Diabetes mellitus is a growing public health concern, presently affecting 25.8 million or 8.3% of the American population. While the availability of novel drugs, techniques, and surgical intervention has improved the survival rate of individuals with diabetes, the prevalence of diabetes is still rising. Type 2 diabetes (T2D) is a result of chronic insulin resistance and loss of -cell mass and function, and it is is always associated with the impairment in energy metabolism, causing increased intracellular fat content in skeletal muscle (SkM), liver, fat, as well as pancreatic islets. As such, the search for novel agents that simultaneously promotes insulin sensitivity and 𝜷-cell survival may provide a more effective strategy to prevent the onset and progression of this disease. Kaempferol is a flavonol that has been identified in many plants and used in traditional medicine. It has been shown to elicit various pharmacological activities in epidemiological and preclinical studies. However, to date, the studies regarding its effect on the pathogenesis of diabetes are very limited. In this dissertation, I explored the anti-diabetic potential of the dietary intake of kaempferol in diet-induced obese mice and insulin-deficient diabetic mice. For the first animal study, kaempferol was supplemented in the diet to determine whether it can prevent insulin resistance and hyperglycemia in high fat (HF) diet-induced obese mice or STZ-induced obese diabetic mice. For the second animal study, kaempferol was administrated once daily via oral gavage to diet-induced obese and insulin-resistant mice or lean STZ-induced diabetic mice to evaluate its efficacy for treating diabetes and further determining the underlying mechanism. The results demonstrated that dietary intake of kaempferol for 5 months (mo) improved insulin sensitivity and glucose tolerances, which were associated with increased Glut4 and AMPKα expression in muscle and adipose tissues in middle-aged mice fed a high-fat (HF) diet. In vitro, kaempferol increased lipolysis and restored chronic high fatty acid-impaired glucose uptake and glycogen synthesis in SkM cells, which were associated with improved AMPKα activity and Glut4 expression. In addition, dietary kaempferol treatment preserved functional pancreatic 𝜷-cell mass and prevented hyperglycemia and glucose intolerance in STZ-induced diabetic mice. Data from the second study show that oral administration of kaempferol significantly improved blood glucose control in obese mice, which was associated with reduced hepatic glucose production and improved whole body insulin sensitivity without altering body weight gain, food consumption, or the adiposity. In addition, kaempferol treatment increased Akt and hexokinase activity, but decreased pyruvate carboxylase and glucose-6 phosphatase activity in the liver homogenate without altering their protein expression. Consistently, kaempferol decreased pyruvate carboxylase activity and suppressed gluconeogenesis in HepG2 cells as well as primary hepatocytes isolated from the livers of obese mice. Kaempferol directly blunted the activity of purified pyruvate carboxylase. In the last study, we found that kaempferol stimulates basal glucose uptake in primary human SkM. In C2C12 mouse myotubes, kaempferol also increased insulin stimulated glycogen synthesis and preserved insulin dependent glycogen synthesis and glucose uptake in the presence of fatty acids. Kaempferol stimulated Akt phosphorylation in a similar time-dependent manner as insulin in human SkM cells. Consistent with this, kaempferol increased Akt and AMPK phosphorylation in isolated murine red SkM tissue. The effect of kaempferol on glucose uptake was blunted in the presence of chemical inhibitors of glucose transporter 4 (Glut4), phosphoinositide 3-kinase (PI3K), glucose transporter 1 (Glut1), and AMPK. The AMPK inhibitor also prevented kaempferol-stimulated Akt phosphorylation. Further, kaempferol improved the stability of insulin receptor substrate-1. Taken together, these studies suggest that the kaempferol is a naturally occurring compound that may be of use in the regulation of glucose homeostasis and diabetes by improving insulin sensitivity and glucose metabolism, as well as by preserving functional 𝜷-cell mass. / Ph. D. / Diabetes mellitus, more commonly referred to as diabetes, is a cause for concern in the context of public health. Currently, 25.8 million or 8.3% of the American population is affected by some type of diabetes. While the development of new drugs, techniques, and surgeries have improved the survival rate of individuals with