Environmental and gene therapy approaches to improve glycemic
control and promote healthy aging
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Travis Blaze McMurphy
Graduate Program in Biomedical Sciences
The Ohio State University
2017
Dissertation Committee:
Dr. Lei Cao Ph.D., Advisor
Dr. A. Courtney DeVries Ph.D.
Dr. Denis C. Guttridge Ph.D.
Dr. F. Kay Huebner Ph.D.
Copyright by
Travis Blaze McMurphy
2017
Abstract
The epidemic of obesity and associated complications comprising metabolic syndrome exact a monumental burden on global public health in both morbidity and cost of treatment. Stressful, sedentary lifestyles coupled with excessive caloric intake contribute to increasing rates of obesity and type II diabetes mellitus worldwide.
Additionally, the likelihood of excess visceral adiposity progressing into metabolic syndrome grows dramatically with age. Obesity-associated insulin resistance commonly precedes the onset of type II diabetes and most treatments for the resulting hyperglycemia mimic, sensitize, or enhance secretion of insulin. Therefore, interventions that improve glycemic control independently of insulin signaling are appealing alternatives. The overall objective of this dissertation is to evaluate the efficacy of environmental enrichment and gene therapy models to improve glycemic control and promote healthy aging.
The ability of pathogenic viruses to alter host metabolism has been recently characterized. Infection by the human adenovirus serotype 36 (AD36) promotes obesity in animal models and correlates to increased adiposity in humans, yet improves glycemic control. Based on in vitro studies, the E4ORF1 protein is responsible for both the adipogenic and insulin sparing properties of AD36 infection via insulin independent Akt activation. We generated a recombinant adeno-associated viral (rAAV) vector to express
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AD36E4ORF1. Through intravenous delivery we expressed AD36E4ORF1 in the livers of diabetic, insulin resistant, and wild-type mice. Hepatic AD36E4ORF1 improved glucose tolerance and attenuated hyperglycemia in obese diabetic Db -/- mice without improving insulin sensitivity. AD36E4ORF1 also reduced hyperinsulinemia and improved glucose tolerance in insulin resistant mice with diet induced obesity (DIO).
Liver specific glucose uptake was increased without improving insulin sensitivity.
Confirming the findings of previous in vitro studies, Akt activity was not only increased but also required for AD36E4ORF1 mediated glucose uptake. AD36E4ORF1 expression is a model of insulin independent AKT activation and provides a novel therapeutic mechanism to improve glycemic control in cases of insulin resistance.
Next, we looked at how environmental factors might contribute to healthy aging and prevention of metabolic syndrome. Animals housed in a larger, more complex enriched environment (EE) are provided with increased somatosensory stimulation, physical exercise, and enhanced social interactions. Together, these stimuli increase expression of brain derived neurotrophic factor (BDNF) in the hypothalamus, activating a hypothalamic sympathoneural-adipocyte axis (HSA). In young animals, HSA activation has been shown to improve glycemic control and overall health but its impact in older animals has not been previously characterized.
Middle-aged 10 month old female mice were housed in EE for 6 weeks and displayed HSA activation, improved glycemic control, and decreased adiposity without a reduction in overall body weight. In a long term study of middle-aged mice housed in EE for 12 months, we observed a metabolic phenotype characteristic of healthy aging and
iii improved glycemic control. The animals housed in EE exhibited improved glucose tolerance, enhanced motor skills, reduced adiposity, increased mitochondrial biogenesis, remodeling and browning of the white adipose tissue, and prevention of age-associated decline in the brown adipose tissue. Remodeling of the adipose tissue was accompanied by an adipose-specific upregulation of the tumor suppressor phosphatase tensin homologue deleted on chromosome ten (PTEN). Activation of the HSA axis was necessary and sufficient to upregulate PTEN in the adipose tissue. Moreover, sympathetic activation of type 1 and 2 β-adrenergic receptors was responsible for increasing PTEN expression. This study is the first to identify a novel physiological mechanism of PTEN upregulation through sympathetic stimulation of the adipose tissue. EE initiated in middle-aged female mice provides a model of improved glycemic control, healthy aging, and physiologically induced PTEN expression in the adipose tissue. Based on these findings, living in enriched environments with increased social and physical interactions promotes healthy aging through activation of a brain-adipocyte connection which reduces risk factors contributing to age-associated pathologies.
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Dedication
I dedicate this document to my girlfriend, Stephanie Steiger, and my family.
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Acknowledgments
I am extremely grateful to everyone who has supported me as I have worked towards this point. To my advisor Dr. Lei Cao, I cannot thank you enough for your guidance, and the opportunity to succeed in your lab. To my committee, Drs. Courtney
DeVries, Denis Guttridge, and Kay Huebner, I thank you for providing invaluable guidance and direction. I would also like to thank my undergraduate mentor, Dr. Matthew
Foradori for granting me my first research opportunity and inspiring me to continue my education. To my colleagues I thank you for your contributions, insight and intelligent discussions. To my friends and family, I greatly appreciate the positive impact you have on my life. Above all I am grateful to my exceedingly supportive girlfriend Stephanie
Steiger, and to my parents Jack and Marcia McMurphy.
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Vita
2004...... Grand Valley High School
2009...... B.S. Marketing Edinboro University of PA
2011...... B.S. Biology Edinboro University of PA
2012 to present ...... Graduate Research Associate, Biomedical
Sciences Graduate Program, The Ohio State
University
Publications
McMurphy, T. B., Huang, W., Xiao, R., Liu, X., Dhurandhar, N. V., & Cao, L. (2017). Hepatic Expression of Adenovirus 36 E4ORF1 Improves Glycemic Control and Promotes Glucose Metabolism Through AKT Activation. Diabetes, 66(2), 358- 371. doi: 10.2337/db16-0876
McMurphy, T., Xiao, R., Magee, D., Slater, A., Zabeau, L., Tavernier, J., & Cao, L. (2014). The anti-tumor activity of a neutralizing nanobody targeting leptin receptor in a mouse model of melanoma. PLoS One, 9(2), e89895. doi: 10.1371/journal.pone.0089895
Huang, W., McMurphy, T., Liu, X., Wang, C., & Cao, L. (2016). Genetic Manipulation of Brown Fat Via Oral Administration of an Engineered Recombinant Adeno- associated Viral Serotype Vector. Mol Ther, 24(6), 1062-1069. doi: 10.1038/mt.2016.34
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During, M. J., Liu, X., Huang, W., Magee, D., Slater, A., McMurphy, T., . . . Cao, L. (2015). Adipose VEGF Links the White-to-Brown Fat Switch With Environmental, Genetic, and Pharmacological Stimuli in Male Mice. Endocrinology, 156(6), 2059-2073. doi: 10.1210/en.2014-1905
Liu, X., McMurphy, T., Xiao, R., Slater, A., Huang, W., & Cao, L. (2014). Hypothalamic gene transfer of BDNF inhibits breast cancer progression and metastasis in middle age obese mice. Mol Ther, 22(7), 1275-1284. doi: 10.1038/mt.2014.45
Liu, X., Magee, D., Wang, C., McMurphy, T., Slater, A., During, M., & Cao, L. (2014). Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype. Mol Ther Methods Clin Dev, 1. doi: 10.1038/mtm.2013.8
Field of Study
Major Field: Biomedical Sciences Program
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Table of Contents
Abstract ...... ii
Dedication ...... v
Acknowledgments...... vi
Vita ...... vii
List of Tables ...... xi
List of Figures ...... xii
Chapter 1: A review of insulin signaling and resistance...... 1
The global epidemics of obesity and metabolic syndrome ...... 2
Insulin mediated glucose uptake ...... 4
The role of the akt in insulin signaling ...... 5
Mechanisms of insulin resistance ...... 8
AD36E4ORF1, a potent activator of Akt ...... 10
PTEN as a regulator of glucose metabolism ...... 12
Chapter 2: Hepatic Expression of Adenovirus 36 E4ORF1 Improves Glycemic Control and Promotes Glucose Metabolism via AKT Activation ...... 16
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Chapter 3: Environmental activation of a hypothalamic-adipocyte axis promotes healthy aging ...... 57
Chapter 4: The anti-tumor activity of a neutralizing nanobody targeting leptin receptor in a mouse model of melanoma ...... 102
Chapter 5: Discussion and Future Directions ...... 127
References ...... 142
x
List of Tables
Table 2.1. Metabolic parameters in db/db mice and DIO mice...... 49
Table 2.2. Metabolic parameters in normal wild type mice...... 50
xi
List of Figures
Figure 2.1. Ad36E4ORF1 alleviates hyperglycemia in db/db mice...... 36
Figure 2.2 Ad36E4ORF1 modulates hepatic gene expression in db/db mice and DIO mice...... 38
Figure 2.3. Signaling pathways and transcriptional factors in livers transduced by
Ad36E4ORF1 in db/db, DIO, and normal wild type mice...... 39
Figure 2.4. Ad36E4ORF1 improves glycemic control in DIO mice...... 40
Figure 2.6. Perifosine attenuates the effects of Ad36E4ORF1 in normal wild type mice.43
Figure 2.7. A Pilot study of intravenous injection of rAAV administered to wild type mice...... 44
Figure 2.8. Ad36E4ORF1 expression improves glycemic control but not insulin sensitivity in db/db mice ...... 45
Figure 2.9. Gene expression of the hypothalamus in Ad36E4ORF1 and Ad5E4ORF1 treated db/db mice...... 46
Figure 2.10. Inflammatory gene expression of the liver in Ad36E4ORF1 and
Ad5E4ORF1 treated db/db, DIO, and normal wild type mice...... 47
Figure 2.11. Gene expression of abdominal fat depot in Ad36E4ORF1 and Ad5E4ORF1 treated wild type mice...... 48
Figure 3.1. Short-term EE activates the HSA axis in 10-month old mice...... 72
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Figure 3.2. Long-term EE initiated at middle age reduces adiposity and improves metabolism...... 73
Figure 3.4. Gene expression profiles of tissues at the age of 22 months after 12-month
EE...... 76
Figure 3.5. Immunohistochemistry of adipose tissues at the age of 22 months after 12- month EE...... 77
Figure 3.6. Hypothalamic BDNF mediates EE-induced adipose remodeling...... 78
Figure 3.7. Intact sympathetic tone is required for EE-induced adipose remodeling...... 79
Figure 3.8. Gene expression profiles after short-term EE in middle-age female mice. .... 80
Figure 3.9. Immunoassays after long-term EE initiated in middle-age female mice...... 81
Figure 3.10. Gene expression profiles after long-term EE in middle-age female mice. ... 82
Figure 3.11. Gene expression profile of rWAT in young male DIO model after 4-week
EE...... 83
Figure 3.12. Gene expression profile of adipocyte and vascular stromal fraction (SVF) after 2-week EE in young male mice on normal diet...... 84
Figure 3.13. Glucose tolerance test after 3-month EE initiated at 18 months of age. n=10 per group...... 85
Figure 3.14. EE enhances adipose glucose uptake without improving insulin sensitivity in
10 month old female mice housed in EE for 90 days...... 86
Figure 4.1. Systemic effects of local administration of 2.17-mAlb adjacent to tumor implantation site...... 115
Figure 4.2. Local administration of 2.17-mAlb inhibited melanoma progression...... 116
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Figure 4.3. Local administration of low dose 2.17-mAlb modulated gene expression in melanoma...... 117
Figure 4.4. Intraperitoneal administration of 2.17-mAlb...... 118
Figure 4.5. Intraperitoneal administration of high-dose 2.17-mAlb increased adiposity.
...... 119
Figure 4.6. Intraperitoneal administration of high-dose 2.17-mAlb affected hypothalamic gene expression...... 120
Figure 4.7. Subcutaneous administration of 2.17-mAlb in the established tumor model when treatment was delayed till palpable tumors appeared...... 121
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Chapter 1: A review of insulin signaling and resistance
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The global epidemics of obesity and metabolic syndrome
Obesity and resulting complications exact a growing and monumental toll on public health. The risk of cardiovascular disease, type II diabetes, cancer, and death from all causes is increased by obesity (Calle, Thun, Petrelli, Rodriguez, & Heath, 1999). The
World Health Organization (WHO) defines obesity based on body mass index (BMI), a simple ratio of height to body weight. A BMI between 25 and 30 is defined as being overweight with a BMI of 30 or greater indicating obesity. As BMI increases further, the risk of obesity associated comorbidities increases substantially. By this definition, more than two billion people are overweight worldwide (Seidell & Halberstadt, 2015). The national health and nutrition examination survey estimated a 36.5% rate of obesity for adults in the United States between 2011 and 2014. Obesity rates were higher still in the middle aged population, at 40.2% (Ogden, Carroll, Fryar, & Flegal, 2015).The financial burden in treatment costs due to obesity is also immense. In the United States alone, the estimated treatment costs of obesity and related comorbidities exceeded 190 billion dollars in 2012 (Cawley & Meyerhoefer, 2012). The overweight and obese populations are increasing in proportion worldwide. By extrapolating current trends, one study estimates that in the United States 86.3% of the population will be overweight or obese by 2030, with associated health care costs skyrocketing to at least 860 billion US dollars.
Given this trajectory, it is increasingly important to research methodologies to abate obesity and its associated health burden on individuals as well as society.
Genetic and environmental factors contribute to development of obesity and metabolic syndrome, but an imbalance of caloric intake to physical activity is the primary
2 driving force (Hruby & Hu, 2015). Chronic stress also contributes to obesity through promoting glucocorticoid release. As over-nutrition and sedentary lifestyles become more prevalent world-wide, the proportion of obese and overweight individuals is rising drastically (Ng et al., 2014). In most cases this increased body weight is due primarily to triglyceride accumulation and hypertrophic expansion in the white adipose tissue.
Pathological overexpansion and immune infiltration of the adipose tissue promotes a state of low grade systemic inflammation and contributes to whole body insulin resistance
(Choe, Huh, Hwang, Kim, & Kim, 2016). In addition ectopic deposition of triglycerides occurs in the skeletal muscle and visceral organs. Increased waist circumference is an effective predictor of excess visceral adiposity, ectopic triglyceride deposition, and obesity related complications (U. Smith, 2015).
Metabolic syndrome, also known as insulin resistance syndrome, is a set obesity associated comorbidities, which greatly increase susceptibility to type two diabetes cardiovascular disease, nonalcoholic fatty liver disease (NAFLD), renal failure, malignancies, and mortality from all causes (Ford, 2004). The threshold criteria defining metabolic syndrome vary slightly among global health organizations; however, all definitions include excess abdominal adiposity, hypertension, hyperglycemia, and dyslipidemia (Beltrán-Sánchez, Harhay, Harhay, & McElligott, 2013; O'Neill &
O'Driscoll, 2015). This definition can also be expanded to include increased inflammation, microalbuminuria, and hypercoagulability (Kaur, 2014). Metabolic syndrome associated dyslipidemia consists of elevated levels of serum triglycerides, free fatty acids, and low density lipoproteins, and reduced levels of high density lipoproteins
3
(HDL), especially in the postprandial state (Kolovou, Anagnostopoulou, & Cokkinos,
2005). The likelihood of developing metabolic syndrome increases dramatically with age
(Desroches & Lamarche, 2007; Ford, Giles, & Dietz, 2002). As metabolic syndrome becomes more common so does type II diabetes, and by the 2030 an estimated 366 million people are projected to be afflicted (Wild, Roglic, Green, Sicree, & King, 2004).
Given the scale of the pandemic comprised of metabolic syndrome, obesity, and type II diabetes, it is clear that alternative treatments and preventative measures are required.
Insulin mediated glucose uptake
Insulin signaling is the principal controller of postprandial glucose disposal and uptake into the skeletal muscle, liver, and adipose tissue (Sharma, Garber, & Farmer,
2008). Additionally, glycogen synthesis, cellular growth and proliferation pathways are activated, while hepatic gluconeogenesis is suppressed. Insulin signaling is also crucial to normal function and regulation of metabolism by the central nervous system
(Kleinridders, Ferris, Cai, & Kahn, 2014). The pancreatic β-cells produce and secrete insulin in response to elevated levels of blood glucose (Fu, Gilbert, & Liu, 2013).
Glucose transporter 4 (GLUT4) is the primary route of cellular glucose influx in insulin responsive tissues, specifically the white adipose tissue, cardiac, and skeletal muscle
(Zisman et al., 2000). GLUT4 is regulated primarily by membrane localization, not expression levels, and in a fasted state resides primarily in cytoplasmic storage vesicles.
In response to insulin mediated stimulation, transit of GLUT4 to the cell membrane is increased (Bogan, 2012). The liver also plays a key role in postprandial glucose
4 homeostasis both by regulating hepatic glucose output and through control of glucose uptake (Moore, Coate, Winnick, An, & Cherrington, 2012). GLUT2 is not only the dominant transporter in hepatic glucose uptake, but also crucial for sensing of glucose levels by pancreatic β-cells and the central nervous system (Thorens, 2015).
The functional insulin receptor (IR) is a membrane bound tyrosine receptor kinase composed of a hetero-tetramer of two external α and two internal β subunits (Tatulian,
2015). In response to insulin or insulin like growth factor docking, the receptor changes conformation and auto-phosphorylates its β subunits, allowing binding and tyrosine phosphorylation of insulin receptor substrate (IRS)(Menting et al., 2013). Tyrosine phosphorylation of IRS allows binding to the p85 regulatory subunit phosphatidyl inositol 3 kinase (PI3K), relieving catalytic inhibition and bringing PI3K into proximity of the plasma membrane (Sanchez-Margalet, Goldfine, Truitt, Imboden, & Sung, 1995).
Insulin receptor substrate activation initiates mitogen activated kinase (MAPK) as well as protein kinase B signaling (Beale, 2013).
The role of the akt in insulin signaling
The Akt pathway, also referred to as protein kinase B (PKB), is the primary mechanism responsible for promoting GLUT4 facilitated glucose uptake in response to insulin signaling (Guo, 2014). Additional Akt phosphorylation targets are involved in angiogenesis, cell growth, proliferation and survival, and regulation of lipogenic and glycolytic pathways (Hemmings & Restuccia, 2012). Additional downstream targets of
Akt include glycogen synthase kinase 3 (GSK)-3, Forkhead box protein O (FOXO), and
5 mechanistic target of rapamycin (mTORC1) through TSC2 inactivation. (Manning &
Cantley, 2007).
The Akt signaling cascade requires generation of the membrane bound lipid, secondary messenger phosphatidylinositol (3,4,5) triphosphate (PIP3) (Franke, Kaplan,
Cantley, & Toker, 1997). Complete activation of Akt is achieved by phosphorylation of both T308 on its t-loop activation site and S473 on its C terminal hydrophobic domain.
While they are catalyzed by separate kinases and occur independently of one another, both phosphorylation events require binding of Akt to PIP3 in response to PI3k signaling.
Phosphorylation of both sites is required for fully activated Akt to properly dissociate from the membrane and phosphorylate downstream targets. Akt enters a catalytically active state when T308 is phosphorylated by PDK1 (T. O. Chan, Rittenhouse, &
Tsichlis, 1999). Prior to its phosphorylation by PDK1, Akt undergoes a conformational change via pleckstrin homology domain mediated binding to PIP3 at the plasma membrane (Milburn et al., 2003). Akt catalytic activity and substrate specificity are further enhanced when mammalian target of rapamycin complex 2 (MTORC2) phosphorylates Akt at S473, an event which also requires association of Akt with PIP3.
(Scheid, Marignani, & Woodgett, 2002) (Riaz, Zeller, & Johansson, 2012). Once fully activated, Akt dissociates from the membrane to phosphorylate its downstream protein targets. Phosphorylation of Akt substrate 160kda (AS160) by Akt initiates transit of
GLUT4 from cytoplasmic vesicles to the cell membrane, increasing cellular glucose uptake (Sano et al., 2003). Phosphorylation of the transcription factor FOXO1 by Akt causes inactivation and nuclear exclusion. FOXO1 activity has been identified as a key
6 regulator of hepatic insulin sensitivity (Lu et al., 2012). Hepatic lipid oxidation is downregulated following deactivation of peroxisome proliferator-activated receptor coactivator 1-α (PGC1-α) via AKT signaling (X. Li, Monks, Ge, & Birnbaum, 2007). De novo lipogenesis is increased through activation of sterol regulatory element-binding protein (SREBP1c) (Yecies et al., 2011).
Akt exists in three tissue specific isoforms with slight structural and functional differences, with Akt1 expressed ubiquitously at low levels and Akt3 primarily in the central nervous system (Mackenzie & Elliott, 2014). The dominant isoform crucial to glucose metabolism in insulin responsive tissues, including the fat, skeletal muscle, and the liver is Akt2 (Gonzalez & McGraw, 2009). Akt1 is expressed ubiquitously throughout the body at lower levels, and due to overlapping function, can compensate for deficiency of the other isoforms (Dummler et al., 2006).
In addition to regulation through availability of PIP3, phosphorylated Akt can be inactivated directly through auto inhibitory negative feedback and exogenous pathways.
Both activation sites required for Akt signaling can be directly targeted by protein phosphatases. Pleckstrin homology domain leucine rich repeat protein phosphatase
(PHLPP) acts on the hydrophobic motif S473 (Newton & Trotman, 2014). Protein phosphatase 2A dephosphorylates AKT at T308 (T. Gao, Furnari, & Newton, 2005).The
Akt pathway self-regulates itself by enacting negative feedback at the level of IRS. The downstream target of Akt, MTORC1 phosphorylates S6K which in turn deactivates IRS to cease the PI3K signaling cascade.
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Mechanisms of insulin resistance
Insulin resistance can be defined as an inability of increasing concentrations of insulin to induce glucose uptake in the skeletal muscle and adipose tissue, while suppressing hepatic glucose production (Guo, 2014). As the chief intermediary of insulin- dependent glucose disposal, the PKB pathway is inhibited by insulin resistance, specifically at the level of insulin receptor substrate interactions. The functionality of IRS is tightly regulated through a complex array of inhibitory serine and threonine phosphorylation sites (Copps & White, 2012). Regulatory phosphorylation of serine and threonine sites also contributes to increased protein degradation of IRS-1(Pederson,
Kramer, & Rondinone, 2001). These sites are targets of kinases involved in regulation of cell growth and metabolism including IKKβ, JNK, MAPK, mTORC1, and S6K (Tanti &
Jager, 2009). The ability of IRS to associate with and activate PI3K in response to insulin receptor activation is diminished in insulin resistance and type II diabetes yet MAPK signaling remains intact (Cusi et al., 2000). Negative feedback through MAPK signaling in response to hyperinsulinemia may contribute significantly to insulin resistance.
Inhibitory phosphorylation of IRS-1 at serine 636 through MAPK signaling interferes with normal activation of PI3K (Bouzakri et al., 2003).
