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Muscle stem cells (MuSCs) enable muscle growth and regeneration after exercise or injury, but how metabolism controls their regenerative potential is poorly understood. We describe that primary metabolic changes can determine murine MuSC fate decisions. We found that glutamine anaplerosis into the tricarboxylic acid (TCA) cycle decreases during MuSC differentiation and coincides with decreased expression of the mitochondrial glutamate deaminase GLUD1. Deletion of Glud1 in proliferating MuSCs resulted in precocious differentiation and fusion, combined with loss of self-renewal in vitro and in vivo. Mechanistically, deleting Glud1 caused mitochondrial glutamate accumulation and inhibited the malate-aspartate shuttle (MAS). The resulting defect in transporting NADH-reducing equivalents into the mitochondria induced compartment-specific NAD+/NADH ratio shifts. MAS activity restoration or directly altering NAD+/NADH ratios normalized myogenesis. In conclusion, GLUD1 prevents deleterious mitochondrial glutamate accumulation and inactivation of the MAS in proliferating MuSCs. It thereby acts as a compartment-specific metabolic brake on MuSC differentiation.
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Provision of amino acids to the liver is instrumental for gluconeogenesis while it requires safe disposal of the amino group. The mitochondrial enzyme glutamate dehydrogenase (GDH) is central for hepatic ammonia detoxification by deaminating excessive amino acids toward ureagenesis and preventing hyperammonemia. The present study investigated the early adaptive responses to changes in dietary protein intake in control mice and liver-specific GDH KO mice (Hep-Glud1-/-). Mice were fed chow diets with a wide coverage of protein contents; i.e., suboptimal 10%, standard 20%, over optimal 30%, and high 45% protein diets; switched every 4 days. Metabolic adaptations of the mice were assessed in calorimetric chambers before tissue collection and analyses. Hep-Glud1-/- mice exhibited impaired alanine induced gluconeogenesis and constitutive hyperammonemia. The expression and activity of GDH in liver lysates were not significantly changed by the different diets. However, applying an in situ redox-sensitive assay on cryopreserved tissue sections revealed higher hepatic GDH activity in mice fed the high-protein diets. On the same section series, immunohistochemistry provided corresponding mapping of the GDH expression. Cosinor analysis from calorimetric chambers showed that the circadian rhythm of food intake and energy expenditure was altered in Hep-Glud1-/- mice. In control mice, energy expenditure shifted from carbohydrate to amino acid oxidation when diet was switched to high protein content. This shift was impaired in Hep-Glud1-/- mice and consequently the spontaneous physical activity was markedly reduced in GDH KO mice. These data highlight the central role of liver GDH in the energy balance adaptation to dietary proteins.
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Proteínas Alimentares , Metabolismo Energético , Glutamato Desidrogenase , Fígado , Animais , Masculino , Camundongos , Proteínas Alimentares/metabolismo , Gluconeogênese , Glutamato Desidrogenase/metabolismo , Glutamato Desidrogenase/genética , Fígado/metabolismo , Camundongos Knockout , Nitrogênio/metabolismoRESUMO
In this study, we identified new lipid species associated with the loss of pancreatic ß-cells triggering diabetes. We performed lipidomics measurements on serum from prediabetic mice lacking ß-cell prohibitin-2 (a model of monogenic diabetes) patients without previous history of diabetes but scheduled for pancreaticoduodenectomy resulting in the acute reduction of their ß-cell mass (â¼50%), and patients with type 2 diabetes (T2D). We found lysophosphatidylinositols (lysoPIs) were the main circulating lipid species altered in prediabetic mice. The changes were confirmed in the patients with acute reduction of their ß-cell mass and in those with T2D. Increased lysoPIs significantly correlated with HbA1c (reflecting glycemic control), fasting glycemia, and disposition index, and did not correlate with insulin resistance or obesity in human patients with T2D. INS-1E ß-cells as well as pancreatic islets isolated from nondiabetic mice and human donors exposed to exogenous lysoPIs showed potentiated glucose-stimulated and basal insulin secretion. Finally, addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. Overall, lysoPIs appear to be lipid species