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1.
Cell Rep ; 31(6): 107623, 2020 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-32402282

RESUMEN

Stem cell-derived ß (SC-ß) cells could provide unlimited human ß cells toward a curative diabetes treatment. Differentiation of SC-ß cells yields transplantable islets that secrete insulin in response to glucose challenges. Following transplantation into mice, SC-ß cell function is comparable to human islets, but the magnitude and consistency of response in vitro are less robust than observed in cadaveric islets. Here, we profile metabolism of SC-ß cells and islets to quantify their capacity to sense glucose and identify reduced anaplerotic cycling in the mitochondria as the cause of reduced glucose-stimulated insulin secretion in SC-ß cells. This activity can be rescued by challenging SC-ß cells with intermediate metabolites from the TCA cycle and late but not early glycolysis, downstream of the enzymes glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. Bypassing this metabolic bottleneck results in a robust, bi-phasic insulin release in vitro that is identical in magnitude to functionally mature human islets.

2.
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.

3.
Methods Mol Biol ; 1846: 325-334, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30242770

RESUMEN

Metabolism is pivotal for formation of the lymphatic vasculature. Understanding metabolism in lymphatic endothelial cells (LECs) requires quantitative characterization of specific metabolic pathways. Here we describe methods for using radioactive tracers to assess flux rates of glycolysis, fatty acid ß-oxidation, glucose oxidation, and glutamine oxidation. We also provide a detailed method for utilizing mass spectrometry (MS) to measure glycolytic intermediates and ATP.


Asunto(s)
Células Endoteliales/metabolismo , Metaboloma , Metabolómica , Adenosina Trifosfato/metabolismo , Cromatografía Liquida , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Glutamina/metabolismo , Glucólisis , Humanos , Metabolómica/métodos , Oxidación-Reducción , Espectrometría de Masas en Tándem
4.
Nature ; 545(7653): 224-228, 2017 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-28467822

RESUMEN

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.


Asunto(s)
Células Endoteliales/citología , Células Endoteliales/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Glucólisis , Neovascularización Fisiológica , Transducción de Señal , Animales , Movimiento Celular , Proliferación Celular , Femenino , Hexoquinasa/metabolismo , Linfangiogénesis , Vasos Linfáticos/citología , Vasos Linfáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/deficiencia , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/deficiencia , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo
5.
J Biol Chem ; 291(23): 12161-70, 2016 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-27002151

RESUMEN

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.


Asunto(s)
Ciclo del Ácido Cítrico/efectos de los fármacos , Hígado/efectos de los fármacos , Redes y Vías Metabólicas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Propionatos/farmacología , Ácido Pirúvico/metabolismo , Animales , Glucemia/metabolismo , Cromatografía Liquida , Epinefrina/sangre , Epinefrina/farmacología , Cromatografía de Gases y Espectrometría de Masas , Glucagón/sangre , Glucagón/farmacología , Gluconeogénesis/efectos de los fármacos , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Insulina/sangre , Hígado/metabolismo , Mitocondrias/metabolismo , Propionatos/administración & dosificación , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem
6.
Cell Metab ; 22(5): 936-47, 2015 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-26411341

RESUMEN

Mass isotopomer multi-ordinate spectral analysis (MIMOSA) is a step-wise flux analysis platform to measure discrete glycolytic and mitochondrial metabolic rates. Importantly, direct citrate synthesis rates were obtained by deconvolving the mass spectra generated from [U-(13)C6]-D-glucose labeling for position-specific enrichments of mitochondrial acetyl-CoA, oxaloacetate, and citrate. Comprehensive steady-state and dynamic analyses of key metabolic rates (pyruvate dehydrogenase, ß-oxidation, pyruvate carboxylase, isocitrate dehydrogenase, and PEP/pyruvate cycling) were calculated from the position-specific transfer of (13)C from sequential precursors to their products. Important limitations of previous techniques were identified. In INS-1 cells, citrate synthase rates correlated with both insulin secretion and oxygen consumption. Pyruvate carboxylase rates were substantially lower than previously reported but showed the highest fold change in response to glucose stimulation. In conclusion, MIMOSA measures key metabolic rates from the precursor/product position-specific transfer of (13)C-label between metabolites and has broad applicability to any glucose-oxidizing cell.


Asunto(s)
Ciclo del Ácido Cítrico/genética , Ácido Cítrico/metabolismo , Insulina/metabolismo , Ácido Oxaloacético/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Acetilcoenzima A/metabolismo , Animales , Isótopos de Carbono , Citratos/metabolismo , Insulina/genética , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Oxidación-Reducción , Consumo de Oxígeno , Piruvato Carboxilasa/genética , Piruvato Carboxilasa/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Ácido Pirúvico/metabolismo , Ratas
7.
Proc Natl Acad Sci U S A ; 112(36): 11330-4, 2015 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-26305973

RESUMEN

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 ∼70% (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 ∼40% (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.