diabetes, the number of diabetes cases continues to rise. Type 2 diabetes (T2D) is a result of the inability of tissues to respond to insulin and a loss of insulin producing β-cell mass and function. T2D is always associated with an impairment in the storage and release of energy, causing increased fat content in skeletal muscle (SkM), liver, and fat cells, as well as pancreatic islets. As such, the search for new agents that simultaneously promotesthe ability of body tissues to respond to insulin and β-cell survival may provide a more effective strategy to prevent the onset and progression of this disease. Kaempferol is a flavonol that has been identified in many plants and used in traditional medicine. It has been shown to elicit various drug-like activities in incidence and distribution studies as well as in preclinical studies. However, to date, the studies regarding its effect on the onset and progression of diabetes are very limited. In this dissertation, I explored the anti-diabetic potential of the dietary intake of kaempferol in diet-induced obese mice and insulin-deficient diabetic mice. For the first animal study, kaempferol was added to the diet to determine whether it can prevent insulin resistance and high blood glucose in high fat (HF) diet-induced obese mice or chemically-induced obese diabetic mice. For the second animal study, kaempferol was given once daily via oral gavage to diet-induced obese and insulin-resistant mice or lean chemically-induced diabetic mice to evaluate its efficacy for treating diabetes and further determining its mechanism. The results demonstrated that dietary intake of kaempferol for 5 months (mo) improved insulin sensitivity and the ability of body tissues to respond to glucose, which were associated with increased expression of the insulin sensitive glucose transporter (Glut4) and a central regulator of metabolism (AMPKα) in muscle and adipose tissues in middle-aged mice fed a high-fat (HF) diet. In cell culture, kaempferol increased triglyceride breakdown and restored the ability of SkM cells to take up glucose and synthesize glycogen following long-term exposure to elevated fatty acids. These results were also associated with an improved AMPKα activity and Glut4 expression. In addition, kaempferol in the diet preserved functional pancreatic β-cell mass and prevented the development of high blood glucose and the inability of body tissues to respond to glucose in chemically-induced diabetic mice. Data from the second study show that oral administration of kaempferol significantly improved blood glucose control in obese mice, which was associated with reduced glucose production in the liver and an improved ability of the whole body to respond to insulin without altering body weight gain, food consumption, or fat storage. In addition, kaempferol treatment increased the activity of the final enzyme in glucose transport (Akt) and first enzyme (hexokinase) in glucose oxidation, but decreased the activity of the first and final regulatory enzymes in glucose production (pyruvate carboxylase and glucose-6 phosphatase respectively) without altering their protein expression. Consistently, kaempferol decreased pyruvate carboxylase activity and suppressed glucose production in HepG2 liver cells as well as primary liver isolated from obese mice. Kaempferol also directly blunted the activity of purified pyruvate carboxylase. In the last study, we found that kaempferol stimulates non-stimulated glucose uptake in primary human SkM. In C2C12 mouse muscle cells, kaempferol also increased insulin stimulated glycogen synthesis and prevented fatty acid impaired glycogen synthesis and glucose uptake stimulated by insulin. Kaempferol stimulated Akt phosphorylation (the active form of the enzyme) in a similar time-dependent manner as insulin in human SkM cells. Consistent with this, kaempferol increased Akt and AMPK phosphorylation in red SkM tissue from mice. The effect of kaempferol on glucose uptake was inhibited in the presence of chemical inhibitors of Glut4, phosphoinositide 3-kinase (an enzyme in the insulin signaling pathway), glucose transporter 1 (a basal glucose transporter), and AMPK. The AMPK inhibitor also prevented kaempferol-stimulated Akt phosphorylation. Further, kaempferol improved the stability of insulin receptor substrate-1. Taken together, these studies suggest that the kaempferol is a naturally occurring compound that may be of use in the regulation of glucose homeostasis and diabetes by improving insulin responsiveness and glucose storage and breakdown, as well as by preserving functional β-cell mass.

