Absence of the Peptide Transporter 1 Induces a Prediabetic and Depressive-Like Phenotype in Mice.

INTRODUCTION: Protein-enriched diets improve glycemic control in diabetes or emotional behavior in depressive patients. In mice, these benefits depend on intestinal gluconeogenesis activation by di-/tripeptides. Intestinal di-/tripeptides absorption is carried out by the peptide transporter 1, PEPT1. The lack of PEPT1 might thus alter glucose and emotional balance. METHODS: To determine the effects of PEPT1 deficiency under standard dietary conditions or during a dietary challenge known to promote both metabolic and cognitive dysfunction, insulin sensitivity, anxiety, and depressive-like traits, hippocampal serotonin (5-HT) and insulin signaling pathway were measured in wild-type (WT) and Pept1-/- mice fed either a chow or a high-fat high-sucrose (HF-HS) diet. RESULTS: Pept1-/- mice exhibited slight defects in insulin sensitivity and emotional behavior, which were aggravated by an HF-HS diet. Pept1-/- mice fed a chow diet had lower hippocampal 5-HT levels and exhibited cerebral insulin resistance under HF-HS diet. These defects were independent of intestinal gluconeogenesis but might be linked to increased plasma amino acids levels. CONCLUSION: Pept1-/- mice develop prediabetic and depressive-like traits and could thus be used to develop strategies to prevent or cure both diseases.

Hepatic deletion of serine palmitoyl transferase 2 impairs ceramide/sphingomyelin balance, bile acids homeostasis and leads to liver damage in mice.

Ceramides (Cer) have been shown as lipotoxic inducers, which disturb numerous cell-signaling pathways, leading to metabolic disorders such as type 2 diabetes. In this study, we aimed to determine the role of de novo hepatic ceramide synthesis in energy and liver homeostasis in mice. We generated mice lacking serine palmitoyltransferase 2 (Sptlc2), the rate limiting enzyme of ceramide de novo synthesis, in liver under albumin promoter. Liver function, glucose homeostasis, bile acid (BA) metabolism and hepatic sphingolipids content were assessed using metabolic tests and LC-MS. Despite lower expression of hepatic Sptlc2, we observed an increased concentration of hepatic Cer, associated with a 10-fold increase in neutral sphingomyelinase 2 (nSMase2) expression, and a decreased sphingomyelin content in the liver. Sptlc2ΔLiv mice were protected against obesity induced by high fat diet and displayed a defect in lipid absorption. In addition, an important increase in tauro-muricholic acid was associated with a downregulation of the nuclear BA receptor FXR target genes. Sptlc2 deficiency also enhanced glucose tolerance and attenuated hepatic glucose production, while the latter effect was dampened in presence of nSMase2 inhibitor. Finally, Sptlc2 disruption promoted apoptosis, inflammation and progressive development of hepatic fibrosis, worsening with age. Our data suggest a compensatory mechanism to regulate hepatic ceramides content from sphingomyelin hydrolysis, with deleterious impact on liver homeostasis. In addition, our results show the involvement of hepatic sphingolipid modulation in BA metabolism and hepatic glucose production in an insulin-independent manner, which highlight the still under-researched role of ceramides in many metabolic functions.

Improvement of energy metabolism associated with NUTRIOSE® soluble fiber, a dietary ingredient exhibiting prebiotic properties, requires intestinal gluconeogenesis.

While the prevalence of obesity progresses worldwide, the consumption of sugars and dietary fiber increases and decreases, respectively. In this context, NUTRIOSE® soluble fiber is a plant-based food ingredient with beneficial effects in Humans. Here, we studied in mice the mechanisms involved, particularly the involvement of intestinal gluconeogenesis (IGN), the essential function in the beneficial effects of dietary fibers. To determine whether NUTRIOSE® exerts its beneficial effects via the activation of IGN, we studied the effects of dietary NUTRIOSE® on the development of obesity, diabetes and non-alcoholic fatty liver disease (NAFLD), which IGN is able to prevent. To assert the role of IGN in the observed effects, we studied wild-type (WT) and IGN-deficient mice. In line with our hypothesis, NUTRIOSE® exerts metabolic benefits in WT mice, but not in IGN-deficient mice. Indeed, WT mice are protected from body weight gain and NAFLD induced by a high calorie diet. In addition, our data suggests that NUTRIOSE® may improve energy balance by activating a browning process in subcutaneous white adipose tissue. While the gut microbiota composition changes with NUTRIOSE®, this is not sufficient in itself to account for the benefits observed. On the contrary, IGN is obligatory in the NUTRIOSE® benefits, since no benefit take place in absence of IGN. In conclusion, IGN plays a crucial and essential role in the set-up of the beneficial effects of NUTRIOSE®, highlighting the interest of the supplementation of food with healthy ingredients in the context of the current obesity epidemic.

