Inhibition of the malate-aspartate shuttle in mouse pancreatic islets abolishes glucagon secretion without affecting insulin secretion.

Altered secretion of insulin as well as glucagon has been implicated in the pathogenesis of Type 2 diabetes (T2D), but the mechanisms controlling glucagon secretion from α-cells largely remain unresolved. Therefore, we studied the regulation of glucagon secretion from αTC1-6 (αTC1 clone 6) cells and compared it with insulin release from INS-1 832/13 cells. We found that INS-1 832/13 and αTC1-6 cells respectively secreted insulin and glucagon concentration-dependently in response to glucose. In contrast, tight coupling of glycolytic and mitochondrial metabolism was observed only in INS-1 832/13 cells. Although glycolytic metabolism was similar in the two cell lines, TCA (tricarboxylic acid) cycle metabolism, respiration and ATP levels were less glucose-responsive in αTC1-6 cells. Inhibition of the malate-aspartate shuttle, using phenyl succinate (PhS), abolished glucose-provoked ATP production and hormone secretion from αTC1-6 but not INS-1 832/13 cells. Blocking the malate-aspartate shuttle increased levels of glycerol 3-phosphate only in INS-1 832/13 cells. Accordingly, relative expression of constituents in the glycerol phosphate shuttle compared with malate-aspartate shuttle was lower in αTC1-6 cells. Our data suggest that the glycerol phosphate shuttle augments the malate-aspartate shuttle in INS-1 832/13 but not αTC1-6 cells. These results were confirmed in mouse islets, where PhS abrogated secretion of glucagon but not insulin. Furthermore, expression of the rate-limiting enzyme of the glycerol phosphate shuttle was higher in sorted primary ß- than in α-cells. Thus, suppressed glycerol phosphate shuttle activity in the α-cell may prevent a high rate of glycolysis and consequently glucagon secretion in response to glucose. Accordingly, pyruvate- and lactate-elicited glucagon secretion remains unaffected since their signalling is independent of mitochondrial shuttles.

Loss of TFB1M results in mitochondrial dysfunction that leads to impaired insulin secretion and diabetes.

We have previously identified transcription factor B1 mitochondrial (TFB1M) as a type 2 diabetes (T2D) risk gene, using human and mouse genetics. To further understand the function of TFB1M and how it is associated with T2D, we created a ß-cell-specific knockout of Tfb1m, which gradually developed diabetes. Prior to the onset of diabetes, ß-Tfb1m(-/-) mice exhibited retarded glucose clearance owing to impaired insulin secretion. ß-Tfb1m(-/-) islets released less insulin in response to fuels, contained less insulin and secretory granules and displayed reduced ß-cell mass. Moreover, mitochondria in Tfb1m-deficient ß-cells were more abundant with disrupted architecture. TFB1M is known to control mitochondrial protein translation by adenine dimethylation of 12S ribosomal RNA (rRNA). Here, we found that the levels of TFB1M and mitochondrial-encoded proteins, mitochondrial 12S rRNA methylation, ATP production and oxygen consumption were reduced in ß-Tfb1m(-/-) islets. Furthermore, the levels of reactive oxygen species (ROS) in response to cellular stress were increased whereas induction of defense mechanisms was attenuated. We also show increased apoptosis and necrosis as well as infiltration of macrophages and CD4(+) cells in the islets. Taken together, our findings demonstrate that Tfb1m-deficiency in ß-cells caused mitochondrial dysfunction and subsequently diabetes owing to combined loss of ß-cell function and mass. These observations reflect pathogenetic processes in human islets: using RNA sequencing, we found that the TFB1M risk variant exhibited a negative gene-dosage effect on islet TFB1M mRNA levels, as well as insulin secretion. Our findings highlight the role of mitochondrial dysfunction in impairments of ß-cell function and mass, the hallmarks of T2D.

Regulation of core clock genes in human islets.

Nearly all mammalian cells express a set of genes known as clock genes. These regulate the circadian rhythm of cellular processes by means of negative and positive autoregulatory feedback loops of transcription and translation. Recent genomewide association studies have demonstrated an association between a polymorphism near the circadian clock gene CRY2 and elevated fasting glucose. To determine whether clock genes could play a pathogenetic role in the disease, we examined messenger RNA (mRNA) expression of core clock genes in human islets from donors with or without type 2 diabetes mellitus. Microarray and quantitative real-time polymerase chain reaction analyses were used to assess expression of the core clock genes CLOCK, BMAL-1, PER1 to 3, and CRY1 and 2 in human islets. Insulin secretion and insulin content in human islets were measured by radioimmunoassay. The mRNA levels of PER2, PER3, and CRY2 were significantly lower in islets from donors with type 2 diabetes mellitus. To investigate the functional relevance of these clock genes, we correlated their expression to insulin content and glycated hemoglobin levels: mRNA levels of PER2 (ρ = 0.33, P = .012), PER3 (ρ = 0.30, P = .023), and CRY2 (ρ = 0.37, P = .0047) correlated positively with insulin content. Of these genes, expression of PER3 and CRY2 correlated negatively with glycated hemoglobin levels (ρ = -0.44, P = .0012; ρ = -0.28, P = .042). Furthermore, in an in vitro model mimicking pathogenetic conditions, the PER3 mRNA level was reduced in human islets exposed to 16.7 mmol/L glucose per 1 mmol/L palmitate for 48 hours (P = .003). Core clock genes are regulated in human islets. The data suggest that perturbations of circadian clock components may contribute to islet pathophysiology in human type 2 diabetes mellitus.