FIGURE 6.1: Adipogenic markers during 3T3-L1 adipogenic differentiation...... 183
SUPPLEMENTARY FIGURE 6.2: Glucose transporter expression during 3T3-L1 adipogenic differentiation ...... 184
SUPPLEMENTARY FIGURE 6.3: Proliferation and differentiation of human primary adipocytes ...... 185
SUPPLEMENTARY FIGURE 6.4: Dose-response effect of exercise serum on GLUT4 protein in human primary adipocytes...... 186
SUPPLEMENTARY FIGURE 6.5: Dose-response effect of exercise serum on Lipogenic markers in human primary adipocytes...... 187
SUPPLEMENTARY FIGURE 6.6: Effect of isoproterenol, IL-6 and FGF-21 treatments on
GLUT4 protein expression ...... 188
XVI
CHAPTER ONE: REVIEW OF LITERATURE
1.1 INTRODUCTION
Dietary modifications such as feeding or fasting, as well as increased energy expenditure during exercise challenge the organism to adapt to changes in nutrient availability in order to preserve metabolic homeostasis. Few physiological parameters are more tightly and acutely regulated in humans than blood glucose, circulating levels of which are co-ordinately regulated by the function of several organs. This includes absorption from the gut in the post-prandial state, glucose-stimulated insulin secretion by β-pancreatic cells, and its disposal and utilization in insulin-sensitive tissues, including cardiac and skeletal muscle, adipose tissue, where it can be either metabolised to produce ATP or stored as glycogen. The brain depends on glucose not only as its main source of energy, but also for neuronal and non-neuronal cellular maintenance, and the synthesis of neurotransmitters (Mergenthaler et al., 2013). Glucose regulates the secretion of insulin by pancreatic β-cells and is re-absorbed by the kidneys. All of these processes rely primarily on the integrity of glucose delivery and transport to the cells, into which glucose is taken up by transporter-mediated diffusion. There are 13 isoforms of facilitative glucose transporters, commonly denominated GLUTs or SLC2A, which are divided into three classes depending on tissue-specific localization, splicing process and main function. Class I comprises transporters GLUT1 to GLUT4, class II GLUT5,
7, 9, and 11, and class III GLUT6, 8, 10, 12, and 13 (Zhao & Keating, 2007). GLUT1 is responsible for basal glucose transport, which occurs independently of insulin, in all insulin-sensitive tissues. GLUT1 and GLU3 take up glucose across the blood-brain barrier (Mergenthaler et al., 2013). GLUT2 is expressed in the liver, intestine, the
1 kidneys, and pancreatic β-cells where it plays a role as glucose sensor allowing glucose- induced release of insulin (Garvey et al., 1992; Watson & Pessin, 2001; Ducluzeau et al., 2002; Watson et al., 2004a; Zorzano et al., 2005). GLUT4 is the only glucose transporter sensitive to the effect of insulin. It is highly expressed in cardiomyocytes, skeletal muscle fibres, and white and brown adipose cells where it confers insulin responsiveness to these tissues (Dalen et al., 2003). Furthermore, the absolute glucose transport rate in those tissues is directly proportional to the total number of GLUT4 molecules at the plasma membrane (Ren et al., 1994; Holloszy, 2005). In these tissues, glucose uptake is stimulated by insulin through its role regulating the translocation of
GLUT4 from intracellular storage sites to the plasma membrane. Similarly, exercise- induced muscle contraction increases skeletal muscle glucose uptake through the activation of different mechanisms from insulin; however, there is convergence of the pathways as insulin sensitivity is enhanced during and after exercise.
GLUT4 expression is highly regulated and can be affected by exercise, nutritional and metabolic states, and hormonal influences. In skeletal muscle, GLUT4 expression is increased immediately after a single bout of exercise and remains elevated as an adaptive response to training, due to the repeated, transient increases in GLUT4 mRNA following each exercise bout, finally leading to the gradual accumulation of GLUT4 protein (Phillips et al., 1996; Kraniou et al., 2000; MacLean et al., 2000; Holmes &
Dohm, 2004; Kraniou et al., 2004, 2006).
It has been argued that glucose flux into adipocytes and the expression of GLUT4 are two important components of a glucose sensing role exerted by the adipose tissue that affects systemic glucose homeostasis (Herman & Kahn, 2006). Adipocytes sense variations in circulating glucose leading to subsequent changes in the expression of
GLUT4, and reciprocally the expression and secretion of adipokines such as RBP4,
2 adiponectin and resistin (Carvalho et al., 2005; Yang et al., 2005; Graham et al., 2006;
Graham & Kahn, 2007).
This present review will summarize the mechanisms that regulate the function and expression of GLUT4, and its relation to diet, exercise and disease states such as insulin resistance and diabetes, with an emphasis on adipose tissue.
1.2 GLUT4 PROTEIN STRUCTURE
GLUT4 protein structure comprises 510 amino acids (43kDa) organized in 12 transmembrane domains (Watson & Pessin, 2001; Watson et al., 2004a), including a major exofacial loop, close to the N-terminal, two substrate binding sites, a major cytoplasmic loop and the C-terminal . GLUT4 contains unique sequences in its N- and
C-terminal cytoplasmic domains that govern kinetic aspects of both endocytosis and exocytosis in a continuously recycling trafficking system (Huang & Czech, 2007). The
N-terminal phenylalanine residue F5QQI and the major cytoplasmic loop are involved in GLUT4 endocytosis and Akt/protein kinase B substrate 160 (AS-160)-dependent intracellular sequestration (Capilla et al., 2007). The N-terminal F5QQI motif serves as a binding site for the medium chain adaptin micro1, a subunit of the AP-1 adaptor complex, whereas the luminal loop contains a binding domain for sortilin, both adaptor proteins playing a role in post-Golgi/endosomal GLUT4 trafficking (Bernhardt et al.,
2009; Pan et al., 2017). The C-terminal includes: 1) a dileucine motif (LL489/490), which regulates cell-surface GLUT4 distribution by exerting an inhibitory role in
GLUT4 internalization (Al-Hasani et al., 2002; Khan et al., 2004), and 2) an insulin- responsive motif, which alteration completely abolished insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes (Song et al., 2008).
3
Following GLUT4 protein synthesis in the ER, GLUT4 is sorted into tubule-vesicular bodies, the insulin-responsive GSV, within Golgi cisternae. This step requires golgin-
160, a Golgi resident protein implicated in maintenance of Golgi structure and function, and cargo protein sorting in Golgi cisternae. In the absence of golgin-160, GLUT4 vesicles relocate to the cell surface and undergo continuous unregulated recycling
(Williams et al., 2006). Once sorted into GSV at the Golgi cisternae, GLUT4 transits along TGN, a process regulated by Golgi-localized-γ-ear-containing Arf-binding protein adaptor protein (GGA) and sortilin. (Watson et al., 2004b; Shi & Kandror, 2005). Here,
GLUT4 protein undergoes several posttranscriptional modifications including Asn57- glycosylation, sialylation, ubiquitination and Ubc9-dependent SUMOylation that are relevant for cellular localization (reviewed elsewhere,Sadler et al., 2013). Fully mature
GLUT4 protein possesses a conserved N-glycosylation consensus site (Asn-Xaa-
Thr/Ser) that is typically positioned in the first or fifth extracellular loop. This site contributes to quality control and overall stability, reducing proteosomal degradation of newly synthesised GLUT4, and intracellular trafficking in response to insulin (Haga et al., 2011; Hirayama et al., 2016).
Under basal (non-insulin stimulated) conditions, the majority of GLUT4 is retained intracellularly by the TUG protein. TUG is a 60-kDa protein presents in the cytosol and on membranes, which binds and anchor GSV to the Golgi matrix through its C-terminal which interacts with an anchoring site composed by golgin-160 and PIST. Insulin stimulation induces TUG site-specific endoproteolytic cleavage, which separates its N- terminal region, containing a GLUT4-binding site, from its anchor N-terminal region, and thereby promotes GLUT4 translocation (Bogan et al., 2012).
It has been shown that prolonged insulin stimulation down-regulates GLUT4 by reducing its synthesis and increasing its degradation rate (Sargeant & Paquet, 1993).
4
Acute insulin stimulation (< 24h) increased GLUT4 protein synthesis by 2-fold in 3T3-
L1 adipocytes. However, extended exposure decreased total cellular GLUT4 content and reduced its half-life from 50 to 15 hours. This effect has been recently attributed to insulin-generated oxidative stress that accelerates GLUT4 degradation through inhibition of retromer in a casein kinase 2 -dependent manner (Ma et al., 2014).
Retromer is a key component of the endosomal protein sorting machinery that mediates the endosome-to-TGN retrograde trafficking of diverse functional transmembrane proteins. Retromer is critical for the stability of the sortilin-GLUT4 complex which is required for the formation of insulin-responsive vesicles in the TGN. Together, sortilin and retromer rescue GLUT4 from lysosomal degradation (Pan et al., 2017). Retromer is composed of three subunits, vacuolar-protein-sorting (Vps) 26, 29 and 35, where Vps35 regulates its membrane association capacity (Yang et al., 2016). Prolonged insulin stimulation in 3T3-L1 adipocytes upregulated NADPH-oxidase 4 (NOX4) that increased H2O2 production which promotes CK2-mediated Vps35 phosphorylation causing disruption of the retromer interaction with sortilin at the GSV, thus switching
GLUT4 sorting to lysosomes (Ma et al., 2014). In summary, adipose tissue and skeletal muscle cellular responsiveness to insulin relies upon proper sorting, intracellular localization and compartmentalization, and trafficking of GLUT4. These processes are tightly regulated during GLUT4 maturation, which involves interaction with adaptor proteins and posttranscriptional modifications that guarantee correct vesicular assembly and GLUT4 functional capacity.
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1.3 INSULIN AND CONTRACTION-STIMULATED GLUT4
TRANSLOCATION
Upon insulin stimulation, GLUT4 is translocated to the plasma membrane from intracellular storage vesicles allowing the entrance of glucose into the cell. Insulin binds to its membrane receptor that induces phosphorylation-mediated receptor activation by its intrinsic tyrosine kinase activity. Activated InsR functions as a docking port for adaptor proteins such as insulin receptor substrate 1 and 2 (IRS-1/2), leucine zipper motif 1 (APPL1) (Ryu et al., 2014; Zhang & Liu, 2014), Shc proteins and Grb2- associated binder (GAB) proteins (Boucher et al., 2014). These proteins act as scaffold to organize and mediate signalling complexes (Boucher et al., 2014). The binding of
IRS1/2 to InsR leads to the recruitment of PI3K. APPL1 forms a complex with IRS1/2 under basal conditions, and this complex is then recruited to the InsR in response to insulin stimulation forming a docking-port for PI3K. The PI3K enzyme catalyses the formation of PIP3 membrane lipids by phosphorylation of PIP2. PIP3 is required for the activation of PDK-1. PDK-1 phosphorylates and activates another serine/threonine kinase protein kinase B (Akt or PKB). When activated by PDK-1, Akt binds ATP and phosphorylates Akt subtrate 160 (AS-160), which interacts with Rab GTPase activating protein (RabGAP) at the surface of GLUT4-containing vesicles (Watson et al., 2004a).
Rab protein in its inactive form binds GDP. Akt-mediated phosphorylation of AS-160 inhibits its GTP-ase activity thus allowing GTP to bind Rab protein on the surface of
GLUT4 vesicles thereby releasing AS-160 inhibitory effect upon Rab which finally drives GLUT4 translocation (Watson et al., 2004a). This pathway is deemed as the most important signal downstream Akt that leads to stimulation of glucose transport (Cho et al., 2001).
6
Similarly, muscle contraction is a powerful stimulator of glucose transport (Wallberg-
Henriksson & Zierath, 2001). The acute metabolic and mechanical demands of exercise require the coordinated regulation of diverse signalling pathways that guarantees the delivery, transport across the muscle-cell surface and intra-myocellular metabolism of glucose (Sylow et al., 2017). In the basal state, GLUT4 is located in two different intracellular pools: 23% as large clusters forming rows surrounding the nuclei and in the core of the fiber, in association with multivesicular bodies, and 77% as small clusters of endosomal vesicles near the Trans-Golgi network (Ploug et al., 1998). Muscle contraction stimulates glucose transport through the activation of several parallel pathways associated with cellular stress, including: 1) changes in energy status during exercise, i.e. increased AMP: ATP ratio, and depletion of glycogen stores, leading to the activation of the energy sensor AMPK which activates vesicle trafficking proteins
(Taylor et al., 2008), 2) transduction of mechanical stress, secondary to cross-bridge cycling, with subsequent increase in extracellular Ca2+ entry and activation of CaMKII
(Jensen et al., 2014), 3) stretch-induced activation of GTPase-Rac1 (Sylow et al., 2016),
4) production of nitric oxide by activation of NOS (Balon & Nadler, 1997), and 5) contraction-stimulated and stretch-stimulated production of reactive oxygen species
(Chambers et al., 2009). In response to contraction-induced stimuli GLUT4 is translocated to both sarcolemma and t-tubule membranes, with similar kinetics, preferentially from endosomal pools. Intravital imaging of GLUT4 translocation in quadriceps muscle of GLUT4-EGFP transfected mice subjected to electrical stimulation showed that GLUT4 appearance at the plasma membrane, and consequently depletion of intracellular vesicle depots, is dependent on the magnitude of muscle contraction, and that the presence of GLUT4 at the surface membrane was increased for more than 2 hours after the cessation of the contraction (Lauritzen et al., 2010).
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In contrast, insulin-stimulated glucose transport responds only to the unidirectional activation of a single pathway, the insulin signalling pathway at the sarcolemma, where local phosphorylation of InsR and IRS1 initiates a cascade of events leading to the activation Akt and subsequent GLUT4 trafficking, docking and fusion into the membrane (Lauritzen et al., 2008). Furthermore, the majority of insulin-mediated
GLUT4 translocation does not involve any high degree of movement of GLUT4 vesicles from the cell interior to the sarcolemma. Instead, insulin recruits and recycles
GLUT4 vesicles from local sub-sarcolemmal and perinuclear pools (Lauritzen et al.,
2008). Next, the insulin signal slowly diffuses towards the t-tubule system, therefore generating a delay in GLUT4 vesicle depletion from intracellular compartments and its appearance at the plasma membrane. This delay has been attributed to spatial conformation of the t-tubule system with surface area 2 to 3-fold larger than sarcolemma, and narrow diameter with invaginations and viscose lumen content
(Lauritzen et al., 2006).
Upon cessation of insulin stimulation and/or muscle contraction, the presence of
GLUT4 at the surface membranes gradually declines whilst intracellular GLUT4 pools gradually re-populate, a process called re-internalization (Lauritzen et al., 2008;
Lauritzen et al., 2010).
1.4 GLUT4 EXPRESSION IN SKELETAL MUSCLE AND ADIPOSE TISSUE
The GLUT4 gene is a single copy 6 kb length gene located in chromosome 17 (Zhao &
Keating, 2007). The promoter region is 895 bp upstream from the transcription initiation site in the 5’-flanking DNA (Zorzano et al., 2005). The transcriptional regulatory function of the promoter region gives tissue-specificity of GLUT4 expression to insulin- responsive tissues (Oshel et al., 2000). Within this region, there are two regulatory sites:
8
Domain I located between – 724 and – 712 bp (Oshel et al., 2000) and the ASE located at – 551 and – 506 bp (Miura et al., 2003). The ASE interacts in a cooperative way with
Domain l to provide full GLUT4 promoter function (Mora et al., 2001; Olson & Knight,
2003; Im et al., 2007). These regulatory regions contain binding sites for a number of nuclear transcription factors (Zorzano et al., 2005), including the GEF that specifically binds to domain I, and the MEF2, which binds to an element located within the ASE.
The expression of these transcription factors overlaps in all GLUT4-expressing tissues suggesting highly regulated GLUT4 gene expression under the control of MEF2 and
Domain l binding proteins (Oshel et al., 2000; Zheng et al., 2001; Im et al., 2007).
1.4.1 Domain 1
This DNA regulatory site is located between –724 and -712 bp upstream from the
GLUT4 transcription initiation site and contains a binding element for the GEF (Oshel et al., 2000). Domain I regulates GLUT4 expression by containing binding sites for transcription factors that interact with GEF (Dowell & Cooke, 2002; Karnieli &
Armoni, 2008) including the IRE, a 30 bp region (-706 to -676 bp) that exclusively binds the NF-1 (Cooke & Lane, 1998, 1999a, b; Gronostajski, 2000). NF-1 belongs to a family of site-specific DNA-binding proteins known as CTF or CAAT box transcription factors (Gronostajski, 2000). Their basic structure contains two domains: DNA- binding/dimerization domain located at the N-terminal and the transcriptional activation/repression domain located at the C-terminal. Phosphorylation-mediated activation of NF-1, downstream cAMP-signaling pathway, represses GLUT4 expression
(Gronostajski, 2000). Likewise, insulin directly, but transiently phosphorylates and activates NF-1, which binds to the IRE leading to the repression of GLUT4 expression.
9
Together, insulin and cAMP pathways potentiate their repression over GLUT4 gene suggesting complementary mechanisms of regulation (Cooke & Lane, 1999a, b).
1.4.2 Adipose tissue-specific element (ASE)
ASE is located within – 551 and – 506 bp in mice (Liu et al., 1992; Liu et al., 1994),-
446 and – 457 bp in rats, and – 463 and – 474 in humans (Mora et al., 2001) of the
GLUT4 promoter region with its sequence (CTGGGCCCAG) well preserved across species. ASE is a MEF2 binding-protein domain that has been described as necessary, but not sufficient, for tissue-specific expression and full function of the GLUT4 promoter (Olson & Knight, 2003; Im et al., 2007). In vitro, transgenic constructs with complete deletion of Domain l showed reduced GLUT4 expression, regardless of the presence of MEF2 (Oshel et al., 2000; Mora et al., 2001; Knight et al., 2003; Sparling et al., 2008).
1.4.3 Myocyte enhancer factors
Myocyte enhancer factors are members of the MADS-box family of transcription factors with a multiplicity of significant biological roles (Shore & Sharrocks, 1995).
Also known as Related to Serum Response Factor (RSRF) subfamily of transcriptional activators, four isoforms have been described: MEF2A/RSRFC4, MEF2B/RSRFR2,
MEF2C, and MEF2D. Their structure is characterized by an 80 – 100 amino acid DNA- binding domain, referred to as core proteins, composed of conserved 56 MADS-box residues and an additional 30 amino acids on the C-terminal. The N-terminal is responsible for sequence-specific DNA contact (Shore & Sharrocks, 1995), whereas the
C-terminal contains the transcriptional activation domain, which is activated by the formation of homo and/or heterodimers. MEF2A is highly expressed in adipose tissue and binds the cis-DNA sequence CTAAAAATAG on the ASE forming either a
10 heterodimer with MEF2D or interacting with GEF. MEF2A and MEF2D are differentially regulated in skeletal muscle and adipose tissues (Mora et al., 2001). While
STZ-induced diabetes markedly reduced MEF2A and GLUT4 expression with no effect on MEF2D in skeletal muscle, in adipose tissue MEF2D protein content was drastically reduced, which impaired the formation of the MEF2A/D complex (Mora et al., 2001).
Although, in the absence of MEF2A, MEF2D can either homodimerize or interact with other factors (Knight et al., 2003), these interactions result in non-functional complexes.
1.4.4 GLUT4 expression is regulated by GEF – MEF2A/MEF2D interaction
In vitro, co-transfection of GEF with either MEF2A or MEF2D resulted in 4.5-fold and
2.4-fold increases in GLUT4 expression, respectively (Knight et al., 2003).
Interestingly, co-transfection of the heterodimer MEF2A/MEF2D complex together with GEF increased GLUT4 transcription significantly less than GEF/MEF2A co- transfection, which could imply that MEF2D competes with MEF2A in activating
GLUT4 promoter, attenuating the functional capacity of GEF/MEF2A/DNA binding sites to interact, hence reducing GLUT4 expression (Knight et al., 2003). Nevertheless, the adipose tissue the MEF2A/MEF2D interaction was required to preserve GLUT4 expression suggesting MEF2D may interact with co-activators or other transcription factors (Mora et al., 2001).
The MEF2A phosphorylation state regulates its DNA binding activity (Ornatsky &
McDermott, 1996; Salt et al., 2000; Sparling et al., 2007). MEF2A in its dephosphorylated state not only has increased affinity for binding its cognate DNA sequence, but also increased affinity for dimerization with GEF and reduced possibilities to form MEF2A/MEF2D heterodimer that reduces its functional transcriptional capacity (Sparling et al., 2008). In its hyper-phosphorylated state,
11
MEF2A dimerized with MEF2D reducing its binding activity (Sparling et al., 2007).
AMPK activation has shown to up-regulate GLUT4 gene expression. AMPK activation with AICAR increased binding activity of MEF2A (Buhl et al., 2001; Zheng et al.,
2001). Once activated, AMPK phosphorylates and activates GEF in cytosol (Holmes et al., 2005). Next, GEF recruits MEF2A and together they translocate to the nucleus, although MEF2A is not directly phosphorylated by AMPK.
It has been speculated that activation of the mitogen-activated protein kinase p38 (p38-
MAPK) could lead to MEF2 hyper-phosphorylation and uncoupling of the
GEF/MEF2A complex (Sparling et al., 2007). Chronic hyperinsulinemia led to activation of p38 MAPK with subsequent reduced GLUT4 protein level in adipocytes from type 2 diabetic patients (Carlson et al., 2003; Carlson & Rondinone, 2005). In contrast, selective pharmacological inhibition of p38MAPK prevented the development of insulin resistance by preserving GLUT4 expression and insulin-stimulated glucose uptake. p38-MAPK has shown to be activated in a cAMP-dependent PKA activation manner following β-adrenergic stimulation (Moule & Denton, 1998). Likewise, FFA released from lipolysis can activate p38MAPK and mediate pro-inflammatory cytokine expression in adipose tissue (Mottillo et al., 2010), like TNF-α, which has also been involved in GLUT4 downregulation (Im et al., 2007).
1.4.5 The role of HDACs in GLUT4 expression
Histone deacetylases mediate the repression of the GLUT4 promoter and this is a major mechanism responsible for regulation of GLUT4 gene expression (Weems & Olson,
2011). These enzymes are members of an ancient protein superfamily, the main role of which is to remove acetyl groups from lysine residues on histones resulting in an increased electrostatic interaction between histones and DNA, thus reducing
12 transcription (Leipe & Landsman, 1997). Eighteen different HDACs have been discovered in mammals, which have been categorized into four distinct families: the class I, II and IV HDAC and the class III NAD-dependant enzymes of the Sirt family
(Kouzarides, 2007). HDAC1, 2, 3, and 8 are members of the class I HDACs; HDAC4,
5, 6, 7, 9, and 10 are class II HDACs; the sirtuin family members, Sirt1, Sirt2, Sirt3,
Sirt4, Sirt5, Sirt6, and Sirt7, are class III HDACs; and HDAC11 is the only class IV
HDAC (Eom & Kook, 2014).
Class IIa HDACs have been shown to negatively influence adipocyte differentiation (Lu et al., 2000; Kim et al., 2009; Chatterjee et al., 2011; Weems & Olson, 2011). GLUT4 appears late during adipocyte differentiation and this has been related to class II
HDAC4, 5, and 9-GLUT4 association in pre-adipocytes (Kim et al., 2009; Weems &
Olson, 2011). GEF and MEF2-mediated GLUT4 expression relies on dissociation of
HDACs (McGee, 2007; Sparling et al., 2008; McGee & Hargreaves, 2011). HDAC5 co- transfection strongly inhibited both GEF and MEF2A-dependent transcriptional activation and reduced GLUT4 expression.
Class II HDACs act as transcriptional repressors mainly by direct association with other transcription factors to their target binding elements in a phosphorylation-dependent manner (Eom & Kook, 2014). In a cardiomyocyte hypertrophy model, synergistic activation of CaMKII and mitogen-activated protein kinase (MAPK) released HDAC repression on MEF2-mediated gene transcription (Lu et al., 2000). During exercise and
AICAR treatment, AMPK translocates into the nucleus of skeletal muscle fibers and phosphorylates HDAC5 on Ser259 and Ser498, both key phosphorylation motifs required for HDAC dissociation from MEF2 (McGee, 2007; McGee & Hargreaves,
2008; McGee et al., 2008; McGee et al., 2009; McGee & Hargreaves, 2010a, b, 2011).
Upon phosphorylation, HDAC is exported from the nucleus by 14-3-3 chaperone
13 protein in a chromosome region maintenance-1-dependent manner, which unlinks
HDAC5 from the GLUT4 promoter leading to its upregulation (McGee et al., 2008;
Sparling et al., 2008). In adipose tissue, AMPK has shown to be activated in response to lipolysis (Gauthier et al., 2008), metformin treatment in T2DM (Boyle et al., 2011), exercise training (Takekoshi et al., 2006), AICAR(Jessen et al., 2003) and isoproterenol treatment (Moule & Denton, 1998).