BACKGROUND: The half-time (HT) period of a soccer match is viewed as a period for recovery. Completely inactive and passive HT has implications on metabolic responses and subsequent performance during the initial phases of the second half. PURPOSE: Determine the effects of an active rewarm-up, compared to a passive period, at halftime on various measures of performance during the first 15-minutes of the second half using global positioning system (GPS) units. Identify the effects of the active versus passive HT period, with CHO beverage supplementation, on blood parameters. METHODS: Crossover design study, twenty collegiate male soccer players participated in two 90-minute soccer matches with passive rest (CON) or a moderate-intensity rewarm-up (RWU) during HT with CHO supplementation. Subjects received five fingerstick blood samples throughout the match (BG) and four subjects had serum insulin/BG taken three times during the match. RESULTS: RWU had significantly (p<0.05) higher measures for total distance, average speed, speed exertion, accelerations, HMP distance, decelerations, and EE during TI-4 half when compared to CON. No subjects experienced hypoglycemia. However, CON did have a significant drop in BG after HT and the lowest mean BG taken at 60-minutes. Tendency for CON and RWU to have HT insulin levels that were elevated and reduced, respectively. CONCLUSION: A passive HT period is not optimal, given its causal role in temporary physical performance deficits in the second half of soccer matches. The results provide a strong rationale for collegiate soccer players and teams to incorporate the 8-minute RWU into the HT regime to optimize second half performance. / Master of Science / The half-time (HT) period of a soccer match is commonly viewed as a period of rest and recovery. A completely inactive HT period has metabolic and performance implications during the initial phases of the second half. A transient reduction in blood glucose (BG) concentrations could contribute to the second half performance decrement. The purpose of this study was to determine the effects of an active rewarm-up, compared to a passive period, at halftime on various measures of performance using global positioning system (GPS) units. Additionally, identify the effects of the active versus passive half-time period, with carbohydrate (CHO) beverage supplementation, on blood parameters. In a crossover design study, twenty collegiate male soccer players participated in two 90-minute soccer matches that included a passive rest (CON) or a moderate-intensity rewarm-up (RWU) during the 15-minute HT period with the consumption of a CHO beverage. Each subject received five finger stick blood samples throughout the match to monitor plasma blood glucose and a subset of four subjects had serum insulin taken three times during the match. The study found that an active HT, compared to passive, mixed with CHO supplementation significantly improved physical performance at the start of the second half. A passive HT period is not advised or optimal, given its causal role in temporary physical performance deficits in the second half of soccer matches. The results from this study provide a strong rationale for collegiate soccer players and teams to incorporate the 8- minute moderate-intensity into the HT regime in order to optimize second half performance.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The STAC3 gene is a functionally undefined gene predicted to encode a protein containing two SH3 domains and one cysteine-rich domain. In this study, we determined the potential role of the STAC3 gene in proliferation and differentiation of bovine satellite cells. We isolated satellite cells from skeletal muscle of adult cattle and transfected them with STAC3 small interfering RNA (siRNA) or scrambled siRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. We assessed the differentiation status of bovine satellite cells by quantifying the expression levels of myogenin and myosin heavy chain genes, and by quantifying fusion index. STAC3 knockdown stimulated mRNA and protein expression of myogenin, and myosin heavy chain 3 and 7, and increased fusion index of bovine satellite cells. These data together suggest that STAC3 inhibits differentiation of bovine satellite cells into myotubes. To determine the underlying mechanism, we identified and validated AP1?1 as a STAC3-interacting protein by yeast two-hybrid screening and co-immunoprecipitation. In C2C12 cells, STAC3 knockdown decreased the expression level of AP1?1 protein. In bovine satellite cells, STAC3 knockdown increased the membrane localization of glucose transporter 4 (GLUT4) and glucose uptake. These data together suggest the following mechanism by which STAC3 inhibits differentiation of bovine satellite cells: STAC3 increases AP1?1 stability in cells; a high level of AP1?1 keeps GLUT4 from translocating to the plasma membrane; reduced membrane localization of GLUT4 impedes glucose uptake; and restricted glucose uptake inhibits differentiation of satellite cells into myotubes. / Master of Science

