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1.
Cell Metab ; 32(5): 751-766.e11, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33147485

RESUMO

The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2-/- mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator in vivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.


Assuntos
Mitocôndrias/metabolismo , Fosfoenolpiruvato/metabolismo , Animais , Homeostase , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piruvato Quinase/metabolismo , Ratos , Ratos Sprague-Dawley
2.
J Lipid Res ; 61(12): 1565-1576, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32907986

RESUMO

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp-/-) mice and age-weight matched wild-type control mice. Young (10-12-week-old) L-Mttp-/- mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp-/- mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKCε activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp-/- mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKCε activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp-/- mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKCε activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp-/- mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp-/- mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp-/- mice.


Assuntos
Proteínas de Transporte/genética , Membrana Celular/metabolismo , Diglicerídeos/metabolismo , Técnicas de Inativação de Genes , Resistência à Insulina , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia
3.
Proc Natl Acad Sci U S A ; 117(14): 8166-8176, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32188779

RESUMO

Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.


Assuntos
Fígado Gorduroso/patologia , Glucoquinase/metabolismo , Glucose/metabolismo , Glicogênio Hepático/biossíntese , Fígado/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Técnicas de Silenciamento de Genes , Glucoquinase/genética , Glucose/administração & dosagem , Glucose-6-Fosfato/análise , Glucose-6-Fosfato/metabolismo , Humanos , Hiperglicemia/etiologia , Hiperglicemia/patologia , Hiperinsulinismo/etiologia , Hiperinsulinismo/patologia , Insulina/metabolismo , Resistência à Insulina , Fígado/patologia , Masculino , Metabolômica , Fosforilação , Ratos
4.
Cell Rep ; 29(11): 3394-3404.e9, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31825824

RESUMO

Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with insulin resistance in rats on a 3 day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure. Instead, we show that cyclin-dependent kinases (CDKs) are activated by the HFD and responsible for PKL phosphorylation at position S113 in addition to other targets. These CDKs control PKL nuclear retention, alter cytosolic PKL activity, and ultimately influence glucose production. These results change our view of PKL regulation and highlight a previously unrecognized pathway of hepatic CDK activity and metabolic control points that may be important in insulin resistance and type 2 diabetes.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Gluconeogênese , Hepatócitos/metabolismo , Piruvato Quinase/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Células Cultivadas , Dieta Hiperlipídica , Glucose/metabolismo , Resistência à Insulina , Masculino , Fosforilação , Piruvato Quinase/química , Ratos , Ratos Sprague-Dawley
5.
FASEB J ; 33(7): 8174-8185, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30922125

RESUMO

The connection between adipose glucocorticoid action and whole-body metabolism is incompletely understood. Thus, we generated adipose tissue-specific glucocorticoid receptor-knockout (Ad-GcR-/-) mice to explore potential mechanisms. Ad-GcR-/- mice had a lower concentration of fasting plasma nonesterified fatty acids and less hepatic steatosis. This was associated with increased protein kinase B phosphorylation and increased hepatic glycogen synthesis after an oral glucose challenge. High-fat diet (HFD)-fed Ad-GcR-/- mice were protected against the development of hepatic steatosis and diacylglycerol-PKCε-induced impairments in hepatic insulin signaling. Under hyperinsulinemic-euglycemic conditions, hepatic insulin response was ∼10-fold higher in HFD-fed Ad-GcR-/- mice. Insulin-mediated suppression of adipose lipolysis was improved by 40% in Ad-GcR-/- mice. Adipose triglyceride lipase expression was decreased and insulin-mediated perilipin dephosphorylation was increased in Ad-GcR-/- mice. In metabolic cages, food intake decreased by 3 kcal/kg per hour in Ad-GcR-/- mice. Therefore, physiologic adipose glucocorticoid action appears to drive hepatic lipid accumulation during stressors such as fasting. The resultant hepatic insulin resistance prevents hepatic glycogen synthesis, preserving glucose for glucose-dependent organs. Absence of adipose glucocorticoid action attenuates HFD-induced hepatic insulin resistance; potential explanations for reduction in hepatic steatosis include reductions in adipose lipolysis and food intake.-Abulizi, A., Camporez, J.-P., Jurczak, M. J., Høyer, K. F., Zhang, D., Cline, G. W., Samuel, V. T., Shulman, G. I., Vatner, D. F. Adipose glucocorticoid action influences whole-body metabolism via modulation of hepatic insulin action.


