RESUMO
Obesity is a major modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), yet how and when obesity contributes to PDAC progression is not well understood. Leveraging an autochthonous mouse model, we demonstrate a causal and reversible role for obesity in early PDAC progression, showing that obesity markedly enhances tumorigenesis, while genetic or dietary induction of weight loss intercepts cancer development. Molecular analyses of human and murine samples define microenvironmental consequences of obesity that foster tumorigenesis rather than new driver gene mutations, including significant pancreatic islet cell adaptation in obesity-associated tumors. Specifically, we identify aberrant beta cell expression of the peptide hormone cholecystokinin (Cck) in response to obesity and show that islet Cck promotes oncogenic Kras-driven pancreatic ductal tumorigenesis. Our studies argue that PDAC progression is driven by local obesity-associated changes in the tumor microenvironment and implicate endocrine-exocrine signaling beyond insulin in PDAC development.
Assuntos
Carcinoma Ductal Pancreático/etiologia , Carcinoma Ductal Pancreático/metabolismo , Obesidade/metabolismo , Animais , Carcinogênese/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Progressão da Doença , Células Endócrinas/metabolismo , Glândulas Exócrinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Obesidade/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais/genética , Microambiente Tumoral/fisiologia , Neoplasias PancreáticasRESUMO
The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.
Assuntos
Neoplasias da Mama/metabolismo , Deleção de Genes , Neoplasias Mamárias Experimentais/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Animais , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Éxons , Feminino , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutação , Metástase Neoplásica , Transplante de Neoplasias , Splicing de RNARESUMO
The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and 13C-glucose and 13C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation.
Assuntos
Protease La , Fator de Necrose Tumoral alfa , Humanos , Fator de Necrose Tumoral alfa/farmacologia , Acetilcoenzima A , Células Endoteliais , Histonas , CitocinasRESUMO
PURPOSE: Metabolic rewiring in malignant transformation is often accompanied by altered expression of metabolic isozymes. Phosphoenolpyruvate carboxykinase-2 (PCK2) catalyzes the rate-limiting step of gluconeogenesis and is the dominant isoform in many cancers including triple-negative breast cancer (TNBC). Our goal was to identify small molecule inhibitors of PCK2 enzyme activity. METHODS: We assessed the impact of PCK2 down regulation with shRNA on TNBC cell growth in vitro and used AtomNet® deep convolutional neural network software to identify potential small molecule inhibitors of PCK2-based structure. We iteratively tested candidate compounds in an in vitro PCK-2 enzyme assay. The impact of the top hit on metabolic flux and cell viability was also assessed. RESULTS: PCK2 downregulation decreased growth of BT-549 and MDA-MB-231 cells and reduced metabolic flux through pyruvate carboxylase. The first AtomNet® in silico structural screen of 7 million compounds yielded 86 structures that were tested in PCK2 enzyme assay in vitro. The top hit (IC50 = 2.4 µM) was used to refine a second round of in silico screen that yielded 82 candidates to be tested in vitro, which resulted in 45 molecules with inhibition > 20%. In the second in vitro screen we also included 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, previously suggested to be PCK2 inhibitor based on structure, which emerged as the top hit. The specificity of this compound was tested in PCK1 and PCK2 enzymatic assays and showed IC50 of 500 nM and 3.5-27 nM for PCK1 and PCK2, respectively. CONCLUSION: 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is a high affinity PCK2 enzyme inhibitor that also has significant growth inhibitory activity in breast cell lines in vitro and represents a potential therapeutic lead compound.
