An Investigation of the Metabolic Health Benefits of Purple Vegetables in an Animal Model of Metabolic Syndrome Using Molecular Approaches

Home , Aldolase B

Hala Mohamed Mahmoud Ayoub

A Thesis presented to The University of Guelph

In partial fulfillment of requirements for the degree of Doctor of Philosophy in Human Health and Nutrition Sciences

Guelph, Ontario, Canada

ABSTRACT

AN INVESTIGATION OF THE METABOLIC HEALTH BENEFITS OF PURPLE VEGETABLES IN AN ANIMAL MODEL OF METABOLIC SYNDROME USING MOLECULAR APPROACHES

Hala Mohamed Mahmoud Ayoub Advisor: University of Guelph, 2018 Professor Kelly A. Meckling

This thesis is an investigation of the benefits of purple vegetables on metabolic health. Consumption of anthocyanin rich plants is positively associated with improvements in various Metabolic Syndrome (MetS) parameters. Purple potatoes (PP) and purple carrots (PC) have higher anthocyanin concentrations and higher biological activity than lightly pigmented cultivars. However, they have not been extensively studied for their potential metabolic health benefits. I hypothesized that the substitution of the majority of carbohydrate in a high fat diet, with PC or PP in obese Zucker rats will

1) A) Improve insulin resistance and hypertension, two main components of MetS, compared to lightly colored varieties of these same vegetables (white potatoes (WP) and orange carrots (OC)) and a sucrose rich diet. B) All the vegetable diets will be superior to the sucrose rich diet. 2) Underlying mechanisms will include modulating adipose and hepatic gene expression. PP and WP consumption improved insulin sensitivity compared to the sucrose rich diet where PP were slightly superior to WP. The two carrot groups showed similar trends to the potatoes but this did not reach statistical significance. Each of the potatoes and the carrots groups improved blood pressure compared to the control group. However, each of the WP and PC were superior to their corresponding color counterpart. Using Western blotting, there were no obvious differences in the expression ii

of a group of the candidate proteins (e.g. FAS, ACC, AMPK, PPAR γ and adiponectin) chosen for the initial mechanistic investigation. Nonetheless, using proteomic analyses, we were able to identify few biological themes modulated by each vegetable in both tissues. We proposed that enhanced protein folding, increased cholesterol efflux and higher free fatty acids re-esterification are mechanisms that participate in the action of PP and PC on MetS pathologies in adipose tissue whereas decreased de novo lipogenesis and oxidative stress to be the suggested mechanisms of action in liver. In conclusion, evidence is provided at both the phenotypic and the molecular level, for the metabolic benefits of purple vegetables. Inclusion of these vegetables in the human diet is suggested as a strategy to improve MetS associated pathologies.

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ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Kelly Meckling, for her guidance, immense knowledge and support throughout my whole program. You are a great professor and an amazing person! I would also like to express my gratitude to our laboratory technician,

Cheryl Cragg, for her mentorship, hard work and patience. I acknowledge the thoughtful discussions and support from all my committee members, Dr. Lindsay Robinson, Dr. J.

Allan Sullivan, Dr. Rong Tsao and Dr. Mary Ruth McDonald.

I would like to acknowledge the most special people in my life:

 Hoda El zalabany and Mohamed Ayoub, my parents: Making you proud of me

has been and will always be a main target in my life.

 Mohammed Abo-Ismail, my husband: I could not have done this without your

continuous support, encouragement, love, pushing me to do better, and even

sometimes our arguments

 Haidy, Mahmoud and Maged, my siblings: you gave me a huge amount of

unconditioned love and support that I am really thankful for

 Habiba, Hala and Hana, my daughters: You are just the fuel that kept me going

every day!

TABLE OF CONTENTS

PAGE

CHAPTER 1 ...... 1

Literature Review ...... 1

1.1 General Introduction ...... 1

1.2 Mechanisms of MetS pathologies ...... 2

1.2.1 Insulin resistance (IR) ...... 2

1.2.1.1 Lipotoxicity ...... 2

1.2.1.2 Endoplasmic reticulum (ER) stress ...... 4

1.2.1.3 Oxidative stress ...... 5

1.2.1.4 Inflammation ...... 6

1.2.1.5 The gut microbiome ...... 7

1.2.1.6 Mitochondrial dysfunction ...... 8

1.2.2 Dyslipidemia in MetS ...... 8

1.2.3 Hypertension in MetS ...... 9

1.3 Dietary sources of polyphenols ...... 9

1.4 Mechanisms by which dietary polyphenols antagonize obesity and insulin

resistance ...... 11

1.4.1 Activation of the transcription factors (PPAR) ...... 11

1.4.2 Enhancement of thermogenesis ...... 15

1.4.3 The effect of polyphenols on lipid metabolism including fatty acid

synthesis and oxidation ...... 17

1.4.4 Effects of polyphenols on pro-inflammatory and anti-inflammatory

cytokines ...... 19

1.5 Evidence from human intervention studies ...... 22

1.5.1 Effects of polyphenols on anthropometric measurements (body

weight, body mass index, waist circumference) and blood pressure

...... 22

1.5.2 Effects of polyphenols on insulin resistance ...... 24

1.5.3 Effects of polyphenols on serum lipid profile ...... 25

1.6 Conclusion ...... 27

1.7 Rational and Hypothesis ...... 28

CHAPTER 2 ...... 36

The Effect of Anthocyanin Rich Purple Vegetable Diets on Metabolic Syndrome

in Obese Zucker Rats 1 ...... 36

2.1. ABSTRACT ...... 36

2.2. INTRODUCTION ...... 37

2.3. MATERIALS AND METHODS ...... 39

2.3.1. Experimental Design ...... 39

2.3.2. Quantification of phenolic and carotenoid content of the experimental

vegetables ...... 40

2.3.3. Glucose tolerance test (GTT) ...... 40

2.3.4. Insulin tolerance test (ITT) ...... 40

2.3.5. Plasma insulin levels ...... 41

2.3.6. Homeostatic model assessment of insulin resistance (HOMA IR) .... 41 vi

2.3.7. Hemodynamic function ...... 41

2.3.8. Western blotting ...... 41

2.3.9. Statistical Analysis ...... 42

2.4. RESULTS ...... 42

2.4.1. Food intake and body weight gain ...... 42

2.4.2. Blood glucose levels during ipGTT ...... 43

2.4.3. Blood glucose levels during ipITT ...... 43

2.4.4. Plasma insulin levels ...... 44

2.4.5. HOMA IR ...... 44

2.4.6. Blood pressure measurements ...... 44

2.4.7. Organs weight ...... 45

2.4.8. Protein expression ...... 45

2.5 DISCUSSION ...... 45

2.6 CONCLUSIONS ...... 50

CHAPTER 3 ...... 64

Proteomic Profiles of the Adipose and Liver Tissues from an Animal Model of

Metabolic Syndrome Fed Purple Vegetables1 ...... 64

3.1. ABSTRACT ...... 64

3.2. INTRODUCTION ...... 65

3.3. Methods ...... 67

3.3.1 Experimental design, sample collection, and tissue homogenization . 67

3.3.2. Sample preparation (denaturation, alkylation, and digestion) and

TMT labeling ...... 67 vii

3.3.3. Liquid chromatography and tandem mass spectrometry (LC-MS/MS)

...... 68

3.3.4. Protein identification and quantitation ...... 69

3.3.5. In-silico functional analyses ...... 69

3.4. RESULTS AND DISCUSSION ...... 70

3.4.1. Purple potato (PP) diet versus control diet ...... 70

3.4.1.1 Adipose tissue protein expression ...... 70

3.4.1.2 Liver protein expression ...... 75

3.4.2. Purple carrots (PC) diet versus control diet ...... 78

3.4.2.1 Adipose tissue protein expression ...... 78

3.4.2.2 Liver protein expression ...... 81

CHAPTER 4 ...... 101

GENERAL DISCUSSION ...... 101

REFERENCES ...... 105

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LIST OF TABLES

Table Title Page Chapter 2. The Effect of Anthocyanin Rich Purple Vegetable Diets on Metabolic Syndrome in Obese Zucker Rats

Table 2.1 Composition of the experimental diets...... 52

Table 2.2 Total Phenolic, Flavonoid, Anthocyanin and Carotenoid content of the experimental vegetables……………………………………...... 53

Table 2.3 Body weight gain and daily food intake of rats fed HFD, WP and PP for 8 wk. ……..……………………………...... 54

Table 2.4 Body weight gain and daily food intake of rats fed HFD, OC and PC for 8 wk……………………………...... 54

Chapter 3. Proteomic Profiles of the Adipose and Liver Tissues from an Animal Model of Metabolic Syndrome Fed Purple Vegetables

Table 3.1 Composition of the experimental diets...... 85

Table 3.2 Differentially expressed proteins in adipose tissue with Purple Potatoes diet...... 86

Table 3.3 Enriched gene ontology biological process terms and KEGG pathways in the list of differentially expressed proteins in adipose tissue that are involved in protein folding, lipid metabolism and cholesterol efflux with Purple Potatoes...... 88

Table 3.4 Differentially expressed proteins in the liver with Purple Potatoes diet…………………………………………………………...………. 89

Table 3.5 Enriched gene ontology biological process terms and KEGG pathways in the list of differentially expressed proteins in the liver that are involved in lipid metabolism and carbohydrate metabolism with Purple Potatoes……...... 91

Table 3.6 Differentially expressed proteins in adipose tissue with Purple Carrots Diet...... 92 Table 3.7 Enriched gene ontology biological process terms and KEGG pathways in the list of differentially expressed proteins in adipose tissue that are involved in lipid metabolism and cholesterol efflux with Purple Carrots …………………………………………………. 96

Table 3.8 Differentially expressed proteins in the liver with Purple Carrots diet…………………………………………………………………… 97

Table 3.9 Enriched gene ontology biological process terms and KEGG pathways in the list of differentially expressed proteins in the liver that are involved in lipid metabolism, carbohydrate metabolism and oxidative stress with Purple Carrots……………………………….. 99

LIST OF FIGURES

Figure Title Page Chapter 1 Literature Review

Figure 1.1 Mechanisms of Metabolic Syndrome pathologies. Mechanisms of insulin resistance appear to be interconnected and mutually related at some points. Obesity and insulin resistance are the main leading factors to hypertension and dyslipidemia...... 29 Figure 1.2 Suggested mechanisms of DAG and ceramides in interrupting the insulin signaling cascade. Adapted from Turban and Hajduch 2012 30 Figure 1.3 Chemical structures of common dietary polyphenols (adapted from Rong Tsao 2010) and examples of some common food sources……………………………………………………………... 31 Figure 1.4 Mechanism of uncoupling proteins (UCP) in thermogenesis. Adapted from Ricquier and Bouillaud 2000………………………. 33 Figure 1.5 Examples of polyphenols/polyphenols rich plants that affect the AMPK pathway at multiple points………………………………… 34 Figure 1.6. Suggested mechanisms of action of dietary polyphenols on Metabolic Syndrome………………………………………………. 35

Chapter 2 The Effect of Anthocyanin Rich Purple Vegetable Diets on Metabolic Syndrome in Obese Zucker Rats

Figure 2 .1. Changes in blood glucose levels during GTT of rats fed (A) HFD, WP and PP (B) HFD, OC and PC for 8 wk. …………………...... 55

Figure 2.2. Total plasma glucose concentration during GTT expressed by glucose AUC of rats fed (A) HFD, WP and PP (B) HFD, OC and PC for 8 wk..………………...... 56

Figure 2.3. Changes in blood glucose levels during ITT of rats fed (A) HFD, WP and PP (B) HFD, OC and PC for 8 wk…...... 57

Figure 2.4 Plasma glucose disposal during ITT expressed by glucose AAC of rats fed (A) HFD, WP and PP (B) HFD, OC, and PC for 8 wk….. 58

Figure 2.5 Fasting insulin levels (ng/mL) and HOMA IR of rats fed (A&B) HFD, WP, and PP (C&D) HFD, OC, and PC for 8 wk ……...... 59

Figure 2.6 Systolic, diastolic, left ventricular pressures and AV difference of rats fed (A&B) HFD, WP and PP (C&D) HFD, OC, and PC for 8 wk. ………………………………………………………………… 60

Figure 2.7 Organ weights’ percentage of the body weight of rats fed (A) xi

HFD, WP, and PP (B) HFD, OC and PC for 8 wk ……...... 61

Figure 2.8 Expression of the proteins of interest in the adipose tissue in rats fed (A)control, WP and PP diets; (B) HFD, OC and PC for 8 wk… 62

Figure 2.9 Expression of the proteins of interest in the liver tissue in rats fed (A)control, WP and PP diets; (B) HFD, OC and PC for 8 wk ...... 63 Figure Title Page Chapter 3. Proteomic Profiles of the Adipose and Liver Tissues from an Animal Model of Metabolic Syndrome Fed Purple Vegetables

Figure 3.1. Suggested mechanisms of action of purple potatoes and purple carrots on Metabolic Syndrome pathologies in the liver and adipose tissue...... 100

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LIST OF ABBREVIATIONS

Abbreviation Term AAC area above the curve ACC Acetyl CoA carboxylase AMPK AMP activated protein kinase AUC area under the curve BW body weight CPT1 carnitine palmitoyl transferase 1 CVD cardiovascular diseases DAVID Database for Annotation, Visualization and Integrated Discovery EGCG epigallocatechin gallate ER stress endoplasmic reticulum stress FAS fatty acid synthase FFA free fatty acids GLUT4 glucose transporter 4 GO gene ontology term HDL high density lipoprotein HFD high fat diet HOMA IR Homeostatic model assessment of insulin resistance IDF International Diabetes Federation IL Interleukin ipGTT intraperitoneal glucose tolerance test ipITT intraperitoneal insulin tolerance test IR Insulin resistance IRS insulin receptor substrate JCE Junipers Chinensis hot water extract JNK c-Jun N-terminal kinases KEGG Kyoto Encyclopedia of Genes and Genomes pathways LC-MS/MS liquid chromatography and tandem mass spectrometry LDL low density lipoprotein LPS Lipopolysaccharides MCP1 monocyte chemoattractant protein1 MetS Metabolic Syndrome NFB nuclear factor B

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Abbreviation Term NLR nod-like receptors NLRP3 NLR family pyrin domain-containing-3 OC orange carrot supplemented high fat diet PAMP pathogen associated molecular pattern PC purple carrot supplemented high fat diet PDK1 phosphoinositide dependent kinase1 PI3K phosphatidylinositol 3 kinase PIP2 phosphatidylinositol biphosphate PIP3 phosphatidylinositol triphosphate PKB/Akt protein Kinase B PKC protein kinase C PP purple potato supplemented high fat diet PP2A protein phosphatase A2 PPAR Peroxisome proliferator-activated receptors PRR pattern recognition receptors RAS renin angiotensin system ROS reactive oxygen species SNS sympathetic nervous system SREBP1c sterol regulatory binding protein 1c T2D type 2 diabetes mellitus TG Triglycerides TLR toll-like receptors TNFα tumor necrosis factor alpha UCPs uncoupling proteins UPR unfolded protein response VLDL very low density lipoprotein

WP white potato supplemented high fat diet

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CHAPTER 1

Literature Review

1.1 General Introduction

Metabolic Syndrome (MetS) is a cluster of metabolic disorders (abdominal obesity, insulin resistance, dyslipidemia and hypertension) that increase the risk of developing cardiovascular diseases (CVD) and type 2 diabetes mellitus (T2D) [1].

Several sets of criteria of identification of MetS have been proposed by different scientific organizations [2]. The most commonly used set is Adult Treatment Panel III criteria where diagnosis of MetS required at least 3 of the following : central obesity: waist circumference ≥ 102 cm or 40 inches (male), ≥ 88 cm or 36 inches(female), dyslipidemia: triglycerides (TG) ≥ 1.7 mmol/L (150 mg/dl), dyslipidemia: high density lipoprotein cholesterol (HDLc) < 1.03 mmol/L (40 mg/dL) (male), < 1.29 mmol/L (50 mg/dL) (female), blood pressure ≥ 130/85 mmHg and/ or fasting plasma glucose ≥ 6.1 mmol/L (110 mg/dL)[3]. This set of criteria was supported by the American Heart

Association/National Heart Blood and Lung Institute except that they modified the threshold of impaired fasting glucose from 6.1 to 5.6 mol/L [4].

In Canada, MetS is of great concern as a cross sectional survey of a sample of the

Canadian population estimated its prevalence to be 19.1% (i.e. about 1 in 5 Canadian adults had MetS)[5]. Life style intervention, including dietary recommendation, is the first line management of MetS that was proven to be more effective than pharmacological therapy [6].

Epidemiological studies support the inverse association between habitual consumption of fruits and vegetables and tea and the prevalence of MetS components

[7,8]. Polyphenols are very common component of foods and beverages of plant origin such as fruits, vegetables, wine and tea [9]. Dietary polyphenols, in several animal and human intervention studies, were reported to have a protective role against MetS. In this review, I will present some reported mechanisms of MetS pathologies (Figure 1.1), common dietary sources of polyphenols, animal and in vitro studies that investigated effects of polyphenols on metabolic health emphasizing some suggested mechanisms of action at the molecular level and finally an evidence from intervention human studies on the metabolic effects of polyphenols.

1.2 Mechanisms of MetS pathologies

1.2.1 Insulin resistance (IR)

1.2.1.1 Lipotoxicity

Accumulation of fatty acids and lipid intermediates has been recognized as an established mechanism of IR in muscle and liver for decades [10]. However, our understanding of the underlying mechanisms has evolved over time from a lipid induced defect in glycolysis (i.e. Randle’s hypothesis) to a lipid induced defect in glucose uptake

[11]. Binding of insulin to its receptor initiates a series of phosphorylation events that eventually lead to glucose uptake into the cell. The activated (phosphorylated) insulin receptor phosphorylates insulin receptor substrates (IRSs) on several tyrosine residues inducing their activation. The activated IRSs interact with and activate phosphatidylinositol 3 kinase (PI3K). Phosphatidylinositol triphosphate (PIP3), generated 2

from phosphatidylinositol biphosphate (PIP2) by the action of PI3K, recruits protein kinase B (PKB/Akt) and atypical protein kinase C (PKC) to the plasma membrane. Also,

PIP3 activates phosphoinositide dependent kinase1 (PDK1) and induces a conformational change in PKB/Akt exposing two regulatory sites. PDK1 activates both atypical PKC and

PKB/Akt by phosphorylating their threonine residues, whereas the serine residue of

PKB/Akt is phosphorylated by mammalian target of rapamycin (mTOR)-rapamycin insensitive companion of mTOR (Rictor) complex. Eventually, both activated aPKC and

PKB/Akt induce the release of glucose transporter 4 (GLUT4) vesicles to the plasma membrane that mediate glucose transport into the cell [12]. Diacylglycerol (DAG) and ceramides are the two lipid molecules that are implicated in interrupting insulin signaling through their effect on PKC isoforms (Figure 1.2)[12]. DAG is thought to activate novel and conventional PKCs that then serine-phosphorylate IRS1 and interrupt the insulin cascade [12], whereas, ceramide activates atypical PKCs that bind PKB/Akt, blocking its recruitment when normally required upon insulin stimulation [12]. Ceramide is also suggested to interrupt insulin signaling by activating protein phosphatase A2 (PP2A) as

PPA2 dephosphorylate PKB/Akt (i.e. inactivate) on both serine and threonine residues[12]. In addition, DAG contributes indirectly to hepatic IR through affecting transcriptional regulation of glucose production [13,14]. DAG-induced suppression of phosphorylation of PKB/Akt, increases the translocation of the gluconeogenic transcription factor, Forkhead box protein O1 (FOXO1), to the nucleus [13,14].

Ectopic lipid accumulation or lipotoxicity is mostly attributed to the imbalance between fatty acid delivery and fatty acid oxidation in liver and muscle[10]. In obesity, increased fatty acid delivery can be due to excess caloric intake [10], impaired storage 3

capacity of the adipose tissue (that may be partly due to a suppressed blood flow) [15] and/or increased lipolysis with high free fatty acids (FFA) release to the circulation [15].

Lipotoxicity is also observed in the disorders of the lack of adipose tissue such as genetic or acquired lipodystrophy or lipoatrophy where fat is redirected to muscles and liver [16].

However, in healthy lean insulin resistant subjects, impaired mitochondrial fatty oxidation rather than increased FFA delivery is thought to be the cause of intramyocellular fat deposition [16].

De novo lipogenesis is also suggested to have a role in promoting fatty liver in muscle IR as glucose is redirected to lipid synthesis in the liver when it fails to be used for glycogen synthesis in the muscle [17].

1.2.1.2 Endoplasmic reticulum (ER) stress

ER stress has an established link with IR [18-21]. Accumulation of misfolded or unfolded proteins induces ER stress that in turn stimulates a stress response known as the unfolded protein response (UPR) [21]. UPR activates 3 proteins (IRE1, PERK and

ATF6). Activation of each protein initiates a cascade of events that eventually lead to reduced protein translation and increased protein folding, thus correcting ER stress [21].

While the three branches of UPR work to fix the ER stress, they activate a number of stress kinases such as nuclear factor kB (NFB) and c-Jun N-terminal kinases (JNK). Those kinases mediate serine phosphorylation of IRS1 thus interrupting insulin signaling [18].

They also promote the synthesis of the proinflammatory cytokines such as tumor necrosis factor alpha (TNFα) and interleukin1 (IL1) [18] that themselves induce IR. In the liver,

ER stress also activates transcription factors involved in lipogenesis such as sterol

regulatory binding protein 1c (SREBP1c) and inhibits very low density lipoprotein

(VLDL) secretion from the liver. So, ER stress-induced hepatic lipogenesis can also promote IR. In adipose tissue, ER stress was shown to decrease perilipin1 expression and increase lipolysis [19]. Increased protein synthesis requirement, adipose tissue hypoxia, energy excess, and inflammation are suggested to be the causes of ER stress in obesity

[18,19]. Inflammation and ER stress are in a vicious cyclic relationship [18,19].