Both in type 1 (T1D) and type 2 diabetes (T2D), the deterioration of glycemic control over time is primarily caused by an inadequate mass and progressive dysfunction of ?-cells, leading to the impaired insulin secretion. Thus, the search for agents to protect b-cell and enhance its function is important for diabetes treatment. Studies have reported that baicalein, a flavone originally isolated from the roots of Chinese herb Scutellaria baicalensis, has various claimed beneficial effects on health, such as anti-oxidant, anti-viral, anti-thrombotic, and anti-inflammatory effects. However, it is unclear whether it exerts an anti-diabetic action. Here, we present evidence that baicalein may be a novel anti-diabetic agent. Specifically, dietary intake of baicalein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in high-fat diet (HFD)-fed middle-aged diabetic mice, which was associated with the improved isle t?-cell survival and mass. Baicalein treatment had no effect on food intake, body weight gain, circulating lipid profile, and insulin sensitivity in HFD-fed mice. In in-vitro studies, baicalein significantly augmented glucose-stimulated insulin secretion in insulin-secreting cells (INS1) and promotes viability of INS1 cells and human islets. These results demonstrate that baicalein may be a naturally occurring anti-diabetic agent by directly modulating pancreatic?-cell function. / Master of Science

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Type 2 diabetes (T2D) is a chronic metabolic disease characterized by hyperglycemia, insulin resistance, and the dysfunction of β-cells. While there are several therapies for T2D, there is no effective treatment that can reverse the functional decline of pancreatic β cells in T2D patients. Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by human intestinal L cells, which can stimulate the proliferation and differentiation of β cells and promote glucose-stimulated insulin secretion (GSIS), thereby playing a critical role in maintaining glycemic homeostasis. Recently, GLP-1-based medications have been developed for treating T2D. However, most of the GLP-1-based drugs are expensive and have significant adverse effects. Therefore, development of safer and more convenient agents that can mimic the physiologically fed state to promote endogenous GLP-1 secretory function of intestinal L-cells to improve glucose homeostasis holds great potential for the prevention and treatment of T2D. This project aimed to examine whether natural compound genipin promotes intestinal GLP-1 secretion and exerts anti-diabetic effects. I found that genipin rapidly increased GLP-1 secretion from intestinal L-cells, with 10 and 100 μM concentration inducing significant incretin hormone release. L-cells exposed to genipin displayed a rapid increase in intracellular [Ca²⁺]i and the activity of phospholipase C (PLC). Inhibition of PLC ablated genipin-stimulated Ca²⁺] increase and GLP-1 secretion, suggesting that genipin-induced GLP-1 release from the cells depends on the PLC/Ca²⁺ pathway. In vivo, genipin reduced the non-fasting and fasting blood glucose levels, improved insulin resistance, and protected again high fat diet-induced liver damage. All together, these data indicate that genipin is a naturally occurring anti-diabetic agent, which could be a pharmaceutical lead for developing anti-diabetic drugs. / M.S. / More than 34 million Americans are suffering from diabetes, with over 90% of these cases being type 2 diabetes (T2D). Loss of β-cell mass and function is central to the deterioration of glycemic control over time in T2D. Therefore, preservation or improvement of β-cell mass and its insulin secretory function could prevent and treat T2D. While there are several pharmaceutical therapies for T2D, no effective treatment is available for reversing functional decline of pancreatic β-cells in T2D patients. It has been well recognized that glucagon-like peptide-1 (GLP-1), which is an incretin hormone secreted from intestinal L-cells, plays a critical role in maintaining glycemic homeostasis via potentiating glucose-stimulated insulin secretion and promoting β-cell proliferation. This present work is to determine whether natural compound genipin promotes intestinal GLP-1 secretion and thus exerts anti-diabetic effect.

The aims of the first study were to determine the effects of early weaning followed by a period of high-grain feeding on signaling protein phosphorylation in muscle and on acetate clearance in growing steers.  Twelve Angus X Simmental steers were used in the study.  Six steers were weaned early (EW) and fed a high-gain diet for 148 d and the remainder were weaned at the normal age (NW).  Longissimus muscle tissue biopsies were collected to determine total and phosphorylated forms of AMP kinase and downstream proteins of the mTOR signaling pathway.   Of the 12, 8 steers were used to assess acetate clearance at different age points.  During early grain feeding, phosphorylated:total ratios of rpS6 and S6K1 were greater in EW steers and they had lower acetate clearance rates and greater synthesis rates.   The objectives of second study were to assess the effects of early grain feeding on acetate and glucose turnover rates, palmitate synthesis, and on acetate and glucose preference by subcutaneous, intramuscular and visceral adipose tissues in finishing steers.  Sixteen Angus x Simmental steers were infused continuously with [2H3] acetate (n = 8) or [U-13C6] glucose (n = 8), over a 12 h period immediately prior to harvest.  Plasma acetate and glucose enrichment, and palmitate enrichment in different adipose tissue locations were determined.  There were no treatment effects on acetate or glucose turnover rates or palmitate fractional synthesis rates (FSR).  Acetate turnover and palmitate FSR from acetate were greater than the corresponding rates from glucose.  There were no differences in preference for acetate or glucose among the fat depots. In conclusion, phosphorylation ratios of signaling proteins were not affected treatment.  Acetate clearance increased when steers were heavier and older.  Thus older calves are able to clear more acetate per unit of time and body mass than younger calves reflective of an enhanced ability to utilize the substrate.  There were no differences in preference for acetate and glucose among the major fat depots.  Thus diets leading to high glucose supply will not preferentially direct energy storage to intramuscular stores. / Ph. D.