RESUMO
Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a class of medications used by individuals with type 2 diabetes that reduce hyperglycaemia by targeting glucose transport in the kidney, preventing its reabsorption, thereby inducing glucosuria. Besides improving HbA1c and reducing body weight and blood pressure, the SGLT2 inhibitors have also been demonstrated to improve cardiovascular and kidney outcomes, an effect largely independent of their effect on blood glucose levels. Indeed, the mechanisms underlying these benefits remain elusive. Treatment with SGLT2 inhibitors has been found to modestly increase systemic ketone levels. Ketone bodies are an ancillary fuel source substituting for glucose in some tissues and may also possess intrinsic anti-oxidative and anti-inflammatory effects. Some have proposed that ketones may in fact mediate the cardio-renal benefits of this drug category. However, a rare complication of SGLT2 inhibition is ketoacidosis, sometimes with normal or near-normal blood glucose concentrations, albeit occurring more frequently in patients with type 1 diabetes who are treated (predominately off-label) with one of these agents. We herein explore the notion that an underpinning of one of the more serious adverse effects of SGLT2 inhibitors may, in fact, explain, at least in part, some of their benefits-a potential 'double-edged sword' of this novel drug category.
Assuntos
Diabetes Mellitus Tipo 2 , Inibidores do Transportador 2 de Sódio-Glicose , Humanos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Cetonas/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , GlicemiaRESUMO
Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glicólise , Neovascularização Fisiológica , Transdução de Sinais , Animais , Movimento Celular , Proliferação de Células , Feminino , Hexoquinase/metabolismo , Linfangiogênese , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Obesity, insulin resistance and the metabolic syndrome are associated with changes to the gut microbiota; however, the mechanism by which modifications to the gut microbiota might lead to these conditions is unknown. Here we show that increased production of acetate by an altered gut microbiota in rodents leads to activation of the parasympathetic nervous system, which, in turn, promotes increased glucose-stimulated insulin secretion, increased ghrelin secretion, hyperphagia, obesity and related sequelae. Together, these findings identify increased acetate production resulting from a nutrient-gut microbiota interaction and subsequent parasympathetic activation as possible therapeutic targets for obesity.
Assuntos
Acetatos/metabolismo , Encéfalo/fisiologia , Microbioma Gastrointestinal/fisiologia , Células Secretoras de Insulina/metabolismo , Síndrome Metabólica/metabolismo , Animais , Dieta Hiperlipídica , Grelina/metabolismo , Glucose/metabolismo , Hiperfagia/metabolismo , Insulina/metabolismo , Secreção de Insulina , Obesidade/metabolismo , Sistema Nervoso Parassimpático/fisiologia , RatosRESUMO
Fatty acid amide hydrolase (FAAH) degrades 2 major classes of bioactive fatty acid amides, the N-acylethanolamines (NAEs) and N-acyl taurines (NATs), in central and peripheral tissues. A functional polymorphism in the human FAAH gene is linked to obesity and mice lacking FAAH show altered metabolic states, but whether these phenotypes are caused by elevations in NAEs or NATs is unknown. To overcome the problem of concurrent elevation of NAEs and NATs caused by genetic or pharmacological disruption of FAAH in vivo, we developed an engineered mouse model harboring a single-amino acid substitution in FAAH (S268D) that selectively disrupts NAT, but not NAE, hydrolytic activity. The FAAH-S268D mice accordingly show substantial elevations in NATs without alterations in NAE content, a unique metabolic profile that correlates with heightened insulin sensitivity and GLP-1 secretion. We also show that N-oleoyl taurine (C18:1 NAT), the most abundant NAT in human plasma, decreases food intake, improves glucose tolerance, and stimulates GPR119-dependent GLP-1 and glucagon secretion in mice. Together, these data suggest that NATs act as a class of lipid messengers that improve postprandial glucose regulation and may have potential as investigational metabolites to modify metabolic disease.
Assuntos
Amidoidrolases/genética , Glicemia/metabolismo , Síndrome Metabólica/metabolismo , Ácidos Oleicos/metabolismo , Taurina/análogos & derivados , Amidoidrolases/metabolismo , Substituição de Aminoácidos , Animais , Glicemia/análise , Modelos Animais de Doenças , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Etanolaminas/sangue , Etanolaminas/metabolismo , Feminino , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Teste de Tolerância a Glucose , Humanos , Injeções Intravenosas , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/genética , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Ácidos Oleicos/administração & dosagem , Ácidos Oleicos/sangue , Período Pós-Prandial/efeitos dos fármacos , Período Pós-Prandial/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Taurina/administração & dosagem , Taurina/sangue , Taurina/metabolismoRESUMO
Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.