1.2.1.3 Oxidative stress

Similar to the ER stress mechanism of IR, the link between oxidative stress and

IR is mostly explained by the activation of stress kinases such as NFB and P38 mitogen- activated protein kinases (P38MAPK) that serine-phosphorylate IRS1 [22,23]. Also, oxidative stress may disrupt the production of cytokines in adipose tissue [24]. In addition, reactive oxygen species (ROS) can directly damage lipids, proteins, and DNA

[22]. High levels of FFA could be the explanation for oxidative stress induced IR. FFA promote oxidative stress by reducing glutathione levels and activating NADPH oxidase

[22,24]. In obesity and overnutrition, excess glucose and FFA delivery to the mitochondria results in increased ROS generation. In this state, IR is seen as a protective mechanism to decrease the nutrient load on the cell [25]. Antioxidants, such as alpha lipoic acid, reduced oxidative stress and improved insulin sensitivity in obese animal models [23]. But on the other side, acute oxidative stress improved insulin sensitivity as a result of keeping the active JNK in the nucleus, rather than the cytoplasm, thereby preventing it from phosphorylating IRS1[26], whereas, chronic oxidative stress had the opposite effect of acute oxidative stress [26].

1.2.1.4 Inflammation

Inflammation is one of the most common explanations for obesity induced IR.

Obesity is a state of chronic inflammation. As obesity progresses, the cellular composition of the adipose tissue changes from an anti-inflammatory profile to a pro- inflammatory profile. Both M1 pro-inflammatory macrophages and the pro-inflammatory adaptive immune cells (CD8+ and T helper cells) are predominant over the anti- inflammatory macrophages (M2) and the anti-inflammatory CD4+ and T regulatory cells

[27,28]. This shift in profile promotes the release of pro-inflammatory cytokines and a chronic inflammatory state [27,28]. Enlarged adipocytes also contribute to increased cytokine release [27]. Obesity triggered inflammation is mainly thought to be due to hypoxia and necrosis of the hypertrophied adipocytes [28]. Both factors attract macrophages to adipose tissue for the disposal of the dead cells and/or creation of new vessels to the hypoxic areas [28].

TNFα, IL6, leptin, adiponectin, monocyte chemoattractant protein1 (MCP1),

IL10, resistin, IL1β are the commonly discussed cytokines in relation to IR [2,15,29,30].

TNFα, IL6, MCP1, resistin, IL1β mediate pro-inflammatory effects whereas adiponectin and IL10 are anti-inflammatory cytokines [29,30]. IL6 has a more complex role, exerting pro-inflammatory effects during its chronic elevation and anti-inflammatory effects with acute elevation as in exercise [29,30]. TNFα interrupts insulin signaling via serine- phosphorylating IRS1 [31], whereas IL6 stimulates suppressor of cytokine signaling

(SOCS) that directly inhibit tyrosine phosphorylation of IRS1 [30]. Pro-inflammatory cytokines also activate stress kinases such as JNK and NFB that also impair IRS1 phosphorylation [30]. Besides their direct effects on insulin signaling, TNFα and IL6 6

induce lipolysis, downregulate adiponectin production and reduce adipogenesis [29].

MCP1 is a chemokine that attracts monocytes to adipose tissue promoting inflammation

[30]. IL1β is secreted from macrophages in response to NLR family pyrin domain- containing-3 (NLRP3) inflammasome activation and it promotes inflammation through activating the NFB pathway and producing other inflammatory cytokines [27]. Both adiponectin and IL10 exert anti-inflammatory and insulin sensitizing effects [29].

Adiponectin stimulates fatty acid oxidation by activating AMP activated kinase and

PPARα [32]. Adiponectin also negatively regulates the release of TNFα [29].

Besides the release of cytokines by the inflamed obese adipose tissue, other cellular stresses such as ER stress, oxidative stress, and saturated fatty acids induce IR through triggering inflammation pathways (see above). Saturated fatty acids are among other metabolic stresses that activate pattern recognition receptors (PRR) such as Toll- like receptors 4 (TLR4) and Nod-like receptors 4 (NLR4) families on immune cells that leads to the production of inflammatory cytokines [27,28].

1.2.1.5 The gut microbiome

In obesity, the impaired barrier function of the intestinal epithelium permits transfer of the gut microbiota to the systemic circulation [33,34]. Lipopolysaccharides

(LPS) of the bacteria trigger an inflammatory response. LPS are considered as pathogen associated molecular pattern (PAMP) molecules that act as a strong immune cells’ PRR activator promoting the production of inflammatory cytokines [33,34]. Besides the direct effect of the inflammatory cytokines on IR, another mechanism of gut microbiota-

induced IR is thought to be nitrosylation of IRS1 as LPS promotes the production of nitric oxide [33].

1.2.1.6 Mitochondrial dysfunction

Mitochondrial dysfunction has been linked to IR through two scenarios.

Mitochondrial dysfunction is probably a primary cause in the first scenario as it is thought to be the reason for the high levels of intracellular lipids and subsequently IR in the muscle of lean insulin resistant subjects and the elderly [16]. In the second scenario, mitochondrial dysfunction is attributed to the reduction of mitochondrial biogenesis as a result of intracellular lipid overload [11]. This dysfunction, in turn, aggravates the intracellular lipid accumulation in a vicious cycle [11]. Another suggested explanation for

IR linkage to mitochondrial dysfunction is that increased fatty acid β-oxidation in IR increases ATP production as a result of mitochondrial overwork. High levels of ATP slow down the mitochondrial function in a negative feedback fashion[35]. The last explanation suggests that mitochondrial dysfunction may be a consequence of IR rather than a cause [35].

1.2.2 Dyslipidemia in MetS

IR seems to be the main factor leading to dyslipidemia in the context of MetS

[17]. In the IR state, glucose tends to be directed to de novo lipogenesis instead of glycogen synthesis [17]. Also, the lack of the inhibitory effect of insulin on hormone sensitive lipase results in higher lipolysis rates in the adipose tissue with an increased

FFA delivery to the liver and other tissues [2]. Both factors induce hepatic TG accumulation and increased VLDL secretion into the circulation [36]. The increased 8

production of apolipoprotein B in IR may contribute to high hepatic VLDL secretion as well. Postprandial high chylomicron levels and decreased TG clearance may also be due to impaired insulin stimulation of lipoprotein lipase activity [37].

High levels of TG rich VLDL triggers an increase in the exchange rate between

TG molecules of VLDL and cholesterol molecules of HDL, resulting in an atherogenic cholesterol rich VLDL and a dissociation susceptible TG rich HDL. Upon hydrolysis of

TG in HDL, apolipoprotein a1 (Apoa1) dissociates from the HDL particle and is excreted by the kidney, causing a decrease in the circulating Apoa1 and HDLc [17,37].

1.2.3 Hypertension in MetS

Obesity and IR are the main contributing elements to the development of hypertension in the context of MetS [2,38-40]. Resistance to insulin action results in inactivation and dephosphorylation of nitric oxide synthase through impairment of the

PI3K signaling pathway with a subsequent reduction in the production of the potent vasodilator, nitric oxide [2,39]. Also, hyperinsulinemia contributes to over activation of the sympathetic nervous system and renin angiotensin system thereby aggravating vasoconstriction. The exaggerated antinatriuretic action of insulin promotes volume expansion, exacerbating hypertension as well [2,40]. In addition, dysregulation of adipose tissue-secreted cytokines like IL6, TNFα and adiponectin seem to induce vasoconstriction as they increase angiotensin II and endothelin I levels[2,38]. Finally, vasoconstrictors, angiotensin II, and endothelin I, secreted from the obese adipose tissue, may be factors that link obesity and hypertension [39].

1.3 Dietary sources of polyphenols 9

Polyphenols are a class of molecules that typically have multiple phenol structural units or may have only one phenol ring such as phenolic acids (Figure 1.3) [9].

Polyphenols are very common components of fruits, vegetables, dry legumes, chocolate and beverages such as tea, coffee and wine [41]. The most common dietary sources of polyphenols are fruits and beverages [9]. The two main groups of polyphenols are flavonoids and phenolic acids [41]. Flavonoids themselves are classified into several subgroups: flavones, flavonols, flavonones, flavononols, flavanols, isoflavones and anthocyanins. Flavonols are commonly found in onions, broccoli, curly kale, leeks and blueberries [9]. Quercetin is the most common flavonol and is most abundant in onions, tea and apples [41]. Flavones are commonly present in parsley and celery [9]. Flavonones are found in high concentration in citrus fruits followed by tomatoes. Examples of flavonones are naringenin in grapefruit, hesperetin in oranges and eriodictyol in lemons

[41]. Soy products are the most common source of isoflavones such as genistein, daidzein, and glycitein [9]. Anthocyanins are abundant in red wine and certain types of vegetables such as cabbage, beans, onions, and radishes, but they are most commonly found in fruits such as black current, blackberries, cherries, raspberries and strawberries

[9,41]. Anthocyanins give fruits, vegetables, and flowers their red, blue and purple colors

[41]. Flavanols are present in two forms; catechins and proanthocyanidins. Catechins are commonly found in fruits such as apricots and cherries whereas the richest sources of catechins are green tea and chocolate [9]. Catechins and epicatechins are mainly found in fruits, whereas gallocatechin, epigallocatechin and epigallocatechin gallate are present in certain legumes, grapes and especially in tea [42]. Proanthocyanidins are responsible for astringency and bitterness of fruits such as grapes, berries, and kakis, beverages such as 10

tea, wine and beer and chocolate [43]. Caffeic acid is one of the most common phenolic acids that is abundant in many fruits such as kiwis whereas ferulic acid is the common phenolic acid found in cereal [9]. Dietary sources and bioavailability of polyphenols are discussed in detail in reviews by Manach et al. [41] and D’Archivio et al. [9]

1.4 Mechanisms by which dietary polyphenols antagonize obesity and insulin resistance

1.4.1 Activation of the transcription factors (PPAR)

Peroxisome proliferator-activated receptors (PPAR) are transcription factors that also have the feature of receptor molecules. Thus, they are able to control gene expression through binding to lipid signaling molecules produced from dietary lipids.

PPAR are known to be master regulators of lipid metabolism [44]. By binding to PPAR

γ, natural or synthetic ligands, increase the expression of networks of target genes leading to an increase in FFA uptake by adipocytes from the circulation. This favorable effect is associated with adipocyte differentiation that prevents adipocyte hypertrophy [45].

Therefore, PPAR γ activation reduces FFA in the circulation, while increasing the ability of adipose tissue to store TG. This decreases lipotoxicity in the muscle and liver, through

“lipid repartitioning”, thus enhancing insulin sensitivity [46]. Thiazolidinedione (TZD), an antidiabetic drug and a PPAR γ selective ligand, was also found to induce adiponectin production [45]. In addition, TZD was reported to have anti-inflammatory effects in the form of reduction of TNFα production in fat cells [47]. PPAR α is another member of

PPAR that is mainly expressed in the liver [48]. Activation of PPAR α induces hepatic fatty acid oxidation. It also decreases liver and muscle steatosis promoting insulin

sensitivity [49]. Together these observations suggest that up regulation of PPAR γ and

PPAR α promote insulin sensitivity.

Many plant polyphenols have been reported to increase PPAR activity and PPAR mRNA expression. High fat diet induced obesity in mice resulted in down regulation of mRNA levels of PPAR γ, whereas, obese mice treated with yerba mate extract for, 8 wk, showed recovery of their mRNA levels of PPAR γ. Yerba mate is a plant with a high polyphenol content including flavonoids (quercetin and rutin) and phenolic acids

(chlorogenic acid and caffeic acid) [50]. A similar favorable effect was observed with

Junipers Chinensis hot water extract (JCE) in rats. Supplementation of a high fat diet with 1% JCE resulted in the restoration of mRNA levels of PPARγ that had been reduced with the high fat diet [51]. The heart wood of JC has been reported to be a source of bioactive polyphenolic compounds such as quercetin, naringenin, taxifolin, aromadendrin and isoquercetin [52]. In Zucker fatty rats, supplementation of a high fat diet with anthocyanin-rich, tart cherry powder for 90 days, increased retroperitoneal fat mRNA levels of PPAR γ and PPAR α compared to the control group [53]. Also, male hamsters fed a high fat diet supplemented with mulberry water extract (0.5%-1.0%-2.0% w/w) experienced significant decreases in body weight, visceral fat, serum TG, serum total cholesterol and liver lipids in a dose-dependent manner compared to hamsters fed the high fat diet, alone. These effects were associated with significantly higher expression of

PPAR α in the liver. Mulberry water extract was reported to contain gallic acid, chlorogenic acid, rutin, and anthocyanins [54]. Moreover, in Dahl salt-sensitive rats with

IR and hyperlipidemia, diets supplemented with freeze dried whole tart cherry (1% w/w), rich in anthocyanins, significantly increased mRNA levels of PPAR α in the liver with a 12

trend toward increasing PPAR γ. The supplemented group showed significantly lower total cholesterol, TG, insulin levels, fasting glucose and hepatic steatosis compared to the control group[55]. In these studies, the other phytochemicals in tart cherry, mulberry,

JCE or yerba mate or at least their synergetic effects with polyphenols may have contributed to the observed effects. In vitro, isolated rat adipocytes treated with cyanidin showed significant increases in PPAR γ mRNA levels [56]. Also, human adipocytes pretreated with quercetin and trans-resveratrol showed decreases in the suppression of

PPAR γ expression mediated by TNFα [57]. Feeding a high fructose diet to rats, for 4 wk, resulted in hyperlipidemia, hyperinsulinemia, and higher IR compared to feeding with purified chow. Supplementation of the high fructose diet with anthocyanin rich extract from black rice, for 4 more wk, significantly improved hyperlipidemia and glucose intolerance. Similar favorable effects were observed with the supplementation of the high fructose diet with the PPAR γ agonist, pioglitazone [58].

In obese hypertensive ovariectomized and intact female rats, genistein treatment, for

4 wk, significantly increased PPAR α and PPAR γ expression in the liver. Genistein treated animals had lower systolic blood pressure, IR and plasma lipids compared to those who were treated with vehicle[59]. In vitro, in preadipocytes and hepatocellular carcinoma cells, isoflavones isolated from Astragalus membranaceus (formononetin and calycosin) and Pueraria thomsonii (daidzein) showed significant increases in the activation of PPARα and PPAR γ compared to other synthetic compounds known to be

PPAR activators. In the same study, two other isoflavones, genistein and biochanin A showed the same effect [60]. When the human hepatoma cell line, HepG2, was treated with genistein (10µm), PPAR α expression was higher than that of the same cells treated 13

with the same dose of the known PPAR α activator, WY14643 [61]. In an in vivo study, mice fed a high fat diet supplemented with high isoflavone (1.82 g/kg diet) soy protein, had lower serum TG compared to a low isoflavone soy protein based diet. A similar effect was observed with fenofibrate, an anti-hypertriglyceridemic drug and a PPAR α agonist, supplemented diet, although the fenofibrate was more potent. In contrast, in

PPAR α knockout mice, fenofibrate did not show an effect on serum TG, whereas the high isoflavone soy protein supplemented diet did decrease serum TG, suggesting that the antiobesity effects of isoflavones are operating through a different mechanism that fenofibrate [62].

In contrast, several investigators reported that dietary polyphenol intake was associated with suppression of PPAR γ activity. Intake of a cocoa powder supplemented high fat diet for 21 days decreased PPAR γ expression in the white adipose tissue of male rats compared to an unsupplemented high fat diet. This effect was associated with a reduction in body weight and mesenteric white adipose tissue [63]. In diet induced obese male mice, consumption of a high fat diet supplemented with epigallocatechin gallate

(EGCG) (0.2-0.5 w/w), for 8 wk, decreased mRNA levels of PPAR γ in epididymal white adipose tissue in a dose dependent manner compared to the high fat diet. This effect was associated with a dose dependent reduction in body weight and adipose tissue weight

[64]. The only limitation of this study that it is not known if the observed effect on mRNA expression is attributed to EGCG supplementation or resulted from the reduction in adipose tissue mass [64]. Klaus et al [65] suggested that studying gene expression earlier, before the obvious reduction in the body fat, can help to confirm this idea.

Similarly, consumption of green tea with a high fat diet for 6 mo was shown to decrease 14

PPAR γ expression in perirenal fat in rats compared to the control (water). This effect was associated with a decrease in body fat and an increase in lean mass[66]. In vitro, during differentiation of human adipocytes, genistein (1µM) treatment was shown to decrease PPAR γ expression. This was associated with a reduction in the number of adipocytes and their lipid droplet content [67]. Similarly, in vitro, in the early phase of differentiation, adding genistein to preadipocytes decreased PPAR γ expression and TG accumulation compared to a vehicle [68].

Although, as previously discussed, PPAR γ activation was shown to improve IR, suppression of PPAR γ activity was shown to decrease adipogenesis. The reduction in adiposity may have indirect positive effects on improving IR.

1.4.2 Enhancement of thermogenesis

Uncoupling proteins (UCPs) are a family of membrane carrier proteins that impair the coupling of ADP phosphorylation to oxidation that occurs in the mitochondrial matrix. Oxidation, driven by the electron transport chain in the mitochondrial matrix, is accompanied by proton pumping from the mitochondrial matrix across the inner mitochondrial membrane whereas ATP synthesis is catalyzed by ATPase which couples the proton transfer across mitochondrial membrane back to mitochondrial matrix to ADP phosphorylation [69]. When the process of oxidation or respiration is not accompanied by

ADP phosphorylation, this results in a proton leak. This dissipated energy is released in the form of heat (Figure 1.4)[69]. This explains the role of UCPs in the reduction of obesity. UCP2 and UCP3 are found to be expressed in the white adipose tissue and several other tissues.

Plants containing polyphenols and isolated polyphenols were shown to enhance the gene expression of UCPs. Mice or rats fed high fat diets supplemented with yerba mate[50] or JCE 1% extract [51] showed higher mRNA expression of UCP 1 with yerba mate and higher UCP2 and UCP3 with JCE, compared to a control group fed the high fat diet, alone. Obese male mice fed a high fat diet (23%fat) supplemented with epigallocatechin-3-gallate (0.2%-0.5%w/w), for 8 wk, showed a significant dose- dependent increase in the mRNA levels of UCP2 compared to a high fat diet control group [64]. Moreover, intake of a high fat diet supplemented with cocoa (50 g cocoa powder/day) increased the gene expression of UCP2 in white adipose tissue in male rats.

This effect was associated with a significant reduction in body weight and mesenteric white adipose tissue and a decreasing trend of serum TG concentrations. There was also a significant increase in HDL-c level [63]. In addition, mice fed a high fat diet supplemented with genistein (0.2% w/w), for 12 wk, showed significantly higher UCP2 expression. They also had significantly lower liver weight, liver TG, epididymal fat weight and cholesterol levels compared to mice fed a high fat diet alone[70]. In high fat diet fed rats, purple potato extract supplementation decreased total body lipids in a therapeutic fashion (i.e. 4 wk after high fat diet feeding), whereas it prevented retroperitoneal lipid accumulation in a preventive designed experiment (i.e. with the start of the high fat diet feeding and for 8 wk). Blood lipid profile (total cholesterol, low density lipoprotein cholesterol (LDL) and total TG) improved in both experiments.

UCP3 was induced in the liver and fat tissues of the treated group whereas UCP2 did not change. In vitro, treatment of the preadipocytes with the same purple potato extract

showed a significant dose-dependent decrease in their growth and their fat accumulation

[71].

1.4.3 The effect of polyphenols on lipid metabolism including fatty acid synthesis and oxidation

AMP activated protein kinase (AMPK) is an enzyme playing a key role in intracellular fatty acid metabolism. Phosphorylated AMPK pathway in fatty acid oxidation is mediated through phosphorylation (inactivation) of acetyl CoA carboxylase

(ACC) which catalyzes the conversion of acetyl CoA to malonyl CoA. Thus, phosphorylation of ACC decreases malonyl CoA. Malonyl CoA inhibits carnitine palmitoyltransferase 1 (CPT1) which transports fatty acids to mitochondria for oxidation.

Thus, AMPK activation increases fatty acid oxidation and reduces lipogenesis [72].

The antiobesity effect of various polyphenols is mediated by influencing the components of AMPK pathway enzymes (AMPK, ACC, and CPT1) and fatty acid synthase (FAS), the enzyme that catalyzes the synthesis of fatty acids from acetyl CoA and malonyl CoA (Figure 1.5). For instance, in the epididymal adipose tissue, AMPK and

ACC phosphorylation, and their protein and mRNA expression, increased in rats fed a high fat diet with added 1% JCE for 79 days compared to the control group fed only a high fat diet. Also, the supplemented group had lower body weight, fat pad weights, insulin and plasma cholesterol levels [51]. Furthermore, in diet induced obese mice, supplementation with epigallocatechin-3- gallate (EGCG) into their high fat diet for 8 wk, was associated with a decrease in the mRNA levels of FAS and an increase in the mRNA levels of CPT1 in the epididymal white adipose tissue compared to the high fat

control diet [64]. This effect was associated with a reduction in the BW and weight of epididymal, subcutaneous, visceral and retroperitoneal adipose tissue. There were also reductions in plasma TG, LDLc, total cholesterol and liver lipids [64]. Administration of green tea and black tea for 6 mo with a 15% fat diet, resulted in an increase in the expression of genes regulating fatty acid oxidation (CPT1, PPAR α) in the liver compared to the control group (water). However, the supplementation increased the expression of genes mediating fatty acid synthesis (FAS, ACC, and SREBP1c) as well.

Thus, there was no change in the liver TG [66].

Consumption of a high fat diet supplemented with a mulberry water extract, rich in polyphenols, significantly increased CPT1 expression while decreasing FAS expression in the liver of male hamsters. Supplemented hamsters had a significant reduction in liver lipids and an improvement in liver functions compared to a high fat diet fed hamsters [54]. Also, for 12 wk, a high fat diet supplemented with purple corn color

(PCC), rich in cyanidin 3-glucoside, did not increase body weight, adipose tissue weight, glucose, and insulin levels compared to an unsupplemented high fat diet in mice. In the white adipose tissue of the mice fed PCC supplemented diet, there was a significant reduction in SREBP and FAS mRNA levels compared to high fat diet fed mice [56].

Moreover, in vitro treatment of isolated rat adipocytes with anthocyanins (cyanidin and cyanidin 3-glucoside) significantly increased phosphorylated AMPK expression [73].

Also, in type II diabetic mice, consumption of an anthocyanin rich bilberry extract supplemented diet, for 5 wk, significantly decreased serum glucose concentrations, serum

TG, and total cholesterol levels and improved IR compared to a control unsupplemented diet. This effect was associated with a significant increase in the phosphorylation of 18

AMPK in white adipose tissue, skeletal muscle, and the liver. Furthermore, the bilberry diet significantly upregulated the expression of CPT1 while inactivating

(phosphorylating) ACC in the liver [74]. Also, incubation of adipocytes with the purple sweet potatoes, for 24 h, down regulated the mRNA expression of FAS and SREBP1c while upregulating CPT1[75]. PSP also significantly decreased adipocytes size and their

TG accumulation [75].