Asunto(s)
Envejecimiento , Glucosa/metabolismo , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Adulto , Anciano , Glucemia/metabolismo , Isótopos de Carbono , Cromatografía Liquida , Citrato (si)-Sintasa/metabolismo , Técnica de Clampeo de la Glucosa , Humanos , Hipoglucemiantes/farmacología , Insulina/sangre , Insulina/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Mitocondrias/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismo , Espectrometría de Masas en Tándem
8.
J Biol Chem ; 289(11): 7257-63, 2014 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-24497630

RESUMEN

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.


Asunto(s)
Regulación Enzimológica de la Expresión Génica , Gluconeogénesis , Péptidos y Proteínas de Señalización Intracelular/fisiología , Hígado/enzimología , Hígado/metabolismo , Mitocondrias/enzimología , Fosfoenolpiruvato Carboxiquinasa (GTP)/fisiología , Alimentación Animal , Animales , Glucemia/metabolismo , Privación de Alimentos , Silenciador del Gen , Glicerol/metabolismo , Glucógeno/metabolismo , Guanosina Trifosfato/metabolismo , Hepatocitos/citología , Homeostasis , Insulina/metabolismo , Isoenzimas/fisiología , Ácido Láctico/metabolismo , Masculino , Mitocondrias/metabolismo , Oligonucleótidos Antisentido/química , Oxígeno/metabolismo , Consumo de Oxígeno , Ratas , Ratas Sprague-Dawley
9.
Proc Natl Acad Sci U S A ; 110(31): 12780-5, 2013 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-23840067

RESUMEN

Hepatic insulin resistance is a principal component of type 2 diabetes, but the cellular and molecular mechanisms responsible for its pathogenesis remain unknown. Recent studies have suggested that saturated fatty acids induce hepatic insulin resistance through activation of the toll-like receptor 4 (TLR-4) receptor in the liver, which in turn transcriptionally activates hepatic ceramide synthesis leading to inhibition of insulin signaling. In this study, we demonstrate that TLR-4 receptor signaling is not directly required for saturated or unsaturated fat-induced hepatic insulin resistance in both TLR-4 antisense oligonucleotide treated and TLR-4 knockout mice, and that ceramide accumulation is not dependent on TLR-4 signaling or a primary event in hepatic steatosis and impairment of insulin signaling. Further, we show that both saturated and unsaturated fats lead to hepatic accumulation of diacylglycerols, activation of PKCε, and impairment of insulin-stimulated IRS-2 signaling. These data demonstrate that saturated fat-induced insulin resistance is independent of TLR-4 activation and ceramides.


Asunto(s)
Grasas Insaturadas en la Dieta/farmacología , Hígado Graso/metabolismo , Resistencia a la Insulina , Hígado/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Diglicéridos/metabolismo , Hígado Graso/inducido químicamente , Hígado Graso/patología , Proteínas Sustrato del Receptor de Insulina/metabolismo , Hígado/patología , Ratones , Ratas , Ratas Sprague-Dawley
10.
Proc Natl Acad Sci U S A ; 109(37): 14966-71, 2012 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-22912404

RESUMEN

Fatty acid amide hydrolase (FAAH) knockout mice are prone to excess energy storage and adiposity, whereas mutations in FAAH are associated with obesity in humans. However, the molecular mechanism by which FAAH affects energy expenditure (EE) remains unknown. Here we show that reduced energy expenditure in FAAH(-/-) mice could be attributed to decreased circulating triiodothyronine and thyroxine concentrations secondary to reduced mRNA expression of both pituitary thyroid-stimulating hormone and hypothalamic thyrotropin-releasing hormone. These reductions in the hypothalamic-pituitary-thyroid axis were associated with activation of hypothalamic peroxisome proliferating-activated receptor γ (PPARγ), and increased hypothalamic deiodinase 2 expression. Infusion of NAEs (anandamide and palmitoylethanolamide) recapitulated increases in PPARγ-mediated decreases in EE. FAAH(-/-) mice were also prone to diet-induced hepatic insulin resistance, which could be attributed to increased hepatic diacylglycerol content and protein kinase Cε activation. Our data indicate that FAAH deletion, and the resulting increases in NAEs, predispose mice to ectopic lipid storage and hepatic insulin resistance by promoting centrally mediated hypothyroidism.