upregulated in the prediabetic stage associated with the loss of ß-cells and that support the secretory function of the remaining ß-cells. ARTICLE HIGHLIGHTS: Circulating lysophosphatidylinositols (lysoPIs) are increased in situations associated with ß-cell loss in mice and humans such as (pre-)diabetes, and hemipancreatectomy. Pancreatic islets isolated from nondiabetic mice and human donors, as well as INS-1E ß-cells, exposed to exogenous lysoPIs exhibited potentiated glucose-stimulated and basal insulin secretion. Addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. LysoPIs appear as lipid species being upregulated already in the prediabetic stage associated with the loss of ß-cells and supporting the function of the remaining ß-cells.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Estado Pré-Diabético , Humanos , Camundongos , Animais , Insulina , Lisofosfolipídeos , Glucose/farmacologia , Insulina Regular HumanaRESUMO
BACKGROUND: The mitochondrial pyruvate carrier (MPC) is a protein complex composed of two subunits, MPC1 and MPC2. This carrier is at the interface between glycolysis and mitochondrial metabolism and plays an essential role in hepatic glucose production. METHODS: Here we describe an in vitro screen for small molecule inhibitors of the MPC using a strain of Lactococcus lactis that has been engineered to co-express the two subunits of the human MPC and is able to import exogenous 14C-pyruvate. We then tested the top candidates for potential antidiabetic effects through the repression of gluconeogenesis. RESULTS: By screening the Prestwick compound library of 1'200 drugs approved by the Food and Drug Administration for inhibitors of pyruvate uptake, twelve hit molecules were identified. In a secondary screen, the most potent inhibitors were found to inhibit pyruvate-driven oxygen consumption in mouse C2C12 muscle cells. Assessment of gluconeogenesis showed that Zaprinast, as well as the established MPC inhibitor UK5099, inhibited in vitro and in vivo hepatic glucose production. However, when tested acutely in mice without the administration of gluconeogenic substrates, MPC inhibitors raised blood glucose levels, pointing to liver-independent effects. Furthermore, chronic treatment with Zaprinast failed to correct hyperglycemia in both lean and obese diabetic mouse models. CONCLUSIONS: New MPC inhibitors have been identified, showing inhibitory effects on hepatic glucose production. GENERAL SIGNIFICANCE: For potential antidiabetic applications, MPC inhibitors should target the liver without undesired inhibition of mitochondrial pyruvate metabolism in the skeletal muscles or pancreatic beta-cells in order to avoid dual effects on glycemia.
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Diabetes Mellitus , Glucose , Estados Unidos , Humanos , Camundongos , Animais , Glucose/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/farmacologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fígado/metabolismo , Diabetes Mellitus/metabolismo , Hipoglicemiantes/farmacologia , Piruvatos/metabolismo , Piruvatos/farmacologiaRESUMO
We identified two NEXMIF variants in two unrelated individuals with non-autoimmune diabetes and autistic traits, and investigated the expression of Nexmif in mouse and human pancreas and its function in pancreatic beta cells in vitro and in vivo. In insulin-secreting INS-1E cells, Nexmif expression increased strongly in response to oxidative stress. CRISPR Cas9-generated Nexmif knockout mice exhibited a reduced number of proliferating beta cells in pancreatic islets. RNA sequencing of pancreatic islets showed that the downregulated genes in Nexmif mutant islets are involved in stress response and the deposition of epigenetic marks. They include H3f3b, encoding histone H3.3, which is associated with the regulation of beta-cell proliferation and maintains genomic integrity by silencing transposable elements, particularly LINE1 elements. LINE1 activity has been associated with autism and neurodevelopmental disorders in which patients share characteristics with NEXMIF patients, and can cause genomic instability and genetic variation through retrotransposition. Nexmif knockout mice exhibited various other phenotypes. Mortality and phenotypic abnormalities increased in each generation in both Nexmif mutant and non-mutant littermates. In Nexmif mutant mice, LINE1 element expression was upregulated in the pancreas, brain, and testis, possibly inducing genomic instability in Nexmif mutant mice and causing phenotypic variability in their progeny.