Assuntos
Gluconeogênese/efeitos dos fármacos , Glicerolfosfato Desidrogenase/antagonistas & inibidores , Metformina/farmacologia , Mitocôndrias/enzimologia , Animais , Glicemia/análise , Glicemia/biossíntese , Células Cultivadas , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/metabolismo , Glicerolfosfato Desidrogenase/deficiência , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ácido Láctico/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Knockout , Oxirredução/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Hypothyroidism, a metabolic disease characterized by low thyroid hormone (TH) and high thyroid-stimulating hormone (TSH) levels in the serum, is strongly associated with nonalcoholic fatty liver disease (NAFLD). Hypothyroidism-induced NAFLD has generally been attributed to reduced TH signaling in the liver with a consequent decrease in lipid utilization. Here, we found that mildly hypothyroid mice develop NAFLD without down-regulation of hepatic TH signaling or decreased hepatic lipid utilization. NAFLD was induced by impaired suppression of adipose tissue lipolysis due to decreased insulin secretion and to a reduced response of adipose tissue itself to insulin. This condition leads to increased shuttling of fatty acids (FAs) to the liver, where they are esterified and accumulated as triglycerides. Lipid accumulation in the liver induces hepatic insulin resistance, which leads to impaired suppression of endogenous glucose production after feeding. Hepatic insulin resistance, synergistically with lowered insulin secretion, increases serum glucose levels, which stimulates de novo lipogenesis (DNL) in the liver. Up-regulation of DNL also contributes to NAFLD. In contrast, severely hypothyroid mice show down-regulation of TH signaling in their livers and profound suppression of adipose tissue lipolysis, which decreases delivery of FAs to the liver. The resulting lack of substrates for triglyceride esterification protects severely hypothyroid mice against NAFLD. Our findings demonstrate that NAFLD occurs when TH levels are mildly reduced, but, paradoxically, not when they are severely reduced. Our results show that the pathogenesis of hypothyroidism-induced NAFLD is both intra- and extrahepatic; they also reveal key metabolic differences between mild and severe hypothyroidism.
Assuntos
Hipotireoidismo/complicações , Resistência à Insulina , Células Secretoras de Insulina/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Tecido Adiposo/metabolismo , Tecido Adiposo/fisiopatologia , Animais , Modelos Animais de Doenças , Hipotireoidismo/etiologia , Insulina/metabolismo , Secreção de Insulina , Metabolismo dos Lipídeos , Lipólise/fisiologia , Fígado/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Simportadores/genéticaRESUMO
A key sensor of cellular energy status, AMP-activated protein kinase (AMPK), interacts allosterically with AMP to maintain an active state. When active, AMPK triggers a metabolic switch, decreasing the activity of anabolic pathways and enhancing catabolic processes such as lipid oxidation to restore the energy balance. Unlike oxidative tissues, in which AMP is generated from adenylate kinase during states of high energy demand, the ornithine cycle enzyme argininosuccinate synthetase (ASS) is a principle site of AMP generation in the liver. Here we show that ASS regulates hepatic AMPK, revealing a central role for ureagenesis flux in the regulation of metabolism via AMPK. Treatment of primary rat hepatocytes with amino acids increased gluconeogenesis and ureagenesis and, despite nutrient excess, induced both AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. Antisense oligonucleotide knockdown of hepatic ASS1 expression in vivo decreased liver AMPK activation, phosphorylation of ACC, and plasma ß-hydroxybutyrate concentrations. Taken together these studies demonstrate that increased amino acid flux can activate AMPK through increased AMP generated by ASS, thus providing a novel link between protein catabolism, ureagenesis, and hepatic lipid metabolism.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Argininossuccinato Sintase/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Ureia/metabolismo , Animais , Ativação Enzimática , Ratos , Ratos Sprague-DawleyRESUMO