The greatest risk factors leading to development of insulin resistance are central obesity, or expansion of the visceral adipose tissue, and lifestyles with little physical activity. (Velasquez-Rodriguez, Velasquez-Villa, Gomez-Ocampo, & Bermudez-
Cardona, 2014). Insulin resistance in the adipose tissue impairs uptake of glucose and lipids, resulting in ectopic deposition of triglycerides in the skeletal muscle and other
8 organs. As the visceral adipose tissue expands, levels cytokines secreted from the adipose tissue, also known as adipokines, are altered. Severe hypertrophy of adipocytes induces hypoxic dysfunction interfering with normal adipokine production and secretion
(Hosogai et al., 2007). Obesity induces a state of chronic systemic inflammation evident by increased macrophage infiltration of the adipose tissue and secretion of inflammatory cytokines, including TNF-α, IL-6, IL-1β PAI, and MCP-1(Shoelson, Lee, & Goldfine,
2006). Serum leptin levels mirror the whole body’s level of adiposity (Frederich et al.,
1995). Expansion of visceral adipose tissue reduces production of adiponectin which plays a significant role in insulin sensitivity (Sevillano, de Castro, Bocos, Herrera, &
Ramos, 2007).
The pro-inflammatory phenotype of obesity is a key component of insulin resistance. Inflammatory cytokine signaling can contribute to inhibitory phosphorylation of IRS, decreasing whole body insulin sensitivity. An elevated concentration of TNF-α promotes insulin resistance by inhibiting AKT signaling at the IRS level (Plomgaard et al., 2005). TNF-α also contributes to insulin resistance by upregulating expression of suppressor of cytokine signaling 3 (SOCS3), which impairs tyrosine phosphorylation of
IRS by IR (Emanuelli et al., 2001). Increased inflammatory cytokine activation of inhibitor kappa B kinase (IKKβ) also induces inhibitory serine phosphorylation of IRS
(Z. Gao et al., 2002).
Ectopic triglyceride deposition in the skeletal muscle coincides with reduced insulin sensitivity in humans (Krssak et al., 1999; Sinha et al., 2002). Whole-body insulin insensitivity is largely due to an inability of insulin to stimulate glucose uptake and
9 glycogen synthesis in the skeletal muscle. As pathogenesis progresses into to a state of hyperglycemia, hepatic triglyceride synthesis is increased while the ability of insulin to regulate glucose transport and glycogen synthesis in the liver is diminished (Samuel &
Shulman, 2016). Sustained hyperglycemia and progression into type II diabetes occurs once the ability of pancreatic β-cells to secrete enough insulin to compensate for reduced insulin sensitivity and augmented hepatic glucose production is exhausted (Stumvoll,
Goldstein, & van Haeften, 2005). Prolonged dyslipidemia and hyperglycemia are detrimental to the function and survival of β-cells and increase apoptosis (Chang-Chen,
Mullur, & Bernal-Mizrachi, 2008). Patients with type II diabetes show a reduction in size and number of pancreatic β-cells (A. E. Butler et al., 2003).
AD36E4ORF1, a potent activator of Akt
The primary concern in hyperglycemia due to insulin resistance is an inability of increased concentrations of serum insulin to produce increased glucose uptake in insulin responsive tissues. Since the PKB pathway is the key regulator of GLUT4 translocation and increased glucose uptake in response to insulin signaling, it is an ideal target for anti- hyperglycemic therapy. By bypassing the necessity of activated IRS signaling, to activate Akt and its downstream targets, AD36E4ORF1 expression provides a novel therapeutic approach for type II diabetes and insulin resistance.
Infection by the human adenovirus type 36 (AD36) correlates with an increased likelihood of obesity in humans (Voss, Atkinson, & Dhurandhar, 2015) and promotes increased adiposity in a variety of model organisms (N. V. Dhurandhar et al., 2000).
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Interestingly, AD36 infection also correlates with improved glycemic control
(Krishnapuram et al., 2011). Both the adipogenic and anti-hyperglycemic properties of adenovirus 36 infection have been attributed entirely to the actions of it early gene 4 open reading frame 1 (AD36E4ORF1) product (E. J. Dhurandhar et al., 2011). The initial characterization of AD36E4ORF1’s metabolic function was derived mainly from work performed on the very closely related AD9E4ORF1. The E4ORF1 products derived from
AD36 AD9 are extremely similar, sharing 92% sequence homology at the amino acid level. AD9E4ORF1 forms a homotrimer and binds to the cell polarity signaling drosophila discs large 1 (DLG1) through a postsynaptic density 95, Dlg-1, Zona occludens 1 (PDZ) binding motif. This complex migrates towards the plasma membrane, where it binds to both the p85 regulatory and p110 catalytic subunits of class 1 PI3K
(Kong, Kumar, Taruishi, & Javier, 2014). The complex relieves functional inhibition of
PI3K by p85 and brings it into close proximity with its substrate PIP2. The end result is an unregulated increase in PIP3 production and constituently active AKT signaling
(Kong et al., 2014). This mechanism was recently confirmed for AD36E4ORF1 as well
(Manish Kumar, Kathleen Kong, & Ronald T. Javier, 2014). Unlike activation of PI3K through IRS, S6K does not impose negative regulation on this system. Negative feedback regulation of IRS through TSC1/2 and MAPK are also avoided. Some in vitro studies suggest a role of increased MAPK activity via increased RAS protein levels (Z.
Q. Wang et al., 2008); however our data and the work done in AD9 do not support this conclusion.
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The E4ORF1 protein has no known extracellular receptor and is not secreted from cells. While this creates the disadvantage of requiring a gene therapy or transgenic model to assess the in-vivo effects, it allows us to easily dissect the tissue specific effects of
E4ORF1 expression. AD36E4ORF1 expression provides a model for continuous activation of downstream PKB signaling without requiring intact insulin signaling.
PTEN as a regulator of glucose metabolism
Phosphatase and tensin homologue deleted on chromosome ten (PTEN) is a dual protein and lipid phosphatase with well documented tumor suppressor functions, reviewed in (J. Li et al., 1997; Nakanishi, Kitagishi, Ogura, & Matsuda, 2014). A loss of
PTEN function is common in cowden syndrome, human cancers and mouse models of tumorigenesis (Hollander, Blumenthal, & Dennis, 2011). PTEN is also a potent regulator of cellular growth, survival, and insulin mediated glucose uptake. The lipid phosphatase function of PTEN is most relevant to insulin dependent glucose uptake. PTEN switches off insulin induced glucose uptake pathways by dephosphorylating PIP3 to its inactive
PIP2 state, preventing sustained stimulation of AKT signaling (Stambolic et al., 1998).
The lipid phosphatase activity of PTEN is negatively regulated through C- terminal phosphorylation by promoting an inactive conformation and preventing translocation to the cell membrane (Chen et al., 2016). Phosphorylation induces a closed conformation, blocking the PDZ binding motif, preventing recruitment to the cell membrane (Vazquez et al., 2001). While phosphorylation of PTEN by casein kinase 2 reduces its lipid phosphatase activity, it enhances protein stability (Torres & Pulido,
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2001). Further, C terminal phosphorylation also promotes binding of PTEN to its negative regulator, phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2
(P-REX2) (Hodakoski et al., 2014). Intriguingly, this inhibitory phosphorylation can be reversed by protein phosphatase activity of PTEN (X. C. Zhang, Piccini, Myers, Van
Aelst, & Tonks, 2012).
Not surprisingly, transgenic animals with tissue-specific PTEN deletion are protected from diabetes and show improved glucose clearance. Hepatic PTEN knockout mice exhibit improved insulin sensitivity at the price of steatosis and hepatomegaly due to triglyceride accumulation within hepatocytes (Peyrou et al., 2015; B. Stiles et al.,
2004). Adipose-specific PTEN deletion improves glucose tolerance and enhances insulin sensitivity in mice (Christine Kurlawalla-Martinez et al., 2005). Muscle-specific ablation of PTEN is sufficient to prevent insulin resistance and increases muscle specific deoxy- glucose uptake in animals maintained on a high fat diet (Wijesekara et al., 2005). A model ablating PTEN in myf5 positive adipocyte precursors increases whole-body adiposity while increasing insulin sensitivity and glucose clearance (Sanchez-Gurmaches et al., 2012). Inhibition of AKT signaling by PTEN may also play a role in insulin secretion. Both pancreatic (Tong et al., 2009) and pancreatic β-cell specific (B. L. Stiles et al., 2006) knockouts of PTEN improve glycemic control and prevent development of diabetes due to streptozotocin treatment.
Maintaining normal protein levels of PTEN is crucial to maintaining normal glucose metabolism. Haplo-insufficiency of PTEN is enough to increase whole body insulin sensitivity and glucose clearance (Wong et al., 2007) while decreasing hepatic
13 lipid content (Schultze et al., 2015). Haplo-insufficiency in humans improves insulin sensitivity while increasing the risk of obesity and cancer (Pal et al., 2012). A reduction in PTEN through RNA interference in OB and DB mice improves glycemic control and diminishes insulin resistance (M. Butler et al., 2002). Physiological regulation of PTEN expression levels remains largely uncharacterized. Post transcriptional regulation is also extremely complex with more than twenty known miRNA’s targeting PTEN (Bermúdez
Brito, Goulielmaki, & Papakonstanti, 2015).
At a glance, the role of PTEN in glucose metabolism appears to be straight forward however; the reality may be much more complex. Studies have definitively shown that PTEN deficiency promotes sustained glucose uptake, and AKT activation; however the reverse does not appear to be true for PTEN overexpression. Whole body overexpression of PTEN improves whole body glycemic regulation, among a plethora of other metabolic benefits (Garcia-Cao et al., 2012; Ortega-Molina & Serrano, 2013). Mice with globally increased PTEN expression demonstrated improvements in energy expenditure, brown adipose tissue function, and glycemic control. These animals were also protected from carcinogenesis, diet induced obesitity, and insulin resistance
(Ortega-Molina et al., 2012). The exact mechanims for improving glycemic control through negatively regulating the PKB pathway are still unclear. It is possible that the protein phosphatase functions of PTEN outside of the PKB pathway counteract its role as a lipid phosphatase. Furthermore, existing models of PTEN overexpression are germline modifications that may alter metabolism through effects on development and growth. An
14 inducible model would permit characterization of the metabolism specific effects of
PTEN overexpression.
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Chapter 2: Hepatic Expression of Adenovirus 36 E4ORF1 Improves Glycemic
Control and Promotes Glucose Metabolism via AKT Activation
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Hepatic Expression of Adenovirus 36 E4ORF1 Improves Glycemic Control and
Promotes Glucose Metabolism via AKT Activation
Travis B. McMurphy 1,2*, Wei Huang 1,2*, Run Xiao 1,2, Xianglan Liu 1,2, Nikhil V.
Dhurandhar 3, and Lei Cao 1,2
Department of Cancer Biology and Genetics
The Comprehensive Cancer Center
The Ohio State University
3. Department of Nutritional Sciences, Texas Tech University, Lubbock, Texas.
Running Title: Adenovirus 36 E4ORF1 improves glycemic control
Key terms: Adenovirus 36, E4orf1, hyperglycemia, liver
Word count: 5412
Correspondence author and person to whom reprint requests should be addressed:
Lei Cao, PhD
460 W 12th Ave, Columbus, OH 43210
Phone: 614-3665679, Fax: 614-2926356
Email: [email protected]
* T. McMurphy and W. Huang contributed equally to this work
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Abstract
Considering that impaired proximal insulin signaling is linked with diabetes, approaches that enhance glucose disposal independent of insulin signaling are attractive. In vitro data indicate that the E4ORF1 peptide derived from human adenovirus 36 (Ad36) interacts with cells from adipose tissue, skeletal muscle and liver to enhance glucose disposal, independent of proximal insulin signaling. Adipocyte-specific expression of
Ad36E4ORF1 improved hyperglycemia in mice. To determine the hepatic interaction of
Ad36E4ORF1 in enhancing glycemic control, we expressed E4ORF1 of Ad36 or adenovirus 5, or fluorescent tag alone using recombinant adeno-associated viral vector in the livers of three mouse models. In diabetic db/db or diet induced obesity (DIO) mice, hepatic expression of Ad36E4ORF1, but not Ad5E4ORF1 robustly improved glycemic control. In normoglycemic wild type mice, hepatic expression of Ad36E4ORF1 lowered nonfasting blood glucose at a high dose of expression. Notably, Ad36E4ORF1 significantly reduced insulin levels in db/db and DIO mice. The improvement in glycemic control was observed without stimulation of the proximal insulin signaling pathway. Collectively, these data indicate that Ad36E4ORF1 is not a typical sensitizer, mimetic, or secretagogue of insulin. Instead, it may have “insulin sparing action” which seems to reduce the need for insulin and hence, reduce insulin levels.
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Introduction
Type 2 diabetes is often associated with resistance to insulin signaling, including impaired response of molecular participants of insulin signaling such as insulin receptor substrate 1 or 2 (IRS-1, IRS-2) (Danielsson et al., 2005; Draznin, 2006; Kovacs et al.,
2003; Rung et al., 2009). This defect in insulin signaling contributes to reduced response to insulin and reduced cellular glucose uptake. Most of the currently available anti- diabetes medications are insulin sensitizers, insulin mimetic or secretagogues, implying their dependence on insulin signaling for optimal benefits. Hence, the class of antidiabetic medications that act independent of IRS-1 or 2 signaling may be more effective, and hence desirable alternatives. To this end, recent in vitro studies described
E4ORF1, a 125 amino acid peptide derived from human adenovirus Ad36, up-regulates glucose uptake in adipocytes and their progenitors and myoblasts, and reduces glucose release from hepatocytes (E. J. Dhurandhar et al., 2011; E. J. Dhurandhar et al., 2012;
Krishnapuram, Dhurandhar, Dubuisson, Hegde, & Dhurandhar, 2013). These studies further indicated that E4ORF1 bypassed IRS1 or 2 signaling, yet improved glucose disposal by up-regulating distal insulin signaling pathway involving phosphatidyl inositol
3-kinase (PI3K), AKT and glucose transporter Glut 4 via Ras activation (N. V.
Dhurandhar, 2013). Collectively, the data indicated that E4ORF1 was not a sensitizer, mimetic or secretagogue of insulin, but had an insulin sparing action (N. V. Dhurandhar,
2013).
Hence, Ad36E4ORF1 is suggested as a novel target to develop anti-hyperglycemic drugs particularly in metabolic syndromes often associated with insulin resistance (N. V.
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Dhurandhar, 2013). However, a number of questions remain. Firstly, a detailed characterization of the in vivo effects of Ad36E4ORF1 is not yet reported. Ad36E4ORF1 is not an endogenous secretory protein and therefore is unlikely to have a receptor on cell surface for its uptake highlighting the challenge in delivery of this peptide. Secondly, whether Ad36E4ORF1 will induce uncontrolled glucose uptake leading to hypoglycemia in vivo is another unanswered question. Thirdly, the potential oncogenic risk associated with Ras induction by Ad36E4ORF1 should be carefully evaluated in vivo. Importantly, although Ad36E4ORF1 interacts with adipose tissue, skeletal muscle and liver to modulate glucose disposal, the interaction of Ad36E4ORF1 with individual tissues involved in systemic glycemic control is unclear. A recent study showed that fat-specific inducible expression of Ad36E4ORF1 improved glycemic control in DIO mice
(Kusminski et al., 2015).
To study the interaction of Ad36E4ORF1 with liver, we used a novel recombinant adeno- associated viral (rAAV) serotype vector Rec2 to deliver Ad36E4ORF1 in vivo.
Intriguingly, adenovirus 5 E4ORF1 (Ad5E4ORF1) has been shown to bind to MYC in the nucleus to activate glycolytic targets that promote a Warburg-like shift to anaerobic glycolysis that converts glucose into nucleotides for viral replication (Thai et al., 2014).
However, the in vivo effects of Ad5E4ORF1 alone are not known. We also generated rAAV to deliver Ad5E4ORF1 in order to determine whether the functions of E4ORF1 are shared across different subfamilies of human adenoviruses.
Research Design and Methods
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Mice
C57Bl/6 mice, six week of age, were purchased from Charles River (Wilmington, MA).
Female db/db mice in a C57/BL6 background, five week of age, were purchased from
Jackson Lab (Bar Harbor, ME). Male DIO mice, 12 weeks of age, were purchased from
Jackson Lab and maintained on high fat diet (60% fat, Research Diet #D12492). All mice were housed in a temperature-controlled room (22 ºC) with a 12 h light-12 h dark cycle, and maintained on a standard rodent diet (7912 rodent chow, Teklad) with ad libitum access to food and water. All use of animals was approved by and in accordance with the
Ohio State University Animal Care and Use Committee. rAAV vector construction and packaging
Addgene plasmid #38063 was used as the template for Ad5E4ORF1. Ad36E4ORF1 plasmid was used as previously reported (E. J. Dhurandhar et al., 2012). Transgenes: dsYFP and Ad5E4ORF1 or Ad36E4ORF1 were inserted into the multiple cloning sites between the CBA promoter (CMV enhancer and chicken β-actin (CBA) promoter) and
WPRE (woodchuck post-transcriptional regulatory element) sequence in the rAAV plasmid. rAAV serotype Rec2 vectors were packaged and purified as described elsewhere
(Liu, Magee, et al., 2014). db/db mice
Female db/db mice, 5 weeks of age, were randomly assigned to receive a dose of 2 x 1010 vg of Rec2-Ad5E4ORF1 (n=8), Ad36E4ORF1 (n=8), dsYFP (n=4), or AAV buffer (n=4) via tail vein injection in 50 l. Body weight and food intake were monitored periodically.
Nonfasting blood glucose was monitored starting 6 days post injection. A glucose
21 tolerance test (GTT) was performed 3 weeks after AAV injection. Mice were sacrificed
29 days after AAV injection. Trunk blood was collected. Liver and abdominal fat were dissected and weighed. In a separate experiment, db/db mice were treated as described above. An insulin tolerance tested was performed 3 weeks after AAV injection.
DIO mice
Male DIO mice, 12 weeks of age, were randomly assigned to receive a dose of 2 x 1010 vg of Rec2-Ad5E4ORF1 (n=8), Ad36E4ORF1 (n=9), or dsYFP (n=8) as described above, and were maintained on HFD. GTT was performed 3 weeks post AAV injection.
Body weight, food intake, and nonfasting blood glucose levels were monitored periodically. Mice were sacrificed 7 weeks post AAV injection.
Wild type mice
Female C57BL/6 mice, 6 weeks of age, were randomly assigned to five groups to receive tail vein injection of rAAV vectors (n=5 per group) at two doses: Rec2-dsYFP: 2 x 109 vg; Rec2-Ad5E4ORF1: 2 x 109 vg, 2 x 1010 vg; Rec2-Ad36E4ORF1: 2 x 109 vg, 2 x 1010 vg. Nonfasting blood glucose was monitored daily starting 3 days post rAAV injection.
Mice were sacrificed 7 days after rAAV injections.
Perifosine experiment
Female C57BL/6 mice, 8 weeks of age, were randomly assigned to receive AAV buffer or Rec2-Ad36E4ORF1 (2 x 1010 vg in 50 l) via tail vein injection. Each vector-injected group was split to two groups to receive perifosine (KareBay BioChem, 36 mg per mouse) or PBS by gavage at day 4 and day 5 after AAV injection. Nonfasting blood
22 glucose was monitored daily starting day 4. Mice were sacrificed day 6 post AAV injection.
Glucose tolerance test
Mice were injected intraperitoneally with glucose solution (1mg glucose per g body weight) after an overnight fast. Blood glucose concentrations were measured with a portable glucose meter (Bayer Contour Next).
Insulin tolerance test
Mice were injected intraperitoneally with insulin (0.75 unit per kg body weight) at 2 pm without a fast. Blood glucose levels were measured with a portable glucose meter.
In vivo glucose uptake
Female db/db mice, 5 weeks of age, were randomly assigned to receive a dose of 2 x 1010 vg of Ad36E4ORF1 (n=4) or AAV buffer (n=4) as described above. The in vivo glucose update during a glucose tolerance test was measured using glucose analog tracer 2-[3H] deoxyglucose (2-DG) 8 weeks post rAAV injection following published method (Boini et al., 2009; Mauvais-Jarvis et al., 2000). Liver glucose uptake is calculated by dividing
[3H]-radioactivity in 2-DG-6-P by mean specific activity of glucose during GTT (120 min) and presented as mmol per mg protein per minute.
Metabolic Parameters
Lipid was extracted from liver by chloroform /methanol (2:1 v/v), followed by rinse in 50 mM NaCl and CaCl (0.36M)/Methanol (1:1 v/v) (Folch, Lees, & Sloane Stanley, 1957).
Non-esterified fatty acid, hepatic triglycerides and cholesterol quantification were measured using WAKO instruments kits, respectively. Serum insulin level was
23 determined with ALPCO mouse insulin ELISA. Serum leptin and adiponectin were measured using DuoSet ELISA Development System (R&D Systems). Serum glucose was measured with QuantiChrom Glucose Assay kit (DIGL-100). Hepatic glycogen content was measured by hydrolysis of liver tissue in acid followed by colorimetric measurement of the resulting glucose (Passonneau & Lauderdale, 1974). Acetyl CoA assay was performed using Acetyl CoA assay kit (Sigma, MAK039).
Glucokinase (GCK) Assay: 50 mg of liver tissue was rinsed with ice-cold PBS, and then was homogenized with 1.5 ml pestle in ice-cold PBS containing phosphatase inhibitor and protease inhibitor cocktail. The homogenates were centrifuged and the protein content was measured using BCA kit (Pierce). The supernatants were immediately subjected to GCK BioAssay ELISA (US Biological Life Sciences).
Western Blot
Liver tissues were homogenized in ice-cold Pierce RIPA buffer containing 1x Roche
Phosstop and Calbiochem protease inhibitor cocktail III. Blots were incubated overnight at 4 C with the following primary antibodies: Ad36E4ORF1 (a gift from Dr.
Dhurandhar, 1:1000), GAPDH (Calbiochem #CB1001, 1:500), Actin (Cell Signaling
#4970, 1:3000), total Ras (Cell Signaling #3965, 1:1000), c- Myc (Cell Signaling #5605,
1:1000), phospho-AKT- Ser473 (Cell Signaling #9271, 1:1000), phospho-AKT-Thr308
(Cell Signaling #9275, 1:1000), total AKT (Cell Signaling #9272, 1:1000), Insulin receptor -subunit (Millipore #05-1104, 1:1000), phospho-insulin receptor (Tyr1322)
(Millipore #04-300, 1:200), IRS-2 (Cell Signaling #4502, 1:1000), phospho-IRS1/2
(Tyr612) (Santa Cruz #sc-17195R, 1:500), mature form SREBP1 (Novus Biologicals
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#NB100-60545, 1:1000), ChREBP (Novus Biologicals #NB400-135, 1:1000), Phospho-
FoxO1 Ser256 (Cell Signaling #9461, 1:1000), FoxO1 (Cell Signaling #2880, 1:1000),
Rictor (Cell Signaling #2140, 1:1000).