Chronic activation of AMPK by injection of 1mg/g body weight of AICAR during 5 consecutive days in mice increased GLUT4 gene expression to a similar extent than endurance training (Holmes et al., 1999). This effect was completely abolished in the absence of PGC-1α, suggesting that AMPK-mediated regulation of GLUT4 expression is exerted through activation of PGC-1α (Leick et al., 2010). It has been shown that
PGC-1α directly induces GLUT4 expression in skeletal muscle (Calvo et al., 2008).
This function is regulated by AMPK, which increases PGC-1α gene expression (Irrcher et al., 2008) and action via direct phosphorylation (Jager et al., 2007). It is well known that adipose and muscle cells in culture have reduced GLUT4 expression level. Ectopic adenoviral expression of PGC1-α in cultured L6 myotubes and C2C12 myoblasts completely rescued GLUT4 mRNA to levels observed in vivo. Transfected C2C12 cells with GLUT4-luciferase reporter gene showed 6-to-10-fold increase in GLUT4 promoter activity exposed to PGC1-α. This effect was mediated by PGC1-α-induced transactivation of the MEF2C isoform (Michael et al., 2001). On the other hand, MEF2 exerts a feedforward regulatory mechanism on PGC1-α by trans-activating its expression. The PGC1-α promoter region contains two binding sites that specifically bind MEF2. This function is repressed by HDAC5 which deacetylates histones at one of the MEF2 binding site in the PGC1-α promoter (Czubryt et al., 2003). . The repressive
14 effect of HDAC5 on PGC1-α is reversed by CaMK-mediated HDAC5 phosphorylation and subsequent inactivation (McKinsey et al., 2000).
1.4.6 Potential involvement of histone acetyltransferases
HATs are enzymes that transfer an acetyl group from acetyl-coA to lysine residues neutralizing positive charges on histones, decreasing the ability of histones to bind to
DNA, thus allowing transcription (Choudhary et al., 2009). Conversely, reversal of acetylation correlates with transcriptional repression (Kouzarides, 2007). Therefore, net transcriptional rate at any given moment is the balance between HDAC and HAT activities (McGee, 2007). To date, GLUT4-specific histone acetyltransferases have not been identified. However, recent reports have described the indirect effect of p300
(Ciccarelli et al., 2016) and GCN5 (Kelly et al., 2009) acetyl transferases in the regulation of GLUT4 in skeletal muscle. The homeotic transcription factor Prep1 has been identified as downstream effector of high glucose-mediated activation of NF-κB - p65. L6-myofibers exposed to a hyperglycaemic environment (25 mmol.l-1 glucose) in vitro as well as in vivo skeletal muscle tissue from STZ-diabetic mice showed activation of NF-κB-p65 which recruited SET7 histone methyltransferase and p300 acetyltransferase to the 5’ region of Prep1, leading to enhanced transcription. Induction of Prep1 led to the recruitment of the repressor complex MEF2/HDAC5 to the GLUT4 promoter reducing its activity (Ciccarelli et al., 2016). Conversely, Prep1 knockout showed altered adipogenic differentiation with increased GLUT4 expression in 3T3-L1 adipocytes (Penkov et al., 2016). In addition to the role of PGC-1α, the β isoform also regulates GLUT4 expression in skeletal muscle, and this function is controlled by
GCN5 acetyl transferase. In human embryonic kidney cells (HEK-293), GCN5 acetylated PGC-1β in several lysine residues which modified its spatial sub-nuclear
15 localization to nuclear foci and repressed its transcriptional co-activation activity on
GLUT4 (Kelly et al., 2009).
1.5 ADIPOSE TISSUE GLUT4 EXPRESSION
In order to understand the mechanisms responsible for the regulation of GLUT4 expression in adipose tissue, it is relevant to consider how glucose and lipid metabolism are linked. Lipid metabolism in adipose tissue is ultimately regulated by synergistic interaction between transcription factors and their gene products, enzymes involved in
FA turnover. Moreover, many of the master regulators of lipid metabolism are also implicated in insulin signalling pathways and GLUT4 regulation, which highlights the close interaction between glucose and lipid metabolism within the adipocyte. The following section will bring together evidence of the role of adipogenic and lipogenic transcription factors in the expression of GLUT4 in the adipose tissue.
1.5.1 SREBP-1c
Also known as adipocyte determination and differentiation dependent factor 1 (ADD1), it is a trans-acting factor that regulates the expression of a range of enzymes required for endogenous cholesterol, FAs, TG and PL synthesis (Eberle et al., 2004; Im et al., 2007) including FAS, GPAT, LPL and leptin (Kim et al., 1998). SREBP-1c is highly expressed in liver, white adipose tissue, skeletal muscle, adrenal glands and the brain
(Eberle et al., 2004). It corresponds to a basic helix-loop-helix-leucine zipper of 1150 amino acids protein, synthesized as an inactive precursor in the ER. It contains three main domains: 1) NH2-terminal transactivation domain that contains a DNA-binding and dimerization region, 2) two hydrophobic transmembrane spanning segments, and 3) the COOH-terminal regulatory domain (Eberle et al., 2004). The inactive precursor protein is cleaved in the ER by a two-step cleavage process releasing the active NH2-
16 terminal domain. In this active form, SREBP-1c is imported into the nucleus binding to an importin carrier. Insulin stimulation induces SREBP-1c processing to its mature form. Nutritional states (fasting/refeeding) in mice (Kim et al., 1998) as well as insulin treatment in 3T3-L1 adipocytes have shown to regulate SREBP-1c mRNA expression
(Griffin & Sul, 2004). In STZ-induced diabetic Sprague-Dawley rats, re-feeding and insulin treatment induced GLUT4 mRNA expression in epididymal WAT mainly through SREBP-1c induction (Im et al., 2006). Chromatin immunoprecipitation experiments localized a putative SREBP-responsive element (SRE) in the human
GLUT4 promoter (Im et al., 2006). This corresponds to a conserved sequence
(TGGGGTGTG) located at – 109/ - 100 bp upstream GLUT4 transcriptional initiation site. In addition, SREBP-1c seems to act synergistically with Sp1, which binding site is located next to the SRE (-132/-122 bp) binding site (Im et al., 2006; Im et al., 2007).
The recruitment of the transcription activator Sp1 to the GLUT4 promoter is critical for
SREBP-1c-mediated GLUT4 induction. Sp1 binding capacity is highly regulated by methylation of a single cytidyl-3', 5'-phospho-guanylyl dinucleotide region (CpG) within the Sp1-binding site, which methylated state is modulated upstream by oestrogen receptor-β (ERβ) (Ruegg et al., 2011).
1.5.2 LXRs
Considered a cholesterol sensor, LXRs are involved in the regulation of adipose tissue
FA metabolism and TG storage (Juvet et al., 2003). LXRs correspond to a nuclear hormone receptor family activated by endogenous ligands, intermediate metabolites or end products of cholesterol metabolism, the cholesterol-derived oxysterols (Dalen et al.,
2003). Two isoforms have been described: LXRα and LXRβ, and both dimerize with
RXR. LXRα is highly expressed in tissues involved in lipid metabolism such as liver,
17 adipose tissue and skeletal muscle and also macrophages and kidneys, whereas LXRβ is ubiquitously localized (Juvet et al., 2003). LXRα functions as a transcription factor, increasing SREBP-1c gene expression (Laffitte et al., 2003). Through its function on
SREBP-1c transcription, LXR increased the expression of lipogenic enzymes involved in FA re-esterification (Tontonoz & Mangelsdorf, 2003). LXRs finely tune glucose metabolism between liver and adipose tissue suppressing genes involved in hepatic gluconeogenesis and increasing GLUT4 expression in adipose tissue, therefore, reducing glucose intolerance in a model of diet-induced insulin resistance (Laffitte et al., 2003). Ligand activation of LXRs increased GLUT4 gene expression in adipose tissue (Dalen et al., 2003). LXR binding element (LXRE) is located between -473 and -
457 bp in humans, and -417 to -402bp in mice GLUT4 promoter (Dalen et al., 2003).
The LXR-RXR heterodimer forms a complex with GLUT4-LXRE. LXRα, but not
LXRβ, selectively increased the basal GLUT4 transcription rate. Interestingly, acute inhibition of FA mitochondrial import resulted in GLUT4 down-regulation in adipose tissue, mainly due to the loss of LXR signalling owing to impaired ligand-mediated
LXR activation (Griesel et al., 2010). In support of that, treatment with pamitoyl-L- carnitine, a CPT-1 substrate and LXR agonist restored GLUT4 levels. In contrast,
Etomoxir, an inhibitor of CPT-1, reduced LXRα association with the GLUT4 promoter and repressed GLUT4 expression (Griesel et al., 2010). In adipose tissue, fasting downregulates GLUT4 expression, effect mediated by the activation of CREB, downstream β-adrenergic-mediated PKA activation. Upon activation, CREB recruits the histones deacetylases HDAC4 and HDAC5 to the LXR-response element on the
GLUT4 promoter. This effect is reversed by agonist-mediated activation of LXR
(Weems et al., 2012). Additionally, LXRs have been described as glucose sensors in the liver (Mitro et al., 2007). Both glucose and glucose-6-phosphate induced LXR/RXR
18 recruitment to LXR target genes, therefore acting as physiological LXR ligands with similar efficacy to that of oxysterols (Mitro et al., 2007). Furthermore, LXRs play a crucial role in the regulation of cellular substrate utilization by switching on lipid and off glucose oxidation (Stenson et al., 2009).
1.5.3 KLF15
KLF15 is a member of the Krüppel-like subfamily of Cys2/His2 zinc finger DNA binding proteins which trans-activates the GLUT4 transcription by binding to a consensus DNA sequence (CACCCCACCG) located at -499/ -503 bp in the proximity of MEF2 binding site (Gray et al., 2002; Banerjee et al., 2003; Mori et al., 2005).
KLF15 and MEF2A work synergistically to induce GLUT4 expression (Mori et al.,
2005).
There is another member of the KLF family, the KLF2 which has been involved in adipocyte differentiation as a negative regulator of adipogenesis, mainly due to directly inhibiting PPAR-γ expression and its function as master regulator of adipogenesis
(Banerjee et al., 2003). In addition, KFL2 inhibited the expression of C/EBPα and
SREBP-1α, both responsible of inducing and maintaining PPAR-γ expression (Banerjee et al., 2003). The latter could be considered a possible indirect mechanism for the delayed appearance of GLUT4 during adipocyte differentiation, since both factors play important roles regulating the expression of GLUT4.
1.5.4 C/EBPα
The C/EBP family of proteins has been described as pleiotropic modulators of energy homeostasis at the gene level (Kaestner et al., 1990) and also as an important regulator of adipocyte differentiation. The C/EBP family is composed by four isoforms: -α, -β, -γ
19 and CHOP10, each one of them expressed at specific times during adipogenesis.
Characteristically, C/EBP family members contain a DNA-binding domain in their C- terminal and a transcriptional activator/repressor section in their N-terminal (Wu et al.,
1999). The C/EBP-α isoform has been related to insulin-stimulated glucose uptake and
GLUT4 trafficking in adipocytes (Hemati et al., 1997; Wertheim et al., 2004).
Adipocytes differentiated from C/EBP (-/-) fibroblasts showed marked decreases in the expression of InsR and IRS-1 mRNA (Wu et al., 1999). C/EBP-α expression is regulated by insulin (Hemati et al., 1997), glucocorticoids (MacDougald et al., 1994) and TNF-α (Stephens & Pekala, 1991, 1992). The GLUT4 promoter contains a functional C/EBP-binding site located at -258 bp upstream from the mouse transcriptional initiation site (Kaestner et al., 1990). Phosphorylation of C/EBP-α on
Ser21 was required for full expression of GLUT4 and adiponectin in adipocytes (Cha et al., 2008). Members of the MAPK family, ERK1/2 and p38, phosphorylate C/EBP-α on
Ser21 (Cha et al., 2008). Single point mutation of this phosphorylation motif to alanine reduced the expression of GLUT4 by 40% in mice, which subsequently developed hyperglycaemia and glucose intolerance (Cha et al., 2008). Although, phosphorylation at other sites on C/EBP-α may change its DNA-binding capacity, it is likely that
C/EBP-α-mediated GLUT4 regulation is dependent on the population of co-activators and co-repressors present in each context (Wertheim et al., 2004). Insulin-mediated activation of p38 MAPK in adipose tissue may induce C/EBP-α phosphorylation at different motifs, other than Ser21, thus attenuating the affinity of C/EBP-α for its DNA- binding site on the GLUT4 promoter, thereby reducing GLUT4 gene expression
(Carlson et al., 2003; Carlson & Rondinone, 2005). Additionally, incubation of 3T3-L1 adipocytes in a hyperinsulinemic media rapidly dephosphorylated C/EBP-α on sites located within p30 and p42, probably mediated by activation of protein phosphatases (1
20 or 2A). Insulin has proven to directly reduce C/EBP mRNA and stimulates the expression of the dominant-negative form of C/EBP, the liver inhibitory protein (LIP) which inactivates C/EBP-α (Hemati et al., 1997).
TNF-α
Tumor necrosis factor alpha (TNF-α) is a cell surface 26 kDa cytotoxic transmembrane protein synthetized from a trimer precursor by TNF-α converting enzyme (Kriegler et al., 1988). Synthesized predominantly by adipocytes, TNF-α exerts an autocrine/paracrine role in adipose tissue as well an endocrine influence acting as a multi-functional regulatory cytokine, implicated in inflammation, cell apoptosis and survival, cytotoxicity, production of other cytokines and reduction of insulin sensitivity
(Ruan & Lodish, 2003, 2004). Its expression has shown to be increased in animal and human models of obesity, where it induces insulin resistance (Hube & Hauner, 1999,
2000). This cytokine binds two different membrane receptors: a 60-kDa TNF receptor subtype (p60-TNFR) and an 80-kDa TNF receptor subtype (p80-TNFR). The signalling pathway from the interaction of TNF-α with its 60 kDa receptor has been suggested to be involved in GLUT4 transcriptional repression (Hube & Hauner, 2000). Three different intracellular mechanisms have been postulated by which TNF-α interferes with
GLUT4 activity and expression (Warzocha et al., 1995). First, TNF-α activates phospholipase A2 (PL-A2) resulting in the production of arachidonic acid (AA), and the activation of phosphatidycholine-specific phospholipase C (PC-PLC) with subsequent production of DG and activation of PKC. This mechanism has been involved in blocking insulin signalling leading to reduced GLUT4 translocation (Corcoran et al.,
2007). Secondly, the activation of the inflammatory pathway through nuclear factor NF-
κB, probably related to FA-induced repression of GLUT4 gene (Shoelson et al., 2003;
Im et al., 2007). Recent evidence has described two NF-kB binding sites in the GLUT4
21 promoter located at -83/-62 and -134/-113 bp (Furuya et al., 2013). Finally, TNF-α impairs the expression of target genes through direct interaction with transcription factors. Acute and/or chronic exposure to TNF-α has been shown to almost completely inhibit GLUT4 protein from either plasma membrane or inner membrane fractions
(Stephens & Pekala, 1992). C/EBP-α and GLUT4 mRNA levels were also decreased; the latter preceded by the first, which implies a loss in the effect of C/EBP-α on GLUT4 expression (Stephens & Pekala, 1991, 1992). Significant reductions in InsR and IRS-1 mRNA were also reported following a short exposition to TNF-α (Stephens & Pekala,
1992).
1.5.5 Peroxisome proliferator-activated receptor-γ
The PPARγ is member of a family of ligand-activated nuclear transcription factors involved in lipid and lipoprotein metabolism, glucose homeostasis, adipocyte differentiation, proliferation and apoptosis. Three PPAR isoforms have been identified:
-α, -β (or – δ) and –γ, each differing in tissue distribution and ligand specificity (Armoni et al., 2003; Armoni et al., 2006; Armoni et al., 2007). PPAR-γ is predominantly expressed in the intestine and adipose tissue (Armoni et al., 2005). Two human PPAR-γ isoforms have been identified: -γ1 and –γ2, PPAR-γ2 being an adipose-specific isoform.
Both –γ isoforms contain a C-terminal ligand binding domain, and the N-terminal containing distinct ligand-independent activation domains, which confers distinct activation capacities to each isoform (Armoni et al., 2006). PPAR-γ has a responsive element (PPARE) on GLUT4 promoter located between -66 and +168 bp at the GLUT4 promoter (Armoni et al., 2003). It exerts a negative effect on GLUT4 expression, which relies on its ligand availability and the presence of an intact MAPK phosphorylation motif on Ser112 (Armoni et al., 2007). In its un-bound state PPAR-γ dimerizes with
22
RXRα, and this complex attaches to its consensus DNA-sequence on the GLUT4 promoter. Next, the PPAR/RXR heterodimer recruits HDAC enzymes forming a co- repressor complex, thereby repressing GLUT4 promoter transcriptional activity
(Armoni et al., 2003). The endogenous PPAR-γ ligand, prostaglandin J2 reinforces this repressive effect on GLUT4 expression. Conversely, under the influence of its synthetic ligand rosiglitazone, PPAR-γ undergoes a conformational change that leads to de- attachment of co-repressors and to the attachment of co-activators, such as HAT enzymes, as well as other transcriptional factors. Finally, PPAR-γ is excluded from its
DNA-binding domain leading to transcriptional activation of GLUT4 gene. This phenomenon explains the anti-diabetic effect of thiazolidinediones, which promote
GLUT4 transcription via effect on PPARγ (Armoni et al., 2003).
1.5.6 Free-fatty acids
FAs have been shown to be negative regulators of the adipocyte glucose transport system (Long & Pekala, 1996b). Chronic exposure of fully differentiated 3T3-L1 adipocytes to arachidonic acid (AA, 20:4), the major polyunsaturated fatty acid present in mammalian cells, resulted in ~70% reduction in GLUT4 gene transcription and destabilization of GLUT4 mRNA (Armoni et al., 2005). Similar effects have been demonstrated utilizing eicosatetraeinoc acid (EYTA, 18:1) which decreased GLUT4 mRNA stability (McGowan et al., 1995; Long & Pekala, 1996a, b). The mechanisms by which AA exert its negative effects involve the production of PGE2. Arachidonic Acid
(AA) can be converted into prostaglandins, PGE2 and PGI2 (prostacyclin) by the COX pathway. PGE2 acts in an autocrine/paracrine fashion binding an specific membrane receptor coupled to an adenylate cyclase that activates protein Gi or Gs decreasing or increasing cAMP levels, respectively (Pegorier et al., 2004). Increased cAMP has been
23 linked to GLUT4 transcriptional repression through activation of NF1 (Cooke & Lane,
1999a). Down-regulation of GLUT4 can also be attributed to direct interaction of AA with yet un-identified transcription factors (Armoni et al., 2005). In addition, counterintuitively FAs can reduce the expression of PPAR-γ, thereby affecting GLUT4 expression. This effect might function as a protective strategy to prevent further dampening of GLUT4 expression. Other mechanisms involved in the negative effects of
FA on GLUT4 expression are: 1) activation of TNF-α (Kriegler et al., 1988; Warzocha et al., 1995; Hube & Hauner, 1999), 2) ligand-mediated activation of Toll-like receptor-
4 (TLR-4) signalling pathway leading to the activation of NF-κB (Schaeffler et al.,
2009), 3) reduction in the nuclear abundance of transcription factors, such as SREBP-1c
(Pegorier et al., 2004) and 4) activation of novel protein kinase theta (PKCθ), leading to insulin resistance (Warzocha et al., 1995).
1.5.7 FOXO1
Members of the FOXO family of proteins are ancient, evolutionarily conserved targets of insulin-like signaling that are important in regulating metabolism in response to changes in nutrient availability and environmental conditions (Barthel et al., 2005). This family of transcription factors is characterized by a 100-aa monomeric DNA-binding domain. It is assembled by three helices and two large loops or wings (the winged-helix structural motif) that are responsible for DNA-binding specificity. On its C-terminal,
FOXO1 contains an acidic serine/threonine-rich region that serves as a DNA-activation domain, whereas the N-terminal contains an alanine-rich region that correspond to a potential transcription repression domain (Armoni et al., 2007). FOXO1 is the most abundant isoform in insulin-responsive tissues such as the liver, adipose tissue, and pancreas. It has been involved in different cellular processes including cell survival, cell
24 cycle progression, DNA repair and insulin sensitivity (Armoni et al., 2006; Armoni et al., 2007; Im et al., 2007). GLUT4 gene expression is directly correlated with FOXO1 levels in insulin target cells. In fully differentiated adipocytes, FOXO1 directly binds to its DNA consensus sequence in the PPAR-γ2 promoter, thus down-regulating the expression of PPAR-γ2. FOXO1 also indirectly represses the PPAR-γ1 gene. Both direct and indirect effects on PPARs will lead to transactivation of GLUT4 gene expression. FOXO1 activity is regulated upstream by insulin-mediated PKB/Akt activation and subsequent phosphorylation of three hierarchical phosphorylation consensus sites, Thr24, Ser256 and Ser319. Once phosphorylated by PKB, FOXO1 is exported from the nucleus resulting in de-repression of PPAR-γ2 transcription and a negative effect on GLUT4 transcriptional rate (Armoni et al., 2006; Armoni et al.,
2007).
1.6 FACTORS INFLUENCING ADIPOSE TISSUE GLUT4 EXPRESSION
Adipose tissue protects the body from lipid accretion in other tissues such as the liver, blood vessels and skeletal muscle, by storing excess fat from the diet. To do so, adipocytes grow in size mainly through enlargement of a single central lipid droplet where newly formed TGs are stored. In normal conditions, TGs are stored in two different fat depots: 1) SCAT, located underneath the skin of the trunk and limbs, and 2)
VAT inside the abdominal cavity in association with internal organs. Each fat depot possesses distinctive morphological and functional characteristics that determine their metabolic and endocrine roles (Lefebvre et al., 1998; Yamamoto et al., 2010). Regional fat depots differ in characteristics such as cellular composition, sympathetic innervation, blood flow, secretion of adipokines (Giorgino et al., 2005), and their capacity for TG accumulation. Fat accumulation in the visceral depot has much more impact on health
25 compared with subcutaneous fat storage (Carey et al., 1997; Wang et al., 2005; Zhang et al., 2008).
TGs can be synthesized by esterification of either incoming FAs or de novo synthesized
FAs. Approximately 30% of lipolysis-derived FAs are re-esterified to TG in the adipose tissue (Edens et al., 1990). FAs are re-esterified to a glyceride-3-phosphate molecule, acting as backbone for the formation of TG. The rate of FA-re-esterification strongly relies on glycerol bioavailability (Ballard et al., 1967). The main source for glycerol production in adipocytes is glucose, which provides glyceraldehyde-3-phosphate for
FA-re-esterification and carbon chains for de novo synthesis of FAs (Flatt & Ball,
1964). This process highlights glucose uptake and metabolism in adipose tissue as important steps for lipogenesis.
Morphological changes involved in expanding fat storage in adipose tissue are commonly associated with functional alterations in the obese state, including: migration and activation of immune cells, which confers a pro-inflammatory state in the tissue, extracellular matrix accumulation & fibrosis, adipocyte cell death and cytokine secretion (Rosen & Spiegelman, 2014), impaired lipolytic activity (Gaidhu et al., 2010) and insulin resistance (Carey et al., 1997). In relation to the latter, recent reports have demonstrated that HFD-induced insulin resistance is a time-dependent, tissue-specific alteration, which initially blunts insulin action in the adipose tissue, then progresses towards compromised insulin action in the liver and skeletal muscle (Turner et al.,
2013). In several models of adipose tissue insulin resistance, including in vitro adipocyte lipid overload, glucose uptake is selectively affected over other functions of insulin including protein synthesis and its anti-lipolytic effect, demonstrating that insulin resistance in the adipose tissue is highly selective for glucose metabolism (Kim et al., 2015; Tan et al., 2015). Although adipose tissue plays a minor part in insulin-
26 mediated glucose disposal, contributing only with 10% of it, its major role in glucose homeostasis seems to rely on integrating glucose flux stimulus into a complex regulatory response that includes changes in its endocrine role, whole body lipid metabolism and insulin sensitivity (Ducluzeau et al., 2002; Herman & Kahn, 2006).
