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2.
Nat Commun ; 13(1): 4278, 2022 07 25.
Article in English | MEDLINE | ID: mdl-35879328

ABSTRACT

Sarcoplasmic/endoplasmic reticulum calcium ATPase SERCA2 mediates calcium re-uptake from the cytosol into sarcoplasmic reticulum, and its dysfunction is a hallmark of heart failure. Multiple factors have been identified to modulate SERCA2 activity, however, its regulation is still not fully understood. Here we identify a Ral-GTPase activating protein RalGAPα1 as a critical regulator of SERCA2 in cardiomyocytes through its downstream target RalA. RalGAPα1 is induced by pressure overload, and its deficiency causes cardiac dysfunction and exacerbates pressure overload-induced heart failure. Mechanistically, RalGAPα1 regulates SERCA2 through direct interaction and its target RalA. Deletion of RalGAPα1 decreases SERCA2 activity and prolongs calcium re-uptake into sarcoplasmic reticulum. GDP-bound RalA, but not GTP-bound RalA, binds to SERCA2 and activates the pump for sarcoplasmic reticulum calcium re-uptake. Overexpression of a GDP-bound RalAS28N mutant in the heart preserves cardiac function in a mouse model of heart failure. Our findings have therapeutic implications for treatment of heart failure.


Subject(s)
Calcium , Heart Failure , Animals , Mice , Calcium/metabolism , Heart Failure/metabolism , Homeostasis , Myocytes, Cardiac/metabolism , ral GTP-Binding Proteins , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
3.
Nat Commun ; 11(1): 2186, 2020 05 04.
Article in English | MEDLINE | ID: mdl-32367034

ABSTRACT

Diabetic cardiomyopathy is a progressive disease in diabetic patients, and myocardial insulin resistance contributes to its pathogenesis through incompletely-defined mechanisms. Striated muscle preferentially expressed protein kinase (SPEG) has two kinase-domains and is a critical cardiac regulator. Here we show that SPEG is phosphorylated on Ser2461/Ser2462/Thr2463 by protein kinase B (PKB) in response to insulin. PKB-mediated phosphorylation of SPEG activates its second kinase-domain, which in turn phosphorylates sarcoplasmic/endoplasmic reticulum calcium-ATPase 2a (SERCA2a) and accelerates calcium re-uptake into the SR. Cardiac-specific deletion of PKBα/ß or a high fat diet inhibits insulin-induced phosphorylation of SPEG and SERCA2a, prolongs SR re-uptake of calcium, and impairs cardiac function. Mice bearing a Speg3A mutation to prevent its phosphorylation by PKB display cardiac dysfunction. Importantly, the Speg3A mutation impairs SERCA2a phosphorylation and calcium re-uptake into the SR. Collectively, these data demonstrate that insulin resistance impairs this PKB-SPEG-SERCA2a signal axis, which contributes to the development of diabetic cardiomyopathy.


Subject(s)
Calcium/metabolism , Diabetic Cardiomyopathies/metabolism , Homeostasis , Insulin Resistance , Muscle Proteins/metabolism , Myosin-Light-Chain Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Diabetic Cardiomyopathies/genetics , Humans , Hypoglycemic Agents/metabolism , Hypoglycemic Agents/pharmacology , Insulin/metabolism , Insulin/pharmacology , Mice, Inbred C57BL , Mice, Transgenic , Muscle Proteins/genetics , Mutation , Myosin-Light-Chain Kinase/genetics , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/genetics , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Signal Transduction/genetics
4.
Sci Adv ; 5(4): eaav4116, 2019 04.
Article in English | MEDLINE | ID: mdl-30989113

