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 secretio

Insulin secretion profiles are modified by overexpression of glutamate dehydrogenase in pancreatic islets

Aims/hypothesis: Glutamate dehydrogenase (GDH) is a mitochondrial enzyme playing a key role in the control of insulin secretion. However, it is not known whether GDH expression levels in beta cells are rate-limiting for the secretory response to glucose. GDH also controls glutamine and glutamate oxidative metabolism, which is only weak in islets if GDH is not allosterically activated by L-leucine or (+/−)-2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH). Methods: We constructed an adenovirus encoding for GDH to overexpress the enzyme in the beta-cell line INS-1E, as well as in isolated rat and mouse pancreatic islets. The secretory responses to glucose and glutamine were studied in static and perifusion experiments. Amino acid concentrations and metabolic parameters were measured in parallel. Results: GDH overexpression in rat islets did not change insulin release at basal or intermediate glucose (2.8 and 8.3mmol/l respectively), but potentiated the secretory response at high glucose concentrations (16.7mmol/l) compared to controls (+35%). Control islets exposed to 5mmol/l glutamine at basal glucose did not increase insulin release, unless BCH was added with a resulting 2.5-fold response. In islets overexpressing GDH glutamine alone stimulated insulin secretion (2.7-fold), which was potentiated 2.2-fold by adding BCH. The secretory responses evoked by glutamine under these conditions correlated with enhanced cellular metabolism. Conclusions/interpretation: GDH could be rate-limiting in glucose-induced insulin secretion, as GDH overexpression enhanced secretory responses. Moreover, GDH overexpression made islets responsive to glutamine, indicating that under physiological conditions this enzyme acts as a gatekeeper to prevent amino acids from being inappropriate efficient secretagogue

Peroxisome proliferator-activated receptor α (PPARα) protects against oleate-induced INS-1E beta cell dysfunction by preserving carbohydrate metabolism

Aims/hypothesis: Pancreatic beta cells chronically exposed to fatty acids may lose specific functions and even undergo apoptosis. Generally, lipotoxicity is triggered by saturated fatty acids, whereas unsaturated fatty acids induce lipodysfunction, the latter being characterised by elevated basal insulin release and impaired glucose responses. The peroxisome proliferator-activated receptor α (PPARα) has been proposed to play a protective role in this process, although the cellular mechanisms involved are unclear. Methods: We modulated PPARα production in INS-1E beta cells and investigated key metabolic pathways and genes responsible for metabolism-secretion coupling during a culture period of 3days in the presence of 0.4mmol/l oleate. Results: In INS-1E cells, the secretory dysfunction primarily induced by oleate was aggravated by silencing of PPARα. Conversely, PPARα upregulation preserved glucose-stimulated insulin secretion, essentially by increasing the response at a stimulatory concentration of glucose (15mmol/l), a protection we also observed in human islets. The protective effect was associated with restored glucose oxidation rate and upregulation of the anaplerotic enzyme pyruvate carboxylase. PPARα overproduction increased both β-oxidation and fatty acid storage in the form of neutral triacylglycerol, revealing overall induction of lipid metabolism. These observations were substantiated by expression levels of associated genes. Conclusions/interpretation: PPARα protected INS-1E beta cells from oleate-induced dysfunction, promoting both preservation of glucose metabolic pathways and fatty acid turnove

Functional significance of repressor element 1 silencing transcription factor (REST) target genes in pancreatic beta cells