1.6.1 The importance of adipose tissue GLUT4
Adipose tissue insulin-mediated glucose uptake and the expression of the glucose transporter GLUT4 have been described to be of importance for both glucose homeostasis and whole-body insulin action. Adipose tissue GLUT4 expression is tightly regulated and is affected by changes in adipocyte cell size (Pedersen et al., 1992; Qi et al., 2009; Favre et al., 2011a), food status (fasting & re-feeding and high fat feeding)
(Berger et al., 1989; Kahn et al., 1989b; Zanquetta et al., 2006; Weems et al., 2012), insulin sensitivity (Kahn et al., 1989a; Garvey et al., 1991; Sparling et al., 2007;
Karnieli & Armoni, 2008), and exercise (Stallknecht et al., 1993; Hussey et al., 2011).
Adipose tissue GLUT4 down-regulation is a common defect in insulin resistant states including obesity, metabolic syndrome, polycystic ovary syndrome and gestational diabetes (Garvey et al., 1991; Pedersen et al., 1992; Carlson et al., 2003; Leguisamo et al., 2012; Chen et al., 2013; Shi et al., 2014; Tumurbaatar et al., 2017). Impaired adipose GLUT4 expression/function, with concomitant reduction in intracellular glucose flux, has been associated with dysregulated adipokine secretion (Graham et al.,
2006; Graham & Kahn, 2007; Qi et al., 2009; Favre et al., 2011a) and the development of hepatic and skeletal muscle insulin resistance (Abel et al., 2001). Adipose-specific
GLUT4 knockout mice developed impaired insulin action in muscle and liver leading to glucose intolerance and hyperinsulinemia (Abel et al., 2001). Conversely, overexpression of adipose GLUT4 reversed insulin resistance and diabetes in mice
27 lacking GLUT4 selectively in muscle (Carvalho et al., 2005). Thus, despite its limited role in glucose disposal, contributing only 10%, adipose tissue insulin-mediated glucose uptake results in critical metabolic cues that drive compensatory homeostatic mechanisms in other tissues to react to changes in adipose insulin sensitivity. It has been postulated that increasing adipose tissue glucose uptake and GLUT4 expression would lead to systemic insulin sensitizing effects by regulating the secretion of adipokines and other active molecules (e.g. adiponectin, retinol binding protein-4, branched FAs esters of hydroxy acids [FA-HA]) which act to enhance insulin action (Herman & Kahn, 2006;
Herman et al., 2012).
1.6.2 Adipose tissue GLUT4 over-expression and insulin action
As mentioned above, the pathogenesis of insulin resistance is characterized by tissue- specific alterations in a time-dependent manner. Most importantly, impaired glucose metabolism in the adipose tissue is associated with either dysfunctional or downregulated glucose transporter GLUT4. On the other hand, transgenic overexpression of GLUT4 selectively in the adipose tissue, although increasing fat mass through adipocyte hyperplasia (Shepherd et al., 1993) and de novo lipogenesis (Herman et al., 2012), prevented the development of glucose intolerance and whole-body insulin resistance (Gnudi et al., 1995; Carvalho et al., 2005). The systemic insulin sensitizing effect of transgenic adipose tissue GLUT4 over-expression can be attributed to two main phenotypic effects: 1) reciprocal regulation of NMNT (Kraus et al., 2014) and 2) activation of the lipogenic transcription factor ChREBP alpha and the induction of its beta isoform which increases de novo synthesis of FAHFAs with potent anti-diabetic effects (Herman et al., 2012; Yore et al., 2014; Moraes-Vieira et al., 2016). Firstly,
NMNT is the most strongly reciprocally regulated gene in white adipose tissue with
28 adipose specific GLUT4 knockout or GLUT4 overexpression in mice. Its expression is increased in WAT and liver of obese and diabetic mice. NMNT inhibition protects against diet-induced obesity by exerting a multilevel regulation of polyamine metabolism, a futile cycle leading to its oxidation and/or excretion, therefore increasing substrate consumption and cellular energy expenditure (Kraus et al., 2014). The second mechanism refers to the glucose-dependent induction of ChREBPα/β transcription factors and upregulation of de novo lipogenesis. Selective adipose GLUT4 over- expression resulted in increased glucose metabolism (glucose incorporation into TG, glyceride-glycerol, glyceride-fatty acids, lactate and CO2), with a differential increase in de novo lipogenesis (Tozzo et al., 1995). Increased GLUT4-mediated intracellular glucose flux increased the expression of ChREBP, but not SREBP-1c, therefore driving upregulation of lipogenic genes fas, acc1, scd1 and elovl6 and “in vivo” de novo lipogenesis in VAT and SCAT (Herman et al., 2012). Glucose-mediated activation of
ChREBPα resulted in transactivation of a novel, potent isoform ChREBPβ that is transcribed from an alternative promoter (exon 1b). This isoform is highly responsive to
GLUT4-mediated changes in glucose flux and a more potent activator of ChREBP transcriptional targets (Herman et al., 2012). Enhanced ChREBP-driven lipogenesis in adipose tissue leads to the production of lipid species characterized by a branched ester linkage between a fatty-acid and a hydroxy-fatty acid or FAHFAs. There is at least 16 different FAHFA members present in serum and in many tissues in humans and rodents
(Yore et al., 2014; Moraes-Vieira et al., 2016). These FAHFAs have proven favourable metabolic effects by stimulating insulin and glucagon-like peptide-1 secretion, and improving glucose tolerance and insulin sensitivity. Most of these effects are mediated by interaction with GRP120, a receptor for long-chain fatty expressed in the gut, adipose tissue, pancreas and immune cells (Oh et al., 2010). In adolescents with
29 impaired glucose tolerance or T2DM, the expression of both ChREBP isoforms has been reported to be decreased in the adipose tissue. Most importantly, improvement of glucose tolerance in this population was associated with increased adipose tissue
ChREBP and GLUT4 expression (Kursawe et al., 2013).
Collectively, these studies showed early involvement of adipose tissue in the development of insulin resistance, which is characterized by downregulation of glucose transporter GLUT4 and defective insulin-stimulated glucose uptake. Conversely, favourable changes in adipose tissue insulin sensitivity, through normalization of glucose uptake, improved de novo lipogenesis, and GLUT4 expression have potential whole-body insulin-sensitizing effects. An intriguing question remains that is to whether whole-body insulin resistance can be prevented by rescuing GLUT4 expression in the adipose tissue despite of obesity.
1.6.3 Nutritional factors influencing adipose tissue GLUT4 expression
Changes in nutrient availability during feeding and fasting transitions challenge the organism to adjust substrate partitioning in order to preserve metabolic homeostasis.
This response is mediated by hormonal regulation that includes sympathetic activation and whole-body insulin resistance during fasting that prioritize glucose utilization to the central nervous system, whilst mobilizing FAs through the stimulation of lipolysis in the adipose tissue. On the other hand, during feeding, or re-feeding after fasting, sympathetic activity is overdriven by increased circulating glucose and insulin levels that increase substrate uptake and utilization for energy production, or energy storage.
In adipose tissue, fasting is associated with a progressive decrease in glucose uptake in adipocytes, which is rapidly restored to normal, or transiently increased upon refeeding
(Kahn et al., 1988; Kahn et al., 1989b). Early studies have described the susceptibility
30 of adipose tissue GLUT4 expression to changes in dietary state (Kahn et al., 1988;
Berger et al., 1989; Kahn et al., 1989b; Camps et al., 1992). Prolong fasting (3 days) reduced GLUT4 protein content between 50% to 80% (Kahn et al., 1988; Berger et al.,
1989; Kahn et al., 1989b), and decreased mRNA to 36% (Kahn et al., 1989b) in rats.
Progression into fasting is characterized by sequential changes in the expression of genes involved in lipid metabolism in adipose tissue and the liver (Palou et al., 2008).
These responses include rapid downregulation of lipogenic genes SREBP1c, FAS and
GPAT in the liver at 4 hours of fasting, followed by the adipose tissue, which responded similarly but only after 8 hr, with marked reduction in the expression of the same set of lipogenic genes, and GLUT4. The effect of fasting on the expression of lipolytic genes also differed between the liver and adipose tissue, with the former showing upregulation of lipolytic genes PPARα, PDK4, FGF21 and CPT1 at 8 hour fasting and the latter responding only after 24 hours with increased gene expression of ATGL, and CPT1.
Lipogenic genes were rapidly up-regulated upon re-feeding in both tissues, while lipolytic genes were blunted. Adipose tissue GLUT4 mRNA was significantly decreased (~60%) at 8 hours fasting, which dropped even further at 24 hours and remained downregulated despite the restoration of food intake after 3-hour re-feeding
(Palou et al., 2008). This suggested the predominance of the “fasting mediators” over refeeding which allows a gradual normalization of glucose uptake in the adipose tissue in an attempt to balance insulin-mediated FAs uptake and re-esterification in the tissue.
In support of this suggestion, rats treated with the β-adrenoceptor antagonist propranolol
(20mg/kg body weight) showed increased adipose tissue GLUT4 expression at the end of 24-hour fasting, whereas during refeeding propranolol attenuated GLUT4 mRNA induction (Zanquetta et al., 2006). Increase in post-prandial adipose tissue blood flow has been described under the regulation of catecholamines and circulating glucose
31 levels (Ardilouze et al., 2004; Manolopoulos et al., 2012). Micro-infusion of 10µmol/L propranolol in SCAT blunted the glucose-stimulated increase in post-prandial blood flow, whereas α-adrenergic blockage did not alter this response, which demonstrated that post-prandial adipose blood flow is under β-adrenergic regulation in vivo in humans (Ardilouze et al., 2004). Together, these data suggest that propranolol treatment during refeeding may have blunted the normal increase in blood flood after meal ingestion occurring locally in the adipose tissue which would have diminished the access of hormones and other mediators that induce in expression of GLUT4. At the onset of refeeding, after prolonged fasting, both insulin, through its downstream transactivation of SREBP-1c (Im et al., 2006; Im et al., 2007), and glucose, by activation of LXR drive the expression of GLUT4 (Dalen et al., 2003; Mitro et al.,
2007).
The mechanisms regulating the expression of adipose GLUT4 during fasting involve recruitment of class II histone deacetylases HDAC4/5 to the LXR binding site on the
GLUT4 promoter, and β-adrenoceptor signalling activation that led to increase intracellular cAMP content and consequent activation of CREB (Weems et al., 2012).
In vitro stimulation with the β-adrenergic agonist isoproterenol, or forskolin, an adenyl cyclase activator, as well as in vivo over-night fasting in C57/BL6 mice showed phosphorylation and activation of CREB, which led to the enrichment of HDAC4/5 at the GLUT4 promoter. HDAC-specific siRNA knockdown abolished the inhibitory effect of increased cAMP on GLUT4 expression. Similarly, treatment with an LXR agonist completely reversed the effect of isoproterenol and HDAC4/5 association to the
GLUT4 promoter, demonstrating that the repressive activity class II histone deacetylases relied on the presence of a functional LXR binding element (Weems et al.,
2012). Additionally, activation of CREB has been reported inducing the expression of
32 the transcriptional repressor ATF3 which targets GLUT4 gene during fasting and in association with obese conditions (Qi et al., 2009).
1.6.4 Insulin resistance, type 2 diabetes mellitus, diet-induced obesity and adipose tissue GLUT4
The pathogenesis of type 2 diabetes mellitus is characterized by the progression of insulin resistance from impaired glucose tolerance and compensatory hyperinsulinemia to disrupted pancreatic β-cell function and finally an overt diabetic state. Insulin resistance is nowadays recognized as a tissue-specific alteration (Kleemann et al.,
2010). Studies analysing the effect of HFD on glucose homeostasis in rodents and humans have shown time-dependent defects in insulin action, with peak insulin resistance in the adipose tissue and the liver within days to weeks on the diet (Kleemann et al., 2010; Turner et al., 2013; Boden et al., 2015; Tan et al., 2015). The adipose- specific insulin resistance has been associated with either downregulated (Garvey et al.,
1991) or dysfunctional (Boden et al., 2015; Kim et al., 2015) GLUT4, in spite of preserved insulin signal transduction (Turner et al., 2013). The sequence of events leading to the development of diet-induced insulin resistance have been studied using the ApoE*3-Leiden (E3L) mouse, a model that resembles human lipoprotein metabolism (Kleemann et al., 2010). These mice were fed a HFD for 12 weeks and developed organ-specific insulin resistance in the liver and adipose tissue shortly after the diet was initiated. Adipose tissue insulin resistance was associated with: 1) marked decrease in glucose uptake, 2) accumulation of saturated FAs, due to up-regulation of cellular processes involved in lipid transport and metabolism, and 3) an inflammatory response characterized by activation of TGFβ, IL-6, TLR-signalling pathways, and increased expression of interleukin-1 receptor antagonist (IL-1r), CCL2/MCP1, PAI-1,
33 fibrinogen-α, β, and γ. Similarly, C57/BL6 mice fed on a HFD for 16 weeks developed early onset insulin resistance in both liver and the adipose tissue within days on HFD, and a much later detriment of insulin action in skeletal muscle (Turner et al., 2013).
Interestingly, although insulin resistance was demonstrated by the blunted inhibition of hepatic glucose production and decreased adipose tissue glucose uptake, insulin signalling, determined by Akt phosphorylation was not affected in all three insulin- sensitive tissues. Instead, impaired insulin sensitivity was accompanied by accumulation of tissue-specific signature of lipid species. While insulin resistant hepatic and skeletal muscle tissues accumulated DGs, adipose tissue preferentially synthesised and stored ceramides and sphingomyelins. Importantly, adipose tissue insulin resistance was characterized by a marked decrease in insulin-stimulated glucose uptake at 1 week, with no further changes over the subsequent 16 weeks on the HFD. Body fat mass and fat pad weight continued increasing throughout the diet intervention, which suggested intact insulin-mediated lipid transport and lipogenesis functions. Previous findings have described FATP1 expression to be elevated after short term HFD (Wiedemann et al.,
2014), with increased insulin-mediated FATP1 translocation (Wu et al., 2006). In support of the former finding of increased fat mass, intact insulin-mediated Akt activation and downstream involvement of mTORC1 signalling pathway may also contribute to the increased fat mass through stimulation of both lipogenesis and adipogenesis (Carnevalli et al., 2010; Ricoult & Manning, 2013). Additionally, insulin resistance has been reproduced in vitro by lipid-overload in 3T3-L1 adipocytes, which depicted impaired insulin action in hypertrophic adipocytes independently of inflammation (Kim et al., 2015). Adipocyte hypertrophy was associated with changes in cell morphology highlighted by enlargement of unilocular lipid droplet that ultimately altered cytosolic and cortical actin-cytoskeleton organization that led to impair insulin-
34 mediated GLUT4 trafficking (Kim et al., 2015). Likewise, insulin-stimulated GLUT4 vesicle tethering and fusion to the plasma membrane have been shown to be markedly impaired in human adipocytes isolated from insulin resistant patients (Lizunov et al.,
2013).
In contrast, calorie restriction has been shown to differentially regulate GLUT4 expression in the adipose tissue after diet-induced obesity in mice (Wheatley et al.,
2011). Eight weeks of HFD induced hyperglycaemia and insulin resistance and these alterations correlated with marked reduction in adipose tissue GLUT4 expression.
Restriction in calorie intake of a 30% improved insulin sensitivity and increased adipose tissue GLUT4 expression, an effect attributed to acetylation of histone 4 at GLUT4 promoter.
Adipose tissue insulin resistance is described to be selective for glucose metabolism in adipocytes, whilst leaving other insulin-mediated functions, i.e. lipolysis inhibition and protein synthesis intact (Tan et al., 2015). Distinctive impaired glucose uptake is observed across different insulin resistance models, including treatments with dexamethasone and TNFα, and siRNA-mediated knockdown of Akt, where insulin resistance is associated with defective activation of the insulin receptor and subsequent impaired signal transduction and altered GLUT4 translocation, despite intact GLUT4 expression. Conversely, marked adipose tissue insulin resistance, despite preserved insulin signalling, was associated with reduced GLUT4 expression in models of in vitro chronic hyperinsulinemia in adipocytes and in genetically obese (ob/ob) mice.
With respect HFD-induced insulin resistance in animal models, the contribution of adipose tissue GLUT4 downregulation to reduced whole-body insulin sensitivity is controversial (Cusin et al., 1990; Le Marchand-Brustel et al., 1990; Assimacopoulos-
35
Jeannet et al., 1995; Park et al., 2005). In Zucker diabetic fatty (ZDF) rats, a monogenic model of obesity-induced type 2 diabetes, where a mutation in the leptin receptor OB-R
(fa/fa) is associated with hyperphagia and leptin resistance, peripheral insulin sensitivity is progressively reduced as fat mass expands (Unger, 1997; Shiota & Printz, 2012).
Animals were completely insulin resistant by 10 weeks of age as demonstrated by marked hyperglycaemia, hyperlipidaemia, elevated expression of inflammatory markers, and increased glucose incorporation into lipids in adipose tissue (Liu et al.,
2002; Leonard et al., 2005). However, a hyper-responsiveness to insulin has been reported early on in life in the adipose tissue of ZDF (Penicaud et al., 1991; Pedersen et al., 1992). This condition occurred in the context of compensatory hyper-insulinaemia and hyperphagia that induced up-regulation of lipogenic genes such as FAS and ACC, and GLUT4 (Penicaud et al., 1991). These adaptations facilitated fat accretion in the adipose tissue. Most importantly, increased adipose tissue GLUT4 in young obese
Zucker rats gradually declined as a function of age and increased cell size. Furthermore, acute diabetes induced by STZ injection in 20-week old ZDF drastically reduced both adipose tissue glucose uptake and GLUT4 expression, thus implying that changes in
GLUT4 expression are potentially a major cause of alterations in insulin-stimulated glucose uptake in adipose tissue throughout the evolution of obesity and diabetes in
ZDF rats (Pedersen et al., 1992; Slieker et al., 1992). Notably, the administration of the insulin sensitizer YM268 (10mg/Kg), an analogue of thiazolidinedione, for 2 weeks ameliorated insulin resistance in 11-week old ZDF rats, and the mechanism by which this compound exerted such effect was through normalizing decreased adipose tissue
GLUT4 content (Shimaya et al., 1997).
In contrast, all human pre-diabetic states, including obesity, impaired glucose tolerance, insulin resistance, polycystic ovary syndrome and gestational diabetes, are characterized
36 by a reduction (~30 to 50%) in adipose tissue GLUT4 protein content (Garvey et al.,
1991; Rosenbaum et al., 1993; Garvey, 1994; Okuno et al., 1995; Marette et al., 1997;
Jensterle et al., 2008; Chen et al., 2013; Kursawe et al., 2013; Shi et al., 2014; Chuang et al., 2015). Adipose tissue GLUT4 protein content is markedly reduced in obese as well as in glucose intolerant and T2DM patients, whereas skeletal muscle GLUT4 is unaffected (Garvey et al., 1991; Garvey et al., 1992; Garvey, 1994). In obesity, adipose tissue GLUT4 downregulation is causative of insulin resistance in adipocytes and worsening of the insulin resistance is due to further reduction in GLUT 4 levels associated with expansion of the fat mass (Garvey et al., 1991). In this context, although
GLUT4 expression is significantly higher in SCAT than in VAT, negative correlation between GLUT4 expression, body fat percentage and insulin action exists in both adipose fat depots (Veilleux et al., 2009). In morbidly obese pre-menopausal women, insulin responsiveness and GLUT4 levels were assessed in three different fat depots:
SCAT, round ligament (deep abdominal pro-peritoneal) and greater omentum (deep abdominal intra-peritoneal). Results showed differential insulin-stimulated glucose uptake and anti-lipolytic effects in isolated adipocytes from these three fat depots with round ligament displaying the greatest insulin sensitivity which paralleled with higher adipose GLUT4 expression, followed by subcutaneous and omental adipose tissues
(Marette et al., 1997). Recently, healthy men subjected to excessive caloric intake
(~6000 Kcal/day) acutely developed marked systemic oxidative stress and insulin resistance (Boden et al., 2015). Impaired whole-body insulin action was attributed to extensive oxidation and carbonylation of adipose tissue GLUT4 that became dysfunctional and subsequently led to systemic insulin resistance (Boden et al., 2015).
One mechanism that has been reported mediating the development of HFD-induced insulin resistance is the activation of CREB. In obese mice, CREB is activated in
37 adipose cells, where it triggered the expression of the transcriptional repressor ATF3 leading to downregulation of target genes adiponectin and GLUT4 (Qi et al., 2009).
Mice fed a HFD as well as VAT from obese humans showed reduced ICER expression associated with an increase in the expression of CREB target gene ATF3 and decreased levels of adiponectin and GLUT4 (Favre et al., 2011a). Conversely, mice over- expressing adipocyte dominant-negative CREB were protected from HFD-induced insulin resistance, showing improved whole-body insulin sensitivity, increased adiponectin secretion, reduced adipose tissue inflammation and hepatic steatosis.
Activation of CREB is mediated by phosphorylation at a single motif, serine 133, by upstream kinase PKA. Previously, it was reported that chronic elevations in circulating insulin paradoxically potentiate catecholamine effect on cAMP-signaling in adipocytes
(Klemm et al., 1998; Reusch et al., 2000; Delghandi et al., 2005; Favre et al., 2011a).
However, evidence have shown that hyperinsulinemia led to the opposite effect interfering with PKA-mediated activation of CREB by either upregulation of phosphodiesterase 3B, therefore reducing intracellular cAMP content, or by weakening of the β2-adrenoceptor/PKA-RIIβ (regulatory subunit) complex through altering anchor protein AKAP, which decreased PKA-mediated CREB-Ser133 phosphorylation
(Enoksson et al., 1998; Zhang et al., 2005). CREB activity is tightly regulated by the level of the inducible cAMP early repressor (ICER) which plays a dominant-negative role by competing with all cAMP-responsive transcriptional activators of CREB for binding to CRE binding sites (Molina et al., 1993). Both factors are induced by increases in intracellular cAMP levels. While CREB rapidly induces the expression of target genes in response to stimuli, the repressor ICER restores their initial expression levels thereby permitting transient induction. Impaired ICER/CREB induction has been reported to promote pancreatic β-cell dysfunction and impair insulin exocytosis
38
(Abderrahmani et al., 2006; Favre et al., 2011b). Furthermore, blunted ICER negative feedback regulation on CREB activity is considered as the primary cause for β-cell dysfunction under conditions of hyperglycaemia (Cho et al., 2012). Restoration of adipose ICER has been considered as a potential therapeutic strategy to combat insulin resistance and its metabolic complications (Brajkovic et al., 2012).
Other anomalies associated with adipose tissue GLUT4 down-regulation in the context of obesity-induced insulin resistance include: 1) accumulation of intermediate lipid metabolites (e.g. prostaglandin E2 and J2, and ceramides) (Tebbey et al., 1994; Long &
Pekala, 1996a, b; Armoni et al., 2003; Armoni et al., 2005), 2) inflammation (Stephens
& Pekala, 1991; Stephens et al., 1997; Lumeng et al., 2007), and 3) altered expression and function of micro-RNAs (e.g. -29a, -93, -133, -222, and -223) (Horie et al., 2009;
Chen et al., 2013; Chuang et al., 2015; Zhou et al., 2016).
1.6.5 Exercise and adipose tissue GLUT4
Exercise training is an effective therapeutic intervention to increase insulin action in skeletal muscle from healthy obese and insulin-resistant individuals (Hawley & Lessard,
2008),(Kraniou et al., 2006; Hussey et al., 2012). Although, exercise-training adaptations occurring in muscle are considered essential for improving insulin sensitivity, its positive effects on glucose homeostasis are not limited to skeletal muscle, and exercise training can also ameliorate the adverse effect of HFD on adipose tissue insulin sensitivity (Gollisch et al., 2009; Higa et al., 2014). These effects could be due to improved adipocyte insulin responsiveness (Rodnick et al., 1987; Peres et al., 2005), mitochondrial biogenesis (Trevellin et al., 2014), improved lipolytic and oxidative capacities (Stephenson et al., 2013; Higa et al., 2014), improved hormonal profile
(Gollisch et al., 2009; Stanford et al., 2015b) and the activation of the thermogenic
39 program of white adipose tissue (Bostrom et al., 2012; Zhang et al., 2014; Stanford et al., 2015a). Exercise training has also been shown to increase adipose tissue GLUT4 expression in rodents and humans (Hirshman et al., 1993; Stallknecht et al., 1993;
Ferrara et al., 1998; Stallknecht et al., 2000; Hussey et al., 2011). Trained rats showed
2-fold increased insulin-stimulated glucose transport in association with improved insulin-to-InsR binding, and improved efficiency in GLUT4 trafficking from intracellular stores to the plasma membrane of fat cells (Vinten & Galbo, 1983; Vinten et al., 1985). Additionally, GLUT4 mRNA and protein levels have been reported to be increased following exercise training in rodents compared with age-matched and young sedentary controls (Hirshman et al., 1993; Stallknecht et al., 1993). In these studies, decreased glucose transport and total transporter number were observed in adipocyte of enlarged size and as animals aged. It was concluded that exercise-induced improvements in insulin sensitivity and GLUT4 levels were secondary to reductions in fat cell size. However, significant improvements in GLUT4 protein amount, maximal insulin-stimulated glucose uptake and GLUT4 translocation with minimal change in adipose cell size compared with age-matched sedentary controls have been reported four days of intense short-term training in trained rats (Ferrara et al., 1998). Thus, changes in adipose tissue GLUT4 expression and glucose metabolism may be consequence of functional adaptations within the cell and not necessarily as result of changes in adipose cell size.
