Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
AN ABSTRACT OF THE DISSERTATION OF Cristal M. Hill, for the Doctor of Philosophy degree in Molecular Biology, Microbiology, and Biochemistry, presented on January 22nd 2016, at Southern Illinois University Carbondale. THE METABOLIC EFFECTS OF DIET-INDUCED OBESITY AND GROWTH HORMONE TREATMENT IN LONG-LIVED MICE WITH ALTERED INSULIN AND INSULIN-LIKE GROWTH FACTOR -1 SIGNALING MAJOR PROFESSOR: Dr. Andrzej Bartke It is well established that high calorie diets providing mostly fat and simple carbohydrates as nutrients promote obesity and are associated with metabolic syndromes such as type 2 diabetes and cardiovascular disease. However, the effects of these types of diets in genetically long lived mice remain to be fully elucidated. The effects of high calorie diets have been reported to induce inflammation and alter longevity. However, when viewed in the context of the growth hormone (GH) pathway, these types of diets that have negative impact on IGF-1 and insulin signaling. To examine high calorie diet and GH-treatment effects in long-lived mice, we designed a three part study using hypopituitary Ames dwarf mice that have primary altered endocrine signaling and Pregnancy Associated Plasma Protein-A knockout mice that have normal endocrine signaling. Most importantly, together these studies investigate the detrimental effects of high energy feeding promoting obesity and influencing adipokine profiles that regulate or alter insulin/ IGF-1 signaling that may possibly impair glucose homeostasis in the context of the GH-axis. Longevity and aging are influenced by common intracellular signals of the insulin/insulin-like growth factor (IGF)-1 (IIS) pathway. Abnormally high levels of bioactive IGF-1 increase the development of various cancers and may contribute to metabolic diseases such as insulin resistance. Enhanced availability of IGF-1 is promoted by cleavage of IGF binding proteins (IGFBPs) by proteases, including the pregnancy associated plasma protein-A (PAPPA). In vitro, PAPPA is regulated by pro-inflammatory cytokines (PICs) such as interleukin (IL)-6 and tumor necrosis factor -a (TNF-a). Mice born with deficiency of the Papp-a gene [PAPP-A knockout (KO) mice] live ∼30–40 % longer than their normal littermates and have decreased bioactive IGF-1 on standard diets. In the first study, our objective was to elucidate how the effects of high-fat, high-sucrose diet (HFHS) promote obesity, induce metabolic dysfunction, and alter systemic cytokine levels in PAPP-A KO and normal mice. We show that PAPP-A KO mice fed HFHS diet for 10 weeks were more glucose tolerant and had enhanced insulin sensitivity compared to normal mice fed identical diet. PAPP-A KO mice fed HFHS diet had lower levels of pro-inflammatory cytokines (IL-2, IL-6, and TNF-α) compared to normal mice fed the same diet. Moreover, anti-inflammatory cytokine (IL-4 and adiponectin) levels were higher in PAPP-A KO mice fed HFHS diet compared to normal mice fed HFHS. Circulating PAPP-A levels were elevated in normal mice fed an HFHS diet compared to normal mice fed a standard, low-fat, low-sucrose (LFLS) diet. Indirect calorimetry, at 10 weeks of feeding HFHS diet, showed significantly increased oxygen consumption (VO2) in PAPP-A KO mice fed HFHS diet compared to normal mice fed the same diet. Furthermore, respiratory quotient (RQ) was significantly lower in PAPP-A KO mice fed HFHS diet compared to normal (N) mice fed HFHS diet indicating PAPP-A KO mice fed HFHS diet are able to rely on fat as their primary source of energy more so than normal controls. We conclude that PAPP-A KO mice are resistant to the HFHS diet induction of metabolic dysfunction associated with higher levels of anti-inflammatory cytokines and have a remarkably metabolically flexible phenotype and that some of the effects of HFHS diet in normal animals may be due to increased levels of PAPP-A. We continued our investigations of high calorie diet effects in long-lived endocrine disrupted Ames dwarf mice. Ames dwarf mice are hypopituitary, thus lacking the production of GH. GH stimulates the production of IGF-1; induces insulin resistance, alters inflammatory cytokine levels and can reduce life expectancy in both humans and mice. Disruption of GH signaling by reducing plasma GH levels significantly or deleting GH receptors extends health span and life span in mice as observed in Ames dwarfs. Metabolic stressors such as high-fat diet (HFD) may alter longevity through the GH signaling pathway. Our objective was to investigate the effects of HFD in Ames dwarf and control mice to elucidate the interactions on environmental (diet) and genetic mechanisms that regulate metabolism in aging processes. We show that Ames dwarf mice fed HFD for 12 weeks are sensitive to weight gain and increase in subcutaneous and visceral adiposity, yet are more insulin sensitive and have higher levels of adiponectin compared to control mice fed either standard diet (STD) or HFD. Interleukin 6 levels were lower in Ames dwarf mice fed HFD than control mice fed either STD or HFD. Energy expenditure was higher in Ames dwarf mice fed HFD than control mice fed STD or HFD. Moreover, we show that transplant of epididymal white adipose tissue (eWAT) from Ames dwarf mice fed HFD is able to improve insulin sensitivity in control mice fed the same diet. We conclude that Ames dwarf mice are resistant to the detrimental metabolic effects of HFD and the visceral adipose tissue of Ames dwarf mice can recuse metabolic dysfunction in control mice. In the third study, we investigated the effects of early-life GH-treatment in Ames dwarf mice starting at 1week of age. The focus of this study was to examine the metabolic effects of GH- treatment and HFD feeding during young age, which is the most critical time for biological maturation. In this study, one week old Ames dwarf and control mice were injected with either GH or saline for 6 weeks and fed STD. At 7 weeks of age, test for insulin sensitivity and calorimetric measurements were performed and the animals were subjected to diet switch from STD to HFD for 12 weeks post GH-treatment. With these preliminary data, we focus on the detrimental effects of GH-treatment during development and on the interaction of the effects of GH and diet. We first show that early-life-GH treatment in hypopituitary Ames dwarf mice induces a slight reduction of insulin sensitivity and decreased use of fatty acids as indicated by indirect calorimetry, thus promoting metabolic dysfunction. In addition, we show that the effects of early-life GH-treatment and high fat diet in Ames dwarf mice worsen insulin sensitivity and impair substrate utilization. We will continue to investigate the expression of genes that are associated with metabolism and longevity in these animals. Inhibition of proteases, such as PAPP-A, may be a therapeutic treatment to decrease the activity of biologically active IGF- to induce protection from metabolic dysfunction, including insulin resistance, in humans. Furthermore, it is not likely to inhibit GH/insulin/ IGF-1 signaling in healthy humans at young age, decreasing the activity of the insulin/ IGF-1 pathway at middle age may be beneficial in human therapies in the aims of protecting against metabolic dysfunction. Combined, these studies provide novel information on the interaction of the GH pathway and diets that induce obesity and metabolic dysfunction. Thus, mice with either primarily altered endocrine signaling or deletion of proteases that increase local IGF-1 signaling are protected from the detrimental effects of high calorie diets on metabolic function and energy expenditure.
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