ABSTRACT

How insulin stimulates postprandial uptake of glucose and long-chain fatty acids (LCFAs) into skeletal muscle and the mechanisms by which these events are dampened in diet-induced obesity are incompletely understood. Here, we show that RalGAPα1 is a critical regulator of muscle insulin action and governs both glucose and lipid homeostasis. A high-fat diet increased RalGAPα1 protein but decreased its insulin-responsive Thr735-phosphorylation in skeletal muscle. A RalGAPα1Thr735Ala mutation impaired insulin-stimulated muscle assimilation of glucose and LCFAs and caused metabolic syndrome in mice. In contrast, skeletal muscle-specific deletion of RalGAPα1 improved postprandial glucose and lipid control. Mechanistically, these mutations of RalGAPα1 affected translocation of insulin-responsive glucose transporter GLUT4 and fatty acid translocase CD36 via RalA to affect glucose and lipid homeostasis. These data indicated RalGAPα1 as a dual-purpose target, for which we developed a peptide-blockade for improving muscle insulin sensitivity. Our findings have implications for drug discovery to combat metabolic disorders.


Subject(s)
GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/metabolism , Glucose/metabolism , Lipid Metabolism , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Animals , Biological Transport , Fatty Acids/metabolism , GTPase-Activating Proteins/genetics , Gene Expression Regulation/drug effects , Gene Knock-In Techniques , Insulin/metabolism , Insulin Resistance/genetics , Metabolic Diseases/etiology , Metabolic Diseases/metabolism , Mice , Models, Biological , Nerve Tissue Proteins/genetics , Obesity/genetics , Obesity/metabolism , Oxidation-Reduction , Phosphorylation , Postprandial Period , ral GTP-Binding Proteins
5.
Diabetes ; 66(9): 2387-2399, 2017 09.
Article in English | MEDLINE | ID: mdl-28696211

ABSTRACT

Skeletal muscle absorbs long-chain fatty acids (LCFAs) that are either oxidized in mitochondria or temporarily stored as triglycerides in lipid droplets (LDs). So far, it is still not fully understood how lipid uptake and storage are regulated in muscle and whether these are important for whole-body lipid homeostasis. Here we show that the small GTPase Rab8a regulates lipid uptake and storage in skeletal muscle. Muscle-specific Rab8a deletion caused hyperlipidemia and exacerbated hepatosteatosis induced by a high-fat diet. Mechanistically, Rab8a deficiency decreased LCFA entry into skeletal muscle and inhibited LD fusion in muscle cells. Consequently, blood lipid levels were elevated and stimulated hepatic mammalian target of rapamycin, which enhanced hepatosteatosis by upregulating hepatic lipogenesis and cholesterol biosynthesis. Our results demonstrate the significance of lipid uptake and storage in muscle in regulating whole-body lipid homeostasis, and they shed light on the roles of skeletal muscle in the pathogenesis of hyperlipidemia and hepatosteatosis.


Subject(s)
Fatty Liver/metabolism , Hyperlipidemias/metabolism , Lipid Metabolism/physiology , Muscle, Skeletal/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Cholesterol/biosynthesis , Gene Expression Regulation/physiology , Hyperlipidemias/blood , Lipid Metabolism/genetics , Mice , Mice, Knockout , Myogenic Regulatory Factor 5/genetics , Myogenic Regulatory Factor 5/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphatidate Phosphatase/genetics , Phosphatidate Phosphatase/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , rab GTP-Binding Proteins/genetics
6.
Diabetologia ; 60(2): 336-345, 2017 02.
Article in English | MEDLINE | ID: mdl-27826658