In humans, exercise training improved adipose tissue insulin-stimulated glucose uptake and insulin-induced anti-lipolysis, and these adaptations were associated with the replenishment of TG stores in adipose tissue between exercise sessions in trained subjects. (Stallknecht et al., 2000). Isolated human adipocytes from trained subjects showed improved insulin-stimulated glucose uptake (Rodnick et al., 1987). Adipose
40 tissue GLUT4 downregulation was recovered to normal levels in patients with type 2 diabetes after 4 weeks of aerobic exercise training (Hussey et al., 2011). Furthermore, adipose tissue GLUT4 levels are inversely correlated with serum adipose-derived RBP4 levels, both reciprocally regulated by exercise training (Yang et al., 2005; Graham et al., 2006). However, the mechanisms by which exercise training increases adipose tissue GLUT4 expression remain to be elucidated. It has been hypothesised that myokines released by contracting skeletal muscles during exercise may influence adipose tissue metabolism and insulin sensitivity (Petersen & Pedersen, 2005; Pedersen,
2006). Myokines including FGF21, IL-15 and irisin were reported to mediate increases in adipose tissue thermogenic and adipogenic capacities, thereby influencing its overall metabolic function, including insulin action (Bostrom et al., 2012; Lee et al., 2014;
Pierce et al., 2015). Recently, it has been shown that transplantation of trained WAT conferred enhanced skeletal muscle glucose uptake and whole-body insulin action in recipient untrained mice even under HFD conditions (Stanford et al., 2015a; Stanford et al., 2015b). These effects were attributed to a marked up-regulation of genes involved in thermogenesis and mitochondrial biogenesis in the trained adipose tissue, but most importantly to the induction of FGF21 (fibroblast growth factor) in the recipient’s adipose tissue (Stanford et al., 2015a), which underscores a critical endocrine/paracrine role of adipose tissue. However, the direct effect of these myo/adipokines on adipose tissue GLUT4 expression has not yet been tested.
41
FIGURE 1.1 Regulatory mechanisms of GLUT4 expression in the adipose tissue.
42
1.7 SUMMARY
In conclusion, adipose tissue is recognized as a target organ for exercise-induced benefits. Adipose tissue glucose metabolism, in particular regulation of GLUT4 expression and function, are key components of feedback mechanisms controlling adipose tissue adipokine production and whole-body glucose homeostasis. Preserving
GLUT4-mediated glucose flux protects against insulin resistance by improving adipose tissue endocrine profile and metabolic function. Exercise training is an effective strategy to recover adipose GLUT4 expression in diabetic individuals, and potentially to slow or reverse the progression from insulin resistance to overt diabetes. The mechanisms involved in exercise-mediated adipose tissue GLUT4 up-regulation may include cross- talk communication between contracting muscles and adipose tissue, direct effects upon activity of transcription factors or other mediators such as microRNA, post- transcriptional modifications, autocrine/paracrine effects of adipokines, or local changes in adipocyte morphology and metabolism.
43
1.8 AIM AND OBJECTIVES
The primary aim of my doctoral studies was to better understand the effects of exercise on adipose tissue glucose transporter GLUT4 expression. A number of models and approaches were utilized in order to address the following objectives:
To investigate whether exercise training and/or increased muscle oxidative capacity
prevent the effect of HFD on adipose tissue GLUT4 in mice.
To determine if short-term training has an effect on the expression of adipose
GLUT4 in healthy individuals.
To assess the effect of exercise serum on the expression of GLUT4 in human
primary adipocytes in vitro, and whether circulating factors mediate the potential
effects of exercise on the expression of adipose tissue GLUT4.
44
2 CHAPTER TWO: DIET AND EXERCISE EFFECTS ON ADIPOSE TISSUE GLUT4
2.1 INTRODUCTION
Adipose tissue insulin-mediated glucose uptake and GLUT4 expression have been described to be of importance for both glucose homeostasis and whole-body insulin action. (Abel et al., 2001; Carvalho et al., 2005; Herman & Kahn, 2006). Despite its limited role in glucose disposal, adipose-specific GLUT4-KO mice developed impaired insulin action in skeletal muscle and liver leading to glucose intolerance and hyperinsulinemia as a direct result of limiting glucose flux into adipocytes (Abel et al.,
2001). These findings have led to the hypothesis that adipose tissue senses changes in circulating glucose and communicates this signal that drives compensatory homeostatic mechanisms in other tissues able to react to changes in adipose tissue insulin sensitivity
(Birnbaum, 2001).
Insulin resistance is recognized as a tissue-specific alteration following HFD (Kleemann et al., 2010) with metabolic compliance in the adipose tissue and the liver quickly compromised showing peak IR for glucose metabolism within weeks on the diet, while additional time is required for insulin action to deteriorate in skeletal muscle (Kleemann et al., 2010; Turner et al., 2013). These early defects in insulin action led to a rapid development of glucose intolerance and hyperinsulinemia (Turner et al., 2013; Boden et al., 2015). In particular, in the adipose tissue insulin-mediated glucose uptake is markedly impaired after one week of HFD, and remained blunted for up to 16 weeks regardless of an intact insulin-signaling pathway (Turner et al., 2013). Interestingly, adipose tissue GLUT4 is down-regulated in all insulin resistant states including obesity,
45 metabolic syndrome, and diabetes (Garvey et al., 1991; Rosenbaum et al., 1993;
Garvey, 1994; Okuno et al., 1995; Marette et al., 1997; Jensterle et al., 2008; Chen et al., 2013; Kursawe et al., 2013; Shi et al., 2014; Chuang et al., 2015). Conversely, favourable changes in insulin sensitivity in the adipose tissue, through normalization of glucose uptake, and GLUT4 expression, have potential systemic insulin-sensitizing effects (Carvalho et al., 2005). Exercise training ameliorates the adverse effect of HFD in adipose tissue (Gollisch et al., 2009; Higa et al., 2014). Isolated human adipocytes from trained subjects showed improved insulin-stimulated glucose uptake (Rodnick et al., 1987). Adipose tissue GLU4 expression is increased following ET in rodents and humans (Hirshman et al., 1993; Stallknecht et al., 1993; Ferrara et al., 1998; Stallknecht et al., 2000; Hussey et al., 2011). However, the mechanism by which ET exerts this effect is unknown. It has been hypothesised that increased muscle oxidative capacity in trained subjects may influence adipose tissue metabolism through a cross-talk mechanism, i.e. the release of cytokines by contracting muscles during exercise –
“exerkines” – that could drive training adaptations in the adipose tissue and potentially improve insulin sensitivity (Petersen & Pedersen, 2005; Pedersen, 2006; Bostrom et al.,
2012). The aim of the present study was to investigate whether GLUT4 is downregulated throughout the time-course of HFD and whether exercise training and/or increased muscle oxidative capacity prevent the effect of HFD on AT-GLUT4 in mice.
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2.2 METHODS:
2.2.1 Adipose tissue samples:
The following experiments were conducted in frozen adipose tissue samples collected in three previously published studies (Turner et al., 2013; Henstridge et al., 2014; Jordy et al., 2015). Briefly, the first study analysed time-dependent effects of HFD on whole- body glucose homeostasis and insulin action (Turner et al., 2013). Epididymal adipose tissue (EPI) samples collected from C57/BL6 mice at 1, 3 and 12 weeks of the diet intervention were analysed for GLUT4 protein content. The second study investigated the effect of exercise training in HFD-induced hepatosteatosis (Jordy et al., 2015).
Adipose tissue samples (EPI) from this study were analysed to determine the effect of incorporating exercise-training to HFD on the expression of adipose tissue GLUT4.
Lastly, the third study examined the potential protective effect of increased muscle oxidative capacity, a major adaptation to exercise training, on HFD-induced IR
(Henstridge et al., 2014). In the present study, EPI samples from wild type and muscle specific HSP72-Tg mice under chow and HFD conditions were analysed. In all studies, male 8-week old C57/BL6 mice were utilized. Mice selectively overexpressing HSP72 in skeletal muscle showed increased running endurance associated with improved muscle oxidative capacity reflected by increased mitochondrial number, basal and maximal mitochondrial respiration and ATP turnover. Most importantly, transgenic mice were protected from HFD-induced obesity, ectopic lipid deposition and IR, exhibited increased energy expenditure, and whole-body and skeletal muscle fat oxidation (Henstridge et al., 2014).
For the HFD time course study, 29 mice were utilized (1week chow, n =5, 1week HFD, n=5, 3week chow, n=5, 3week HFD, n=5, 12week chow, n=5, 12 week HDF, n=4). For
47 the HFD and exercise intervention a total number of 36 mice were utilized (chow-sed, n=10, HFD-sed, n=10, chow-4week training, n=8, HFD-4week training, n=8). For the
HSP72-Tg study, 20 mice were utilized (WT-chow, n=5, Tg-chow, n=5, WT-HFD, n=5,
Tg-HFD, n =5). Chow diet (5% energy from fat, Specialty Feeds, Glen Forrest, WA,
Australia) and HFD (43% or 45% energy from fat, Specialty Feeds) were utilized across the three studies.
The duration of HFD intervention was as follow: Study 1, HFD time course: 12 weeks of HFD (45% energy from fat), study 2: HFD and exercise intervention, 8weeks of HFD
43% energy from fat (42.9% saturated, 35.24% monounsaturated, and 21.86% polyunsaturated fatty acids), and study 3: HSP72-Tg: 10 weeks of HFD (45% energy from fat). Animals were bred in-house (AMREP Animal Services, Melbourne, VIC,
Australia), and maintained at 22±1°C on a 12/12 h light/dark cycle, with free access to their respective diets and water.
2.2.2 Adipose tissue homogenisation:
Approximately 150 mg of frozen tissue samples were homogenized in 250 µl of lysis buffer (20mM HEPES, pH 7.4, 2M EGTA, 50mM β-glycerophosphate, 1mM DTT,
1mM Sodium orthovanadate, 10% Glycerol and 1% Triton X, 0.1mM NaF, 0.1mM
PMSF) supplemented with proteases (Complete®, Roche, Inc.) and phosphatases
(PhosSTOP®, Roche, Inc.) inhibitor cocktail tablets. Tissue samples were homogenized on ice using an electronic homogenizer (Polytron, PT 1200E)) in 2x 20 seconds bursts.
Whole tissue homogenates were then centrifuged at 13,000 rpm for 10 min at 4oC. The upper fat layer was discarded and supernatants were transferred to new pre-chilled microtubes. Protein concentration in whole tissue homogenates was determined using
48 the bicinchoninic acid protein assay (BCA) (Pierce, Rockford, IL, USA) according to manufacturer directions.
2.2.3 Immunoblotting:
Whole-tissue lysates were solubilized in 4x Laemelli’s buffer containing 400mM DTT.
A solution of 1 mg.ml-1 concentration was prepared per sample. Protein lysates were then heated at 95oC for 5 min and spun at 13,000 rpm for one min. Proteins were separated and identified using SDS-PAGE. Same protein concentrations (15 to 30 µg per lane) were loaded into 4-20% Criterion TGX stain-free pre-cast gels (Bio-Rad, Inc.,
USA) and run at 150V for 60 min. Proteins were semi dried transferred onto Midi 0.2
μm PVDF membranes using the Bio-Rad Trans Blot® TurboTM system. Subsequently, membranes were blocked in blocking buffer (Tris-buffer saline with 0.1% (v/v) Tween-
20 [TBST]) containing either 5% BSA or 5% non-fat skim milk powder for one hour with continuous agitation, and then incubated overnight in primary antibodies.
Membranes were then washed 3 x 10 min in TBST and incubated in secondary antibody for 1 hour at room temperature (RT). Immunoblots were developed using enhanced chemiluminescence reagents (Amersham ECL Prime Western Blotting Detection
Reagent) in the ChemiDoc MP system and analysed using the Image Lab 4.0 software
(Bio- Rad, Inc., US). For phospho-proteins, the same membranes were stripped in 0.2M sodium hydroxide (NaOH) for 10 min, then washed 2x5min in TBST, blocked in blocking buffer for 1 hr and RT, and probed overnight with phospho-primary polyclonal antibodies. The light density of the proteins of interest was normalized over either β- actin as housekeeping loading control or total protein content, determined by total staining. Protein loading and transferring efficacy were verified by quantification of lane density in stain-free gels, ponceau staining or total protein staining of membranes.
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2.2.4 Primary antibodies:
Anti-GLUT4 rabbit polyclonal (1:1000 dilution, Thermo Fisher Scientific), anti-NF-κB
(p65) and anti-phospho NF-κB (Ser 536) rabbit monoclonal (1:500 dilution, Cell
Signaling Technology), β-actin polyclonal (1:1000 dilution, Cell Signaling
Technology), anti-ATF-3 rabbit polyclonal (1:500 dilution, Santa Cruz Biotechnology),
CREB/phosphor-CREB (Ser 133) (1:500 dilution, Cell Signaling Technology). All primary antibodies were prepared in 1x TBST containing 5% BSA (w/v) and 0.01% sodium azide (v/v). Secondary antibody: anti-rabbit IgG-horse radish peroxidase conjugated (Sigma-Aldrich Co., USA) 1:5000 in 1x TBST + 5% BSA (w/v).
2.2.5 Statistical analysis:
Data are presented as means + SEM of protein contents in adipose tissue samples across interventions and phenotypes. Effect of diet, exercise training and HSP72 genotype were compared using 2-way ANOVA and Bonferroni post hoc test. Statistical significance level was set at p<0.05.
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2.3 RESULTS:
2.3.1 Effect of HFD on body weight and fat mass gains
As reported in the primary studies the weight and fat gains were: In study 1, HFD
time-course: Body mass was not different between chow-fed and HFD mice after 1
week, although it increased significantly after from week 3 and thereafter. In
contrast, epididymal fat pad mass was increased by 3 days of the HFD and was not
further increased until 12 weeks of HFD. In study 2, wild type and HSP72Tg mice
were fed on chow and a HFD (45% of energy from fat) for 10 weeks. This dietary
intervention significantly induced a 30% body weight gain and a 3-fold increase in
fat pad mass in wild type mice. Ten weeks of HFD also induced fasting
hyperglycaemia and hyperinsulinemia in these animals. These effects were
completely prevented in HSP72Tg mice. In study 3, HFD and exercise intervention.
At the end of 4 weeks of HFD, body weight increased ~10%, whereas fat mass and
body fat percentage showed a marked ~2-fold increase. At the end of the 8-week
HFD intervention, body weight increased up to ~20%, and fat mass and percentage
of body fat up to 3-fold compared with chow fed sedentary mice. Conversely,
exercise training effective slowed the progression of body weight and fat gains in
HFD-fed and exercise trained mice.
2.3.2 Effect of HFD on epididymal adipose tissue GLUT4:
High fat diet markedly decreased GLUT4 content in EPI adipose tissue at 1, 3 and 12 weeks of HFD, with no difference between time points (9.4 + 1.2 vs 4.6 + 1.0 AU; 8.0 +
1.8 vs 4.1 + 0.4 AU, and 10.3 + 1.9 vs 5.5 + 0.7 AU at 1 week, 3 week and 12 weeks respectively, diet effect: p < 0.0001, Fig-2.1-A-B).
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2.3.3 Effect of HFD on CREB/ATF3 pathway:
Next, the protein content and phosphorylation status of CREB and ATF3 were analysed as a putative mechanism involved in GLUT4 downregulation (Qi et al., 2009). A significant increase in total CREB protein content was observed in HFD-fed animals at
12 weeks (Figure-2.2-A), although overall, there was no change in CREB phosphorylation state (Ser-133) (Figure-2.2-B). Total ATF3 protein content was reduced in fat fed animals when compared with control chow fed age-matched mice throughout the 12-week diet intervention (main diet effect, p = 0.0002, Figure-2.2-C).
2.3.4 Effect of HFD on TLR4/NF-κβ pathway:
Another mechanism that has been involved in adipose tissue GLUT4 downregulation is obesity-induced inflammation and downstream activation of NF-κβ through FFA- induce activation of toll-like receptor 4 (TLR4) (Shoelson et al., 2003; Schaeffler et al.,
2009). HFD transiently increased TLR4 protein content at 3 weeks (diet effect, p =
0.0072, time effect, p = 0.0002), which was significantly decreased later during the diet intervention (p < 0.0001, 3-week HFD vs 12-week HFD, Figure-2.3-A). Acute phosphorylation-mediated activation of NF-κβ was observed at 1 week HFD (p =
0.0405), however this response was lost in the following weeks (Figure-2.3-B).
2.3.5 Effect of 4 weeks of exercise-training on adipose tissue GLUT4 protein in mice fed on chow and HFD diets:
There were no significant changes in GLUT4 protein content in EPI following 8 weeks of HFD. Similarly, there was no difference in GLUT4 protein between sedentary and trained mice fed on normal chow. However, four weeks of exercise training
52 significantly increased adipose tissue GLUT4 protein content in EPI fat (p < 0.0001,
Figure-2.4-A) in HFD-fed mice.
2.3.6 Effect of muscle-specific HSP72-Tg over-expression on adipose tissue
GLUT4:
There was a marked HFD effect on adipose GLUT4 protein content in both wild type and HSP72-Tg mice (p < 0.05, Figure-2.5-A-B). Adipose tissue GLUT4 protein content tended to be reduced in transgenic mice fed a chow diet, although it did not reach statistical significance (p = 0.0866).
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FIGURE-2.1: Time-course effect of HFD in adipose tissue GLUT4 protein.
A) Quantification of GLUT4 protein content in EPI of C57/BL6 mice at 1, 3 and 12 weeks of chow and HFD. Values represent mean + SEM of GLUT4/β-actin ratio. B): Representative GLUT4 and β-actin immunoblots. Two-way ANOVA, (****): main diet effect, p < 0.0001.
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FIGURE-2.2: Time-course effect of HFD in CREB, p-CREB (ser133) and ATF3 protein
A) Quantification of CREB, and B) p-CREB/total protein, and C) ATF-3/ b-actin ratio in EPI of C57/BL6 mice at 1, 3, and 12 weeks of chow and HFD. Bottom panels: representative immunoblots for CREB and p-CREB-Ser133, total protein staining, ATF3 and β-actin. Protein abundance was normalized over total protein staining. Values represent mean + SEM of protein contents. Two-way ANOVA, Bonferroni post-hoc test, (a): Diet main effect, p < 0.0001, (b): time main effect, p < 0.0001, (c): interaction, p < 0.0001, (****): chow vs HFD at 12 weeks. (***): Chow vs HFD, diet effect: p = 0.0002.
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FIGURE-2.3: Time-course effect of HFD in the activation of TLR4/NF-κβ
Quantification of A): TLR-4/β-actin, and B): phospho-NF- κβ/total ratio in EPI of C57/BL6 mice at 1, 3 and 12 weeks of chow and HFD. Values represent mean + SEM. Two-way ANOVA, Bonferroni post-hoc test, (a): diet main effect, p = 0.0072, (b) time main effect, p = 0.0002, (**): 3-week HFD vs 1-week chow and 12-week HFD and (*) 3-week HFD vs 12-week chow. (*) HFD vs Chow at 1-week, p = 0.0405.
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FIGURE-2.4: Effect of High fat diet and exercise-training on adipose GLUT4 protein.
Quantification of GLUT4/total protein ratio in EPI SCAT of 8-week old C57/B6 mice fed a HFD and exercise trained compared with sedentary and chow fed controls. Bottom panel: representative immunoblots of GLUT4 and respective total protein staining. Values represent mean + SEM. Two-way ANOVA, (*) training effect, p = 0.0299.
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FIGURE-2.5: Effect of muscle-specific HSP72-Tg on adipose tissue GLUT4 protein.
A): Quantification of GLUT4/total protein ratio in EPI of wild type (WT) and muscle- specific HSP72-Tg mice (HSP-Tg) under chow and HFD diets. B): Representative Immunoblots of GLUT4 and β-actin, and respective total protein staining. Values represent mean + SEM. Two-way ANOVA, (*): main diet effect, p < 0.05.
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2.4 DISCUSSION
Data in the present study showed that HFD rapidly reduced adipose tissue GLUT4 in
mice. After one week of HFD, GLUT4 protein was decreased to approximately 50%
of its content, compared with chow fed animals. The initial study, from where the
frozen adipose tissue samples were taken, described defective insulin-stimulated
glucose uptake in the adipose tissue within the first week of HFD, which occurred
despite an intact insulin signalling pathway (Turner et al., 2013). Together, these
results suggest that HFD-induced adipose-specific IR, reflected as blunted insulin-
stimulated adipose tissue glucose uptake, is mainly influenced by a rapid reduction in
adipose tissue GLUT4 protein that is maintained at lower levels throughout the diet
intervention. A plausible explanation for this effect is the continuous presence of the
main dietary intervention, a 43% HFD. While this effect may have interfered with
GLUT4 mRNA expression, the rate of protein turn-over should have remained
constant; thereby protein abundance would have been maintained at a lower level.
Interestingly, the magnitude of the observed decrease in adipose tissue GLUT4
protein abundance at each time point throughout the course of HFD intervention is
similar to the reduced adipose tissue GLUT4 protein expression levels reported in
human obese insulin resistant subjects (Garvey et al., 1991). The latter is further
decreased in patients with non-insulin-dependent DM. Therefore, the 50% decrease
observed in the present study marks the appearance of adipose tissue insulin
resistance and in the absence of an overt diabetic state, adipose tissue GLUT4 protein
abundance is not further decrease.
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One of the mechanisms that has been implicated in HFD-induced IR in the adipose tissue is the activation of CREB, and the CREB-mediated recruitment of transcription repressor ATF3 to the GLUT4 and adiponectin gene promoter regions (Qi et al., 2009).
Activation of CREB has been shown to be induced under obesogenic conditions (Favre et al., 2011a). Elevated intracellular cAMP levels increase PKA kinase activity, which activates CREB by selective phosphorylation of a single motif at serine 133.
Phosphorylation of CREB can also be mediated by time-delayed PKA-dependent p38-
MAPK/MSK-1 pathway activation (Delghandi et al., 2005). In the present study, immunoblotting data showed no significant changes in CREB, phospho-CREB (Ser-
133) and ATF3 protein content throughout the diet intervention. The absence of increased phosphorylation of CREB after 12 weeks of HFD suggests that upstream mechanisms leading to its activation are not involved in the observed adipose tissue
GLUT4 downregulation following HFD, at least within the time-frame considered in this investigation. A similar decrease in adipose tissue GLUT4 has been described previously in 5-month old female Sprague Dawley rats, although following 6 weeks of
60% HFD (Gorres et al., 2011). Such an effect was attributed to decreased oestrogen receptor alpha (ERα) and HSP72 protein contents in adipose tissue. Interestingly, these alterations were associated to whole-body glucose intolerance in spite of intact insulin- stimulated glucose uptake and GLUT4 expression in skeletal muscles.
In relation to HFD-induced activation of inflammatory mediators, an early activation of
NF-κβ, determined by phosphorylation of its p65 subunit, at one week of the HFD was observed. However, this response occurred in the absence of increased TLR4 protein content. Furthermore, a transient increase in TLR4 protein was observed at week 3, but this effect was dissociated from NF-κβ activation. TLR4 is highly expressed in human adipose tissue in comparison with other members of the TLR family (TLR 1, 2, 7 and 8)
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(Vitseva et al., 2008). The TLR4-signaling pathway is activated in vitro by nutritional
FAs, and in vivo in murine models of obesity (Schaeffler et al., 2009; Kim et al., 2012).
Stimulation of TLR4 has been associated with NF-κB p65 nuclear activation and nuclear translocation, followed by upregulation of pro-inflammatory cytokines and chemokines including TNF-a, IFN-a/b, IL-6, IL-12-p40, CXCL2 (Vitseva et al., 2008;
Schaeffler et al., 2009; Kim et al., 2012). The present study showed TLR4 protein content to be transiently activated at 3 weeks, and decreased at 12 weeks of HFD in
C57/BL6 mice. This transient activation was not linked to the observed rapid activation of NFkB following one week of HFD.