Assuntos
Tecido Adiposo/metabolismo , Glucocorticoides/metabolismo , Resistência à Insulina , Insulina/metabolismo , Lipólise , Fígado/metabolismo , Animais , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Glucocorticoides/genética , Insulina/genética , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Camundongos , Camundongos Knockout , Receptores de Glucocorticoides/deficiência , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo
6.
Metabolism ; 93: 1-9, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30576689

RESUMO

CONTEXT: Excessive adipose glucocorticoid action is associated with insulin resistance, but the mechanisms linking adipose glucocorticoid action to insulin resistance are still debated. We hypothesized that insulin resistance from excess glucocorticoid action may be attributed in part to increased ectopic lipid deposition in liver. METHODS: We tested this hypothesis in the adipose specific 11ß-hydroxysteroid dehydrogenase-1 (HSD11B1) transgenic mouse, an established model of adipose glucocorticoid excess. Tissue specific insulin action was assessed by hyperinsulinemic-euglycemic clamps, hepatic lipid content was measured, hepatic insulin signaling was assessed by immunoblotting. The role of hepatic lipid content was further probed by administration of the functionally liver-targeted mitochondrial uncoupler, Controlled Release Mitochondrial Protonophore (CRMP). FINDINGS: High fat diet fed HSD11B1 transgenic mice developed more severe hepatic insulin resistance than littermate controls (endogenous suppression of hepatic glucose production was reduced by 3.8-fold, P < 0.05); this was reflected by decreased insulin-stimulated hepatic insulin receptor kinase tyrosine phosphorylation and AKT serine phosphorylation. Hepatic insulin resistance was associated with a 53% increase (P < 0.05) in hepatic triglyceride content, a 73% increase in diacylglycerol content (P < 0.01), and a 66% increase in PKCε translocation (P < 0.05). Hepatic insulin resistance was prevented with administration of CRMP by reversal of hepatic steatosis and prevention of hepatic diacylglycerol accumulation and PKCε activation. CONCLUSIONS: These findings are consistent with excess adipose glucocorticoid activity being a predisposing factor for the development of lipid (diacylglycerol-PKCε)-induced hepatic insulin resistance.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Tecido Adiposo/enzimologia , Resistência à Insulina , Metabolismo dos Lipídeos , Fígado/metabolismo , Animais , Dieta Hiperlipídica , Glucocorticoides/metabolismo , Camundongos , Camundongos Transgênicos
7.
FASEB J ; 31(7): 2916-2924, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28330852

RESUMO

Lipodystrophy is a rare disorder characterized by complete or partial loss of adipose tissue. Patients with lipodystrophy exhibit hypertriglyceridemia, severe insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). Efforts to ameliorate NASH in lipodystrophies with pharmacologic agents have met with limited success. We examined whether a controlled-release mitochondrial protonophore (CRMP) that produces mild liver-targeted mitochondrial uncoupling could decrease hypertriglyceridemia and reverse NASH and diabetes in a mouse model (fatless AZIP/F-1 mice) of severe lipodystrophy and diabetes. After 4 wk of oral CRMP (2 mg/kg body weight per day) or vehicle treatment, mice underwent hyperinsulinemic-euglycemic clamps combined with radiolabeled glucose to assess liver and muscle insulin responsiveness and tissue lipid measurements. CRMP treatment reversed hypertriglyceridemia and insulin resistance in liver and skeletal muscle. Reversal of insulin resistance could be attributed to reductions in diacylglycerol content and reduced PKC-ε and PKC-θ activity in liver and muscle respectively. CRMP treatment also reversed NASH as reflected by reductions in plasma aspartate aminotransferase and alanine aminotransferase concentrations; hepatic steatosis; and hepatic expression of IL-1α, -ß, -2, -4, -6, -10, -12, CD69, and caspase 3 and attenuated activation of the IRE-1α branch of the unfolded protein response. Taken together, these results provide proof of concept for the development of liver-targeted mitochondrial uncoupling agents as a potential novel therapy for lipodystrophy-associated hypertriglyceridemia, NASH and diabetes.-Abulizi, A., Perry, R. J., Camporez, J. P. G., Jurczak, M. J., Petersen, K. F., Aspichueta, P., Shulman, G. I. A controlled-release mitochondrial protonophore reverses hypertriglyceridemia, nonalcoholic steatohepatitis, and diabetes in lipodystrophic mice.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipertrigliceridemia/tratamento farmacológico , Lipodistrofia/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Ionóforos de Próton/uso terapêutico , Animais , Preparações de Ação Retardada , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Resistência à Insulina , Masculino , Camundongos , Mitocôndrias Hepáticas/efeitos dos fármacos , Ionóforos de Próton/administração & dosagem , Distribuição Aleatória
8.
J Clin Invest ; 127(2): 657-669, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-28112679