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Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Variação Biológica da População , Instabilidade Genômica , Genômica , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos KnockoutRESUMO
CONTEXT: During an asymptomatic prediabetic state, the functional ß-cell mass decreases to a critical threshold, triggering diabetes and related symptoms. To date, there are no reliable readouts able to capture in vivo a potential drop of the ß-cell mass. OBJECTIVE: Beside its use as a short-term marker of glycemic control, the deoxyhexose 1,5-anhydroglucitol was identified in rodents as a circulating biomarker of the functional ß-cell mass already in the asymptomatic prediabetic stage. The present study investigated the putative corresponding relevance of circulating 1,5-anhydroglucitol in different human cohorts. METHODS: We analyzed clinical and blood parameters in patients with established type 2 diabetes and subjects considered at high risk of developing diabetes, as well as patients with no history of diabetes scheduled for pancreaticoduodenectomy. RESULTS: Circulating 1,5-anhydroglucitol was reduced in type 2 diabetic patients, negatively correlating with fasting plasma glucose (Pâ <â 0.0001) and hemoglobin A1c (Pâ <â 0.0001). In healthy subjects, 1,5-AG levels positively correlated with body mass index (Pâ =â 0.004) and Homeostatic Model Assessment of Insulin Resistance %S (Pâ <â 0.03) and was particularly high in nondiabetic obese individuals, potentially accounting for compensatory ß-cell expansion. Patients with no history of diabetes undergoing pancreaticoduodenectomy exhibited a 50% reduction of circulating 1,5-anhydroglucitol levels following surgery leading to an acute loss of their ß-cell mass (Pâ =â 0.002), regardless their glucose tolerance status. CONCLUSION: In summary, plasma concentration of 1,5-anhydroglucitol follows the ß-cell mass and its noninvasive monitoring may alert about the loss of ß cells in subjects at risk for diabetes, an event that cannot be captured by other clinical parameters of glycemic control.
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Diabetes Mellitus Tipo 2 , Estado Pré-Diabético , Biomarcadores , Glicemia , Desoxiglucose , Hemoglobinas Glicadas/análise , Humanos , Fenótipo , Estado Pré-Diabético/diagnóstico , Sujeitos da PesquisaRESUMO
AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to high glucose and fatty acids has been proposed to induce glucolipotoxicity. However, contradictory results suggest adaptations of the beta cells, which might be instrumental for partial preservation of the secretory response. In this context, we delineated the expression pattern of genes related to lipid pathways along with fat storage/mobilisation during glucose-stimulated insulin secretion. METHODS: Insulin-secreting cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mmol/l) without or with BSA-complexed 0.4 mmol/l palmitate and oleate. Then, transcriptomic analyses of lipid pathways were performed in human islets by RNA-Seq and in INS-1E cells and rat islets by quantitative RT-PCR. Storage of fat was assessed in INS-1E cells by electron microscopy and Bodipy staining, which was also used for measuring lipid mobilisation rate. The secretory response was monitored during acute 15 mmol/l glucose stimulation using online luminescence assay for INS-1E cells and by radioimmunoassay for rat islets. RESULTS: In human islets, chronic exposure to palmitate and oleate modified expression of a panel of genes involved in lipid handling. Culture at 25 mmol/l glucose upregulated genes encoding for enzymes of the glycerolipid/NEFA cycle and downregulated receptors implicated in fatty acid signalling. Similar results were obtained in INS-1E cells, indicating enhanced capacity of the glycerolipid/NEFA cycle under glucotoxic conditions. Exposure to unsaturated C18:1 fatty acid favoured intracellular lipid accumulation in a glucose-dependent way, an effect also observed with saturated C16:0 fatty acid when combined with the panlipase inhibitor Orlistat. After the glucolipotoxic culture, intracellular fat mobilisation was required for acute glucose-stimulated secretion, particularly in oleate-treated cells under glucotoxic culture conditions. The lipid mobilisation rate was governed chiefly by the levels of stored fat as a direct consequence of the culture conditions rather than energetic demands, except in palmitate-loaded cells. CONCLUSIONS/INTERPRETATION: Glucolipotoxic conditions promote the capacity of the glycerolipid/NEFA cycle thereby preserving part of the secretory response. The cycle of fat storage/mobilisation emerges as a mechanism helping the beta cell to cope with glucotoxic conditions.
Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Ácidos Graxos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Glucose/metabolismo , Glucose/toxicidade , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ácido Oleico/farmacologia , Palmitatos/metabolismo , Palmitatos/toxicidade , RatosRESUMO
Intestinal surface changes in size and function, but what propels these alterations and what are their metabolic consequences is unknown. Here we report that the food amount is a positive determinant of the gut surface area contributing to an increased absorptive function, reversible by reducing daily food. While several upregulated intestinal energetic pathways are dispensable, the intestinal PPARα is instead necessary for the genetic and environment overeating-induced increase of the gut absorptive capacity. In presence of dietary lipids, intestinal PPARα knock-out or its pharmacological antagonism suppress intestinal crypt expansion and shorten villi in mice and in human intestinal biopsies, diminishing the postprandial triglyceride transport and nutrient uptake. Intestinal PPARα ablation limits systemic lipid absorption and restricts lipid droplet expansion and PLIN2 levels, critical for droplet formation. This improves the lipid metabolism, and reduces body adiposity and liver steatosis, suggesting an alternative target for treating obesity.
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Fígado Gorduroso/genética , Intestinos/metabolismo , PPAR alfa/genética , Perilipina-2/genética , Adiposidade/genética , Animais , Dieta/métodos , Ingestão de Alimentos/fisiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Regulação da Expressão Gênica , Humanos , Absorção Intestinal/fisiologia , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Transgênicos , PPAR alfa/deficiência , PPAR alfa/metabolismo , Perilipina-2/metabolismo , Período Pós-Prandial , Transdução de Sinais , Triglicerídeos/metabolismoRESUMO
Macrophages rely on tightly integrated metabolic rewiring to clear dying neighboring cells by efferocytosis during homeostasis and disease. Here we reveal that glutaminase-1-mediated glutaminolysis is critical to promote apoptotic cell clearance by macrophages during homeostasis in mice. In addition, impaired macrophage glutaminolysis exacerbates atherosclerosis, a condition during which, efficient apoptotic cell debris clearance is critical to limit disease progression. Glutaminase-1 expression strongly correlates with atherosclerotic plaque necrosis in patients with cardiovascular diseases. High-throughput transcriptional and metabolic profiling reveals that macrophage efferocytic capacity relies on a non-canonical transaminase pathway, independent from the traditional requirement of glutamate dehydrogenase to fuel É-ketoglutarate-dependent immunometabolism. This pathway is necessary to meet the unique requirements of efferocytosis for cellular detoxification and high-energy cytoskeletal rearrangements. Thus, we uncover a role for non-canonical glutamine metabolism for efficient clearance of dying cells and maintenance of tissue homeostasis during health and disease in mouse and humans.
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Aminação , Glutamina/metabolismo , Fosforilação Oxidativa , Animais , Camundongos , FagocitoseRESUMO
Impaired liver function may lead to hyperammonemia and risk for hepatic encephalopathy. In brain, detoxification of ammonia is mediated mainly by glutamine synthetase (GS) in astrocytes. This requires a continuous de novo synthesis of glutamate, likely involving the action of both pyruvate carboxylase (PC) and glutamate dehydrogenase (GDH). An increased PC activity upon ammonia exposure and the importance of PC activity for glutamine synthesis has previously been demonstrated while the importance of GDH for generation of glutamate as precursor for glutamine synthesis has received little attention. We therefore investigated the functional importance of GDH for brain metabolism during hyperammonemia. To this end, brain slices were acutely isolated from transgenic CNS-specific GDH null or litter mate control mice and incubated in aCSF containing [U-13C]glucose in the absence or presence of 1 or 5 mM ammonia. In another set of experiments, brain slices were incubated in aCSF containing 1 or 5 mM 15N-labeled NH4Cl and 5 mM unlabeled glucose. Tissue extracts were analyzed for isotopic labeling in metabolites and for total amounts of amino acids. As a novel finding, we reveal a central importance of GDH function for cerebral ammonia fixation and as a prerequisite for de novo synthesis of glutamate and glutamine during hyperammonemia. Moreover, we demonstrated an important role of the concerted action of GDH and alanine aminotransferase in hyperammonemia; the products alanine and α-ketoglutarate serve as an ammonia sink and as a substrate for ammonia fixation via GDH, respectively. The role of this mechanism in human hyperammonemic states remains to be studied.