Exercise training has shown to be effective in improving GLUT4 expression levels in both skeletal muscle and adipose tissue in patients with type 2 diabetes (O'Gorman et al., 2006; Hussey et al., 2011). Thus, the present study sought to examine whether exercise training could counteract the detrimental effect of HFD on adipose tissue
GLUT4. Four weeks of exercise training significantly increased adipose tissue GLUT4 protein content in mice previously exposed to HFD for four weeks. The changes in body weight and fat mass following the 8-week HFD alone and in combination with exercise have been reported elsewhere (Jordy et al., 2015), and showed a significant ~10% overall increase in the initial body mass with the diet, and the same relative reduction in body weight and fat mass gains following four weeks exercise training in the fat fed group. Thus, concurrent exercise training effectively modulated body weight and fat mass gains, and this effect was associated with increased adipose tissue GLUT4 content in the present study.
Contrary to the expected HFD effect on adipose tissue GLUT4, as reported in time- course study of this thesis, and despite the significant increases in body and fat mass reported previously (Jordy et al., 2015), no diet effect was observed on adipose tissue
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GLUT4 in the HFD sedentary group (Figure 2.5). This was an intriguing finding considering the robust HFD effect seen in the other two studies, and also taking into consideration that all the animals in the three studies received the same housing conditions and dietary composition, for both chow and HFD diets (refer to methods).
Likewise, it is worth noting that the early downregulation of adipose tissue GLUT4, observed during the first week of HFD (Figure 2.1), occurred despite of minimal changes in the fat mass at this point during the HFD, as reported in the primary study
(Turner et al., 2013). In view of this discrepancy, changes in adipose tissue GLUT4 protein have been inversely correlated to age and adipose cell volume/size, with reduced expression of adipose tissue GLUT4 protein in hypertrophied adipocytes of older obese animals in comparison with control young mice (Pedersen et al., 1992). Furthermore, the same pattern has been observed in humans, where obese subjects with preserved insulin sensitivity showed greater adipocyte cell number, smaller cell size and increased expression of differentiation markers and GLUT4, despite the enlargement of adipose mass (McLaughlin et al., 2007). Although speculative, it is possible that that the observed lack of downregulation of adipose tissue GLUT4, despite increased body weight and fat mass (Jordi et al., 2015) was related to differential contribution of either cell volume (hypertrophy) or cell number (hyperplasia) following HFD; hence a larger fat mass, endorsed by increased cell number rather that enlargement of individual cells, would result in a relative greater GLUT4 content per gram of fat mass compared with increased fat mass due to fewer but enlarged adipocytes. Taken together, data presented in this thesis and previous reports suggest variability in body weight and fat mass phenotypes in relation to HFD, and more importantly, that changes in the GLUT4 content in a particular fat pad may be related to overall changes in cell size and number irrespective of variations in fat pad mass.
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Exercise training significantly increased adipose tissue GLUT4 protein in the HFD trained group. Early reports have shown increased GLUT4 content in adipose tissue after 6 weeks (Hirshman et al., 1993) or 10 weeks (Stallknecht et al., 1993) of exercise training in Sprague Dawley rats. Interestingly, significant changes in insulin-stimulated glucose uptake, GLUT4 translocation and protein content have been described following only 4 days of swimming training in rats, and this response occurred with a minimal change in adipose cell size (Ferrara et al., 1998). In addition, another intriguing finding of the present study was the absence of a response to exercise training on adipose tissue GLUT4 in CHOW fed animals. As mentioned above, previous findings have described exercise training effects on adipose tissue GLUT4 expression in sedentary healthy rats, although these findings were associated with prolonged and relatively more intense exercise regimes: 1) six weeks of running distance-controlled training (Hirshman et al., 1993), and 2) 10 weeks, five days/week of gradually increased (up to six h/day) swimming training (Stallknecht et al., 1993). Moreover, the short-term training effect described previously was also related to a more intense exercise with four days of 2x3h/day swimming training (Ferrara et al., 1998).
Notwithstanding the obvious differences between murine species (mouse versus rat), it is likely that the lack of a training effect on adipose tissue GLUT4 from chow fed mice in the present study, was associated with a shorter (four weeks versus six and ten weeks), less intense (five day/week, 30 to 60 min at 50% to 80% of max speed incremental treadmill running) (Jordy et al., 2015) exercise training.
Improvements in the capacity to utilize energy substrates during exercise in skeletal muscle, i.e. increased oxidative capacity, is one of the most important adaptations that occur in relation to exercise training. Notably, increased cellular energy status and substrate turnover, both outcomes of the trained status, occur in parallel with the
63 expansion of mitochondria and an increased functional capacity of oxidative enzymes.
In conjunction, these adaptations elicit an improved metabolic adjustment in skeletal muscle to the energy demands imposed by the physical effort. Furthermore, improved skeletal muscle oxidative capacity may have implications beyond the muscle itself, and convey the muscle's higher metabolic requirements through a signal towards the rest of the body that communicates its need for blood and nutrients supplies. It has been hypothesised that increased skeletal muscle oxidative capacity may mediate some of the adaptations occurring in the adipose tissue following exercise training (Bostrom et al.,
2012; Villarroya, 2012). For instance, PGC-1α is described as playing a pivotal role in the regulation of oxidative metabolism in skeletal muscle (Baar et al., 2002; Puigserver
& Spiegelman, 2003; Olesen et al., 2010; Perez-Schindler et al., 2014). The expression and secretion of the myokine “irisin” is dependent on the activation of muscle PGC-1α during exercise, which has the potential to re-model adipose tissue metabolism by increasing both oxidative and thermogenic capacities (Bostrom et al., 2012; Zhang et al., 2014).
In the context of this thesis, and considering the accessibility to tissue samples from wild type and HSP72Tg mice, it was interesting to explore whether an increased muscle oxidative capacity generated a phenotype on the adipose tissue, in particular on GLUT4.
With this purpose, EPI adipose tissue samples from muscle-specific HSP72-Tg mice were analysed and compared with samples from wild type mice. It has been shown that these mice display increased skeletal muscle mitochondrial number, and improved oxidative and endurance capacities (Henstridge et al., 2014). Moreover, these mice were protected against HFD-induced increases in body weight and fat mass, inflammation and insulin resistance (Chung et al., 2008; Henstridge et al., 2014). In the present study, however, this phenotype did not prevent HFD-induced downregulation of adipose tissue
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GLUT4 protein. Of note, adaptations associated with increased muscle oxidative capacity driven by muscle-specific HSP72 over-expression were not related to changes in PGC-1α expression (Henstridge et al., 2014). Thus, it is possible that the beneficial effects occurring in the adipose tissue following exercise training, associated with increased muscle oxidative capacity, were related to the activation of other mediators in skeletal muscle, e.g. PGC-1α, that potentially have a more relevant role on cross-talk communication mechanisms toward adipose tissue. Regardless of the potential intervention of a myokine on the regulation of adipose tissue GLUT4, the local obesogenic microenvironment imposed by the HFD in the adipose tissue may have overdriven the endocrine effect of such mediator.
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2.5 CONCLUSION
Taken together, data in the present study showed that HFD rapidly reduced GLUT4 protein content in adipose tissue which may explain the development of adipose tissue
IR. Neither CREB/ATF3 nor TLR4/NF-κβ pathways were activated following 12 weeks of HFD in mice; therefore, these mechanisms cannot account for HFD-mediated adipose
GLUT4 downregulation. Concurrent exercise-training increased GLUT4 protein content in adipose tissue, although this effect was present only in HFD-fed animals. This training response was absent from chow fed mice, which suggest that such effect may require longer and/or more intense exercise training. Lastly, increased muscle oxidative capacity in response to selective HSP72 over-expression in skeletal muscle did not prevent HFD-induced downregulation of adipose tissue GLUT4, which suggest the participation of other mediator(s) of muscle oxidative capacity is required in order to induce an endocrine effect on adipose tissue.
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3 CHAPTER THREE: SHORT-TERM EXERCISE TRAINING AND ADIPOSE TISSUE GLUT4 IN HUMANS
3.1 INTRODUCTION
Of the numerous benefits of physical activity on health, one of the most important is the ability of exercise training to improve insulin action. Training-induced adaptations in skeletal muscle are considered central to this effect, due to the major role of this tissue in glucose disposal. These adaptations occur rapidly following the onset of training
(Kraniou et al., 2004, 2006; Winnick et al., 2008), and include increased GLUT4 translocation and expression. Increased muscle GLUT4 protein is mediated by the cumulative effect of transient increases in GLUT4 mRNA following each exercise bout, which finally leads to gradual accumulation of GLUT4 protein (Phillips et al., 1996;
Kraniou et al., 2000; Kraniou et al., 2004). In contrast to training adaptations in skeletal muscle where increase in GLUT4 content occurs rapidly following a single bout of exercise, adaptations in adipose tissue do not seem to follow the same timeline.
Preliminary results from this lab showed no change in adipose GLUT4 expression and protein content immediately and 3 hours after a single bout of exercise (Boland et.al. unpublished data). However, adipose tissue GLUT4 expression has been previously reported to be increased after 4 weeks of aerobic exercise training in patients with type
2 diabetes (Hussey et al., 2011). Similarly, data presented previously in this thesis showed increased adipose tissue GLUT4 protein content after 4 weeks of exercise training in mice fed a HFD, although not following similar exercise training in chow-fed mice. Studies in rodents have shown increased adipose tissue GLUT4 expression and improve in insulin sensitivity following short-term training (Ferrara et al., 1998).
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Although adipose tissue GLUT4 expression does not appear to respond to acute exercise, exercise training in subjects with diabetes increased adipose tissue GLUT4.
Whether adipose tissue GLUT4 expression is increased, after short-term exercise training in healthy humans, has not yet been studied. Therefore, the aim of this study was to investigate the effect of short-term training on GLUT4 protein content in adipose tissue of healthy individuals.
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3.2 METHODS
3.2.1 Subjects:
Ten healthy, untrained subjects (age: 27 + 2 yrs, body mass: 75 + 5 kg, height: 175 + 2
-2 -1 cm, and BMI: 24.3 + 1.4 kg.m , VO2 peak: 3.1 l.min ) volunteered to participate in the study. The untrained characteristic is described as the absence of endurance exercise training at least 1 hr, three times per week in the last 6 months previous to the commencement of the intervention.
Participants received information about the study and associated risks, completed a medical questionnaire and provided written informed consent before participation. All experimental procedures were approved by the University of Melbourne Health
Sciences Human Research Ethics Committee.
3.2.2 Peak pulmonary oxygen consumption (VO2 peak):
At least 7 days before the first training session, all subjects performed an incremental cycling test to exhaustion on an electronically braked cycle-ergometer (Lode, Corival,
The Netherlands). On test day, subjects arrived to the laboratory after overnight fasting.
VO2 peak was determined using the Parvo Medics True One 2400 metabolic measurement system (Sandy, Utah, USA). The test protocol consisted of a two min 50 watts (W) warm-up step, followed by a second 2-min step at 100W. From here, resistance was increased 25W every minute until volitional fatigue or until a 60- revolution per minute (rpm) cadence could no longer be maintained. Measurement of expired air composition and flow were obtained every 15 seconds in the metabolic cart, and VO2 peak was determined as the mean of the total VO2 values obtained during the
70 last minute until exhaustion. The effect of training on VO2 peak was determined by a second test performed during the first half of the last training session.
3.2.3 Anthropometric measurements:
All parameters were measured before the first and at the last training sessions. Body mass (kg) and height (cm) were determined using a calibrated scale (Seca, Germany) and a stadiometer, respectively, and BMI (kg.m-2) was calculated. Waist and hip circumferences (cm) were measured using a measuring tape, and skinfolds (mm) were obtained using a Harpenden caliper. Body fat percentage was estimated from the sum of skinfolds as previously determined (Durnin & Womersley, 1974). Trunk skinfold summation included subscapular, suprailliac, supraspinale and para-umbilical skinfolds.
Limb skinfold summation included biceps, triceps, anterior thigh and medial calf skinfolds.
3.2.4 Exercise training:
Subjects exercise trained for 10 days in two blocks of five consecutive sessions over two weeks. Training sessions consisted of cycling on a cycle-ergometer alternating between two modalities: 1) 6 x 1h sessions of continuous cycling at 75% VO2 peak in non-consecutive days, and 2) 4 x 6 x 5 min bouts at 90% VO2 peak HIIT, with 3 min active recovery at 40% VO2 peak between each HIIT bout (18min). Target intensities
(40%, 75% and 90% VO2 peak) and power output (W) were determined by the linear relationship between oxygen uptake and power output. Expired air analyses were performed during sessions to ensure that subjects were exercising at the target exercise intensities. All training sessions were performed in the laboratory under constant supervision.
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3.2.5 Muscle and adipose tissue biopsies:
Tissue samples were collected at baseline (SED) and 24h after the last training session
(TRAINED). Samples were obtained under local anaesthesia (1% xylocaine) from vastus lateralis and para-umbilical abdominal SCAT using the percutaneous needle biopsy technique, with suction as previously described (Evans et al., 1982). Samples were immediately frozen in liquid nitrogen and stored.
3.2.6 Adipose tissue homogenization:
Approximately 200mg of frozen adipose tissue samples were lysed in three volumes of lysis buffer (as described in chapter 2) supplemented with 10µl.ml-1 proteases inhibitor cocktail (Sigma Aldrich, cat#P2714), and 10µl.ml-1 phosphatases inhibitor cocktail #2
(Sigma Aldrich, cat#P5726) and 10µl.ml-1 cocktail #3 (Sigma Aldrich, cat#P0044).
Samples were homogenized using the Precellys 24 automated biological sample lyser with CK-14 beads vials (Ozyme, SaintQuentin, France) in two cycles at 4,500g for 20 sec with 45 sec pause. Lysates were spun for five min at 5,000g at 4oC. The upper fat layer was discarded and infranatants were transferred to a new set of microtubes and spun at 13,000g for 10 min at 4oC. Protein concentration in whole tissue homogenates was determined using the BCA protein assay (Pierce, Rockford, IL, USA) according to manufacturer directions.
3.2.7 Skeletal muscle homogenization:
Approximately 20mg of frozen muscle were homogenized in ten volumes of lysis buffer
(25mM Tris-HCl, pH 6.8, 5mM EGTA, 1mM sodium Orthovanadate, 50mM Sodium
Fluoride, 10% Glycerol, 1% Triton-x-100, 1% PMSF) supplemented as described above. Tissue samples were homogenized with an electronic homogenizer (Polytron) by
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3 x 15 sec bursts on ice. The homogenates were spun in a centrifuge for 5 min at 1000g at 4oC. Supernatants were recovered and stored for later analysis.
3.2.8 Immunoblotting:
Muscle and adipose tissue protein lysates were prepared and separated by electrophoresis as described in chapter 2, with modifications. Briefly, a solution of 1.5 mg/ml concentration was prepared per sample. Protein lysates were heated at 37oC for
10 min and spun for 1 min at 13,000g at room temperature. Same protein concentration
(22.5 µg protein) was loaded per lane in the gels. Gels were imaged prior to and after transferring to verify protein loading and transfer efficacy. In adipose tissue samples, the following primary antibodies were probed: GLUT4 (1:1000 anti-GLUT4 rabbit polyclonal AB, Thermo Scientific), GLUT1 (1:500 anti-GLUT1(N-20) goat polyclonal,
Santa Cruz, sc#1603), FABP4 (1:500 FABP4-recombinant Oligoclonal antibody- purified, Novex by Life technologies to 1ug.ml-1 final concentration), COX IV (1:1000 rabbit polyclonal AB, Cell Signaling, #4844), MEF2A (H-300) (1:1000 anti-MEF2A rabbit polyclonal, Santa Cruz, sc-#10734), HDAC5 (1:1000 HDAC5-mouse mAB,
Santa Cruz, sc#133106). In muscle samples primary antibodies for GLUT4, COXIV, and HDAC5 were probed. Additionally, adipose tissue samples were probed with total
OXPHOS antibody (1:500 total OXPHOS rodent WB antibody cocktail, Abcam, ab#110413). This antibody contains 5 mouse mAbs, one each against complex-I subunit
NDUFB8 (ab110242), complex-II-SDHB-30kDa (ab14714), complex III-Core protein
2[UQCRC2] (ab14745), complex-IV subunit I [MTCO1] (ab14705) and complex-V alpha subunit [ATP5A] (ab14748). After overnight incubation in primary AB, membranes were washed 3x10 min in TBST and exposed to secondary AB for 1h at RT.
Membranes were washed again in TBST and incubated for 1 min in chemiluminescence
73 reagents (ECL) (GE Healthcare/Amersham, Uppsala, Sweden) or SuperSignalTM west
Femto miximum sensitivity substrate (Thermo ScientificTM, cat#34094). Antibody binding was visualized and quantified using the ChemiDoc MPTM system and analysed using the Image Lab 4.0 software (Bio-Rad, Inc., US). The light density of the proteins of interest was normalized over β-actin as loading control or total protein content, determined from stain-free gels.
3.2.9 Adipose tissue histological sections:
A fraction of the adipose tissue biopsies was separated for cell counting. Fresh samples were washed in PBS and placed in 10% formalin for 24 hours. Samples were then desiccated and preserved in 70% ethanol until paraffin processing. Five 5-micrometre- thick tissue sections per sample were sliced with a microtome from paraffin blocks every 50µm and stained with haematoxylin-eosin. Nine out of ten paired samples were analysed.
3.2.10 Adipocyte size:
Relative adipocyte size was determined at 10x magnification of haematoxylin-eosin stained sections. Automated cell count was carried out using Cell Profiler, cell image analysis software (Carpenter Lab, Broad Institiute, US; version 2.2.0). Approximately
300 cells were counted per image in three or four representative sections per sample.
Adipocyte cell size was determined based on cell diameter measured as the mean
Feret’s diameter, also known as maximum caliper diameter, which corresponds to the perpendicular distance between parallel tangents touching opposite sides of the perimeter of an object (Walton, 1952), in this case a cell.
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3.2.11 Statistical analysis:
All values are reported as means + SEM. Relative protein contents in immunoblotting experiments were determined by arbitrary units of blots band density normalized over total protein loading. Beta-actin immunoblot was used as to confirm even protein loading in adipose tissue samples. The mean Feret’s diameter was calculated from minimal and maximal values determined by the software and average value accounted for cell size and is expressed in micrometres. Sample cell size represents the average cell size of the total number of cells per section counted per sample. Anthropometric measurements as well as molecular and histological analyses of sedentary and trained means were compared using a paired t-test with a significance level of p < 0.05.
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3.3 RESULTS
3.3.1 Characteristics of the subjects:
No changes in body mass or percentage of body fat mass were observed (BM: 75 + 5 and 75 + 4.3 Kg, 22 + 2 and 21 + 2%, sedentary and trained, respectively).
Hip circumference (98 + 3 cm to 97 + 3 cm, p = 0.019) and limb skinfold summation
(39 + 4 to 37 + 3 mm, p = 0.015) were significantly reduced following short-term training. VO2 peak was increased by ~6% (3.1 + 0.15 l.min-1 to 3.3 + 0.2 l.min-1, p =
0.0425). Peak power output was also increased ~6% (15W, p = 0.012). Main characteristics are summarized in Table 3.1.
3.3.2 Short-term training adaptations in skeletal muscle:
The muscle protein abundance of GLUT4 and COX-IV increased significantly following the training (47% and 39%, respectively, p < 0.05; Figures 3.1 and 3.2).
3.3.3 Short-term training adaptations in adipose tissue:
Total protein content of adipose tissue GLUT4, GLUT1 (Figure 3.3), HDAC5, MEF2A
(Figure 3.4), COX-IV, FABP4 (Figure 3.5) and OXPHOS (Figure 3.6) were not affected by the training. Mean adipocyte cell size in abdominal para-umbilical SCAT samples was unchanged following the training (Figure 3.7).
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Age (years) 27.2 + 1.8 Height (cms) 175.6 + 1.7
Body composition Sed Trained p value Body mass (kg) 75 + 5 75 + 4.3 0.204
BMI (kg.m-2) 24 + 1.4 24 + 1.3 0.133
Body fat mass (%) 22 + 2 21 + 2 0.060
Waist circumference (cm) 83 + 3.2 82 + 3.2 0.088
Hip circumference (cm) 98 + 3 97 + 3 0.019 * waist/hip ratio 0.84 + 0.01 0.85 + 0.01 0.223
∑4-skinfold-trunk (mm) 77 + 13 75 + 13 0.090
∑4-skinfold-lower limbs (mm) 21 + 2 19 + 1 0.016 *
Exercise performance: -1 VO2 peak (l.min ) 3.1 + 0.2 3.3 + 0.2 0.043 * -1 -1 VO2 peak (ml.kg .min ) 42 + 2 44 + 3 0.045 * PPO (W) 260 + 13 275 + 14 0.012 * HR max (bpm) 179 + 3 173 + 3 0.064
TABLE 3.1: Characteristics of the subjects
Values presented as means + SEM (n = 10). (*) Paired T-test: Sed vs Trained, p < 0.05.
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FIGURE 3.1: Effect of short-term training on skeletal muscle GLUT4 protein.
A) Representative immunoblot of GLUT4 for 2 subjects at baseline (SED) and after 2 weeks of exercise training (TRAINED). B) Quantification of GLUT4 protein abundance, and C) individual changes in GLUT4 protein content in skeletal muscle in response to training. All values are mean + SEM of GLUT4/total protein ratio. (*): paired t-test, p = 0.0195.
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FIGURE 3.2: Effect of short-term training on skeletal muscle COX-IV protein.
A) Representative immunoblot of COX-IV for 2 subjects at baseline (SED) and after 2 weeks of exercise training (TRAINED). B) Quantification of COX-IV protein abundance, and C) individual changes in COX-IV protein content in skeletal muscle in response to training. All values are mean + SEM of COX-IV/total protein ratio. (*): paired t-test, p = 0.0484.
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FIGURE 3.3: Effect of short-term training on adipose tissue GLUT4 and GLUT1 proteins
Upper panels: Representative immunoblots: A) GLUT4 and B) GLUT1 at baseline (SED) and after 2 weeks of exercise training (TRAINED). Each panel includes representative total protein and β-actin immunoblot. Bottom panels: Quantification of GLUT4 (A) and GLUT1 (B) protein abundance in adipose tissue in response to training. All values are means + SEM of blot/total protein ratio.
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FIGURE 3.4: Effect of short-term training on adipose tissue MEF2A and HDAC5 proteins.
Upper panels: Representative immunoblots: A) MEF2A and B) HDAC5 at baseline (SED) and after 2 weeks of exercise training (TRAINED). Each panel includes representative total protein and β-actin immunoblot. Bottom panels: Quantification of MEF2A (A) and HDAC5 (B) protein abundance in adipose tissue in response to training. All values are mean + SEM of blot/total protein ratio
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FIGURE 3.5: Effect of short-term training on adipose tissue COX-IV and FABP4 proteins.
Upper panels: Representative immunoblots: A) COX-IV and B) FABP4 at baseline (SED) and after 2 weeks of exercise training (TRAINED). Each panel includes representative total protein and β-actin immunoblot. Bottom panels: Quantification of COX-IV (A) and FABP4 (B) protein abundance in adipose tissue in response to training. All values are mean + SEM of blot/total protein ratio.
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FIGURE 3.6: Effect of short-term training on adipose tissue OXPHOS proteins.
Upper panels: A): Representative immunoblots of total OXPHOS and β-actin, and total protein loading. B-F): Quantification of OXPHOS protein abundance in adipose tissue in response to training. All values are mean + SEM of OXPHOS/total protein ratio
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FIGURE 3.7 Effect of short-term training on adipocyte cell size.
Five micron-thick paraffin sections were stained with H&E and cell size was measured. The diameters of approximately 300 cells/image in four section/subject were analysed using Cell profiler. A) Quantification of mean cell size and individual average diameter per subject in response to exercise training. Bottom: representative sections for baseline (B) and trained (C) conditions. All values are mean of cell size, determined as Feret’s diameter.
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3.4 DISCUSSION
In the present study, 10 sessions of intense endurance training, involving a combination of continuous moderate and HIIT cycling, significantly increased VO2 peak on average by approximately 6%, with individual increments ranging from 1 to 21%. A similar increment in VO2 peak was observed after comparable training schemes over two weeks
(McConell et al., 2005), 18 sessions of HIIT over six weeks involving 5 x 60s bouts at
130% of maximal load (Larsen et al., 2015), and following 6 x 8 to 12 x 60s interval sessions at 100% peak power output during two weeks (Jacobs et al., 2013). Likewise,
PPO increased ~6% in the present study, similar to increments reported previously following short-term training (Jacobs et al., 2013).