Quantitative RT-PCR
Total RNA was isolated using RNeasy Mini Kit plus RNase-free DNase treatment
(Qiagen). First-strand cDNA was generated using TaqMan Reverse Transcription
Reagent. Quantitative PCR was carried out using StepOne Plus Real-Time PCR System
(Applied Biosystems) with the Power SYBR Green PCR Master Mix (Applied
Biosystems). Primer sequences are available upon request. Data were calibrated to endogenous control Actb or Hprt1 and the relative gene expression was quantified using the 2 -CT method (Livak & Schmittgen, 2001).
Hepatocytes isolation and glucose output assay
Mouse hepatocytes were isolated from chow-fed WT mice 4 days after tail-vein injection of Rec2-Ad36E4ORF1 (2 x 1010 vg per mouse) or AAV buffer as a control according to published method with modifications (Bocharov et al., 1995). Briefly, mice were anesthetized and perfused at a rate of 3 ml/min via cannulation through the inferior vena cava with perfusion buffer (115 mM NaCl, 5mM KCl, 25mM HEPES, 0.5mM EGTA, and 25mM D-glucose, pH 7.4), followed by Liver Digestion Medium (Invitrogen). The portal vein was cut to allow the flow of the solution through liver. The perfusion temperature was kept at 37oC. The liver was dissected, and capsule peeled off.
Hepatocytes were dispersed by mechanical dissociation, and filtered through 100 µm strainer. The hepatocytes were rinsed twice in ice-cold DMEM supplemented with 10%
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FBS, Penicillin/Streptomycin, 10 nM insulin and 100 nM Dexamethasone and re- suspended in the same culture medium, then seeded in collagen-coated 6-well plate. The cell culture was continued for 24 h before starved overnight starved in serum-free
DMEM without insulin and Dexamethasone. The glucose output was measured in glucose- and phenol red-free DMEM supplemented with 20 mM sodium lactate and 2 mM sodium pyruvate. After 5-h incubation, supernatant medium was collected, and briefly spun. The glucose concentration in medium was determined with a colorimetric glucose assay kit I (Eton Bioscience, CA). The glucose read-out was normalized to the total protein content from each well cell lysate.
Oil Red-O staining
Lipids in liver were stained on frozen sections using an Oil Red-O solution (Sigma).
Statistical analysis
Data are expressed as means.e.m. We used JMP software to analyze the following:
Student’s t test for comparison between two groups; one-way ANOVA for multiple group data followed by a post hoc test. Multivariate ANOVA was used to analyze quantitative
RT-PCR data.
Results
The effects of Ad36E4ORF1 in diabetes model db/db mice
In a pilot study we injected rAAV-GFP and rAAV-Ad36E4ORF1 to mice via a single tail vein injection (2x1010 vg per mouse) and sacrificed mice 6 days post injection.
Intravenous delivery of AAV serotype Rec2 preferentially transduced liver. GFP fluorescence was observed in the liver (Fig 7A) and Ad36E4ORF1 expression was
26 confirmed by western blot (Fig 7B). No Ad36E4ORF1 was found in heart, kidney, skeletal muscle, or white fat (Fig 7B).
To investigate the therapeutic potential of Ad36E4ORF1, we injected rAAV-
Ad36E4ORF1 to the genetic model of diabetes and obesity db/db mice (2x1010 vg per mouse, single dose, tail vein injection). To study whether the effects were conserved among the E4ORF1 genes of other human adenovirus, we generated rAAV vector to express Ad5E4ORF1 with the same expression cassette. Destablized YFP (dsYFP) and
AAV buffer were used as controls. No difference was observed between dsYFP and
AAV buffer, and therefore the two groups were combined as a control group (data not shown). The mice treated with Ad5E4ORF1 showed decreased body weight by day 16
(Fig 1A). Both Ad5E4ORF1 and Ad36E4ORF1 treated mice showed slight but significant reduced food intake (Fig 1B). Nonfasting blood glucose level was monitored periodically starting from day 6. Ad36E4ORF1 treated mice showed significantly reduced blood glucose level by day 13 and the normalized blood glucose level was maintained throughout the rest of the experiment (Fig 1C). In contrast Ad5E4ORF1 treatment did not relieve hyperglycemia over the 4 weeks of experiment (Fig 1C). At 3 weeks post AAV injection, a glucose tolerance test (GTT) was performed after overnight fast. Ad36E4ORF1 treatment significantly reduced fasting blood glucose level and robustly improved glycemic control (Fig 1D). Most of the Control and Ad5E4ORF1- treated mice had blood glucose levels exceeding the glucose meter range and therefore assigned 600 mg/dL at 30 min after glucose injection. Thus, the extent of reduced blood glucose level by Ad36E4ORF1 was underestimated at this time point. In addition,
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Ad36E4ORF1-treated mice showed reduced insulin levels at all time points during GTT
(Fig 1E). Amount of plasma insulin per unit amount of glucose was significantly lower in the Ad36E4ORF1 group compared to control at 30, 60 and 90 minutes on GTT (Fig 1F).
Ad36E4ORF1 treatment did not change liver mass or fat mass (Fig 1G). We repeated the experiment to measure the in vivo glucose uptake during a GTT using glucose analog tracer 2-[3H] deoxyglucose (2-DG) 8 weeks post rAAV injection. Ad36E4ORF1 treatment significantly increased the liver glucose uptake but not the skeletal muscle or fat (Fig 1H). We repeated the experiment once more to measure blood glucose level more accurately using a glucose meter up to 900 mg/dL. Hepatic expression of Ad36E4ORF1 robustly improved GTT (Fig 8A). An insulin tolerance test (ITT) was performed (Fig 8B,
C). Ad36E4ORF1 treatment showed no effect on normalized blood glucose level in ITT
(Fig 8C) consistent with insulin-sparing action.
The serum levels of metabolites were measured at sacrifice 4 weeks post rAAV injection.
Ad36E4ORF1 group showed significant reduction of glucose, insulin and adiponectin while a trend of reduction of triglyceride and elevation of cholesterol in the circulation
(Table 1). No changes were found in the serum level of free fatty acid or leptin (Table 1).
Liver function panel was shown in Table S1. Among the liver metabolites measured,
Ad36E4ORF1 treatment significantly increased Acetyl-CoA level (Fig 1I).
Ad36E4ORF1 modulated hepatic gene expression
We selected genes involved in glucose metabolism, lipid metabolism, and inflammation for the qRT-PCR analysis on liver samples from Control, Ad5E4ORF1, and
Ad36E4ORF1-treated db/db mice (Fig 2A). No changes of the expression of insulin
28 receptor (Insr), glucose transporter type 2 (Glut2) or glucose transporter type 4 (Glut4) were observed (Fig 2A). Ad36E4ORF1 selectively upregulated the expression of genes involved in glycolysis (Hk2 encoding hexokinase 2 and Pgam2 encoding phosphoglycerate mutase 2) while did not change the expression of genes involved in gluconeogenesis (G6pc encoding glucose-6-phosphatase and Pck1 encoding phosphoenolpyruvate carboxykinase-1), in glycogen synthesis (Gys encoding glycogen synthase 2), in pentose phosphate pathway (G6pdx encoding glucose-6-phosphate dehydrogenase X-linked, Pgd encoding phosphogluconate dehydrogenase, Rpe encoding ribulose-5-phosphate-3-epimerase). Pyruvate dehydrogenase kinase isoenzyme 4
(encoded by Pdk4) involved in the conversion of lactate to acetyl CoA was upregulated by Ad5E4ORF1 but not Ad36E4ORF1 (Fig 2A). Glucokinase (Gck) expression at mRNA level was upregulated by Ad36E4ORF1 (Fig 2A), and the protein level of this enzyme that is critically involved in controlling blood glucose levels was significantly increased more than 2 fold in Ad36E4ORF1-treated mice (Fig 1J). Ad36E4ORF1 modulated a number of genes involved in lipogenesis and lipid oxidation (Fig 2A). Two major transcription factors known to regulate de novo lipogenesis (DNL) enzymes, sterol regulatory-element-binding-protein 1c (encoded by Srebf1c) and carbohydrate- responsive-element-binding protein (ChREBP, also known as MLXIPL) were measured
(Horton, Goldstein, & Brown, 2002; Iizuka, Bruick, Liang, Horton, & Uyeda, 2004).
Ad36E4ORF1 significantly downregulated both the canonical ChREBP- and the more potent isoform ChREBP- (Fig 2A). This downregulation of ChREBP was associated with downregulation of its lipogenic targets including Acaca (encoding acetyl-CoA
29 carboxylase alpha) and Gpd1 (encoding glycerol-3-phosphate dehydrogenase 1) that is required for triglyceride synthesis (Sato, Morita, Mori, & Miura, 2014). In addition,
Acox1 (encoding acyl-CoA oxidase 1), Cpt1a (encoding carnitine palmitoyltransferase
1A), and Ppara (encoding peroxisome proliferastor-activated receptor alpha), genes involved in -oxidation, were downregulated in Ad36E4ORF1-treated mice (Fig 2A).
Ad5E4ORF1 treatment showed significant downregulation of lipogenic transcription factors Chrebpa, Chrebpb, and Srebf1c, and lipogenic target Gpat (encoding glycerol-3- phosphate acyltransferase) (Fig 2A). Both Ad5E4ORF1 and Ad36E4ORF1 were associated with induction of genes associated with inflammation including Pai1
(encoding plasminogen activator inhibitor-1) and Ccl2 (encoding monocyte chemotactic protein 1, MCP-1) (Fig 10A). Hypothalamic expression of the genes involved in regulation of energy balance was profiled by qRT-PCR (Fig 9).
Ad36E4ORF1 activated AKT independent of Ras or Myc
In vitro data suggest that Ad36E4ORF1 upregulates Ras leading to the activation of PI3K and AKT2 (E. J. Dhurandhar et al., 2012). We investigated the signaling pathways in the livers by western blot. Ad36E4ORF1 transduction had no significant impact on insulin receptor, IRS2, Ras, Myc, rictor, or phosphor-Foxo1 (Fig 3A). However, phospho-AKT at both Ser473 and Thr308 were significantly induced in Ad36E4ORF1-transduced liver
(Fig 3A).
The effects of Ad36E4ORF1 in DIO mice
Next we investigated the effects of Ad36E4ORF1 in DIO model that was insulin resistant, but not hyperglycemic. The obese DIO mice were randomly assigned to receive
30 rAAV-Ad36E4ORF1, Ad5E4ORF1, or dsYFP (2x1010 vg per mouse, single dose, tail vein injection). Ad36E4ORF1 treatment progressively decreased body weight by week 5 after rAAV injection (Fig 4A) with no change in food intake (Fig 4B). Nonfasting blood glucose level was monitored starting from 2 weeks post AAV injection. Ad36E4ORF1 treatment slightly but significantly reduced blood glucose level compared to control mice at some time points (Fig 4C). A GTT was performed 3 weeks after AAV injection.
Fasting blood glucose level in Ad36E4ORF1-treated mice was decreased compared to control mice. Furthermore Ad36E4ORF1 mice displayed significantly improved glycemic control during GTT (Fig 4D). Ad5E4ORF1-treated mice also showed improved
GTT compared to control mice although to a milder degree than Ad36E4ORF1. When the blood glucose changes were calibrated to baseline, only Ad36E4ORF1 mice showed significant difference (Fig 4E). Mice were sacrificed 7 weeks post AAV injection.
Ad36E4ORF1 mice showed increased liver weight whereas decreased adiposity (Fig 4F).
Ad36E4ORF1 treatment resulted in significant reduction of serum levels of glucose, insulin, leptin, adiponectin, and triglyceride (Table 1). However triglyceride level in liver was significantly increased in Ad36E4ORF1-treated mice consistent to enlarged liver
(Table 1). Liver glycogen level was reduced in Ad36E4ORF1 mice (Table 1). The expression of the same set of genes as the db/db mice study was analyzed by qRT-PCR
(Fig 2B). In DIO model, Ad36E4ORF1 transduction led to upregulation of genes involved in glycolysis including Hk2, Pgam2, and Pfkfb3 (encoding 6-phosphofructo-2- kinase/fructose-2,6-biphosphatase 3). Ad36E4ORF1 downregulated G6pc (Fig 2B) which was not observed in db/db mice (Fig 2A). The contribution of suppression of
31 gluconeogenesis to the improvement of glycemic control requires further investigation. In contrast to db/db mice, Ad36E4ORF1-treated DIO mice showed selective induction of lipogenic genes including Scd2 (encoding stearoyl-coenzyme A desaturase 2) and Gpd1
(Fig 2B). Inflammation markers were induced in Ad36E4ORF1-transduced liver (Fig
10B). Similar to db/db mice, Ad36E4ORF1 transduction activated AKT signaling independent of insulin receptor or Ras (Fig 3B).
The effects of Ad36E4ORF1 in normal wild type mice
To further study the efficacy of Ad36E4ORF1 dose, we tested two doses in normal wild type mice (2x109, 2x1010 vg per mouse). Body weight was monitored daily and showed no changes (Fig 5A). Nonfasting blood glucose level was monitored starting day 5 after
AAV injection. Only high dose (2x1010 vg per mouse) Ad36E4ORF1 mice showed progressive decrease of blood glucose level (Fig 5B). One week after rAAV injection, mice were sacrificed. Only the high dose Ad36E4ORF1 showed a significant increase of liver mass (Fig 5C). No change in the abdominal fat pad weight was observed (Fig 5C).
Dose-dependent Ad36E4ORF1 expression was confirmed by western blot (Fig 3C). And dose-dependent effects on serum metabolites were observed. High dose Ad36E4ORF1 mice showed significantly decreased glucose whereas increased fatty acid and triglyceride levels among all groups (Table 2). Leptin level was not changed (Table 2), which is consistent with minimal transgene expression in the adipose tissue at the doses used (data not shown) and lack of impact on adipose tissue (Fig 11). We repeated the high dose Ad36E4ORF1 experiment to isolate hepatocyte for glucose output assay.
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Ad36E4ORF1 expression was confirmed by western blot (data not shown).
Ad36E4ORF1 expression did not change hepatocyte glucose output (Fig 5D).
Ad5E4ORF1 showed no changes in liver mass or serum metabolites (Fig 5C, Table 2) even at high dose although the transgene expression level was comparable to that of
Ad36E4ORF1 at the same dose as measured by gene expression of WPRE element in all rAAV vectors (data not shown).
Liver cholesterol levels were not changed among all groups (Table 2). The liver triglyceride level was increased in high dose Ad36E4ORF1 mice by approximately 4.5 fold compared to dsYFP control, indicating that the lipid associated with fatty liver was primarily triglyceride (Table 2). High dose Ad36E4ORF1 led to increased fatty acid while reducing glycogen levels in the liver (Table 2). Interestingly Ad5E4ORF1 at high dose elevated fatty acid levels in the liver (Table 2).
Gene expression profiling was performed on liver samples from dsYFP, high dose
Ad5E4ORF1, and both doses of Ad36E4ORF1 (Fig 5E). High dose Ad36E4ORF1 showed significant downregulation of Insr consistent with the suppression of proximal insulin signaling by Ad36 viral infection (Krishnapuram et al., 2011). High dose
Ad36E4ORF1 upregulated the expression of genes involved in glycolysis and downregulated genes involved in gluconeogenesis. Pdk4 involved in lactate conversion was upregulated by high dose Ad36E4ORF1 (Fig 5E). Moreover, Gys2 was downregulated consistent with the reduced level of glycogen in the livers transduced by high dose Ad36E4ORF1 (Fig 5E, Table 2). G6pdx involved in pentose phosphate pathway was downregulated (Fig 5E). Among genes involved in lipid metabolism or
33 associated with fatty liver, the effects of Ad36E4ORF1 were also dose-dependent (Fig
5E). Low dose Ad36E4ORF1 significantly upregulated ChREBP- (Fig 5E). In the adipose tissue, the expression of the canonical ChREBP- isoform is not regulated by glucose flux. However, glucose-induced ChREBP- transcriptional activity increases
ChREBP- expression. And ChREBP- alone responds robustly to GLUT4-mediated changes in glucose flux (Herman et al., 2012). This upregulation of ChREBP- by
Ad36E4ORF1 was associated with a collective upregulation of its lipogenic targets including Fasn (encoding fatty acid synthase), Acaca, Gpat, Gpd1, and Scd2 (Fig 5E).
Furthermore, western blot confirmed the increase of ChREBP and mature form of
SHREBP proteins (Fig 3C). With the occurrence of hepatic steatosis in high dose
Ad36E4ORF1 mice, the expression of lipogenic transcription factors and fatty acid synthetic enzymes were downregulated suggesting a negative feedback to the accumulation of triglyceride (Fig 5E). In addition, Acox1, Cpt1a, and Ppara, genes involved in -oxidation, were downregulated (Fig 5E). The hepatic steatosis in high dose
Ad36E4ORF1 was associated with robust induction of inflammatory genes including
Pai1 and Ccl2 (Fig 10C). In contrast to Ad36E4ORF1, high dose Ad5E4ORF1 led to no induction of glycolytic genes (Fig 5E), limited induction of lipogenic genes (Fig 5E) or inflammatory genes (Fig 10C).
We investigated the signaling pathways in the livers of low dose Ad36E4ORF1,
Ad5E4ORF1, and dsYFP controls by western blot (Fig 3C). Phospho-AKT at Ser473 and
Thr308 was significantly increased in Ad36E4ORF1 (Fig 3C). Furthermore, MYC was not upregulated by Ad5E4ORF1, which was inconsistent to in vitro data (Thai et al., 34
2014). Ad5E4ORF1 also increased phospho-AKT although the magnitude was less than
Ad36E4ORF1 (Fig 3C). Phospho-Foxo1 was increased in Ad36E4ORF1-transduced liver
(Fig 3C).
AKT blockade attenuates Ad36E4ORF1 effects
To investigate the role of AKT activation in mediating Ad36E4ORF1 effects, perifosine, an oral bioactive alkylphospholipid, was used to inhibit AKT activation (Hideshima et al., 2006). Wild type mice of normal weight were randomly assigned to receive rAAV-
Ad36E4ORF1 (high dose of 2x1010 vg per mouse) or AAV buffer. Perifosine was given orally on day 4 and day 5 to half of Ad36E4ORF1 mice or control mice. Nonfasting blood glucose levels were monitored daily starting before the first dose of perifosine on day 4. Ad36E4ORF1-treated mice receiving oral PBS showed significantly decreased blood glucose level on day 5 and day 6. Perifosine prevented the lowering of glucose in
Ad36E4ORF1 treated mice on day 5 and substantially attenuated the drop of blood glucose level on day 6 (Fig 6A). Mice were sacrificed day 6 after AAV injection. H&E and Trichrome staining showed no hepatic toxicity associated with perifosine treatment
(Fig 6D). Perifosine significantly blocked the hepatomegaly induced by high dose
Ad36E4ORF1 (Fig 6B) and substantially attenuated liver steatosis revealed by Oil-Red O staining (Fig 6C, D). Western blot confirmed the inhibition of AKT activation by perifosine (Fig 6E).
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Continued Figure 2.1. Ad36E4ORF1 alleviates hyperglycemia in db/db mice. (A) Body weight of mice treated with rAAV-Ad36E4ORF1 (n=8), rAAV-Ad5E4ORF1 (n=8) or control (n=8, combination of dsYFP and AAV buffer). * P<0.05 Ad36E4ORF1 compared to Control mice. (B) Food intake. (C) Nonfasting blood glucose levels. (D) GTT after overnight fast. (E) Serum insulin during GTT. (F) Insulin to glucose ratio, (G) Tissue weights. Data are means SEM. n=8 per group. (H) Glucose update assay in liver tissues from db/db mice 8 weeks post rAAV-Ad36E4ORF1 injection in a separate experiment. n=4 per group. (I) Acetyle-CoA level in liver tissues. n= 8 per group. (J) Glucokinase assay of liver. n=4 Control, n=3 Ad5E4ORF1, n=4 Ad36E4ORF1. * P<0.05, ** P<0.01, *** P<0.001.
36
Figure 2.1 Continued
37
Figure 2.2 Ad36E4ORF1 modulates hepatic gene expression in db/db mice and DIO mice. (A) db/db mice. n=8 Control, n=5 Ad5E4ORF1, n=5 Ad36E4ORF1. (B) DIO mice. n=5 per group. Data are means SEM. * P<0.05, ** P<0.01, *** P<0.001, + P=0.06.
38
Figure 2.3. Signaling pathways and transcriptional factors in livers transduced by Ad36E4ORF1 in db/db, DIO, and normal wild type mice. (A) db/db mice. (B) DIO mice. (C) Normal wild type mice.
39
Figure 2.4. Ad36E4ORF1 improves glycemic control in DIO mice. (A) Body weight. (B) Food intake. (C) Nonfasting blood glucose levels. (D) GTT after overnight fast. (E) The change of blood glucose calibrated to baseline during GTT. (F) Tissue weight at sacrifice. Data are means SEM. n=8 Control, n=8 Ad5E4ORF1, n=9 Ad36E4ORF1, * P<0.05, ** P<0.01, *** P<0.001, # P=0.07 compared to Control.
40
Continued Figure 2.5. Dose-dependent effects of Ad36E4ORF1 in normal wild type mice. (A) Body weight. (B) Nonfasting blood glucose level. (C) Tissue weights aft sacrifice. (D) Hepatocyte glucose output assay. (E) Hepatic gene expression. n=5 per group. Data are means SEM. Low: 2x109, high: 2x1010 vg per mouse. * P<0.05, ** P<0.01, *** P<0.001 compared to YFP.
41
Figure 2.5 Continued
42
Figure 2.6. Perifosine attenuates the effects of Ad36E4ORF1 in normal wild type mice. (A) nonfasting blood glucose levels. (B) Liver weight calibrated to body weight at sacrifice. (C) Liver triglyceride concentration. (D) H&E, Trichrome, and Oil-Red O staining of livers. (E) Western blots of livers. Data are means SEM. n=4-6 per group. P values of significance or trend of significance are listed above the bars.
43
Figure 2.7. A Pilot study of intravenous injection of rAAV administered to wild type mice. (A) GFP fluorescence in the liverof mice receiving rAAV-GFP. Scale bar: 100 μm. (B) Western blots of Ad36E4ORF1 in various tissues of amouse receiving rAAV- Ad36E4ORF1.
44
Figure 2.8. Ad36E4ORF1 expression improves glycemic control but not insulin sensitivity in db/db mice. (A) Glucose tolerance test. (B, C) Insulin tolerance test. n=8 per group. Data are means ± SEM. * P<0.05, ** P<0.01, *** P<0.001.
