Spinal muscular atrophy (SMA) is a devastating recessive neurological disorder thought to be affecting primarily the motor neurons. As such, paralysis, motor weakness and death ensue. While SMA is most commonly seen in infants and children, it can span all ages. Its genetic etiology revolves around the homozygous deletion or mutation of the SMN1 gene, whose product (SMN protein) has critical and ubiquitous roles in mRNA splicing, amongst various other functions in mRNA metabolism. As such, SMN depletion in other non-neuronal cells type is likely to have physiological repercussions, and perhaps modulate the SMA phenotype. Herein, we identify the molecular pathways of atrophy in skeletal and cardiac muscle of two mouse models of SMA and their therapeutic modulation via the histone deacetylase inhibitor trichostatin A. We also identify dramatic changes in immune organs in mouse models of SMA, which could impact susceptibility to infections. Furthermore, we establish the presence of important defects in fatty acid homeostasis in the liver and plasma seen in both mouse models and SMA patients. Finally, we provide the first mild mouse model of SMA that reliably reproduces canonical features of SMA, permitting aging studies. This model presents with a prominent myopathic phenotype prior to motor neuron death, without extra-neuronal involvement during the course of its lifespan. Overall, our work shows multiple potentially clinically relevant defects in extra-neuronal organs, provides ways to abrogate them and provides a framework to study them over the course of aging.

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Indiana University-Purdue University Indianapolis (IUPUI) / Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that primarily regulates glycolytic and lipogenic enzymes such as L-pyruvate kinase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and mitochondrial glycerol-3-phosphate acyltransferase 1. SREBP-1c activity is higher in the liver of human obese patients, as well as ob/ob and db/db mouse models of obesity and type 2 diabetes, underscoring the role of this transcription factor as a contributor to hepatic steatosis and insulin resistance. Nonetheless, SREBP-1 deficient ob/ob mice, do not display improved glycemia despite a significant decrease in hepatic lipid accumulation, suggesting that SREBP-1 might play a role at regulating carbohydrate metabolism. By silencing SREBP-1 in the liver of normal and type 2 diabetes db/db mice, we showed that indeed, SREBP-1 is needed for appropriate regulation of glycogen synthesis and gluconeogenesis enzyme gene expression. Depleting SREBP-1 activity more than 90%, resulted in a significant loss of glycogen deposition and increased expression of Pck1 and G6pc. Hence, the benefits of reducing de novo lipogenesis in db/db mice were offset by the negative impact on gluconeogenesis and glycogen synthesis. Some studies had also indicated that SREBP-1 regulates the insulin signaling pathway, through regulation of IRS2 and a subunit of the PI3K complex, p55g. To gain insight on the consequences of silencing SREBP-1 on insulin sensitivity, we analyzed the insulin signaling and mTOR pathways, as both are interconnected through feedback mechanisms. These studies suggest that SREBP-1 regulates S6K1, a downstream effector of mTORC1, and a key molecule to activate the synthesis of protein. Furthermore, these analyses revealed that depletion of SREBP-1 leads to reduced insulin sensitivity. Overall, our data indicates that SREBP-1 regulates pathways important for the fed state, including lipogenesis, glycogen and protein synthesis, while inhibiting gluconeogenesis. Therefore, SREBP-1 coordinates multiple aspects of the anabolic response in response to nutrient abundance. These results are in agreement with emerging studies showing that SREBP-1 regulates a complex network of genes to coordinate metabolic responses needed for cell survival and growth, including fatty acid metabolism; phagocytosis and membrane biosynthesis; insulin signaling; and cell proliferation.