In relation to the effect of short-term training in the adipose tissue, no changes were observed in adipose tissue total protein content of GLUT4 and other protein of interest including GLUT1, HDAC5, MEF2A, COX-IV, FABP4, and total OXPHOS. This is the first evidence that GLUT4 protein expression is not modified in the adipose tissue after two weeks of endurance exercise training in humans. Conversely, studies in rodents have demonstrated a rapid increase, ranging from 30 to 60% in adipose tissue GLUT4 expression following training lasting from 4 days to 10 weeks. (Hirshman et al., 1993;
Stallknecht et al., 1993; Ferrara et al., 1998). Increased GLUT4 in the adipose tissue is associated with improved insulin sensitivity and increased basal and insulin-mediated glucose disposal in the tissue, in the absence of an effect in GLUT1 abundance
(Hirshman et al., 1993; Stallknecht et al., 1993). The latter is in line with present data showing no effect on GLUT1 protein content.
HDAC5 and MEF2A have been associated with the regulation of GLUT4 in adipocytes in vitro (Sparling et al., 2008) and in vivo following fasting in mice (Weems et al.,
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2012). Data in the current study showed protein content of both HDAC5 and MEF2A unaffected following 2 weeks of training. In skeletal muscle, both transcription factors are critical in the exercise-mediated regulation of GLUT4. In the adipose tissue, however, the participation of these markers has not been investigated previously.
Nevertheless, both factors have been described relevant for the regulation of adipose tissue GLUT4 during adipogenic differentiation and fasting in mature adipocytes
(Weems & Olson, 2011; Weems et al., 2012). The lack of an exercise training effect on adipose tissue GLUT4 following 10 days of intense exercise training can be partially explained by the absence of a response in the total abundance of adipose tissue HDAC5 and MEF2.
Changes in COX-IV protein content and/or activity are considered measurements of mitochondrial content and mitochondrial oxidative capacity and together with changes in GLUT4 protein abundance are consider favourable metabolic adaptations in skeletal muscle in response to training (Egan et al., 2013; Jacobs et al., 2013). Therefore, the analysis of changes in both proteins brings a thorough understanding of adaptation occurring in a tissue in association with the increased energy demands associated with the acquired training state.
In relation to exercise-induced adaptations in the adipose tissue, previous studies have not been able to demonstrate improved adipose tissue oxidative capacity after similar training interventions (Camera et al., 2010; Larsen et al., 2015). In this regard, the present data of unaltered COX-IV and total OXPHOS protein content is in line with previous findings showing no changes in fat oxidation rate, maximal citrate synthase activity and mitochondrial volume (Camera et al., 2010), as well as mitochondrial content and total OXPHOS capacity (Larsen et al., 2015). Together, these findings
86 suggest a longer exposure to the exercise stimulus may be needed in order to induce changes in adipose tissue oxidative capacity and mitochondrial function.
The adipogenic differentiation is regulated by PPARγ. Subsequently, formation of lipid droplets and lipid-filling capacity rely on induction of C/EBP and the expression of FA transporter proteins (FABP4), FAS and the insulin responsive glucose transporter
GLUT-4 (Hamm et al., 1999; Schmid et al., 2005; Fernyhough et al., 2007). Moreover, expression of GLUT4 in adipocytes regulates fatty acid synthesis and has been involved in adipocyte hyperplasia (Shepherd et al., 1993; Tozzo et al., 1995; Moraes-Vieira et al.,
2016). In the context of adaptations to exercise training in the adipose tissue, it is important to determine whether this response includes changes in the expression not only of GLUT4 but also other adipogenic markers, such as FABP4. The adipose- specific FABP4, also known as aP2, is considered an adipogenic marker and its expression is under the control of adipogenic master regulators PPARγ and C/EBPα transcription factors (Ross et al., 1990; Sen et al., 2001; Joosen et al., 2006). It has been previously shown that 10 weeks of exercise training significantly increased FABP4 gene expression in epididymal and retroperitoneal adipose tissue of obese Zucker rats
(Krskova et al., 2012). However, in the present study, no change in FABP4 protein content was observed.
Long-term exercise interventions have been associated with morphological changes in the adipose tissue, i.e. reduction in adipocyte cell size, especially in overweight/obese populations (Larson-Meyer et al., 2006; You et al., 2006). Almost all the available data regarding the effect of life-style interventions in fat mass loss and the reduction in adipose cell size combines the effect of low-calorie diets with different modalities of exercise training (Murphy et al., 2017), and it is the additive effect of both strategies that renders more significant effects. There is only one study analysing the isolated
87 effect of exercise training on adipocyte cell volume (Schwartz, 1987). In this study, 13 weeks of moderate intensity (40 min/day at 70% - 85% HR reserve) walk/jog exercise,
3 to 5 days per week reduced adipocyte cell volume (µg TG) 8% and 13% in gluteo- femoral and abdominal SCAT, respectively, effects that were associated with a beneficial change in the lipid profile of healthy obese subjects. In the present study, mean adipocyte cell size was not affected by the training intervention. Notably, however, quantification of cell diameter in 6 from 9 paired samples analysed was reduced ranging from -0.3% to -25.0% (mean 9.1%). The Feret’s diameter has been recently utilized for the determination of size of Oil-red-O stained lipid droplet in differentiated 3T3-L1 adipocytes (Rizzatti et al., 2013). In addition, significant decreases in hip circumference and skinfolds in lower limbs were observed in the present study. These effects may have been mediated by increased local lipolytic activity in peripheral adipose tissue surrounding active muscles in the legs. Both, adipose tissue blood flow and lipolysis increased in fat tissue adjacent to active muscles during high intensity one-legged knee extension exercise (Stallknecht et al., 2007).
The training scheme utilized in this study has previously been shown to induce training adaptations in muscle (McConell et al., 2005; Camera et al., 2010). In the present study an improved metabolic response to exercise in skeletal muscle, reflected by increased
COX-IV and GLUT4 protein contents, was observed. Muscle COX-IV protein content increased ~39%, which represents expanded mitochondrial content (MacInnis & Gibala,
2017). This finding is similar to previous reports showing 25 to 35 % increase after 5 to
6 sessions of HIIT training in male subjects (Gibala et al., 2006; Perry et al., 2010;
MacInnis et al., 2017). These results agree with training-induced increase in COX-IV enzyme activity (Gibala et al., 2006; Jacobs et al., 2013). COX-IV protein and activity
88 have been reported similarly increased following 2 weeks of HIIT or endurance trainings (Gibala et al., 2006; MacInnis et al., 2017).
Increased muscle GLUT4 gene expression can be seen immediately after an acute bout of exercise and its level remains elevated for up to 3 hours (Kraniou et al., 2000).
GLUT4 protein content is significantly increased following one week of endurance exercise training (Kraniou et al., 2004; Egan et al., 2013) . The 47% increase in muscle
GLUT4 protein content in the present study is in line with previous findings showing rapid upregulation of muscle GLUT4 protein in response to training (Gulve & Spina,
1995; Houmard et al., 1995; Phillips et al., 1996). In the present study, inter-subject variability in muscle GLUT4 protein increments ranged from 3 to 150% at the end of the two-week intervention. In the reviewed literature, relative increases in muscle
GLUT4 protein content vary between ~0.4 to 3.4-fold following short-term training
(Gulve & Spina, 1995; Houmard et al., 1995; Phillips et al., 1996; Kawanaka et al.,
1997; Terada et al., 2001; Langfort et al., 2003; Kraniou et al., 2004; Egan et al., 2013).
Biological as well as technical variability may account for these differences.
Interestingly, GLUT4 protein content has been reported increased ~36% following seven days of endurance training with no further increase with prolonged training (4 weeks) at progressively increasing workloads (Langfort et al., 2003). Despite the inter- study variability in the relative increase in GLUT4 protein in skeletal muscle following training, there is a consensus that this adaptation translates improve glucose metabolism in skeletal muscle.
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3.5 CONCLUSION
Short-term adaptations to exercise training included increased VO2 peak and PPO at
the end of the two week intervention. In skeletal muscle, increased GLUT4 and
COXIV are consistent with improved metabolic response to exercise. In adipose
tissue, however, these adaptations do not seem to follow that same timeline as in
skeletal muscle. No training effect was observed in adipose tissue GLUT4 and other
proteins of interest, including COX-IV as a measurement of adipose tissue oxidative
capacity. As discussed in chapter 2, previous findings have described exercise
training effects on adipose tissue GLUT4 of rats after prolonged and intense exercise
regimes (Hirshman et al., 1993; Stallknecht et al., 1993; Ferrara et al., 1998).
Similarly, in humans increased adipose tissue GLUT4 expression was observed only
after 4 weeks of training in patients with type 2 DM (Hussey et al., 2011). Taken
together, these reports suggest 10 days of endurance exercise training in the present
study was insufficient to induce the expected training effect on adipose tissue
GLUT4 previously observed in rats and diabetic patients, thus exercise-induced
adaptations in the adipose tissue may require a longer exposure to the exercise
stimulus.
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4 CHAPTER FOUR: EXERCISE SERUM EFFECTS ON GLUT4 IN HUMAN PRIMARY ADIPOCYTES
4.1 INTRODUCTION
Exercise training improves insulin responsiveness for glucose metabolism in adipose tissue of rodents and humans (Rodnick et al., 1987; Peres et al., 2005; Gollisch et al.,
2009; Higa et al., 2014). Adipose tissue insulin-stimulated glucose transport is increased by ~2-fold in trained rats, in association with improved InsR binding and
GLUT4 translocation into the plasma membrane (Vinten & Galbo, 1983; Vinten et al.,
1985). Similarly, isolated human adipocytes from trained subjects showed improved glucose uptake in significantly smaller cells compared with sedentary controls (Rodnick et al., 1987). Adipose tissue GLUT4 expression is increased in rats following exercise training, effect mostly attributed to reduced fat cell size (Hirshman et al., 1993;
Stallknecht et al., 1993). However, GLUT4 expression has been described to be increased in spite of unchanged cell size following short-term training (Ferrara et al.,
1998). Thus, changes in adipose tissue GLUT4 may be consequence of functional adaptations within the cell and not necessarily the result of changes in adipose cell size.
It has been hypothesised that cytokines released by contracting-skeletal muscles during exercise, “exerkines”, may influence adipose tissue metabolism (Petersen & Pedersen,
2005; Pedersen, 2006), and its adaptive response to metabolic cues such as exercise and glucose (Lee & Fried, 2006; Brandt et al., 2012; Keipert et al., 2014; Knudsen et al.,
2014). The mechanisms by which exercise training potentially increases adipose tissue
GLUT4 expression are currently unknown, although findings in this thesis and previous studies have shown an exercise training effect in conditions where GLUT4 is downregulated at baseline (Hussey et al., 2011) or in the context of HFD (Figure 2.4).
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The activity of the master adipogenic transcription factor PPARγ is induced by ligand- dependent mechanisms. The endogenous PPAR-γ ligand, prostaglandin J2 binds to
PPARγ C-terminal ligand binding domain inducing its activation. Upon activation,
PPAR-γ binds responsive element (PPARE) on GLUT4 promoter leading to repression of GLUT4 mRNA transcription. In fully mature adipocytes, PPAR-γ expression is under the upstream regulation of insulin through phosphorylation-mediated nuclear export of FOXO1 which results in de-repression of PPAR-γ2 transcription and subsequently increase in GLUT4 transcriptional rate (Armoni et al., 2003; Armoni et al.,
2007). The latter fact makes the PPARγ expression a relevant marker to be investigated in relation to changes in adipose tissue GLUT4.
Previous findings have described changes in GLUT4 protein content occurring in association with changes in either adipose tissue mass or adipocyte volume (Pedersen et al., 1992; Stallknecht et al., 1993). As mentioned above, exercise training reduces adipocyte cell size and improves insulin sensitivity in human adipocytes. Adipocyte cell size may be affected by lipolytic stimuli inducing lipid mobilization during exercise and following exercise training. The main lipolytic mediators are circulating catecholamines, although these hormones have shown to decrease GLUT4 expression.
Nevertheless, and considering that exercise trained adipocytes are smaller, potentially due to improved lipolytic state, but develop improved insulin responsiveness for glucose uptake, mainly secondary to increased GLUT4 expression and activity, it is possible that the exercise-induced effect on adipose tissue GLUT4 may be mediated by circulating factors. Therefore, the aim of this study was to investigate the effect of exercise serum, and by implication circulating factors, on the expression of GLUT4 in human primary adipocytes in vitro.
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4.2 METHODS:
4.2.1 Subjects:
Eight healthy, untrained subjects (age: 21.4 + 0.6 y, body mass: 71 + 4 kg, BMI: 22 + 1 kg.m-2) volunteered to participate in the study. Participants received information about the study and associated risks, completed a medical questionnaire and provided written informed consent before takin part in the study. Subjects performed a VO2 peak test one week before commencing the exercise trial, as described in Chapter 3. On the experimental day, subjects arrived after an overnight fast (~8hr), exercised for an hour on a cycle-ergometer at an intensity of 70 - 75% VO2 peak. All experimental procedures were approved by the University of Melbourne Health Sciences Human Research Ethics
Committee. Characteristics of the subjects are summarized in Table 4.1.
4.2.2 Blood sampling and serum extraction:
An intravenous catheter (22G x 1” Terumo® Surflo® I.V. catheter, Phillipines) was inserted into the cephalic vein on the right forearm while subjects rested for at least 10 min. Exercise samples were collected during the last minute of the exercise bout, while the subjects were still pedalling. Blood samples were collected into 5 ml BD-
Vacutainer© Rapid serum tubes [RST]-blood collection tubes (Franklin Lakes, NJ, US) containing thrombin. Tubes were gently inverted 8 times immediately after collection and kept at room temperature for no longer than 30 min. Samples were then spun at
5000 rpm [1328 g] x 10 min at 4oC. Serum was divided into 1 ml aliquots in 1.2 ml
CORNING® polypropylene sterile cryogenic vials (Corning Incorporated, #430487,
Corning, NY) and immediately stored at -80oC.
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4.2.3 Adipose tissue biopsy:
Subcutaneous abdominal, para-umbilical adipose tissue biopsies were collected from two healthy volunteers (age: 31 & 35 yrs, 71 & 94 kg, BMI: 25 & 27 kg.m-2). Local anaesthesia (2% Xylocaine, no epinephrine) was administered subcutaneously, three cm lateral from the umbilicus. A small incision was made in site and adipose tissue was obtained using a 14 G, 3 1/8” needle (Braun Sterican®) attached to a Hepafix® Syringe from a depth of ~50 mm under the skin. The SVF was isolated from whole-tissue samples by collagenase digestion and centrifugation processes as described previously
(Lee & Fried, 2014; Tsiloulis et al., 2016).
4.2.4 SVF-derived adipocyte proliferation and differentiation:
SVF-derived cells were differentiated into mature adipocytes. Cells (passage #0, P0) were quickly thawed and re-suspended in growth media MEM-α (Minimum Essential
Medium, GibcoTM) containing 10% FBS (GibcoTM) and 1% penicillin/streptomycin
(1:1, 100 U.ml-1, GIBCO®) in T-75mm flasks (P1). To avoid the deleterious effect of
DMSO on freezing media (10% DMSO in growth media), the first media change was performed at 24hr after seeding, followed by media changes every second day. Cells proliferated until ~100% confluent in approximately 7 to 10 days. Upon confluency, monolayer cells were detached from the surface of the plate by enzymatic digestion with 0.05% trypsin (Trypsin, EDTA, phenol red, GibcoTM), pelleted by centrifugation
(2,000rpm, 5 min at 4oC), re-suspended in small volume of media and counted for viable cells in a suspension using 1:1 trypan blue in a haemocytometer. Cells were seeded at a density of 20,000 – 30,000 cells per ml in 12-well plates as requested by experiments. Cells in plate (P2) proliferated until fully confluent before differentiation induction. Adipocyte differentiation was performed as previously described by Lee and
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Fried (Lee et al., 2012; Lee & Fried, 2014) with some modifications. Briefly, the day after reaching confluency (Diff. day 0), cells were exposed to adipogenic agents in serum-free media (Dulbecco’s modified Eagle’s medium [DMEM]/F12 + GlutaMAX®,
GibcoTM), containing 1% P/S. Induction cocktail includes 0,2 nM triiodothyronine (T3),
20 nM human Insulin (Actrapid® HM, Novo Nordisk), 250 nM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 2 µM rosiglitazone, 1,7 µM pantothenate,
3,3 µM biotin, 10 µg.ml-1 transferrin. On day three, media were replaced by differentiation media containing an identical hormonal formulation except rosiglitazone and maintained until day seven. From day seven onwards, differentiation media was replaced by maintenance media lacking rosiglitazone and IBMX (see Table 4.2). On day
10, insulin was withdrawn from media. On day 12, cells were ready for experiments.
4.2.5 Dose response to exercise-conditioned serum (ECS):
On differentiation day 12, cells were exposed to increasing concentrations of ECS (5%,
10%, 20% and 50%) added to maintenance media. Wells were washed with warm
DPBS followed by replenishment of serum-containing media. Gene expression of adipogenic genes, including GLUT4 were determined following 6h, 12h and 24h, whilst protein content was assessed after 48h exposure. Treatments were performed in duplicate in two independent experiments. At differentiation day 14, upon completion of the 48h treatment, cells were washed in PBS and plates were frozen for later analyses.
4.2.6 Treatment with ECS, IL-6, FGF21 and isoproterenol:
The effect of 10% ECS in human SVF-derived adipocytes was compared with the effect of selected myokines: interleukin-6 (IL-6, Human recombinant Interleukin-6, expressed in E. coli, Sigma-Aldrich®, St. Louis, MO 63103, USA) and fibroblast-growth-factor-
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21(FGF21, Human recombinant FGF21 expressed in E. coli, Sigma-Aldrich®, St.
Louis, MO 63103, USA), and the β-adrenoceptor agonist isoproterenol. The 10% concentration of ECS in media was determined from dose-response experiment on
GLUT4 protein content (supplementary information and suppl. Figure 6.4). Cells in duplicate wells were treated with 10% pre-exercise serum (PRE) or 10% ECS, 1µM isoproterenol, 20ng.ml-1 IL-6 and 100ng.ml-1 FGF21. Treatment concentrations for IL-6,
FGF21 and isoproterenol were determined based on previous reports (Ji et al., 2011;
Lee et al., 2014).
4.2.7 Gene expression in human primary adipocytes:
Expression of the adipogenic markers PPARγ, C/EBPa, FABP4, and glucose transporter
GLUT4 (TaqMan probes, Thermo-Fisher Scientific, see Table 4.3) was determined by real time qPCR. The housekeeping gene TATA-box binding protein 1 (TBP1, TaqMan probe, Thermo-Fisher Scientific) was utilized to determine relative expression of genes of interest (GOI). Experiments were performed twice and each treatment in duplicate.
Cell samples for gene expression were collected at 6, 12 and 24hr exposure to treatments. Sample homogenization and RNA purification were assessed using
PureLink® RNA Mini Kit (Ambion®, Life Technologies ®) according to manufacturer’s directions. Purified RNA was reconstituted in 30µl nuclease-free water and assessed for RNA quality and quantification using nanodrop.
4.2.8 cDNA synthesis and RT-qPCR:
Single strand cDNA chains were synthesised from purified RNA using iSCRIPTTM cDNA synthesis kit (Bio-Rad, Hercules CA, USA) according to manufacturer’s specifications to a final concentration of 2.5 ng.µl-1. Real-time quantification of RNA targets was performed using AmpliTaq Gold® DNA Polymerase (Applied Biosystem®
97 by Life TechnologiesTM) for experiments using TaqMan probes. Relative expression of genes of interest in pre-designed probes determined by TaqMan technology was assessed using the ∆∆Ct method comparing treated groups with control-untreated group and normalizing over the expression of the housekeeping gene TBP1).
4.2.9 Immunoblotting:
After 48h ECS treatment, cells were washed with PBS and re-suspended in 100µl RIPA buffer (supplemented with phosphatases and proteases inhibitors + 0.01% DTT). Cell monolayers were disrupted in well using plastic cell lifters, then lysates were transferred to 1.7ml eppendorf tubes and homogenized in situ using 0.3ml syringe. Protein concentration of whole cell lysates was determined by BCA assay and absorbance was measured at 562nm. Sample solubilisation and SDS-PAGE were performed as described in chapter 2. The following primary antibodies were used: GLUT4: 1:1000 anti-GLUT4 rabbit polyclonal AB, Thermo-Fisher Scientific); FABP4: 1:500 FABP4- recombinant oligoclonal antibody-purified (Novex by Life technologies, initial concentration 0.5 mg.ml-1 diluted to 1µg.ml-1); PPARγ: 1:200 PPARγ (H-100): sc-7196, rabbit polyclonal against amino acids 8-106 human PPARγ (Santa Cruz, initial concentration 200µg.ml-1 diluted to 1µg.ml-1).
4.2.10 Glucose uptake:
Cells were assayed on day 14 after 48h treatments. Media were removed and cells washed in PBS once. Cells were incubated for 10 min at 37oC in 0.5 ml per well basal-
“cold” media (DMEM/F12 serum-free media, 1M 2-deoxy-glucose and 0.1% BSA) with or without 100µM Insulin (Actrapid). Next, cold buffer was replaced by “hot” media containing 1µCi.ml-1 2-[1,2-3H(N)]-2-deoxy glucose (NET549A001MC,
1mCi.ml-1) and cells were incubated for 10 min in the presence or absence of 100uM
98 insulin. Hot buffer was stored for total radioactivity counting. The reaction was stopped by placing the plates in ice and washing the cells in ice-cold PBS three times. Next, cells were lysed in 200µl PBS containing 0.1% Triton-X-100 using plastic cell lifters. A
30µl aliquot of the cell homogenates was separated for protein assay. Radioactivity in the lysates was determined by liquid scintillation counting (Tri-Card 2810TR;
PerkinElmer).
4.2.11 Oil-red-O staining (ORO):
In order to determine lipid-loading capacity of mature human primary adipocytes, lipid content was stained with ORO staining on day 12. A stock solution of 0.5% ORO is prepared in 60% triethyl phosphate. Three parts of stock solution were dissolved in two parts of distilled water and filtered through Whatman #1 filter paper, followed by a 0.22
μm syringe filter on the day of experiment. 300 μl of 10% formalin were added to each well and incubated 5 min at RT. Next, wells were washed 3 x 5 min with 1 ml PBS and incubated then in 400 μl of ORO working solution at RT for 5 min, followed by another set of 3 x 5 min wash. Meyer’s Haematoxylin was carefully added so the surface of the plate was fully covered. A final set of washes was repeated before imaging.
4.2.12 Statistical analysis:
Relative gene expression in time-course experiments was compared using One-way analysis of variance (ANOVA). Significant difference between time-points was identified by Turkey’s multiple comparisons test. The effect of ECS treatment and gene induction time were analysed using two-way ANOVA. The same analysis was performed for glucose uptake assay. Significant differences were identified by
Bonferroni’s multiple comparisons test. Relative protein content in immunoblotting experiments was determined by arbitrary units of blots band density normalized over
99 total protein loading of PRE and ECS or treatments. Means values were compared using a paired t-test with a significance level of p < 0.05.
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4.3 RESULTS
4.3.1 Time-response effect of exercise serum on GLUT4 mRNA expression in human primary adipocytes:
Human primary adipocytes were harvested at 6, 12 and 24 hr during incubation in 10% serum obtained before (PRE) or after (ECS) exercise and analysed for GLUT4 gene expression by real time qPCR. GLUT4 mRNA was significantly increased by ~3-fold at
12 hr with no effect at 6 or 24 hr during the exposure (p < 0.05, Figure 4.1).
4.3.2 Effect of ECS on GLUT4 expression levels in human primary adipocytes:
Adipose cells were harvested at the end of 48 hr incubation in 10% PRE and ECS from eight subjects and analysed for GLUT4 protein content by Western blotting. On average, there was a small (~15%), but significant increase in GLUT4 protein content in cells incubated with ECS (p=0.0439, Figure 4.2). Similarly, a ~3-fold induction of
GLUT4 gene was observed at 12 h in ECS compared with PRE (p = 0.0365; Figure
4.3).
4.3.3 Effect of exercise serum on adipogenic markers:
There was a significant increase in the expression of adipogenic markers PPARγ,
C/EBPα, and PLIN1 at 12hr of ECS exposure (p < 0.05, Figure 4.4). Protein content of
PPARγ did not change following 48hr serum exposure, despite early increase in gene expression (Figure 4.4). FABP4 mRNA (Figure 4.4) and protein (Figure 4.5) levels did not change with the exposure at selected time points.
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4.3.4 Effect of exercise serum on insulin-stimulated glucose uptake:
Insulin significantly stimulated glucose uptake in both PRE and ECS treatment groups, with no statistical difference between groups (main effect of insulin, p < 0.05; Figure
4.6).
4.3.5 Effects of Isoproterenol, IL-6 and FGF21 on GLUT4:
A significant increase in GLUT4 mRNA was observed in cells treated with 1µM isoproterenol at 6 hr (p = 0.0219); however, this effect was lost at 12 and 24h. Neither
IL-6 nor FGF21 treatments showed an effect on GLUT4 mRNA level at the examined time points (Figure 4.7). There was no significant change in GLUT4 protein content in adipocytes exposed to selected myokines IL-6, FGF21 and β-adrenergic agonist isoproterenol at 48h exposure (Supplementary Figure. 6.6).