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Over the last decades, lipotoxicity and glucotoxicity emerged as established mechanisms participating in the pathophysiology of obesity-related type 2 diabetes in general, and in the loss of ß-cell function in particular. However, these terms hold various potential biological processes, and it is not clear what precisely they refer to and to what extent they might be clinically relevant. In this review, we discuss the basis and the last advances of research regarding the role of free fatty acids, their metabolic intracellular pathways, and receptor-mediated signaling related to glucose-stimulated insulin secretion, as well as lipid-induced ß-cell dysfunction. We also describe the role of chronically elevated glucose, namely, glucotoxicity, which promotes failure and dedifferentiation of the ß cell. Glucolipotoxicity combines deleterious effects of exposures to both high glucose and free fatty acids, supposedly provoking synergistic defects on the ß cell. Nevertheless, recent studies have highlighted the glycerolipid/free fatty acid cycle as a protective pathway mediating active storage and recruitment of lipids. Finally, we discuss the putative correspondence of the loss of functional ß cells in type 2 diabetes with a natural, although accelerated, aging process.
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Adaptação Biológica , Glucose/toxicidade , Secreção de Insulina , Células Secretoras de Insulina/patologia , Lipídeos/farmacologia , Adaptação Biológica/efeitos dos fármacos , Animais , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/ultraestrutura , Modelos BiológicosRESUMO
The endothelial cells of the blood-brain barrier participate in the regulation of glutamate concentrations in the brain interstitial fluid by taking up brain glutamate. However, endothelial glutamate metabolism has not been characterized, nor is its role in brain glutamate homeostasis and endothelial energy production known. The aim of this study was to investigate endothelial glutamate dehydrogenase (GDH) expression and glutamate metabolism and probe its functional significance. The primary brain endothelial cells were isolated from bovine and mouse brains, and human brain endothelial cells were derived from induced pluripotent stem cells. GDH expression on the protein level and GDH function were investigated in the model systems using western blotting, confocal microscopy, 13 C-glutamate metabolism, and Seahorse assay. In this study, it was shown that GDH was expressed in murine and bovine brain capillaries and in cultured primary mouse and bovine brain endothelial cells as well as in human-induced pluripotent stem cell-derived endothelial cells. The endothelial GDH expression was confirmed in brain capillaries from mice carrying a central nervous system-specific GDH knockout. Endothelial cells from all tested species metabolized 13 C-glutamate to α-ketoglutarate, which subsequently entered the tricarboxylic acid (TCA)-cycle. Brain endothelial cells maintained mitochondrial oxygen consumption rates, when supplied with glutamate alone, whereas glutamate supplied in addition to glucose did not lead to additional oxygen consumption. In conclusion, brain endothelial cells directly take up and metabolize glutamate and utilize the resulting α-ketoglutarate in the tricarboxylic acid cycle to ultimately yield ATP if glucose is unavailable.
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Trifosfato de Adenosina/metabolismo , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Glutamato Desidrogenase/biossíntese , Ácido Glutâmico/metabolismo , Ácidos Tricarboxílicos/metabolismo , Animais , Encéfalo/citologia , Bovinos , Células Cultivadas , Humanos , Hipoglicemia/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Chronic exposure of ß-cells to nutrient-rich metabolic stress impairs mitochondrial metabolism and its coupling to insulin secretion. We exposed isolated human islets to different metabolic stresses for 3 days: 0.4 mM oleate or 0.4 mM palmitate at physiological 5.5 mM glucose (lipotoxicity), high 25 mM glucose (glucotoxicity), and high 25 mM glucose combined with 0.4 mM oleate and/or palmitate (glucolipotoxicity). Then, we profiled the mitochondrial carriers and associated genes with RNA-Seq. Diabetogenic conditions, and in particular glucotoxicity, increased expression of several mitochondrial solute carriers in human islets, such as the malate carrier DIC, the α-ketoglutarate-malate exchanger OGC, and the glutamate carrier GC1. Glucotoxicity also induced a general upregulation of the electron transport chain machinery, while palmitate largely counteracted this effect. Expression of different components of the TOM/TIM mitochondrial protein import system was increased by glucotoxicity, whereas glucolipotoxicity strongly upregulated its receptor subunit TOM70. Expression of the mitochondrial calcium uniporter MCU was essentially preserved by metabolic stresses. However, glucotoxicity altered expression of regulatory elements of calcium influx as well as the Na+/Ca2+ exchanger NCLX, which mediates calcium efflux. Overall, the expression profile of mitochondrial carriers and associated genes was modified by the different metabolic stresses exhibiting nutrient-specific signatures.
