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The metabolic pathways that are involved in regulating insulin secretion from pancreatic β-cells are still incompletely understood. One potential regulator of the metabolic phenotype of β-cells is the transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor (HIF)-1β. ARNT/HIF-1β levels are profoundly reduced in islets obtained from type 2 diabetic patients. However, no study to date has investigated key pathways involved in regulating insulin release in β-cells that lack ARNT/HIF-1β. In this study, we confirm that siRNA-mediated knockdown of ARNT/HIF-1β inhibits glucose-stimulated insulin secretion. We next investigated the metabolic consequence of the loss of ARNT/HIF-1β knockdown. We demonstrate that β-cells with reduced ARNT/HIF-1β expression levels exhibit a 31% reduction in glycolytic flux without significant changes in glucose oxidation or the ATP:ADP ratio. Metabolic profiling of β-cells treated with siRNAs against the ARNT/HIF-1β gene revealed that glycolysis, anaplerosis, and glucose-induced fatty acid production were down-regulated, and all are key events involved in glucose-stimulated insulin secretion. In addition, both first and second phase insulin secretion in islets were significantly reduced after ARNT/HIF-1β knockdown. Together, our data suggest an important role for ARNT/HIF-1β in anaplerosis, and it may play a critical role in maintaining normal secretion competence of β-cells. [ABSTRACT FROM AUTHOR]

Tre-2/USP6, BUB2, cdc16 domain family, member 1 (TBC1D1), a Rab-GTPase activating protein, is a paralogue of AS160, and has been implicated in the canonical insulin-signaling cascade in peripheral tissues. More recently, TBC1D1 was identified in rat and human pancreatic islets; however, the islet function of TBC1D1 remains not fully understood. We examined the role of TBC1D1 in glucose homeostasis and insulin secretion utilizing a rat knockout (KO) model. Chow-fed TBC1D1 KO rats had improved insulin action but impaired glucose-tolerance tests (GTT) and a lower insulin response during an intraperitoneal GTT compared with wild-type (WT) rats. The in vivo data suggest there may be an islet defect. Glucose-stimulated insulin secretion was higher in isolated KO rat islets compared with WT animals, suggesting TBC1D1 is a negative regulator of insulin secretion. Moreover, KO rats displayed reduced β-cell mass, which likely accounts for the impaired whole-body glucose homeostasis. This β-cell mass reduction was associated with increased active caspase 3, and unaltered Ki67 or urocortin 3, suggesting the induction of apoptosis rather than decreased proliferation or dedifferentiation may account for the decline in islet mass. A similar phenotype was observed in TBC1D1 heterozygous animals, highlighting the sensitivity of the pancreas to subtle reductions in TBC1D1 protein. An 8-week pair-fed high-fat diet did not further alter β-cell mass or apoptosis in KO rats, suggesting that dietary lipids per se, do not lead to a further impairment in glucose homeostasis. The present study establishes a fundamental role for TBC1D1 in maintaining in vivo β-cell mass. [ABSTRACT FROM AUTHOR]

Type 2 diabetes is associated with impaired nutrient-regulated anaplerosis and insulin secretion in pancreatic β-cells. One key anaplerotic substrate that may be involved in regulating insulin release is α-ketoglutarate (αKG). Since prolyl hydroxylase domain proteins (PHDs) can metabolize cytosolic αKG, we sought to explore the role of this enzyme in the regulation of β-cell function. The oxygen-sensing PHDs regulate the stability of hypoxia-inducible factor 1α (HIF1α) as well as other proline-containing proteins by catalyzing the hydroxylation of proline residues. This reaction is dependent on sufficient levels of oxygen, iron, and αKG. In the present study, we utilized both pharmacological and genetic approaches to assess the impact of inhibiting PHD activity on β-cell function. We demonstrate that ethyl-3,4-dihydroxybenzoate (EDHB), a PHD inhibitor, significantly blunted glucose-stimulated insulin secretion (GSIS) from 832/13 clonal cells, rat, and human islets. EDHB reduced glucose utilization, ATP/ADP ratio, and key TCA cycle intermediates such as pyruvate, citrate, fumarate, and malate. siRNA-mediated knockdown of PHD1 and PHD3 inhibited GSIS, whereas siRNA-mediated knockdown of PHD2 had no effect on GSIS. Taken together, the current results demonstrate an important role for PHDs as mediators of islet insulin secretion. [ABSTRACT FROM AUTHOR]

Summary Increased protein synthesis plays an etiologic role in diverse cancers. Here, we demonstrate that METTL13 (methyltransferase-like 13) dimethylation of eEF1A (eukaryotic elongation factor 1A) lysine 55 (eEF1AK55me2) is utilized by Ras-driven cancers to increase translational output and promote tumorigenesis in vivo. METTL13-catalyzed eEF1A methylation increases eEF1A's intrinsic GTPase activity in vitro and protein production in cells. METTL13 and eEF1AK55me2 levels are upregulated in cancer and negatively correlate with pancreatic and lung cancer patient survival. METTL13 deletion and eEF1AK55me2 loss dramatically reduce Ras-driven neoplastic growth in mouse models and in patient-derived xenografts (PDXs) from primary pancreatic and lung tumors. Finally, METTL13 depletion renders PDX tumors hypersensitive to drugs that target growth-signaling pathways. Together, our work uncovers a mechanism by which lethal cancers become dependent on the METTL13-eEF1AK55me2 axis to meet their elevated protein synthesis requirement and suggests that METTL13 inhibition may constitute a targetable vulnerability of tumors driven by aberrant Ras signaling. Graphical Abstract Highlights • METTL13 is the physiologic eEF1A lysine 55 dimethyltransferase • METTL13 dimethylation of eEF1A stimulates protein synthesis in cancer cells • The METTL13-eEF1A methylation axis fuels Ras-driven tumorigenesis in vivo • METTL13 depletion sensitizes cancer cells to PI3K and mTOR pathway inhibitors Ras-driven cancers ramp up protein synthesis by increasing the GTPase activity of a translation elongation factor through a mechanism that involves METTL13-catalyzed eEF1A dimethylation [ABSTRACT FROM AUTHOR]