4.3.6 Effects of Isoproterenol, IL-6 and FGF21 on adipogenic markers:
Treatment with 1µM isoproterenol significantly decreased the expression of PPARγ,
PLIN1, and C/EBPα with no significant effect on FABP4 (Figure 4.8). Treatments with
IL-6 and FGF21 did not significant change the expression of adipogenic markers.
Protein expression of adipogenic markers was unchanged at the end of 48h treatments
(data not shown).
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Parameter (units) Values (n = 8)
Anthropometric
Age (years) 21 + 1
Height (cm) 180 + 3
Body mass (kg) 71 + 4
BMI (kg.m-2) 22 + 1
Exercise performance
Heart rate max (bpm) 179 + 3
-1 VO2 peak (l.min ) 3.2 + 0.3
-1 -1 VO2 peak (ml.kg .min ) 43 + 2
PPO (watts) 299 + 18
TABLE 4.1: Characteristics of the subjects
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Induction Differentiation Maintenance Reagent (*) Stock Final conc. 0-3 days 3-7 days 7-14 days T3 200nM 200 pM + + + Transferrin 10mg.ml-1 10 μg.ml-1 + + + Biotin 3.3mM 3.3 μM + + Pantothenic acid 17mM 17 μM + + + Human Insulin 100 UI 20 nM + + + (Actrapid ®) Dexamethasone 5mM 0.25 μM + + + IBMX 250mM 500 μM + + - Rosiglitazone 10mM 2 μM + - - TABLE 4.2: Human primary adipocytes differentiation protocol (*) Basic culture media is composed by DMEM-F12 + 1% P/S.
Gene name Probe number
GLUT4 Hs00168966 TBP1 Hs99999910 PPARγ Hs00234592 FABP4 Hs01086177 PLIN1 Hs00160173 C/EBPα Hs00269972 TABLE 4.3: Adipogenic genes TaqMan probes
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FIGURE 4.1: Time course of exercise serum effects on GLUT4 mRNA in human primary adipocytes.
GLUT4 mRNA in human primary adipocytes exposed to 10% serum obtained before (PRE) or after (ECS) exercise (n=4). Cells were harvested and analysed for GLUT4 mRNA at 6, 12 and 24h during exposure. Values represent means + SEM of fold change calculated from ∆∆Ct of GLUT4mRNA related to housekeeping TBP1 mRNA; (*):Two-way ANOVA, Turkey post hoc test, p < 0.05 PRE vs ECS and 12h vs 6h and 24h.
105
FIGURE 4.2: Effect of ECS on GLUT4 protein in human primary adipocytes.
GLUT4 protein content in human primary adipocytes exposed to 10% serum obtained before (PRE) of after (ECS) exercise for 48hr (n=8). Bottom panel: representative GLUT4 blots for cells treated with PRE (2) and ECS (2). Values represent means + SEM of GLUT4/total protein staining ratio, arbitrary units. (*): Paired t-test, p < 0.05 PRE vs ECS.
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FIGURE 4.3: Effect of exercise serum on GLUT4 mRNA expression in human primary adipocytes.
GLUT4 mRNA in human primary adipocytes exposed to 10% serum obtained before (PRE) or after (ECS) exercise for 12hr (n=6). Values represent means + SEM of fold change calculated from ∆∆Ct of GLUT4 mRNA related to housekeeping TBP1 mRNA (*): Paired t-test, p < 0.05 PRE vs ECS.
107
FIGURE 4.4: Effect of exercise serum on adipogenic marker mRNA expression in human primary adipocytes.
Gene expression levels of adipogenic markers in human primary adipocytes exposed to 10% serum obtained before (PRE) or after (ECS) exercise (n=4) as indicated in methods.Values represent mean + SEM fold change of mRNA expression calculated from ∆∆Ct of indicated gene related to housekeeping TBP1 mRNA. (*): Paired t-test, p < 0.05.
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FIGURE 4.5: Effect of exercise serum on adipogenic marker protein content in human primary adipocytes.
Protein expression level of adipogenic markers PPARγ and FABP4 in human primary adipocytes exposed to 10% serum obtained before (PRE) or after (ECS) exercise (n=4) as indicated in methods. Upper panels: Quantification of PPARγ and FABP4 protein contents. Bottom panel: representative GLUT4 blots for cells treated with PRE (2) and ECS (2). Values represent mean + SEM of protein expression normalized over total protein content. (*): Paired t-test, p < 0.05.
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FIGURE 4.6: Effect of exercise serum on insulin-stimulated glucose uptake in human primary adipocytes.
Glucose uptake in human primary adipocytes, with and without insulin, after 48 hr exposure to 10% serum obtained before (PRE) or after (ECS) exercise (n=8). Values are means + SEM of incorporation of 2-deoxy-glucose expressed in ρmol.min-1 and normalized over protein concentration. (*): Two-way ANOVA, p < 0.05, main effect Ins(+) vs Ins(-).
110
FIGURE 4.7: Effect of isoproterenol, IL-6 and FGF-21 treatments on GLUT4 mRNA expression.
Expression levels of GLUT4 in human primary adipocytes exposed to 1 µM isoproterenol, 20ng/mL IL-6 and 100 ng/mL FGF-21 as indicated in methods. Values represent mean + SEM fold change of GLUT4 mRNA expression calculated from ∆∆Ct related to housekeeping TBP1 mRNA. (*): Two-way ANOVA, time, serum treatment, and interaction effects, p < 0.005.
111
FIGURE 4.8: Effect of isoproterenol, IL-6, FGF-21, and 10% FBS treatments on adipogenic marker mRNA expression.
Expression levels of adipogenic markers in human primary adipocytes exposed to 10%FBS, 1µM isoproterenol, 20ng/mL IL-6 and 100ng/mL FGF21, as indicated in methods. A) Values represent mean + SEM fold change of mRNA expression calculated from ∆∆Ct of indicated mRNA related to housekeeping TBP1 mRNA. (*): One-way ANOVA, p < 0.05.
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4.4 DISCUSSION
Human exercise serum was used in an attempt to explore the possibility that changes observed in adipose tissue GLUT4 following exercise training may be induced by circulating factors, potentially muscle-derived secreted proteins with the capacity to improve adipose tissue insulin responsiveness and GLUT4 expression. Human serum is composed of more than four thousand compounds including, but not limited to, metabolites, several lipid species, ketones, glycoproteins, vitamins, enzymes, hormones, and neurotransmitters (Psychogios et al., 2011; Boudah et al., 2014). Inter-subject variation in serum composition is attributed to differences in gender, age, BMI, and health status of the donor (Dunn et al., 2015). In recent years, several studies have used human serum as to more accurately replicate the endogenous biochemical milieu of an in vivo approach into in vitro conditions. Human serum has proven to be effective in the expansion of multipotent stem cells and differentially reduced the expression of adhesion and extracellular matrix-associated proteins (Bieback et al., 2010) that may contribute to the therapeutic potential of cell therapy (Dreher et al., 2013). Autologous conditioned serum may also be beneficial for the treatment of degenerative diseases in humans, such as osteoarthritis (Frizziero et al., 2013).
Serum obtained from exercised subjects represents a biochemical milieu that contains circulating factors from contracting skeletal muscle, cytokines released from other organs in response to exercise, as well as hormones liberated by SNS-stimulated activation of secretory glands, mainly cortisol, catecholamines, and growth factors
(Maling et al., 1966; Pritzlaff et al., 1999; Zouhal et al., 2008). Exercise-conditioned serum has been shown to decrease cell proliferation and increase apoptosis rates in mammary and prostate cancer cell lines (Barnard et al., 2006; Hojman et al., 2011;
113
Soliman et al., 2011). Through a similar approach, conditioned serum from exercised healthy subjects, as well as conditioned media from electrically stimulated human skeletal muscle cells, prevented pro-inflammatory cytokine-induced pancreatic β-cell apoptosis and reduced β-cell insulin secretion, effects that were partially attributed to muscle-derived IL-6 (Christensen et al., 2015). Human ligament constructs, bioengineered from cells isolated from human anterior cruciate ligament, cultured in media supplemented with resistance exercise-conditioned serum showed significant increases in ligament mechanical proprieties and enhanced collagen content compared with serum obtained at rest (West et al., 2015; Lee-Barthel et al., 2017). Additionally, other sources of serum conditioning (e.g. calorie-restriction, and remote ischemic preconditioning) have reported similar results in vitro, overall providing cytoprotective effects to cells in culture (Zitta et al., 2012; de Cabo et al., 2015; Weber et al., 2015).
Data in the present study showed increased GLUT4 expression in human primary adipocytes exposed to exercise serum that was both dose and time-dependent. GLUT4 mRNA levels were increased ~3-fold at 12 hr, but were unchanged at 6 and 24 hr of exposure, whereas GLUT4 protein content was increased by 15–20% at 48hr compared with exposure to 10% serum obtained at rest before exercise. In spite of the increase in adipocyte GLUT4 expression, there was no change in insulin-stimulated glucose uptake after 48hr exposure to ECS. It is possible that circulating metabolites and/or hormones in the exercise serum may have exerted an inhibitory effect on insulin stimulation of glucose uptake that offset any potential benefit of increased GLUT4 protein expression.
Exercise serum has been shown to reduce insulin secretion in vitro from INS1 cells and ex vivo from pancreatic islets (Christensen et al., 2015). The composition of human serum is dominated by DGs, TGs, phospholipids, FAs, steroids and steroid derivatives
(Psychogios et al., 2011) that may interfere with insulin action in adipocytes leading to
114 reduced insulin-stimulated glucose uptake. Furthermore, intense exercise acutely increases the release of catecholamines into circulation by 3 to 9-fold (Kindermann et al., 1982; Stich et al., 2000), which overdrives the hormonal control of insulin on glucose regulation during exercise in the post-absorptive state (Sigal et al., 1996).
Exposure to ECS serum also induced a transient increase in the expression of adipogenic (PPARγ, C/EBPα, PLIN1) genes at 6hr of the treatment. This response may have been driven by the presence of certain metabolites in the serum, such as FAs, which can regulate gene expression in a hormonal-independent manner (Pegorier et al.,
2004). The plasma concentration of non-esterified fatty acids is reported to be increased by three to five-fold during moderate intensity exercise (Horowitz & Klein, 2000; van
Loon et al., 2005), reaching peak concentrations in blood at the end of an exercise bout
(Mulla et al., 2000). It is likely that increased concentration of FAs in the exercise serum may have generated an input that stimulated gene transcription in the human primary adipocytes. This effect has been extensively reviewed elsewhere (Duplus et al.,
2000; Fernyhough et al., 2007; Georgiadi & Kersten, 2012). However, and in contrast to
GLUT4 protein content, no change in the protein concentration of PPARγ and FABP4 was observed at 48h of the ECS exposure. These findings suggest an inhibitory effect associated with a prolonged exposure that compensated the transient transcriptional activation observed early on during the treatment, as has been previously described
(Armoni et al., 2005). Alternatively, differences in the gene expression pattern and protein turn-over may also explain the early induction and absence of protein accumulation later on during the treatment observed in the present study. This regulatory mechanism has been previously described for other mediators of adipogenesis (Lechner et al., 2013). Notably, GLUT4 protein half-life is described to be
115
~50hr and is significantly reduced by chronic insulin treatment (Sargeant & Paquet,
1993).
Next, the effect of β-adrenergic agonist isoproterenol on GLUT4 expression in human primary adipocytes was explored. Beta-adrenergic activity has been reported modulating GLUT4 expression during fasting and re-feeding. This effect is characterized by marked repression of GLUT4 gene and protein during fasting and rapid upregulation over refeeding, with the latter response being blunted by beta-adrenoceptor blockade (Zanquetta et al., 2006). In the present study, there was an early induction of
GLUT4 mRNA at 6 hr during 1µM isoproterenol treatment; however, this response did not reflect an increased GLUT4 protein content at later time points.
The present study also sought to explore the role of IL-6 and FGF21 on the expression of GLUT4 in human primary adipocytes, since both cytokines have been reported to potentially mediate metabolic adaptations in adipose tissue (Petersen et al., 2005; Ji et al., 2011; Brandt et al., 2012; Kim et al., 2013; Knudsen et al., 2014; Lee et al., 2014;
Mottillo et al., 2017). Neither IL-6 (20ng.ml-1) nor FGF21 (100ng.ml-1) treatments induced the expression of GLUT4 and adipogenic genes (data not shown). The same IL-
6 treatment concentration has been reported to exert a direct lipolytic effect and induce mitochondrial dysfunction, although without direct effect on insulin-stimulated glucose transport in 3T3-L1 adipocytes (Ji et al., 2011). Although, whole-body IL-6 knockout has shown to impair PPARγ, TNF-α, leptin and UCP-1 expression and function in the adipose tissue of mice in response to either exercise training or cold exposure (Brandt et al., 2012; Knudsen et al., 2014) suggesting auto/paracrine effects, muscle-specific overexpression of IL-6 did not exert a favourable endocrine role in the adipose tissue
(Brandt et al., 2012). The latter agrees with unaffected GLUT4 and adipogenic gene expression in the present study. In relation to FGF21, FGF21 receptor (FGFR1c) and its
116 obligate co-receptor, b-klotho (KLB) are expressed in human adipose stem cell-derived adipocytes, conferring the cells with the capacity to respond to pharmacologically administered FGF21. FGF21 treatment has been reported exerting synergistic effect to insulin on glucose uptake, which was associated with a marked increase in Akt-Ser-473 phosphorylation (Lee et al., 2014). However, in the present study, no change was observed in GLUT4 expression in human primary adipocytes exposed to 100ng.ml-1
FGF21. Taken together, these finding suggest that the effect of exercise serum on
GLUT4 expression is not mediated by IL-6 or FGF21.
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4.5 CONCLUSION
The findings reported in this chapter showed a time-dependent effect of exercise serum in human primary adipocytes, with an early, but transient (at 6hr), activation of adipogenic genes, including PPARγ, C/EBPα, and PLIN1, which may have, at least in part, driven GLUT4 expression later on during the serum exposure (at 12hr), leading to increased GLUT4 protein content at the end of the 48h serum exposure. Most importantly, data presented here strongly suggest the presence of (a) humoral mediator(s) released during exercise, (an) “exerkines(s)” with the capacity to drive the metabolic response to exercise in adipocytes.
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5 CHAPTER FIVE: SUMMARY AND FINAL CONCLUSIONS
The regulation of circulating glucose levels as well as the capacity to detect and respond to changes in intracellular glucose flux, i.e. glucose sensing, are tightly controlled metabolic responses. Such control can protect against dysfunction such as loss of consciousness and fatigue due to hypoglycaemia during prolonged exercise and toxicity to peripheral tissues in response to the chronic hyperglycaemia of diabetes (Huang &
Czech, 2007). The glucose sensing function is not restricted to pancreatic β-cells and neurons, but tissues such as the liver and adipose tissue may also sense variations in circulating glucose levels and then communicate this “message” to other tissues through cross-talk mechanisms, i.e. the secretion of hormones, cytokines or circulating metabolites (Herman & Kahn, 2006). These processes rely on the integrity of glucose transport into cells. GLUT4 is the major insulin-sensitive glucose transporter expressed predominantly in skeletal and cardiac muscles, and adipose tissues, where it carries glucose inside the cells in response to insulin and muscle contraction. Despite a quantitatively minor role of adipose tissue in whole-body glucose disposal, adipose tissue insulin-mediated glucose uptake and the expression of GLUT4 have been described to be of importance for both glucose homeostasis and whole body insulin action (Abel et al., 2001; Birnbaum, 2001; Herman & Kahn, 2006). It has been previously shown that selective ablation of adipose tissue GLUT4 impaired insulin action in skeletal muscle and the liver leading to glucose intolerance and hyperinsulinemia (Abel et al., 2001), whereas its over-expression prevents the development of insulin resistance (Carvalho et al., 2005). In humans, adipose tissue
GLUT4 expression is down-regulated in all insulin resistant states including obesity,
120 metabolic syndrome, and type 2 diabetes mellitus, while muscle GLUT4 remains unaffected. Interestingly, exercise training increased GLUT4 expression in the adipose tissue of patients with type 2 diabetes to levels similar to those seen in healthy subjects
(Hussey et al., 2011).
Whether the down-regulation of adipose tissue GLUT4 is a primary defect in the pathogenesis of insulin resistance, and whether exercise training can alleviate this alteration were questions that were further explored in the present study. In Chapter 2, we had the possibility to analyse frozen adipose tissue samples from a study that examined the time-course of HFD effects on whole-body and tissue-specific insulin sensitivity (Turner et al., 2013). In this study, impaired glucose tolerance appeared in parallel with an increase in fat mass during the initial weeks on the diet. Most importantly, the reduction in systemic insulin sensitivity, reflected by impaired glucose infusion rate and disposal during hyperinsulinaemic-euglycaemic clamp, was observed after only a week on a HFD, and it was associated with blunted adipose tissue insulin- stimulated glucose uptake. We analysed the GLUT4 protein content in EPI fat by immunoblotting, and we found that adipose tissue GLUT4 was markedly down- regulated, and that this alteration occurred early on during the diet and remained reduced throughout the course of the intervention. This pattern matched the blunted insulin-stimulated glucose uptake. Taking into consideration that insulin-signalling pathway remained intact and that there was no evidence of inflammation occurring at this early stage, findings that have been also observed by others (Kleemann et al., 2010;
Kim et al., 2015), it is likely that the down-regulation of GLUT4 was the main factor mediating the reduced insulin action occurring rapidly after HFD in the adipose tissue, therefore being a major contributor to HFD-induced adipose tissue-specific insulin resistance. Interestingly, once GLUT4 downregulation develops, it plateaus and is
121 maintained over the course of the HFD intervention suggesting that this defect is secondary to a direct effect of the diet and is not affected by the gradual accumulation of bioactive lipid species, i.e. ceramides and sphingomyelin (as described in the primary,
Turner et al., 2013), which is another alteration described in the time course of HFD- induced IR that has been implicated in the pathogenesis of insulin resistance (Bruce et al., 2012; Samuel & Shulman, 2012). If the accumulation of lipid species in the adipose tissue would have repressed GLUT4 expression levels, GLUT4 protein content would have been gradually lost, coupling with the increasing lipid concentration.
Next, we asked the question whether an exercise training intervention prevented the down-regulation of adipose tissue GLUT4 in the context of HFD-induced IR. We studied changes in adipose tissue GLUT4 protein content in EPI fat samples collected from exercise-trained C57/Bl6 mice following HFD (Jordy et al., 2015). In these animals, obesity and IR were induced by HFD for 4 wk. Mice then underwent an exercise training program for another 4 weeks. In this study, we found that EPI fat samples from exercise trained mice showed an increased GLUT4 protein, although this effect was only evident under HFD conditions with no effect in mice fed a chow diet
(Figure 2.4). Although unexpected, this finding agreed with inconsistent exercise training effect on adipose tissue GLUT4 previously reported in rats fed either a normal or a high fat diet (Hirshman et al., 1993; Gollisch et al., 2009). Recently, it has been suggested that fasting and/or feeding conditions may interfere with the effect of exercise on adipose tissue (Chen et al., 2017). Although speculative, one explanation for the lack of an exercise training effect in chow fed mice is, potentially, the undeliberate access to food or an extended fasting period in this group before the animals were culled for the experiments.
122
Also, and to our surprise, we did not find a HFD effect on adipose tissue GLUT4 of sedentary mice, despite the rapid HFD-induced reduction in adipose tissue GLUT4 protein from early stages and throughout the diet intervention reported previously in this thesis (Figure 2.1). This was a puzzling finding, which we were unable to explain, given that the animals in both studies were housed under the same conditions, and that the
HFD interventions in both studies were exactly the same.
One of the most important adaptations following exercise training in skeletal muscle is the increase in muscle oxidative capacity. This effect is characterized by increased expression and functions of mitochondrial enzymes, increased mitochondrial mass and number and angiogenesis, which together increase metabolic function and capacity for substrate turn-over in skeletal muscle. In view of the predominant role of skeletal muscle in insulin-stimulated glucose disposal, these improvements mediate the beneficial effect of exercise training in whole body insulin sensitivity and glucose homeostasis. It has been previously shown that high intrinsic muscle oxidative capacity has systemic metabolic effects that protect against diet-induced IR (Morris et al., 2014;
Vieira-Potter et al., 2015; Matthew Morris et al., 2016). Furthermore, the training- induced adaptation in muscle oxidative capacity is deemed essential for the therapeutic effect of exercise training in the treatment of metabolic alterations including insulin resistance, obesity and diabetes in humans (Reitman et al., 1984; Segal et al., 1991).
Thus, we hypothesised that an increased muscle oxidative capacity is also necessary to induce a training effect on adipose tissue GLUT4 expression. To investigate this hypothesis, we had access to adipose tissue samples from transgenic mice selectively over-expressing HSP72 in skeletal muscle (Henstridge et al., 2014). These animals displayed marked increases in muscle oxidative and endurance running capacities that were associated with protective effects against HFD-induced IR, and body mass and fat
123 gains. These mice also showed an increased insulin-stimulated glucose clearance in muscle and adipose tissues. In the present study, however, this phenotype did not prevent HFD-induced adipose tissue GLUT4 down-regulation (Figure 2.5). From this result, we concluded that perhaps any effect of increased muscle oxidative capacity upon adipose tissue GLUT4 expression requires the intervention of another mediator which was not directly affected by the over-expression of HSP72. One of the key regulators of muscle mitochondrial biogenesis and oxidative capacity is the transcriptional regulator PGC-1α (Olesen et al., 2010). Most of the muscle adaptations following exercise training have been associated with PGC-1α (Lira et al., 2010; Perez-
Schindler et al., 2014). Moreover, PGC-1α has been related to the expression and secretion of the myokine irisin, which drives endocrine effects on adipose tissue by inducing browning of WAT and thermogenesis (Bostrom et al., 2012). Interestingly, the expression of PGC-1α was not differentially regulated in the muscle-specific HSP72 transgenic model (Henstridge et al., 2014). This finding is particularly important, since it appears to dissociate the muscle oxidative phenotype from PGC-1α, at least in terms of potential effects on the adipose tissue phenotype. The latter led us to reformulate our discussion as to consider that, perhaps, a cross-talk mechanism is necessary in order to adipose tissue GLUT4 to be upregulated following exercise training, and that this mechanism may be associated with muscle PGC-1α or with a PGC-1α-related myokine, secreted during exercise, and not directly mediated by an increased muscle oxidative capacity.
Another interesting aspect to consider in relation to the effect of exercise training on adipose tissue GLUT4 expression is the time course of this response. As mentioned above, preliminary results from our lab have shown no change in adipose GLUT4 expression acutely after a single bout of exercise in healthy subjects (Boland et.al.
124 unpublished data). On the other hand, four weeks of exercise training increased adipose tissue GLUT4 proteins levels, albeit in a clinical population of type 2 diabetes patients
(Hussey et al., 2011). Studies in rats have shown increased GLUT4 expression and function following long-term exercise training (6 to 8 weeks) (Hirshman et al., 1993;
Stallknecht et al., 1993). There is also evidence of increased adipose tissue GLUT4 after short-term training (four days) in rats (Ferrara et al., 1998). However, the short-term effect of exercise training has not been explored in humans. Therefore, in my second study, we aimed to determine the effect of short-term 10-day training on adipose tissue
GLUT4 expression in a cohort of healthy, but sedentary, subjects. In this study, the expected adaptations to exercise training, e.g. increases in VO2 peak and PPO (Table
3.1), were observed in association with improved metabolic response to exercise in skeletal muscle, as reflected by increased GLUT4 and COX-IV protein contents
(Figures 3.1 and 3.2). However, there was no exercise training effect on adipose tissue
GLUT4 protein content following 10 days of exercise training. Additionally, glucose transporter GLUT1, transcription factor MEF2A, class II HDAC5, adipogenic marker
FABP4 and markers of oxidative capacity COX-IV and total OXPHOS were also unchanged by short-term intense endurance training. In agreement with previous findings (Camera et al., 2010; Larsen et al., 2015), we concluded that a longer exposure to the exercise stimulus may be required in order to induce an exercise training effect on adipose tissue.
At this point in the present investigation, our results showed adipose tissue GLUT4 protein content to be increased in high fat fed mice following four weeks of exercise training, although this effect did not appear to be associated with increased muscle oxidative capacity, at least as driven by muscle-specific HSP72 over-expression, and that an exercise effect on adipose tissue GLUT4 expression was absent after short-term
125 training in healthy humans. Nevertheless, studies in rodents have shown that exercise training increases adipose tissue GLUT4. We were interested to determine whether this effect may be mediated by circulating factors released during exercise and tested this hypothesis directly in cultured human primary adipocytes using human exercise serum.