Heat stress is detrimental across many, if not most aspects of animal agriculture. Heat stress can have direct effects on the animal, effects on their environment and effects on the feedstuffs they consume. The work presented here will address direct effects on the animal (using dairy cattle) and effects on their feed (using pigs). Dairy cattle were chosen to study direct effects on the animal because they exhibit unique metabolic adaptions during heat stress. One adaptation of interest is a heat-stress induced state of hyperinsulinemia and hypoglycemia while self-limiting feed intake. In regards to effects on feedstuffs, high ambient temperatures are associated with elevated concentrations of mycotoxins in cereal grains. Mycotoxins are problematic because, at sufficient concentrations, they induce specific, toxic effects. Zearalenone is one such mycotoxin that is particularly problematic for swine, as it is a potent phytoestrogen. As such, it can reduce productivity by disrupting reproductive processes. Although these topics are disparate, the overarching goal of this work is to improve agricultural animal productivity during periods of heat stress. Overall, the three objectives of this thesis were to: 1) isolate the production-related effects of hyperinsulinemia with hypoglycemia from heat stress, 2) determine whether glucose supplementation during heat stress could improve or rescue milk production during heat stress, and finally, 3) investigate reproductive tract morphology following different durations of zearalenone consumption in peri-pubertal gilts. The first experiment compared the effects of thermoneutral, thermoneutral + hyperinsulinemic-hypoglycemic clamp (HHC), environmental heat stress, and heat stress + euglycemic clamp (EC) on milk production, milk components, and circulating glucose concentrations. Milk production, as expected, did decrease under heat stress when compared to thermoneutral conditions. Milk production during the HHC was intermediate, which was likely the result of the induced hypoglycemia. Interestingly, the glucose infused during the EC did not improve milk production over heat stress. Baseline blood glucose concentrations declined during heat stress, and those concentrations measured during the thermoneutral period were correlated with the change in blood glucose (from thermoneutral to heat stress). In the second experiment, zearalenone (6 mg) was fed to peri-pubertal gilts for 0, 7 or 21 days. Gilts were monitored daily for observable signs of zearalenone toxicity, and tissues were harvested after 21 days of treatment. No differences in the lengths or weights of any portions of the reproductive tract were observed. Likewise, there were no differences in body weight, carcass weight, dressing percent or liver weight. In summary, heat stress has varied effects on livestock production and the ultimate outcomes are not always predictable. Future work will be aimed at defining the factors that contribute to this variability. / Master of Science / Heat stress can have direct and indirect consequences that affect the efficiency of livestock production. The overall objective of this work is to improve agricultural animal productivity during periods of heat stress. The first study compared milk production, milk characteristics and blood glucose concentrations in the presence or absence of heat stress. Aspects of productivity were also measured when insulin and/or glucose were administered in a manner that mimicked either heat stress or thermoneutral conditions. Heat stress did cause a decline in circulating blood glucose, feed intake, and milk production. Infusion of insulin during thermoneutral conditions caused a decline in milk production but not in feed intake. As a way to compensate for the naturally low circulating glucose during heat stress, glucose was infused during heat stress in the last portion of the experiment. Simply infusing glucose to return the animal to pre-heat stress concentrations did not improve milk production. Further work is needed to determine factors (other than feed intake) that limit milk production during heat stress. The second study investigated whether the mycotoxin, zearalenone, affects the size or weight of the pig's reproductive tract. If differences existed, they would be indicative of potential problems with the function of those tissues. Young female pigs consumed zearalenone for 0, 7 or 21 days and tissues were evaluated 21 days after the experiment began. No changes in tissue weights or lengths were observed. Future experiments should be aimed at determining the concentration and duration of exposure at which zearalenone becomes problematic for young, female pigs.