In diabetic fatty male Zucker rats with dyslipidemia and mild hypertension, consumption of high isoflavone soy protein diet (432 mg aglycone/kg diet), for 11 wk, significantly decreased FAS activity in adipose tissue compared to a control casein diet.

The isoflavone diet fed rats also had lower liver weights and TG and better glycemic control than the control casein fed rats [76]. In vitro, treatment of the human hepatoma cell line (HepG2 cells) with genistein (1-10 µm) showed a significant dose-dependent enhancement of CPT1 expression after 24 h compared to vehicle. This effect persisted even in the presence of an estrogen inhibitor [61]. In vitro, incubation of 3T3-L preadipocytes with genistein at different doses, EGCG or capsaicin (red pepper polyphenol) showed a significant increase in AMPK and ACC phosphorylation [77].

Also, significant reductions in FAS and SREBP expression in white adipose tissue and

FAS expression in the liver were reported with the intake of cocoa supplemented high fat diet for 3 wk in male rats [63].

1.4.4 Effects of polyphenols on pro-inflammatory and anti-inflammatory cytokines

Consumption of dietary polyphenols was shown to reduce the expression of some adipose tissue released proinflammatory cytokines (e.g. TNFα, IL6, and MCP1) and increase anti-inflammatory cytokines (e.g. adiponectin).

In obese Zucker rats, administration of quercetin (10 mg/ kg.BW), for 10 wk, increased plasma adiponectin level and reduced TNFα in the visceral adipose tissue [78].

This was associated with a reduction in the body weight, plasma concentration of TG,

FFA, total cholesterol, plasma insulin and systolic blood pressure. At a lower dose (2 mg/ kg BW), the same effects were observed but it did not affect the level of adiponectin or

TNFα [78].

In obese mice, treatment with yerba mate (1g/ kg) extract, for 8 wk, significantly reduced mRNA expression of TNFα, IL6 and MCP1 and increased mRNA expression of adiponectin in the white depose tissue. This effect was associated with a reduction in the body weight, epidydimal fat pad weight and TG, LDLc, and glucose levels compared to the placebo group. A marked decrease in the macrophage infiltration in the white adipose tissue was also observed with the yerba mate ingestion [50].

When Zucker rats were fed a high fat diet supplemented with a whole tart cherry powder, rich in anthocyanins, for 90 days, they showed a reduction in plasma IL6 and

TNFα and the mRNA expression of IL6 and TNFα in the retroperitoneal fat. This effect was attributed to the reduction of NFB activity where NFB is a transcription factor that plays a key role in the regulation of inflammation [53]. In addition, this effect was associated with lower body weight, percentage of fat mass, total cholesterol, TG, insulin, and glucose compared to the rats fed a high fat diet only [53]. In the white adipose tissue of male mice, the increase in TNFα induced by a high fat diet was reversed by adding 20

purple corn color to the high fat diet [56]. Similarly, consumption of a high fat diet by male mice, for 8 wk, upregulated the expression of the inflammatory genes, TNFα, IL6 and MCP1. This effect was reversed by adding 4% anthocyanin rich blue berry powder to the high fat diet of the mice. In addition, the mice fed the high fat diet with the blue berry powder had better insulin sensitivity [79]. However, in these studies, it is not known if the observed reduction in the inflammatory cytokines levels is attributed to the direct effect of polyphenols or the reduction in adipose tissue mass. However, in vitro, adipocytes treated with purple sweet potato extracts, showed downregulated expression of both MCP1 and IL6 [75].

Mice fed EGCG (3.2 g/kg diet) supplemented high fat diet for 16 wk had a significantly reduced MCP1 concentration that was associated with an improvement in

IR. The lowering effect of EGCG on MCP1 may be attributed to the reduction in adipose tissue as supplemented mice showed a significant reduction in body weight, percentage body fat and total visceral adipose tissue. Moreover, EGCG supplementation decreased liver weight, liver TG and lipid hepatic cellular accumulation. The EGCG supplemented group had higher fecal lipids compared to low fat and high fat diets [80]. This suggested that EGCG mediates its antiobesity effect, at least partly, by decreasing intestinal lipid absorption. Also, IR improvement could partly have contributed to the observed positive effect of EGCG on hepatic steatosis [80].

In vitro, treatment of isolated rat adipocytes with cyanidin increased adiponectin secretion at 7 h and adiponectin mRNA levels at 24 h [73]. Moreover, human adipocytes treated with grape seed procyanidins (GSPE) showed an enhanced expression of adiponectin and a reduced expression of IL6 and MCP1, alone, and in the presence of an 21

inflammatory stimulus (TNFα). In addition, treatment of macrophage like cells with

GSPE decreased MCP1 expression in the presence of an inflammatory stimulus (LPS). In the two cell lines, pre-treatment with GSPE, before incubation with TNFα, was associated with inhibition of NFB activation (i.e. translocation to the nucleus). This activation was induced by incubating the cells with TNFα [81]. Similarly, human macrophages pretreated with grape powder extract showed a reduced expression of

TNFα, IL6, and IL8 and a suppressed activation of NFB after incubation with LPS. In this study, grape powder extract was shown to have a high concentration of quercetin-3- glucoside [82]. Also pre-treatment of primary human adipocytes with quercetin and trans-resveratrol, for 1 h before treatment with TNFα, decreased expression of IL6, IL8, and MCP1. It was also shown to decrease serine phosphorylation of IRS1 induced by

TNFα, whereas only quercetin was shown to decrease NFB activity induced by TNFα

[57].

1.5 Evidence from human intervention studies

Many human studies have been conducted to investigate the effects of dietary polyphenols on parameters of MetS. In the next part of this review, I will present the results of recent human studies emphasizing the effects of various dietary polyphenols on the metabolic risk factors.

1.5.1 Effects of polyphenols on anthropometric measurements (body weight, body mass index, waist circumference) and blood pressure

Dietary polyphenols were shown to affect body weight and/or blood pressure. In a study done on obese subjects with MetS, supplementation of green tea beverage or green 22

tea extract capsules for 8 wk significantly decreased body weight and body mass index

(BMI) compared to a control group, whereas there was no effect on blood pressure [83].

However, the effects of green tea catechins, in this study, may have been confounded with the caffeine content of the tea. In contrast, in another study that used only EGCG capsules (800 mg) in obese or overweight subjects, there was no effect of the supplement on BMI or waist circumference but there was a significant reduction of the diastolic blood pressure compared to the placebo group [84]. Also, for 16 wk, supplementation of a decaffeinated green tea extract (EGCG=856 mg), in obese subjects with T2D, did not show a significant difference in waist circumference compared to a placebo group but the supplemented group had significantly lower waist circumference compared to the base line [85]. Consumption of 2 cups of a blueberry beverage daily, for 8 wk, did not affect body weight but significantly reduced blood pressure in obese subjects with MetS [86].

Also, consumption of 100 g berries (whole bilberries , blackcurrent or strawberry puree) and small glass of berry juice (cold press chokeberry and raspberry ) daily , for 8 wk, significantly decreased systolic blood pressure without affecting body weight in subjects with CVD risk factors (mild hypertension, high blood glucose and/or high cholesterol levels) [87]. In patients with hypertension and impaired glucose tolerance, there was a significant reduction in systolic and diastolic blood pressure with flavanol rich dark chocolate supplementation (100 g /day), for 15 days, compared to the baseline [88]. In contrast, there was no effect of dark chocolate supplementation (50 g), for 12 wk, on blood pressure or waist circumference in prehypertensive subjects [89]. Also, there was only a decreasing trend in the blood pressure of healthy subjects supplemented with dark chocolate (100 g) for 15 days [90]. Supplementation of isoflavonoids (glycitein, 23

daidzein, genistein) for 3 mo, did not affect blood pressure or body weight in postmenopausal women [91].

The lack of significant effect of dietary polyphenols on weight may be due to the short durations of the studies. More significant effects may have been seen if the studies were conducted for longer periods. Also, the significant effect on body weight seen with the green tea may be attributed to the caffeine content of the tea rather than the polyphenol content. In addition, the effects of polyphenols on blood pressure may be affected by the health of the test subjects as the lowering effect on the blood pressure was more evident in patients with hypertension rather than healthy or pre-hypertensive subjects.

1.5.2 Effects of polyphenols on insulin resistance

Dietary polyphenols were shown to have an equivocal effect on IR in various studies. Daily treatment of postmenopausal women with genistein (54 mg/day) resulted in a significant reduction in IR after 12 and 24 mo compared to a placebo [92]. Also, ingestion of decaffeinated green tea extract, during the 24 h before oral glucose tolerance test, showed insulin sensitivity improvement in healthy young men compared to a placebo [93]. In addition, a significant decrease in IR and an increase in β cell function were observed after consumption of flavanol rich dark chocolate for 15 days compared to flavanol free white chocolate in patients with impaired glucose tolerance [88]. Similar favorable effects of dark chocolate on IR was reported in healthy subjects in an earlier study [90]. In contrast, consumption of 2 cups of strawberry beverage [94], 2 cups of blueberry beverage [86], for 8 wk, or intake of anthocyanin capsules (320 mg), for 12 wk,

[95] were shown not to affect serum glucose concentrations. Also, in obese nondiabetic subjects, green tea, green tea extract [83] or EGCG supplementation [84] did not affect

IR. Moreover, treatment of postmenopausal women with isoflavonoids (114mg/day of glyctein, daidzein and genistein) was shown not to affect IR [91].

The observed inconsistency between these studies could be explained by the variations in polyphenol content and specific phytochemical species present in each study mixture. Also, there may have been differences in the doses of the supplements and in the overall health of the test subjects, that may have contributed to the observed variations in the effects of dietary polyphenols on IR.

1.5.3 Effects of polyphenols on serum lipid profile

The effect of dietary polyphenols on the serum lipid profile was investigated in several studies. Anthocyanin supplementation, for 12 wk, was shown to significantly decrease LDLc while increasing HDLc in patients with dyslipidemia. In the same study, in vitro, anthocyanin supplemented subjects’ serum showed a significant increase in cholesterol efflux from macrophages which contributes to reverse cholesterol transport

(i.e. cholesterol is transferred back to the liver from cells). The possible mechanism of this effect was suggested to be due to a reduction in the activity of cholesterol ester transfer protein (CETP) as in vitro, CETP activity decreased in a dose-dependent manner in HepG2 cells treated with cyanidin 3-O-β-glucosides [95]. CETP is a protein that facilitates the transport of cholesterol esters from HDL and exchanges them with TG from LDL and VLDL thus decreasing HDL and increasing LDL [96]. Consumption of 2 cups of freeze dried strawberry beverage, for 8 wk, significantly decreased LDLc and

total cholesterol without affecting HDLc or TG [94]. Also, supplementation with 100 g berries and a small cup of berry juice , for 8 wk, showed significant elevation of HDLc with no change in the total cholesterol and TG in subjects with CVD disease risk factors

(i.e. subjects with mild hypertension, high total cholesterol and/or low HDLc levels) [87].

Cranberry extract capsule supplementation, for 12 wk, significantly decreased LDLc and total cholesterol: HDLc ratio compared to a placebo [97]. In contrast, in subjects with normal baseline values of the serum lipids, consumption of 2 cups of blueberry beverage, for 8 wk, did not affect serum lipids [86]. In these studies, the effect of polyphenols on serum lipids may have been affected by the magnitude of dyslipidemia of the test subjects at baseline.

Treatment of perimenopausal healthy women with soy isoflavone extract, for 4 wk, significantly decreased serum LDL and cholesterol compared to the baseline with no change in HDLc, VLDL, or TG [98]. It is worth noting that the supplement used had some soy protein, so it is possible that at least some of the observed effect was due to the soy protein rather than the isoflavones. This is supported by the results from two other studies, that used only genistein [92] and only isoflavonoids [91], and found no effect on serum lipids.

In hypertensive patients, daily consumption of flavanol rich dark chocolate (100 g), for 15 days, resulted in a significant reduction of LDLc and total cholesterol compared to the baseline, whereas flavanol free white chocolate had no effect on the serum lipid profile [88]. Also, consumption of high polyphenol dark chocolate (45 g), for 8 wk, significantly increased HDLc with no significant changes in LDLc and TG compared to low polyphenol chocolate in diabetic patients [99]. In contrast, in healthy subjects, 26

consumption of dark chocolate, for 15 days, did not affect serum lipids [90]. The lack of significant effect in this study may be attributed to the absence of dyslipidemia or any other metabolic risk factor in the test subjects. However, supplementation with green tea, green tea extract [83] or decaffeinated green tea extract [85] did not show any effect on serum lipid profile either in obese subjects with MetS or T2D.

1.6 Conclusion

There was some inconsistency and sometimes a selective improvement in the human studies regarding the effects of polyphenols on MetS parameters. This is mainly due to variations in the types of the supplemented polyphenols in these studies. Dietary polyphenols show variation in their bioactivity and bioavailability which is a substantial determinant of their health effects [40]. Also, there were differences in the doses and forms of the supplementations. Some studies used plants with known rich polyphenol content, whereas others used isolated polyphenols or pure compounds rather than the intact plant. The health of the test subjects varied among the studies between healthy, obese subjects with MetS or subjects with clinically overt T2D or hypertension. Thus, this could have contributed to the differences in the observed effects of polyphenols in these studies.

At the molecular and cellular level, animal and in vitro studies are more consistent and provide accumulating data that dietary polyphenols are able to target several mechanisms involved in MetS pathologies (Figure 1.6). Overall, a growing body of evidence now exists that leads nutritionists and health promotion specialists, to

recommend including polyphenol rich plants in the human diet, as one of several ways to improve metabolic health and combat the risk of chronic disease development.

1.7 Rational and Hypothesis

Accumulating evidence showed that polyphenols rich plants and more specifically anthocyanin rich plants have favorable effects on MetS parameters, however in most of the studies they have been compared to a non-plant diet so it was hard to dissect the effect of the purple color from the positive effects of the plants as a whole.

Purple potatoes and purple carrots have higher anthocyanin concentrations and higher biological activity compared to their less pigmented counterparts, white potatoes and orange carrots [100-102]. However, they have not been extensively studied for their direct effects on metabolic health, and the possible molecular mechanisms for these activities are poorly studied. So in this thesis, we aim at studying the effects of the purple varieties of these two very commonly consumed vegetables (i.e. potatoes and carrots) to their lightly colored varieties and a sucrose rich diet on metabolic health in attempt to investigate the potential benefits of adding these vegetables to the diet (i.e. vegetable effect) and also whether adding extra purple color will confer additional benefits (i.e. color effect).

We hypothesize that the substitution of the majority of the carbohydrates, in a high fat diet, with the purple colored varieties of potatoes and carrots will 1) A) Improve insulin resistance and hypertension, two main components of MetS, compared to lightly colored varieties of these same vegetables and a sucrose rich diet. B) All the vegetable

diets will be superior to the sucrose rich diet. 2) Underlying mechanisms will include modulating adipose and hepatic gene expression

Figure 1.1 Mechanisms of Metabolic Syndrome pathologies. Mechanisms of insulin resistance appear to be mutually related at some points. Insulin resistance is the main leading factors to hypertension and dyslipidemia.

Figure 1.2. Suggested mechanisms of DAG and ceramides in interrupting the insulin signaling cascade. Adapted from Turban and Hajduch 2012[12]. IRS: insulin receptor substrate, PI3K: phosphatidylinositol 3 kinase, PIP2: phosphatidylinositol biphosphate,

PIP3: phosphatidylinositol triphosphate, PDK1: phosphoinositide dependent kinase 1,

PKB: protein kinase B, aPKC: atypical protein kinase C, n/c PKC: novel and conventional protein kinase C, PP2A: protein phosphatase A2, GLUT4: glucose transporter 4, DAG: diacylglycerol, P: phosphorylation reaction, flat ended arrow means inhibition.

Polyphenols

I. Phenolic acids

a) Benzoic acids b) Cinnamic acids

II. Flavonoids

a) Flavones b) Flavonols c) Flavonones

d) Flavononols e) Flavanols

g) Isoflavones f) Anthocyanins

Figure 1.3. Chemical structures of common dietary polyphenols (adapted from Rong

Tsao 2010[103]) and examples of some common food sources

Figure 1.4. Mechanism of uncoupling proteins (UCP) in thermogenesis. Adapted from

Ricquier and Bouillaud 2000[69].

Figure 1.5. Examples of polyphenols/polyphenols rich plants that affect the AMPK pathway at multiple points. AMPK: AMP activated protein kinase. ACC: acetyl CoA carboxylase, FAS: fatty acid synthase, CPT1: carnitine palmitoyl transferase1, SREBP1: sterol regulatory element binding protein1, JCE: Junipers chinensis hot water extract,

PSP: purple sweet potaoes, EGCG: epigallocatechine gallate. P means phosphorylated. A plus sign means that it increases the expression whereas a minus sign means it decreases the expression. Flat ended arrow means inhibition.

Figure 1.6. Suggested mechanisms of action of dietary polyphenols on Metabolic

Syndrome

CHAPTER 2

The Effect of Anthocyanin Rich Purple Vegetable Diets on Metabolic Syndrome in

Obese Zucker Rats 1

2.1. ABSTRACT

Consumption of highly colored fruits and vegetables rich in anthocyanins has been associated with numerous health benefits. Purple carrots (PC) and purple potatoes

(PP) have higher anthocyanins concentrations and higher biological activities compared to less pigmented cultivars. We hypothesized that substitution of the majority of carbohydrate in a high fat diet with PP or PC, for 8 wk, would improve insulin resistance and hypertension, major components of Metabolic Syndrome (MetS), compared to orange carrots (OC), white potatoes (WP) or a control, high fat, sucrose-rich diet (HFD) in obese Zucker rats. After 8 wk of feeding, intraperitoneal glucose tolerance (ipGTT), intraperitoneal insulin tolerance (ipITT), and invasive hemodynamic tests were performed. The PP group had better glucose tolerance compared to the WP and the HFD groups and higher insulin sensitivity as measured by the ipITT and HOMAIR (P=0.018) compared to the HFD without having any effect on blood pressure. The PC reduced left

------1 This chapter has been presented at two conferences and published in Journal of Medicinal Food: Hala M. Ayoub, McDonald MR, Sullivan JA, Tsao R, Platt M, Simpson J, Meckling KA. The Effect of Anthocyanin-Rich Purple Vegetable Diets on Metabolic Syndrome in Obese Zucker Rats. J Med Food. 2017. doi: 10.1089/jmf.2017.0025 Hala M. Ayoub, Jordan Yarr, Heba Salim, Mary Ruth McDonald, J. Allan Sullivan, Rong Tsao and Kelly A. Meckling. The Effect of Anthocyanin Rich Purple Vegetable Diets on Insulin Resistance in a Model of Metabolic Syndrome. Annual Canadian Nutrition Society proceeding 2015, Winnipeg, Manitoba, Canada. Hala M. Ayoub, Mary Ruth McDonald, J. Allan Sullivan, Rong Tsao and Kelly A. Meckling. The Effect of Anthocyanin Rich Purple vegetables on Insulin Resistance in a Model of Metabolic Syndrome. The 37th International Carrot Conference on Sept. 16, 2015, Alliston, Ontario, Canada.

ventricular pressure compared to both the HFD (P=0.01) and the OC (P=0.049) groups and reduced systolic and diastolic blood pressures compared to the HFD group (P=0.01 and <0.0001 respectively) without having any effect on glucose homeostasis. The PC animals consumed more and were more obese than the HFD, possibly obscuring any benefit in this vegetable on glucose tolerance. The bioactives in the vegetables, responsible for blood pressure and glucose homeostasis could be different, and their effects independent of each other. The specific bioactives of each vegetable and their molecular targets remain to be identified. None-the-less, incorporation of purple vegetables in functional food products may provide metabolic/cardiovascular benefits in the background of a high fat diet that promotes obesity.

Keywords: polyphenols, flavonoids, insulin resistance, glucose tolerance test, cardiovascular disease, hypertension, diabetes, overweight, high fat diet

2.2. INTRODUCTION

MetS is a cluster of metabolic risk factors (abdominal obesity, insulin resistance, dyslipidemia, and hypertension) that is associated with an increased risk of developing

CVD and T2D [1]. MetS is a growing global health problem. According to the

International Diabetes Federation (IDF), about 25% of the adult population in the world has MetS. In Canada, the prevalence of MetS was recently estimated to be 19.1% [5] and

18.3% [104].

Epidemiologic evidence supports an inverse association between consumption of diets rich in fruits and vegetables and the prevalence of MetS [7]. Plants often contain

several bioactive phytochemicals that have multiple biological activities, giving them many mechanisms by which to delay or reverse MetS associated pathologies [105].

Anthocyanins are water soluble pigments that give fruits and vegetables their red, blue and purple colors [106], and are commonly found in the human diet [9,41]. In several recent animal and human studies, anthocyanin-rich plants were shown to positively modify MetS biomarkers. Whole tart cherry [53,55], purple corn [56], mulberry water extract [54], blueberry powder [79], bilberry extract [74], blood orange juice [107], blueberry beverage [86], strawberry beverage [94], black rice [58], black soy bean [108] and anthocyanin supplementation [95] were reported to decrease and/or improve one or more of the metabolic risk factors such as body weight, adipose tissue weight, dyslipidemia, hyperinsulinemia, hyperglycemia, glucose intolerance, insulin resistance, hepatic steatosis and/or blood pressure. Several mechanisms were suggested to explain these favorable effects of anthocyanins on MetS. In these studies, anthocyanins were mainly shown to modulate the expression of genes that regulate lipid metabolism, inflammation, and energy homeostasis, all of which are thought to be critical to the pathogenesis of obesity and insulin resistance.

The color in purple carrots is attributed to their high content of anthocyanins

[100,109]. Purple carrot juice was shown to have higher antioxidant, anti-inflammatory, and hypolipidemic effects compared to β-carotene in high-carbohydrate high-fat diet-fed rats [102]. Higher concentrations of antioxidants in purple potato cultivars compared to white potato cultivars are mainly attributed to their anthocyanin content [110]. Purple potatoes were shown to have anti-obesity and hypolipidemic effects in high fat diet-fed rats [71] and antioxidative and anti-inflammatory effects in men [101]. 38

Given the reported favorable effects of anthocyanin-rich plants on MetS, the high concentration of anthocyanins in purple carrots and purple potatoes and their reported higher biological activity compared to their less pigmented counterparts, we hypothesize that the substitution of the majority of carbohydrate in a high fat diet, with purple carrots or purple potatoes, for 8 weeks, would 1) Improve insulin resistance and hypertension compared to lightly colored varieties of these same vegetables and a sucrose rich diet and

2) All vegetable diets would perform better than the typical sucrose-rich, high fat diet known to produce the MetS in animals.