Additionally, we sought to compare the effect of exercise serum with well-known myokines IL-6 and FGF21, the expression of which is acutely increased in skeletal muscle after exercise (Catoire et al., 2014), and the β-adrenoceptor agonist isoproterenol, which has been implicated in the regulation of GLUT4 during feeding and fasting (Zanquetta et al., 2006). The finding of an increased GLUT4 expression in human primary adipocytes cultured in exercise serum suggests the presence of (a) circulating factor(s), other than known myokines IL-6 and FGF21, which effects on
GLUT4 expression were induced time and dose-dependently (Figures 4.1, 4.2, and 4.3).
Notwithstanding the beneficial effect of exercise serum on GLUT4 expression in human primary adipocytes, no effect was observed in insulin-stimulated glucose uptake (Figure
4.6). This finding dissociates the biological effect of a circulating factor, present in the exercise serum, on GLUT4 expression from the potential functional relevance of increasing the expression of glucose transporters on insulin action in adipocytes. A study in mature human adipocytes differentiated from SVF-derived precursor cells showed higher absolute rate of insulin-stimulated glucose uptake but similar insulin sensitivity (% maximal response) in cells cultured in 3% foetal bovine serum compared with cells cultured in media without serum (Lee et al., 2012). Our results showed similar insulin-stimulated glucose uptake in cells exposed to 10% serum collected before (PRE) and after (ECS) an acute exercise bout in humans (Figure 4.6). Despite of increased total abundance of GLUT4 protein in human primary adipocytes exposed to
ECS compared with cells exposed to PRE, the absence of a differential glucose uptake
126 response to insulin stimulation suggest that translocation of GLUT4 vesicles to the plasma membrane might be compromised by factors present in the ECS.
Here, it is worth commenting on the limitations associated with this study. Due to a constrained number of human primary cultures, we had to prioritize our resources and efforts to the most critical experiments. The optimization and preliminary experiments were included in this thesis as supplementary information (Chapter 6). Some of the proposed experiments that could not be carried out aimed to: 1) extend the study of the time-course effect during ECS exposure, 2) explore different candidate circulating factors, 3) modify the serum milieu, e.g. lipid depletion, antibody-mediated neutralization of cytokines, serum de-proteinization, etc. These approaches may be considered in future studies.
The exercise serum is likely to be enriched with exercise-responsive hormones, cytokines and other mediators that make it an optimal milieu in vitro to replicate the complexity of the endogenous biochemical conditions in vivo during exercise.
Nonetheless, it is the same complexity of its composition that makes it difficult to isolate a potential individual factor, and therefore to address its specific biological effect contributing to the findings observed in the present study. Challenging as it may be, the identification of circulating mediators would eventually require a broader spectrum of approaches aiming to screening for potential candidates, as well as exploring sources/tissues of origin, mechanisms of production and secretion (e.g. cleavage and release of mature proteins or vesicle transport), transport and delivery (i.e. association with transport proteins in plasma), and signal transduction of this/those candidate factor(s). One possibility would be to utilize the advantages of “multi-omics” analyses including non-targeted proteomics, metabolomics and lipidomics in exploring the exercise signature effects on adipose tissue. We have carried out preliminary studies
127 using new generation RNA sequencing in muscle biopsies, collected before and after an acute exercise bout, in association with bioinformatics analyses in order to identify novel exercise-induced myokines. The next steps into this explorative investigation would be cross-referencing the list of up-regulated, potentially secreted, muscle protein candidates with “omics” analyses in plasma or alternatively multiplex immunoassay for myokine profiling (Schipper et al., 2010). These approaches would pinpoint potential candidates, which would be followed by proof of principle experiments in in vitro as well as in vivo models.
In terms of exploring mechanisms of action of any factor(s) potentially involved in the regulation of adipose tissue GLUT4, these experiments should include the study of class
II HDAC5 enzymatic function, a well-known mechanism that has been described playing a critical role in mediating the regulation of GLUT4 expression in skeletal muscle during exercise (McGee & Hargreaves, 2004; McGee et al., 2006; McGee et al.,
2008; McGee et al., 2009), and in adipocytes during adipogenic differentiation and following fasting (Weems & Olson, 2011; Weems et al., 2012).
In summary, conclusions that can be drawn from the studies in this thesis are:
1. Murine adipose tissue rapidly responds to a HFD with reduced GLUT4 levels, which is associated with reduced insulin-stimulated glucose uptake.
2. Exercise training increases adipose tissue GLUT4 protein content in trained
HFD-fed mice, but not in chow fed mice.
3. Increased muscle oxidative capacity does not prevent HFD-induced GLUT4 down-regulation in mouse adipose tissue.
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4. In humans, short-term (10 d) exercise training did not increase adipose tissue
GLUT4 levels, despite an increase in skeletal muscle GLUT4 and oxidative capacity.
This implies differential time course and regulation of GLUT4 expression in adipose tissue and skeletal muscle and that a longer term exercise stimulus may be required to increased adipose tissue GLUT4 levels.
5. Exercise serum increased GLUT4 expression levels in human primary adipocytes, suggesting that circulating factor(s) may potentially mediate an exercise- induced increase in adipose tissue GLUT4 levels. The underlying regulatory mechanisms and identification of potential factor(s) and their tissue of origin warrant further investigation.
An intriguing question that still remains from the present series of studies is whether preventing the down-regulation of adipose tissue GLUT4, and the development of adipose-specific IR, could dampen the progression of systemic insulin resistance. If indeed increased adipose tissue GLUT4 expression has this potential benefit, then further identification of regulatory mechanisms and their relation to exercise, in future studies is important for the optimization of exercise interventions and the identification of novel therapeutic strategies that target adipose tissue for the treatment and management of metabolic disease.
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6 CHAPTER 6: SUPPLEMENTARY INFORMATION
6.1.1 Preliminary experiments:
The initial approach for this part of the present investigation was to utilize a well- characterized model of adipocyte biology in culture, the 3T3-L1 cells. This model has been extensively utilized for the study of adipocyte differentiation and glucose metabolism (Stephens & Pekala, 1991; Hemati et al., 1997; Klemm et al., 1998;
Yokomori et al., 1999; Reusch et al., 2000; Dowell & Cooke, 2002; Gao et al., 2011;
Ling et al., 2012; Shen et al., 2013; Vishwanath et al., 2013). Pilot experiments were performed in order to profile the expression of GLUT4 in association with classic adipogenic markers (see supplementary information, chapter 6). However, for the purpose of this research, it was decided not to continue with this cell line for the experiments involving culturing the cells in human ECS owing to previous evidence describing that differentiated 3T3-L1 adipocytes display: 1) mixed adipocyte linage features between white and brown adipocytes, 2) high susceptibility to catecholamine- induced thermogenesis, and 3) low beige gene expression profile (Morrison & McGee,
2015). Experiments at this stage supported the development of technical skills and knowledge, which were applied later in the design and planning of experiments in human primary adipocytes.
6.2 Characterization of GLUT4 and adipogenic genes expression in 3T3-L1 cells during adipogenic differentiation.
The time-sequence of GLUT4 gene expression was assessed in vitro in murine 3T3-L1 fibroblast. 3T3-L1 fibroblasts (American Type Culture Collection [ATCC], passage 15)
175 were cultured to confluence in proliferation media (D-Modified Eagle’s Medium
[DMEM], high glucose [25 mM], 4mM L-glutamine, 1mM pyruvate, Life
Technologies-GIBCO®), supplemented with 10% (v/v) heat-inactivated foetal bovine serum [FBS] (Life Technologies-GIBCO®) and 1% (v/v) penicillin/streptomycin (1:1,
-1 o 100U.ml , GIBCO®) at 37 C and 10% CO2. Passage #15 cells proliferated until ~95% confluent, then passaged again (P#16) by enzymatic de-attachment using 30µl.cm-2 TEP
(0.25% Trypsin, 0.5M EDTA prepared in PBS), and seeded accordingly in 12-well plates for differentiation. Fully confluent cells were differentiated to adipocytes by exposing the cells to adipogenic inducers: 2µg.ml-1 insulin (Humulin), 2.54µM dexamethasone (Sigma Aldrich) and 0.5mM IBMX (Sigma Aldrich). At day 3 post- induction, cells were maintained in DMEM media containing 2µg.ml-1 insulin until experimental day.
6.3 Gene expression of adipogenic markers in 3T3-L1 cells during differentiation:
Gene expression of adipogenic genes (PPARγ, C/EBPα, FAS and FABP4) and glucose transporters GLUT1 and GLUT4 was assessed at days 0, 1, 2, 4, 6, 8, and 10 of the adipogenic differentiation of 3T3-L1 pre-adipocytes by real time qPCR. Cells were seeded in 6-well plates, each time point in triplicate. At each time-point, cells were lysed in well in 500 µl Trizol, and mechanically disrupted using a cell scraper (lifter), and thoroughly homogenized by pipetting in a 0.3ml syringe. 100 µl chloroform were added to the lysates and mixed vigorously for 15 sec, and incubated for another two min at RT. Lysates were spun for 15 min x 12,000g at 4oC. Next, RNA-containing supernatant was transferred into a new set of microtubes. Equal volume of 70% ethanol was added to the samples, mix by vortexing, quickly spun and transferred to RNA
176 elution columns (RNeasy mini columns, Qiagen). Samples were then subjected to serial centrifugations and washes to finally elute the RNA. Eluted RNA was recovered into a final volume of 100 µl RNase-free water. Quantity and quality of the RNA were determined using the Agilent Bioanalyser 2100.
Reverse transcription for real time qPCR was carried out using the SuperScript® III
Reverse Transcriptase kit (Thermo-Fisher Scientific) according to manufacturer’s instructions. Briefly, RNA samples were thawed in ice while reaction mix was prepared.
Reaction mix contains 10 mM dNTP mix (10 mM each dATP, dGTP, dCTP and dTTP at neutral pH) and Oligo(dT)18 primer. 10 µl of isolated RNA were added into the reaction mix. All components were mixed, briefly spun and incubated at 65C for 5 min, then incubated on ice for at least 1 min prior to the addition of the SuperScript™ III RT
(200 units per μl) mix that also contains First-strand buffer, 0.1M DTT and
RNaseOUT™ Recombinant RNase Inhibitor (40 units per μl). 20 µl final reaction mix was incubated in the thermocycler as follows: 1) enzyme activation: 30 min at 50C, 2)
Inactivation by heating at 70C for 15 min, 3) 5 min at 4C. Next, cDNA quantification was carried out using the Quant-iT™ OliGreen® ssDNA Assay Kit.
Synthetized cDNA was diluted 1:10 in nuclease-free water (provided in the kit).
Samples were loaded in a black-96-well fluoro plate together with oligo standards in triplicate. Oligo standard serial dilution from 100 µg.ml-1 was prepared as follows: 15
µg.ml-1, 12.5 µg.ml-1, 10 µg.ml-1, 7.5 µgml-1, 5 µg.ml-1, 2.5 µg.ml-1 and 1.25 µg.ml-1 in
1X TE buffer. Primers sequences are detailed in Table 4.2.
All values are reported as means + SEM. For the characterization of GLUT4 expression in 3T3-L1 adipocytes, relative expression of genes of interest was determined in relation
177 to Ct values obtained at day 0. Fold change was calculated and this value represented relative to day 0.
6.4 Differentiation of human SVF-derived pre-adipocytes and exposure to conditioned serum:
Cells proliferated in approximately 10 to 14 days until reaching confluency. Then, pharmacological differentiation was induced for 7 days as described in methods. Insulin was maintained in the media for another 3 days. On day 10, cells were insulin starved for 48 hr. On day 12, cells were treated with conditioned serum (PRE and ECS), isoproterenol and selected myokines IL-6 and FGF21. On day 14, after 48-hour exposure, cell morphology was characterized by ORO staining and featured increased cell volume, with abundant intracellular lipid droplets of different sizes (Supplementary
Figure 6.3).
6.5 Lipogenic gene expression in human primary adipocytes
The gene expression of lipogenic markers ChREBP, ChREBPα, ChREBPβ, SREBP-1c, and FAS was determined by real time qPCR using qPCR probes (Sigma®,
Supplementary Table 6.2). Housekeeping gene hypoxanthine phosphoribosyltransferase
1 (HPRT1) was utilized to determine relative expression of genes of interest (GOI).
Primer sequences for lipogenic genes were determined as previously described
(Kursawe et al., 2013). Experiments were performed twice and each treatment in duplicate. Cell samples were exposed to increasing concentrations of exercise serum for
12hrs. Sample homogenization and RNA purification were assessed using PureLink®
RNA Mini Kit (Ambion®, Life Technologies ®) according to manufacturer’s directions. Purified RNA was reconstituted in 30 µl nuclease-free water and assessed for
RNA quality and quantification using nanodrop.
178
Single strand cDNA chains were synthesised from purified RNA using iSCRIPTTM cDNA synthesis kit (Bio-Rad, Hercules CA, USA) according to manufacturer’s specifications to a final concentration of 2.5 ng.µl-1. Real-time quantification was performed using the QuantiNovaTM SYBR® Green PCR Kit (Qiagen). Amplification efficiency of target (ChREBP total, alpha and beta, SREBP1c, and FAS) and reference
HPRT1 genes was determined by standard curve fitting using gradual dilution of cDNA
(1:10, 1:20, 1:50, 1:100 and 1:500) on each primer set. Then, relative gene expression was determined using the delta-delta Ct method (∆∆Ct) comparing serum treated groups with control-10% FBS treated group and normalizing over the expression of the housekeeping gene HPRT1.
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6.6 SUPPLEMENTARY RESULTS
6.6.1 Expression of adipogenic markers in 3T3-L1 adipocytes during differentiation:
On differentiation day 10, cells were fully mature and featured functional characteristics of adipocytes, reflected as lipid-loading capacity and insulin responsiveness (ORO staining and glucose uptake, data not shown). Expression of both adipogenic regulators
C/EBPα and PPARγ was significantly increased at day 4 compared with day 0 (p < 0.05 and p < 0.01, respectively), reaching peak values at day 6 (p < 0.0001). Induction of these genes was progressively downregulated after day 6 (day 6 vs day 8, p< 0.005
C/EBPα and p< 0.0005 PPARγ; day 6 vs day 10, p < 0.0005 C/EBPα and p < 0.0001
PPARγ; Supplementary Figure 6.1-A-B). Similar expression profile was observed in adipogenic genes FAS, FABP4, which reached peak values on day 6 compared with day
0 (p < 0.005 FAS and p < 0.0001 FABP4). FABP4 expression was induced on day 4 and stayed increased until day 8 (p < 0.0005 compared to day 0 and 1, and p < 0.005 compared with day 2), whereas FAS expression was significantly decreased after day 6
(p < 0.005 day 6 vs day 10; Supplementary Figure 4.1-C-D).
6.6.2 Expression of glucose transporters expression in 3T3-L1 adipocytes during differentiation:
GLUT4 gene expression was significantly increased by 6-fold at day 6 (p < 0.0001 compared with days 0, 1 and 2, and p < 0.0005 with day 4). Expression was maintained significantly increased until day 8 (p < 0.005) but markedly reduced by day 10 (p <
0.005, compared with day 6). The expression of GLUT1 was also significantly increased by days 6 and day 8 compared with day 0 (p < 0.0005 and p < 0.005, respectively). Peak value at day 6 was also significantly higher compared to day 1, 2
180 and 4 (p < 0.005 day 6 vs day 1, p < 0.05 day 6 vs day 2 and 4). By day 10, GLUT1 gene expression was markedly downregulated when compared with day 6 (p < 0.05)
(Supplementary Figure 6.2-A-B).
6.6.3 Dose-response effect of exercise serum on GLUT4 protein in human primary adipocytes:
Forty eight hours exposure to 10% exercise serum in the media significantly increased
GLUT4 protein content in human primary adipocytes serum compared to untreated cells
(p < 0.05) with no further effect in cells treated with higher serum concentrations. Based on this observation, a 10% serum concentration was utilized in following experiments
(Supplementary Figure 6.4-A-B).
6.6.4 Dose-dependent effect of exercise serum on lipogenic markers:
Exposure to increasing concentrations of exercise serum in the media increased the expression of lipogenic genes ChREBP isoforms α and β, SREBP-1c and FAS compared to 10% FBS. 20% ECS in the media significantly increased ChREBPs and
FAS mRNA expression, whereas 10% ECS increased SREBP-1 (p < 0.05).
181
Gene Forward Reverse
CEBPα 5’-CGCAAGAGCCGAGATAAAGC-3’ 5’-GTCATTGTCACTGGTCAACTCC-3’
FASN TCGGGTGTGGTGGGTTTGG 5’-GCGTGAGATGTGTTGCTGAGG-3’
PPARγ 5’-GGAAGCCCTTTGGTGACTTTATGG-3’ 5’-GCAGCAGGTTGTCTTGGATGTC-3’
GLUT1 5’-CAGCAGCAAGAAGGTGACG-3’ 5’-TGGTGAGTGTGGTGGATGG-3’
GLUT4 5’-CCAGCCTACGCCACCATAG-3’ 5’-TTCCAGCAGCAGCAGAGC-3’
FABP4 5’-ACACCGAGATTTCCTTCAAACTGG-3’ 5’-TCTTCACCTTCCTGTCGTCTGC-3’
SUPPLEMENTARY TABLE 6.1: Primer sequence for genes of interest in 3T3-L1
Primer name Primer Sequence
F 5’-GAGACAAGATCCGCCTGAAC-3’ ChREBP total R 5’-GTCACGAAGCCACACACG-3’ F 5’-AGTGCTTGAGCCTGGCCTAC-3’ ChREBPα R 5’-TTGTTCAGGCGGATCTT GTC-3’ F 5’-AGCGGATTCCAGGTGAGG-3’ ChREBPβ R 5’-TTGTTCAGGCGGATCTTGTC-3’ F 5’-CGGAACCATCTTGGCAACA-3’ SREBP1c R 5’-GCCGGTTGATAGGCAGCTT-3’ F 5’-CGCTCGGCATGGCTATCT-3’ FAS R 5’-CTCGTTGAAGAACGCATCCA-3’ F 5' -TCCTCCTCAGACCGCTTTT-3' HPRT1 R 5' -CCTGGTTCATCATCGCTAATC-3' SUPPLEMENTARY TABLE 6.2: Lipogenic genes, primers sequence
182
SUPPLEMENTARY FIGURE 6.1: Adipogenic markers during 3T3-L1 adipogenic differentiation.
Adipogenic program was pharmacologically induced for 3 days in 3T3-L1 cells. All values represent mean + SEM of Ct/cDNA relative to day 0 or indicated comparisons in brackets. A) PPARγ, B) C/EBP1α, C) FAS, and D) FABP4. One-way ANOVA, Turkey post-hoc test, (*): p < 0.05, (**): p < 0.01, (***): p < 0.0005, (****): p < 0.0001.
183
SUPPLEMENTARY FIGURE 6.2: Glucose transporter expression during 3T3-L1 adipogenic differentiation
Adipogenic program was pharmacologically induced for 3 days in 3T3-L1 cells. All values represent mean + SEM of Ct/cDNA relative to day 0 or indicated comparisons in brackets; A) GLUT4, and B) GLUT1. One-way ANOVA, Turkey post-hoc test, (*): p < 0.05, (**): p < 0.01, (***):p < 0.0005, (****):p < 0.0001.
184
SUPPLEMENTARY FIGURE 6.3: Proliferation and differentiation of human primary adipocytes
A) Representative light microscopy images (10x, bar: 400µm) of SVF-derived cells at days 2, 4, and 10 during proliferation. At day 10, adipogenic differentiation was induced as described in methods. B) Mature human primary adipocytes on days 12 and C) day 14 during differentiation. Lipid content in mature human primary adipocytes was stained using ORO staining. Representative 10x (bar: 400µm) and 40x (bar: 100µm) images are shown.
185
SUPPLEMENTARY FIGURE 6.4: Dose-response effect of exercise serum on GLUT4 protein in human primary adipocytes.
Mature human primary adipocytes were exposed to 5%, 10%, 20% and 50% exercise serum for 48h as described in methods. Cells were harvested and analysed for GLUT4 protein content by immunoblotting. A): Values represent mean + SEM of arbitrary units of GLUT4/total protein ratio; B): representative GLUT4 immunoblot and corresponding total protein staining; (*) One-way ANOVA, Bonferroni post hoc test, p < 0.05.
186
SUPPLEMENTARY FIGURE 6.5: Dose-response effect of exercise serum on Lipogenic markers in human primary adipocytes.
Mature human primary adipocytes were exposed to 5%, 10%, 20% and 50% exercise serum for 6hr as described in methods. Cells were harvested and analysed for lipogenic genes expression. Values represent mean + SEM of fold change mRNA expression calculated from ∆∆Ct of indicated mRNA related to housekeeping HPRT mRNA; A) total ChREBP, B) ChREBPα, C) ChREBPβ, D) SREBP-1c, and E) FAS. (*): Two-way ANOVA, Bonferroni post hoc test, p < 0.05.
187
SUPPLEMENTARY FIGURE 6.6: Effect of isoproterenol, IL-6 and FGF-21 treatments on GLUT4 protein expression
Protein expression levels of GLUT4 in human primary adipocytes exposed to 1 µM isoproterenol, 20ng.ml-1 IL-6 and 100 ng.ml-1 FGF21 as indicated in methods. Upper panel: Quantification of GLUT4 protein content. Values represent mean + SEM of protein expression normalized over total protein content. Bottom panel: representative immunoblot and total protein. (*): Two-way ANOVA, time, serum treatment, and interaction effects, p < 0.005.
188
6.7 GLUT4 expression and adipogenic profile in 3T3-L1 adipocytes
In the first part of the present study, the gene expression profile of adipogenic markers and glucose transporters was analysed during the differentiation of murine 3T3-L1 fibroblasts to adipocytes. Expression of adipogenesis master regulators PPARγ and
C/EBPα appeared elevated 96hr after the adipogenic induction was initiated, which agreed with previously reported findings (Jefcoate et al., 2008). This process is deemed critical for the sequential activation of a large number of downstream target genes during terminal differentiation whose expression determines the adipocyte phenotype and insulin-mediated lipogenesis (Rosen et al., 2002; Tang et al., 2005; Wang et al.,
2006).
Data in the present study showed expression of FABP4 to be increased by day 4 (~50- fold), followed by induction of FAS and glucose transporters GLUT1 and GLUT4 by day 6, all of which are characteristic of terminal adipogenic differentiation (Spiegelman et al., 1983; Gregoire et al., 1998). Increasing intracellular accumulation of lipids during differentiation relies on the expression and function of fatty acid transporters
(e.g. FABP4), lipogenic enzymes (e.g. FAS) and the capacity to response to insulin, through the expression of insulin receptor and insulin-sensitive glucose transporter
GLUT4 (Rosen & Spiegelman, 2000). In this regard, FABP4 expression is important due to its critical role in intracellular FA trafficking from the lipid droplet to the plasma membrane and its involvement in FA re-uptake and re-esterification after lipolysis
(Stahl et al., 1999). Also, FABP4 interacts with hormone-sensitive lipase allowing its translocation to the surface of the lipid droplet upon PKA activation (Smith et al.,
2004), therefore eliciting lipolysis. Likewise, the expression of the lipogenic enzyme
FAS is critical for the acquisition of adipocyte lipid mass through the “de novo”
189 synthesis of saturated fatty acids during lipogenesis. Moreover, pharmacological inhibition or mRNA silencing of FAS has shown to prevent 3T3-L1 pre-adipocyte differentiation (Schmid et al., 2005). In the present study, FAS gene expression reached peak values at day 6 (~3-fold), which coupled with the induction of GLUT4 expression translate the capacity of the adipocytes to accumulate lipid in response to insulin. The expression of GLUT1 was also induced late during differentiation, although its relative expression is much lower compared with GLUT4.
Expression of GLUT4 is needed for optimal insulin responsiveness and glucose metabolism in adipocytes, responses that are driven by the synergistic activity of adipogenic regulators PPARγ and C/EBPα (Hamm et al., 1999; Fernyhough et al.,
2007). GLUT4 appears late during 3T3-L1 adipogenic differentiation, and several factors have been involved in the tight regulation of its expression including: class II
HDAC (HDAC4, 5, and 9), KLF15, and DNA methylation (Yokomori et al., 1999;
Gray et al., 2002; Mori et al., 2005; Kim et al., 2009; Weems & Olson, 2011).
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Minerva Access is the Institutional Repository of The University of Melbourne
Author/s: Flores Opazo, Marcelo Alejandro
Title: Exercise and adipose tissue GLUT4
Date: 2017
Persistent Link: http://hdl.handle.net/11343/192983
File Description: Exercise and adipose tissue GLUT4
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