RESUMO

The mechanism by which leptin reverses diabetic ketoacidosis (DKA) is unknown. We examined the acute insulin-independent effects of leptin replacement therapy in a streptozotocin-induced rat model of DKA. Leptin infusion reduced rates of lipolysis, hepatic glucose production (HGP), and hepatic ketogenesis by 50% within 6 hours and were independent of any changes in plasma glucagon concentrations; these effects were abrogated by coinfusion of corticosterone. Treating leptin- and corticosterone-infused rats with an adipose triglyceride lipase inhibitor blocked corticosterone-induced increases in plasma glucose concentrations and rates of HGP and ketogenesis. Similarly, adrenalectomized type 1 diabetic (T1D) rats exhibited decreased rates of lipolysis, HGP, and ketogenesis; these effects were reversed by corticosterone infusion. Leptin-induced decreases in lipolysis, HGP, and ketogenesis in DKA were also nullified by relatively small increases (15 to 70 pM) in plasma insulin concentrations. In contrast, the chronic glucose-lowering effect of leptin in a STZ-induced mouse model of poorly controlled T1D was associated with decreased food intake, reduced plasma glucagon and corticosterone concentrations, and decreased ectopic lipid (triacylglycerol/diacylglycerol) content in liver and muscle. Collectively, these studies demonstrate marked differences in the acute insulin-independent effects by which leptin reverses fasting hyperglycemia and ketoacidosis in a rodent model of DKA versus the chronic pleotropic effects by which leptin reverses hyperglycemia in a non-DKA rodent model of T1D.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Cetoacidose Diabética/tratamento farmacológico , Insulina/metabolismo , Leptina/farmacologia , Lipólise/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Cetoacidose Diabética/induzido quimicamente , Cetoacidose Diabética/metabolismo , Cetoacidose Diabética/patologia , Diglicerídeos/metabolismo , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Triglicerídeos/metabolismo
9.
J Clin Invest ; 126(11): 4361-4371, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27760050

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet-induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance.


Assuntos
Gorduras na Dieta/efeitos adversos , Resistência à Insulina , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Gorduras na Dieta/farmacologia , Glicogênio/biossíntese , Glicogênio/genética , Fígado/patologia , Camundongos , Camundongos Mutantes , Mutação de Sentido Incorreto , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Fosforilação , Proteína Quinase C-épsilon/genética , Proteína Quinase C-épsilon/metabolismo , Receptor de Insulina/genética
10.
Am J Physiol Endocrinol Metab ; 311(1): E105-16, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27166280

RESUMO

Mitochondrial dysfunction is associated with many human diseases and results from mismatch of damage and repair over the life of the organelle. PARK2 is a ubiquitin E3 ligase that regulates mitophagy, a repair mechanism that selectively degrades damaged mitochondria. Deletion of PARK2 in multiple in vivo models results in susceptibility to stress-induced mitochondrial and cellular dysfunction. Surprisingly, Park2 knockout (KO) mice are protected from nutritional stress and do not develop obesity, hepatic steatosis or insulin resistance when fed a high-fat diet (HFD). However, these phenomena are casually related and the physiological basis for this phenotype is unknown. We therefore undertook a series of acute HFD studies to more completely understand the physiology of Park2 KO during nutritional stress. We find that intestinal lipid absorption is impaired in Park2 KO mice as evidenced by increased fecal lipids and reduced plasma triglycerides after intragastric fat challenge. Park2 KO mice developed hepatic steatosis in response to intravenous lipid infusion as well as during incubation of primary hepatocytes with fatty acids, suggesting that hepatic protection from nutritional stress was secondary to changes in energy balance due to altered intestinal triglyceride absorption. Park2 KO mice showed reduced adiposity after 1-wk HFD, as well as improved hepatic and peripheral insulin sensitivity. These studies suggest that changes in intestinal lipid absorption may play a primary role in protection from nutritional stress in Park2 KO mice by preventing HFD-induced weight gain and highlight the need for tissue-specific models to address the role of PARK2 during metabolic stress.


Assuntos
Peso Corporal/genética , Dieta Hiperlipídica , Resistência à Insulina/genética , Absorção Intestinal/genética , Metabolismo dos Lipídeos/genética , Ubiquitina-Proteína Ligases/genética , Animais , Metabolismo Energético , Ácidos Graxos/farmacologia , Fígado Gorduroso/genética , Fezes/química , Infusões Intravenosas , Mucosa Intestinal/metabolismo , Lipídeos/análise , Lipídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mitofagia/genética , Triglicerídeos/sangue , Aumento de Peso/genética
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