Métabolisme du glucose et du glycérol dans la cellule pancréatique β et les hépatocytes et identification des voies de détoxification du glucose

Excessive intake of calorigenic nutrients and low levels of physical activity contribute to the rapid increase in obesity and related metabolic diseases such as metabolic syndrome and type 2 diabetes (T2D). Insulin secreted by the β cells of the islets of Langerhans, is a key hormone in energy regulation of the organism. Plasma glucose is the main stimulator of insulin secretion via the production of metabolic coupling factors (MCF), which are key molecules mediating the link between glucose metabolism by β cell and the exocytosis of insulin. Other calorigenic nutrients such as fatty acids and certain amino acids amplify glucose-induced insulin secretion (GSIS), partly via their intracellular metabolism. Nevertheless, the contribution of MCFs in GSIS and the related mechanisms remain partly unknown. Our laboratory has highlighted the contribution of the glycerolipids/free fatty acids (GL/FFA) cycle in insulin secretion induced by glucose and amplified by fatty acids, and this cycle, which encompasses lipogenesis and lipolysis, plays an important role in the overall regulation of glucose and lipid metabolism. In addition, it takes part in regulating the GSIS via MCF production such as monoacylglycerol and in the intracellular availability of nutrients and their derivatives. Disturbed GL/FFA cycle contributes to the pathogenesis of T2D because of its role in lipid signaling and calorigenic nutrients detoxification. Indeed, chronic exposition to elevated glucose and FFA cause pancreatic β-cell dysfunction and apoptosis as well. Nevertheless, β cells must continually take-up glucose for promoting insulin secretion. To cope with the cellular toxicity caused by excessive intake of calorigenic nutrients, cells use metabolic detoxification pathways to convert calorigenic nutrients into more inert molecules and eventually export them extracellularly.  Cells have various mechanisms and strategies to eliminate "foreign" molecules such as toxins, drugs and other xenobiotics. However, the mechanisms involved in the detoxification of excess nutrients and their derivatives are mostly unknown whatever the cell type.  In the first part of the thesis, we hypothesized the existence of metabolic pathways for calorigenic nutrients detoxification. To highlight these pathways, we evaluated the destination of glucose carbon towards different metabolite candidates. More specifically, one way to identify pathways possibly involved in glucose excess detoxification is to measure metabolic pathway activities and metabolite levels at various concentrations of glucose, and identify those that increase further beyond the maximum glucose concentrations for GSIS. Furthermore, in contrast to most GSIS studies, we used a nutrient enriched incubation medium with a basal level of 4 mmol/l glucose supplemented with glutamine (2 mmol/l) and carnitine (50 µmol/l) when using isolated Langerhans islets. In fact, we realized that the basal level of 2-3 mmol/l glucose alone used in most studies, results in cellular energy depletion.  Overall results indicate that detoxification processes could involve different forms of energy storage (TG, glycogen and cholesterol esters) and the extracellular release of metabolites derived from glucose (glycerol, cholesterol and FFA). Furthermore, data indicate that a major fate of glucose carbons is their transformation and released into the medium in the form of glycerol.  In the second part of the thesis, we sought to identify metabolic pathways and MCF candidates that correlate best with insulin secretion at various concentrations of glucose. This was done in isolated rat Langerhans islets and also using a targeted metabolomic approach in INS 832/13 cells. Several metabolites correlate well with GSIS, particularly several Krebs cycle intermediates, malonyl-CoA and also a lowering in the levels of ADP. Taken together, these data highlight the role of different mechanisms in β-cell metabolic signaling in response to glucose, such as oxidative mitochondrial metabolism, anaplerosis, malonyl-CoA/lipid signaling, and suggest that a lowering in ADP levels may play an important role in GSIS. In the third part of the thesis, given that glycerol 3-phosphate (Gro3P) and glycerol levels increase dramatically in response to glucose in β cells, we sought to determine the process at the source of this glycerol production. In the first study, results suggested that the glycerol production could originate from the GL/FFA cycle but also from an unknown source also associated with extracellular glucose levels. We hypothesized that an unknown enzyme in mammals could directly transform Gro3P into glycerol. Using an in silico analysis approach, we realized that human and mouse phosphoglycolate phosphatase (PGP) share significant amino acid sequence homologies with lower organism enzymes, which hydrolyze Gro3P into glycerol. Thus, we studied in detail the role of PGP in the metabolism of the β cell and hepatocytes. We have determined that PGP has an important Gro3P phosphatase (G3PP) activity in mammals that can hydrolyze Gro3P into glycerol. We hypothesized that PGP effectively acts as a G3PP and plays a central role in the regulation of glucose and lipid metabolism and signaling, as well as in response to metabolic stress. Using molecular biology and biochemistry approaches, we observed that PGP/G3PP, by controlling glycolysis-derived Gro3P intracellular levels, regulates glycolysis flux as well as many metabolic pathways and cellular functions in β cells and hepatocytes. PGP/G3PP also controls the synthesis of glycerolipids, fatty acids oxidation, redox state and mitochondrial energy metabolism in the pancreatic β cell and hepatocytes, the GSIS and the response to metabolic stress in the β cell, and gluconeogenesis in hepatocytes. In summary, we have obtained evidences on the role of various metabolic pathways and MCF in β cell metabolic signaling of GSIS and gluco-detoxification processes. More specifically, we have shed light on metabolic pathways that may be quantitatively important and could play a key role in the detoxification of nutrient excess. The data indicate that β cells convert a significant amount of glucose into glycerol, which is exported out of the cell.  This transformation of the glucose carbons acts probably as a gluco-detoxification process and also could serve to modulate metabolic pathways such as glycolysis. In addition, we discovered a new metabolic pathway in β cells and hepatocytes with the identification of a G3PP that is able to directly transform Gro3P into glycerol. The identification of a previously unknown G3PP in mammalian cells is an important addition to our understanding of the regulation of intermediary metabolism at large, metabolic signaling and glucose excess detoxification. Thus, G3PP is an interesting new therapeutic targets for cardio-metabolic diseases-related disorders such as T2D and obesity.

Keyphrases: glycérol-3-phosphate phosphatase, Glucose and lipid metabolism, Mitochondrial metabolism, pancreatic β-cell, hepatocyte, insulin secretion, gluco-detoxification, glucolipotoxicity, glycerol-3-phosphate phosphatase, type 2 diabetes, metabolic coupling factors