Mitochondrial damages and the regulation of insulin secretion.

Pancreatic beta-cells are able to respond to nutrients, principally glucose, as the primary stimulus for insulin exocytosis. This unique feature requires translation of metabolic substrates into intracellular messengers recognized by the exocytotic machinery. Central to this signal transduction mechanism, mitochondria integrate and generate metabolic signals, thereby coupling glucose recognition with insulin secretion. In response to a glucose rise, nucleotides and metabolites are generated by mitochondria and participate, together with cytosolic Ca2+, in the stimulation of insulin exocytosis. Mitochondrial defects, such as mutations and ROS (reactive oxygen species) production, might be associated with beta-cell failure in the course of diabetes. mtDNA (mitochondrial DNA) mutation A3243G is associated with MIDD (mitochondrial inherited diabetes and deafness). A common hypothesis to explain the link between the genotype and the phenotype is that the mutation might impair mitochondrial metabolism expressly required for beta-cell functions, although this assumption lacks direct demonstration. mtDNA-deficient cellular models are glucose-unresponsive and are defective in mitochondrial function. Recently, we used clonal cytosolic hybrid cells (namely cybrids) harbouring mitochondria derived from MIDD patients. Compared with control mtDNA from the same patient, the A3243G mutation markedly modified metabolic pathways. Moreover, cybrid cells carrying patient-derived mutant mtDNA exhibited deranged cell Ca2+ handling and elevated ROS under metabolic stress. In animal models, transgenic mice lacking expression of the mitochondrial genome specifically in beta-cells are diabetic and their islets are incable of releasing insulin in response to glucose. These various models demonstrate the fragility of nutrient-stimulated insulin secretion, caused primarily by defective mitochondrial function.

Diabetes-associated mitochondrial DNA mutation A3243G impairs cellular metabolic pathways necessary for beta cell function.

AIMS/HYPOTHESIS: Mitochondrial DNA (mtDNA) mutations cause several diseases, including mitochondrial inherited diabetes and deafness (MIDD), typically associated with the mtDNA A3243G point mutation on tRNALeu gene. The common hypothesis to explain the link between the genotype and the phenotype is that the mutation might impair mitochondrial metabolism expressly required for beta cell functions. However, this assumption has not yet been tested. METHODS: We used clonal osteosarcoma cytosolic hybrid cells (namely cybrids) harbouring mitochondria derived from MIDD patients and containing either exclusively wild-type or mutated (A3243G) mtDNA. According to the importance of mitochondrial metabolism in beta cells, we studied the impact of the mutation on key parameters by comparing stimulation of these cybrids by the main insulin secretagogue glucose and the mitochondrial substrate pyruvate. RESULTS: Compared with control mtDNA from the same patient, the A3243G mutation markedly modified metabolic pathways leading to a high glycolytic rate (2.8-fold increase), increased lactate production (2.5-fold), and reduced glucose oxidation (-83%). We also observed impaired NADH responses (-56%), negligible mitochondrial membrane potential, and reduced, only transient ATP generation. Moreover, cybrid cells carrying patient-derived mutant mtDNA exhibited deranged cell calcium handling with increased cytosolic loads (1.4-fold higher), and elevated reactive oxygen species (2.6-fold increase) under glucose deprivation. CONCLUSIONS/INTERPRETATION: The present study demonstrates that the mtDNA A3243G mutation impairs crucial metabolic events required for proper cell functions, such as coupling of glucose recognition to insulin secretion.

Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli.

The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid-induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K(i) (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH-dependent. Kinetics of AtPUMP1-mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.