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The prevalence of non-alcoholic fatty liver disease (NAFLD) in children has increased over the past decades, creating a need for animal models that recapitulate the features of the pediatric disease. Iberian pigs have a leptin-resistant phenotype characterized by hyperleptinemia, hyperphagia, and extreme adipogenesis. We hypothesized that neonatal Iberian pigs fed a high fat high-fructose (HFF) diet will develop a pattern of liver injury resembling pediatric NAFLD. In addition, we sought to determine if a mixture of probiotics would prevent the disease. Animals were fed 1 of 4 diets containing (g/kg body weight × d) 0 g fructose, 11 g fat and 199 kcal (CON-N; n=8), 22 g fructose, 16 g fat and 300 kcal (HFF2-N; n=8), CON + probiotic (CON-P; n=6), or HFF2 + probiotic (HFF2-P; n=6) every 6 h for 70 d. The probiotic mixture (6.2 × 104 cfu/mL) contained Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum and Bacillus amyloliquefaciens. Body weight was recorded every 3 d. Serum markers of liver injury and dyslipidemia were measured on d 40 and 65 at 2 h post feeding. Fasting leptin, insulin, glucose and homeostatic model assessment (HOMA) values were assessed on d 70. Liver and skeletal muscle (longissimus dorsi) were collected on d 70 for histology, triacylglyceride (TAG) quantification, relative gene expression, and Western blot analysis. Metabolomic analysis was performed on liver tissue and plasma. Body weight was not significantly greater in HFF fed pigs compared to CON. Leptin, alanine and aspartate aminotransferases, alkaline phosphatase, lactate dehydrogenase and total bilirubin were increased (P ≤ 0.001), and high and low density lipoproteins decreased (P ≤ 0.05) in HFF2-N and HFF2-P. Livers in HFF2-P and HFF2-N had higher relative weight and TAG (P ≤ 0.001), micro and macrovesicular steatosis, ballooning degeneration, Mallory-denk bodies, inflammation and necrosis, increased gene expression of TNFα, TGFβ, IL1α and PPARγ (P ≤ 0.001), and decreased ChREBP (P ≤ 0.001). A probiotic affect was seen as pigs fed CON-P and HFF2-P had higher insulin and HOMA values were increased (P ≤ 0.05). Western blot analysis showed dysregulation of autophagy in liver of pigs fed CON-P and HFF2-P, and in skeletal muscle of pigs fed CON-N and HFF2-N. Metabolomic analysis demonstrated dysregulation of one-carbon metabolism, the tricarboxylic acid cycle (TCA), the urea cycle, and amino acid metabolism of pigs fed HFF2 diets compared to CON diets. In conclusion, Iberian pigs fed a HFF diet recapitulate many pediatric NAFLD-associated features, in the absence of obesity and independently of probiotic supplementation, suggesting a potentially suitable model for pediatric NAFLD research. Furthermore, probiotic supplementation did not ameliorate the onset of NAFLD when fed in conjunction with a HFF diet.

The obesity epidemic is an important global health concern. Obesity is associated with a number of diseases including type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease, and some cancers. Insulin resistance, a precursor to type 2 diabetes, is defined as an unresponsiveness of metabolic tissues to insulin, leading to long-term hyperglycemia and hyperinsulinemia. The fatty acid-induced model of insulin resistance indicates that an accumulation of lipid intermediates interferes with insulin signal transduction leading to insulin resistance. It is, therefore, important to examine means by which these lipid intermediates can be reduced to alleviate interferences in insulin signaling in the treatment of insulin resistance and type 2 diabetes. Exercise and metformin are two common interventions in patients with type 2 diabetes and obesity. They both commonly activate AMP-activated protein kinase (AMPK). AMPK contributes to a number of metabolic processes including increased glucose and fatty acid oxidation. However, the effects of AMPK activation on insulin sensitivity are currently not fully understood. This compilation of studies examined the insulin sensitizing effects of AMPK activation via metformin, exercise, and novel AMPK activator R419 in obese mice. In Chapter 2 we show that metformin increases AMPK phosphorylation of acetyl-CoA carboxylase (ACC) 1 Ser79 and ACC2 Ser212, resulting in increased fatty acid oxidation, decreased lipid content and improvements in hepatic insulin sensitivity. In Chapter 3 we show that exercise-induced improvements in insulin sensitivity occur independent of AMPK phosphorylation of ACC phosphorylation sites and independent of lipid content in the liver. Finally, in Chapter 4 we show that R419 improves skeletal muscle insulin sensitivity independent of AMPK and lipid content but improves exercise capacity via a skeletal muscle AMPK-dependent pathway in obese mice. These findings suggest that future studies examining the effects of AMPK activation in obesity will aid in our understanding of the mechanisms of insulin resistance and introduce methods of prevention and treatment of obesity and type 2 diabetes. / Thesis / Doctor of Philosophy (PhD)

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京都大学 / 新制・課程博士 / 博士(農学) / 甲第25344号 / 農博第2610号 / 新制||農||1107(附属図書館) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 太田 毅, 教授 横井 伯英, 教授 舟場 正幸 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

This thesis focuses on the effects of n-3 polyunsaturated fatty acids (n-3 PUFA) on development of non-alcoholic fatty liver disease (NAFLD) in experiment, on prevalence of this condition in patients with type 2 diabetes mellitus and metabolic syndrome and also on non-invasive diagnostics. The aim was to study the effect of n-3 PUFA on NAFLD development in an experimental model and based on analysis of a group of patients with type 2 diabetes and metabolic syndrome to assess the prevalence of this condition. Lastly we aimed to evaluate non-invasive diagnostic methods of liver fibrosis and NASH. We demonstrated beneficial effects of n-3 PUFA administration on NAFLD development in a C57/Bl6 mice high fat methionin-cholin defficient dietary model of NAFLD. n-3 PUFA administration led to biochemical improvement, decrease of lipid accumulation in the liver as well as improvement of histology. These effects are determined by complex modulation of lipid metabolism, mainly due to decrease in availability of fatty acids for triglyceride synthesis in the liver, changes of adipokine levels and amelioration of proinflammatory status in the liver. In a group of type 2 diabetics we found NAFLD prevalence of almost 80%, 14% of these patients had also signs of liver fibrosis or cirrhosis. Non-invasive methods...