2.3. MATERIALS AND METHODS

2.3.1. Experimental Design

Seventy-five obese susceptible Zucker male rats were obtained from Charles

River Laboratories at 4 wk of age and housed, two rats per cage, at 23± 3°C under an automatic lighting schedule (0800–2000 h light). Animals were acclimated to regular chow for 1 wk and then randomized to 5 experimental diets (15 rats/group): control (high fat diet, HFD), white potato supplemented high fat diet (WP), orange carrot supplemented high fat diet (OC), purple potato supplemented high fat diet (PP) or purple carrot supplemented high fat diet (PC) for 8 wk. The five experimental diets were prepared by Research Diets Inc (New Brunswick, NJ, USA), using a modified high fat

AIN-93M diet as the base (135 calories provided from fat/ 100 g diet). Each diet was the same except for the type of carbohydrates. The control group had sucrose, whereas the other four groups had one of each vegetable, as the main sources of carbohydrates. The composition of the pelleted experimental diets is shown in Table 1. Body weight and

food intake were measured twice a week throughout the study. This protocol was approved by the Animal Care Committee of the University of Guelph (Animal Utilization

Protocol #12R012) in accordance with the guidelines from the Canadian Council on

Animal Care (CCAC).

2.3.2. Quantification of phenolic and carotenoid content of the experimental vegetables

Polyphenol and carotenoid extracts were prepared from freeze dried vegetable powder as previously described [111]. Total phenolic content (TPC) was determined using Folin-Ciocalteu’s phenol reagent (FCR) method and expressed as milligram Gallic acid equivalent per gram dry weight [111]. Total anthocyanins content (TAC) was quantified using the pH differential method and expressed as milligram cyanide- 3- glucoside equivalent per gram dry weight [111]. Total flavonoid (TFC) and carotenoid

(TCC) contents were also estimated [112,113] and expressed as milligram catechin equivalent and microgram β carotene equivalent per gram dry weight, respectively.

2.3.3. Glucose tolerance test (GTT)

After 8 wk of feeding, an intraperitoneal glucose tolerance test (ipGTT) was performed on overnight fasted rats. The glucose level was measured from the whole tail blood at 0, 10, 20, 30, 60, 90 and 120 min following the ip injection of 40% glucose solution (2 g/kg BW), using a glucometer [114]. Glucose area under the curve (AUC) above the baseline measurement was calculated using these values [115].

2.3.4. Insulin tolerance test (ITT)

Three days after the ipGTT, blood glucose measures were obtained from the tail at 0, 5, 10, 15, 30 and 45 min following ip injection of human insulin (1 unit /kg BW)

[79]. The values obtained were used to calculate glucose area above the curve (AAC) that is below the baseline measurement [115].

2.3.5. Plasma insulin levels

Blood samples were obtained by cardiac puncture on anesthetized rats after hemodynamic measurements were taken (see below). Fasting plasma insulin levels were determined by ELISA using a commercial assay kit (Rat insulin ELISA kit, EMD

Millipore, USA).

2.3.6. Homeostatic model assessment of insulin resistance (HOMA IR)

The following formula was used to calculate HOMA IR: fasting plasma glucose

(mmol/L)* fasting plasma insulin (mIU/l)/22.5 [116].

2.3.7. Hemodynamic function

The rats were anesthetized with an isoflurane/oxygen mix (2.25%/100%) and maintained at 37°C throughout the procedure. A 1.2F catheter (FTS-1211B-0018,

Scisense Inc.) was inserted through the right carotid artery and into the left ventricle.

Blood pressure readings were digitized at a sampling rate of 2000 Hz and recorded by a computer using iWorx® analytic software (Labscribe2, Dover, NH, USA). Values were obtained during a period of ~5min of stable function and averaged from a 10 s sample.

2.3.8. Western blotting

Liver and adipose tissue (epididymal fat depot) samples were homogenized (Fast

Prep ®24; MP biomedical, Santa Ana, CA) using NP40 cell lysis buffer (Invitrogen, CA)

(3 volumes for adipose and 30 volumes for liver samples) supplemented with protease inhibitor cocktail and phenylmethylsulfonyl fluoride (Sigma-Aldrich). The lysates were centrifuged at 5000 g for 10 min. at 4oC [117,118]. The total protein content of the 41

infranatant was determined using BCA protein assay kit (Thermo Fisher Scientific). 20

µg protein samples were separated on 4-10% gradient SDS-PAGE gels then transferred onto a nitrocellulose membrane using a wet transfer technique at 100 V for 1 h.

Membranes were blocked with 5% BSA in 0.05% TBST for 1 h and then incubated with the appropriate primary antibody overnight at 4o C. The membranes were washed several times with TBST then incubated with the appropriate horseradish peroxidase conjugated secondary antibody for 1 h. at room temperature. The signals were visualized using enhanced chemiluminescence. Densitometry was used for band quantification using

Alpha View software of the Flourchem HD imaging system (Alpha Innotech, Santa

Clara, CA). Phosphorylated proteins were normalized to the corresponding total. All other proteins were normalized to GAPDH. Primary antibodies were from Cell Signaling.

ACC (#3676), phospho ACC (# 11818), AMPK alpha (#5831), phospho AMPK alpha

(#2535), AMPK beta (#4150), phospho AMPK beta (#4181), adiponectin (# 2789), FAS

(# 3180), PPAR gamma (# 2435), perilipin (#9349) and secondary antirabbit (#7074).

2.3.9. Statistical Analysis

Data were analyzed using the PROC MIXED of SAS 9.4 software [119]. Two- tailed t-tests on all pairs (i.e. pairwise contrast) after ANOVA were used. IpGTT and ipITT data were analyzed using repeated measures ANOVA. The differences among the means with P-values ≤ 0.05 were considered significant.

2.4. RESULTS

2.4.1. Food intake and body weight gain

Rats fed the WP diet had significantly higher body weight gain (P= 0.04) and higher food intake (P<0.0001) compared to the rats fed the PP or control HFD diets 42

(Table 2.3). Both carrot groups ate more (P= 0.0005) and had higher body weights

(P=0.001) than the HFD group, but there were no differences between cultivars (Table

2.4).

2.4.2. Blood glucose levels during ipGTT

Blood glucose concentrations of the PP group were not significantly different from those of the WP or the HFD group at min 0, 10, 20 and 30, during the ipGTT.

However, at 60 min, glucose levels of the PP group were significantly lower than the WP group (P= 0.049) and at the last 2 time points of the test, 90 min and 120 min, the PP group had significantly lower blood glucose concentrations compared to both the HFD and the WP groups (at min 90, P= 0.009 and P=0.0066, at min 120, P< 0.0001 and

P=0.04 respectively) (Figure 2.1A). The PP group had numerically smaller AUC compared to both the HFD and the WP groups but it did not reach significance (P=0.089)

(Figure 2.2A).

There were no differences in the blood glucose levels between the PC group and the HFD or the OC groups at any time point of the test (Figure 2.1B). Also, there were no differences in glucose AUC among the 3 groups (Figure 2.2B).

2.4.3. Blood glucose levels during ipITT

Blood glucose concentrations of the PP group were not significantly different from that of the WP or the HFD group at 0, 5, 10 and 15 min during the ipITT. However, at 30 min and 45 min, the PP group showed significantly lower blood glucose concentrations compared to the HFD group (P=0.02 and P=0.006 respectively) and the

WP group was also significantly lower than the HFD at 45 min post-insulin injection

(Figure 2.3A). Glucose AAC did not significantly differ among the 3 groups (Figure

2.4A).

There were no significant differences in the blood glucose levels of the PC group compared to the HFD or the OC groups at any time point of the test (Figure 2.3B), nor were there differences in glucose AAC among the HFD or either carrot cultivar group

(Figure 2.4B).

2.4.4. Plasma insulin levels

Rats fed both potato cultivars had significantly lower fasting plasma insulin levels compared to the HFD group (Figure 2.5A). For the carrot cultivars, the trend was similar, in that the more highly colored variety appeared to have lower insulin values than the lightly colored and both carrot cultivars were lower than the HFD; however, this did not reach statistical significance (Figure 2.5C).

2.4.5. HOMA IR

The PP group had significantly lower HOMA IR values compared to the HFD group (P=0.018) (Figure 2.5B). For the WP, the HOMA-IR was intermediate between the

HFD and PP groups and not significantly different from either. For the carrots, again the trends were similar to the potatoes but again these differences were not statistically significant. (Figure 2.5D).

2.4.6. Blood pressure measurements

The systolic and diastolic blood pressures of the WP group were significantly lower than the HFD. In this case, the PP pressures were intermediate between the WP and HFD groups and not statistically different from either (Figure 2.6A). In contrast to the results of absolute blood pressures, the PP had significantly lower AV differences 44

than that of the HFD (P= 0.02) and the WP group was intermediate and not different from

HFD or PP (Figure 2.6B). Left ventricular pressure (LVP) was not significantly different among the 3 groups (Figure 2.6A).

In the carrot arms of the study, the group fed PC demonstrated significantly lower systolic, diastolic and peak LVP compared to the HFD group (P=0.01, <0.0001 and 0.01 respectively). For the OC group, the reduction in systolic blood pressure was not as large as for the PC and not significantly different from either the HFD or PC groups. However, for diastolic blood pressure, the reduction was more marked and resulted in a significantly lower blood pressure than the HFD that was similar to the PC (Figure 2.6C).

Again, though the AV differences appeared lower for both carrot groups compared to the

HFD, this too did not reach statistical significance. (Figure 2.6D).

2.4.7. Organs weight

Organs weights, expressed as a percentage of body weight, were compared between the groups (Figure 2.7). All vegetable groups had significantly lower liver weights than the HFD, however, the light and highly colored cultivars behaved equally well. All other organ weights did not differ among groups.

2.4.8. Protein expression

No significant differences were seen among the carrot or the potato groups in the expression of the studied proteins in either the adipose or the liver tissues (Figures 2.8 and 2.9).

2.5 DISCUSSION

The purpose of this study was to examine the potential benefits of substituting vegetables for sucrose in a high fat diet that produces profound obesity and MetS in 45

susceptible animals. Obese Zucker rats develop obesity rapidly, even on a moderate diet, and quickly develop hypertension and symptoms of T2D. Here we showed that substituting purple or white potatoes for sucrose as the major source of carbohydrate, improved insulin sensitivity, with purple potatoes being superior to white potatoes.

Comparing the impaired glucose tolerance among the groups can be done by either using “the time course of the blood glucose measurements” or the glucose AUC

[120]. In our study, we used both ways to express ipGTT and ipITT results. During ipGTT, blood glucose levels of the PP group were significantly lower at the last 2 time points of the test compared to both the WP and the HFD groups. This indicated that the glucose clearance from the circulation was faster with the PP compared to the other 2 groups. Glucose tolerance is a reflection of two factors: 1) insulin sensitivity of the target tissues and 2) insulin secretion from the pancreatic β cells in response to glucose [120].

Therefore the ipGTT does not determine the underlying mechanisms for the observed changes in the glucose tolerance (i.e. the causative mechanism can be either changes in insulin sensitivity, insulin secretion or a combination of both). In our experiment, we did not measure insulin levels at each time point during the ipGTT, so we are unable to discriminate between these possibilities (i.e the WP and the HFD may have had worse insulin sensitivity, worse insulin secretion or a combination of both).

Based on ipITT results, both the PP and WP had faster glucose clearance from the circulation compared to the HFD group (manifested by significantly lower glucose levels at the last two time points of the test for the PP and only at the last time point for the WP compared to the HFD group). So the lower glucose tolerance of the WP, compared to the

PP could be due to impaired insulin secretion rather than impaired insulin sensitivity 46

(after eliminating insulin secretion as a confounding factor, glucose clearance of the WP became better than the control group). Impaired pancreatic function is typically seen as a late event in this pathologic condition, that occurs after the pancreas gets exhausted trying to compensate for insulin resistance [121]. However, these animals were still relatively young so pancreatic exhaustion seems unlikely.

We also used fasting plasma insulin levels and HOMA IR to assess insulin resistance. HOMA IR is an accepted, reliable, surrogate measure of insulin resistance in rodents [122] that correlates well with results from the hyperinsulinemic euglycemic clamp [123]. According to both assays, the PP and the WP groups were more insulin sensitive compared to the control group and this is also consistent with the findings of the ipITT. These findings are in agreement with several studies that reported an insulin sensitizing effect of other anthocyanin-rich plants [53,55,56,58,74,79].

In contrast to the potatoes, carrots substituted for sucrose in a high fat diet did not exert a statistically significant positive effect on glucose homeostasis or insulin sensitivity, although the trends were very similar for carrots and potatoes. The effect of obesity on insulin resistance could be the explanation for the lack of a positive effect on insulin resistance with the carrot groups. The carrot groups had significantly higher food intake and body weight gain compared to the HFD group (i.e. they ate more and were more obese, Table 2.4). The additional obesity could have counteracted any possible benefit of carrots on glucose homeostasis. This hypothesis is further supported by the potato arms of the study. WP was less effective at improving glucose tolerance than PP and the WP group, like the carrots, also had higher food intake and body weight gain over the course of the study (Table 2.3). Another possible explanation of the differential 47

effects of both purple vegetables on glucose tolerance is the bio-accessibility of their anthocyanins. In a recent in vitro study, the bio-accessibility of anthocyanins derived from the PP was higher than that of the PC [124]. The absorbed anthocyanins were also different between the two vegetables [124].

High blood pressure is one of the major components of MetS and a significant risk factor for the development of CVD [1,3]. In a recent SPRINT study, lowering blood pressure in nondiabetic, high risk CVD patients reduced major CVD events and overall mortality [125]. Obesity and insulin resistance are recognized as the main factors contributing to the development of hypertension in MetS by inducing endothelial dysfunction, sympathetic nervous and renin-angiotensin system overactivity, and high levels of inflammatory cytokines [38,39,126]. In our study, despite the lack of a significant positive effect of the PC on insulin variables, PC did show significantly lower

LVP compared to both the HFD and the OC groups and lower systolic and diastolic pressure than the HFD group. The OC group did have improvements in diastolic blood pressure that were similar to PC, but the effects on systolic blood pressure and LV pressure were less obvious than for the PC. A selective improvement of blood pressure without affecting insulin sensitivity was previously reported with blueberry beverage supplementation in obese humans [86]. In spite of the marked obesity and insulin resistance, PC were still able to reduce blood pressure. This means that lowering blood pressure can be achieved independently from processes that impact insulin resistance.

Although in our study, the underlying mechanism is still unknown, in other in vitro and ex vivo studies, anthocyanins increased NO synthase levels in both human and bovine cells while decreasing endothelin 1 production [127,128]. Also, blueberry diets decreased 48

the vasoconstrictor response in the aortic rings of rats via a NO synthase-dependent mechanism [129].

Unlike the significant favorable effect on glucose tolerance, the PP diet did not affect blood pressure as dramatically as the WP. In fact, it was intermediate between the

HFD and WP groups. This suggests that the bioactives responsible for improvements in glucose homeostasis may be different from those that affect hemodynamic parameters.

Clearly, anthocyanins are not the only important phytochemicals in these vegetables. It is worth mentioning that the reduction of the AV difference by the PP could be a sign of healthier aortic valves. Aortic valve stenosis or calcification results in left ventricular pressure overload and a high pressure gradient across the valve [130].

Both purple vegetables and their lightly colored counterparts significantly decreased liver weight compared to the HFD. This could be a sign of normalizing the hepatic enlargement induced by fat deposition. Fatty liver or hepatic steatosis is a condition that is positively correlated with obesity and MetS [131]. An improvement in hepatic steatosis, inflammation and enzyme activity was reported with tart cherries [55] and purple carrot juice [102] in animal studies. We did not histologically examine the livers to examine lipid deposition or other evidence of steatosis, although this would be something interesting to do in the future.

To begin an investigation of possible mechanisms for the observed effects on glucose homeostasis and blood pressure, we chose a few candidate genes to explore. These included proteins known to affect lipid homeostasis and inflammation; e.g. PPAR γ, adiponectin, perilipin, FAS, phosphorylated ACC and phosphorylated AMPK. PPAR γ is a master regulator of lipid metabolism that promotes adipocyte differentiation and the fat 49

storage ability of the adipose tissue, thereby enhancing insulin sensitivity [44].

Adiponectin is an anti-inflammatory adipose derived cytokine that also has insulin sensitizing effects [132], whereas ACC is a key enzyme in fatty acid synthesis. Its phosphorylation (i.e. inactivation) by phosphorylated AMPK increases fatty acid oxidation by affecting CPT1 activity [72]. However, none of the studied proteins’ expression was modulated by the experimental vegetables. This probably means that the vegetables, in our experiment, exerted the observed effects through other mechanisms that still need to be explored.

2.6 CONCLUSIONS

Feeding diets containing white or purple potatoes as replacements for sucrose

(simple sugars) can reduce the severity of risk factors associated with MetS in an animal model. Both potato cultivars improved blood pressure and improved insulin sensitivity with the purple potatoes being slightly better at improving insulin sensitivity and the white potatoes were better at improving hemodynamic variables. On the other hand, carrots as substitutes for sucrose had lesser effects on glucose tolerance while retaining the benefits of vegetable consumption on blood pressure. The reduced benefit of white potatoes on insulin sensitivity compared to purple potatoes could be an effect of anthocyanins (higher in purple cultivar) or a result of the higher obesity in WP compared to PP fed animals. The lack of benefit of carrots on glucose tolerance is most likely confounded by their significantly higher obesity than either HFD or potato fed animals.

In contrast to the possible role of anthocyanins in glucose regulation, they are unlikely to be the bioactives in these vegetables responsible for the blood pressure lowering activity of both vegetable types and both high and low anthocyanin varieties. The examination of 50

total phenolic, flavonoid, anthocyanin and carotenoid content do not point to any particular class of compound and thus a more thorough characterization of the individual species and their bioactivities will be necessary to identify the critical bioactives. It is also highly likely that the compounds are acting in additive or synergistic ways that cannot be achieved by feeding purified compounds or extracts. None-the-less, our study supports the inclusion of whole cooked potatoes or raw carrots in the human diet as part of a strategy to decrease obesity related disorders including hypertension and T2D.

Table 2.1 Composition of the experimental diets

Component in g/kg diet Control WP1 PP2 OC3 PC4 Casein (protein) 140 140 140 140 140 L- Cystine 1.8 1.8 1.8 1.8 1.8 Lard 120 120 120 120 120 Soybean Oil 40 40 40 40 40 Maltodextrin 10 150 150 150 150 150 Sucrose 450 - - 150 150 Freeze dried baked white potato - 450 - - - Freeze dried baked purple potato - - 450 - - Freeze dried raw orange carrot - - - 300 - Freeze dried raw purple carrot - - - - 300 Cellulose, BW200 50 50 50 50 50 Vitamin Mix v10037 10 10 10 10 10 Mineral Mix s10022M 35 35 35 35 35 Choline bitartrate 2.5 2.5 2.5 2.5 2.5

1 WP is high fat diet supplemented with white potatoes, 2 PP is high fat diet supplemented with purple potatoes, 3 OC is high fat diet supplemented with orange carrots, and 4 PC is high fat diet supplemented with purple carrots.

Table 2.2 Total Phenolic, Flavonoid, Anthocyanin and Carotenoid content of the experimental vegetables1.

Vegetable TPC2 TFC3 TAC4 TCC5 White Potatoes 2.38± 0.02a 0.43± 0.07a _ 1.02± 0.27a Purple Potatoes 5.2± 0.01b 2.08± 0.14b 1.47± 0.06a 2.87± 0.33a Orange Carrots 1.6± 0.03c 0.57± 0.06a _ 52.9± 6.25b Purple Carrots 9.92± 0.42d 5.27± 0.16c 3.35± 0.22b 145.03± 6.25c

1Values are means ± SD, no=3. Means within column without a common letter differ. P <0.05.

2Total phenolic content. Values are expressed as milligram Gallic acid equivalent per gram of dry weight (mg GAE/g DW).

3Total flavonoid content. Values are expressed as milligram Catchin equivalent per gram of dry weight.

4Total anthocyanin content. Values are expressed as milligram cyanidin-3-glucoside equivalent per gram of dry weight (mg C3G/g DW).

5Total carotenoid content. Values are expressed as µg β carotene equivalent per gram of dry weight.

Table 2.3. Body weight gain and daily food intake of rats fed HFD, WP and PP for 8 wk1

Diet Body weight gain, g Food intake per day, g HFD 392.0 ± 10.8b 28.3 ± 0.5b WP 435.2 ± 10.8a 30.7 ± 0.5a PP 403.8 ± 10.4b 27.5 ± 0.5b 1Values are least square means (LSM) ± standard error (SE), n = 14-15. Means within column without a common letter differ. P ˂ 0.05. ; HFD, high fat diet; WP, white potato supplemented high fat diet; PP, purple potato supplemented high fat diet.

Table 2.4. Body weight gain and daily food intake of rats fed HFD, OC and PC for 8 wk1

Diet Body weight gain, g Food intake per day, g HFD 392.0 ± 12.0b 28.3 ± 0.6b OC 436.1 ± 11.6a 30.3 ± 0.6a PC 449.3 ± 11.6a 31.6 ± 0.6a 1Values are least square means (LSM) ± standard error (SE), n = 14-15. Means within column without a common letter differ. P ˂ 0.05. ; HFD, high fat diet; OC, orange carrot supplemented high fat diet; PC, purple carrot supplemented high fat diet

A 35 HFD WP 30

PP a a a a 25 a a a a a ab a a 20 b a a

15 a b

10 a b

(mmoL) levels glucose Blood b 5 ab

0 0 10 20 30 60 90 120

Time Postglucose Injection (min.)

B HFD 30 a a a OC a a a a

25 PC a a a a a a a 20 a a a a 15

10 a a

Blood glucose levels (mmoL) levels glucose Blood 5 a

0 0 10 20 30 60 90 120 Time Postglucose Injection (min.)