A caveolin-1 dependent glucose-6-phosphatase trafficking contributes to hepatic glucose production.

OBJECTIVE: Deregulation of hepatic glucose production is a key driver in the pathogenesis of diabetes, but its short-term regulation is incompletely deciphered. According to textbooks, glucose is produced in the endoplasmic reticulum by glucose-6-phosphatase (G6Pase) and then exported in the blood by the glucose transporter GLUT2. However, in the absence of GLUT2, glucose can be produced by a cholesterol-dependent vesicular pathway, which remains to be deciphered. Interestingly, a similar mechanism relying on vesicle trafficking controls short-term G6Pase activity. We thus investigated whether Caveolin-1 (Cav1), a master regulator of cholesterol trafficking, might be the mechanistic link between glucose production by G6Pase in the ER and glucose export through a vesicular pathway. METHODS: Glucose production from fasted mice lacking Cav1, GLUT2 or both proteins was measured in vitro in primary culture of hepatocytes and in vivo by pyruvate tolerance tests. The cellular localization of Cav1 and the catalytic unit of glucose-6-phosphatase (G6PC1) were studied by western blotting from purified membranes, immunofluorescence on primary hepatocytes and fixed liver sections and by in vivo imaging of chimeric constructs overexpressed in cell lines. G6PC1 trafficking to the plasma membrane was inhibited by a broad inhibitor of vesicular pathways or by an anchoring system retaining G6PC1 specifically to the ER membrane. RESULTS: Hepatocyte glucose production is reduced at the step catalyzed by G6Pase in the absence of Cav1. In the absence of both GLUT2 and Cav1, gluconeogenesis is nearly abolished, indicating that these pathways can be considered as the two major pathways of de novo glucose production. Mechanistically, Cav1 colocalizes but does not interact with G6PC1 and controls its localization in the Golgi complex and at the plasma membrane. The localization of G6PC1 at the plasma membrane is correlated to glucose production. Accordingly, retaining G6PC1 in the ER reduces glucose production by hepatic cells. CONCLUSIONS: Our data evidence a pathway of glucose production that relies on Cav1-dependent trafficking of G6PC1 to the plasma membrane. This reveals a new cellular regulation of G6Pase activity that contributes to hepatic glucose production and glucose homeostasis.

Portal Glucose Infusion, Afferent Nerve Fibers, and Glucose and Insulin Tolerance of Insulin-Resistant Rats.

BACKGROUND: The role of hepatoportal glucose sensors is poorly understood in the context of insulin resistance. OBJECTIVES: We assessed the effects of glucose infusion in the portal vein on insulin tolerance in 2 rat models of insulin resistance, and the role of capsaicin sensitive nerves in this signal. METHODS: Male Wistar rats, 8 weeks old, weighing 250-275 g, were used. Insulin and glucose tolerance were assessed following a 4-hour infusion of either glucose or saline through catheterization in the portal vein in 3 paradigms. In experiment 1, for diet-induced insulin resistance, rats were fed either a control diet (energy content: proteins = 22.5%, carbohydrates = 64.1%, and lipids = 13.4%) or a high-fat diet (energy content: proteins = 15.3%, carbohydrates = 40.3%, and lipids =44.4%) for 4 months. In experiment 2, for centrally induced peripheral insulin resistance, catheters were inserted in the carotid artery to deliver either an emulsion of triglycerides [intralipid (IL)] or saline towards the brain for 24 hours. In experiment 3, for testing the role of capsaicin-sensitive nerves, experiment 2 was repeated following a periportal treatment with capsaicin or vehicle. RESULTS: In experiment 1, when compared to rats fed the control diet, rats fed the high-fat diet exhibited decreased insulin and glucose tolerance (P ≤ 0.05) that was restored with a glucose infusion in the portal vein (P ≤ 0.05). In experiment 2, infusion of a triglyceride emulsion towards the brain (IL rats) decreased insulin and glucose tolerance and increased hepatic endogenous production when compared to saline-infused rats (P ≤ 0.05). Glucose infusion in the portal vein in IL rats restored insulin and glucose tolerance, as well as hepatic glucose production, to controls levels (P ≤ 0.05). In experiment 3, portal infusion of glucose did not increase insulin tolerance in IL rats that received a periportal pretreatment with capsaicin. CONCLUSIONS: Stimulation of hepatoportal glucose sensors increases insulin tolerance in rat models of insulin resistance and requires the presence of capsaicin-sensitive nerves.

mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease.

Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade®/modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a.

Dietary Fibers and Proteins Modulate Behavior via the Activation of Intestinal Gluconeogenesis.

INTRODUCTION: Several studies have suggested that diet, especially the one enriched in microbiota-fermented fibers or fat, regulates behavior. The underlying mechanisms are currently unknown. We previously reported that certain macronutrients (fermentable fiber and protein) regulate energy homeostasis via the activation of intestinal gluconeogenesis (IGN), which generates a neural signal to the brain. We hypothesized that these nutriments might control behavior using the same gut-brain circuit. METHODS: Wild-type and IGN-deficient mice were fed chow or diets enriched in protein or fiber. Changes in their behavior were assessed using suited tests. Hippocampal neurogenesis, extracellular levels of serotonin, and protein expression levels were assessed by immunofluorescence, in vivo dialysis, and Western blotting, respectively. IGN was rescued by infusing glucose into the portal vein of IGN-deficient mice. RESULTS: We show here that both fiber- and protein-enriched diets exert beneficial actions on anxiety-like and depressive-like behaviors. These benefits do not occur in mice lacking IGN. Consistently, IGN-deficient mice display hallmarks of depressive-like disorders, including decreased hippocampal neurogenesis, basal hyperactivity, and deregulation of the hypothalamic-pituitary-adrenal axis, which are associated with increased expression of the precursor of corticotropin-releasing hormone in the hypothalamus and decreased expression of the glucocorticoid receptor in the hippocampus. These neurobiological alterations are corrected by portal glucose infusion mimicking IGN. CONCLUSION: IGN translates nutritional information, allowing the brain to finely coordinate energy metabolism and behavior.

Calcitonin Gene-Related Peptide-Induced Phosphorylation of STAT3 in Arcuate Neurons Is a Link in the Metabolic Benefits of Portal Glucose.

INTRODUCTION: Intestinal gluconeogenesis (IGN) exerts metabolic benefits in energy homeostasis via the neural sensing of portal glucose. OBJECTIVE: The aim of this work was to determine central mechanisms involved in the effects of IGN on the control of energy homeostasis. METHODS: We investigated the effects of glucose infusion into the portal vein, at a rate that mimics IGN, in conscious wild-type, leptin-deficient Ob/Ob and calcitonin gene-related peptide (CGRP)-deficient mice. RESULTS: We report that portal glucose infusion decreases food intake and plasma glucose and induces in the hypothalamic arcuate nucleus (ARC) the phosphorylation of STAT3, the classic intracellular messenger of leptin signaling. This notably takes place in POMC-expressing neurons. STAT3 phosphorylation does not require leptin, since portal glucose effects are observed in leptin-deficient Ob/Ob mice. We hypothesized that the portal glucose effects could require CGRP, a neuromediator previously suggested to suppress hunger. In line with this hypothesis, neither the metabolic benefits nor the phosphorylation of STAT3 in the ARC take place upon portal glucose infusion in CGRP-deficient mice. Moreover, intracerebroventricular injection of CGRP activates hypothalamic phosphorylation of STAT3 in mice, and CGRP does the same in hypothalamic cells. Finally, no metabolic benefit of dietary fibers (known to depend on the induction of IGN), takes place in CGRP-deficient mice. CONCLUSIONS: CGRP-induced phosphorylation of STAT3 in the ARC is part of the neural chain determining the hunger-modulating and glucose-lowering effects of IGN/portal glucose.