45
Figure 2.9. Gene expression of the hypothalamus in Ad36E4ORF1 and Ad5E4ORF1 treated db/db mice. n=4 per group. * P<0.05 compared to YFP control. Npy, neuropeptide Y; Pomc, proopiomelanocortin; Cartpt, cocaine-amphetamine-regulated transcript; Crh, corticotropin-releasing hormone; Trh, thyrotropin-releasing hormone; Insr, insulin receptor; Mc4r, melanocortin-4 receptor.
46
Figure 2.10. Inflammatory gene expression of the liver in Ad36E4ORF1 and Ad5E4ORF1 treated db/db, DIO, and normal wild type mice. (A) db/db mice. n=8 Control, n=5 Ad5E4ORF1, n=5 Ad36E4ORF1. (B) DIO mice. n=5 per group. (C) Normal wild type mice. n=5 per group. Low: 2x109, high: 2x1010 vg per mouse. Data are means ± SEM. * P<0.05, ** P<0.01, *** P<0.001 compared to YFP control.
47
Figure 2.11. Gene expression of abdominal fat depot in Ad36E4ORF1 and Ad5E4ORF1 treated wild type mice. n=5 per group. Data are means ± SEM. high: 2x1010 vg per mouse. Glut4, glucose transporter type 4; Insr, insulin receptor; Lep, leptin; Cpt1a, carnitine palmitoyltransferase 1A; Lpl, lipoprotein lipase; Saa3,serum amyloid A3; Srebf1c, sterol regulatory element-binding protein.
48
db/db DIO Control Ad5E4 Ad36E4 Control Ad5E4 Ad36E4 Serum 47061.2 42451.6 29232.4* 225.916.0 182.311.3* 172.814.8* glucose (mg/dl) Serum 17.74.2 5.01.4* 5.52.1* 1.730.22 1.010.28 0.370.21*** insulin (ng/ml) Serum 80.59.4 83.28.4 106.49.8 176.918.3 139.612.2 168.415.5 cholesterol (mg/dl) Serum 159.136.9 102.89.4 79.09.4 99.89.4 92.710.3 59.77.3** triglyceride (mg/dl) Serum 0.450.05 0.440.06 0.410.03 0.360.03 0.310.04 0.300.04 NEFA (mEQ/l) Serum 49.34.3 43.84.3 51.25.6 20.01.0 14.21.4** 3.81.0*** leptin (ng/ml) Serum 6.40.6 5.40.5 4.40.4* 12.00.6 10.40.4 8.20.6*** adiponectin (g/ml) Liver 10.31.3 13.51.1 13.31.0 15.63.0 14.83.1 16.12.4 cholesterol (mg/g) Liver 149.19.4 173.49.4 186.69.4 113.938.5 136.839.3 262.521.8* triglyceride * (mg/g) Liver 0.240.02 0.200.01 0.210.3 0.160.01 0.160.01 0.150.01 NEFA (mEQ/g) Liver 7.490.73 5.940.15 6.200.28 5.400.38 4.540.34 2.860.10** glycogen (mg/g) Table 2.1. Metabolic parameters in db/db mice and DIO mice. Data are means SEM. n=8 per group in db/db experiment. n=8 Control, n=8 Ad5E4ORF1, n=9 Ad36E4ORF1 in DIO experiment. * P<0.05, ** P<0.01, *** P<0.001 compared to Control.
49
YFP Ad5E4 low Ad5E4 high Ad36E4 Ad36E4 high low Serum 226.518.8 193.112.3 210.18.3 201.48.6 146.620.5** glucose (mg/dl) Serum 0.710.35 0.610.15 0.530.14 0.650.17 0.290.08 insulin (ng/ml) Serum 76.36.7 90.511.0 80.37.8 98.69.6 73.711.0 cholesterol (mg/dl) Serum 75.611.6 54.65.6 74.68.9 61.04.2 137.914.7** triglyceride (mg/dl) Serum 0.410.04 0.500.04 0.480.05 0.540.12 0.800.08 NEFA (mEQ/l) Serum 1.200.29 1.730.25 1.390.35 1.950.34 1.220.38 leptin (ng/ml) Liver 0.910.07 0.960.10 1.040.15 0.740.07 1.160.25 cholesterol (mg/g) Liver 1.010.11 1.300.24 1.510.21 1.740.26 4.680.82** triglyceride (mg/g) Liver 0.0170.002 0.0140.001 0.0350.002* 0.0120.001 0.0410.003** NEFA (mEQ/g) Liver 83.83.2 70.45.4 75.75.1 74.53.1 51.13.6** glycogen (mg/g) Table 2.2. Metabolic parameters in normal wild type mice. Data are means SEM. Low: 2x109, high: 2x1010 vg per mouse. n=5 per group. * P<0.05, ** P<0.01, compared to YFP.
50
Discussion
Ad36 is one of the 56 serotypes in 7 subgroups of human adenoviruses. Ad36 was the first human adipogenic virus reported and the most investigated up to date. In various animal models, experimental infection of Ad36 increases obesity but improves glycemic control (N. V. Dhurandhar, 2011). Studies from many countries show that prior Ad36 exposure in humans, as determined by the presence of neutralizing antibodies to Ad36 is associated with increased risk of obesity (Aldhoon-Hainerova et al., 2014; Almgren et al.,
2012; Atkinson et al., 2005; Atkinson, Lee, Shin, & He, 2010; Gabbert, Donohue,
Arnold, & Schwimmer, 2010; Na et al., 2010; Trovato et al., 2009; Trovato et al., 2010), better glycemic control (Krishnapuram et al., 2011; W. Y. Lin et al., 2013) and lower hepatic lipid levels (Trovato et al., 2009; Trovato et al., 2010; Trovato et al., 2012).
Subsequent in vitro studies attribute these effects of Ad36 to its E4ORF1 gene (E. J.
Dhurandhar et al., 2012; Krishnapuram et al., 2013). These studies indicate the potential of E4ORF1 as a template for developing anti-diabetic therapies (E. J. Dhurandhar et al.,
2011; E. J. Dhurandhar et al., 2012; N. V. Dhurandhar, 2013; Krishnapuram et al., 2011).
Improvement in glycemic control
We tested the effect of hepatic expression of Ad36 E4ORF1 in genetic model of diabetes
(db/db mice), dietary model of insulin resistance (DIO mice) and normoglycemic mice
(WT). In the diabetes model rAAV-Ad36E4ORF1 treatment completely alleviated hyperglycemia (>70%, Figure 1C) and robustly improved glycemic control during GTT without significantly increasing hepatic steatosis. In the DIO model, Ad36E4ORF1 reduced the blood glucose level but did not cause hypoglycemia, and significantly
51 improved GTT. Finally, in normoglycemic mice, hepatic expression of Ad36E4ORF1 at higher level lowered nonfasting blood glucose. Notably, Ad36E4ORF1 significantly reduced insulin levels in db/db and DIO mice and showed a trend in WT mice. The improvement in glycemic control was observed without stimulation of the proximal insulin signaling (IR, IRS-2). Collectively, these data indicate that Ad36E4ORF1 is not a typical sensitizer, mimetic, or secretagogue of insulin. Instead, it appears to have “insulin sparing action” (Kusminski et al., 2015) which reduces the need for insulin and hence, reduce insulin levels.
Although Ad36 E4ORF1 does not induce proximal insulin signaling, it activates the distal insulin signaling involving AKT via the activation of Ras in vitro (E. J. Dhurandhar et al., 2012). However Ad36E4ORF1 expression in the livers of all mouse models did not up-regulate Ras. Yet, in agreement with in vitro data, among all the models tested,
Ad36E4ORF1 activated AKT independent of proximal insulin receptor signaling.
Requirement of AKT for Ad36E4ORF1-induced modulation of glucose levels was demonstrated by inhibiting AKT activation with perifosine, which attenuated the lowering of glucose. The mechanisms of Ad36E4ORF1 induction of p-AKT independent of Ras remain to be elucidated. Complete activation of AKT is achieved by phosphorylation of both Thr308 and Ser473. While they are catalyzed by separate kinases and occur independently of one another, both phosphorylation events require binding of
AKT to PIP3 in response to PI3K signaling. Phosphorylation of both sites is required for catalytically active AKT to properly dissociate from the membrane and phosphorylate downstream targets. AKT enters a catalytically active state when Thr308 is
52 phosphorylated by PDK1 (T. O. Chan et al., 1999). AKT catalytic activity and substrate specificity are further enhanced when mTORC2 phosphorylates AKT at Ser473, an event that also requires association of AKT with PIP3 (Scheid et al., 2002). Rictor is a core component of mTORC2 and required for Ser473 phosphorylation (Sarbassov, Guertin,
Ali, & Sabatini, 2005). Two core proteins of mTORC2, rictor and SIN, are not only required for mTORC2 assembly but also prevent degradation of one another (Oh &
Jacinto, 2011). Therefore measurement of either provides a valid assessment of mTORC2 abundance. Our data showed that levels of rictor were unchanged in response to hepatic expression of Ad36E4ORF1 (Fig 3). Moreover it is reported that liver-specific rictor knockout mice display dramatic reduced AKT phosphorylation at Ser473 but not at
Thr308 indicating only Ser473 is a target of mTOC2 (Hagiwara et al., 2012). Our data showed that hepatic Ad36E4ORF1 expression increased phosphorylation of AKT at
Thr308 and Ser473 suggesting that it is unlikely that mTORC2 function is increased to mediate Ad36E4ORF1-induced AKT activation. Taken together, it is likely that phosphorylation of AKT is due to elevated PIP3 levels in response to E4ORF1 mediated
PI3K activation similar to in vitro findings of highly homologous E4ORF1 protein from
Adenovirus type 9 (AD9) (Frese et al., 2003; Kong et al., 2014).
Hepatic steatosis
Ad36E4ORF1 increased liver weight and triglycerides in DIO or WT mice, but not in db/db mice. Based on the data of gene expression profiling and liver metabolites, we propose the actions of Ad36E4ORF1 on cellular metabolism observed in the transduced liver of normoglycemic mice. Ad36E4ORF1 elevates glycolysis. The glycolytic
53 intermediates are shunted mainly to triacylglycerol synthesis whereas glycogen, lactate or
ATP production and nucleotides synthesis remain either slightly reduced or unchanged.
Ad36E4ORF1 induces the expression of ChREBP- that is highly responsive to glucose influx, and subsequently induce the lipogenic and glycolytic genes. The accumulation of triacylglycerol leads to steatosis.
Whether the effects on glycemic control and liver DNL can be dissociated could be possibly dependent on the basal AKT activation status in the liver as well as the presence and severity of insulin resistance. For example, in diabetic db/db mice, AKT activity was decreased compared to wild type mice (Shao, Yamashita, Qiao, & Friedman, 2000).
Therefore the Ad36E4ORF1 treatment could normalize the abnormally low AKT activity and subsequently improve glycemic control. However, in normal animals, overshoot of
AKT activation by Ad36E4ORF1 could lead to hepatic steatosis and hypoglycemia, a phenomenon similar to the PTEN mutants (He et al., 2010). Ad36E4ORF1 activated
AKT leading to an increase in hepatic glycolysis. Yet glucose output, FOXO1 and its target gene PCK1 were unchanged. One would anticipate that activation of AKT would phosphorylate its target protein FOXO1, therefore decrease FOXO1 activity and reduce gluconeogenesis. However, Ad36E4ORF1 up-regulated inflammatory cytokines (Fig 10) in addition to steatosis. It has been shown that the expression and activity of FOXO1 are increased in nonalcoholic steatohepatitis (Valenti L, 2008). Therefore, it is possible that the steatosis and increase of inflammatory cytokines associated with hepatic
Ad36E4ORF1 expression offset the effect of AKT activation on FOXO1.
Ad36 and E4ORF1
54
All serotypes of adenovirus have the E4ORF1 gene that shares conserved amino acids domain with dUTPase, but has distinct functionality (Weiss, Lee, Prasad, & Javier,
1997). Ad5E4ORF1 is well characterized and mediates the reprogramming of cellular metabolism that mirrors the Warburg effect in cancer by enhancing MYC transcriptional activation of glycolytic genes and promote glycolysis, resulting in increased nucleotide biosynthesis (Thai et al., 2014). The in vitro findings have yet been confirmed in vivo.
The amino acid homology between Ad5E4ORF1 and Ad36E4ORF1 is approximately
45%. Our data demonstrate that the effects of Ad36E4ORF1 are different from those of
Ad5E4ORF1. In contrast to Ad36E4ORF1, Ad5E4ORF1 did not decrease blood glucose level in normal animals or in db/db mice. Hepatic expression of Ad5E4ORF1 did not replicate the increase of MYC observed in cell culture (Thai et al., 2014). The discrepancy could be due to the difference of transduced tissue and cell types.
Interestingly, both Ad36E4ORF1 and Ad5E4ORF1 significantly activated AKT and promoted hepatic glycolysis in normal animals. Activation of PI3K/AKT pathway is a well-documented function of E4ORF1 shared by several human adenoviruses and may contribute to tumorigenic property, promotion of survival, viral replication, or reprograming of host cell metabolism depending on adenovirus types and infected cells and tissues (E. J. Dhurandhar et al., 2012; O'Shea et al., 2005; Seandel et al., 2008; Thai et al., 2014). E4ORF1 from Ad9 and Ad5 interacts with a select group of PDZ domain- containing cellular products (Chung, Frese, Weiss, Prasad, & Javier, 2007; Kong et al.,
2014; Weiss & Javier, 1997). The extreme carboxy-terminal of E4ORF1 contains the crucial PDZ domain-binding motif essential for activation of PI3/AKT signaling (Frese et
55 al., 2003; Thai et al., 2014; Thomas, Schaack, Vogel, & Javier, 2001; Weiss, Gold,
Vogel, & Javier, 1997). Because the consensus X-S/T-X-(VLI) is conserved in Ad5, Ad9, and Ad36 E4ORF1 (Frese et al., 2003), it is possible that the same motif in Ad36 participates in PI3/AKT signaling. The Ad 36 infection has not been linked to an increased risk of tumor formation in humans or animals. Additionally, expression of
Ad36E4ORF1 in the adipose tissue is not tumorigenic (Kusminski et al., 2015). However, given the potential tumorigenic effect of Ad9E4ORF1, it is important to investigate whether the Ad36E4ORF1 and Ad9E4ORF1, who share a 92% amino acid homology, also share similar effects on cellular metabolism as well as oncogenesis. Our rAAV vector provides a powerful tool to study the short-term and long-term effects of adenoviral genes in various tissues in vivo.
This proof-of-concept study shows that predominant hepatic expression of Ad36E4ORF1 alleviates hyperglycemia in genetic models of diabetes and diet-induced insulin resistance, and this property may not be shared by the E4ORF1 genes of all human adenoviruses. The insulin-sparing action of Ad36E4ORF1 is particularly attractive. It is unknown how the hepatic response would be modulated, due to secondary input from other tissues, if they were to also express E4ORF1. Nonetheless, the amount of E4ORF1 expression in liver appears to have a threshold that should not be exceeded. Future studies with detailed analysis on glycemic control in animal models of hyperglycemia should further characterize the phenomenon.
56
Chapter 3: Environmental activation of a hypothalamic-adipocyte axis promotes
healthy aging
57
Environmental activation of a hypothalamic-adipocyte axis promotes healthy aging
Travis McMurphy, Wei Huang, Xianglan Liu, Kyle Widstrom, Run Xiao, Nicholas
Queen, Jason Siu & Lei Cao*
Department of Cancer Biology & Genetics, College of Medicine
The Ohio State University, Columbus, OH 43210
*Correspondence: [email protected] Phone: 614-3665679, Fax: 614-6888675
Running title: Hypothalamic-adipocyte axis improves healthy aging
Summary
With the aging of population, it is vital to understand the dynamics of aging, their interaction with lifestyle factors, and the connections to age-related disease processes.
Our recent work on environmental enrichment (EE), a housing environment boosting mental health, has revealed a novel anticancer and anti-obesity phenotype mediated by a brain-fat axis, the hypothalamic-sympathoneural-adipocyte (HSA) axis. Here we investigated the role of the HSA axis in the aging process. Short-term EE of six weeks activated the HSA axis in aged mice. Long-term EE of twelve months reduced adiposity without weight loss, improved glucose tolerance, decreased leptin level, enhanced motor abilities, and inhibited anxiety. The EE-induced adipose remodeling was associated with upregulation of PTEN that was independent of gender or age and also mediated by the
HSA axis. These data suggest that EE induces metabolic and behavioral adaptations that are shared by those factors known to increase healthspan and lifespan.
58
Introduction
The incidence of age-related diseases such as cancer, cardiovascular disorders and neurodegenerative diseases rises as life expectancy increases. Therefore, it is vital to understand more about the dynamics of aging, particularly how they interact with various environmental and lifestyle factors, and the connections between disease processes and aging in order to develop more effective strategies to prevent, diagnose and treat age- related diseases. Numerous studies on model organisms have revealed an interaction between genes and environment in determining healthy lifespan, i.e. the age free of significant diseases (Fontana, Partridge, & Longo; D. L. Smith, Jr., Nagy, & Allison).
Calorie restriction (CR) without malnutrition (often reduced calorie intake by 40% compared to ad libitum) is, by far, the most robust and reproducible approach to delay the onset of age-related disorders and extend lifespan in a wide range of model organisms from yeast to monkeys (Fontana et al.). Intermittent fasting (IF; a diet with reduced meal frequency, for example, alternative-day feeding) also induces resistance to toxicity and stress, and extends lifespan (Mattson, 2005). The metabolic and physiological characteristics of CR that may contribute to its anti-aging capacity include reduced adiposity, higher insulin sensitivity, improved lipid profiles, reduced oxidative stress and inflammation (Anderson, Shanmuganayagam, & Weindruch, 2009; Colman et al., 2009;
Fontana & Klein, 2007; Fontana, Meyer, Klein, & Holloszy, 2004; Heilbronn et al.,
2006). In the past few years, we used a eustress model, environmental enrichment (EE) that has a profound impact on brain structure and function, to study how physical and social environments modulate physiology and disease risk and progression. We
59 demonstrate that EE leads to leanness, resistance to diet-induced obesity (DIO), and inhibition of melanoma and colon cancer (Cao et al., 2011; Cao et al., 2009; L. Cao et al.,
2010). The anticancer effects of EE have been confirmed and expanded by other researchers to breast cancer (Nachat-Kappes et al., 2012), pancreatic cancer (G. Li et al.,
2015), and glioma (S. Garofalo et al., 2015). Our mechanistic studies elucidate one key underlying mechanism: the activation of a specific neuroendocrine brain-adipocyte axis, the hypothalamic-sympathoneural-adipocyte (HSA) axis (Cao & During, 2012). The physical, social, and cognitive stimuli provided by EE induces brain-derived neurotrophic factor (BDNF) in the hypothalamus and thereby elevates the sympathetic tone preferentially to the adipose tissue. The adipose tissue remodeling, including the white- to-brown phenotypic switch and the suppression of leptin, results in anti-obesity and anticancer phenotype. Some of the features of EE overlap with CR and/or protein phosphatase and tensin homologue (PTEN) transgenic mice (Ptentg), or fat-specific insulin receptor knock-out (FIRKO) mice with extended lifespan (Bluher et al., 2002;
Katic et al., 2007; Ortega-Molina et al., 2012). The following characteristics suggest that
EE may regulate aging and age-related diseases and the HSA axis may mediate this anti- aging effect. 1) HSA axis activation leads to a robust reduction in fat mass with little change in body weight (Cao et al., 2009; L. Cao et al., 2010). Several genetically modified mouse models link reduced adiposity to longevity, including the translational inhibitor 4E-BP1 (Eif4ebp1-/-) knock-out (Tsukiyama-Kohara et al., 2001), C/EBPß knock-in (ß/ß) (Chiu, Lin, Huang, & Lee, 2004), and c-Cbl knock-out (Molero et al.,
2004). The FIRKO mice with fat-specific disruption of the insulin receptor gene have
60
50% reduced fat mass, improved whole body insulin sensitivity, and an extended lifespan
(18%) (Bluher, Kahn, & Kahn, 2003; Bluher et al., 2002). EE initiated in young male mice led to over 60% reduction of abdominal fat when mice were fed with normal chow diet (NCD) whereas body weight was identical to the mice with standard laboratory environment (SE) (Cao et al., 2011). Muscle mass was increased in EE and hypothalamic
BDNF overexpressing mice. Both environmental and genetic activation of the HSA axis is particularly efficient in decreasing adiposity, allowing the dissociation of fat loss from weight loss which is difficult to achieve with other interventions (Heitmann & Garby,
2002). 2) HSA axis activation is associated with increased whole body metabolic rate, increased oxygen consumption in fat, increased mitochondrial content, and upregulation of genes involved in mitochondrial biogenesis and activity (Cao et al., 2011), resembling
FIRKO and Ptentg mice. 3) HSA axis activation alleviates obesity-associated insulin resistance, hyperglycemia and dyslipidemia (Cao et al., 2011; Cao et al., 2009), mimicking CR. 4) HSA axis activation reduces serum IGF-1 levels in male mice, also similar to CR. The IGF-1 /growth hormone pathway is one of the most conserved pathways implicated in aging (Brown-Borg, 2009) with IGF-1 deficiency leading to smaller body size, delayed age-related pathology, and an extended lifespan (Brown-Borg,
2009). 5) HSA axis activation alters adipokine levels with higher adiponectin and lower leptin expression in adipose tissue as well as in the circulation (Cao et al., 2009; L. Cao et al., 2010), again mimicking CR. 6) HSA axis activation suppresses tumor progression and metastasis (L. Cao et al., 2010; Liu, McMurphy, et al., 2014). Epidemiological and experimental studies have revealed that lifestyle and environmental factors can influence
61 cancer initiation, promotion and progression. Targeting the major players such as obesity, lack of physical activity, and unhealthy diet could prevent many cancers (Calle & Kaaks,
2004; Hursting, Slaga, Fischer, DiGiovanni, & Phang, 1999). CR is broadly effective in cancer prevention in rodents and monkeys and CR-induced metabolic adaptations are thought to be responsible for its anticancer phenotype (Albanes, 1987; Colman et al.,
2009; Longo & Fontana, 2010). Our studies have shown that EE and BDNF overexpression markedly suppresses tumor growth via metabolic and immune modulations (L. Cao et al., 2010; Liu, McMurphy, et al., 2014; Xiao et al., 2016). 7) HSA axis activation is associated with enhanced immunocompetence. Aging-associated immune dysfunction exerts a strong influence on age-related morbidity and mortality
(Wayne, Rhyne, Garry, & Goodwin, 1990) including low lymphoproliferative response to mitogens and low natural killer (NK) cytotoxicity (De la Fuente & Miquel, 2009;
DelaRosa et al., 2006; Guayerbas & De La Fuente, 2003). We and others have demonstrated that EE and BDNF overexpression in young mice increase lymphocyte proliferation in response to the mitogen Concavalin A, enhance NK cell activity
(Benaroya-Milshtein et al., 2004; L. Cao et al., 2010), and increase cytotoxicity of T cells after tumor implantation (L. Cao et al., 2010; Xiao et al., 2016). 8) EE leads to a modest increase in serum corticosterone consistent with a mild stress. The exposure to the mild, nonaversive challenges of EE may condition the stress system to be more adaptive and may therefore buffer the reaction to subsequent major external stressors (Benaroya-
Milshtein et al., 2004; L. Cao et al., 2010; Larsson, Winblad, & Mohammed, 2002).