Previous studies have implicated age-associated reductions in mitochondrial oxidative phosphorylation activity in skeletal muscle as a predisposing factor for intramyocellular lipid (IMCL) accumulation and muscle insulin resistance (IR) in the elderly. To further investigate potential alterations in muscle mitochondrial function associated with aging, we assessed basal and insulin-stimulated rates of muscle pyruvate dehydrogenase (VPDH) flux relative to citrate synthase flux (VCS) in healthy lean, elderly subjects and healthy young body mass index- and activity-matched subjects. VPDH/VCS flux was assessed from the (13)C incorporation from of infused [1-13C] glucose into glutamate [4-13C] relative to alanine [3-13C] assessed by LC-tandem MS in muscle biopsies. Insulin-stimulated rates of muscle glucose uptake were reduced by 25% (P<0.01) in the elderly subjects and were associated with â¼70% (P<0.04) increase in IMCL, assessed by 1H magnetic resonance spectroscopy. Basal VPDH/VCS fluxes were similar between the groups (young: 0.20±0.03; elderly: 0.14±0.03) and increased approximately threefold in the young subjects following insulin stimulation. However, this increase was severely blunted in the elderly subjects (young: 0.55±0.04; elderly: 0.18±0.02, P=0.0002) and was associated with an â¼40% (P=0.004) reduction in insulin activation of Akt. These results provide new insights into acquired mitochondrial abnormalities associated with aging and demonstrate that age-associated reductions in muscle mitochondrial function and increased IMCL are associated with a marked inability of mitochondria to switch from lipid to glucose oxidation during insulin stimulation.
Assuntos
Envelhecimento , Glucose/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Adulto , Idoso , Glicemia/metabolismo , Isótopos de Carbono , Cromatografia Líquida , Citrato (si)-Sintase/metabolismo , Técnica Clamp de Glucose , Humanos , Hipoglicemiantes/farmacologia , Insulina/sangue , Insulina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Mitocôndrias/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Espectrometria de Massas em TandemRESUMO
In mammals, pyruvate kinase (PK) plays a key role in regulating the balance between glycolysis and gluconeogenesis; however, in vivo regulation of PK flux by gluconeogenic hormones and substrates is poorly understood. To this end, we developed a novel NMR-liquid chromatography/tandem-mass spectrometry (LC-MS/MS) method to directly assess pyruvate cycling relative to mitochondrial pyruvate metabolism (VPyr-Cyc/VMito) in vivo using [3-(13)C]lactate as a tracer. Using this approach, VPyr-Cyc/VMito was only 6% in overnight fasted rats. In contrast, when propionate was infused simultaneously at doses previously used as a tracer, it increased VPyr-Cyc/VMito by 20-30-fold, increased hepatic TCA metabolite concentrations 2-3-fold, and increased endogenous glucose production rates by 20-100%. The physiologic stimuli, glucagon and epinephrine, both increased hepatic glucose production, but only glucagon suppressed VPyr-Cyc/VMito These data show that under fasting conditions, when hepatic gluconeogenesis is stimulated, pyruvate recycling is relatively low in liver compared with VMito flux and that liver metabolism, in particular pyruvate cycling, is sensitive to propionate making it an unsuitable tracer to assess hepatic glycolytic, gluconeogenic, and mitochondrial metabolism in vivo.
