Human Pancreatic Islets React to Glucolipotoxicity by Secreting Pyruvate and Citrate.

Progressive decline in pancreatic beta-cell function is central to the pathogenesis of type 2 diabetes (T2D). Here, we explore the relationship between the beta cell and its nutritional environment, asking how an excess of energy substrate leads to altered energy production and subsequent insulin secretion. Alterations in intracellular metabolic homeostasis are key markers of islets with T2D, but changes in cellular metabolite exchanges with their environment remain unknown. We answered this question using nuclear magnetic resonance-based quantitative metabolomics and evaluated the consumption or secretion of 31 extracellular metabolites from healthy and T2D human islets. Islets were also cultured under high levels of glucose and/or palmitate to induce gluco-, lipo-, and glucolipotoxicity. Biochemical analyses revealed drastic alterations in the pyruvate and citrate pathways, which appear to be associated with mitochondrial oxoglutarate dehydrogenase (OGDH) downregulation. We repeated these manipulations on the rat insulinoma-derived beta-pancreatic cell line (INS-1E). Our results highlight an OGDH downregulation with a clear effect on the pyruvate and citrate pathways. However, citrate is directed to lipogenesis in the INS-1E cells instead of being secreted as in human islets. Our results demonstrate the ability of metabolomic approaches performed on culture media to easily discriminate T2D from healthy and functional islets.

Warburg-associated acidification represses lactic fermentation independently of lactate, contribution from real-time NMR on cell-free systems.

Lactate accumulation and acidification in tumours are a cancer hallmark associated with the Warburg effect. Lactic acidosis correlates with cancer malignancy, and the benefit it offers to tumours has been the subject of numerous hypotheses. Strikingly, lactic acidosis enhances cancer cell survival to environmental glucose depletion by repressing high-rate glycolysis and lactic fermentation, and promoting an oxidative metabolism involving reactivated respiration. We used real-time NMR to evaluate how cytosolic lactate accumulation up to 40 mM and acidification up to pH 6.5 individually impact glucose consumption, lactate production and pyruvate evolution in isolated cytosols. We used a reductive cell-free system (CFS) to specifically study cytosolic metabolism independently of other Warburg-regulatory mechanisms found in the cell. We assessed the impact of lactate and acidification on the Warburg metabolism of cancer cytosols, and whether this effect extended to different cytosolic phenotypes of lactic fermentation and cancer. We observed that moderate acidification, independently of lactate concentration, drastically reduces the glucose consumption rate and halts lactate production in different lactic fermentation phenotypes. In parallel, for Warburg-type CFS lactate supplementation induces pyruvate accumulation at control pH, and can maintain a higher cytosolic pyruvate pool at low pH. Altogether, we demonstrate that intracellular acidification accounts for the direct repression of lactic fermentation by the Warburg-associated lactic acidosis.

Role of mitochondria-associated endoplasmic reticulum membrane (MAMs) interactions and calcium exchange in the development of type 2 diabetes.

Glucotoxicity-induced ß-cell dysfunction in type 2 diabetes is associated with alterations of mitochondria and the endoplasmic reticulum (ER). Mitochondria and ER form a network in cells that controls cell function and fate. Mitochondria of the pancreatic ß cell play a central role in the secretion of insulin in response to glucose through their ability to produce ATP. Both organelles interact at contact sites, defined as mitochondria-associated membranes (MAMs), which were recently implicated in the regulation of glucose homeostasis. Here, we review MAM functions in the cell and we focus on the crosstalk between the ER and Mitochondria in the context of T2D, highlighting the pivotal role played by MAMs especially in ß cells through inter-organelle calcium exchange and glucotoxicity-associated ß cell dysfunction.

Loss and gain of function of Grp75 or mitofusin 2 distinctly alter cholesterol metabolism, but all promote triglyceride accumulation in hepatocytes.

In the liver, contact sites between the endoplasmic reticulum (ER) and mitochondria (named MAMs) may be crucial hubs for the regulation of lipid metabolism, thus contributing to the exacerbation or prevention of fatty liver. We hypothesized that tether proteins located at MAMs could play a key role in preventing triglyceride accumulation in hepatocytes and nonalcoholic fatty liver disease (NAFLD) occurrence. To test this, we explored the role of two key partners in building MAM integrity and functionality, the glucose-regulated protein 75 (Grp75) and mitofusin 2 (Mfn2), which liver contents are altered in obesity and NAFLD. Grp75 or Mfn2 expression was either silenced using siRNA or overexpressed with adenoviruses in Huh7 cells. Silencing of Grp75 and Mfn2 resulted in decreased ER-mitochondria interactions, mitochondrial network fusion state and mitochondrial oxidative capacity, while overexpression of the two proteins induced mirror impacts on these parameters. Furthermore, Grp75 or Mfn2 silencing decreased cellular cholesterol content and enhanced triglyceride secretion in ApoB100 lipoproteins, while their overexpression led to reverse effects. Cellular phosphatidylcholine/phosphatidylethanolamine ratio was decreased only upon overexpression of the proteins, potentially contributing to altered ApoB100 assembly and secretion. Despite the opposite differences, both silencing and overexpression of Grp75 or Mfn2 induced triglyceride storage, although a fatty acid challenge was required to express the alteration upon protein silencing. Among the mechanisms potentially involved in this phenotype, ER stress was closely associated with altered triglyceride metabolism after Grp75 or Mfn2 overexpression, while blunted mitochondrial FA oxidation capacity may be the main defect causing triglyceride accumulation upon Grp75 or Mfn2 silencing. Further studies are required to decipher the link between modulation of Grp75 or Mfn2 expression, change in MAM integrity and alteration of cholesterol content of the cell. In conclusion, Grp75 or Mfn2 silencing and overexpression in Huh7 cells contribute to altering MAM integrity and cholesterol storage in opposite directions, but all promote triglyceride accumulation through distinct cellular pathways. This study also highlights that besides Mfn2, Grp75 could play a central role in hepatic lipid and cholesterol metabolism in obesity and NAFLD.

Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems.

A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.