Atrial Natriuretic Peptide Orchestrates a Coordinated Physiological Response to Fuel Non-shivering Thermogenesis.

Atrial natriuretic peptide (ANP) is a cardiac hormone controlling blood volume and pressure in mammals. It is still unclear whether ANP controls cold-induced thermogenesis in vivo. Here, we show that acute cold exposure induces cardiac ANP secretion in mice and humans. Genetic inactivation of ANP promotes cold intolerance and suppresses half of cold-induced brown adipose tissue (BAT) activation in mice. While white adipocytes are resistant to ANP-mediated lipolysis at thermoneutral temperature in mice, cold exposure renders white adipocytes fully responsive to ANP to activate lipolysis and a thermogenic program, a physiological response that is dramatically suppressed in ANP null mice. ANP deficiency also blunts liver triglycerides and glycogen metabolism, thus impairing fuel availability for BAT thermogenesis. ANP directly increases mitochondrial uncoupling and thermogenic gene expression in human white and brown adipocytes. Together, these results indicate that ANP is a major physiological trigger of BAT thermogenesis upon cold exposure in mammals.

The role of kidney in the inter-organ coordination of endogenous glucose production during fasting.

OBJECTIVE: The respective contributions to endogenous glucose production (EGP) of the liver, kidney and intestine vary during fasting. We previously reported that the deficiency in either hepatic or intestinal gluconeogenesis modulates the repartition of EGP via glucagon secretion (humoral factor) and gut-brain-liver axis (neural factor), respectively. Considering renal gluconeogenesis reportedly accounted for approximately 50% of EGP during fasting, we examined whether a reduction in renal gluconeogenesis could promote alterations in the repartition of EGP in this situation. METHODS: We studied mice whose glucose-6-phosphatase (G6Pase) catalytic subunit (G6PC) is specifically knocked down in the kidneys (K-G6pc-/- mice) during fasting. We also examined the additional effects of intestinal G6pc deletion, renal denervation and vitamin D administration on the altered glucose metabolism in K-G6pc-/- mice. RESULTS: Compared with WT mice, K-G6pc-/- mice exhibited (1) lower glycemia, (2) enhanced intestinal but not hepatic G6Pase activity, (3) enhanced hepatic glucokinase (GK encoded by Gck) activity, (4) increased hepatic glucose-6-phosphate and (5) hepatic glycogen spared from exhaustion during fasting. Increased hepatic Gck expression in the post-absorptive state could be dependent on the enhancement of insulin signal (AKT phosphorylation) in K-G6pc-/- mice. In contrast, the increase in hepatic GK activity was not observed in mice with both kidney- and intestine-knockout (KI-G6pc-/- mice). Hepatic Gck gene expression and hepatic AKT phosphorylation were reduced in KI-G6pc-/- mice. Renal denervation by capsaicin did not induce any effect on glucose metabolism in K-G6pc-/- mice. Plasma level of 1,25 (OH)2 D3, an active form of vitamin D, was decreased in K-G6pc-/- mice. Interestingly, the administration of 1,25 (OH)2 D3 prevented the enhancement of intestinal gluconeogenesis and hepatic GK activity and blocked the accumulation of hepatic glycogen otherwise observed in K-G6pc-/- mice during fasting. CONCLUSIONS: A diminution in renal gluconeogenesis that is accompanied by a decrease in blood vitamin D promotes a novel repartition of EGP among glucose producing organs during fasting, featured by increased intestinal gluconeogenesis that leads to sparing glycogen stores in the liver. Our data suggest a possible involvement of a crosstalk between the kidneys and intestine (via the vitamin D system) and the intestine and liver (via a neural gut-brain axis), which might take place in the situations of deficient renal glucose production, such as chronic kidney disease.

Hepatocytes contribute to residual glucose production in a mouse model for glycogen storage disease type Ia.

