MicroRNA-200 Is Induced by Thioredoxin-interacting Protein and Regulates Zeb1 Protein Signaling and Beta Cell Apoptosis

Small noncoding microRNAs have emerged as important regulators of cellular processes, but their role in pancreatic beta cells has only started to be elucidated. Loss of pancreatic beta cells is a key factor in the pathogenesis of diabetes, and we have demonstrated that beta cell expression of thioredoxin-interacting protein (TXNIP) is increased in diabetes and causes beta cell apoptosis, whereas TXNIP deficiency is protective against diabetes. Recently, we found that TXNIP also impairs beta cell function by inducing microRNA (miR)-204. Interestingly, using INS-1 beta cells and primary islets, we have now discovered that expression of another microRNA, miR-200, is induced by TXNIP and by diabetes. Furthermore, we found that miR-200 targeted and decreased Zeb1 (zinc finger E-box-binding homeobox 1) and promoted beta cell apoptosis as measured by cleaved caspase-3 levels, Bax/Bcl2 ratio, and TUNEL. In addition, Zeb1 knockdown mimicked the miR-200 effects on beta cell apoptosis, suggesting that Zeb1 plays an important role in mediating miR-200 effects. Moreover, miR-200 increased beta cell expression of the epithelial marker E-cadherin, consistent with inhibition of epithelial-mesenchymal transition, a process thought to be involved in beta cell expansion. Thus, we have identified a novel TXNIP/miR-200/Zeb1/E-cadherin signaling pathway that, for the first time, links miR-200 to beta cell apoptosis and diabetes and also beta cell TXNIP to epithelial-mesenchymal transition. In addition, our results shed new light on the regulation and function of miR-200 in beta cells and show that TXNIP-induced microRNAs control various processes of beta cell biology.     

Targeted Metabolomics Connects Thioredoxin-interacting Protein (TXNIP) to Mitochondrial Fuel Selection and Regulation of Specific Oxidoreductase Enzymes in Skeletal Muscle

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIP^SKM−/−) Txnip deficiency. Compared with littermate controls, both TKO and TXNIP^SKM−/− mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability.     

Hyperplasia of Pancreatic Beta Cells and Improved Glucose Tolerance in Mice Deficient in the FXYD2 Subunit of Na,K-ATPase

Restoration of the functional potency of pancreatic islets either through enhanced proliferation (hyperplasia) or increase in size (hypertrophy) of beta cells is a major objective for intervention in diabetes. We have obtained experimental evidence that global knock-out of a small, single-span regulatory subunit of Na,K-ATPase, FXYD2, alters glucose control. Adult Fxyd2^−/− mice showed significantly lower blood glucose levels, no signs of peripheral insulin resistance, and improved glucose tolerance compared with their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the Fxyd2^−/− mice compared with the wild type littermates, compatible with an observed increase in the level of circulating insulin. No changes were seen in the exocrine compartment of the pancreas, and the mice had only a mild, well-adapted renal phenotype. Morphometric analysis revealed an increase in beta cell mass in KO compared with WT mice. This appears to explain a phenotype of hyperinsulinemia. By RT-PCR, Western blot, and immunocytochemistry we showed the FXYD2b splice variant in pancreatic beta cells from wild type mice. Phosphorylation of Akt kinase was significantly higher under basal conditions in freshly isolated islets from Fxyd2^−/− mice compared with their WT littermates. Inducible expression of FXYD2 in INS 832/13 cells produced a reduction in the phosphorylation level of Akt, and phosphorylation was restored in parallel with degradation of FXYD2. Thus we suggest that in pancreatic beta cells FXYD2 plays a role in Akt signaling pathways associated with cell growth and proliferation.