Project description:Obesity-linked type 2 diabetes (T2D) is a major health problem of global epidemic proportions. The onset of T2D is marked by an eventual failure in pancreatic β-cell function and mass that is no longer able to compensate for the inherent insulin resistance and increased metabolic load intrinsic to obesity. However, β-cells have an inbuilt adaptive flexibility enabling them to effectively adjust insulin production rates relative to the metabolic demand. In a commonly used model of obesity-linked T2D, the db/db mouse, we have recently shown that so-called β-cell dysfunction is symptomatic of a marked increase in insulin production attempting to compensate for increased metabolic load. Pancreatic β-cells from these animals have markedly reduced intracellular insulin stores, yet high rates of (pro)insulin secretion (illustrated by severe chronic hyperinsulinemia), together with a substantial increase in proinsulin biosynthesis highlighted by expanded rough endoplasmic reticulum and Golgi apparatus. However, when the metabolic overload and/or hyperglycemia is normalized, β-cells from db/db mice quickly restore their insulin stores and normalize secretory function. This demonstrates the β-cell’s adaptive flexibility and indicates that therapeutic approaches applied to encourage β-cell rest are capable of restoring endogenous β-cell function. However, mechanisms that regulate β-cell adaptive flexibility are essentially unknown. To gain deeper mechanistic insight into the molecular events underlying β-cell adaptive flexibility in db/db β-cells, we conducted a combined proteomic and post-translational modification specific proteomic (PTMomics) approach on islets from db/db mice and wild-type controls (WT) with or without prior exposure to normal glucose levels. We identified differential modifications of proteins involved in cell redox homeostasis, protein refolding, protein K48-linked deubiquitination, mRNA/protein export along the microtubules, focal adhesion, ERK1/2 signaling, and renin-angiotensin-aldosterone signaling, as well as phosphorylation-regulated sialyltransferase activity, associated with β-cell adaptive flexibility. These proteins are all related to proinsulin biosynthesis and processing, maturation of insulin secretory granules, and vesicular trafficking—core pathways involved in the adaptation of insulin production to meet metabolic demand. Collectively, this study outlines a novel and comprehensive global proteome and PTMome signaling map that highlights important molecular mechanisms related to the adaptive flexibility of β-cell function, providing improved insight into disease pathogenesis of T2D.

Project description:A better understanding of the molecular mechanisms underlying the development and progression of diabetic neuropathy (DN) is essential for the design of mechanism-based therapies. We examined changes in global gene expression to define pathways regulated by diabetes in peripheral nerve. Microarray data for 24 week-old BKS db/db and db/+ mouse sciatic nerve were analyzed to define significantly differentially expressed genes (DEGs); DEGs were further used for functional enrichment analysis and network analysis to identify biological processes and pathways differentially regulated in the db/db nerve. Expression profile clustering was performed to identify co-expressed DEGs. A set of co-expressed lipid metabolism genes was used for promoter sequence analysis.We identified 4,017 DEGs; 2,122 genes were up-regulated and 1,895 genes were down-regulated in the db/db relative to the db+ samples. Over-represented biological processes identified by the functional enrichment analysis include cell cycle, lipid metabolic process, lipid transport, carbohydrate metabolic process, response to stress, apoptosis, axonogenesis and cell adhesion. Pathways regulated in the db/db nerve include lipid metabolism, carbohydrate metabolism, energy metabolism, PPAR signaling, apoptosis, and axon guidance. The majority of DEGs in the glycolysis, TCA cycle, oxidative phosphorylation, fatty acid metabolism, glycerolipid metabolism, mitochondrial fatty acid elongation, lipid transport, adipocytokine signaling, PPAR signaling, and apoptosis pathways are up-regulated, whereas most of the axonogenesis-related genes are down-regulated in db/db nerve. A network of DEGs based on their co-citation in literature identified regulatory relationship between Tnf-α and key genes from the regulated pathways. Promoter sequence analysis identified over-represented transcription factor binding site (TFBS) motifs in the promoter regions of twenty two co-expressed lipid metabolism-related genes suggesting coordinated regulation of these genes by multiple transcription factors (TF). Furthermore, TF binding to these TFBS and differentially regulated in our data are annotated with nervous system development and immune response suggesting possible co-regulation of lipid metabolism, nervous system development and stress response genes. Gene expression changes in our data are consistent with pathological characteristics observed in DN including axon degeneration and demyelination, and support existing hypotheses regarding hyperglycemia mediated nerve damage in DN. Our findings support the role of hyperglycemia-induced oxidative stress and ischemia in nerve injury. Our results also support the hypothesis of oxidized lipid-mediated nerve injury and increased mitochondrial oxidative stress in dyslipidemia. Moreover, our analyses revealed a possible co-regulation mechanism connecting hyperlipidemia, stress response and axonal degeneration. Microarray data for 24 week-old BKS db/db and db/+ mouse sciatic nerve were analyzed to define significantly differentially expressed genes

Project description:A better understanding of the molecular mechanisms underlying the development and progression of diabetic neuropathy (DN) is essential for the design of mechanism-based therapies. We examined changes in global gene expression to define pathways regulated by diabetes in peripheral nerve. Microarray data for 24 week-old BKS db/db and db/+ mouse sciatic nerve were analyzed to define significantly differentially expressed genes (DEGs); DEGs were further used for functional enrichment analysis and network analysis to identify biological processes and pathways differentially regulated in the db/db nerve. Expression profile clustering was performed to identify co-expressed DEGs. A set of co-expressed lipid metabolism genes was used for promoter sequence analysis.We identified 4,017 DEGs; 2,122 genes were up-regulated and 1,895 genes were down-regulated in the db/db relative to the db+ samples. Over-represented biological processes identified by the functional enrichment analysis include cell cycle, lipid metabolic process, lipid transport, carbohydrate metabolic process, response to stress, apoptosis, axonogenesis and cell adhesion. Pathways regulated in the db/db nerve include lipid metabolism, carbohydrate metabolism, energy metabolism, PPAR signaling, apoptosis, and axon guidance. The majority of DEGs in the glycolysis, TCA cycle, oxidative phosphorylation, fatty acid metabolism, glycerolipid metabolism, mitochondrial fatty acid elongation, lipid transport, adipocytokine signaling, PPAR signaling, and apoptosis pathways are up-regulated, whereas most of the axonogenesis-related genes are down-regulated in db/db nerve. A network of DEGs based on their co-citation in literature identified regulatory relationship between Tnf-α and key genes from the regulated pathways. Promoter sequence analysis identified over-represented transcription factor binding site (TFBS) motifs in the promoter regions of twenty two co-expressed lipid metabolism-related genes suggesting coordinated regulation of these genes by multiple transcription factors (TF). Furthermore, TF binding to these TFBS and differentially regulated in our data are annotated with nervous system development and immune response suggesting possible co-regulation of lipid metabolism, nervous system development and stress response genes. Gene expression changes in our data are consistent with pathological characteristics observed in DN including axon degeneration and demyelination, and support existing hypotheses regarding hyperglycemia mediated nerve damage in DN. Our findings support the role of hyperglycemia-induced oxidative stress and ischemia in nerve injury. Our results also support the hypothesis of oxidized lipid-mediated nerve injury and increased mitochondrial oxidative stress in dyslipidemia. Moreover, our analyses revealed a possible co-regulation mechanism connecting hyperlipidemia, stress response and axonal degeneration.

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Characterization of Signaling Pathways Associated with Pancreatic β-cell Adaptive Flexibility in Compensation of Obesity-linked Diabetes in <i>db/db</i> Mice.

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