The mitochondrial fission proteins Fis1 and Drp1 are important for glucose-induced insulin secretion in glucose-responsive INS1 832/13 cells

Abstract

Background and aims: Mitochondria in living cells exist as a dynamic network that continuously cycle through fusion and fission events. Mitochondrial fusion is regulated by the Mitofusins 1 and 2 (Mfn1 and Mfn2) and the optic atrophy protein 1 (Opa1), whereas fission is mainly controlled by the fission protein 1 (Fis1) and the dynamin related protein 1 (Drp1). Dysfunctional parts of the mitochondrial network are sorted out by mitophagy to support mitochondrial function and vitality. Whether both fission proteins, Fis1 and Drp1, are essential to maintain mitochondrial function and glucose-induced insulin secretion in pancreatic beta cells, is currently being discussed. Therefore the aim of this study was to investigate alterations in mitochondrial dynamics and cellular function in glucose-responsive INS1 832/13 cells after knockdown of Fis1 and Drp1. Materials and methods: Down-regulation of Fis1 and Drp1 were achieved by using the GIPZ Lentiviral short hairpin RNAmir expression system. INS1 832/13 cells were infected with lentiviral particles containing shRNA for Fis1 or Drp1 and a non-silencing shRNA as control. Gene and protein expression were analysed by Real-Time PCR, western blot, and immunofluorescence analyses. The ATP content was measured using the ATPlite assay. Glucose-induced insulin secretion was determined by ELISA. Mitochondrial membrane potential was determined by TMRE staining. Mitochondrial morphology was analysed by fluorescence microscopy after transfection with the pTag BFP Mito plasmid. Results: Knockdown of Fis1 and Drp1 in glucose-responsive INS1 823/13 cells resulted in a significantly diminished gene and protein expression compared to control infected cells. The mitochondrial membrane potential was significantly decreased in shFis1 and shDrp1 cells compared to control cells by 40 and 20%, respectively. A homogenous mitochondrial network structure was observed in INS1 823/13 control cells. Knockdown of Fis1 and Drp1 resulted in a significantly higher mean mitochondrial area compared to control cells. However, whereas the Fis1 knockdown especially increased the value of elongated mitochondria in INS1 823/13 cells, knockdown of Drp1 resulted in an increasing number of clustered mitochondria. In both, shFis1 and shDrp1 cells the ATP content and glucose-induced insulin secretion was significantly reduced compared to control INS1 823/13 cells. Conclusion: Our results suggest that both proteins, Fis1 and Drp1, are important for proper glucose-induced insulin secretion coupling process in pancreatic beta cells. Because the reduced expression of Drp1 and Fis1 resulted in a different mitochondrial structure, we propose for both proteins specific mechanisms on mitochondrial dynamics and cellular function in INS1 beta-cells.