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1.
Nature ; 579(7798): 279-283, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32132708

RESUMEN

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.


Asunto(s)
Glucagón/farmacología , Gluconeogénesis/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Hígado/efectos de los fármacos , Acetilcoenzima A/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Diabetes Mellitus Tipo 2/fisiopatología , Activación Enzimática/efectos de los fármacos , Glucagón/sangre , Receptores de Inositol 1,4,5-Trifosfato/genética , Lipasa/metabolismo , Lipólisis/efectos de los fármacos , Lipólisis/genética , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Oxidación-Reducción/efectos de los fármacos
2.
Proc Natl Acad Sci U S A ; 117(14): 8166-8176, 2020 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-32188779

RESUMEN

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.


Asunto(s)
Hígado Graso/patología , Glucoquinasa/metabolismo , Glucosa/metabolismo , Glucógeno Hepático/biosíntesis , Hígado/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Hígado Graso/etiología , Técnicas de Silenciamiento del Gen , Glucoquinasa/genética , Glucosa/administración & dosificación , Glucosa-6-Fosfato/análisis , Glucosa-6-Fosfato/metabolismo , Humanos , Hiperglucemia/etiología , Hiperglucemia/patología , Hiperinsulinismo/etiología , Hiperinsulinismo/patología , Insulina/metabolismo , Resistencia a la Insulina , Hígado/patología , Masculino , Metabolómica , Fosforilación , Ratas
3.
Sci Transl Med ; 11(512)2019 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-31578240

RESUMEN

Nonalcoholic fatty liver disease (NAFLD) is estimated to affect up to one-third of the general population, and new therapies are urgently required. Our laboratory previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides. Although we previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, hepatic inflammation, and fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess its safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of longer-term CRMP treatment on hepatic mitochondrial oxidation and on the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in high-fat, fructose-fed cynomolgus macaques (n = 6) and spontaneously obese dysmetabolic rhesus macaques (n = 12). Using positional isotopomer nuclear magnetic resonance tracer analysis (PINTA), we demonstrated that acute CRMP treatment (single dose, 5 mg/kg) increased rates of hepatic mitochondrial fat oxidation by 40%. Six weeks of CRMP treatment reduced hepatic triglycerides in both nonhuman primate models independently of changes in body weight, food intake, body temperature, or adverse reactions. CRMP treatment was also associated with a 20 to 30% reduction in fasting plasma triglycerides and low-density lipoprotein (LDL)-cholesterol in dysmetabolic nonhuman primates. Oral administration of CRMP reduced endogenous glucose production by 18%, attributable to a 20% reduction in hepatic acetyl-coenzyme A (CoA) content [as assessed by whole-body ß-hydroxybutyrate (ß-OHB) turnover] and pyruvate carboxylase flux. Collectively, these studies provide proof-of-concept data to support the development of liver-targeted mitochondrial uncouplers for the treatment of metabolic syndrome in humans.

4.
Cell Rep ; 28(3): 759-772.e10, 2019 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-31315053

RESUMEN

Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.

5.
Nat Med ; 25(3): 526-528, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30733621

RESUMEN

In the version of this article originally published, the VPC and VCS flux data shown in Fig. 6e,f were inadvertently duplicated from Fig. 5j,k. The correct data are now shown in Fig. 6e,f. In these corrected data, VPC flux in response to chronic oral metformin treatment was still significantly decreased (Fig. 6e), and there was still no impact of metformin on VCS flux (Fig. 6f). Therefore, the text describing these data remains the same and this correction does not change the conclusion of this study.

6.
Nat Med ; 24(9): 1384-1394, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30038219

RESUMEN

Metformin, the universal first-line treatment for type 2 diabetes, exerts its therapeutic glucose-lowering effects by inhibiting hepatic gluconeogenesis. However, the primary molecular mechanism of this biguanide remains unclear, though it has been suggested to act, at least partially, by mitochondrial complex I inhibition. Here we show that clinically relevant concentrations of plasma metformin achieved by acute intravenous, acute intraportal or chronic oral administration in awake normal and diabetic rats inhibit gluconeogenesis from lactate and glycerol but not from pyruvate and alanine, implicating an increased cytosolic redox state in mediating metformin's antihyperglycemic effect. All of these effects occurred independently of complex I inhibition, evidenced by unaltered hepatic energy charge and citrate synthase flux. Normalizing the cytosolic redox state by infusion of methylene blue or substrates that contribute to gluconeogenesis independently of the cytosolic redox state abrogated metformin-mediated inhibition of gluconeogenesis in vivo. Additionally, in mice expressing constitutively active acetyl-CoA carboxylase, metformin acutely decreased hepatic glucose production and increased the hepatic cytosolic redox state without altering hepatic triglyceride content or gluconeogenic enzyme expression. These studies demonstrate that metformin, at clinically relevant plasma concentrations, inhibits hepatic gluconeogenesis in a redox-dependent manner independently of reductions in citrate synthase flux, hepatic nucleotide concentrations, acetyl-CoA carboxylase activity, or gluconeogenic enzyme protein expression.