FIGURE 2.1. Changes in blood glucose levels during GTT of rats fed (A) HFD, WP and

PP (B) HFD, OC and PC for 8 wk. n= 14-15. Values are means ±SE. Means within each time point without a common letter differ. P<0.05. HFD, high fat diet; WP, white potato supplemented high fat diet;

OC, orange carrot supplemented high fat diet; PP, purple potato supplemented high fat diet; PC, purple carrot supplemented high fat diet 55

A HFD

2500 WP ANOVA P=0.089 PP 2000

1500

1000

500

0 GTT_AUC

B HFD 2000 OC 1950 PC 1900

1850

1800

1750

1700

1650 AUC_GTT

FIGURE 2.2 Total plasma glucose concentration during GTT expressed by glucose

AUC of rats fed (A) HFD, WP and PP (B) HFD, OC and PC for 8 wk. n=14-15. Values are means ±SE. HFD, high fat diet; WP, white potato supplemented high fat diet; OC, orange carrot supplemented high fat diet; PP, purple potato supplemented high fat diet; PC, purple carrot supplemented high fat diet.

HFD A 12 WP a PP

a a a a a a a 8 a a a ab a b a 6 a b b

4 Blood glucose levels (mmoL) levels glucose Blood 2

0 0 5 10 15 30 45 Time Postinsulin Injection (min.)

HFD B 14 OC 12 a a

a a a PC 10 a a a a a a 8 a a a a a a a 6

4 Blood glucose levels (mmoL) levels glucose Blood 2

0 0 5 10 15 30 45 Time Postinsulin Injection (min.)

FIGURE 2.3. Changes in blood glucose levels during ITT of rats fed (A) HFD, WP and PP (B) HFD, OC and PC for 8 wk. n= 14-15. Values are means ±SE. Means within each time point without a common letter differ. P<0.05. HFD, high fat diet; WP, white potato supplemented high fat diet; OC, orange carrot supplemented high fat diet; PP, purple potato supplemented high fat diet; PC, purple carrot supplemented high fat diet 57

A HFD 120 WP

100 PP

0 AAC_ITT

B HFD

OC 100 PC 90

80 70

50 40

10 0

AAC_ITT

FIGURE 2.4. Plasma glucose disposal during ITT expressed by glucose AAC of rats fed

(A) HFD, WP and PP (B) HFD, OC and PC for 8 wk. n= 14-15. Values are means ±SE. HFD, high fat diet; WP, white potato supplemented high fat diet; OC, orange carrot supplemented high fat diet;

PP, purple potato supplemented high fat diet; PC, purple carrot supplemented high fat diet. 58

HFD HFD A C 50 a WP 50 OC 45 45 PP PC 40 40

35 35

30 b 30

25 b 25 ng/mL ng/mL 20 20 15 15 10 10

5 5 0 0 fasting plasma insulin fasting plasma insulin

B HFD D HFD

a 400 WP 400 OC 350 350 ab PP PC 300 300

250 250 b 200 200 150 150

100 100

50 50 0 0

HOMA IR HOMA IR

FIGURE 2.5. Fasting insulin levels (ng/mL) and HOMA IR of rats fed (A&B) HFD, WP and PP (C&D) HFD, OC and PC for 8 wk. n= 7-8. Values are means± SE. Bars without a common letter differ. P ≤ 0.05. HFD, high fat diet; WP, white potato supplemented high fat diet; OC, orange carrot supplemented high fat diet; PP, purple potato supplemented high fat diet; PC, purple carrot supplemented high fat diet.

FIGURE 2.6. Systolic, diastolic, left ventricular pressures and AV difference of rats fed (A&B) HFD, WP and PP (C&D) HFD, OC and PC for 8 wk. n= 9-14. Values are means± SE. Bars without a common letter differ. P ≤ 0.05. HFD, high fat diet; WP, white potato supplemented high fat diet; OC, orange carrot supplemented high fat diet; PP, purple potato supplemented high fat diet; PC, purple carrot supplemented high fat diet.

A HFD 6 a WP PP

b 4 b

weight body the of %

muscle heart epi. Fat liver

HFD B 6 OC a PC 5 b b 4

weight body the of % 1

0 muscle heart epi. Fat liver

FIGURE 2.7 Organ weights’ percentage of the body weight of rats fed (A) HFD, WP and PP (B) HFD, OC and PC for 8 wk. n= 14-15. Values are means± SE. Bars without a common letter differ. P ≤ 0.05. HFD, high fat diet; WP, white potato supplemented high fat diet; OC, orange carrot supplemented high fat diet; PP, purple potato supplemented high fat diet; PC, purple carrot supplemented high fat diet

A 4.5 4 3.5

2.5 Control

2 WP protein level protein 1.5 PP 1 0.5 0 AMPK alpha AMPK beta Adiponectin PPAR FAS Perillipin phos phos gamma

B 4.5 4 3.5

2.5 Control 2 OC

protein level protein 1.5 PC 1 0.5 0 AMPK alpha AMPK beta Adiponectin PPAR FAS Perillipin phos phos gamma

FIGURE 2.8. Expression of the proteins of interest in the adipose tissue in rats fed

(A)control, WP and PP diets (B) HFD, OC and PC for 8 wk. n= 7. Values are means±

SE. Proteins are expressed relative to the corresponding total or GAPDH. HFD, high fat diet;

WP, white potato supplemented high fat diet; OC, orange carrot supplemented high fat diet; PP, purple potato supplemented high fat diet; PC, purple carrot supplemented high fat diet.

A 12

6 Control

WP protien level protien 4 PP

0 ACC phosph ampk alpha Ampk beta FAS phospho phospho

B 12

6 Control

OC protein level protein 4 PC

0 ACC phosph ampk alpha Ampk beta FAS phospho phospho

FIGURE 2.9. Expression of the proteins of interest in the liver tissue in rats fed

(A)control, WP and PP diets (B) HFD, OC and PC for 8 wk. n= 7. Values are means±

SE. Proteins are expressed relative to the corresponding total or GAPDH. HFD, high fat diet;

WP, white potato supplemented high fat diet; OC, orange carrot supplemented high fat diet; PP, purple potato supplemented high fat diet; PC, purple carrot supplemented high fat diet.

CHAPTER 3

Proteomic Profiles of the Adipose and Liver Tissues from an Animal Model of Metabolic Syndrome Fed Purple Vegetables1

3.1. ABSTRACT

Metabolic Syndrome is a complex disorder that predisposes to CVD and type 2

DM. Proteomics and bioinformatics have proven as an effective tool to study complex diseases and mechanisms of action of nutrients. Since proteins are the most common end products of changes in gene expression, their levels and/or activity are better reflections of actual cellular biological processes, than studying mRNA levels. We previously showed that substitution of the majority of carbohydrate in a high fat diet by purple potatoes (PP) or purple carrots (PC) improved insulin sensitivity and hypertension in an animal model of Metabolic Syndrome (obese Zucker rats) compared to a control sucrose rich diet but we failed to identify possible mechanisms using western blotting of a set of candidate proteins. In the current study, we used TMT 10plex mass tag combined with

LC/MSMS technique to study proteomic modulation in the liver (n= 3 samples/group) and adipose tissue (n=3 samples/group), of the PP and PC fed rats, in a trial to find the molecular mechanisms of the phenotypic effects of these purple vegetables. We also performed functional enrichment analysis (using DAVID) to find out the enriched biological processes and pathways in the obtained list of the differentially expressed

------1 This chapter has been published in Nutrients: Hala M Ayoub, Mary Ruth McDonald, James Alan Sullivan, Rong Tsao and Kelly A Meckling. Proteomic Profiles of Adipose and Liver Tissues from an Animal Model of Metabolic Syndrome Fed Purple Vegetables. Nutrients 2018, 10(4), 456; doi: 10.3390/nu10040456

proteins by the 2 vegetables. Protein folding, lipid metabolism, and cholesterol efflux were identified as main modulated biological themes in adipose tissue whereas, in the liver, lipid metabolism, carbohydrate metabolism and oxidative stress were the main modulated themes. We propose that enhanced protein folding, increased cholesterol efflux and higher free fatty acid (FFA) re-esterification are mechanisms by which PP and

PC affect Metabolic Syndrome pathologies in adipose tissue, whereas, a decreased de novo lipogenesis, oxidative stress and FFA uptake to be the suggested mechanisms of action in the liver. In conclusion, we provide molecular evidence for the reported metabolic benefits of these purple vegetables. We validate and support our previous study’s recommendation that inclusion of these purple vegetables in the human diet is a good choice for better metabolic health.

Key words: Cardiovascular disease, hypertension, insulin resistance, high fat diet, purple carrots and potatoes, proteomic analyses

3.2. INTRODUCTION

MetS is a complex disorder that predisposes to T2D and CVD. IR is frequently identified as a leading factor in these pathologies [2]. Use of proteomic and bioinformatics tools in protein expression studies enables greater understanding of biological mechanisms of complex diseases and also mechanisms of action of drugs or nutrients [133,134]. Proteins are the final and active product of most of the genome and thus the most accurate reflection of what is happening at the molecular level. Poor correlation between mRNA and protein expression attributed to impaired translation

efficiency [135] emphasizes the significance of directly determining the protein abundance. Western blotting has been used as an effective tool for studying the protein expression for the last 30 years. However, it does not give the complete picture. It only allows for investigating a group of candidate proteins that may represent one side of the story.

Proteomic analysis studies reported hepatic proteome changes in MetS induced by high fat and high fructose diets in rodents [136,137]. The modulated proteins were involved in glucose metabolism, lipid metabolism, oxidative stress and ER stress.

Polyphenols and anthocyanin rich plants modified the protein and/or mRNA expression of several genes known to be involved in the processes of lipid metabolism, inflammation, and energy homeostasis in the liver and/or adipose tissue. That was associated with an improvement in various metabolic risk factors such as glucose tolerance, insulin sensitivity, and hepatic steatosis [53-56,73,74,79]. However, to our knowledge, there has not been a study that looked at the whole proteome changes in response to anthocyanin rich plants-supplemented diets. Studying the whole proteome will enable formulating a fair, unbiased and comprehensive picture of the molecular mechanisms responsible for these plants’ biological activity.

We previously showed that obese Zucker rats fed a high fat diet supplemented with purple potatoes (PP) or purple carrots (PC) as major sources of carbohydrate had better insulin sensitivity and lower blood pressure compared to control unsupplemented high fat diet fed rats [138]. The current study aimed to examine the proteomic changes in the liver and adipose tissues of a MetS animal model in response to PP, PC or sucrose containing diets feeding using TMT10 plex mass tag labeling combined with LC/MSMS technique. 66

This technique enables the concurrent identification and comparative quantitation of the peptides from 10 different samples. These profiles are then used to generate potential molecular mechanisms for the observed phenotypic changes induced by these vegetables

(i.e. improvement in insulin sensitivity and blood pressure).

3.3. Methods

3.3.1 Experimental design, sample collection, and tissue homogenization

Liver and adipose tissue (epididymal fat depot) samples were collected from rats ad libitum fed 3 exact experimental modified high fat AIN-93M diets (n=15 rats/diet) that only differed for the carbohydrate source for 8 weeks (Table 1). The control diet had sucrose whereas PP and PC diets had purple potatoes and purple carrots, respectively, as main sources of carbohydrate. A subsample of the liver (n= 3 per diet group) and adipose tissues (n= 3 per diet group) were randomly selected and homogenized (Fast Prep ®24;

MP biomedical, Santa Ana, CA) using NP40 cell lysis buffer (Invitrogen, CA) (3 volumes for adipose and 30 volumes for liver samples) supplemented with protease inhibitor cocktail and phenyl methyl sulfonyl fluoride (Sigma-Aldrich). The lysates were centrifuged at 5000 g for 10 min. at 4o C [117,118]. The total protein content of the infranatant was determined using a BCA protein assay kit (Thermo Fisher Scientific).

The lysates were then sent to the SPARC BioCentre, SickKids Hospital (Toronto,

Ontario) to perform the TMT labeling and LC/MSMS analyses.

3.3.2. Sample preparation (denaturation, alkylation, and digestion) and TMT labeling

The samples were solubilized with 1% sodium dodecyl sulfate (SDS) and 8M urea with sonication. The proteins were reduced in 1mM dithiothreitol (DTT) for 1 h at 56 °C 67

and the free cysteine residues were alkylated by incubating with iodoacetamide for 30 min. protected from light at room temperature. The proteins were precipitated with 5 volumes of prechilled acetone overnight at - 20 °C. The samples were centrifuged at 8000

× g for 10 minutes at 4°C. The pellets were allowed to dry for 2-3 min before dissolved with triethylammonium bicarbonate (TEAB). The samples were then digested with trypsin 2.5 µg for 100 µg of protein overnight at 37oC. Fifty μg of protein from each sample was labeled using 0.4 mg of TMT 10plex (ThermoFisher) by incubating at room temperature for 1 h. The labeling reaction was stopped using 5% hydroxylamine. The peptides were mixed and the solvent removed under vacuum.

3.3.3. Liquid chromatography and tandem mass spectrometry (LC-MS/MS)

The peptides were analyzed on an Orbitrap analyzer (Q-Exactive, ThermoFisher, San

Jose, CA) outfitted with a nanospray source and EASY-nLC nano-LC system

(ThermoFisher, San Jose, CA). a 75μm x 50 cm PepMax RSLC EASY-Spray column filled with 2μM C18 beads (ThermoFisher San, Jose CA) was used to load the peptide mixture at a pressure of 800 Bar. Peptides were then subjected to a stepwise gradient elution over 240 min at a rate of 250nl/min (0%-4% acetonitrile containing 0.1% formic acid over 2 minutes; 4%-28% acetonitrile containing 0.1% formic acid over 226 minutes,

28%-95% acetonitrile containing 0.1% formic acid over 2 minutes, constant 95% acetonitrile containing 0.1% formic acid for 10 minutes). In the Q-Exactive mass spectrometer (Thermo-Fisher), one MS full scan (525–1600 m/z) was performed with an automatic gain control (AGC) target of 1e6, maximum ion injection time of 120 ms and a resolution of 35 000 with subsequent 15 data-dependent MS/MS scans with a resolution

of 35,000, an AGC target of 1e6, maximum ion time of 120ms, and one microscan. The intensity threshold required to trigger a MS/MS scan was at an underfill ratio of 0.2%. In the higher energy collision dissociation (HCD) trap, normalized collision energy of 30V was used for the fragmentation. The dynamic exclusion was applied with an exclusion period of 40 s [139].

3.3.4. Protein identification and quantitation

The MS/MS data were searched against the Rat UniProt database using Proteome

Discoverer version 1.4 (Thermo-Fisher Scientific) which also extracted the quantitation data from the 10 TMT tags. The data was imported into Scaffold Q+ (Proteome

Software) for label based quantitative analysis. Protein identifications were accepted if they contained at least 2 identified peptides above 95% tandem mass spectrometry confidence (with 0% decoy false discovery rate (FDR)). Differentially expressed proteins were determined by applying T-Test with unadjusted significance level p < 0.05 corrected by Benjamini-Hochberg.

3.3.5. In-silico functional analyses

We performed in silico functional analyses of the differentially expressed proteins in order to explore the biological meaning behind the modulation of expression of these proteins by the purple vegetable diets. The Database for Annotation, Visualization and

Integrated Discovery (DAVID) [140] was used to perform functional enrichment analyses. The enriched (i.e. overrepresented) Kyoto Encyclopedia of Genes and

Genomes (KEGG) pathways and gene ontology (GO) terms biological processes component in the list of the differentially expressed proteins were identified. To account

for multi-hypotheses testing, the P-values of the enrichment analyses were adjusted using

Benjamini-Hochberg (P< 0.05).

3.4. RESULTS AND DISCUSSION

3.4.1. Purple potato (PP) diet versus control diet

3.4.1.1 Adipose tissue protein expression

A total of 1944 proteins were identified in the adipose tissue of the rats fed the PP and the control diets (Supplemental Table 1), in which 85 proteins were differentially expressed. Forty-six proteins were down regulated and 39 proteins were upregulated with the PP diet (Table 3.2). Three KEGG pathways and 220 biological processes were enriched in the proteins list (at Benjamini P-value <0.05) (Supplemental Table 2). Some of the enriched pathways and processes observed were mainly involved in protein folding, lipid metabolism and cholesterol efflux (Table 3.3).

Protein folding and endoplasmic reticulum (ER) stress. “Protein processing in ER” pathway and “protein folding” biological process were strongly enriched in the differentially expressed protein list (Table 3.3). All the proteins involved in both the pathway and the biological process were upregulated with the PP diet. UDP-glucose glycoprotein glucosyltransferase 1(Uggt1), calnexin (Canx) and calreticulin (Calr) are involved in quality control process of protein folding in ER through recognizing, retaining and refolding the immaturely folded proteins [141]. Uggt1 recognizes proteins with folding defects, retains them and directs them to Canx/Calr cycle to be refolded properly. Heat shock protein family A member 5(Hspa5), PDI family (pdia 3,4 &6) and heat shock protein 90, beta, member 1(Hsp90b1) are also recognized as major molecular

chaperones [142]. Both Hspa5 and Hsp90b1 catalyze protein folding while PDI family catalyzes the formation of disulfide bonds and thereby regulating protein folding as well.

Accumulation of misfolded or unfolded proteins results in ER stress. ER stress response or UPR is known as a common mechanism of the pathogenesis of insulin resistance. For instance, UPR recruits and activates a number of stress kinases that eventually impair the insulin signaling pathway through inducing serine phosphorylation of IRS1. Moreover, activation of the stress kinases promotes proinflammatory cytokines synthesis that also negatively affects insulin signaling [21].

Lipid metabolism

Lipid synthesis. Both “fatty acid biosynthetic” and “lipid biosynthetic” processes were enriched in the differentially expressed protein list (Table 3.3). Acetyl CoA carboxylase alpha (Acaca) and fatty acid synthase (Fasn) were down regulated with the

PP diet. This indicated that de novo fatty acid synthesis was probably down regulated with the PP diet. However, the upregulation of phosphoenolpyruvate carboxykinase

1(Pck1) could be an indication of an increased fatty acid re-esterification, that could be coupled with the increased glyceroneogenesis. In adipose tissue, cytosolic Pck1 is a key enzyme in glyceroneogenesis that involves the synthesis of glycerol 3 phosphate (G-3-P) by decarboxylating amino acids to phosphoenolpyruvate (PEP) that then converts to dihydroxyacetone phosphate (DHAP), a precursor of G-3-P [143]. The synthesized G-3-P is utilized for fatty acid re-esterification and TG synthesis in white adipose tissue [143].

In fact, overexpression of Pck1 in adipose tissue of mice was shown to increase FFA re- esterification, glyceroneogenesis and obesity while decreasing circulating FFA levels and preserving glucose tolerance and whole body insulin sensitivity [144]. Lipid localization 71

to the adipose tissue will probably decrease the lipid accumulation in other tissues (i.e. lipotoxicity). Intracellular accumulation of lipid intermediates like DAG and ceramides are known to interrupt insulin signaling [12]. Both apolipoprotein C1 (Apoc1) and apolipoprotein C2 (Apoc2) were upregulated with the PP diet. Apoc2 is required for lipoprotein lipase (LPL) activation. The LPL hydrolyzes TG to FFA that are uptaken and deposited to the adipose tissue [145]. However, Apoc1 exerts the opposite effect of Apoc2 on LPL activity. So it is not clear if LPL is activated or inhibited.

Hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) downregulation is an indication of a probable decrease in ketogenesis with the PP diet. Hmgcs2 is a rate-limiting enzyme of the ketone bodies biosynthesis [146]. It is a mitochondrial form of the enzyme that catalyzes the condensation of acetyl CoA with acetoacetyl CoA to form HMGCOA

[146]. Ketogenesis is induced in long fasting, prolonged exercise, and diabetes. Ketone bodies are used as fuels in these cases [146]. Also, Hmgcs2 expression increased with starvation and decreased in response to insulin [146]. So it seems that PP fed rats did not need ketone bodies for energy compared to the control group. Or perhaps they just had less acetyl CoA generated from β-oxidation that led to less ketone bodies synthesis.

Lipid catabolism. “Lipid catabolic” process and both “TG catabolic” and “TG metabolic” processes were enriched in this protein list. Upregulation of both plin1 and ces1d proteins could be indicative of higher lipolysis activity with the PP diet. Both perilipin 1(Plin1) and carboxylesterase 1D (Ces1d) are known to be lipolytic proteins

(Table 3.3). However, Plin1 has a complex role in lipolysis as it exerts opposing effects on basal and catecholamine stimulated lipolysis. Under the basal state, Plin1 decreases lipolysis through coating the lipid droplets and preventing the access of the lipolytic 72

enzymes (e.g. hormone sensitive lipase) to the stored lipids. At the same time, TG levels are relatively unchanged. Most of the liberated FFA, resulting from TG hydrolysis, are actually being recycled to TG [147] whereas, under stimulated conditions (i.e. during fasting or exercise), the phosphorylated Plin1 gives access to hormone sensitive lipase and TG lipase to the lipid core allowing lipolysis [147]. Perillpin ablation in mice resulted in higher basal lipolysis and lower stimulated lipolysis. Perilipin null mice were lean but less glucose tolerant [148]. Ces1d was identified as a major lipolytic enzyme in mice

[149]. However, it was not confirmed that it has the same effect on the lipolytic activity in human adipose tissue [150]. Furthermore, the concomitant upregulation of Apoc2 and

Pck1 may support the idea that the liberated FFA are not released to the circulation and instead they may actually be re-esterified and deposited to the adipose tissue. However, the complement C3 (C3) downregulation can be associated with a decrease in TG synthesis. Although C3a is thought to be a lipogenic factor that stimulates TG synthesis, due to the discrepancy in the C3 deficient mice studies, its role on lipid metabolism and

IR is still not clear [151].

So generally we can see some evidence of lower FFA release and lower de novo fatty acid synthesis with the PP diet that may explain the higher insulin sensitivity with this diet.