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Diabetes Mellitus/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Mitocôndrias/metabolismo , Estresse Fisiológico , Adulto , Transporte Biológico , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Diabetes Mellitus/genética , Feminino , Glucose/metabolismo , Humanos , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genéticaRESUMO
Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells1-4. Macrophages and satellite cells communicate in different ways1-5, but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles.
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Glutamina/metabolismo , Macrófagos/metabolismo , Músculo Esquelético/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Envelhecimento/metabolismo , Sistema ASC de Transporte de Aminoácidos/antagonistas & inibidores , Sistema ASC de Transporte de Aminoácidos/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Glutamato Desidrogenase/deficiência , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamato-Amônia Ligase/metabolismo , Macrófagos/enzimologia , Masculino , Camundongos , Antígenos de Histocompatibilidade Menor/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Oxirredução , Células Satélites de Músculo Esquelético/citologia , Serina-Treonina Quinases TORRESUMO
Chronic exposure of pancreatic ß-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in ß-cells under conditions of nutrient deprivation, while little is known on AMPK responses to metabolic stresses. Here, we first reviewed recent studies on the role of AMPK activation in ß-cells. Then, we investigated the expression profile of AMPK pathways in ß-cells following metabolic stresses. INS-1E ß-cells and human islets were exposed for 3 days to glucose (5.5-25 mM), palmitate or oleate (0.4 mM), and fructose (5.5 mM). Following these treatments, we analyzed transcript levels of INS-1E ß-cells by qRT-PCR and of human islets by RNA-Seq; with a special focus on AMPK-associated genes, such as the AMPK catalytic subunits α1 (Prkaa1) and α2 (Prkaa2). AMPKα and pAMPKα were also evaluated at the protein level by immunoblotting. Chronic exposure to the different metabolic stresses, known to alter glucose-stimulated insulin secretion, did not change AMPK expression, either in insulinoma cells or in human islets. Expression profile of the six AMPK subunits was marginally modified by the different diabetogenic conditions. However, the expression of some upstream kinases and downstream AMPK targets, including K-ATP channel subunits, exhibited stress-specific signatures. Interestingly, at the protein level, chronic fructose treatment favored fasting-like phenotype in human islets, as witnessed by AMPK activation. Collectively, previously published and present data indicate that, in the ß-cell, AMPK activation might be implicated in the pre-diabetic state, potentially as a protective mechanism.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose , Regulação Enzimológica da Expressão Gênica , Ilhotas Pancreáticas/enzimologia , Adulto , Animais , Glicemia/análise , Feminino , Frutose/metabolismo , Perfilação da Expressão Gênica , Homeostase , Humanos , Insulina/metabolismo , Insulinoma/enzimologia , Masculino , Pessoa de Meia-Idade , Ácido Oleico/análise , Ácido Palmítico/análise , Fenótipo , RNA-Seq , Ratos , Estresse FisiológicoRESUMO
BACKGROUND: Gain-of-function mutations in the GLUD1 gene, encoding for glutamate dehydrogenase (GDH), result in the hyperinsulinism/hyperammonemia HI/HA syndrome. HI/HA patients present with harmful hypoglycemia secondary to protein-induced HI and elevated plasma ammonia levels. These symptoms may be accompanied by seizures and mental retardation. GDH is a mitochondrial enzyme that catalyzes the oxidative deamination of glutamate to α-ketoglutarate, under allosteric regulations mediated by its inhibitor GTP and its activator ADP. The present study investigated the functional properties of the GDH-G446V variant (alias c.1496G > T, p.(Gly499Val) (NM_005271.4)) in patient-derived lymphoblastoid cells. RESULTS: The calculated energy barrier between the opened and closed state of the enzyme was 41% lower in GDH-G446V compared to wild-type GDH, pointing to altered allosteric regulation. Computational analysis indicated conformational changes of GDH-G446V in the antenna region that is crucial for allosteric regulators. Enzymatic activity measured in patient-derived lymphoblastoid cells showed impaired allosteric responses of GDH-G446V to both regulators GTP and ADP. In particular, as opposed to control lymphoblastoid cells, GDH-G446V cells were not responsive to GTP in the lower range of ADP concentrations. Assessment of the metabolic rate revealed higher mitochondrial respiration in response to GDH-dependent substrates in the GDH-G446V lymphoblastoid cells compared to control cells. This indicates a shift toward glutaminolysis for energy provision in cells carrying the GDH-G446V variant. CONCLUSIONS: Substitution of the small amino acid glycine for the hydrophobic branched-chain valine altered the allosteric sensitivity to both inhibitory action of GTP and activation by ADP, rendering cells metabolically responsive to glutamine.