Assuntos
Ciclo do Ácido Cítrico/efeitos dos fármacos , Fígado/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Propionatos/farmacologia , Ácido Pirúvico/metabolismo , Animais , Glicemia/metabolismo , Cromatografia Líquida , Epinefrina/sangue , Epinefrina/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Glucagon/sangue , Glucagon/farmacologia , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Insulina/sangue , Fígado/metabolismo , Mitocôndrias/metabolismo , Propionatos/administração & dosagem , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
Prolyl endopeptidase (PREP) has been implicated in neuronal functions. Here we report that hypothalamic PREP is predominantly expressed in the ventromedial nucleus (VMH), where it regulates glucose-induced neuronal activation. PREP knockdown mice (Prep(gt/gt)) exhibited glucose intolerance, decreased fasting insulin, increased fasting glucagon levels, and reduced glucose-induced insulin secretion compared with wild-type controls. Consistent with this, central infusion of a specific PREP inhibitor, S17092, impaired glucose tolerance and decreased insulin levels in wild-type mice. Arguing further for a central mode of action of PREP, isolated pancreatic islets showed no difference in glucose-induced insulin release between Prep(gt/gt) and wild-type mice. Furthermore, hyperinsulinemic euglycemic clamp studies showed no difference between Prep(gt/gt) and wild-type control mice. Central PREP regulation of insulin and glucagon secretion appears to be mediated by the autonomic nervous system because Prep(gt/gt) mice have elevated sympathetic outflow and norepinephrine levels in the pancreas, and propranolol treatment reversed glucose intolerance in these mice. Finally, re-expression of PREP by bilateral VMH injection of adeno-associated virus-PREP reversed the glucose-intolerant phenotype of the Prep(gt/gt) mice. Taken together, our results unmask a previously unknown player in central regulation of glucose metabolism and pancreatic function.
Assuntos
Glucagon/metabolismo , Hipotálamo/enzimologia , Insulina/metabolismo , Serina Endopeptidases/metabolismo , Animais , Glicemia/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Técnica Clamp de Glucose , Intolerância à Glucose/enzimologia , Intolerância à Glucose/etiologia , Hipotálamo/fisiologia , Indóis/farmacologia , Secreção de Insulina , Canais Iônicos/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Pâncreas/metabolismo , Fosforilação , Prolil Oligopeptidases , Receptor de Insulina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Inibidores de Serina Proteinase/farmacologia , Tiazolidinas/farmacologia , Proteína Desacopladora 1 , Núcleo Hipotalâmico Ventromedial/enzimologia , Núcleo Hipotalâmico Ventromedial/fisiologiaRESUMO
Imeglimin is a promising new oral antihyperglycemic agent that has been studied in clinical trials as a possible monotherapy or add-on therapy to lower fasting plasma glucose and improve hemoglobin A1c (1-3, 9). Imeglimin was shown to improve both fasting and postprandial glycemia and to increase insulin secretion in response to glucose during a hyperglycemic clamp after 1-wk of treatment in type 2 diabetic patients. However, whether the ß-cell stimulatory effect of imeglimin is solely or partially responsible for its effects on glycemia remains to be fully confirmed. Here, we show that imeglimin directly activates ß-cell insulin secretion in awake rodents without affecting hepatic insulin sensitivity, body composition, or energy expenditure. These data identify a primary amplification rather than trigger the ß-cell mechanism that explains the acute, antidiabetic activity of imeglimin.
Assuntos
Glicemia/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Triazinas/farmacologia , Animais , Glicemia/metabolismo , Dieta Hiperlipídica , Jejum , Glucose/metabolismo , Técnica Clamp de Glucose , Resistência à Insulina , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Período Pós-Prandial , Ratos , Ratos Sprague-DawleyRESUMO
Synthesis of phosphoenolpyruvate (PEP) from oxaloacetate is an absolute requirement for gluconeogenesis from mitochondrial substrates. Generally, this reaction has solely been attributed to the cytosolic isoform of PEPCK (PEPCK-C), although loss of the mitochondrial isoform (PEPCK-M) has never been assessed. Despite catalyzing the same reaction, to date the only significant role reported in mammals for the mitochondrial isoform is as a glucose sensor necessary for insulin secretion. We hypothesized that this nutrient-sensing mitochondrial GTP-dependent pathway contributes importantly to gluconeogenesis. PEPCK-M was acutely silenced in gluconeogenic tissues of rats using antisense oligonucleotides both in vivo and in isolated hepatocytes. Silencing PEPCK-M lowers plasma glucose, insulin, and triglycerides, reduces white adipose, and depletes hepatic glycogen, but raises lactate. There is a switch of gluconeogenic substrate preference to glycerol that quantitatively accounts for a third of glucose production. In contrast to the severe mitochondrial deficiency characteristic of PEPCK-C knock-out livers, hepatocytes from PEPCK-M-deficient livers maintained normal oxidative function. Consistent with its predicted role, gluconeogenesis rates from hepatocytes lacking PEPCK-M are severely reduced for lactate, alanine, and glutamine, but not for pyruvate and glycerol. Thus, PEPCK-M has a direct role in fasted and fed glucose homeostasis, and this mitochondrial GTP-dependent pathway should be reconsidered for its involvement in both normal and diabetic metabolism.