Indeed, CR (Patel & Finch, 2002) and regular physical exercise and cognitive stimulation
62 are beneficial on health, and they too increase cortisol levels (Gotthardt et al., 1995; Witt,
Snook, O'Dorisio, Zivony, & Malarkey, 1993), suggesting that eustress may increase resistance to disease and be beneficial for health and longevity. To date, the majority of studies on EE and aging examine the effects on cognitive decline and neurodegenerative diseases showing that EE could reverse age-related neural, cognitive and behavioral impairments (Goldberg, Haack, & Meshul, 2011; Harburger, Lambert, & Frick, 2007;
Mattson, Duan, Lee, & Guo, 2001; Segovia, del Arco, & Mora, 2009). However, scarce evidence is available on the effects of EE on peripheral systems and healthspan or lifespan. Here, we investigated the effects of EE on healthy aging from a unique perspective of the recently characterized HSA axis.
Results
Short-term EE activates the HSA axis in middle age female mice
Our previous studies on EE were performed on young male mice often initiated immediately after weaning at the age of 3 weeks. To prevent the risk of fighting in group housed older male mice, we used female mice in the aging studies. First we randomly assigned female mice at the age of 10 months to live in SE or EE for 6 weeks. Body weight and food intake were monitored weekly. EE slightly decreased weight (Figure
1A) and increased food consumption (Figure 1B) but neither was significant. In contrast, robust reduction of adiposity was observed in EE mice with both brown adipose tissue
(BAT) and the three white adipose (WAT) depots: the subcutaneous inguinal WAT
(iWAT), abdominal retroperitoneal WAT (rWAT) and gonadal WAT (gWAT) (Figure
1C). Visceral WAT showed largest reduction up to approximately 60% consistent with
63 the activation of the HSA axis observed in young male mice (Figure 1C) (Cao et al.,
2011). The signature of serum biomarkers often associated with EE in young male mice was examined in the middle age female mice (Figure 1D). The decrease of IGF-1 and increase of adiponectin found in young male mice were not observed in the middle age female mice (Figure 1D). However, EE resulted in a significant and large drop of leptin by 60% consistent with the finding in young mice that leptin is the most pronounced change in serum biomarkers responding to EE (Cao et al., 2011; L. Cao et al., 2010).
Real-time quantitative RT-PCR was used to profile the expression of genes involved in energy homeostasis and inflammation. EE significantly upregulated Bdnf expression indicating the HSA axis activation. The expressions of BDNF receptor TrkB (Ntrk2) and its upstream melanocortin-4 receptor (Mc4r) were not changed (Figure 1E). EE upregulated both the orexigenic neuropeptide Y (Npy) and the anorexic proopiomelanocortin (Pomc). Insulin receptor (Insr) and leptin receptor (Lepr) expression were also upregulated by EE (Figure 1E). Among the inflammatory genes profiled, only interleukin-1b (Il1b) was significantly downregulated (Figure 1E). Consistent to young mice results (Cao et al., 2011), rWAT showed most changes in gene expression profile in response to EE while BAT and gWAT showed very limited changes and no change was found in liver (Figure 8). Although the effects of EE in middle age female mice did not completely match those in young male mice, the key features of HSA axis activation namely upregulation of hypothalamic BDNF, reduction of adiposity, and drop of leptin were confirmed allowing the investigation of EE’s impact on healthy aging.
Long-term EE improves metabolism
64
In a long-term EE study, 10-months old female mice were randomly assigned to live in
EE or SE for 12 months. Mice were subjected to a series of metabolic measurements and behavior tests as indicated in Figure 2A. Weekly weight monitoring showed that EE reduced weight between 14 to 21 weeks and there was no difference at the end of the study of 12-month EE (Figure 2B). Food intake was monitored for 10 weeks (8~10.5- month EE). EE mice tended to eat more but not significant (Figure 2C). Rectal temperature was measured after 39-week EE and no significant change was observed
(SE: 36.83±0.13 °C; EE: 37.16±0.13 °C, P=0.10). A glucose tolerance test (GTT) performed at 32-week EE showed substantial improvement (Figure 2F). Similar to short- term EE, adiposity was greatly reduced at the end of the study of 12-month EE (Figure
2G) and serum leptin level was approximately 50% lower in EE mice (Figure 2H).
Furthermore serum glucose level was also decreased in EE (Figure 2H). In a separate cohort of mice, GTT at 2-month EE also showed improved glucose tolerance (Figure
2D). We measured the in vivo glucose uptake during a GTT using glucose analog tracer
2-[3H] deoxyglucose (2-DG) at 3-month EE. EE significantly increased the adipose glucose uptake (Fig 1E).
In contrast to the strong metabolic effects, long-term EE had no effects on either the proliferative response of splenic lymphocytes to the T cell mitogen Concavalin A (Figure
9A), or the NK cell cytotoxicity against melanoma cells (Figure 9B).
EE improves motor behavior and reduces anxiety
EE has profound influences on brain structure and function and is often associated with neuroprotection against a variety of toxin- and genetically-induced models of
65 neurological diseases (Nithianantharajah & Hannan, 2006). Rotarod treadmill test measures motor abilities including balance, coordination, physical condition, and motor- planning (Jones & Roberts, 1968). After living in EE for 9 months, mice were subjected to rotarod treadmill test. EE greatly prolonged the time the mice were able to remain on the rod (Figure 3A) and increased the rotating speed at the first fall (Figure 3B) indicating significant improvement in motor abilities. In addition we performed a battery of anxiety and depression behavior tests. Open field (OF) test is classically used to assess exploratory behavior, general locomotion, and anxiety (Ramos, 2008; Stanford, 2007).
OF draws on the natural conflict between the tendency to explore a new environment and to avoid an exposed open area (Crawley, 1985). An increase in time spent in the center of the open field is considered to reflect reduced anxiety level. Living in EE for 2 months (a separate cohort of mice) significantly increased the time spent in the center of arena
(Figure 3C) and the portion of distance that the animal traveled in the center zone (Figure
3D) suggesting an anxiolytic effect. Interestingly enriched mice exhibited less locomotion (Figure 3E), which may indicate an enhanced habituation reflecting a more efficient information processing by EE mice likely a consequence of their greater experience dealing with a new and changing environment (E. J. Lin, Choi, Liu, Martin, &
During, 2011; Varty, Paulus, Braff, & Geyer, 2000). Novelty suppressed feeding (NSF) test assesses hyponeophagia, in which exposure to a novel environment suppresses feeding behavior (Samuels & Hen, 2011). NSF has been used to study anxiety- and depression-related behaviors since it is sensitive to anxiolytic and chronic antidepressant treatments. In the NSF assessed after 10-month EE, the latency to eat was significantly
66 reduced in EE mice suggesting reduced anxiety (Figure 3F). This anxiolytic effect was not due to an enhanced appetitive drive (Figure 3G). In another anxiety behavior test cold-induced defecation (CID) (Barone et al., 2008), a trend of but not significant decrease was observed in EE mice (Figure 3H). Forced swim test is one the most commonly used rodent behavioral tests for screening antidepressant drugs (Cryan &
Mombereau, 2004). No significant effect was observed in FST (Figure 3I, J).
Long-term EE modulates adipose phenotype
Long-term EE of 12 months led to a pattern of hypothalamic gene expression changes different to that after short-term EE (Figure 4A). The changes in BDNF and other genes involved in energy homeostasis found in 6-week EE mice (Figure 1E) were not detected.
Instead a cluster of genes involved in inflammation including Il1b, Il6, Ccl2 (encoding monocyte chemoattractant protein-1 MCP-1), Nfkbia (encoding nuclear factor of light polypeptide gene enhance in B cells inhibitor ), and Socs3 (encoding suppressor cytokine signaling 3) were collectively downregulated in EE mice (Figure 4A). In contrast Pten was significantly upregulated. This induction of Pten was not found in the amygdala while both Ccl2 and Nfkbia were downregulated in this brain area involved in emotionality including anxiety (Figure 10A). Aging is associated with a decline of BAT activity (Enerback, 2010). The BAT of 22-months old mice in SE appeared pale whereas the BAT in EE mice was darker. H&E staining showed the BAT of EE mice maintained typical BAT morphology of younger mice and was absent of white adipocyte infiltration often associated with aging (Figure 5A). In contrast to the mild change in gene expression after 6-week EE (Figure 8A), long-term EE robustly modulated BAT gene
67 expression (Figure 4B). Leptin expression was reduced by over 80% while adiponectin expression showed a trend of increase. Glucose transporter type 4 (Glut4), the major type of glucose transporter in adipose tissue, was significantly induced together with Insr by
EE (Figure 4B). Both lipolytic gene Lpl (encoding lipoprotein lipase) and lipogenic gene
Gpat (encoding glycerol-3-phosphate acyltransferase) were upregulated. BAT dissipates energy via releasing chemical energy from mitochondria in the form of heat. This process is primarily mediated by uncoupling protein-1 (UCP1) that is a specific BAT marker
(Enerback et al., 1997). Ucp1 was significantly upregulated by EE suggesting the preservation of proper BAT functions against aging-related loss. The transcriptional coactivator PGC-1 switch cells from energy storage to energy expenditure by inducing mitochondrial biogenesis and genes involved in thermogenesis (Puigserver et al., 1998).
Ppargc1a (encoding PGC-1) was increased over 3 folds in EE BAT (Figure 4B). EE similarly induced Ppargc1a expression in rWAT and gWAT (Figure 4C, Figure 10B) but not in liver (Figure 4D) or muscle (Figure 4E). Fh1 (encoding mitochondrial fumarate hydratase) and Parp1 (encoding poly ADP-ribose polymerase 1) are associated with CR- induced metabolic adaption (Mitchell et al., 2016). EE induced both Fh1 and Parp1 in
BAT and WAT (Figure 4B, C, Figure 10B). Pten was highly induced by EE in all adipose depots (Figure 4B, C, Figure 10B) but not in liver or muscle (Figure 4D, E). Sirtuins are associated with longevity (Giblin, Skinner, & Lombard, 2014). EE stimulated Sirt1 expression in BAT, rWAT and liver (Figure 4B, C, D). Consistent with the upregulation of mitochondrial genes transcription, mitochondrial DNA contents were increased in adipose tissue and liver (Figure 4F) indicating increased mitochondrial biogenesis.
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Histology showed that the size of white adipocyte in EE mice was much smaller than that in SE mice (Figure 5B, C). Immunohistochemistry demonstrated higher levels of PGC1-
, PTEN, and UCP1 in adipose tissues of EE mice consistent to the gene expression profiling (Figure 5A, B, C).
The HSA axis mediates EE’s induction of adipose Pten
Ptentg mice overexpressing Pten in all tissues have extended lifespan and their metabolic phenotypes overlap with those associated with EE (Ortega-Molina et al., 2012). Here we found that EE significantly upregulated Pten expression in all adipose depots examined but not in liver or muscle (Figure 4, Figure 10). Next we investigated whether adipose
Pten induction was mediated by the HSA axis. To assess whether the Pten induction was dependent on gender or diet, we randomly assigned male C57Bl/6 mice, 3 weeks of age, to live in SE or EE. The diet was changed to high fat diet (HFD, 45% fat) immediately following the initiation of EE and maintained on HFD till the end of the study of 4-week
EE. As reported previously, EE inhibited DIO, elevated energy expenditure, prevented
DIO-associated hyperinsulinemia, hyperleptinemia, hyperglycemia, and dyslipidemia
(Cao et al., 2011). EE increased the expression of Adrb3 (encoding 3 adrenergic receptor), Glut4, Insr, and Ppargc1a (Figure 11) similar to the changes in aged female mice after long-term EE (Figure 4C). Pten expression was significantly upregulated by
EE over 3-fold (Figure 11) suggesting the Pten induction independent of gender or diet.
EE robustly suppressed inflammatory gene expression including macrophage marker
Emr1 (encoding F4/80), Ccl2, and Mgl1 (encoding macrophage galactose-type C-type lectin 1) in WAT (Figure 11).
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To investigate the source of the Pten upregulation in adipose tissue, we isolated adipocytes and stromal vascular fractions (SVF) of epididymal WAT (eWAT) and iWAT after 2-week EE on NCD. In visceral eWAT, Pten expression was induced in both adipocytes and SVFs by EE. In subcutaneous iWAT, Pten was only upregulated in adipocytes but not in the SVFs (Figure 12).
BDNF has been identified as the key mediator of EE-induced WAT remodeling (Cao et al., 2011). Here we investigated the role of hypothalamic BDNF in the regulation of adipose Pten. rAAV-mediated overexpression of BDNF in the hypothalamus upregulated
Pten expression and reproduced the gene expression signatures associated with EE
(Figure 6A, B, Figure 4C). Conversely, BDNF heterozygous mice with hypothalamic
BDNF protein levels approximately 40% lower than wild type show an increase of adiposity prior to significant body weight difference occurs (Cao et al., 2011). The molecular features of WAT were a complete reversal of that found in EE or BDNF- overexpressing mice including a suppression of brown gene program. Our new data showed that Pten was strongly downregulated together with Glut4, Insr, and Ppargc1a in eWAT (Figure 6C) in BDNF heterozygous mice whereas Pten was not suppressed in the muscle of the same mouse (relative expression WT: 1.010.28, BDNF+/-: 1.540.18,
P=0.22). Furthermore, a dominant-negative trunked form of the high-affinity BDNF receptor (TrkB.T1) (Cao et al., 2011) was used to specifically antagonize BDNF signaling the hypothalamus. AAV-TrkB.T1 led to a complete reversal of the gene expression signatures associated with EE or BDNF overexpression (Figure 6D). Pten was sharply downregulated by 75% in eWAT of TrkB.T1 mice (Figure 6D).
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To examine whether intact sympathetic signaling is required for the EE’s induction of adipose Pten, mice were housed in SE or EE and -blocker propranolol was supplied to the drinking water (Cao et al., 2011). Propranolol completely blocked the EE-induced
Pten induction and other molecular features (Figure 7A). EE leads to a mild but significant increase of serum corticosterone (L. Cao et al., 2010). Therefore we investigated the role of hypothalamus pituitary adrenal (HPA) axis using adrenalectomized mice. Adrenalectomy did not block the gene expression signature of
EE. Pten was still highly upregulated together with Glut 4, Insr, and Ppargc1a (Figure
7B).
Our previous study shows that EE induces adipose vascular endothelial growth factor
(VEGF) and VEGF signaling is required for browning induced by diverse physiological and pharmacological approaches (During et al., 2015). Thus we investigated whether similar phenomenon existed for adipose Pten. Daily injection of 3-agonist CL-316243 and the peroxisome proliferator-activated receptor (PPAR)- ligand rosiglitazone for 9 days led to strong induction of browning (During et al., 2015). Both CL-316243 and rosiglitazone robustly induced the expression of Glut4, Insr, and Ppargc1a but failed to induce Pten expression (Figure 7C). Mice living in a larger space with access to running wheels showed beige cell induction (During et al., 2015). Interestingly, although running greatly upregulated Glut4 and Ppargc1a, exercise significantly reduced Pten expression in rWAT (Figure 7D).
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Figure 3.1. Short-term EE activates the HSA axis in 10-month old mice. (A) Body weight (n=10 per group). (B) Cumulative food intake. (C) Body and tissue weight at sacrifice after 6-week EE (n=10 per group). (D) Serum biomarkers at sacrifice (n=10 per group). (E) Gene expression profile of the hypothalamus after 6-week EE (n=5 per group). * P<0.05, ** P<0.01, + P=0.06. Values are means ± SEM.
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Figure 3.2. Long-term EE initiated at middle age reduces adiposity and improves metabolism. (A) Time line. (B) Body weight (n=10 per group). (C) Cumulative food intake. (D) Glucose tolerance test at 8-week in EE (n=10 per group). (E) Glucose uptake assay in adipose tissues at 12-week in EE in a separate experiment (n=5 per group). (F) Glucose tolerance test at 32-week in EE (n=10 per group). (E) Body and tissue weight at the age of 22 months after 12-month EE (n=8 per group). (F) Serum biomarkers at sacrifice (n=8 per group). * P<0.05, ** P<0.01, *** P<0.001. Values are means ± SEM.
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Continued Figure 3.3. EE improves motor behavior and reduces anxiety. (A, B) Rotarod treadmill test at the age of 19 months after 9-month EE (n=9 per group). Time remaining on rod (A), Speed at fall (B). (C-E) Open field test at the age of 12 months after 2-month EE (n=10 per group). Time spent at the center of arena (C), ratio of distance travelled in center to total distance (D), total distance travelled (E). (F, G) Novelty suppressed feeding test at the age of 20 months after 10-month EE (n=10 SE, n=9 EE). Latency to consumption (F), food consumption in standard cage after the test (G). (H) Cold induced defecation test at the age of 19.5 months after 9.5-month EE (n=10 SE, n=9 EE). (I, J) Forced swim test at
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Figure 3.3 Continued
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Figure 3.4. Gene expression profiles of tissues at the age of 22 months after 12- month EE. (A) Hypothalamus (n=8 per group). (B) BAT (n=6 per group). (C) rWAT (n=6 per group). (D) Liver (n=6 per group). (E) Skeletal muscle (n=6 per group). (F) Mitochondrial DNA content (rWAT: n=8 per group, gWAT: n=7 per group, liver: n=4 per group). * P<0.05, ** P<0.01, *** P<0.001. Values are means ± SEM.
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Figure 3.5. Immunohistochemistry of adipose tissues at the age of 22 months after 12-month EE. (A) BAT. (B) rWAT. (C) gWAT. Scale bar, 100 µm in H&E, 50 µm in UCP1, PTEN, and PGC-1α staining.
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Figure 3.6. Hypothalamic BDNF mediates EE-induced adipose remodeling. (A) Gene expression of rWAT in rAAV-BDNF injected mice overexpressing BDNF in the hypothalamus. (B) Gene expression of eWAT in mice overexpressing BDNF. n=5 per group. (C) Gene expression of eWAT in BDNF+/- mice (n=4 per group). (D) Gene expression of eWAT in rAAV-TrkB.T1 injected mice (n=4 per group). * P<0.05, ** P<0.01, *** P<0.001, + P=0.069. Values are means ± SEM.
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Figure 3.7. Intact sympathetic tone is required for EE-induced adipose remodeling. (A) Gene expression of rWAT in mice treated with oral propranolol and housing in EE for 4 weeks (n=5 per group). (B) Gene expression of rWAT in adrenalectomized mice housing in EE for 5 weeks (n=5 per group). (C) Gene expression of rWAT after chronic injection of CL316,243 (CL) or rosiglitazone (Rosi) for 9 days (n=5 per group). (D) Gene expression of rWAT after 3 weeks of wheel running (n=5 per group). * P<0.05, ** P<0.01, *** P<0.001, + P=0.065. Values are means ± SEM.
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Figure 3.8. Gene expression profiles after short-term EE in middle-age female mice. (A) BAT. (B) rWAT. (C) gWAT. (D) Liver. n=5 per group. * P<0.05, ** P<0.01, + P=0.06. Values are means ± SEM.
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Figure 3.9. Immunoassays after long-term EE initiated in middle-age female mice. (A) The proliferative response of splenic lymphocytes to the T cell mitogen Con A. (B) NK cell cytotoxicity. n=5 per group. Values are means ± SEM.
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Figure 3.10. Gene expression profiles after long-term EE in middle-age female mice. (A) Amygdala, n=8 per group. (B) gWAT, n=6 per group. * P<0.05, ** P<0.01, + P=0.06. Values are means ± SEM.
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Figure 3.11. Gene expression profile of rWAT in young male DIO model after 4- week EE. n=5 per group. * P<0.05, ** P<0.01, *** P<0.001, + P=0.06. Values are means ± SEM.
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Figure 3.12. Gene expression profile of adipocyte and vascular stromal fraction (SVF) after 2-week EE in young male mice on normal diet. n=5 per group. * P<0.05, ** P<0.01. Values are means ± SEM.
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Figure 3.13. Glucose tolerance test after 3-month EE initiated at 18 months of age. n=10 per group. * P<0.05. Values are means ± SEM.
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Figure 3.14. EE enhances adipose glucose uptake without improving insulin sensitivity in 10 month old female mice housed in EE for 90 days. (A) Insulin tolerance testing (B) Glucose uptake assay of gonadal adipose tissue (C) Glucose uptake assay of intrascapular brown adipose tissue. n=10 per group.Values are means ± SEM.
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Discussion
The evolutionary theory of aging states that the mechanisms beneficial to coping with environmental demands and resistance to disease are beneficial on lifespan. Moreover, these mechanisms are conserved across species (Mattson, Maudsley, & Martin, 2004).
There is little doubt that the brain plays a commanding role in these lifespan-determining pathways. However, how these neuronal pathways convey signals to the periphery to improve the healthspan of many different organ systems is poorly understood. We propose that the newly defined HSA axis may provide one mechanistic explanation. The upstream key component BDNF, highly responsive to environmental stimuli, could control the HSA axis activity and thereby regulate the phenotype and function of adipose tissue. Adipose tissue, as the principal responsive organ in the periphery of this regulation network, is able to subsequently influence multiple organ systems to change body composition, metabolism, insulin sensitivity, hormones and growth factors, immune functions, and cancer and ultimately healthspan or lifespan. Our data demonstrated that middle-age female mice were readily stimulated by EE exhibiting a robust activation of the HSA axis. We are currently conducting an experiment subjecting 18 months old mice to EE. The preliminary data showed that aged mice were still highly responsive to EE with a significantly improved GTT observed after 3-month EE (Figure 13). This study and our previous investigation demonstrate that the key EE-induced features independent of gender and age include upregulation of BDNF in the hypothalamus, reduction of adiposity, drop of leptin, improved glycemic control, and remodeling of adipose tissues.
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Several effects of EE found in young male animals were not reproduced in older female mice such as decreased serum IGF-1, increased serum adiponectin, and enhanced NK cell activity. The modulations of IGF-1 and adiponectin by EE are likely more related to gender since both factors were not significantly changed in young female wild type or spontaneous breast cancer mouse model (data not shown, manuscript under review). The immune analyses of this study were limited to splenic lymphocyte proliferation and NK cell cytotoxicity. Our recent studies using young animals have revealed new effects of EE on immune functions including regulation of thymus and T cell development and modulation of immune cells residing in adipose tissues (manuscripts in preparation). It is particularly intriguing to investigate how EE regulates different types of immune cells residing in the adipose tissues in aged animals since these cells could play different, even possibly opposite, roles in metabolic adaption along aging process (Bapat et al., 2015;
Kolodin et al., 2015).