Asunto(s)
Amidohidrolasas/genética , Metabolismo Energético/fisiología , Hipotiroidismo/complicaciones , Hipotiroidismo/genética , Resistencia a la Insulina/fisiología , Amidohidrolasas/deficiencia , Análisis de Varianza , Animales , Ácidos Araquidónicos/administración & dosificación , Cromatografía Liquida , Endocannabinoides/administración & dosificación , Metabolismo Energético/genética , Etanolaminas/administración & dosificación , Hipotiroidismo/enzimología , Immunoblotting , Ratones , Ratones Noqueados , PPAR gamma , Ácidos Palmíticos/administración & dosificación , Reacción en Cadena de la Polimerasa , Alcamidas Poliinsaturadas/administración & dosificación , Espectrometría de Masas en Tándem , Tirotropina/metabolismo , Hormona Liberadora de Tirotropina/metabolismo , Tiroxina/sangre , Triyodotironina/sangre
11.
Methods Mol Biol ; 810: 281-309, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22057574

RESUMEN

Nuclear magnetic resonance (NMR) spectroscopy is a technique with an increasing importance in the study of metabolic diseases. Its initial important role in the determination of chemical structures (1, 2) has been considerably overcome by its potential for the in vivo study of metabolism (3-5). The main characteristic that makes this technique so attractive is its noninvasiveness. Only nuclei capable of transitioning between energy states, in the presence of an intense and constant magnetic field, are studied. This includes abundant nuclei such as proton ((1)H) and phosphorous ((31)P), as well as stable isotopes such as deuterium ((2)H) and carbon 13 ((13)C). This allows a wide range of applications that vary from the determination of water distribution in tissues (as obtained in a magnetic resonance imaging scan) to the calculation of metabolic fluxes under ex vivo and in vivo conditions without the need to use radioactive tracers or tissue biopsies (as in a magnetic resonance spectroscopy (MRS) scan). In this chapter, some technical aspects of the methodology of an NMR/MRS experiment as well as how it can be used to study mitochondrial bioenergetics are overviewed. Advantages and disadvantages of in vivo MRS versus high-resolution NMR using proton high rotation magic angle spinning (HRMAS) of tissue biopsies and tissue extracts are also discussed.


Asunto(s)
Metabolismo Energético/fisiología , Espectroscopía de Resonancia Magnética/métodos , Mitocondrias Cardíacas/metabolismo , Animales , Isótopos de Carbono/metabolismo , Espectroscopía de Resonancia Magnética/instrumentación , Ratones , Protones , Ratas
12.
Endocrinology ; 153(2): 583-91, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22147010

RESUMEN

Nonalcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disease in the United States and is strongly associated with hepatic insulin resistance. We examined whether the thyroid hormone receptor-α (Thra) would be a potential therapeutic target to prevent diet-induced NAFLD and insulin resistance. For that purpose, we assessed insulin action in high-fat diet-fed Thra gene knockout (Thra-0/0) and wild-type mice using hyperinsulinemic-euglycemic clamps combined with (3)H/(14)C-labeled glucose to assess basal and insulin-stimulated rates of glucose and fat metabolism. Body composition was assessed by (1)H magnetic resonance spectroscopy and energy expenditure by indirect calorimetry. Relative rates of hepatic glucose and fat oxidation were assessed in vivo using a novel proton-observed carbon-edited nuclear magnetic resonance technique. Thra-0/0 were lighter, leaner, and manifested greater whole-body insulin sensitivity than wild-type mice during the clamp, which could be attributed to increased insulin sensitivity both in liver and peripheral tissues. Increased hepatic insulin sensitivity could be attributed to decreased hepatic diacylglycerol content, resulting in decreased activation of protein kinase Cε and increased insulin signaling. In conclusion, loss of Thra protects mice from high-fat diet-induced hepatic steatosis and hepatic and peripheral insulin resistance. Therefore, thyroid receptor-α inhibition represents a novel pharmacologic target for the treatment of NAFLD, obesity, and type 2 diabetes.


Asunto(s)
Dieta/efectos adversos , Grasas de la Dieta/efectos adversos , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Receptores alfa de Hormona Tiroidea/genética , Receptores alfa de Hormona Tiroidea/metabolismo , Animales , Técnica de Clampeo de la Glucosa , Insulina/metabolismo , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Noqueados , Obesidad/genética , Transducción de Señal
13.
Cell Metab ; 14(2): 184-95, 2011 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-21803289

RESUMEN

Reduced expression of the Indy (I'm Not Dead, Yet) gene in D. melanogaster and its homolog in C. elegans prolongs life span and in D. melanogaster augments mitochondrial biogenesis in a manner akin to caloric restriction. However, the cellular mechanism by which Indy does this is unknown. Here, we report on the knockout mouse model of the mammalian Indy (mIndy) homolog, SLC13A5. Deletion of mIndy in mice (mINDY(-/-) mice) reduces hepatocellular ATP/ADP ratio, activates hepatic AMPK, induces PGC-1α, inhibits ACC-2, and reduces SREBP-1c levels. This signaling network promotes hepatic mitochondrial biogenesis, lipid oxidation, and energy expenditure and attenuates hepatic de novo lipogenesis. Together, these traits protect mINDY(-/-) mice from the adiposity and insulin resistance that evolve with high-fat feeding and aging. Our studies demonstrate a profound effect of mIndy on mammalian energy metabolism and suggest that mINDY might be a therapeutic target for the treatment of obesity and type 2 diabetes.


