Ames dwarf mice have a spontaneous homozygous Prophet of Pituitary Factor 1 (Prop1) loss-of-function mutation. The Prop1 mutation results in a lack of differentiation of lactotrophs, thyrotrophs, and somatotrophs in the anterior pituitary. Without these endocrine cell types, Ames dwarf mice have essentially no circulating levels of growth hormone (GH), thyroid-stimulating hormone (TSH), and prolactin, and exhibit downstream hormonal deficiencies including insulin-like growth factor 1 (IGF-1), 3’,3,5-triiodothyronine (T3), and thyroxine (T4). Ames dwarf mice are exceptionally long-lived (40% to over 60% depending on sex and diet). They are also extremely insulin sensitive, have a delayed incidence of cancer, and have improved energy metabolism. While the extended lifespan and the many characteristics of an extended healthspan have been known for some time in Ames dwarf mice, the revelation that dwarf mice have improved energy metabolism was less than a decade ago. This finding came about at the molecular level (improved efficiency of the electron transport chain) and at the whole-animal level (increased oxygen consumption and decreased respiratory quotient). To date, however, few studies have been directed at furthering our understanding of the possible mechanism(s) by which Ames dwarf mice have altered energy metabolism. The goal of the studies presented in this dissertation is to delineate these mechanisms and to lay the groundwork for future studies that broaden our understanding of the role(s) of energy metabolism in the aging process. Project 1 examines the effects of early-life T4 replacement therapy in Ames dwarf mice. Previous work established that life-long T4 replacement therapy shortens lifespan in Snell dwarf mice (these mice have endocrine deficits that are essentially identical to those of Ames dwarf mice), while short-term replacement therapy during the early postnatal period of Ames dwarf mice does not. We hypothesized that T4 replacement therapy causes transient impairment of energy metabolism, which is why long-term T4 replacement therapy shortens longevity, and short-term replacement therapy does not. Supporting our hypothesis, we showed that short-term T4 replacement therapy during the early postnatal period transiently impaired energy metabolism as measured by indirect calorimetry. Following early-life T4 replacement therapy, we also observed an accelerated rate of sexual development, as well as lasting effects on bone physiology. Project 2 continued our investigation of energy metabolism by examining a highly metabolic tissue: brown adipose tissue (BAT), which is responsible for non-shivering thermogenesis. Our laboratory has already demonstrated functional alterations in visceral adipose tissue of Ames dwarf mice, and given the altered energy metabolism of Ames dwarf mice, we hypothesized that BAT may also be functionally unique compared to their normal littermates. Supporting our hypothesis, we observed alterations in gene expression, relative weight, and histological structure of BAT in Ames dwarf mice. Moreover, surgical removal of the interscapular BAT depot resulted in a unique physiological response, where Ames dwarf mice lost adiposity in their subcutaneous, perirenal, and epididymal white adipose tissue depots, thus contrasting with normal mice that gained adiposity. Project 3 built upon the findings of our second study, where we continued to examine the role of non-shivering thermogenesis and core body temperature in Ames dwarf mice. To further understand the role of non-shivering thermogenesis in glucose homeostasis and energy metabolism, we housed a cohort of Ames dwarf mice and their normal littermates at room temperature (23˚C), and another cohort at thermoneutrality (for mice this is 30˚C). We found that Ames dwarf mice placed at thermoneutrality had impaired glucose homeostasis and energy metabolism. This is an important finding because we and others believe both of these metabolic processes are important factors for longevity. Taken together, these studies indicate that the improved energy metabolism in Ames dwarf mice is dependent upon several factors, including a loss of thyroid hormone signaling and improved non-shivering thermogenesis.

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.