Asunto(s)
Gluconeogénesis/efectos de los fármacos , Metformina/farmacología , Acetil-CoA Carboxilasa/metabolismo , Adenilato Quinasa/metabolismo , Animales , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Dihidroxiacetona/metabolismo , Modelos Animales de Enfermedad , Inyecciones Intravenosas , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Metformina/administración & dosificación , Ratones , Oxidación-Reducción , Fosforilación/efectos de los fármacos , Ácido Pirúvico/metabolismo , Ratas Sprague-Dawley , Estreptozocina
7.
Nat Commun ; 9(1): 498, 2018 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-29386503

RESUMEN

The originally published version of this Article contained an error in Equation 30, which was inadvertently introduced during the production process. This has now been corrected in the PDF and HTML versions of the Article.

8.
Nat Commun ; 8(1): 798, 2017 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-28986525

RESUMEN

Hepatic mitochondria play a central role in the regulation of intermediary metabolism and maintenance of normoglycemia, and there is great interest in assessing rates of hepatic mitochondrial citrate synthase flux (V CS) and pyruvate carboxylase flux (V PC) in vivo. Here, we show that a positional isotopomer NMR tracer analysis (PINTA) method can be used to non-invasively assess rates of V CS and V PC fluxes using a combined NMR/gas chromatography-mass spectrometry analysis of plasma following infusion of [3-13C]lactate and glucose tracer. PINTA measures V CS and V PC fluxes over a wide range of physiological conditions with minimal pyruvate cycling and detects increased hepatic V CS following treatment with a liver-targeted mitochondrial uncoupler. Finally, validation studies in humans demonstrate that the V PC/V CS ratio measured by PINTA is similar to that determined by in vivo NMR spectroscopy. This method will provide investigators with a relatively simple tool to non-invasively examine the role of altered hepatic mitochondrial metabolism.Liver mitochondrial metabolism plays an important role for glucose and lipid homeostasis and its alterations contribute to metabolic disorders, including fatty liver and diabetes. Here Perry et al. develop a method for the measurement of hepatic fluxes by using lactate and glucose tracers in combination with NMR spectroscopy.


Asunto(s)
Citrato (si)-Sintasa/metabolismo , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Piruvato Carboxilasa/metabolismo , Acetatos , Animales , Isótopos de Carbono , Espectroscopía de Resonancia Magnética con Carbono-13 , Cromatografía de Gases y Espectrometría de Masas , Ácido Glutámico , Humanos , Ácido Láctico , Espectroscopía de Resonancia Magnética , Masculino , Ácido Pirúvico/metabolismo , Ratas
9.
J Clin Invest ; 126(11): 4361-4371, 2016 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-27760050

RESUMEN

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.


Asunto(s)
Grasas de la Dieta/efectos adversos , Resistencia a la Insulina , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Sustitución de Aminoácidos , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Grasas de la Dieta/farmacología , Glucógeno/biosíntesis , Glucógeno/genética , Hígado/patología , Ratones , Ratones Mutantes , Mutación Missense , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/genética , Fosforilación , Proteína Quinasa C-epsilon/genética , Proteína Quinasa C-epsilon/metabolismo , Receptor de Insulina/genética
10.
Nat Commun ; 7: 12639, 2016 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-27577745

RESUMEN

Insulin resistance is a key driver of type 2 diabetes (T2D) and is characterized by defective insulin receptor (INSR) signalling. Although surface INSR downregulation is a well-established contributor to insulin resistance, the underlying molecular mechanisms remain obscure. Here we show that the E3 ubiquitin ligase MARCH1 impairs cellular insulin action by degrading cell surface INSR. Using a large-scale RNA interference screen, we identify MARCH1 as a negative regulator of INSR signalling. March1 loss-of-function enhances, and March1 overexpression impairs, hepatic insulin sensitivity in mice. MARCH1 ubiquitinates INSR to decrease cell surface INSR levels, but unlike other INSR ubiquitin ligases, MARCH1 acts in the basal state rather than after insulin stimulation. Thus, MARCH1 may help set the basal gain of insulin signalling. MARCH1 expression is increased in white adipose tissue of obese humans, suggesting that MARCH1 contributes to the pathophysiology of T2D and could be a new therapeutic target.


Asunto(s)
Antígenos CD/metabolismo , Diabetes Mellitus Tipo 2/patología , Resistencia a la Insulina/fisiología , Insulina/metabolismo , Obesidad/patología , Receptor de Insulina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Tejido Adiposo Blanco/patología , Adolescente , Animales , Antígenos CD/genética , Biopsia , Línea Celular , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/terapia , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Obesidad/sangre , Obesidad/etiología , Obesidad/terapia , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Fosforilación , ARN Interferente Pequeño/metabolismo , Receptor de Insulina/genética , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Regulación hacia Arriba
11.
Am J Physiol Endocrinol Metab ; 311(1): E105-16, 2016 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-27166280

RESUMEN

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.


Asunto(s)
Peso Corporal/genética , Dieta Alta en Grasa , Resistencia a la Insulina/genética , Absorción Intestinal/genética , Metabolismo de los Lípidos/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Metabolismo Energético , Ácidos Grasos/farmacología , Hígado Graso/genética , Heces/química , Infusiones Intravenosas , Mucosa Intestinal/metabolismo , Lípidos/análisis , Lípidos/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Mitofagia/genética , Triglicéridos/sangre , Aumento de Peso/genética
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