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Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Guanosina Trifosfato/metabolismo , Hiperinsulinismo/patologia , Linfócitos/patologia , Mutação , Adulto , Regulação Alostérica , Estudos de Casos e Controles , Feminino , Glutamato Desidrogenase/química , Humanos , Hiperinsulinismo/genética , Recém-Nascido , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Conformação ProteicaRESUMO
AMP-activated protein kinase (AMPK) is an important energy sensor located in cells throughout the human body. From the periphery, AMPK is known to be a metabolic master switch controlling the use of energy fuels. The energy sensor is activated when the energy status of the cell is low, initiating energy-producing pathways and deactivating energy-consuming pathways. All brain cells are crucially dependent on energy production for survival, and the availability of energy substrates must be closely regulated. Intriguingly, the role of AMPK in the regulation of brain cell metabolism has been sparsely investigated, particularly in astrocytes. By investigating metabolism of 13 C-labeled energy substrates in acutely isolated hippocampal slices and cultured astrocytes, with subsequent mass spectrometry analysis, we here show that activation of AMPK increases glycolysis as well as the capacity of the TCA cycle, that is, anaplerosis, through the activity of pyruvate carboxylase (PC) in astrocytes. In addition, we demonstrate that AMPK activation leads to augmented astrocytic glutamate oxidation via pyruvate recycling (i.e., cataplerosis). This regulatory mechanism induced by AMPK activation is mediated via glutamate dehydrogenase (GDH) shown in a CNS-specific GDH knockout mouse. Collectively, these findings demonstrate that AMPK regulates TCA cycle dynamics in astrocytes via PC and GDH activity. AMPK functionality has been shown to be hampered in Alzheimer's and Parkinson's disease and our findings may therefore add to the toolbox for discovery of new metabolic drug targets.
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Proteínas Quinases Ativadas por AMP , Astrócitos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Astrócitos/metabolismo , Respiração Celular , Ciclo do Ácido Cítrico , Glutamato Desidrogenase , Camundongos , Estresse OxidativoRESUMO
Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, leading to insulin secretion defects and eventually beta-cell failure. Using a semi-high throughput approach applied to INS-1E beta-cells, we tested multiple conditions of chronic exposure to basal, intermediate and high glucose, combined with saturated versus mono- and polyunsaturated fatty acids in order to assess cell integrity, lipid metabolism, mitochondrial function, glucose-stimulated calcium rise and secretory kinetics. INS-1E beta-cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mM) without or with BSA-complexed 0.4 mM saturated (C16:0 palmitate), monounsaturated (C18:1 oleate) or polyunsaturated (C18:2 linoleate, C18:3 linolenate) fatty acids, resulting in 0.1-0.5 µM unbound fatty acids. Accumulation of triglycerides in cells exposed to fatty acids was glucose-dependent, oleate inducing the strongest lipid storage and protecting against glucose-induced cytotoxicity. The combined chronic exposure to both high glucose and either palmitate or oleate altered mitochondrial function as well as glucose-induced calcium rise. This pattern did not directly translate at the secretory level since palmitate and oleate exhibited distinct effects on the first and the second phases of glucose-stimulated exocytosis. Both fatty acids changed the activity of kinases, such as the MODY-associated BLK. Additionally, chronic exposure to fatty acids modified membrane physicochemical properties by increasing membrane fluidity, oleate exhibiting larger effects compared to palmitate. Chronic fatty acids differentially and specifically exacerbated some of the glucotoxic effects, without promoting cytotoxicity on their own. Each of the tested fatty acids functionally modified INS-1E beta-cell, oleate inducing the strongest effects.