Assuntos
Regulação Enzimológica da Expressão Gênica , Gluconeogênese , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Fígado/enzimologia , Fígado/metabolismo , Mitocôndrias/enzimologia , Fosfoenolpiruvato Carboxiquinase (GTP)/fisiologia , Ração Animal , Animais , Glicemia/metabolismo , Privação de Alimentos , Inativação Gênica , Glicerol/metabolismo , Glicogênio/metabolismo , Guanosina Trifosfato/metabolismo , Hepatócitos/citologia , Homeostase , Insulina/metabolismo , Isoenzimas/fisiologia , Ácido Láctico/metabolismo , Masculino , Mitocôndrias/metabolismo , Oligonucleotídeos Antissenso/química , Oxigênio/metabolismo , Consumo de Oxigênio , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Plasma glucose levels are tightly regulated within a narrow physiologic range. Insulin-mediated glucose uptake by tissues must be balanced by the appearance of glucose from nutritional sources, glycogen stores, or gluconeogenesis. In this regard, a common pathway regulating both glucose clearance and appearance has not been described. The metabolism of glucose to produce ATP is generally considered to be the primary stimulus for insulin release from beta-cells. Similarly, gluconeogenesis from phosphoenolpyruvate (PEP) is believed to be the primarily pathway via the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). These models cannot adequately explain the regulation of insulin secretion or gluconeogenesis. SCOPE OF REVIEW: A metabolic sensing pathway involving mitochondrial GTP (mtGTP) and PEP synthesis by the mitochondrial isoform of PEPCK (PEPCK-M) is associated with glucose-stimulated insulin secretion from pancreatic beta-cells. Here we examine whether there is evidence for a similar mtGTP-dependent pathway involved in gluconeogenesis. In both islets and the liver, mtGTP is produced at the substrate level by the enzyme succinyl CoA synthetase (SCS-GTP) with a rate proportional to the TCA cycle. In the beta-cell PEPCK-M then hydrolyzes mtGTP in the production of PEP that, unlike mtGTP, can escape the mitochondria to generate a signal for insulin release. Similarly, PEPCK-M and mtGTP might also provide a significant source of PEP in gluconeogenic tissues for the production of glucose. This review will focus on the possibility that PEPCK-M, as a sensor for TCA cycle flux, is a key mechanism to regulate both insulin secretion and gluconeogenesis suggesting conservation of this biochemical mechanism in regulating multiple aspects of glucose homeostasis. Moreover, we propose that this mechanism may be important for regulating insulin secretion and gluconeogenesis compared to canonical nutrient sensing pathways. MAJOR CONCLUSIONS: PEPCK-M, initially believed to be absent in islets, carries a substantial metabolic flux in beta-cells. This flux is intimately involved with the coupling of glucose-stimulated insulin secretion. PEPCK-M activity may have been similarly underestimated in glucose producing tissues and could potentially be an unappreciated but important source of gluconeogenesis. GENERAL SIGNIFICANCE: The generation of PEP via PEPCK-M may occur via a metabolic sensing pathway important for regulating both insulin secretion and gluconeogenesis. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.