Cardiovascular diseases (CVDs) have been recorded as the number one cause of death worldwide, accounting for 32% of total deaths annually. More than two-thirds of all CVD cases are associated with atherosclerosis, which is the accumulation of fats and other substances causing plaque formation in the interior walls of major arteries. This leads to narrowing of the lumen and hardening of the arteries, ultimately resulting in angina, heart attack and/or stroke. Studies have shown that the pathogenesis of atherosclerosis and associated CVDs is strongly linked to elevated secretion of liver-specific lipoproteins called very-low-density-lipoprotein (VLDL). VLDLs are crucial lipoproteins responsible for transportation of triacylglycerides (TAGs), chemically inert particles that are physiologically significant for their energy storing capacity, from the liver to peripheral tissues. These VLDL particles are synthesized in the lumen of the endoplasmic reticulum (ER) of hepatocytes, transported from the ER to the cis-Golgi in special transport vesicles called VLDL-transport-vesicles (VTVs) and secreted into plasma through a highly regulated secretory pathway. Previous studies from our laboratory have shown that VTV-mediated ER-to-Golgi VLDL trafficking is the rate-limiting step in overall VLDL secretion from hepatocytes into plasma. In this project, we investigated intracellular VLDL trafficking and VLDL secretion in Ames dwarf (Prop1df, df/df) mice, a mutant mouse model homozygous for a recessive mutation at Prop1 gene locus (Prop1df) having deficiency of growth hormone (GH), thyroid stimulating hormone (TSH) and prolactin (PRL). This model is characteristic of prolonged longevity (~50% longer) and improved insulin sensitivity in comparison to their wild-type (N) counterparts. Ames dwarf (df/df) mice have recently been shown to have highly reduced plasma TAG levels, associating them with reduced susceptibility to atherosclerosis and associated CVDs. The underlying mechanism responsible for reduced VLDL secretion in Ames dwarf mice is yet to be characterized. We hypothesize that VTV-mediated trafficking of VLDL is reduced in Ames dwarf mice because of reduced expression of proteins regulating VLDL and VTV formation. To test our hypothesis, we first performed VTV-budding assay using cellular fractions isolated separately from Ames dwarf (df/df) and wild-type (N) mice livers. Our results show a significant (45%) reduction in VTV-budding process in Ames dwarf (df/df) mice compared to wild-type (N). Next we performed 2-dimensional differential gel electrophoresis (2-DIGE) on VTV and whole cell lysate (WCL) samples in order to examine the differences in protein expression and to have highly specific protein separation. ExPASy database was used to analyze protein spots that allowed us in identifying proteins specifically expressed in each of the mouse groups. Employing western blotting, samples (ER, cytosol, VTV and WCL) from both sets of mice were tested for expression levels of VLDL and VTV associated proteins (ApoB100, Sec22b, CideB, MTP, Apo-A1 and Apo-AIV) with ?-actin as the loading control. Significant differences in expression level of these proteins were observed which strongly suggest that the formation of VTV from ER in male Ames dwarf (df/df) mice is reduced compared to wild-type (N). Overall, we conclude that the differential expression of proteins required for VLDL transport causes reduced VLDL secretion in male Ames dwarf (df/df) mice.

Ames dwarf (df/df) mice are deficient in anterior pituitary hormones: growth hormone (GH), thyroid stimulating hormone (TSH), and prolactin (PRL) due to a spontaneous, homozygous mutation of prop1[superscript df] gene. These dwarf mice exhibit characteristics such as delayed growth and development coupled with delayed aging, increased lifespan, overall increased insulin sensitivity, as well as resistance to certain diseases and cancers. The mutant mice possess low blood glucose, low serum insulin, and lower body temperature. Their enhanced longevity (about 40-60% longer lifespan than normal mice) is associated with their GH deficiency and disruption in the somatotropic axis (GH/IGF-1 hormonal pathway) as well as increased insulin sensitivity, which is supported by other mutant mouse models for longevity like Snell dwarfs and growth hormone receptor knock-out (GHRKO) mice. When young male Ames dwarf mice were treated with GH replacement therapy, they showed increased body growth to nearly match the normal mouse phenotype. In conjunction to an increase in physical growth, however, GH treatment also decreases the longevity and insulin sensitivity that are characteristic of these mice to levels seen in normal mice. Because of the lack of TSH, they also have undetectable levels of Thyroxine (T4). While T4 treatment didn't increase bodyweight of dwarfs to the same extent as GH treatment, the T4 treated mice retained their enhanced lifespan. Although df/df mice have enhanced whole-body insulin sensitivity, the male skeletal muscle was previously shown to be less responsive to insulin than their liver. In our study we analyzed the insulin signaling pathway in skeletal muscle from female mice after treatment with GH or GH combined with T4. Gene expression and protein expression were investigated in the skeletal muscle of female Ames dwarf mice that were treated with GH or GH and T4 therapy. Real Time Polymerase Chain Reaction (RT-PCR) was used to analyze the expression of mRNA involved with insulin and GH signaling, while western blots were used to analyze protein expression. This project found that female Ames skeletal muscle didn't respond to GH treatment to the same extent as males, and that GH and T4 treatment tends to neutralize the effects seen in GH-only treatment.