Cholesterol efflux/ reverse cholesterol transport (RCT). Both “cholesterol efflux” and “RCT” processes are enriched in this differentially expressed proteins list (Table

3.3). Apolipoprotein A1 (Apoa1), Apoa2, Apoc1, and Apoc2 were upregulated while apolipoprotein E (Apoe) was down regulated with the PP diet. Since Apoa1 and Apoa2 are the most abundant apolipoproteins in HDLc [145], perhaps the higher protein 73

abundance is simply an indication of overall higher HDLc levels with the PP compared to the control diet. As reported previously, the PP group was more insulin sensitive than the control group; it would not be surprising to see an associated improved lipid profile (i.e. higher HDLc). The association of dyslipidemia with IR is thought to be due to the high

VLDL hepatic secretion and the high postprandial chylomicron levels coupled with the exchange of cholesterol esters from HDLc with TG from TG rich lipoproteins. This leaves a more hydrolysis and dissociation prone TG rich HDL particle, and thus reduces the number of HDL particles [37]. Apoa1 transcription was shown to be modulated by dietary and hormonal factors [152]. Also increased human Apoa1 expression in transgenic mice increased HDLc levels and inhibited atherosclerosis [152]. So at this point, it is not clear if the high Apoa1 and Apoa2 are the result of a higher insulin sensitivity and higher HDLc with the PP diet or due to a direct effect of the PP on the expression of Apoa1 and Apoa2. Also since Apoe is typically found on TG rich lipoproteins (chylomicrons, IDL, VLDL) [145], its decreased expression may be just a reflection of lower levels of these lipoproteins with the PP diet. Apoa1 has a major role in cholesterol efflux (i.e. cholesterol acceptor) and is also a main lecithin cholesterol acyl transferase (LACT) activator that catalyzes cholesterol esterification and promotes more cholesterol uptake by HDL particles [145]. However, Apoa2, Apoc1, Apoc2, and Apoe were all shown to be involved in cholesterol efflux as well [153]. This strongly suggests that cholesterol efflux is enhanced with the PP diet. Cholesterol efflux is the first step of

RCT that involves the removal of the excess cholesterol from the tissues and delivering it back to the liver for excretion [145].

Cholesterol efflux capacity was progressively reduced in patients with MetS with the increasing number of MetS components [154]. It also was negatively correlated with fasting blood glucose and systolic blood pressure [154]. Efflux capacity is inversely associated with the risk of coronary heart disease (CHD) [155]. Although the capacity is positively correlated with the APOA1 concentration, it is the capacity, rather than the concentration, that is suggested to be the accurate predictor of CHD [155].

Taken together, these data suggest that reduced ER stress, decreased de novo lipogenesis, a decrease in basal lipolysis, increased fatty acid re-esterification, and probably increased cholesterol efflux in adipose tissue, each contributes to the mechanisms responsible for improving MetS pathologies (insulin sensitivity and hypertension), with PP feeding (Figure 3.1) .

3.4.1.2 Liver protein expression

A total of 941 proteins were identified in the livers of rats fed the PP and the control diets (Supplemental Table 3) of which 69 proteins were differentially expressed. 37 proteins were down regulated and 32 proteins were upregulated with the PP diet (Table

3.4). A total of 26 KEGG pathways and 134 biological processes were enriched in the proteins list (at Benjamini P-value <0.05) (Supplemental Table 4). Some of the enriched pathways and processes were involved in lipid and carbohydrate metabolism (Table 3.5).

Lipid metabolism

Lipid synthesis. Both “fatty acid biosynthetic” and “lipid biosynthetic” processes are enriched in the list of the differentially expressed proteins (Table 3.5). Downregulation of

Fasn, pyruvate carboxylase (Pc) and ATP citrate lyase (Acly) with PP likely indicates a

decrease in the de novo fatty acid synthesis. Pc catalyzes the conversion of pyruvate to oxaloacetate that condenses with acetyl CoA to produce citrate. In the cytoplasm, Acly converts citrate back to acetyl CoA which is then used in fatty acid synthesis [156]. In db/db mice, ablation of hepatic citrate lyase prevented de novo lipogenesis and hepatic steatosis and promotes insulin sensitivity in muscle [157].

Farnesyl diphosphate synthase (Fdps) and solute carrier family 27 member 5

(Slc27a5) were both upregulated. Fdps catalyzes the formation of farnesyl pyrophosphate that constitutes a branching point of the isoprenoid pathway that yields both sterol and non-sterol metabolites [158]. Slc27a5 is a bile acyl CoA synthase that is involved in bile acid conjugation and activation before excretion into the bile canaliculi [159]. So even though the upregulation of Fdps can be a sign of increased de novo cholesterol synthesis, the upregulation of slc27a5 suggests an increased incorporation of the synthesized cholesterol in the bile acid synthesis. Bile acid formation from cholesterol is a main cholesterol excretion route [158]. Both acyl-CoA synthetase long-chain family member

1(Acsl1) and acyl CoA synthetase long-chain family member 5(Acsl5) are upregulated.

Long chain acyl CoA synthases are a group of enzymes that catalyze the formation of acyl CoAs that can then be directed to either lipid synthesis or oxidation [160]. Acsl1 is suggested to be mainly involved in TG synthesis whereas Acls5 is suggested to be involved in β-oxidation [160]. However, data from a loss of function in vitro study observed a role for Acsl5 in directing fatty acids to TG synthesis [161]. In another loss of function study, hepatic Acsl1 was suggested to have a role in both β-oxidation and TG synthesis [162]. Because both pathways may be activated, it would be important to know

the relative activation of one pathway over the other (i.e. enzyme activities and/or metabolite levels) to determine whether there would be overall change.

Lipid catabolism. “Lipid catabolic” and “fatty acid beta-oxidation” processes were enriched in the list of the differentially expressed proteins extracted from the liver tissues of the PP group (Table 3.5). Upregulation of acyl CoA oxidase 3, pristanoyl (Acox3) and cytosolic isocitrate dehydrogenase (Idh1) is probably a sign of higher peroxisomal fatty acid β-oxidation in the liver with the PP diet. Acox3 is a rate-limiting enzyme in the β- oxidation pathway of the peroxisomes as it catalyzes the oxidation of methyl-branched fatty acyl CoAs and to a lesser extent straight chain fatty acids[163]. Also, cytosolic Idh was shown to be necessary for peroxisomal β-oxidation of unsaturated fatty acids in rat liver cells through providing NADPH [164]. Upregulation of fatty acid binding protein

1(Fabp1) can be an indirect sign of increased fatty acid β-oxidation. Fabp1 binds long chain fatty acids and facilitates their delivery to the peroxisome or the mitochondria for oxidation thus minimizing their toxic effects [165]. In contrast, acyl CoA dehydrogenase, long chain (Acadl) was found to be down regulated which may be a sign of a decrease in the mitochondrial fatty acid oxidation [163]. The decrease in the mitochondrial oxidation could be due to the decrease in the abundance of the fatty acids as a result of the reduced de novo lipogenesis.

Carbohydrate metabolism. “Carbohydrate catabolic” process and “pentose phosphate” KEGG pathway were enriched in this list as well (Table 3.5). Glycolysis seems to be decreased with the PP diet as glucose-6-phosphate isomerase (Gpi), fructose- bisphosphate aldolase B (Aldob) and pyruvate kinase (Pklr), 3 main enzymes of the glycolytic pathway [166], were all downregulated with the PP diet. Also, 77

dihydrolipoamide S-acetyltransferase (Dlat) was downregulated. Dlat is a component of pyruvate dehydrogenase complex that converts pyruvate to acetyl CoA that gets directed to citric acid cycle or used for denovo lipogenesis. While glycolysis seems to be decreased, glycogen synthesis pathway proteins (i.e. glycogen synthase) do not seem to be higher in PP livers compared to control liver. However, it does seem that glucose is being directed to the pentose phosphate pathway as glucose 6 phosphate dehydrogenase is upregulated. It is true that transketolase (Tkt) is down regulated but it is more involved in the non-oxidative part of the pathway that produces more glycolytic intermediates. The main products of the pentose phosphate pathway are NADPH and ribose 5 phosphates.

NADPH is known to be used in fatty acid and cholesterol biosynthesis and the reduction of oxidized glutathione [167]. Reduced glutathione may confer antioxidant protective effects as it reduces oxidized glutathione peroxidase [168]. It is worth noting that

Glutathione peroxidase (Gpx1), the enzyme that reduces H2O2, is also among the upregulated proteins in this list [168].

Taken together, these data suggest that a decrease in hepatic de novo lipogenesis, a probable increase in the peroxisomal fatty acid oxidation and a decrease in the fatty acid delivery to the liver from the adipose tissue, each contributes to the mechanisms responsible for improving MetS pathologies with PP feeding (Figure 3.1). All of these are indicative of mechanisms involved in hepatic lipid accumulation [36]. A decrease in hepatic de novo lipogenesis improves hepatic insulin sensitivity [36].

3.4.2. Purple carrots (PC) diet versus control diet

3.4.2.1 Adipose tissue protein expression

We identified 1944 proteins in the adipose tissue of the rats fed the PC and the control diets (Supplemental Table 1) where 224 proteins were differentially expressed. A total of

118 proteins were down regulated and 106 proteins were upregulated with the PC diet

(Table 3.6). 24 KEGG pathways and 405 biological processes GO terms were enriched in the proteins list (Benjamini P-value <0.05) (Supplemental Table 5). Some of the enriched pathways and processes observed were mainly involved in lipid metabolism and cholesterol efflux (Table 3.7).

Lipid metabolism

Lipid synthesis. “Fatty acid biosynthetic” and “lipid biosynthetic” biological processes are enriched in the list of the differentially expressed proteins (Table 3.7).

Among the proteins involved in these BP GO terms are Acaca and Fasn that were downregulated with the PC diet, whereas ER lipid raft associated 2 (Erlin2) was upregulated. This can be an indication of a decreased de novo fatty acid synthesis. Erlin2 depletion was shown to activate SREBP genes and subsequently increasing fatty acid and cholesterol biosynthesis [169]. Hmgcs2 and aldo-keto reductase family 1, member

D1(Akr1d1) were also down regulated that is probably accompanied with a reduction in the biosynthesis of ketone bodies [146] and steroids [170]. Although there seems to be a decrease in the de novo fatty acid synthesis, there may be an increase in fatty acid re- esterification and TG synthesis. Glycerol-3-phosphate acyltransferase 3(Gpat3) and Pck1 were both upregulated. Gpat3 is the first enzyme of the TG de novo synthesis pathway.

Its increased expression increases TG formation [171].

Lipid catabolism. Both “lipid catabolic” and “fatty acid catabolic” processes were enriched in the protein list (Table 3.7). Acetyl CoA acyltransferase 2 (Acaa2) and 79

trifunctional protein (Hadha & Hadhb) were downregulated. They are the enzymes catalyzing the last steps of the mitochondrial fatty acid β-oxidation. Also, peroxisomal bifunctional protein (Ehhadh) is downregulated and which is involved in peroxisomal fatty acid β-oxidation [163]. The decrease in the fatty acid oxidation may be due to either the reduced abundance of the newly synthesized fatty acids or directing fatty acids to the re-esterification pathway.

“Regulation of lipolysis in adipocytes” KEGG pathway is also enriched in this list with GNAS complex locus (Gnas), abhydrolase domain containing 5(Abhd5), hormone sensitive lipase (Lipe), cAMP-activated protein kinase (Prkaca) and Plin1 (Table 3.7).

They were all upregulated with the PC diet. This can be an indication of increased stimulated lipolysis in this group. Under catecholamine stimulation and during fasting,

Gnas activates adenylate cyclase with a subsequent increase in cAMP. High levels of cAMP activate Prkaca that phosphorylates both Lipe and Plin1 with a subsequent TG hydrolysis [172]. Plin1 phosphorylation induces a conformational change that gives lipolytic enzymes more access to the adipocytes allowing lipolysis [173]. Abhd5 also positively regulate lipolysis via activating adipose triglyceride lipase (ATGL). ATGL hydrolyzes TG releasing FFA and DAG [174]. However, also only under the stimulated lipolysis state and Plin1 phosphorylation, Abhd5 gets released from its binding with Plin1 which allows its action on ATGL [147]. During fasting or exercise, the liberated FFA are needed and directed to other tissues to be oxidized for energy. Also, Plin1 upregulation may indicate less basal lipolysis. Higher basal lipolysis is suggested to be the cause of insulin resistance in Plin1 null mice with low stimulated lipolysis [148]. However, more

studies on differentiating the role of stimulated lipolysis versus the role of basal lipolysis in insulin resistance are needed.

Cholesterol efflux. Apoa2, Apoa4, Apoc2 and Apoc3 and caveolin1 (Cav1) are all upregulated with the PC diet (Table 3.7). And they are all shown to promote cholesterol efflux in vitro [153,175]. However, NPC intracellular cholesterol transporter 2 (Npc2) is down regulated. NPC2 is involved in the cholesterol egress from the lysosomes to the plasma membrane for removal. Npc2 deficiency is associated with abnormal intracellular cholesterol accumulation [176].

Taken together, similar to PP, a decreased de novo lipogenesis, increased fatty acid re-esterification, and a probable increase in the cholesterol efflux are suggested as mechanisms contributing to improving MetS in adipose tissue with PC feeding (Figure

3.1).

3.4.2.2 Liver protein expression

The results indicated that 941 proteins were identified in the liver of the rats fed the

PC and the control diets (Supplemental Table 3) where 62 proteins were differentially expressed. 29 proteins were down regulated and 33 proteins were upregulated with the

PC diet (Table 3.8). A total of 20 KEGG pathways and 130 biological processes were enriched in the proteins list (Benjamini P-value <0.05) (Supplemental Table 6). Some of the enriched pathways and processes were mainly involved in lipid metabolism, carbohydrate metabolism and oxidative stress (Table 3.9).

Lipid metabolism. “Fatty acid catabolic” and “acyl CoA biosynthetic” processes were enriched in the list of the differentially expressed proteins (Table 3.9). Acly, Fasn and

pyruvate dehydrogenase alpha 1(Pdha1) are all down regulated. This probably indicated a decrease in de novo fatty acid synthesis. Acly and Pdha1 are both acetyl CoA sources.

Also, fatty acid β-oxidation also seems to be downregulated as Acaa2, Acadl and carnitine palmitoyltransferase 1A (Cpt1a) are all downregulated. Acadl and Acaa2 catalyze the first and the last steps of β-oxidation pathway respectively whereas Cpt1a is the enzyme that is responsible for transporting fatty acids to the mitochondria for oxidation [163]. However, peroxisomal fatty acid β-oxidation is probably increased as d bifunctional protein (Hsd17b4) is upregulated. Hsd17b4 is involved in peroxisomal fatty acid β-oxidation and bile acid synthesis [177]. Primary bile acid synthesis was also enriched in this list (Table 3.9). However, even though Hsd17b4 is upregulated, Akr1d1 and sterol carrier protein 2 (Scp2) are downregulated. Both are also involved in bile acid biosynthesis [178,179]. So no conclusion on bile acid synthesis can be made.

Carbohydrate metabolism. Down regulation of both Pklr and Pdha1 was a probable sign of decreased glycolysis (Table 3.9). UDP-glucose pyrophosphorylase 2 (Ugp2) was upregulated that may be a probable indication of increased glycogen synthesis. Ugp2 catalyzes the reversible synthesis of UDP glucose which is the immediate precursor of glycogen synthesis [180]. Sorbitol dehydrogenase (Sord) is downregulated. Sord is the second enzyme of the polyol pathway where glucose is converted to sorbitol then fructose by the action of Sord. However, its catalytic action is suggested to contribute to oxidative stress by producing NADH that produces ROS by the action of NADH oxidase

[181].

Oxidative stress. “Response to oxidative stress” and “hydrogen peroxide catabolic” biological processes (Table 3.9) are enriched in this list. Upregulation of catalase (Cat), 82

the enzyme catalyzing the conversion of H2O2 to water and 02 [182], can be a sign of antioxidant protective effects. Also, downregulation of both hemoglobin subunit beta and hemoglobin alpha 1(Hbb and Hba1) may be a sign of less oxidative stress with PC group.

The expression of both proteins was higher in fatty liver disease that was suggested to be due to the associated higher oxidative stress [183]. Similarly, heat shock protein family A

(Hsp70) and heat shock protein family D member 1(Hspd1) expression and phosphorylation respectively were induced in response to oxidative stress [184,185].

Parkinsonism associated deglycase (Park7) is a redox sensitive protein that was shown to be upregulated in vitro under oxidative stress conditions [186]. So down regulation of

Hspa8, Hspd1, and Park7 may also be a sign of less oxidative stress with PC. Oxidative stress is an established player in promoting insulin resistance [187] and hypertension

[188]. In fact, oxidative stress may be one of the links between fat accumulation in the liver and insulin resistance [189]. Oxidative stress interrupts insulin signaling through activating stress kinases and serine-phosphorylating IRS1 [189]. Furthermore, ROS induces endothelial dysfunction as one way of developing hypertension [188]. Oxidative stress is seen as a common pathological mechanism between fatty liver and CVD [190].

Taken together, similar to PP, decreased de novo lipogenesis, increased peroxisomal fatty acid oxidation and reduced oxidative damage may be contributing to improving

MetS in the liver with PC feeding (Figure 3.1).

4.6. Conclusions

There are some obvious similarities between the two purple vegetables in the enriched biological processes, the involved proteins and finally in the main suggested mechanisms of action in each of the liver and adipose tissue. In adipose tissue, lipid 83

metabolism and cholesterol efflux were modulated by both vegetables whereas ER stress was changed by PP alone. Proteins involved in de novo lipogenesis (Fasn and Acaca) were down regulated with both vegetables while Pck1 and Gpat3, proteins involved in

FFA re-esterification, were upregulated with both vegetables and PC alone respectively.

Both vegetables also shared the upregulation of the lipolytic proteins (Plin1 and Ces1d) as well as the cholesterol efflux proteins (Apoa2 and Apoc2). In liver, lipid metabolism was also modulated by both vegetables. De novo lipogenesis proteins were downregulated (Acly with both vegetables, Fasn and Pdha1 with PC alone and Pc with

PP alone). Both vegetables also seem to modulate oxidative stress proteins. Gpx1 and Cat were upregulated with PP and PC respectively. Overall, we provided a molecular basis of the metabolic benefits of these purple vegetables that substantiate the results of our previous study on the metabolic phenotypic parameters. Since the lightly colored varieties were not included in this study, we cannot make a conclusion on the differential effect of the purple color versus the light color.

TABLE 3.1 Composition of the experimental diets

Component in g/kg diet Control PP1 PC2 Casein (protein) 140 140 140 L- Cystine 1.8 1.8 1.8 Lard 120 120 120 Soybean Oil 40 40 40 Maltodextrin 10 150 150 150 Sucrose 450 - 150 Freeze dried baked purple potato - 450 - Freeze dried raw purple carrot - - 300 Cellulose, BW200 50 50 50 Vitamin Mix v10037 10 10 10 Mineral Mix s10022M 35 35 35 Choline bitartrate 2.5 2.5 2.5 1 PP is high fat diet supplemented with purple potatoes. 2 PC is high fat diet supplemented with purple carrots.

TABLE 3.2. Differentially expressed proteins in adipose tissue with Purple Potatoes diet

Differentially Expressed Proteins Gene Name P Log2 Up or value1 Fold Down Change2 Regulated Serum albumin precursor Alb 0.0001 -0.17 down Serotransferrin precursor Tf 0.0001 -0.33 down Fatty acid synthase Fasn 0.0001 -0.26 down Myosin-9 Myh9 0.0001 -0.14 down Alpha-1-macroglobulin precursor A1m 0.0001 0.32 up Fibrillin-1 isoform X1 Fbn1 0.0001 -0.12 down Filamin-A isoform X2 Flna 0.0001 -0.06 down Spectrin beta chain, non-erythrocytic 1 isoform X1 SPTBN1 0.0001 0.07 up 78 kda glucose-regulated protein precursor Hspa5 0.0001 0.3 up Membrane primary amine oxidase Aoc3 0.0001 0.11 up Calreticulin precursor Calr 0.0001 0.33 up Transketolase isoform X1 Tkt 0.0001 -0.19 down Endoplasmin precursor Hsp90b1 0.0001 0.33 up Inter-alpha-trypsin inhibitor heavy chain H4 precursor Itih4 0.0001 0.12 up Carboxylesterase 1D precursor Ces1d 0.0001 0.38 up Pyruvate kinase PKM isoform X2 Pkm 0.0001 -0.11 down Apolipoprotein A-I preproprotein Apoa1 0.0001 0.26 up Hemopexin precursor Hpx 0.0001 -0.3 down Cofilin-1 Cfl1 0.0001 -0.16 down Vitamin D-binding protein precursor Gc 0.0001 -0.14 down Fibrinogen beta chain precursor Fgb 0.0001 0.2 up Myoferlin Myof 0.0001 -0.18 down Hypoxia up-regulated protein 1 isoform X1 Hyou1 0.0001 0.38 up Plastin-3 isoform X2 Pls3 0.0001 0.29 up Complement factor B precursor Cfb 0.0001 -0.25 down Carbamoyl-phosphate synthase [ammonia], mitochondrial Cps1 0.0001 -0.38 down precursor Fibrinogen gamma chain isoform X1 Fgg 0.0001 0.22 up Dolichyl-diphosphooligosaccharide--protein Rpn2 0.0001 0.26 up glycosyltransferase subunit 2 isoform X1 UDP-glucose:glycoprotein glucosyltransferase 1 precursor Uggt1 0.0001 0.15 up Protein disulfide-isomerase A6 precursor Pdia6 0.0001 0.31 up Dolichyl-diphosphooligosaccharide--protein Rpn1 0.0001 0.22 up glycosyltransferase subunit 1 precursor Adipocyte plasma membrane-associated protein isoform X2 Apmap 0.0001 0.16 up Acetyl-coa carboxylase 1 Acaca 0.0001 -0.26 down Apolipoprotein E precursor Apoe 0.0001 -0.4 down Fibrinogen alpha chain isoform 2 precursor Fga 0.0001 0.21 up Catechol O-methyltransferase isoform X1 Comt 0.0001 -0.21 down Peroxiredoxin-5, mitochondrial precursor Prdx5 0.0001 -0.29 down Phosphoenolpyruvate carboxykinase, cytosolic [GTP] Pck1 0.0001 0.19 up Coronin-1A isoform X1 CORO1A 0.0001 -0.24 down Hydroxymethylglutaryl-coa synthase, mitochondrial isoform Hmgcs2 0.0001 -0.25 down X1 Complement component C7 isoform X1 C7 0.0001 -0.25 down Perilipin-2 Plin2 0.0001 -0.4 down Galectin-3 Lgals3 0.0001 -0.44 down Integrin alpha-M isoform X1 Itgam 0.0001 -0.3 down Brain acid soluble protein 1 Basp1 0.0001 0.19 up 86