The activation of the HSA axis is a potent model to decrease fat mass with little or no impact on body weight. EE provides a physiological model to clarify controversies in aging research, e.g. whether weight loss is beneficial to lifespan and whether fat loss with no loss of lean mass is required (Allison et al., 1999). Although maximum lifespan was not assessed in this study, EE initiated at middle age promoted healthy aging associated with some metabolic adaptations overlapping with CR. There are differences. CR requires sustained reduction of food consumption that is difficult to achieve outside of laboratory. EE led to leanness and resistance to DIO with no suppression of food consumption and instead via increasing energy expenditure. In contrast, CR is associated
88 with reduced metabolic rate. Physical exercise, a model with decreased adiposity but increased energy expenditure, has been shown to be beneficial on healthy aging but unable to extend maximum lifespan (Huffman, 2010). EE provides opportunity of physical exercise. However our previous studies have demonstrated that voluntary running is not sufficient to activate the HSA axis and EE has stronger anticancer and anti- obesity effects than running alone even with overall lower physical activity (Cao et al.,
2011; L. Cao et al., 2010). Thus EE is a new model to study the relationship between energy expenditure and aging.
The majority of studies on EE and aging investigate behaviors and neurological diseases.
Our study is the first assessing EE-induced metabolic adaptations in aging. Nevertheless we examined a battery of anxiety and depression behavior tests and showed that EE initiated at middle age significantly reduced anxiety. The mechanisms underlying this anxiolytic effect could be multifactorial and require further investigations. EE can modulate limbic systems that are involved in anxiety. Hypothalamus, an area integrating metabolism, stress, and immune functions, is highly responsive to EE and could also contribute to the anxiolytic effect. In young animals, EE upregulated BDNF expression in arcuate nucleus as well as ventromedial (VMH) and dorsomedial (DMH) hypothalamus
(L. Cao et al., 2010). DMH is a brain area not only involved in physiological functions such as metabolism and environmental threats, but also critically involved in behavioral regulation, particularly fear, anxiety and panic-like disorders (Canteras, 2002; Shekhar,
Sims, & Bowsher, 1993; Silva et al., 2014). Obesity has been linked to neuropsychiatric and anxiety disorders including generalized anxiety disorder, panic disorder, post-
89 traumatic stress disorder, emotional reactivity and cognitive dysfunctions (de Noronha et al., 2016; Gariepy, Nitka, & Schmitz, 2010). Our data demonstrate that EE induces an anti-obesity and anxiolytic phenotype. DMH could be a target to study whether the anti- obesity and anxiolytic effects are linked. It is also possible that global improvement of metabolism associated with the HSA axis activation indirectly influences brain functions and behaviors including anxiety. Genetically activating the HSA axis via AAV-mediated hypothalamic BDNF overexpression reproduces EE’s metabolic effects (Cao et al.,
2011). We are investigating whether hypothalamic BDNF overexpression could also mimic EE’s effects on behaviors.
One new finding of this study is the induction of PTEN in adipose tissue by EE. Ptentg mice globally overexpressing Pten have extended lifespan associated with decreased adiposity, increased energy expenditure, induction of beige cells, and improved GTT
(Ortega-Molina et al., 2012). In contrast to the artificial genetic model, EE is a physiological paradigm, a life style manipulation that was shown to induce Pten preferentially in the adipose tissue independent of gender or age. Our mechanistic studies demonstrated that the HSA axis mediated the EE’s Pten induction. Hypothalamic overexpression of BDNF reproduced adipose Pten induction. Conversely, both global
BDNF deficiency in BDNF+/- mice and the inhibition of BDNF signaling specifically in the hypothalamus via dominant negative TrkB, suppressed adipose Pten expression.
Furthermore, blocker prevented EE’s Pten induction while adrenalectomy had no effect, suggesting intact SNS but not the HPA axis is required for EE’s modulation of adipose Pten. Intriguingly, chronic use of -3 agonist CL-316243 failed to induce adipose
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Pten suggesting that the -adrenergic receptor signaling is necessary but not sufficient to induce adipose Pten. Future studies are needed to identify additional factors mediating the EE’s Pten induction. Although EE induces beige cells in rWAT in young mice and
Ptentg mice show WAT browning, the EE-induced Pten is unlikely essential for browning because Pten was upregulated in visceral fat where no browning was observed. And several pharmacological and physiological approaches (-agonist, PPAR ligand, running) robustly induced browning but failed to induce Pten. The functions of adipose
Pten remain controversial (C. Kurlawalla-Martinez et al., 2005; Morley, Xia, & Scherer,
2015). Adipose-specific Pten knockout induced in adult mice results in enhancement of insulin sensitivity and systemic metabolic improvement (Morley et al., 2015). EE in adult and aged mice leads to an opposite phenotype characterized as upregulation of adipose
Pten, increased adipose glucose uptake, and improved glucose tolerance, which is consistent to Ptentg mice. The inducible adipose-specific Pten knockout mouse (Morley et al., 2015) is a valuable tool to investigate the role of adipose Pten in EE-induced metabolic adaptations. Moreover, most studies on PTEN use genetic models (mutations, knockout, knock-in etc.) because physiological regulations of PTEN remain unknown.
EE can serve as a physiological approach to study PTEN in metabolism, cancer, and aging (Ortega-Molina & Serrano, 2013).
In summary, EE activated the HSA axis in aged mice leading to metabolic adaptations including reduced adiposity, improved glycemic control, increased mitochondrial biogenesis/function, decreased leptin, and adipose remodeling (including upregulation of
Pten). EE also enhanced motor abilities and reduced anxiety. These data suggest that a
91 physically, mentally, and socially active environment can promote healthy aging and a specific brain-fat axis may be one of the underlying mechanisms. Further characterization of the HSA axis and identification additional mechanisms may reveal potential targets for the prevention and treatment of age-related diseases.
Experimental Procedures
EE protocol with normal chow diet. We housed female 10-month old C57Bl/6 mice
(from National Institute on Aging, Aged Rodent Colonies) in large cages (63 cm x 49 cm x 44 cm, 5 mice per cage) supplemented with running wheels, tunnels, igloos, huts, retreats, wood toys, a maze, and nesting material in addition to standard lab chow and water. We housed control mice under standard laboratory conditions (5 mice per cage).
All use of animals was approved by, and in accordance with the Ohio State University
Animal Care and Use Committee. Mice were housed in temperature (22-23 ºC) and humidity controlled rooms with food and water ad libitum. We fed the mice with normal chow diet (NCD, 11% fat, caloric density 3.4kcal/g, Teklad). In the short term EE study, mice were sacrificed 6-week after EE housing. In the long term EE study, body weight was monitored weekly until the end of the study at the age of 22 month. Food intake was monitored for 10 weeks as the total food consumption of each cage and represented as the average of food consumption per mouse per day. Rectal temperature was measured 39- week in EE.
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Glucose tolerance test. Glucose tolerance test was performed 32 weeks after the initiation of EE. Mice were injected intraperitoneally with glucose solution (2mg glucose per kg body weight) after an overnight fast. Blood was obtained from the tail at 15, 30,
60, 90, and 120 min after glucose injection. Blood glucose concentrations were measured with a portable glucose meter (Bayer Contour Next).
Insulin tolerance test. Insulin tolerance test was performed 13 weeks in EE. Mice were injected intraperitoneally with insulin (0.75 unit per kg body weight) at 2 PM without a fast. Blood was obtained from the tail and blood glucose level was measured as described above. rAAV vector construction and packaging. The rAAV plasmid contains a vector expression cassette consisting of the CMV enhancer and chicken β-actin (CBA) promoter, woodchuck post-transcriptional regulatory element (WPRE) and bovine growth hormone poly-A flanked by AAV2 inverted terminal repeats. Transgenes: Human
TrkB isoform 1 (TrkB.T1) cDNA, human BDNF, destabilized YFP were inserted into the multiple cloning sites between the CBA promoter and WPRE sequence. rAAV serotype 1 vectors for TrkB.T1, YFP, or BDNF were packaged and purified as described elsewhere
(Cao et al., 2004).
AAV1 mediated BDNF overexpression in hypothalamus. Male C57Bl/6 mice, 6 weeks of age, were purchased from Charles River and randomly assigned to receive AAV1-
BDNF or AAV1-YFP. Mice were anaesthetized with a single dose of ketamine/xylazine
(100mg/kg and 20mg/kg; i.p.) and secured via ear bars and incisor bar on a Kopf stereotaxic frame. A mid-line incision was made through the scalp to reveal the skull and
93 two small holes were drilled into the skull with a dental drill above the injection sites (-
0.8AP, ±0.3ML, -5.0DV; mm from bregma). rAAV vectors (1x109 genomic particles per site) were injected bilaterally into the hypothalamus the at a rate of 0.1L/min using a
10L Hamilton syringe attached to Micro4 Micro Syringe Pump Controller (World
Precision Instruments Inc., Sarasota, USA). At the end of infusion, the syringe was slowly removed from the brain and the scalp was sutured. Animals were placed back into a clean cage and carefully monitored post-surgery until fully recovered from anesthesia.
We monitored body weight every 5-7 days and recorded the food intake. Mice were maintained on NCD in SE until the end of the study (4 weeks after surgery).
AAV mediated overexpression of dominant negative TrkB.T1. We randomly assigned
8-week old male C57Bl/6 mice to receive AAV-TrkB.T1 or AAV-YFP. We injected
AAV vectors bilaterally to the hypothalamus (-1.2AP, ±0.5ML, -6.2DV; mm from bregma, 5.0x109 genomic particles per site). We monitored body weight every 5-7 days and recorded the food intake. We kept the mice on NCD till the end of the study 4 weeks after surgery.
Quantitative RT-PCR. We dissected brown and white adipose tissues, amygdala, and hypothalamus and isolated total RNA using RNeasy Lipid Kit plus RNase-free DNase treatment (Qiagen). We generated first-strand cDNA using TaqMan Reverse
Transcription Reagent (Applied Biosystems) and carried out quantitative PCR using
StepOnePlus Real-Time PCR System (Applied Biosystems) with the Power SYBR Green
PCR Master Mix (Applied Biosystems). Primer sequences are available on request. We
94 calibrated data to endogenous control Actb or Hprt1 and quantified the relative gene expression using the 2 -CT method (Livak & Schmittgen, 2001).
Mitochondrial DNA measurement. Total DNA was isolated using the AllPrep
DNA/RNA/Protein Mini Kit (Qiagen). Mitochondrial mass was determined by measuring mitochondrial DNA-encoded cytochrome c oxidase subunit I (Cox I) by qPCR. Cox I levels were normalized to Bdnf encoded by nuclear DNA.
Serum harvest and biomarkers measurement. Trunk blood was collected at euthanasia. We prepared serum by allowing the blood to clot for 30 min on ice followed by centrifugation. Serum was at least diluted 1:5 in serum assay diluent and assayed using the following DuoSet ELISA Development System (R&D Systems): mouse IGF-1,
Adiponectin, and Leptin. Glucose and triglycerides were measured using Cayman
Chemical colorimetric assay kits. Total cholesterol, and non-esterified fatty acid levels were measured using Wako instruments kits.
BDNF heterozygous mice. We used male BDNF +/- mice provided by Dr. F. Lee of
Weill Medical College of Cornell University to breed a BDNF +/- colony. We dissected brown and white fat pads of male BDNF heterozygous mice (n=4) and age matched
C57BL/6 wild type littermates (n=4) at the age of 2 months.
Immunohistochemistry. We cut paraffin-embeded sections (4 μm) of adipose tissues and subjected the sections to citrate-based antigen retrieval following by incubations with antibodies against UCP1 (Abcam ab10983, 1:1000), PGC-1α (Abcam ab54481, 1:250), or PTEN (Cell Signaling 138G6, 1:200). The sections were visualized with DAB and counterstained with hematoxylin.
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Propranolol experiment. We randomly assigned male C57BL/6 mice (from Charles
River), 3 weeks of age, to live in EE or SE supplied with propranolol in drinking water
(0.5 g/L). Fat pads were dissected after 5 weeks EE.
Adrenalectomy. Male adrenalectomized mice, 4 weeks of age, were purchased from
Charles River and randomly assigned to live in EE or SE supplied with 0.9% sodium chloride in drinking water. Fat pads were collected and analyzed after 5-week EE.
EE protocol with HFD. We randomly assigned 20 C57Bl/6 mice to live in EE or SE for
4 weeks. We switched the diet from NCD to high fat diet (HFD, 45% fat, caloric density
4.73kcal/g, Research Diets, Inc.) when EE was initiated.
Isolation of adipocytes and stromal vascular fractions (SVF). Based on previously described methods (Horton, Shimomura, Ikemoto, Bashmakov, & Hammer, 2003;
Sackmann-Sala, Berryman, Munn, Lubbers, & Kopchick, 2012), adipose tissues were dissected and transferred to 12 well culture plate containing Krebs-Ringer HEPES buffer
(5mM D-glucose, 2% BSA, 135 mM NaCl, 2.2 mM CaCl2, 1.25mM MgSO4, 0.45mM
KH2PO4, 2.17 mM Na2HPO4, and 10mM HEPES (pH 7.4)), then minced to a fine consistency. Collagenase II (Sigma) at 1.2 mg/ml was added and the fat pads mixture was incubated at 370C with shaking at 90 RPM for 45 min. The mixture was spun at 800 rpm for 5 min after passed through strainer (100µm mesh size). The mature adipocytes floating at the top and SVF pellets at the bottom were collected separately, and washed with above-mentioned buffer twice. SVFs were treated with red blood cell lysis solution
(Sigma) for 5 min at room temperature and washed one more time with the buffer. The
96 adipocytes and SVFs were immediately subjected to RNA isolation with RNeasy Micro kit (QIAGEN).
CL-316,243 and rosiglitazone treatment. We randomly assigned male C57BL/6 mice
(from Charles River) to receive vehicle, CL316,243 (Sigma, 1 mg/kg, i.p., daily), rosiglitazone (10 mg/kg, i.p., daily). Fat pads were dissected and analyzed after 9 days injection of CL316,243 or rosiglitazone.
Voluntary running. We housed male 3-week-old C57BL/6 mice in rat cages with a small plastic running wheel, 3 mice per cage. Fat pads were dissected and analyzed after
3 weeks voluntary running. Sedentary control mice were housed in cages without running wheel.
Splenocyte proliferation assay. We harvested splenocytes from mice of the 12-month
EE at sacrifice. Single-cell splenocyte suspensions were prepared by teasing spleens and passing through 40 m Cell Strainer. Erythrocytes were depleted with Red Blood Cell
Lysis Buffer (Sigma). Splenocytes were washed 3 times and the viability was assessed by
Trypan blue exclusion (usually >90%). Splenocytes were seeded in 96-well plates in complete medium (RPMI1640, 25mM HEPES, 2mM L-glutamine, 50M - mercaptoethanol, 2g/L sodium bicarbonate, 5% FBS). Quardruplicate of cells from each mouse spleen were stimulated with 0 g/ml of mitogen or 5 g/ml Concanavalin-A
(Sigma) and cultured for 48-72 hrs. Cell proliferation was determined using the CellTiter
96Aquesous One Solution Cell Proliferation Assay (Promega). Data were expressed as
Stimulation Index = mean OD of wells with Concanavalin-A stimulation/mean OD of the wells without stimulation.
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Cell-mediated cytotoxicity assay. We assayed immune cell cytotoxicity using the
CytoTox96 Assay (Promega) according to the manufacturer’s instruction. For NK cell activity, splenocytes were prepared from mice that undergone 12-month EE. Splenocytes were incubated with B16 melanoma cells at various effector: target ratios, the effector
NK cells lysed the target cells and LDH release was measured. Each reaction was performed in quadruplicate. The data were calculated using the following formula: % cytotoxicity = (Experimental release- Effector spontaneous release-Target spontaneous release)/(Target maximum release-Target spontaneous release) x 100.
In vivo glucose uptake
The in vivo glucose update during a glucose tolerance test was measured using glucose analog tracer 2-[3H] deoxyglucose (2-DG) in mice 2 weeks post initiation of EE following published method (Boini et al., 2009; Mauvais-Jarvis et al., 2000). Briefly 2-
DG (PerkinElmer, MA) was mixed with regular D-glucose (10µCi/mouse) and injected intraperitoneally. 10-15 µl of blood was collected for glucose specific activity calculation at 0, 15, 30, 60, 90 and 120 min meanwhile blood glucose level was measured with glucometer at each time point of GTT. Then mice were euthanized and liver, adipose tissue and muscle were collected and snap-frozen for further analysis. Adipose tissues
(300 mg per mouse) were homogenized in deironed water, and the proteins were precipitated with 7% ice-cold perchloric acid, and then neutralized by 2.2 M potassium bicarbonate. The aliquot of supernatant was passed through an anion exchange column
(Ag-1 x8, Bio-Rad) to trap 2-DG-P. The column was eluted, 2-DG-P was collected, and counted for [3H] –radioactivity (LS-6500, Beckman Counter). For blood sample, 2 µl of
98 serum was deproteinized in 3.5% ice-cold perchloride acid (200µl), then neutralized with
2.2 M potassium bicarbonate. The supernatant was counted for [3H] –radioactivity.
Glucose specific activity (GSC) is calculated by dividing sample radioactivity by glucose concentration. Adipose glucose uptake is calculated by dividing [3H]-radioactivity in 2-
DG-6-P by mean specific activity of glucose during GTT (120 min) and presented as mmol per mg protein per minute.
Open field test (OF). To assess exploration and general motor activity, mice were placed individually intot the center of an open square arena (60 cm x 60 cm, enclosed by walls of 48 cm). Each mouse was allowed 10 min in the arena, during which time its activity was recorded and analyzed by TopScan (Clever Sys Inc). Specifically the parameters measured include distances traveled in the periphery and in the center of the arena (36 cm x 36 cm), the total distance traveled, and the time spent in the center of the arena. The total distance traveled provides a measure of exploratory activity while the time and distance ration of arena center exploration provide an indication of anxiety. The arena was cleaned with 30% ethanol between trials to remove any odor cues.
Novelty suppressed feeding test (NSF). Mice were fasted overnight with food removed at 1700h. The testing phase was conduced the next morning at 1000h. Mouse was individually placed into a brightly lit novel open cage (40 cm x 28 cm x 20 cm). A piece of white filter paper (7 cm diameter) was placed in the center of the cage with a single pre-weighed food pellet. The latency to consumption (first bite of the food pellet) was recorded. The cut-off time was 10 min. To assess if there was any difference in consumptive drive, each mouse was placed in a standard cage with the pre-weighed food
99 pellet after its first bite or at cut-off time if it failed to each within 10 min. The amount of food consumed in 5 min was measured.
Forced swim test (FST). Mice were placed individually in a transparent cylinder (21 cm diameter, 24 cm height) containing water (25 ± 2 ºC) to a depth of 15 cm for 6 min. At the end of each trial, mice were dried and returned to their home cage. Trials were video- recorded and a blinded experimenter scored the amount of time mice remained immobile as a measure of depressive-like behavior.
Cold-Induced defecation (CID). A large container was filled halfway with ice. A novel cage smaller than the standard ventilated cage was placed on top of the ice. Mouse was placed into the smaller cage and a lid was placed on top. After 20 min, the mouse was removed and the number of fecal matter was counted. Mice were allowed to recover in a cage partially on a heating pad for 1 h prior to returning to its home cage. All cages used were cleaned with Spor-Klenz before using with a new animal.
Rotarod treadmill test. Mice were placed on a rod 3 cm in diameter and 6 cm long and elevated 30 cm from the base of the apparatus. The rod was rotating at 4 rotations per minute (RPM) when a mouse was placed on a rod. Rotation acceleration was set to 20
RPM and after 10 s of the mouse being on the rod the acceleration started. The time on the rod and the speed at which the mouse fell off were recorded and the mouse would be removed from the apparatus at a cut off time of 5 min. Each mouse was subjected to 2 trials. The apparatus was cleaned with 70% ethanol after each test.
Statistical analysis. Data are expressed as means.e.m. We used JMP software to analyze the following: two-way analysis of variance for body weight, food intake, insulin
100 tolerance, and glucose tolerance; Student’s t test for adiposity, body temperature, behavior, and quantitative RT-PCR data.
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Chapter 4: The anti-tumor activity of a neutralizing nanobody targeting leptin
receptor in a mouse model of melanoma
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The anti-tumor activity of a neutralizing nanobody targeting leptin receptor in a mouse model of melanoma
Travis McMurphy1,2, Run Xiao 1,2, Daniel Magee 1,2, Andrew Slater1,2, Lennart Zabeau3,
Jan Tavernier3, and Lei Cao 1,2 *
1. Department of Molecular Virology, Immunology, and Medical Genetics
2. The Comprehensive Cancer Center
The Ohio State University, Columbus, USA
3. Flanders Institute for Biotechnology, Department of Medical Protein Research,
Faculty of Medicine and Health Sciences,
Ghent University, Ghent, Belgium
*Correspondence: [email protected], Phone: 614-2472792, Fax: 614-2926356
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Abstract
Environmental and genetic activation of a brain-adipocyte axis inhibits cancer progression. Leptin is the primary peripheral mediator of this anticancer effect in a mouse model of melanoma. In this study we assessed the effect of a leptin receptor antagonist on melanoma progression. Local administration of a neutralizing nanobody targeting the leptin receptor at low dose adjacent to tumor decreased tumor mass with no effects on body weight or food intake. In contrast, systemic administration of the nanobody failed to suppress tumor growth. Daily intraperitoneal injection of high-dose nanobody led to weight gain, hyperphagia, increased adiposity, hyperleptinemia, and hyperinsulinemia, and central effects mimicking leptin deficiency. The blockade of central actions of leptin by systemic delivery of nanobody may compromise its anticancer effect underscoring the need to develop peripherally acting leptin antagonists coupled with efficient cancer- targeting delivery.
Introduction
We recently report that living in an enriched housing environment that provides physical, social, and cognitive stimuli reduces tumor growth and increases remission in mouse models of melanoma and colon cancer (L. Cao et al., 2010). Our mechanistic studies have elucidated one key mechanism underlying the anti-cancer effect of environmental enrichment (EE): the activation of a previously poorly understood neuroendocrine hypothalamic-sympathoneural-adipocyte axis (HSA). The complex environmental stimuli induce the expression of brain-derived neurotrophic factor (BDNF)
104 in the hypothalamus and the ensuing increase in sympathetic tone to white adipose tissue.