It is a long-standing enigma how glycogen storage disease (GSD) type I patients retain a limited capacity for endogenous glucose production despite the loss of glucose-6-phosphatase activity. Insight into the source of residual endogenous glucose production is of clinical importance given the risk of sudden death in these patients, but so far contradictory mechanisms have been proposed. We investigated glucose-6-phosphatase-independent endogenous glucose production in hepatocytes isolated from a liver-specific GSD Ia mouse model (L-G6pc-/- mice) and performed real-time analysis of hepatic glucose fluxes and glycogen metabolism in L-G6pc-/- mice using state-of-the-art stable isotope methodologies. Here we show that G6pc-deficient hepatocytes are capable of producing glucose. In vivo analysis of hepatic glucose metabolism revealed that the hepatic glucokinase flux was decreased by 95% in L-G6pc-/- mice. It also showed increased glycogen phosphorylase flux in L-G6pc-/- mice, which is coupled to the release of free glucose through glycogen debranching. Although the ex vivo activities of debranching enzyme and lysosomal acid maltase, two major hepatic α-glucosidases, were unaltered in L-G6pc-/- mice, pharmacological inhibition of α-glucosidase activity almost completely abolished residual glucose production by G6pc-deficient hepatocytes. CONCLUSION: Our data indicate that hepatocytes contribute to residual glucose production in GSD Ia. We show that α-glucosidase activity, i.e. glycogen debranching and/or lysosomal glycogen breakdown, contributes to residual glucose production by GSD Ia hepatocytes. A strong reduction in hepatic GCK flux in L-G6pc-/- mice furthermore limits the phosphorylation of free glucose synthesized by G6pc-deficient hepatocytes, allowing the release of glucose into the circulation. The almost complete abrogation of GCK flux in G6pc-deficient liver also explains the contradictory reports on residual glucose production in GSD Ia patients. (Hepatology 2017;66:2042-2054).

Gut-Brain Glucose Signaling in Energy Homeostasis.

Intestinal gluconeogenesis is a recently identified function influencing energy homeostasis. Intestinal gluconeogenesis induced by specific nutrients releases glucose, which is sensed by the nervous system surrounding the portal vein. This initiates a signal positively influencing parameters involved in glucose control and energy management controlled by the brain. This knowledge has extended our vision of the gut-brain axis, classically ascribed to gastrointestinal hormones. Our work raises several questions relating to the conditions under which intestinal gluconeogenesis proceeds and may provide its metabolic benefits. It also leads to questions on the advantage conferred by its conservation through a process of natural selection.

The suppression of hepatic glucose production improves metabolism and insulin sensitivity in subcutaneous adipose tissue in mice.

AIMS/HYPOTHESIS: Despite the strong correlation between non-alcoholic fatty liver disease and insulin resistance, hepatic steatosis is associated with greater whole-body insulin sensitivity in several models. We previously reported that the inhibition of hepatic glucose production (HGP) protects against the development of obesity and diabetes despite severe steatosis, thanks to the secretion of specific hepatokines such as fibroblast growth factor 21 (FGF21) and angiopoietin-related growth factor. In this work, we focused on adipose tissue to assess whether liver metabolic fluxes might, by interorgan communication, control insulin signalling in lean animals. METHODS: Insulin signalling was studied in the adipose tissue of mice lacking the catalytic subunit of glucose 6-phosphatase, the key enzyme in endogenous glucose production, in the liver (L-G6pc -/- mice). Morphological and metabolic changes in the adipose tissues were characterised by histological analyses, gene expression and protein content. RESULTS: Mice lacking HGP exhibited improved insulin sensitivity of the phosphoinositide 3-kinase/Akt pathway in the subcutaneous adipose tissue associated with a browning of adipocytes. The suppression of HGP increased FGF21 levels in lean animals, and increased FGF21 was responsible for the metabolic changes in the subcutaneous adipose tissue but not for its greater insulin sensitivity. The latter might be linked to an increase in the ratio of monounsaturated to saturated fatty acids released by the liver. CONCLUSIONS: Our work provides evidence that HGP controls subcutaneous adipose tissue browning and insulin sensitivity through two pathways: the release of beneficial hepatokines and changes in hepatic fatty acids profile.

Post-Translational Regulation of the Glucose-6-Phosphatase Complex by Cyclic Adenosine Monophosphate Is a Crucial Determinant of Endogenous Glucose Production and Is Controlled by the Glucose-6-Phosphate Transporter.

