MiD51 Is Important for Maintaining Mitochondrial Health in Pancreatic Islet and MIN6 Cells

Background: Mitochondrial dynamics are important for glucose-stimulated insulin secretion in pancreatic beta cells. The mitochondrial elongation factor MiD51 has been proposed to act as an anchor that recruits Drp1 from the cytosol to the outer mitochondrial membrane. Whether MiD51 promotes mitochondrial fusion by inactivation of Drp1 is a controversial issue. Since both the underlying mechanism and the effects on mitochondrial function remain unknown, this study was conducted to investigate the role of MiD51 in beta cells. Methods: Overexpression and downregulation of MiD51 in mouse insulinoma 6 (MIN6) and mouse islet cells was achieved using the pcDNA expression vector and specific siRNA, respectively. Expression of genes regulating mitochondrial dynamics and autophagy was analyzed by quantitative Real-Time PCR, glucose-stimulated insulin secretion by ELISA, and cellular oxygen consumption rate by optode sensor technology. Mitochondrial membrane potential and morphology were visualized after TMRE and MitoTracker Green staining, respectively. Immunofluorescence analyses were examined by confocal microscopy. Results: MiD51 is expressed in insulin-positive mouse and human pancreatic islet and MIN6 cells. Overexpression of MiD51 resulted in mitochondrial fragmentation and cluster formation in MIN6 cells. Mitochondrial membrane potential, glucose-induced oxygen consumption rate and glucose-stimulated insulin secretion were reduced in MIN6 cells with high MiD51 expression. LC3 expression remained unchanged. Downregulation of MiD51 resulted in inhomogeneity of the mitochondrial network in MIN6 cells with hyperelongated and fragmented mitochondria. Mitochondrial membrane potential, maximal and glucose-induced oxygen consumption rate and insulin secretion were diminished in MIN6 cells with low MiD51 expression. Furthermore, reduced Mfn2 and Parkin expression was observed. Based on MiD51 overexpression and downregulation, changes in the mitochondrial network structure similar to those in MIN6 cells were also observed in mouse islet cells. Conclusion: We have demonstrated that MiD51 plays a pivotal role in regulating mitochondrial function and hence insulin secretion in MIN6 cells. We propose that this anchor protein of Drp1 is important to maintain a homogeneous mitochondrial network and to avoid morphologies such as hyperelongation and clustering which are inaccessible for degradation by autophagy. Assuming that insulin granule degradation frequently suppresses autophagy in beta cells, MiD51 could be a key element maintaining mitochondrial health

The novel GCK variant p.Val455Leu associated with hyperinsulinism is susceptible to allosteric activation and is conducive to weight gain and the development of diabetes

Aims/hypothesis!#!The mammalian enzyme glucokinase (GK), expressed predominantly in liver and pancreas, plays an essential role in carbohydrate metabolism. Monogenic GK disorders emphasise the role of GK in determining the blood glucose set point.!##!Methods!#!A family with congenital hyperinsulinism (CHI) was examined for GCK gene variants by Sanger sequencing. A combined approach, involving kinetic analysis (also using GK activators and inhibitors), intracellular translocation assays, insulin secretion measurements and structural modelling, was used to investigate the novel variant compared with known variants.!##!Results!#!We report on the novel gain-of-function GCK variant p.Val455Leu (V455L), inherited as an autosomal dominant trait in a German family with CHI and concomitant obesity (fasting blood glucose 2.1 mmol/l, BMI 45.0 kg/m!##!Conclusion/interpretation!#!We provide new knowledge on the structure-function relationship of GK, with special emphasis on enzyme activation, potentially yielding fresh strategic insights into breaking the vicious circle of fluctuating blood glucose levels and the attendant risk of long-lasting metabolic changes in both CHI and type 2 diabetes

Precise expression of Fis1 is important for glucose responsiveness of beta cells

Mitochondrial network functionality is vital for glucose-stimulated insulin secretion in pancreatic beta cells. Altered mitochondrial dynamics in pancreatic beta cells are thought to trigger the development of type 2 diabetes mellitus. Fission protein 1 (Fis1) might be a key player in this process. Thus, the aim of this study was to investigate mitochondrial morphology in dependence of beta cell function, after knockdown and overexpression of Fis1. We demonstrate that glucose-unresponsive cells with impaired glucose-stimulated insulin secretion (INS1-832/2) showed decreased mitochondrial dynamics compared with glucose-responsive cells (INS1-832/13). Accordingly, mitochondrial morphology visualised using MitoTracker staining differed between the two cell lines. INS1-832/2 cells formed elongated and clustered mitochondria, whereas INS1-832/13 cells showed a homogenous mitochondrial network. Fis1 overexpression using lentiviral transduction significantly improved glucose-stimulated insulin secretion and mitochondrial network homogeneity in glucose-unresponsive cells. Conversely, Fis1 downregulation by shRNA, both in primary mouse beta cells and glucose-responsive INS1-832/13 cells, caused unresponsiveness and significantly greater numbers of elongated mitochondria. Overexpression of FIS1 in primary mouse beta cells indicated an upper limit at which higher FIS1 expression reduced glucose-stimulated insulin secretion. Thus, FIS1 was overexpressed stepwise up to a high concentration in RINm5F cells using the RheoSwitch system. Moderate FIS1 expression improved glucose-stimulated insulin secretion, whereas high expression resulted in loss of glucose responsiveness and in mitochondrial artificial loop structures and clustering. Our data confirm that FIS1 is a key regulator in pancreatic beta cells, because both glucosestimulated insulin secretion and mitochondrial dynamics were clearly adapted to precise expression levels of this fission protein