The preferential sympathetic activation of white adipose tissue suppresses leptin expression and release via action on -adrenergic receptors leading to a robust drop of leptin level in circulation. Our pharmacological and genetic studies demonstrate that leptin is the key peripheral effector in the HSA axis mediating the anti-cancer effect of
EE (L. Cao et al., 2010). We have developed a molecular therapy to treat both obesity and cancer by neurosurgical delivering a recombinant adeno-associated virus (rAAV) vector in order to overexpress BDNF in the hypothalamus. This gene therapy reproduces the anti-obesity and anti-cancer effects of EE (Cao et al., 2009; L. Cao et al., 2010). In this study we investigated the effect of pharmacological blockade of leptin in the same mouse model of melanoma.
Leptin (encoded by Ob gene) is a pleotropic hormone primarily produced in adipose tissue. Leptin plays a crucial role in energy homeostasis by acting in the central nervous system (CNS) to increase energy expenditure and decrease feeding via a host of autonomic and neuroendocrine processes (Coppari & Bjorbaek, 2012; Y. Zhang et al.,
1994). In addition to its central effects in the CNS, leptin exhibits a large number of peripheral actions including modulation of immune system (Batra et al., 2010; Lam,
Wang, Ko, Kincade, & Lu, 2010), regulation of liver and muscle lipid oxidation and glucose metabolism (Bates, Kulkarni, Seifert, & Myers, 2005; Huo et al., 2009;
Minokoshi et al., 2002), and regulation of pancreatic -cell function (Covey et al., 2006;
Gray, Donald, Jetha, Covey, & Kieffer, 2010; Levi et al., 2011; Morioka et al., 2007).
Leptin mediates its effects upon binding and activation of the leptin receptor (LepR)
105 encoded by the Db gene (Tartaglia et al., 1995). Six LepR isoforms have been characterized: a long form (LepRb or LepRlo), four short forms (LepRa, c, d, and f), and a soluble form (LepRe or sLepR) (Ge, Huang, Pourbahrami, & Li, 2002). The long form
LepRb is considered to possess full signaling capacity (Myers, Cowley, & Munzberg,
2008). All isoforms have an identical extracellular domain consisting of two CRH
(cytokine receptor homology) domains, CRH1 and CRH2, both separated by an immunoglobulin-like domain, and followed by two additional membrane-proximal fibronectin type III domains. To investigate the potential of leptin antagonists in cancer treatment, choosing a neutralizing antibody targeting the LepR instead of leptin could restrict leptin blockade to the periphery because the antibody most likely does not cross the blood-brain barrier (BBB). Zabeau et al generated neutralizing nanobodies targeting
LepR (Zabeau et al., 2012). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family (Hamers-
Casterman et al., 1993). The cloned variable domain is a stable polypeptide harboring the full antigen-binding capacity of the original heavy-chain antibody (Coppieters et al.,
2006; van der Linden et al., 2000). The advantages of nanobodies compared to classical antibodies include improved tissue penetration, stability, easier genetic manipulation and production in bacteria. Nanobody 2.17 directly against the CRH2 domain of LepR blocks leptin binding to the receptor. To improve in vivo use, the nanobody targeting LepR was converted into a bi-specific format by fusing it to a nanobody that targets mouse serum albumin (mAlb). Binding to endogenous serum albumin greatly prolonged half-life of the
106 bi-specific nanobody in the circulation (Zabeau et al., 2012). Here we assessed the effects of the bi-specific nanobody 2.17-mAlb in the highly aggressive B16 melanoma model.
Materials and Methods
Mice
Male C57BL/6J mice, 6 weeks of age, were purchased from Charles River. All protocols were approved by the Institutional Animal Ethics Committees of the Ohio State
University and were in accordance with NIH guidelines.
Bispecific nanobody
The construction, production, and purification of bi-specific nanobody 2.17-mAlb were described in detail before (Zabeau et al., 2012).
Melanoma implantation and nanobody treatment
We single housed mice for melanoma implantation and treatment of 2.17-mAlb. In local administration experiment, mice were shaved at the right flank. A syngeneic melanoma cell line B16 (ATCC) was subcutaneously implanted on the right flank (1x 105 cells per mouse). 2.17-mAlb (10 g per mouse per injection), or PBS as a control, was injected subcutaneously adjacent to the tumor cell implantation site at day 1, 7, and 14 after tumor cell implantation. We measured the size of tumor using a caliber and calculated the tumor volume by the formula for ellipsoid (V=length x width2 x /6). Mice were sacrificed 18 days after tumor implantation. In systemic administration experiment, B16 cells were implanted to the right flank of mice as described above. The mice were randomized to three groups: PBS, low-dose 2.17-mAlb, and high-dose 2.17-mAlb. 2.17-mAlb or PBS
107 was injected intraperitoneally immediately following tumor cell implantation (100 g per mouse per injection). Low-dose 2.17-mAlb mice received 2.17-mAlb twice weekly.
High-dose 2.17-mAlb mice received daily injection. Mice were sacrificed 16 days after tumor cell implantation. We dissected out the tumors from neighboring tissues and measured the weight at the time of sacrifice. In the established tumor model experiment,
B16 cells were implanted to the right flank of mice as described above. On day 5 after tumor cell implantation when tumors became palpable, the mice were randomized to four groups: PBS, three doses of 2.17-mAlb treatment: 10 g, 50 g, and 100 g per mouse per injection. The mice received PBS or 2.17-mAlb injections subcutaneously adjacent to the tumor implantation site on day 5, day 8, day 12 and day 15. Mice were sacrificed day
18 after tumor cell implantation.
Body weight and food consumption
We maintained the mice on a normal 12 h /12 h light/dark cycle with food and water ad libitum throughout the experiment. Body weight of individual mouse was recorded twice weekly. Food consumption was recorded twice weekly as the total food consumption and represented as the average of food consumption per mouse per day.
Serum harvest and biomarkers measurement
Blood was collected following decapitation. We prepared serum by allowing the blood to clot for 30 min on ice followed by centrifugation. Serum was at least diluted 1:5 in serum assay diluent and assayed using DuoSet ELISA Development System (R&D Systems) for mouse leptin, adiponectin, IGF-1, and soluble leptinR. Insulin was measured using
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Mercodia ultrasensitive mouse insulin ELISA (ALPCO Diagnostic). Glucose was measured using QuantiChrom Glucose Assay (BioAssay Systems).
Hypothalamic dissection
Brains were quickly isolated on ice. The hypothalamus was dissected from 2 mm-thick- coronal sections (-0.7~-2.7 mm from bregma, 1.5 mm dorsal to the bottom of the brain, 1 mm bilateral to the midline) under a dissection scope and stored at -80 ºC for further analysis.
Quantitative RT-PCR
We dissected epididymal adipose tissues and isolated total RNA using RNeasy Lipid Kit plus RNase-free DNase treatment (Qiagen). Tumor RNA and hypothalamic RNA were isolated using RNeasy mini kit plus RNase-free DNase treatment. We generated first- strand cDNA using TaqMan Reverse Transcription Reagent (Applied Biosystems) and carried out quantitative PCR using Light Cycler (Roche) with the Power SYBR Green
PCR Master Mix (Applied Biosystems). We designed primers to detect the following mouse mRNAs: Agrp, Cartp, Npy, Mc4r, Pomc, Insr, Leprb, Lep, Adipoq, Ap2, Fasn,
Cpt1a, Cd31, Vegf, Kdr, Mitf, Tyrp2, and Magea4. Primer sequences are available on request. We calibrated data to endogenous control Actb or Hprt1 and quantified the
(CT,R-CT,T) relative gene expression using the equation T0/R0=K x 2 . T0 is the initial number of target gene mRNA copies, R0 is the initial number of internal control gene mRNA copies, CT,T is the threshold cycle of the target gene, CT,R is the threshold cycle of the internal control gene and K is a constant.
Cell proliferation.
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We cultured B16 melanoma cells (5000 cells/well in 96-well plate) with DMEM medium plus 1% mouse serum with or without 2.17-mAlb (50 g/ml) for 3 days. Proliferation was measured using the CellTiter 96Aquesous One Solution Cell Proliferation Assay
(Promega).
Western blot
The dissected tumors were lysed in 100 l RIPA buffer containing 1% proteinase inhibitor (Calbiochem 539134) by sonication. Rabbit Anti-CD31 (Abcam ab28367,
1:300), rabbit Anti-VEGF (Abcam ab46154, 1:1000), mouse Anti-GAPDH (Calbiochem
CB1001, 1:1000) were used in western blot analysis.
Statistical analysis
Values are expressed as mean SD. We used JMP software to analyze the following: repeated measures MANOVA for food intake, weight gain, and tumor volume; one-way
ANOVA for serum biomarker measurements, tumor weight and adipose tissue weight, quantitative RT-PCR data, western blot quantification.
Results
Local administration of a nanobody targeting LepR
We firstly assessed the effect of nanobody 2.17-mAlb on melanoma progression when injected adjacent to the tumor implantation site. B16 melanoma cells were injected subcutaneously to the flank of male C57BL/6J mice. One day after tumor cell implantation, a low-dose of nanobody 2.17-mAlb (10 g/mouse) or PBS was injected 110 subcutaneously adjacent to the tumor cell implantation site. The nanobody or PBS control was injected at day 7 and day 14 at the same dose and the experiment was terminated at day 18 after tumor cell implantation. The nanobody 2.17-mAlb treatment did not affect weight gain (Fig 1A) or food intake (Fig 1B) indicating the absence of central effects. We observed a signature biomarker change in the serum associated with EE-induced inhibition of melanoma including decreased leptin, increased adiponectin, and decreased
IGF-1 (L. Cao et al., 2010). The subcutaneous administration of low-dose 2.17-mAlb had no significant effects on circulating leptin, adiponectin, or IGF-1 (Fig 1C). Leptin inhibits insulin expression and secretion and affects -cell mass (Marroqui et al., 2012). The low- dose 2.17-mAlb had no significant effect on serum insulin while decreased blood glucose levels were observed (Fig 1C). Interestingly, 2.17-mAlb significantly increased sLepR level in the circulation (Fig 1C). Local administration of low-dose 2.17-mAlb (30
g/mouse the whole course) significantly slowed the melanoma growth (Fig 2A) and decreased melanoma mass by 33.17.9% (Fig 2B). Quantitative RT-PCR was used to measure relative expression levels of transcription factors and antigens which have been associated with melanocyte differentiation and progression including microphthalmia- associated transcription factor (Mitf), silver gp100, tyrosinase, tyrosinase related protein
1, and 2 (Tyrp), as well as melanoma antigen family A2 and A4 (Mage). MITF, the transcription factor regulating the development and differentiation of melanocytes (Vance
& Goding, 2004) was significantly elevated in 2.17-mAlb treated mice, as was TYRP-2
(Fig 3A). MITF leads to differentiation, pigmentation and cell-cycle arrest in melanocytes. Progression of melanoma is associated with decreased differentiation and
111 lower expression of MITF although its function may not be the same in melanoma as in normal melanocytes (Miller & Mihm, 2006). The increase in MITF and the genes in its pathway found in 2.17-mAlb treated animals may indicate more differentiated and less progressive tumor. Similar molecular changes were found in EE-induced inhibition of melanoma progression including increased Mitf, Maega4 and Tyrp2 (Data not shown).
Leptin plays a role in modulating angiogenesis. 2.17-mAlb decreased the expression of vascular marker CD31 and the key VEGF receptor KDR that is critical to tumor angiogenesis (Fig 3A) suggesting that the nanobody suppressed angiogenesis. Western blot showed that the VEGF protein level was significantly reduced by 60.312.7%
(P=0.042) (Fig 3B). In an in vitro experiment, the expression of LepR in B16 melanoma cells was confirmed by RT-PCR. In a cell proliferation experiment, B16 melanoma cells were cultured with mouse serum. 2.17-mAlb substantially attenuated the effect of mouse serum on tumor cell proliferation (Fig 2C). These results showed that the nanobody targeting LepR efficiently inhibited melanoma proliferation in vitro and tumor progression in vivo possibly via direct effect on cancer cell proliferation and indirect effects on tumor angiogenesis.
Systemic administration of nanobody targeting LepR
We next evaluated the effects of nanobody when administrated systemically. The B16 melanoma cells were implanted to the flank of mice and the 2.17-mAlb was injected intraperitoneally (i.p. 100 g/mouse) immediately following the tumor cell implantation.
In the low-dose group, nanobody was injected twice weekly (5 injections in total). In the high-dose group, nanobody was injected daily till the end of the experiment at day 16.
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Intraperitoneal administration of nanobody showed dose-dependent effects on weight gain and food intake. High-dose nanobody led to accelerated weight gain (Fig 4A) and hyperphagia (Fig 4B) while low-dose nanobody showed no significant changes. In contrast to local administration, intraperitoneal administration of nanobody failed to inhibit melanoma growth (Fig 4D). High-dose nanobody markedly increased the adiposity with visceral fat pad increased by 51.36.6% (Fig 5A). Consistent with the increased fat mass, serum leptin level was increased in the high-dose group while adiponectin and IGF-1 were not affected (Fig 4C). Insulin level was significantly increased in the high-dose group (Fig 4C). The hyperleptinemia and hyperinsulinemia could compromise the anti-cancer effect of 2.17-mAlb. The sLepR level was substantially increased in both low-dose and high-dose 2.17-mAlb treated mice (Fig 4C).
The increase of sLepR was dose-dependent with high-dose i.p. 2.17-mAlb showing the largest increase while low-dose 2.17-mAlb injected locally showing the smallest change
(Fig 4C, Fig 1C). We examined the gene expression of visceral fat by quantitative RT-
PCR. High-dose 2.17-mAlb increased leptin expression in the adipose tissue (Fig 5B).
Ap2, an adipocyte differentiation marker was also increased consistent with the expansion of fat depot (Kajimura et al., 2008). Leprb, the long-form leptin receptor, showed a trend of increase (Fig 5B) probably indicating an adaptive response to the antagonism to LepR. The accelerated weight gain and hyperphagia suggested that high- dose intraperitoneal administration of 2.17-mAlb antagonized central actions of leptin.
Leptin acts on two populations of neurons in the arcuate nucleus of hypothalamus, with one population expressing Pro-opiomelanocortin (POMC), the other co-expressing
113 neuropeptide Y (NPY) and agouti-related peptide (AgRP) (Elmquist, Coppari, Balthasar,
Ichinose, & Lowell, 2005; Morton, Cummings, Baskin, Barsh, & Schwartz, 2006). We profiled gene expression in the hypothalamus by quantitative RT-PCR (Fig 6). The orexigenic neuropeptides NPY and AgRP were significantly induced consistent with the increase in food intake. The anorexigenic POMC and CART prepropeptide (Cartpt) as well as the melanocortin 4 receptor (MC4R), a key pathway regulating energy balance
(Flier, 2004), were not affected (Fig 6).
Nanobody targeting LepR in established tumor model
We next tested the efficacy of nanobody targeting LepR in the established melanoma model. The B16 cells were implanted to the flank of the mice. Local subcutaneous nanobody treatment was delayed to day 5 after tumor cells implantation when tumors became palpable. Three dose levels (10 g, 50 g, and 100 g per mouse per injection) were used. Low dose nanobody (10 g per injection, 40 g the whole course) had no effects on weight gain (Fig 7A), food intake (Fig 7B), or adiposity (Fig 7C). Low dose nanobody significantly decreased tumor mass even with shorter window of treatment (Fig
7D). In contrast, subcutaneous injection of high dose nanobody failed to inhibit tumor growth (Fig 7D). High dose nanobody treatment (s.c. 100 g per injection, 400 g the whole course) led to accelerated weight gain (Fig 7A), increased food intake (Fig 7B), increased fat pad mass (Fig 7C), elevated leptin and insulin levels in the circulation (Fig
7E). These changes were similar to the intraperitoneal administration of high dose nanobody (daily i.p. 100 g per injection) although to a smaller degree (Fig 4).
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Figure 4.1. Systemic effects of local administration of 2.17-mAlb adjacent to tumor implantation site. (A) Body weight (PBS: n=17, 2.17-mAlb: n=23). (B) Food intake (PBS: n=17, 2.17-mAlb: n=23). (C) Biomarkers in serum 18 days after 3 injections of 2.17-mAlb (total dose 30 g per mouse). n=10 per group, * P<0.05. Data are meansSD
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Figure 4.2. Local administration of 2.17-mAlb inhibited melanoma progression. (A) Tumor volume (P<0.05. PBS: n=17, 2.17-mAlb: n=23). (B) Tumor weight (PBS: n=17, 2.17-mAlb: n=23. * P<0.05). (C) 2.17-mAlb inhibited B16 melanoma growth in vitro when cultured with mouse serum (n=4. * P<0.05). Data are meansSD. 116
Figure 4.3. Local administration of low dose 2.17-mAlb modulated gene expression in melanoma. (A) Gene expression in tumor (n=5 per group. * P<0.05). Mitf, microphthalmia-associated transcription factor; Tyrp2, tyrosinase related protein 2; Magea4, melanoma antigen family A4. Data are meansSD. (B) Western blot of tumors.
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Figure 4.4. Intraperitoneal administration of 2.17-mAlb. (A) High-dose intraperitoneal administration of 2.17-mAlb accelerated weight gain (n=10 per group, P<0.05 high-dose 2.17-mAlb compared to PBS and low-dose 2.17-mAlb. No significance between low-dose 2.17-mAlb and PBS). (B) High-dose 2.17-mAlb increased food intake (n=10 per group, P<0.05 high-dose 2.17-mAlb compared to PBS and low- dose 2.17-mAlb. No significance between low-dose 2.17-mAlb and PBS). (C) Biomarkers in serum (n=10 per group. Bars not connected by same letter are significantly different. (D) Tumor weight (n=10 per group). Data are meansSD.
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Figure 4.5. Intraperitoneal administration of high-dose 2.17-mAlb increased adiposity. (A) Subcutaneous and visceral fat pad weight (n=10 per group. * P<0.05). (B) Gene expression profile of epididymal fat (n=5 per group. * P<0.05, + P=0.08). Data are meansSD.
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Figure 4.6. Intraperitoneal administration of high-dose 2.17-mAlb affected hypothalamic gene expression. n=5 per group. * P<0.05. Data are meansSD.
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Figure 4.7. Subcutaneous administration of 2.17-mAlb in the established tumor model when treatment was delayed till palpable tumors appeared. (A) Weight gain (n=9 per group, P<0.05 2.17-mAlb 100 g compared to PBS and 2.17-mAlb 10 g. No significance between other groups). (B) Food intake (n=9 per group, P<0.05 2.17-mAlb 100 g compared to PBS and 2.17-mAlb 10 g. No significance between other groups). (C) Epididymal fat pad weight (n=9 per group, bars not connected by same letter are significantly different). (D) Tumor weight (n=9 per group, bars not connected by same letter are significantly different). (E) Biomarkers in serum (n=9 per group, bars not connected by same letter are significantly different). Data are meansSD.
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Discussion
Leptin is not only the metabolic switch that conveys metabolic information to the brain but is also involved in multiple pathways affecting many peripheral organs as a mitogen, metabolic regulator, survival or angiogenic factor depending on the tissue type
(Wauters, Considine, & Van Gaal, 2000). Clinical reports link elevated serum leptin levels to an increased risk of certain cancers including prostate (C. Garofalo & Surmacz,
2006), breast (Cirillo, Rachiglio, la Montagna, Giordano, & Normanno, 2008), and melanoma (Gogas et al., 2008). In vitro and preclinical in vivo data suggest that leptin acts as a mitogenic agent to promote prostate, breast, and ovarian cancer cell growth and/or enhances cancer angiogenesis and migration (Choi, Choi, Auersperg, & Leung,
2004; Frankenberry, Skinner, Somasundar, McFadden, & Vona-Davis, 2006; Ray &
Cleary, 2010). Thus leptin antagonists hold potential for future therapeutic use in cancer.
A few anti-LepR antibodies have been generated and tested in models of heart failure
(Purdham et al., 2008), multiple sclerosis (Matarese et al., 2005), and autoimmune encephalomyelitis (De Rosa et al., 2006). An anti-rat LepR mAb reduced the growth of bone marrow leukemic cells with concomitant decrease in angiogenesis, and prolonged survival (Iversen, Drevon, & Reseland, 2002). A pegylated leptin peptide antagonist
(LPA) significantly inhibited breast cancer xenografts hosted by immunodeficient mice without affecting energy balance (Rene Gonzalez et al., 2009).
In this study we assessed the effects of a neutralizing anti-LepR nanobody in a mouse model of melanoma. Local subcutaneous administration of low-dose 2.17-mAlb significantly inhibited melanoma growth associated with decreased angiogenesis in the
122 tumor. The absence of effects on weight and food intake suggested that the central actions of leptin were not disrupted by low-dose 2.17-mAlb although the low-dose nanobody administered adjacent to the tumor was sufficient to decrease the growth of a highly aggressive melanoma by 33%. These results further support our finding that the
EE-induced anti-cancer effect was mediated, at least in part, by leptin.
The effects of high dose 2.17-mAlb are more complex. The intraperitoneal injection of
2.17-mAlb at high-dose (100 g/mouse, daily) resulted in weight gain, hyperphagia, increased adiposity, hyperleptinemia, and hyperinsulinemia indicating efficient blockade of leptin signaling in CNS. On the other hand, low-dose 2.17-mAlb (i.p. 100 g/mouse, twice weekly, 500 g the whole course) showed neither significant metabolic effects nor anti-cancer effect suggesting that the antagonist availability and activity were insufficient at the respective sites of action. Therefore the overall impact of 2.17-mAlb on tumor growth was determined not only by the direct effects of LepR antagonist on tumor cells and/or other cells supporting tumor growth, but also by other systemic factors such as insulin metabolism that are regulated by leptin. In the context of cancer, insulin signaling and thus the role of leptin in the regulation of pancreatic -cell functions are of importance. Our previous data have shown that obesity increases B16 melanoma growth in obese leptin-deficient ob/ob mice consistent with other reports (Brandon et al., 2009;
L. Cao et al., 2010). Prevention of the obesity by pair feeding ob/ob mice dramatically reduces tumor weight to a level significantly lower than wild-type mice of the same weight (Brandon et al., 2009). Our leptin replacement data also showed that exogenous leptin increased melanoma mass in ob/ob mice by 140% compared to pair-fed saline-
123 infused mice with identical body weight and fat mass (L. Cao et al., 2010). These data all support the role of leptin in promoting melanoma growth. The hyperinsulinemia associated with leptin deficiency in ob/ob mice may underlie the accelerated tumor growth in ob/ob mice and similarly could counteract the anti-cancer effect of 2.17-mAlb in the high-dose administration experiment. Although leptin modulates glucose metabolism via central and peripheral mechanisms, the pancreatic -cells is a critical target of leptin actions (Marroqui et al., 2012). LepRs are expressed in the -cells and their activation directly inhibits insulin secretion. Long-term leptin deficiency inhibits insulin gene expression and -cells mass (Marroqui et al., 2012). Therefore the adverse effects on -cells and insulin require attention for the development and application of leptin antagonists.