Carbonyl reductase [NADPH] 1 LOC102556347 0.0001 0.27 up Apolipoprotein C-II precursor Apoc2 0.0001 0.46 up Laminin subunit alpha-4 precursor Lama4 0.0002 0.1 up Protein disulfide-isomerase A3 precursor Pdia3 0.0002 0.22 up Cathepsin D precursor Ctsd 0.0002 -0.23 down Macrophage mannose receptor 1 precursor Mrc1 0.0003 -0.14 down Filamin-B Flnb 0.0003 0.11 up 3-ketoacyl-coa thiolase, mitochondrial Acaa2 0.0003 -0.14 down Chloride intracellular channel protein 1 Clic1 0.0003 -0.15 down Integrin beta-2 precursor Itgb2 0.0003 -0.26 down Cystatin-B Cstb 0.0003 -0.23 down Von Willebrand factor A domain-containing protein 5A LOC108348048 0.0003 -0.16 down isoform X2 Apolipoprotein A-II isoform X1 Apoa2 0.0003 0.41 up Neutral alpha-glucosidase AB isoform X1 Ganab 0.0004 0.14 up Transaldolase Taldo1 0.0004 -0.14 down Tissue alpha-L-fucosidase precursor Fuca1 0.0004 0.76 up Phosphatidylethanolamine-binding protein 1 Pebp1 0.0004 0.36 up Apolipoprotein C-I precursor Apoc1 0.0005 0.39 up Protein disulfide-isomerase A4 precursor Pdia4 0.0006 0.29 up Selenium-binding protein 1 isoform X1 LOC103689947 0.0006 -0.12 down Heat shock 70 kda protein 1A Hspa1b 0.0006 0.12 up Ester hydrolase c11orf54 homolog RGD1309534 0.0006 -0.15 down Complement C3 precursor C3 0.0007 -0.15 down Reticulocalbin-1 precursor Rcn1 0.0007 0.29 up Histidine--trna ligase, cytoplasmic Hars 0.0007 0.27 up Transmembrane glycoprotein NMB precursor Gpnmb 0.0009 -0.3 down Rho GDP-dissociation inhibitor 2 isoform X1 Arhgdib 0.0010 -0.22 down Granulins isoform a precursor Grn 0.0011 -0.23 down Betaine--homocysteine S-methyltransferase 1 Bhmt 0.0011 -0.32 down Plastin-2 isoform X2 Lcp1 0.0012 -0.14 down Transgelin-2 isoform X1 Tagln2 0.0012 -0.15 down Calnexin isoform X1 Canx 0.0013 0.13 up Nucleolin Ncl 0.0016 -0.13 down Prothymosin alpha Ptma 0.0016 -0.14 down ATP synthase subunit d, mitochondrial Atp5h 0.0017 -0.11 down Alpha-1-acid glycoprotein precursor Orm1 0.0017 -0.36 down Perilipin-1 isoform X1 Plin1 0.0018 0.1 up NAD(P)H-hydrate epimerase Naxe 0.0018 0.18 up Fructose-bisphosphate aldolase A isoform X2 Aldoa 0.0019 -0.09 down Cysteine sulfinic acid decarboxylase isoform X1 Csad 0.0019 0.12 up 1 P value of the T-test less than 5% Benjamini-Hochberg threshold (0.0022)

2 Log2 Fold Change by Category (Purple Potatoes / Control)

TABLE 3.3 Enriched gene ontology biological process terms and KEGG pathways in the list of differentially expressed proteins in adipose tissue that are involved in protein folding, lipid metabolism and cholesterol efflux with Purple Potatoes diet

Biological Theme GO (BP) and KEGG Pathway1 P-value2 Genes Names3 Protein Folding GO:0006457~protein folding 1.46E-06 Uggt1, Canx, Calr, Hspa1b, Hsp90b1, Pdia3, Pdia4, Pdia6 rno04141~Protein processing in 7.66E-10 Uggt1, Canx, Calr, Ganab, endoplasmic reticulum Hspa1b, Hsp90b1, Hspa5, Hyou1, Pdia3, Pdia4, Pdia6, Rpn1, Rpn2 Lipid Metabolism GO:0006633~fatty acid biosynthetic 2.95E-03 Acaca, Apoa1, Apoc1, Apoc2, process Fasn GO:0008610~lipid biosynthetic process 2.29E-03 Hmgcs2, Acaca, Apoa1, Apoc1, Apoc2, Apoe, C3, Fasn, Pck1 GO:0016042~lipid catabolic process 2.71E-04 Apoa1, Apoa2, Apoc1, Apoe, Cps1, Ces1d, Plin1 GO:0006641~triglyceride metabolic 2.65E-07 Apoa1, Apoc1, Apoc2, Apoe, process Cps1, C3, Plin1, Pck1

Cholesterol efflux GO:0033344~cholesterol efflux 3.68E-05 Apoa1, Apoa2, Apoc1, Apoc2, Apoe GO:0043691~reverse cholesterol transport 1.34E-03 Apoa1, Apoa2, Apoe

1 GO (BP) is Gene Ontology (GO) biological process component (BP) and KEGG pathway is Kyoto Encyclopedia of Genes and Genomes biological pathway

2 P-value of the enrichment analyses is significant at Benjamini < 0.05

3 Genes names in bold are upregulated with Purple Potatoes diet while the un-bold names are downregulated with the Purple Potatoes diet in adipose tissue

TABLE 3.4 Differentially expressed proteins in the liver with Purple Potatoes diet

Log2 Up or Gene P Fold Down Differentially Expressed Proteins Name Value1 Change2 Regulated Carbamoyl-phosphate synthase [ammonia], mitochondrial Cps1 0.0001 0.17 up Fatty acid-binding protein, liver Fabp1 0.0001 0.27 up Long-chain-fatty-acid--CoA ligase 1 Acsl1 0.0001 0.1 up Bucs1 protein Acsm1 0.0001 0.19 up 3-alpha-hydroxysteroid dehydrogenase Akr1c9 0.0001 0.17 up Aldh4a1 protein (Fragment) Aldh4a1 0.0001 0.13 up Alpha-aminoadipic semialdehyde dehydrogenase Aldh7a1 0.0001 0.17 up Cystathionine gamma-lyase Cth 0.0001 0.2 up Microsomal triglyceride transfer protein Mttp 0.0001 0.15 up Long-chain-fatty-acid--CoA ligase 5 Acsl5 0.0001 0.24 up Bile acyl-CoA synthetase Slc27a5 0.0001 0.22 up Alcohol sulfotransferase A St2a2 0.0001 0.43 up Aldose reductase-related protein 1 Akr1b7 0.0001 1.41 up Fatty acid synthase Fasn 0.0001 -0.18 down Pyruvate carboxylase, mitochondrial Pc 0.0001 -0.09 down Serum albumin Alb 0.0001 -0.13 down Triokinase/FMN cyclase Tkfc 0.0001 -0.14 down Transketolase Tkt 0.0001 -0.13 down ATP-citrate synthase Acly 0.0001 -0.27 down Serotransferrin Tf 0.0001 -0.24 down Pyruvate kinase Pklr 0.0001 -0.18 down Selenium-binding protein 1 Selenbp1 0.0001 -0.14 down Glucose-6-phosphate isomerase Gpi 0.0001 -0.18 down Purine nucleoside phosphorylase Pnp 0.0001 -0.12 down Malate dehydrogenase, mitochondrial Mdh2 0.0001 -0.22 down Keratin, type II cytoskeletal 8 Krt8 0.0001 -0.2 down Glycerol kinase Gk 0.0001 -0.16 down Cytochrome P450 2C11 Cyp2c11 0.0001 -0.37 down Keratin, type I cytoskeletal 18 Krt18 0.0001 -0.23 down Phosphate carrier protein, mitochondrial Slc25a3 0.0001 -0.2 down Isoform 2 of Fibrinogen beta chain Fgb 0.0001 0.27 up Acyl-coenzyme A synthetase ACSM5, mitochondrial Acsm5 0.0001 -0.35 down Farnesyl pyrophosphate synthase 1 Fdps 0.0001 0.21 up Protein disulfide-isomerase P4hb 0.0002 0.1 up Choline dehydrogenase, mitochondrial Chdh 0.0002 -0.13 down Carboxylesterase 1D Ces1d 0.0002 0.36 up Malate dehydrogenase, cytoplasmic Mdh1 0.0003 0.15 up

Malic enzyme Me1 0.0003 -0.15 down Glutathione peroxidase Gpx1 0.0003 0.17 up Aflatoxin B1 aldehyde reductase member 3 Akr7a3 0.0004 -0.26 down Lactamase, beta Lactb 0.0004 -0.14 down Alpha-aminoadipic semialdehyde synthase, mitochondrial Aass 0.0005 0.22 up Perilipin 2 Plin2 0.0005 -0.41 down Acyl-coenzyme A oxidase Acox3 0.0005 0.09 up Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial Aadat 0.0005 0.18 up Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Dlat 0.0006 -0.17 down Carboxylic ester hydrolase (Fragment) Ces2e 0.0009 0.49 up Cytochrome P450 2B3 Cyp2b3 0.0009 0.18 up Estrogen sulfotransferase, isoform 3 Ste 0.001 -0.46 down Glucose-6-phosphate 1-dehydrogenase G6pdx 0.001 0.35 up Alcohol dehydrogenase 1 Adh1 0.0012 0.08 up Isocitrate dehydrogenase [NADP] cytoplasmic Idh1 0.0012 0.09 up Glutathione S-transferase alpha-4 Gsta4 0.0012 0.13 up Myosin, heavy polypeptide 9, non-muscle Myh9 0.0012 -0.1 down Protein deglycase DJ-1 Park7 0.0012 -0.26 down Transgelin-2 Tagln2 0.0013 -0.21 down Phosphoenolpyruvate carboxykinase, cytosolic [GTP] Pck1 0.0014 0.11 up Long-chain specific acyl-CoA dehydrogenase, mitochondrial Acadl 0.0014 -0.1 down Voltage-dependent anion-selective channel protein 3 Vdac3 0.0017 -0.27 down Alpha-1-macroglobulin A1m 0.0018 0.14 up Aflatoxin B1 aldehyde reductase member 2 Akr7a2 0.0019 -0.15 down Fructose-bisphosphate aldolase Aldob 0.0021 -0.11 down Epoxide hydrolase 1 Ephx1 0.0021 -0.11 down UDP-glucuronosyltransferase 2B2 Ugt2b 0.0023 0.17 up 3 beta-hydroxysteroid dehydrogenase type 5 Hsd3b5 0.0024 -0.24 down 3-hydroxyisobutyryl-CoA hydrolase, mitochondrial Hibch 0.0027 -0.16 down Cytosol aminopeptidase Lap3 0.0028 -0.08 down UDP-glucuronosyltransferase 2B17 OS Ugt2b17 0.003 0.27 up Biliverdin reductase A Blvra 0.0033 -0.15 down 1 1 P value of the T test less than 5% Benjamini-Hochberg threshold (0.0037)

2 Log2 Fold Change by Category (Purple Potatoes / Control)

TABLE 3.5 Enriched gene ontology biological process terms and KEGG pathways in the list of differentially expressed proteins in liver that are involved in lipid metabolism and carbohydrate metabolism with Purple Potatoes

Biological theme GO (BP) and KEGG P- Genes names3 pathway1 value2 Lipid Metabolism GO:0006633~fatty acid 1.58E- Acly, Acadl, Acsm1, Acsm5, Fasn biosynthetic process 03

GO:0008610~lipid 6.00E- Hsd3b5, Acly, Acadl, Acsl1, Acsl5, biosynthetic process 08 Acsm1, Acsm5, Fdps, Fasn, G6pd, Idh1, Pck1, Pc, Slc27a5 GO:0016042~lipid catabolic 7.64E- Hibch, Acadl, Acox3, Acsl5, Cps1, process 05 Ces1d, Fabp1, Idh1

GO:0006635~fatty acid beta- 1.32E- Hibch, Acadl, Acox3, Acsl5, Ces1d, oxidation 04 Fabp1

Carbohydrate Metabolism GO:0016052~carbohydrate 1.42E- Aldob, Cps1, Gpi, Gk, Pklr catabolic process 03

rno00030: Pentose phosphate 1.19E- Aldob, G6pd, Gpi, Tkt pathway 03

1 GO (BP) is Gene Ontology (GO) biological process component (BP) and KEGG pathway is Kyoto Encyclopedia of Genes and Genomes biological pathway

2 P-value of the enrichment analyses is significant at Benjamini < 0.05

3 Genes names in bold are upregulated with Purple Potatoes diet while the un-bold names are downregulated with the Purple Potatoes diet in liver

TABLE 3.6 Differentially expressed proteins in adipose tissue with Purple Carrots Diet

Log2 Down or P Differentially Expressed Proteins Gene Name Fold Up value1 Change2 Regulated Serum albumin precursor Alb 0.0001 -0.19 down Serotransferrin precursor Tf 0.0001 -0.22 down Fatty acid synthase Fasn 0.0001 -0.15 down Myosin-9 Myh9l1 0.0001 -0.07 down Elongation factor 1-alpha 1 Eef1a1 0.0001 -0.11 down Filamin-A isoform X2 Flna 0.0001 -0.09 down Alpha-enolase Eno1 0.0001 -0.15 down Ribosome-binding protein 1 isoform X4 Rrbp1 0.0001 -0.16 down Plastin-2 isoform X2 Lcp1 0.0001 -0.17 down Aldehyde dehydrogenase, mitochondrial precursor Aldh2 0.0001 -0.16 down Collagen alpha-1(XIV) chain precursor Col14a1 0.0001 -0.44 down ATP-citrate synthase isoform X1 Acly 0.0001 -0.26 down Glutamate dehydrogenase 1, mitochondrial precursor Mrc1 0.0001 -0.14 down Carbamoyl-phosphate synthase [ammonia], mitochondrial precursor Cps1 0.0001 -0.75 down Heterogeneous nuclear ribonucleoprotein U Hnrnpu 0.0001 -0.2 down Serine protease inhibitor A3N Serpina3n 0.0001 -0.27 down Decorin isoform X1 Dcn 0.0001 -0.4 down Glutathione S-transferase alpha-3 Gsta1 0.0001 -0.27 down Prolargin isoform X3 Prelp 0.0001 -0.29 down 3-ketoacyl-coa thiolase, mitochondrial Acaa2 0.0001 -0.29 down Acetyl-coa carboxylase 1 Acaca 0.0001 -0.18 down Aspartate aminotransferase, mitochondrial Got2 0.0001 -0.21 down Heterogeneous nuclear ribonucleoprotein K isoform X2 Hnrnpk 0.0001 -0.18 down ATP synthase subunit d, mitochondrial Atp5h 0.0001 -0.14 down Catechol O-methyltransferase isoform X1 Comt 0.0001 -0.34 down Nucleolin Ncl 0.0001 -0.32 down Hydroxymethylglutaryl-coa synthase, mitochondrial isoform X1 Hmgcs2 0.0001 -0.47 down Complement component C7 isoform X1 C7 0.0001 -0.21 down Galectin-3 Lgals3 0.0001 -0.34 down Biglycan precursor Bgn 0.0001 -0.24 down Granulins isoform a precursor Grn 0.0001 -0.33 down Ezrin Ezr 0.0001 -0.24 down Nucleophosmin Npm1 0.0001 -0.33 down Elongation factor Tu, mitochondrial precursor Tufm 0.0001 -0.12 down Beta-2-glycoprotein 1 precursor Apoh 0.0001 -0.37 down Betaine--homocysteine S-methyltransferase 1 Bhmt 0.0001 -0.69 down Obg-like atpase 1 Ola1 0.0001 -0.14 down Glutathione S-transferase Mu 1 Gstm1 0.0001 -0.62 down High mobility group box 1 like Hmg1l1 0.0001 -0.4 down Alcohol dehydrogenase 1 Adh1 0.0001 -0.75 down Fatty acid-binding protein, liver Fabp1 0.0001 -0.77 down Von Willebrand factor A domain-containing protein 5A isoform X2 LOC108348048 0.0001 -0.17 down Serine/threonine-protein kinase N3 Pkn3 0.0001 -0.26 down Heterogeneous nuclear ribonucleoprotein M isoform b Hnrnpm 0.0001 -0.22 down Argininosuccinate synthase isoform X1 Ass1 0.0001 -0.53 down Fructose-bisphosphate aldolase B Aldob 0.0001 -0.65 down LIM and senescent cell antigen-like-containing domain protein 1 Lims1 0.0001 -0.17 down Arginase-1 Arg1 0.0001 -0.5 down Sorbitol dehydrogenase Sord 0.0001 -0.31 down Carbonic anhydrase 3 isoform X1 Car3 0.0001 0.15 up Vimentin Vim 0.0001 0.23 up Long-chain-fatty-acid--coa ligase 1 isoform X1 Acsl1 0.0001 0.12 up Alpha-1-macroglobulin precursor Pzp 0.0001 0.21 up Fibrillin-1 isoform X1 Fbn1 0.0001 0.15 up Complement C3 precursor C3 0.0001 0.14 up 92

Spectrin beta chain, non-erythrocytic 1 isoform X1 Sptbn1 0.0001 0.1 up Plectin isoform 1 Plec 0.0001 0.06 up Membrane primary amine oxidase Aoc3 0.0001 0.21 up All-trans-retinol 13,14-reductase precursor Retsat 0.0001 0.17 up Collagen alpha-3(VI) chain isoform X4 Col6a3 0.0001 0.12 up Vinculin Vcl 0.0001 0.12 up Carboxylesterase 1D precursor Ces1d 0.0001 0.32 up Perilipin-1 isoform X1 Plin1 0.0001 0.19 up Complement C4 precursor C4a 0.0001 0.25 up Malate dehydrogenase, cytoplasmic isoform Mdh1 Mdh1 0.0001 0.12 up EH domain-containing protein 1 Ehd1 0.0001 0.13 up Catalase Cat 0.0001 0.14 up Laminin subunit alpha-4 precursor Lama4 0.0001 0.27 up Laminin subunit beta-1 isoform X2 Lamb1 0.0001 0.26 up Laminin subunit gamma-1 precursor Lamc1 0.0001 0.21 up Aldose reductase Akr1b1 0.0001 0.17 up Periostin isoform X2 Postn 0.0001 0.25 up Hormone-sensitive lipase Lipe 0.0001 0.18 up L-lactate dehydrogenase B chain isoform Ldhb Ldhb 0.0001 0.15 up Succinyl-coa:3-ketoacid coenzyme A transferase 1, mitochondrial Oxct1 0.0001 0.15 up precursor Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit Rpn2 0.0001 0.12 up 2 isoform X1 Cysteine sulfinic acid decarboxylase isoform X1 Csad 0.0001 0.16 up Cell surface glycoprotein MUC18 isoform 1 precursor Mcam 0.0001 0.24 up Adipocyte plasma membrane-associated protein isoform X2 Apmap 0.0001 0.19 up Alanine aminotransferase 1 isoform X1 Gpt 0.0001 0.19 up Nidogen-1 isoform X2 Nid1 0.0001 0.18 up Fibrinogen alpha chain isoform 2 precursor Fga 0.0001 0.17 up Annexin A3 isoform X1 Anxa3 0.0001 0.16 up Glutathione peroxidase 3 precursor Gpx3 0.0001 0.2 up Phosphoenolpyruvate carboxykinase, cytosolic [GTP] Pck1 0.0001 0.32 up Perilipin-4 isoform X2 Plin4 0.0001 0.21 up Laminin subunit alpha-2 isoform X1 Lama2 0.0001 0.23 up Heat shock protein beta-1 Hspb1 0.0001 0.27 up Integrin alpha-7 isoform X1 Itga7 0.0001 0.18 up Acetolactate synthase-like protein Ilvbl 0.0001 0.21 up Caveolin-1 isoform alpha Cav1 0.0001 0.29 up Ras-related protein Rab-18 isoform X1 Rab18 0.0001 0.16 up Apolipoprotein A-II isoform X1 Apoa2 0.0001 0.37 up 1-acyl-sn-glycerol-3-phosphate acyltransferase gamma Agpat3 0.0001 0.18 up GNAS isoform GNASL Gnas 0.0001 0.22 up Chloride intracellular channel protein 1 Clic1 0.0001 -0.15 down Neprilysin isoform X1 Mme 0.0001 0.24 up Creatine kinase B-type Ckb 0.0001 -0.15 down Protein S100-B isoform X1 S100b 0.0001 0.17 up Fibrinogen beta chain precursor Fgb 0.0001 0.14 up Calumenin isoform a precursor Calu 0.0001 0.15 up T-complex protein 1 subunit zeta Cct6a 0.0001 -0.12 down Hepatoma-derived growth factor Hdgf 0.0001 -0.3 down Transaldolase Taldo1 0.0002 -0.13 down Sorbin and SH3 domain-containing protein 2 Sorbs2 0.0002 0.29 up Fibrinogen gamma chain isoform X1 Fgg 0.0002 0.14 up Dysferlin Dysf 0.0002 0.16 up Aminoacyl trna synthase complex-interacting multifunctional protein 1 Aimp1 0.0002 -0.32 down Apolipoprotein C-III precursor Apoc3 0.0002 0.25 up Heat shock 70 kda protein 1A Hspa1b 0.0002 0.11 up Transmembrane protein 43 Tmem43 0.0002 0.14 up Monoglyceride lipase isoform X1 Mgll 0.0002 0.14 up Apolipoprotein A-IV precursor Apoa4 0.0002 0.15 up 93