The excessive endogenous glucose production (EGP) induced by glucagon participates in the development of type 2 diabetes. To further understand this hormonal control, we studied the short-term regulation by cyclic adenosine monophosphate (cAMP) of the glucose-6-phosphatase (G6Pase) enzyme, which catalyzes the last reaction of EGP. In gluconeogenic cell models, a 1-h treatment by the adenylate cyclase activator forskolin increased G6Pase activity and glucose production independently of any change in enzyme protein amount or G6P content. Using specific inhibitors or protein overexpression, we showed that the stimulation of G6Pase activity involved the protein kinase A (PKA). Results of site-directed mutagenesis, mass spectrometry analyses, and in vitro phosphorylation experiments suggested that the PKA stimulation of G6Pase activity did not depend on a direct phosphorylation of the enzyme. However, the temperature-dependent induction of both G6Pase activity and glucose release suggested a membrane-based mechanism. G6Pase is composed of a G6P transporter (G6PT) and a catalytic unit (G6PC). Surprisingly, we demonstrated that the increase in G6PT activity was required for the stimulation of G6Pase activity by forskolin. Our data demonstrate the existence of a post-translational mechanism that regulates G6Pase activity and reveal the key role of G6PT in the hormonal regulation of G6Pase activity and of EGP.

A gut-brain neural circuit controlled by intestinal gluconeogenesis is crucial in metabolic health.

OBJECTIVES: Certain nutrients positively regulate energy homeostasis via intestinal gluconeogenesis (IGN). The objective of this study was to evaluate the impact of a deficient IGN in glucose control independently of nutritional environment. METHODS: We used mice deficient in the intestine glucose-6 phosphatase catalytic unit, the key enzyme of IGN (I-G6pc (-/-) mice). We evaluated a number of parameters involved in energy homeostasis, including insulin sensitivity (hyperinsulinemic euglycaemic clamp), the pancreatic function (insulin secretion in vivo and in isolated islets) and the hypothalamic homeostatic function (leptin sensitivity). RESULTS: Intestinal-G6pc (-/-) mice exhibit slight fasting hyperglycaemia and hyperinsulinemia, glucose intolerance, insulin resistance and a deteriorated pancreatic function, despite normal diet with no change in body weight. These defects evoking type 2 diabetes (T2D) derive from the basal activation of the sympathetic nervous system (SNS). They are corrected by treatment with an inhibitor of α-2 adrenergic receptors. Deregulation in a key target of IGN, the homeostatic hypothalamic function (highlighted here through leptin resistance) is a mechanistic link. Hence the leptin resistance and metabolic disorders in I-G6pc (-/-) mice are corrected by rescuing IGN by portal glucose infusion. Finally, I-G6pc (-/-) mice develop the hyperglycaemia characteristic of T2D more rapidly under high fat/high sucrose diet. CONCLUSIONS: Intestinal gluconeogenesis is a mandatory function for the healthy neural control of glucose homeostasis.

A link between hepatic glucose production and peripheral energy metabolism via hepatokines.

Type 2 diabetes is characterized by a deterioration of glucose tolerance, which associates insulin resistance of glucose uptake by peripheral tissues and increased endogenous glucose production. Here we report that the specific suppression of hepatic glucose production positively modulates whole-body glucose and energy metabolism. We used mice deficient in liver glucose-6 phosphatase that is mandatory for endogenous glucose production. When they were fed a high fat/high sucrose diet, they resisted the development of diabetes and obesity due to the activation of peripheral glucose metabolism and thermogenesis. This was linked to the secretion of hepatic hormones like fibroblast growth factor 21 and angiopoietin-like factor 6. Interestingly, the deletion of hepatic glucose-6 phosphatase in previously obese and insulin-resistant mice resulted in the rapid restoration of glucose and body weight controls. Therefore, hepatic glucose production is an essential lever for the control of whole-body energy metabolism during the development of obesity and diabetes.

Glucotoxicity induces glucose-6-phosphatase catalytic unit expression by acting on the interaction of HIF-1α with CREB-binding protein.