ABSTRACT

AIMS/HYPOTHESIS: TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab GTPase-activating protein (RabGAP) that has been implicated in regulating GLUT4 trafficking. TBC1D1 can be phosphorylated by the AMP-activated protein kinase (AMPK) on Ser231, which consequently interacts with 14-3-3 proteins. Given the key role for AMPK in regulating insulin-independent muscle glucose uptake, we hypothesised that TBC1D1-Ser231 phosphorylation and/or 14-3-3 binding may mediate AMPK-governed glucose homeostasis. METHODS: Whole-body glucose homeostasis and muscle glucose uptake were assayed in mice bearing a Tbc1d1 Ser231Ala-knockin mutation or harbouring skeletal muscle-specific Ampkα1/α2 (also known as Prkaa1/2) double-knockout mutations in response to an AMPK-activating agent, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR). Exercise-induced muscle glucose uptake and exercise capacity were also determined in the Tbc1d1 Ser231Ala-knockin mice. RESULTS: Skeletal muscle-specific deletion of Ampkα1/a2 in mice prevented AICAR-induced hypoglycaemia and muscle glucose uptake. The Tbc1d1 Ser231Ala-knockin mutation also attenuated the glucose-lowering effect of AICAR in mice. Glucose uptake and cell surface GLUT4 content were significantly lower in muscle isolated from the Tbc1d1 Ser231Ala-knockin mice upon stimulation with a submaximal dose of AICAR. However, this Tbc1d1 Ser231Ala-knockin mutation neither impaired exercise-induced muscle glucose uptake nor affected exercise capacity in mice. CONCLUSIONS/INTERPRETATION: TBC1D1-Ser231 phosphorylation and/or 14-3-3 binding partially mediates AMPK-governed glucose homeostasis and muscle glucose uptake in a context-dependent manner.


Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Exercise/physiology , GTPase-Activating Proteins/genetics , Glucose/metabolism , Ribonucleotides/metabolism , 14-3-3 Proteins/genetics , 14-3-3 Proteins/metabolism , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Aminoimidazole Carboxamide/metabolism , Animals , Biological Transport , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , Humans , Immunoblotting , Immunoprecipitation , Mice , Muscle, Skeletal/metabolism , Mutation/genetics , Phosphorylation , Ribonucleotides/genetics , Signal Transduction/genetics , Signal Transduction/physiology
7.
Proc Natl Acad Sci U S A ; 113(26): 7219-24, 2016 06 28.
Article in English | MEDLINE | ID: mdl-27307439

ABSTRACT

Tre-2/USP6, BUB2, cdc16 domain family member 1 (the TBC domain is the GTPase activating protein domain) (TBC1D1) is a Rab GTPase activating protein that is phosphorylated on Ser(231) by the AMP-activated protein kinase (AMPK) in response to intracellular energy stress. However, the in vivo role and importance of this phosphorylation event remains unknown. To address this question, we generated a mouse model harboring a TBC1D1(Ser231Ala) knockin (KI) mutation and found that the KI mice developed obesity on a normal chow diet. Mechanistically, TBC1D1 is located on insulin-like growth factor 1 (IGF1) storage vesicles, and the KI mutation increases endocrinal and paracrinal/autocrinal IGF1 secretion in an Rab8a-dependent manner. Hypersecretion of IGF1 causes increased expression of lipogenic genes via activating the protein kinase B (PKB; also known as Akt)-mammalian target of rapamycin (mTOR) pathway in adipose tissues, which contributes to the development of obesity, diabetes, and hepatic steatosis as the KI mice age. Collectively, these findings demonstrate that the AMPK-TBC1D1 signaling nexus interacts with the PKB-mTOR pathway via IGF1 secretion, which consequently controls expression of lipogenic genes in the adipose tissue. These findings also have implications for drug discovery to combat obesity.


Subject(s)
AMP-Activated Protein Kinases/metabolism , GTPase-Activating Proteins/metabolism , Insulin-Like Growth Factor I/metabolism , Obesity/metabolism , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , Adipocytes/metabolism , Adipose Tissue/metabolism , Animals , Cells, Cultured , Chondrocytes/metabolism , GTPase-Activating Proteins/genetics , Gene Expression Regulation , Glucose/metabolism , HEK293 Cells , Hep G2 Cells , Hepatocytes/metabolism , Humans , Liver/metabolism , Male , Mice, Transgenic , Muscle Contraction , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiology , Obesity/genetics , Phosphorylation , Serine/metabolism , Triglycerides/metabolism
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