High dose nanobody targeting LepR blocked leptin signaling in the hypothalamus as evidenced by induction of orexigenic NPY and AgRP as well as hyperphagia and increased adiposity. There is little evidence from the literature that nanobodies are actively or passively transported across BBB (Zabeau et al., 2012). The only two nanobodies known to transmigrate in an in vitro human BBB model and in vivo were generated by enrichment of a phage-display nanobody library with human cerebromicrovascular endothelial cells (Muruganandam, Tanha, Narang, & Stanimirovic,
2002). One explanation might be that the leptin-sensing neurons in the arcuate nucleus could make direct contact with the blood circulation (Cheunsuang & Morris, 2005;
Faouzi et al., 2007; Munzberg, Flier, & Bjorbaek, 2004). Another idea is that the nanobodies targeting LepR could disrupt the transportation of leptin across BBB. In this
124 study, we observed a robust increase of sLepR in 2.17-mAlb treated mice even when low-dose of nanobody was used. sLepR deriving from shedding of the extracellular domain is the main binding protein for leptin in the blood and modulates the bioavailability of leptin (Ge et al., 2002; Lammert, Kiess, Bottner, Glasow, & Kratzsch,
2001; Maamra et al., 2001). Experimental and clinical studies demonstrate an important role of sLepR as modulator of leptin action (Aleksandrova et al., 2013; J. L. Chan et al.,
2002; Reinehr, Kratzsch, Kiess, & Andler, 2005; Sun et al., 2010). The regulatory mechanisms for the generation of sLepR are not well understood. A recent report suggests that lipotoxicity and apoptosis increase LepR cleavage via ADAM10 (A
Disintegrin and Metalloproteinase 10) as a major protease (Schaab et al., 2012). sLepR mainly originates from short LepR isoforms (De Ceuninck, Wauman, Masschaele,
Peelman, & Tavernier, 2013; Schaab et al., 2012). Leptin transport across BBB is thought to be dependent on short LepR isoforms (Bjorbaek et al., 1998; Hileman et al., 2002;
Kastin, Pan, Maness, Koletsky, & Ernsberger, 1999). The increase in sLepR could indicate elevated shedding of short LepR isoforms and therefore could restrain leptin transport and subsequently impair central action of leptin (Tu, Kastin, Hsuchou, & Pan,
2008). An alternative explanation for the increase of sLepR level in nanobody-treated mice could be that the sLepR is bound by 2.17-mAlb and thereby is retained from clearance from circulation. Therefore more research is needed to understand the regulatory mechanisms of the expression of LepR isoforms and the constitutive shedding of the extracellular domain as well as the roles of these isoforms in controlling leptin transport, bioavailability, and binding and activating signaling pathways in order to
125 design LepR antagonists as potential therapeutics. The idea that large molecules such as nanobodies or antibodies cannot cross the BBB and therefore can restrict their actions to the periphery seems overly simplistic. Our data raise several questions in targeting leptin signaling as a treatment for cancer: how to restrict antagonizing actions to the periphery; how to prevent adverse effects such as hyperinsulinemia; how to improve bioavailability to cancer. Coupling the nanobody to the agents specifically targeting the tumor (antibody or drug conjugates) (Simon, Stefan, Pluckthun, & Zangemeister-Wittke, 2013) may enhance the anti-cancer efficacy while prevent adverse peripheral and central effects of leptin deficiency.
In summary, we demonstrated the anti-cancer effect of a neutralizing nanobody targeting LepR in a mouse model of melanoma. Systemic administration of high dose nanobody led to blockade of central actions of leptin and may compromise the anti- cancer effect of the nanobody. These data provide insights for development of LepR antagonists as treatment for cancer.
126
Chapter 5: Discussion and Future Directions
127
Hepatic Expression of AD36E4ORF1
AD36E4ORF1 expression is a model of insulin independent Akt activation and provides a novel therapeutic mechanism to improve glycemic control in cases of insulin resistance. Our findings confirmed the results of in-vitro studies demonstrating that the
AD36E4ORF1 protein is a potent activator of PI3K and protein kinase B signaling, without requiring upstream insulin signaling. Hepatic expression of AD36E4ORF1 was sufficient to improve glycemic control both insulin resistant and diabetic animals. In
Db/Db mice, Hepatic AD36E4ORF1expression enhanced glycemic control and attenuated hyperglycemia and hyperinsulinemia without provoking serious deleterious side effects. In obese insulin resistant animals maintained on a high fat diet (DIO)
AD36E4ORF1 expression also attenuated hyperinsulinemia and improved glucose tolerance; however it contributed to liver pathology. Recombinant adeno-associated virus
(rAAV) mediated expression of AD36E4ORF1 provides a tissue specific model to study perpetually active protein kinase B signaling, in vivo. Before determining the feasibility of AD36E4ORF1 as a therapeutic agent, there are serious concerns and limitations that must be addressed.
While this study demonstrated proof of efficacy, there are dangers to over activating Akt as an intervention for hyperglycemia. Non-alcoholic fatty liver disease
(NAFLD) is already a frequent comorbidity of metabolic syndrome, as insulin resistance fails to suppress hepatic glucose output but hyperinsulinemia still promotes increased hepatic lipogenesis. While not significantly contributing to an increase in hepatic triglyceride content in Db/Db mice, AD36E4ORF1 expression did nothing to improve the
128 already serious steatosis in these animals. In DIO animals, visceral adiposity was reduced, provoking increased hepatic triglyceride content and hepatomegaly. In lean wildtype animals, elevated hepatic Akt activity promoted de novo lipogenesis and decreased lipid oxidation. This over-activation of Akt signaling was pathological, inducing hypoglycemia and fatty liver. Another concern is that AD36E4ORF1 expression increases inflammation. AD36E4ORF1 expression increased mRNA levels of inflammatory genes in the livers of wildtype, DIO, and Db/Db mice.
The differential effects on liver composition between wildtype and hyperglycemic animals may reflect a restoration of Akt activity to normal levels in insulin resistant animals. Other feedback mechanisms due to already significant steatosis in Db/Db and
DIO mice may also be responsible for the differential effects on liver composition.
AD36E4ORF1 expression increased expression of the key regulators of liver lipid metabolism CHREBP and SREBP1c in wildtype animals, while reducing it in Db/Db mice (Fig 2-3). Because of these dangers and the potential oncogenic risk of unregulated growth factor signaling, further studies are required to assess the safety of AD36E4ORF1 expression.
Mechanism of Action
Based on our results, it is very evident that the dosage of AD36E4ORF1 expression must be tightly regulated. As E4ORF1 is not secreted from cells and acts entirely via intracellular pathways, a large amount of tissue transduction is required to have a marked impact on systemic metabolism. Under physiological conditions, insulin
129 mediated glucose uptake through PKB signaling is controlled by a vast array of negative feedback and self-inhibition. Under normal conditions the phosphorylation state of Akt mirrors concentrations of serum glucose and inulin, with very little activity in a fasted state. AD36E4ORF1 is completely exogenous in nature and thus uninhibited by cellular regulatory mechanisms. The in-vivo stability and half-life of AD36E4ORF1 are also unknown. For AD36E4ORF1 to have any real therapeutic potential its activity or expression level must be regulated in some way.
Our results support the insulin independent mechanism of Akt activation proposed by in vitro studies. AD36E4ORF1 required PI3K facilitated activation of Akt to function; however it does not appear to preferentially activate a specific isoform of Akt in the liver.
Prior to employing the PI3K inhibitor perifosine to blockade PI3K activity, we conducted a study of hepatic AD36E4ORF1 in Akt2 -/- animals maintained on normal chow diet.
Total Akt levels were reduced by roughly 85% in the livers of Akt2 -/- animals yet the ability of AD36E4ORF1 to cause hypoglycemia and fatty liver was not diminished.
Further analysis revealed little difference in Akt phosphorylation between AD36E4ORF1 treated WT and Akt -/- animals. These results can be explained at least in part due to the functional redundancy between Akt isoforms and by low basal phosphorylation levels of
Akt in a fasted state. Inhibition of PI3K function through perifosine treatment severely attenuated the ability of AD36E4ORF1 to increase Akt phosphorylation and prevented both hypoglycemia and hepatic accumulation in WT animals.
A role of RAS signaling has been proposed for AD36E4ORF1 facilitated PI3k activation however little evidence supports this conclusion. The most current and
130 complete model of function is that a ternary complex of DLG1, AD36E4ORF1, and PI3K migrates to the cell membrane, relieving catalytic inhibition of PI3K to increase PIP3 production and increase Akt signaling. Ras was not implicated in this in-vitro mechanistic model (M. Kumar, K. Kong, & R. T. Javier, 2014). Further examination is required to clarify the exact role and necessity of MAPK signaling in
AD36E4ORF1function. Due to differential pathway activation, insulin resistance promotes elevated MAPK activation without activation of PKB and increased MAPK signaling co-occurs with hyperinsulinemia. If AD36E4ORF1 expression further enhances
MAPK activation it may diminish its usefulness in models of hyperinsulinemia. Our study and proposed mechanism of action does not implicate any functional activation of
RAS or involve the MAPK pathway. This discrepancy can easily be addressed by quantifying levels of phosphorylated downstream targets of RAS, specifically pMAPK also known as pERK. Phosphorylation states are a much better indicator of pathway activity than total protein content. If any change is present it is likely that phosphorylation and therefore activity of MAPK signaling is decreased due to reduced concentrations of serum insulin.
Oncogenic Potential of AD36E4ORF1
While we did not observe any oncogenic growth in a five month study in Db/Db mice, the carcinogenic potential of AD36E4ORF1 expression cannot be ruled out entirely at this point. The very closely related adenovirus 9 (AD9) is considered to be oncogenic, producing mammary tumors specifically in rats. While AD9E4ORF1 has been shown to
131 be necessary for AD9 mediated mammary tumorigenesis in rats (Thomas et al., 2001;
Thomas et al., 1999), it has not been shown to be sufficient to do so alone. Furthermore the Akt pathway is abnormally active in a variety of human cancers and cancer cell lines
(Cheung & Testa, 2013). If not solely sufficient to induce carcinogenesis, it is very likely that AD36E4ORF1 expression can contribute to neoplastic growth in predisposed tissues.
Proteins containing PDZ motifs play a key role in regulating cellular transport, junctions and adhesion, and are often considered to be tumor suppressors. Sequestration through binding of E4ORF1 may interfere with the normal functions of these proteins.
DLG1, the key recruiter of AD36E4ORF1 to the cell membrane, functions to maintain cellular polarity, junctions, and may have tumor suppressor functions. Additionally
DLG1 interacts with and contributes to the functions of the tumor suppressors APC,
PTEN and β-catenin (Roberts, Delury, & Marsh, 2012). PTEN has also been found to associate with DLG1 as part of its membrane transport (Sotelo, Valiente, Gil, & Pulido,
2012). The phospholipase phosphatase activity of PTEN is dependent on PDZ mediated membrane recruitment so it is possible that saturation of PDZ domains by E4ORF1 may interfere with normal PTEN functions. This potential interaction may contribute to sustained activation of Akt by AD36E4ORF1.
Future studies of tissue selective AD36E4ORF1 expression
The liver was selected for this first study as it is a large metabolically active organ and effectively of transduced through intravenous viral administration. This study demonstrated the efficacy of AD36E4ORF1 expression to improve glycemic control;
132 however unregulated and sustained activation of Akt signaling may be deleterious to normal liver functions. The other insulin responsive tissues, specifically the brown and white adipose depots or skeletal muscle may provide more feasible therapeutic targets.
The REC2 serotyped recombinant adeno-associated viral vectors (rAAV) used in this study not only have high tissue tropism for the liver but can effectively transduce both brown and white adipose tissue. In this study we expressed E4ORF1 under control of a chicken beta-actin (CBA) promoter with a cytomegalovirus (CMV) enhancer sequence with an added woodchuck post transcriptional regulatory element (WPRE) sequence for added mRNA stability. The end result of this system is high levels of transgene expression with little to no tissue specificity. With this vector system, avoiding off target expression, specifically in the liver is nearly impossible regardless of delivery method.
Our lab has recently conceived a novel approach to almost entirely prevent hepatic transgene expression, regardless of delivery method. We have generated a new viral construct with an additional expression cassette containing a self-targeting miRNA under the albumin promoter. By targeting the exogenous WPRE sequence, unique to transgene mRNA, off target interference with physiological pathways is avoided. By incorporating this new regulatory (-AMW) cassette, with the albumin promoter driving transcription of a miRNA targeting the WPRE region of transgene mRNA, tissue selective expression can finally be achieved.
The white adipose tissue may present a better therapeutic target than the liver, although the properties of AD36E4ORF1 expression have been somewhat characterized.
A recent study by our collaborators utilizing an inducible adipocyte specific expression of
133
AD36E4ORF1 model demonstrated improved glycemic control in DIO animals. This improvement was paired with an expansion, inflammation, and fibrosis of the brown and white adipose depots(Kusminski et al., 2015). Our AAV-mediated gene therapy approach would allow selective AD36E4ORF1 expression in the white adipose, without disrupting normal physiology of the brown adipose tissue.
The skeletal muscle makes up the largest and most metabolically significant portion of insulin responsive tissue and may be an ideal therapeutic target for
AD36E4ORF1 expression. Over stimulation of PI3K by AD36E4ORF1 may also have differential downstream effects in various tissues. In the skeletal muscle, PI3K activation promotes muscular hypertrophy (Glass, 2010). It is possible that myocyte specific expression of AD36E4ORF1 would dispose of excess glucose uptake through hypertrophy and increased glycogen synthesis. As the REC2 serotyped vectors utilized in our previous work are not effective in the skeletal muscle, an alternative approach is required. AAV8 serotyped vectors effectively transduce the skeletal muscle (L. Wang et al., 2016). By repackaging our dual cassette AD36E4ORF1-AMW backbone into an
AAV8 serotyped capsid and performing localized injections, we will have an effective and selective approach to analyze the properties of AD36E4ORF1 expression in the skeletal muscle.
134
Environmental enrichment activates a hypothalamic adipocyte axis to promote
healthy aging.
While previous studies have characterized the neural benefits of enrichment in aged animals, our work is the first to focus on the metabolic benefits and role in healthy aging. A short term study of 10 month old female mice demonstrated the efficacy of environmental enrichment (EE) to activate the HSA in middle aged animals. EE increased hypothalamic expression of BDNF and genes involved in energy homeostasis.
In a one year study of middle aged animals housed in enrichment of middle-aged mice housed in EE we observed a metabolic phenotype characteristic of healthy aging and improved glycemic control. The animals housed in EE exhibited improved glucose tolerance, enhanced motor skills, reduced adiposity, increased mitochondrial biogenesis, remodeling and browning of the white adipose tissue, reduced anxiety, and prevention of age-associated decline in the brown adipose tissue. While animals housed in EE maintained an equivalent body weight to controls, they had less adipose tissue and slightly increased lean muscle mass. Remodeling of the adipose tissue was accompanied by an adipose-specific upregulation of the tumor suppressor phosphatase tensin homologue deleted on chromosome ten (PTEN). EE initiated in middle-aged female mice provides a model of improved glycemic control, healthy aging, and physiologically induced PTEN expression in the adipose tissue.
The most consistent and striking metabolic effect of housing in EE for both young and aged animals is an improvement in glucose tolerance. Surprisingly there was no change in insulin sensitivity as measured by insulin tolerance testing (Fig 3-14A). In both
135 free-fed and fasted states, serum levels of insulin were lower for animals housed in EE.
Moreover, while they were reduced in size, the white adipose depots themselves exhibited elevated glucose uptake (Fig 3-14B,C). Together, these results indicate enhancement of insulin independent pathways modulating glucose uptake into the white adipocytes. Intensified β-adrenergic stimulation of the adipose tissue may be the chief underlying cause of enhanced glucose transport into adipocytes.
Along with increased neural stimulation to the adipose tissue through the HSA,
EE provides animals with additional physical exercise compared to control housed animals. Physical activity is increased through voluntary running and more opportunities for exploratory behavior. At this time the metabolic improvements specific to hypothalamic sympathoneural-adipocyte stimulation cannot be clearly delineated.
Myokine release from skeletal muscle has been demonstrated to induce browning of the white adipose tissue in response to physical exercise.
To elucidate the exact contribution of HSA and the metabolic phenotype observed in EE, further studies will be required. By activating the HSA through overexpression of
BDNF in the hypothalamus of aged animals, we can more precisely dissect the role of
HSA activation in healthy aging and metabolism. We will also examine the requirement of intact HSA activation for environmental enrichment to improve glycemic control and promote healthy aging. Through expression of a dominant negative isoform of TRKB,
BDNF signaling can be attenuated significantly. To blockade HSA activation, we will surgically deliver a rAAV vector to express this dominant negative TRKB in the
136 hypothalamus of middle aged mice. Following this procedure animals will be placed into control or enriched housing.
Lifespan and Healthspan Extension
Several methods proven to extend maximum lifespan and promote healthy aging produce similar metabolic phenotypes to what we have observed in EE. Caloric restriction, essential amino acid restriction, fat specific insulin receptor knockout
(FIRKO), and PTEN overexpression animals all show a reduction in total adiposity and inflammation. Given these similarities, there is a potential that environmental EE may extend lifespan in both disease models and normal animals. However, EE increases energy expenditure, lean muscle mass and may slightly increase food intake while caloric restriction reduces them. The ten month old C57/Bl6 mice employed in our study represent a model of middle aged animals and do not exhibit an abundance of deleterious age associated changes compared to young animals. Our next step towards assessing the ability of EE to enhance lifespan or health span will be to evaluate the efficacy of EE in a cohort of animals of more advanced age. We are currently expanding environmental enrichment to elderly animals, aged 18 months. A long-term study to ascertain the efficacy of environmental enrichment to extend healthy lifespan is underway.
Upregulation of PTEN expression through HSA activation
Activation of the HSA through environmental enrichment provides a physiological model for healthy remodeling of the adipose tissue coupled with increased
137
PTEN expression. This study is the first to identify an adipose specific increase in PTEN expression due to adrenergic signaling. Moreover, a reduction in sympathetic tone to the adipose tissue through genetic inactivation of the HSA reduced PTEN expression below normal levels (Figure 3-6). Interestingly, while beta-3 receptors are the most common form in the adipose tissue, administration of a β-3 adrenergic CL-316,243 agonist did not upregulate PTEN upregulation. However, treatment with the β-1/2 agonist isoproterenol, increased PTEN expression in the adipose tissue to a similar extent as housing EE or hypothalamic BDNF overexpression. Whether or not PTEN upregulation in response to adrenergic signaling in other tissues is a conserved mechanism remains to be seen.
Previous work in our lab demonstrated that genetic or environmental activation of the
HSA preferentially elevates norepinephrine concentrations in the adipose tissue with little to no change in the serum or skeletal muscle (Lei Cao et al., 2010). Unlike HSA activation, β-adrenergic agonist treatment non-selectively activates receptors systemically, including those in the skeletal and cardiac muscle. Further examination of tissues from isoproterenol treated animals will be reveal whether adrenergic stimulation is sufficient to upregulate PTEN in other tissues.
It is still not clear whether PTEN overexpression is a critical regulator of the phenotype observed in EE or merely a consequence of adipose remodeling. Insulin stimulation promotes triglyceride accumulation and hypertrophic expansion of the white adipose tissue, while adrenergic stimulation promotes lipolysis and reduces adiposity
(Bartness, B, H, J, & K, 2010). As a negative regulator of distal insulin signaling, PTEN
138 overexpression may contribute to sustained lipolysis and the reduction in adiposity observed in EE animals.
Further studies are required to delineate the exact functions of PTEN upregulation in adipose tissue remodeling and metabolism. To evaluate the necessity of PTEN for EE to remodel the white adipose tissue, we will employ an adipose specific deletion of PTEN in animals prior to housing them in EE. To conditionally ablate PTEN in the adipose tissue of adult animals we will make use of a rAAV mediated vector to express Cre recombinase in the adipose tissue of PTEN floxxed +/+ animals. Through intraperitoneal viral delivery of this vector, PTEN will be deleted specifically in the gonadal, mesenteric, and to an extent in the retroperitoneal adipose depots.
The specific functions of PTEN overexpression in the adipose tissue remain to be elucidated. We have produced a rAAV vector to express PTEN while almost entirely avoiding expression in the liver and pancreas, by incorporating an AMW regulatory cassette. Using this approach we can effectively transduce, the gonadal and mesenteric adipose tissue through a single intraperitoneal injection. The brown adipose tissue can be preferentially transduced via oral gavage of rec 2 serotyped vectors (Huang, McMurphy,
Liu, Wang, & Cao, 2016). Through minor surgical procedures unilateral or bilateral delivery of AAV vectors can be achieved to the subcutaneous inguinal adipose tissue or intra-scapular brown adipose tissue. While the visceral adipose tissue is more relevant to metabolic syndrome, the subcutaneous adipose tissue is more metabolically active and its transduction may be have a larger impact on systemic glycemic control.
139
A gene therapy approach can also be utilized to increase the quantity and function of PTEN systemically. Recently, a longer alternatively spliced variant of PTEN was identified, consisting of 576 amino acids opposed to the 403 of normal PTEN. This
Long-PTEN performs the same intracellular functions as the standard isoform; however it is also secreted exosomes and taken up by other cells (Putz et al., 2012). Intraperitoneal protein administration of Long-PTEN was sufficient to reduce PI3K activation and suppress tumorigenesis in a study of nude mice (Hopkins et al., 2013). PTEN-long expression has potential as a model of inducible PTEN expression in a range of tissues.
Treatment with a non-selective rAAV vector expressing long-PTEN may provide a therapeutic route to reduce adiposity, and improve glycemic control, while increasing the anti-tumor activity of PTEN in multiple tissues.
Applicability to human health
Due at least in part to differences in body size, the brown adipose in adult humans is much less metabolically active than that of rodents during non-shivering thermogenesis. Adult humans do have brown adipose tissue which can be activated through adrenergic stimulation from the SNS however; its metabolic impact may be limited (Bartness et al., 2010). Unlike rodents, humans lack beta-3 receptors entirely in their white adipose depots. Human white adipose tissue is still innervated by the SNS but only in the form of β-1 and β-2 receptors. Unfortunately β-adrenergic receptors are also expressed by the cardiac muscle and chronic stimulation can lead to devastating consequences including congestive heart failure (Triposkiadis et al., 2009). As a result,
140 there are no adrenergic agonists capable of selectively targeting the white adipose tissue in humans. Therefore an approach which stimulates the white adipose specifically through activation of the SNS is ideal. Through further characterization of the benefits of
EE to metabolism and healthy aging and their underlying mechanisms, we hope to have a positive impact on human health.
141
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