The activation of glucose-6-phosphatase (G6Pase), a key enzyme of endogenous glucose production, is correlated with type 2 diabetes. Type 2 diabetes is characterized by sustained hyperglycemia leading to glucotoxicity. We investigated whether glucotoxicity mechanisms control the expression of the G6Pase catalytic unit (G6pc). We deciphered the transcriptional regulatory mechanisms of the G6pc promoter by glucotoxicity in a hepatoma cell line then in primary hepatocytes and in the liver of diabetic mice. High glucose exposure induced the production of reactive oxygen species (ROS) and, in parallel, induced G6pc promoter activity. In hepatocytes, glucose induced G6pc gene expression and glucose release. The decrease of ROS concentrations by antioxidants eliminated all the glucose-inductive effects. The induction of G6pc promoter activity by glucose was eliminated in the presence of small interfering RNA, targeting either the hypoxia-inducible factor (HIF)-1α or the CREB-binding protein (CBP). Glucose increased the interaction of HIF-1α with CBP and the recruitment of HIF-1 on the G6pc promoter. The same mechanism might occur in hyperglycemic mice. We deciphered a new regulatory mechanism induced by glucotoxicity. This mechanism leading to the induction of HIF-1 transcriptional activity may contribute to the increase of hepatic glucose production during type 2 diabetes.

New insights into the organisation and intracellular localisation of the two subunits of glucose-6-phosphatase.

Glucose-6 phosphatase (G6Pase), a key enzyme of glucose homeostasis, catalyses the hydrolysis of glucose-6 phosphate (G6P) to glucose and inorganic phosphate. A deficiency in G6Pase activity causes type 1 glycogen storage disease (GSD-1), mainly characterised by hypoglycaemia. Genetic analyses of the two forms of this rare disease have shown that the G6Pase system consists of two proteins, a catalytic subunit (G6PC) responsible for GSD-1a, and a G6P translocase (G6PT), responsible for GSD-1b. However, since their identification, few investigations concerning their structural relationship have been made. In this study, we investigated the localisation and membrane organisation of the G6Pase complex. To this aim, we developed chimera proteins by adding a fluorescent protein to the C-terminal ends of both subunits. The G6PC and G6PT fluorescent chimeras were both addressed to perinuclear membranes as previously suggested, but also to vesicles throughout the cytoplasm. We demonstrated that both proteins strongly colocalised in perinuclear membranes. Then, we studied G6PT organisation in the membrane. We highlighted FRET between the labelled C and N termini of G6PT. The intramolecular FRET of this G6PT chimera was 27%. The coexpression of unlabelled G6PC did not modify this FRET intensity. Finally, the chimera constructs generated in this work enabled us for the first time to analyze the relationship between GSD-1 mutations and the intracellular localisation of both G6Pase subunits. We showed that GSD1 mutations did neither alter the G6PC or G6PT chimera localisation, nor the interaction between G6PT termini. In conclusion, our results provide novel information on the intracellular distribution and organisation of the G6Pase complex.

Involvement of ATP-sensitive potassium (K(ATP)) channels in the loss of beta-cell function induced by human islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is a major component of amyloid deposition in pancreatic islets of patients with type 2 diabetes. It is known that IAPP can inhibit glucose-stimulated insulin secretion; however, the mechanisms of action have not yet been established. In the present work, using a rat pancreatic beta-cell line, INS1E, we have created an in vitro model that stably expressed human IAPP gene (hIAPP cells). These cells showed intracellular oligomers and a strong alteration of glucose-stimulated insulin and IAPP secretion. Taking advantage of this model, we investigated the mechanism by which IAPP altered beta-cell secretory response and contributed to the development of type 2 diabetes. We have measured the intracellular Ca(2+) mobilization in response to different secretagogues as well as mitochondrial metabolism. The study of calcium signals in hIAPP cells demonstrated an absence of response to glucose and also to tolbutamide, indicating a defect in ATP-sensitive potassium (K(ATP)) channels. Interestingly, hIAPP showed a greater maximal respiratory capacity than control cells. These data were confirmed by an increased mitochondrial membrane potential in hIAPP cells under glucose stimulation, leading to an elevated reactive oxygen species level as compared with control cells. We concluded that the hIAPP overexpression inhibits insulin and IAPP secretion in response to glucose affecting the activity of K(ATP) channels and that the increased mitochondrial metabolism is a compensatory response to counteract the secretory defect of beta-cells.