Alcohol dehydrogenase [NADP(+)] Akr1a1 0.0002 -0.13 down Glucose-6-phosphate isomerase Gpi 0.0002 0.13 up Lumican precursor Lum 0.0003 -0.18 down Glutamine synthetase Glul 0.0003 -0.22 down PDZ and LIM domain protein 1 Pdlim1 0.0003 0.25 up Filamin-B Flnb 0.0003 0.1 up Legumain precursor Lgmn 0.0003 -0.17 down RNA-binding protein FUS isoform X1 Fus 0.0003 -0.2 down Septin-9 isoform 2 Sept9 0.0003 -0.17 down Delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrial Aldh4a1 0.0003 -0.32 down Cadherin-13 precursor Cdh13 0.0003 0.25 up Apolipoprotein C-II precursor Apoc2 0.0003 0.34 up Protein-glutamine gamma-glutamyltransferase 2 Tgm2 0.0003 -0.3 down Glutathione S-transferase Mu 2 Gstm2 0.0004 -0.25 down 60S ribosomal protein L5 Rpl5 0.0004 -0.18 down Transketolase isoform X1 Tkt 0.0005 -0.1 down Synapse-associated protein 1 isoform X1 Syap1 0.0005 0.2 up Sulfated glycoprotein 1 isoform X1 Psap 0.0005 -0.32 down Camp-dependent protein kinase type II-beta regulatory subunit Prkar2b 0.0005 0.15 up Proliferation-associated protein 2G4 Pa2g4 0.0005 -0.27 down L-lactate dehydrogenase A chain isoform X1 Ldha 0.0005 -0.14 down Unconventional myosin-Ic Myo1c 0.0006 0.07 up Prelamin-A/C Lmna 0.0006 0.1 up Phosphoserine aminotransferase Psat1 0.0006 0.15 up Isocitrate dehydrogenase [NADP], mitochondrial precursor Idh2 0.0006 -0.23 down Reticulon-4 Rtn4 0.0006 0.18 up Transmembrane glycoprotein NMB precursor Gpnmb 0.0006 -0.27 down Nucleobindin-1 isoform X1 Nucb1 0.0006 0.13 up Retinol dehydrogenase 11 precursor Rdh11 0.0006 0.28 up Poly [ADP-ribose] polymerase 3 Parp3 0.0007 -0.19 down Hsc70-interacting protein St13 0.0007 0.11 up 40S ribosomal protein S19 Rps19 0.0007 -0.23 down Alpha-actinin-4 Actn4 0.0007 0.08 up Serine hydroxymethyltransferase, cytosolic Shmt1 0.0008 -0.25 down Cofilin-1 Cfl1 0.0009 -0.12 down Lamin-B1 Lmnb1 0.0010 0.17 up Heterogeneous nuclear ribonucleoprotein A3 isoform a Hnrnpa3 0.0010 -0.26 down Polymerase I and transcript release factor Ptrf 0.0010 0.12 up Ras gtpase-activating-like protein IQGAP1 Iqgap1 0.0011 -0.07 down Probable ATP-dependent RNA helicase DDX5 isoform X1 Ddx5 0.0011 -0.14 down Eukaryotic initiation factor 4A-II isoform X1 Eif4a2 0.0011 0.12 up Moesin Msn 0.0012 -0.14 down Ribonuclease UK114 Rida 0.0012 -0.32 down Dynactin subunit 2 Dctn2 0.0012 0.1 up Splicing factor U2AF 65 kda subunit isoform X1 U2af2 0.0013 -0.18 down Annexin A1 isoform X2 Anxa1 0.0013 0.11 up ATP synthase subunit O, mitochondrial precursor Atp5o 0.0014 -0.13 down Uncharacterized protein LOC315963 RGD1310507 0.0014 -0.14 down Coagulation factor XIII A chain F13a1 0.0014 0.16 up 1-acylglycerol-3-phosphate O-acyltransferase ABHD5 Abhd5 0.0014 0.16 up Receptor of activated protein C kinase 1 Rack1 0.0015 -0.16 down Ethylmalonyl-coa decarboxylase isoform X2 Echdc1 0.0015 0.15 up Peptidyl-prolyl cis-trans isomerase FKBP9 precursor Fkbp9 0.0015 0.2 up Glutathione S-transferase Mu 5 Got2 0.0016 -0.43 down ATP synthase-coupling factor 6, mitochondrial isoform X2 Atp5j 0.0016 -0.13 down Epididymal secretory protein E1 precursor Npc2 0.0016 -0.15 down Glycerol-3-phosphate acyltransferase 3 isoform X1 Gpat3 0.0016 0.27 up 60S ribosomal protein L4 Rpl4 0.0017 -0.13 down Carbonyl reductase [NADPH] 1 LOC102556347 0.0017 0.24 up Transmembrane protein 120A Tmem120a 0.0017 0.33 up 94

Annexin A5 Anxa5 0.0019 0.12 up Trifunctional enzyme subunit alpha, mitochondrial precursor Hadha 0.0021 -0.08 down Sorbin and SH3 domain-containing protein 1 isoform X6 Sorbs1 0.0021 0.16 up Long-chain fatty acid transport protein 3 precursor Slc27a3 0.0021 0.22 up Ceruloplasmin isoform 1 precursor Cp 0.0022 -0.08 down Heterogeneous nuclear ribonucleoproteins C1/C2-like isoform X5 LOC100911576 0.0022 -0.13 down Peroxisomal bifunctional enzyme Ehhadh 0.0022 -0.29 down Fructose-1,6-bisphosphatase 1 Fbp1 0.0024 -0.52 down Aconitate hydratase, mitochondrial precursor Aco2 0.0025 0.08 up General vesicular transport factor p115 isoform X1 Uso1 0.0025 0.14 up Antigen-presenting glycoprotein CD1d precursor Cd1d1 0.0025 0.18 up Bifunctional glutamate/proline--trna ligase isoform X1 Eprs 0.0027 -0.12 down Alpha-2-HS-glycoprotein precursor Ahsg 0.0027 -0.27 down Macrophage mannose receptor 1 precursor Mrc1 0.0028 -0.09 down Peptidyl-prolyl cis-trans isomerase B precursor Ppib 0.0028 -0.17 down 40S ribosomal protein S9 LOC103689992 0.0028 -0.13 down Aldehyde dehydrogenase family 8 member A1 Aldh8a1 0.0028 -0.8 down Erlin-2 isoform X1 Erlin2 0.0028 0.1 up Peroxiredoxin-5, mitochondrial precursor Prdx5 0.0029 -0.18 down Pantetheinase precursor Vnn1 0.0029 0.24 up Adenosylhomocysteinase Ahcy 0.0030 -0.18 down 3-oxo-5-beta-steroid 4-dehydrogenase Akr1d1 0.0030 -0.44 down Septin-11 Sept11 0.0032 -0.14 down Cathepsin D precursor Ctsd 0.0033 -0.16 down ATP synthase subunit delta, mitochondrial isoform X1 Atp5d 0.0033 0.1 up Coronin-1A isoform X1 Coro1A 0.0034 -0.14 down Calcium-binding mitochondrial carrier protein Aralar2 isoform X1 Slc25a13 0.0034 -0.33 down Annexin A6 Anxa6 0.0034 0.08 up 40S ribosomal protein S15 Rps15 0.0034 0.23 up Mitochondrial dicarboxylate carrier Slc25a10 0.0035 -0.12 down Serum deprivation-response protein Sdpr 0.0035 0.12 up Ras-related protein Rab-2A Rab2a 0.0035 0.12 up Platelet endothelial cell adhesion molecule precursor Pecam1 0.0036 0.17 up Glyceraldehyde-3-phosphate dehydrogenase Gapdh 0.0038 -0.09 down Peptidyl-prolyl cis-trans isomerase A LOC100360977 0.0039 -0.14 down Actin-related protein 2/3 complex subunit 1B Arpc1b 0.0040 -0.17 down Thiosulfate sulfurtransferase Tst 0.0040 -0.2 down Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 Gnb1 0.0040 0.14 up Phenylalanine-4-hydroxylase Pah 0.0046 -0.4 down Talin-1 Tln1 0.0048 -0.05 down 60S ribosomal protein L30 Rpl30 0.0048 -0.14 down Erythrocyte band 7 integral membrane protein Stom 0.0048 0.26 up Camp-dependent protein kinase catalytic subunit alpha Prkaca 0.0050 0.23 up Calcineurin B homologous protein 1 Chp1 0.0050 0.15 up Trifunctional enzyme subunit beta, mitochondrial isoform X2 Hadhb 0.0052 -0.1 down Transmembrane 9 superfamily member 3 isoform X2 Tm9sf3 0.0052 -0.22 down Peroxiredoxin-1 Prdx1 0.0053 -0.12 down UDP-glucuronosyltransferase 2B2 precursor Ugt2b 0.0053 -0.64 down Carbonyl reductase [NADPH] 3 Cbr3 0.0055 0.14 up Guanylate-binding protein 4 isoform X1 LOC685067 0.0056 0.15 up Creatine kinase M-type Ckm 0.0057 0.19 up 1 1 P value of the T-test less than 5% Benjamini-Hochberg threshold (0.0058)

2 Log2 Fold Change by Category (Purple Carrots / Control)

TABLE 3.7 Enriched gene ontology biological process terms and KEGG pathways in the list of differentially expressed proteins in adipose tissue that are involved in lipid metabolism and cholesterol efflux with Purple Carrots.

Biological GO (BP) and KEGG Pathway1 P-value2 Genes Names3 Theme Lipid Metabolism GO:0006633~fatty acid 9.44E- Erlin2, Acaca, Anxa1, Apoa4, biosynthetic process 04 Apoc2, Apoc3, Fasn, Mgll GO:0008610~lipid biosynthetic 1.14E- Hmgcs2, Erlin2, Abhd5, Acaca, process 03 Acsl1, Aldh8a1 , Akr1d1, Anxa1, Apoa4, Apoc2, Apoc3, C3, Fasn , Gpat3, Mgll, Pck1 GO:0016042~lipid catabolic 3.35E- Abhd5, Acaa2, Akr1d1, Apoa2, process 08 Apoa4, Apoc2, Apoc3, Apoh, Cps1, Ces1d, Ehhadh, Fabp1, Hadha, Hadhb, Lipe, Mgll, Plin1, Prkaca GO:0009062~fatty acid catabolic 5.28E- Acaa2, Ces1d, Ehhadh, Fabp1, process 04 Hadha, Hadhb, Lipe

rno04923: Regulation of lipolysis 5.01E- Gnas, Abhd5, Lipe, Mgll, Plin1, in adipocytes 03 Prkaca Cholesterol efflux GO:0033344~cholesterol efflux 1.20E- Npc2, Apoa2, Apoa4, Apoc2, Apoc3, 04 Cav1

1 GO (BP) is Gene Ontology (GO) biological process component (BP) and KEGG pathway is Kyoto Encyclopedia of Genes and Genomes biological pathway

2 P-value of the enrichment analyses is significant at Benjamini < 0.05

3 Genes names in bold are upregulated with Purple Carrots diet while the un-bold names are downregulated with the Purple Carrots diet in adipose tissue

TABLE 3.8 Differentially expressed proteins in the liver with Purple Carrots diet

Log2 Up or Differentially Expressed Proteins Gene Name P value1 Fold Down Change2 Regulated Carbamoyl-phosphate synthase [ammonia], mitochondrial Cps1 0.0001 0.05 up Cytosolic 10-formyltetrahydrofolate dehydrogenase Aldh1l1 0.0001 0.14 up Catalase Cat 0.0001 0.15 up Cytochrome P450 2C7 Cyp2c7 0.0001 0.29 up Alcohol dehydrogenase 1 Adh1 0.0001 0.14 up Alpha-1-macroglobulin A1m 0.0001 0.14 up Epoxide hydrolase 1 Ephx1 0.0001 0.21 up Cystathionine gamma-lyase Cth 0.0001 0.19 up 4-hydroxyphenylpyruvate dioxygenase Hpd 0.0001 0.25 up Glutathione S-transferase Gsta5 0.0001 0.46 up Protein Sar1a Sar1a 0.0001 0.18 up Aflatoxin B1 aldehyde reductase member 3 Akr7a3 0.0001 0.45 up Histidine ammonia-lyase Hal 0.0001 0.35 up Carboxylesterase 1D Ces1d 0.0001 0.54 up Fatty acid synthase Fasn 0.0001 -0.13 down Aldehyde dehydrogenase, mitochondrial Aldh2 0.0001 -0.25 down 3-ketoacyl-CoA thiolase, mitochondrial Acaa2 0.0001 -0.27 down 60 kDa heat shock protein, mitochondrial Hspd1 0.0001 -0.07 down Transketolase Tkt 0.0001 -0.25 down ATP-citrate synthase Acly 0.0001 -0.3 down Malate dehydrogenase, mitochondrial Mdh2 0.0001 -0.22 down Keratin, type II cytoskeletal 8 Krt8 0.0001 -0.13 down Sorbitol dehydrogenase Sord 0.0001 -0.14 down Aldehyde dehydrogenase X, mitochondrial Aldh1b1 0.0001 -0.46 down Protein LOC679794 LOC679794 0.0001 -0.33 down UDP-glucuronosyltransferase 2B2 Ugt2b 0.0002 0.18 up Hemoglobin subunit beta-1 Hbb 0.0002 -0.24 down Pyruvate kinase Pklr 0.0002 -0.12 down Protein Ugp2 Ugp2 0.0002 0.25 up Isoform 2 of Fibrinogen beta chain Fgb 0.0002 0.21 up UDP-glucuronosyltransferase 2B15 Ugt2b15 0.0003 0.13 up Alpha-aminoadipic semialdehyde synthase, mitochondrial Aass 0.0004 0.17 up Cytochrome P450 2C23 Cyp2c23 0.0004 0.2 up Argininosuccinate synthase Ass1 0.0004 0.11 up Pyruvate dehydrogenase E1 component subunit alpha Pdha1 0.0004 -0.22 down Keratin, type I cytoskeletal 18 Krt18 0.0006 -0.17 down 3-oxo-5-beta-steroid 4-dehydrogenase Akr1d1 0.0006 -0.08 down 3-alpha-hydroxysteroid dehydrogenase Akr1c9 0.0007 0.1 up Perilipin 2 Plin2 0.0007 -0.31 down Hemoglobin subunit alpha-1/2 Hba1 0.0007 -0.22 down Long-chain specific acyl-CoA dehydrogenase, mitochondrial Acadl 0.0008 -0.1 down Carnitine O-palmitoyltransferase 1, liver isoform Cpt1a 0.0009 -0.22 down L-gulonolactone oxidase Gulo 0.0009 -0.17 down Retinol dehydrogenase 7 Rdh7 0.0010 0.15 up Protein deglycase DJ-1 Park7 0.0010 -0.17 down Peroxisomal multifunctional enzyme type 2 Hsd17b4 0.0015 0.07 up 60S ribosomal protein L14 Rpl14 0.0015 -0.17 down Glutathione S-transferase Gsta2 0.0017 0.53 up Malate dehydrogenase, cytoplasmic Mdh1 0.0017 0.13 up 97

Probable 2-oxoglutarate dehydrogenase E1 component Dhtkd1 0.0017 0.12 up DHKTD1, mitochondrial 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 Hmgcs2 0.0017 -0.07 down (Mitochondrial) Pterin-4-alpha-carbinolamine dehydratase Pcbd1 0.0017 -0.2 down Heat shock cognate 71 kDa protein Hspa8 0.0018 -0.06 down Non-specific lipid-transfer protein Scp2 0.0020 -0.15 down Carbonic anhydrase 3 Ca3 0.0022 0.48 up Protein LOC100911833 LOC297568 0.0023 0.14 up Cytochrome P450 2A2 Cyp2a2 0.0023 0.38 up Cullin-associated NEDD8-dissociated protein 1 Cand1 0.0023 -0.2 down Eukaryotic translation elongation factor 1 beta 2 Eef1b2 0.0023 -0.16 down Ectonucleoside triphosphate diphosphohydrolase 5 Entpd5 0.0027 0.17 up Glutathione S-transferase alpha-5 Gsta5 0.0027 0.33 up Formimidoyltransferase-cyclodeaminase Ftcd 0.0033 0.06 up 1 1 P value of the T-test less than 5% Benjamini-Hochberg threshold (0.00336)

2 Log2 Fold Change by Category (Purple Carrots / Control)

TABLE 3.9 Enriched gene ontology biological process terms and KEGG pathways in the list of differentially expressed proteins in the liver that are involved in lipid metabolism, carbohydrate metabolism and oxidative stress with Purple Carrots.

Biological GO (BP) and KEGG Pathway1 P- Genes Names3 Theme value2 Lipid Metabolism GO:0009062~fatty acid catabolic 2.46E- Acaa2, Acadl, Ces1d, Cpt1a, process 04 Hsd17b4

GO:0071616~acyl-CoA 1.85E- Acly, Fasn, Pdha1, Pdha1l1 biosynthetic process 04

rno00120: Primary bile acid 4.77E- Akr1d1, Hsd17b4, Scp2 biosynthesis 03

Carbohydrate Metabolism GO:0005975~carbohydrate 8.83E- Ugp2, Cps1, Cpt1a, Dhtkd1, Entpd5, metabolic process 04 Mdh1, Mdh2, Pdha1, Pklr, Sord

Oxidative Stress GO:0006979~response to oxidative 1.60E- Park7, Car3, Cat, Hsp70, Hspa8, stress 03 Hspd1, Hbb, Hba1 GO:0042744~hydrogen peroxide 2.55E- Cat, Hbb, Hba1 catabolic process 03

1 GO (BP) is Gene Ontology (GO) biological process component (BP) and KEGG pathway is Kyoto Encyclopedia of Genes and Genomes biological pathway

2 P-value of the enrichment analyses is significant at Benjamini < 0.05

3 Genes names in bold are upregulated with Purple Carrots diet while the un-bold names are downregulated with the Purple Carrots diet in liver

Figure 3.1 Suggested mechanisms of action of purple potatoes and purple carrots on

Metabolic Syndrome pathologies in the liver and adipose tissue.

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CHAPTER 4

GENERAL DISCUSSION

In this project, we aimed to explore the metabolic benefits of purple vegetables and possible mechanistic explanations for their activities. There is accumulating evidence that polyphenol rich plants in general, and more specifically anthocyanins rich plants, improve metabolic parameters associated with MetS. In previous studies anthocyanin rich plant diets were typically compared to low plant or non-plant based diets, therefore it would be difficult to separate out the effects of the polyphenols from the more general benefits of plant-based diets. To more specifically assess the highly colored anthocyanins, we designed our experiment to compare two color varieties, purple and non-purple, of two very commonly consumed vegetables, potatoes and carrots to each other and furthermore to a semi-purified, non-plant diet. As predicted, the more highly colored cultivars performed better than the non-plant diet in two main elements of MetS with slight additional benefits of PP and PC over their lightly colored counterparts, on glucose homeostasis and hypertension, respectively. However, it is possible that the difference between PP and WP in terms of glucose homeostasis was exacerbated by the additional obesity in the WP group. Thus, it is not possible with this design to dissect out the roles of anthocyanins per se, from the impact of obesity itself.

Although it is possible that anthocyanins play a major role in glucose homeostasis, they are unlikely to be responsible on their own, for the improvements in blood pressure seen with the various vegetable diets. In fact, WP feeding resulted in better antihypertensive activity than the PP feeding. It could be another potato

101

phytochemical component, other than anthocyanins, that is responsible for the favorable blood pressure responses in the potato diets. With respect to blood pressure, the PC group did do better than the OC and it is possible that anthocyanins are at least partially responsible, however, the PC cultivar also had higher carotenoids, phenolic and flavonoid content than the orange cultivar. So it is difficult to attribute the benefits to anthocyanins alone. Since we are studying the whole vegetable and not the extracted phytochemicals, the effects cannot be attributed to a specific bioactive, but are more likely a result of interactions and synergism between phytochemicals and other food components (i.e. resistant starch).

Not pair-feeding was a limitation to this study; obesity may have confounded insulin sensitivity benefits with WP, OC and/or PC. Despite this limitation, we were able to confirm that obesity is a critical risk factor for the development of IR. Thus, while adding vegetables can improve metabolic risk with respect to IR, without targeting the obesity itself, these improvements will be modest. In contrast, the benefits of the vegetables on hypertension appeared to be dissociated from the obesity. Even most obese groups, WP, OC, and PC, showed significantly reduced hypertension compared to the non-vegetable diet. This suggests that the mechanisms driving IR may be distinct from those driving hypertension.

Based on previously reported potential mechanisms of action of polyphenols on

MetS, we chose to examine a group of candidate proteins for our initial mechanistic investigation. However using Western blotting, we were unable to find statistically significant differences between the vegetables and the control diets, for any of these targeted proteins. Western blotting success requires that you know ahead of time which 102

proteins are important targets. A negative result is not very helpful in this respect as it does not reveal potential new mechanisms of bioactivity [191]. Although extremely expensive and time-consuming because of the very large data set that is generated, we chose to examine the proteome in two key tissues relevant to MetS pathogenesis, the liver, and adipose. As a starting point, we decided only to look at differences between the purple cultivars and the control diet from the completed feeding trial. From this simplified dataset we proposed a few mechanisms by which purple vegetables could antagonize MetS pathologies. These included enhancing protein folding, FFA re- esterification and cholesterol efflux in adipose tissue while reducing oxidative stress and de novo lipogenesis in the liver. ER stress, lipotoxicity, oxidative stress and increased hepatic de novo lipogenesis are key players in the development of MetS. Even though both purple vegetables varied in their potency on the studied metabolic parameters (i.e. insulin sensitivity and hypertension), they seemed to share a lot of similarities in the mechanisms of action at the molecular level.

Whereas few proteins such as FAS, ACC and PLIN did not significantly differ based on western blotting results, we observed significant differences in these proteins using proteomic analysis. However, proteomic analysis is a more robust technique and superior to western blotting. LC/MSMS is a quantitative technique versus a semi- quantitative nature of the western blotting. Also in western blotting, the specificity of the antibodies is questionable when we see false positive nonspecific bands [192] (which we did see with FAS and ACC). The only way, to know if the antibody is specific, is usually that the obtained band corresponds to the molecular weight of the target protein [192]. On the other side, LC/MSMS technique possesses multiple points to verify and check for the 103

quality of the results (i.e. multiple precursor ions and fragment ions per peptide and multiple peptides per protein) [192]. We also used target decoy strategy to exclude false positive results. Any match between an observed peptide and the decoy database (which is the reverse of the target database) is considered false positive and was excluded.

The obvious limitation of the proteomic technique is that it does not provide an idea about the protein activity. We only measured protein amounts and not the activity and we do not know how well they correlate. We also did not determine the post- translational modifications, such as phosphorylation, acetylation, methylation, and ubiquitination, which are determinants of the protein activity and function [193].

In conclusion, our research provided a proof of the metabolic benefits of these purple vegetables at both phenotypic and molecular levels. Here we give a scientific basis for the consumers to include these vegetables in their diet, for the food industry to include them in functional food products and for the farmers to produce more of these cultivars.

However, we need to emphasize that, based on the results from our first study; there are no large differences between the two color varieties of each vegetable. The observed positive effects with these purple vegetables are probably due to the vegetable effect rather than the color effect which makes the consumption of both the highly (purple) and the lightly (white and orange) colored varieties equally advantageous.

Overall, this research added to the growing body of knowledge that purple plants have beneficial metabolic health effects and that they act through protein expression modulation. The next step will be validation and replication of these findings in human intervention studies.

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CHAPTER 5

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