Medicine (St Vincent's) - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
ItemAdipose-derived mesenchymal cell derivation, characterization and differentiation for potential use in cell replacement therapy for diabetes( 2013)Type 1 diabetes (T1D) is characterized by the loss of insulin-producing β-cells in the pancreas. T1D can be treated using cadaveric islet transplantation, but this therapy is severely limited by a lack of donor pancreas. To develop an alternative cell therapy, candidate populations were identified through epigenetic characterization of multiple tissues. Histone modification status at the promoter region of key endocrine pancreatic genes was assessed using chromatin immunoprecipitation sequencing (ChIP-seq) and validated using promoter-specific TaqMan-based quantitative PCR (qPCR). Visceral fat was identified as a tissue retaining epigenetic signatures similar to those observed in the pancreas. Human adipose-derived mesenchymal cells (AMCs) were characterized using flowcytometry, confocal microscopy, qPCR, in situ PCR and next generation sequencing technologies. Multiple transcription factor-encoding adenoviruses (e.g. Pdx1, MafA, Ngn3) were employed to determine the differentiation potential of these cells. Analysis of multiple pancreatic hormones and transcription factors in these samples demonstrated consistent differentiation. The differentiation potential was further explored using AMCs isolated from transgenic mice that express GFP under the regulation of Pdx1 (pancreatic and duodenal homeobox 1) or insulin-1 gene promoters. GFP expression was quantitated as an index of gene promoter activity during differentiation to insulin-producing cells, in the presence of various pro-differentiation small molecules. Human AMCs were exposed to a standard differentiation protocol and seen to migrate to form islet-like cell aggregates (ICAs), showing significant increases in islet hormone transcripts in vitro. These adipose-derived ICAs were transplanted into immunocompromised animals using two models of transplantation. Cells were transplanted in a Theracyte immunoisolation device into the peritoneum, and within a blood clot under the kidney capsule. Transplanted cells maintained expression of endocrine pancreatic transcription factors and did not undergo a regressive mesenchymal transition following surgery. Circulating blood samples collected from peripheral circulation of these mice, following a glucose injection, showed that differentiated and engrafted human AMCs could sense, transcribe, translate, package and secrete insulin in response to a glucose stimulus. These studies indicate that human AMCs can differentiate into insulin-producing cells in vitro and have potential for cell replacement therapy in diabetes.
-
ItemRole of AMP-activated protein kinase in regulating skeletal muscle metabolism during exercise( 2013)AMP-activated protein kinase (AMPK) αβγ heterotrimer is an evolutionary conserved serine/ threonine stress sensing kinase that once activated, by low energy status (nutrient deprivation, exercise), restores energy balance by switching off ATP consuming pathways (fatty acid and cholesterol synthesis) and switching on ATP generating pathways (fatty acid oxidation, glucose uptake, mitochondrial biogenesis). Acetyl-CoA carboxylase (ACC; 1 and 2) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA; a precursor for fatty acid synthesis, and an inhibitor of carnitine palmitoyltransferase-1, which controls transport of fatty acids into mitochondria for oxidation. ACC was one of the first identified substrates of AMPK, and phosphorylation of ACC1 at S79 (equivalent S212 site on ACC2) inhibits enzyme activity, reduces malonyl-CoA and suppresses de novo fatty acid synthesis in liver, where ACC1 is predominantly expressed. No studies have assessed the contribution of ACC2 S212 phosphorylation in regulating skeletal muscle fatty acid oxidation, where ACC2 is predominantly expressed. Skeletal muscle is a major contributor to whole-body energy expenditure and is responsible for ~80% of insulin-stimulated glucose disposal; therefore, metabolic alterations in this tissue could influence whole-body insulin sensitivity and substrate utilization. Pharmacological activation of AMPK with aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) enhances fatty acid oxidation, glucose uptake, insulin sensitivity and mitochondrial biogenesis in skeletal muscle; however, AMPK deficient mouse models where a single subunit has been mutated or deleted show a relatively minor role for AMPK in regulating these processes at rest and during exercise. An important caveat of these studies is that AMPK activity is partially suppressed due to presence of the alternative subunit isoform. Interleukin-6 (IL-6) is produced and released from skeletal muscle during exercise, and like AICAR and contraction, increases AMPK activity and is Geffects of IL-6 on substrate utilization during exercise and insulin-sensitivity post-exercise. Therefore, to assess the contribution of AMPK, ACC2 and IL-6 in regulating substrate utilization at rest and during exercise we generated muscle-specific AMPK β1β2 null (M-KO) and whole-body ACC2 S212A knockin (KI) mice and utilized IL-6 KO mice. Under resting conditions, all mice had similar body and tissue weights, oxygen consumption, substrate utilization and activity levels compared to WT littermates. AMPK β1β2 M-KO mice had normal insulin sensitivity despite reduced mitochondria content (~30%) and impaired exercise tolerance (~95%) and glucose uptake during exercise/ muscle contraction (~55 to 70%). IL-KO mice had similar exercise tolerance and muscle glucose clearance during steady-state submaximal treadmill exercise. Muscles from ACC2 S212A KI mice tended to oxidize less fatty acids, which resulted in greater accumulation of muscle ceramides and whole-body and skeletal muscle insulin resistance. ACC2 KI mice were also resistant to AICAR effects on lowering ACC2 activity and enhancing fatty acid oxidation. When challenged with a high-fat diet (HFD) for 12 weeks or exercise, ACC2 KI responded similar to WT, suggesting that AMPK phosphorylation of ACC2 S212 is not essential for metabolic control in response exercise or a HFD. Collectively, these studies highlight that AMPK is important for mitochondrial content, exercise capacity and insulin-independent regulation of glucose uptake during exercise; however, IL-6 or AMPK/ACC2 signaling is not essential for regulating substrate utilization during exercise, and alternative pathways are involved.