Assuntos
Glucose/metabolismo , Mitocôndrias/enzimologia , Fosfoenolpiruvato Carboxiquinase (GTP)/fisiologia , Animais , Gluconeogênese/fisiologia , Homeostase/fisiologia , Humanos , Células Secretoras de Insulina/fisiologia , Isoenzimas/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/fisiologiaRESUMO
An agreed-upon consensus model of glucose-stimulated insulin secretion from healthy ß-cells is essential for understanding diabetes pathophysiology. Since the discovery of the KATP channel in 1984, an oxidative phosphorylation (OxPhos)-driven rise in ATP has been assumed to close KATP channels to initiate insulin secretion. This model lacks any evidence, genetic or otherwise, that mitochondria possess the bioenergetics to raise the ATP/ADP ratio to the triggering threshold, and conflicts with genetic evidence demonstrating that OxPhos is dispensable for insulin secretion. It also conflates the stoichiometric yield of OxPhos with thermodynamics, and overestimates OxPhos by failing to account for established features of ß-cell metabolism, such as leak, anaplerosis, cataplerosis, and NADPH production that subtract from the efficiency of mitochondrial ATP production. We have proposed an alternative model, based on the spatial and bioenergetic specializations of ß-cell metabolism, in which glycolysis initiates insulin secretion. The evidence for this model includes that 1) glycolysis has high control strength over insulin secretion; 2) glycolysis is active at the correct time to explain KATP channel closure; 3) plasma membrane-associated glycolytic enzymes control KATP channels; 4) pyruvate kinase has favorable bioenergetics, relative to OxPhos, for raising ATP/ADP; and 5) OxPhos stalls before membrane depolarization and increases after. Although several key experiments remain to evaluate this model, the 1984 model is based purely on circumstantial evidence and must be rescued by causal, mechanistic experiments if it is to endure.
Assuntos
Glucose , Secreção de Insulina , Células Secretoras de Insulina , Insulina , Canais KATP , Fosforilação Oxidativa , Células Secretoras de Insulina/metabolismo , Humanos , Glucose/metabolismo , Canais KATP/metabolismo , Canais KATP/genética , Secreção de Insulina/fisiologia , Animais , Insulina/metabolismo , Glicólise/fisiologia , Modelos Biológicos , Trifosfato de Adenosina/metabolismoRESUMO
Midlobular hepatocytes are proposed to be the most plastic hepatic cell, providing a reservoir for hepatocyte proliferation during homeostasis and regeneration. However, other mechanisms beyond hyperplasia have been little explored and the contribution of other hepatocyte subpopulations to regeneration has been controversial. Thus, re-examining hepatocyte dynamics during regeneration is critical for cell therapy and treatment of liver diseases. Using a mouse model of hepatocyte- and non-hepatocyte- multicolor lineage tracing, we demonstrate that midlobular hepatocytes also undergo hypertrophy in response to chemical, physical, and viral insults. Our study shows that this subpopulation also combats liver impairment after infection with coronavirus. Furthermore, we demonstrate that pericentral hepatocytes also expand in number and size during the repair process and Galectin-9-CD44 pathway may be critical for driving these processes. Notably, we also identified that transdifferentiation and cell fusion during regeneration after severe injury contribute to recover hepatic function.
Assuntos
Hepatopatias , Regeneração Hepática , Animais , Regeneração Hepática/fisiologia , Fígado/metabolismo , Hepatócitos/metabolismo , Hepatopatias/metabolismo , Modelos Animais de Doenças , Proliferação de CélulasRESUMO
Uncoupling protein-3 (UCP3) is a mitochondrial transmembrane protein highly expressed in the muscle that has been implicated in regulating the efficiency of mitochondrial oxidative phosphorylation. Increasing UCP3 expression in skeletal muscle enhances proton leak across the inner mitochondrial membrane and increases oxygen consumption in isolated mitochondria, but its precise function in vivo has yet to be fully elucidated. To examine whether muscle-specific overexpression of UCP3 modulates muscle mitochondrial oxidation in vivo, rates of ATP synthesis were assessed by 31 P magnetic resonance spectroscopy (MRS), and rates of mitochondrial oxidative metabolism were measured by assessing the rate of [2-13 C]acetate incorporation into muscle [4-13 C]-, [3-13 C]-glutamate, and [4-13 C]-glutamine by high-resolution 13 C/1 H MRS. Using this approach, we found that the overexpression of UCP3 in skeletal muscle was accompanied by increased muscle mitochondrial inefficiency in vivo as reflected by a 42% reduction in the ratio of ATP synthesis to mitochondrial oxidation.