Asunto(s)
Adiposidad/genética , Metabolismo Energético/genética , Resistencia a la Insulina/genética , Metabolismo de los Lípidos/genética , Simportadores/biosíntesis , Envejecimiento , Animales , Restricción Calórica , Transportadores de Ácidos Dicarboxílicos , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Obesidad/genética , Simportadores/deficiencia , Simportadores/genética
14.
Hepatology ; 53(4): 1175-81, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21400553

RESUMEN

UNLABELLED: Pyruvate dehydrogenase plays a critical role in the regulation of hepatic glucose and fatty acid oxidation; however, surprisingly little is known about its regulation in vivo. In this study we examined the individual effects of insulin and substrate availability on the regulation of pyruvate dehydrogenase flux (V(PDH) ) to tricarboxylic acid flux (V(TCA) ) in livers of awake rats with lipid-induced hepatic insulin resistance. V(PDH) /V(TCA) flux was estimated from the [4-(13) C]glutamate/[3-(13) C]alanine enrichments in liver extracts and assessed under conditions of fasting and during a hyperinsulinemic-euglycemic clamp, whereas the effects of increased plasma glucose concentration on V(PDH) /V(TCA) flux was assessed during a hyperglycemic clamp in conjunction with infusions of somatostatin and insulin to maintain basal concentrations of insulin. The effects of increases in both glucose and insulin on V(PDH) /V(TCA) were examined during a hyperinsulinemic-hyperglycemic clamp. The effects of chronic lipid-induced hepatic insulin resistance on this flux were also examined by performing these measurements in rats fed a high-fat diet for 3 weeks. Using this approach we found that fasting V(PDH) /V(TCA) was reduced by 95% in rats with hepatic insulin resistance (from 17.2 ± 1.5% to 1.3 ± 0.7%, P < 0.00001). Surprisingly, neither hyperinsulinemia per se or hyperglycemia per se were sufficient to increase V(PDH) /V(TCA) flux. Only under conditions of combined hyperglycemia and hyperinsulinemia did V(PDH) /V(TCA) flux increase (44.6 ± 3.2%, P < 0.0001 versus basal) in low-fat fed animals but not in rats with chronic lipid-induced hepatic insulin resistance. CONCLUSION: These studies demonstrate that the combination of both hyperinsulinemia and hyperglycemia are required to increase V(PDH) /V(TCA) flux in vivo and that this flux is severely diminished in rats with chronic lipid-induced hepatic insulin resistance.


Asunto(s)
Grasas/metabolismo , Glucosa/metabolismo , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Animales , Glucemia/metabolismo , Ciclo del Ácido Cítrico/efectos de los fármacos , Grasas de la Dieta/administración & dosificación , Técnica de Clampeo de la Glucosa , Hígado/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley
15.
Cell Metab ; 12(6): 668-74, 2010 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-21109199

RESUMEN

Aging-associated muscle insulin resistance has been hypothesized to be due to decreased mitochondrial function, secondary to cumulative free radical damage, leading to increased intramyocellular lipid content. To directly test this hypothesis, we examined both in vivo and in vitro mitochondrial function, intramyocellular lipid content, and insulin action in lean healthy mice with targeted overexpression of the human catalase gene to mitochondria (MCAT mice). Here, we show that MCAT mice are protected from age-induced decrease in muscle mitochondrial function (∼30%), energy metabolism (∼7%), and lipid-induced muscle insulin resistance. This protection from age-induced reduction in mitochondrial function was associated with reduced mitochondrial oxidative damage, preserved mitochondrial respiration and muscle ATP synthesis, and AMP-activated protein kinase-induced mitochondrial biogenesis. Taken together, these data suggest that the preserved mitochondrial function maintained by reducing mitochondrial oxidative damage may prevent age-associated whole-body energy imbalance and muscle insulin resistance.


Asunto(s)
Envejecimiento/metabolismo , Catalasa/metabolismo , Metabolismo Energético/fisiología , Resistencia a la Insulina/fisiología , Mitocondrias/metabolismo , Mitocondrias/fisiología , Músculo Esquelético/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Catalasa/genética , Daño del ADN , Humanos , Insulina/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructura , Estrés Oxidativo/fisiología , Consumo de Oxígeno/fisiología , Especies Reactivas de Oxígeno/metabolismo
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