Aims/hypothesis: The expression of several neuronal genes in pancreatic beta cells is due to the absence of the transcription factor repressor element 1 (RE-1) silencing transcription factor (REST). The identification of these traits and their functional significance in beta cells has only been partly elucidated. Herein, we investigated the biological consequences of a repression of REST target genes by expressing REST in beta cells. Methods: The effect of REST expression on glucose homeostasis, insulin content and release, and beta cell mass was analysed in transgenic mice selectively expressing REST in beta cells. Relevant target genes were identified in INS-1E and primary beta cells expressing REST. Results: Transgenic mice featuring a beta cell-targeted expression of REST exhibited glucose intolerance and reduced beta cell mass. In primary beta cells, REST repressed several proteins of the exocytotic machinery, including synaptosomal-associated protein (SNAP) 25, synaptotagmin (SYT) IV, SYT VII, SYT IX and complexin II; it impaired first and second phases of insulin secretion. Using RNA interference in INS-1E cells, we showed that SYT IV and SYT VII were implicated in the control of insulin release. Conclusions/interpretation: The data document the critical role of REST target genes in pancreatic beta cells. Specifically, we provide evidence that the downregulation of these genes is detrimental for the exocytosis of large dense core vesicles, thus contributing to beta cell dysfunction and impaired glucose homeostasi

Spin density wave induced disordering of the vortex lattice in superconducting La2−x_{2-x}Srx_xCuO4_4

We use small angle neutron scattering to study the superconducting vortex lattice in La$_{2-x}$Sr$_x$CuO$_4$ as a function of doping and magnetic field. We show that near optimally doping the vortex lattice coordination and the superconducting coherence length $\xi$ are controlled by a van-Hove singularity crossing the Fermi level near the Brillouin zone boundary. The vortex lattice properties change dramatically as a spin-density-wave instability is approached upon underdoping. The Bragg glass paradigm provides a good description of this regime and suggests that SDW order acts as a novel source of disorder on the vortex lattice.Comment: Accepted in Phys. Rev.

Postmortem examination of patient H.M.’s brain based on histological sectioning and digital 3D reconstruction

Modern scientific knowledge of how memory functions are organized in the human brain originated from the case of Henry G. Molaison (H.M.), an epileptic patient whose amnesia ensued unexpectedly following a bilateral surgical ablation of medial temporal lobe structures, including the hippocampus. The neuroanatomical extent of the 1953 operation could not be assessed definitively during H.M.’s life. Here we describe the results of a procedure designed to reconstruct a microscopic anatomical model of the whole brain and conduct detailed 3D measurements in the medial temporal lobe region. This approach, combined with cellular-level imaging of stained histological slices, demonstrates a significant amount of residual hippocampal tissue with distinctive cytoarchitecture. Our study also reveals diffuse pathology in the deep white matter and a small, circumscribed lesion in the left orbitofrontal cortex. The findings constitute new evidence that may help elucidate the consequences of H.M.’s operation in the context of the brain’s overall pathology

M19 Modulates Skeletal Muscle Differentiation and Insulin Secretion in Pancreatic β-Cells through Modulation of Respiratory Chain Activity

Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic β-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic β-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.Peer reviewe

Where have all the beetles gone? Long‐term study reveals carabid species decline in a nature reserve in Northern Germany

1. The drastic insect decline has received increasing attention in scientific as well as in public media. Long-term studies of insect diversity trends are still rare, even though such studies are highly important to assess extent, drivers and potential consequences of insect loss in ecosystems. 2. To gain insights into carabid diversity trends of ancient and sustainably managed woodlands, we analysed data of carabid beetles from a trapping study that has been run for 24 years in an old nature reserve of Northern Germany, the Luneburg Heath. We examined temporal changes in several diversity measures € (e.g. biomass, species richness, functional diversity and phylogenetic diversity) and tested diverse species traits as predictor variables for species occurrence. 3. In contrast to recently published long-term studies of insect diversity, we did not observe a decline in biomass, but in species richness and phylogenetic diversity in carabids at our study site. Additionally, hibernation stage predicted the occurrence probability of carabids: Species hibernating as imagines or both imagines and larvae and breeding in spring showed strongest declines. 4. We assume the detected trends to be the result of external effects such as climate change and the application of pesticides in the surrounding. Our results suggest that the drivers for the insect decline and the responses are multifaceted. This highlights the importance of long-term studies with identification of the catches to, at best, species level to support the understanding of mechanisms driving changes in insect diversity and abundance