Showing papers by "Michael F. Hirshman published in 2007"

Ca2+/Calmodulin-Dependent Protein Kinase Kinase-α Regulates Skeletal Muscle Glucose Uptake Independent of AMP-Activated Protein Kinase and Akt Activation

Abstract: Studies in nonmuscle cells have demonstrated that Ca 2+ /calmodulin-dependent protein kinase kinases (CaMKKs) are upstream regulators of AMP-activated protein kinase (AMPK) and Akt. In skeletal muscle, activation of AMPK and Akt has been implicated in the regulation of glucose uptake. The objective of this study was to determine whether CaMKKα regulates skeletal muscle glucose uptake, and whether it is dependent on AMPK and/or Akt activation. Expression vectors containing constitutively active CaMKKα (caCaMKKα) or empty vector were transfected into mouse muscles by in vivo electroporation. After 2 weeks, caCaMKKα was robustly expressed and increased CaMKI (Thr 177/180 ) phosphorylation, a known CaMKK substrate. In muscles from wild-type mice, caCaMKKα increased in vivo [ 3 H]-2-deoxyglucose uptake 2.5-fold and AMPKα1 and -α2 activities 2.5-fold. However, in muscles from AMPKα2 inactive mice (AMPKα2i), caCaMKKα did not increase AMPKα1 or -α2 activities, but it did increase glucose uptake 2.5-fold, demonstrating that caCaMKKα stimulates glucose uptake independent of AMPK. Akt (Thr 308 ) phosphorylation was not altered by CaMKKα, and caCaMKKα plus insulin stimulation did not increase the insulin-induced phosphorylation of Akt (Thr 308 ). These results suggest that caCaMKKα stimulates glucose uptake via insulin-independent signaling mechanisms. To assess the role of CaMKK in contraction-stimulated glucose uptake, isolated muscles were treated with or without the CaMKK inhibitor STO-609 and then electrically stimulated to contract. Contraction increased glucose uptake 3.5-fold in muscles from both wild-type and AMPKα2i mice, but STO-609 significantly decreased glucose uptake (∼24%) only in AMPKα2i mice. Collectively, these results implicate CaMKKα in the regulation of skeletal muscle glucose uptake independent of AMPK and Akt activation.

Adrenaline is a critical mediator of acute exercise-induced AMP-activated protein kinase activation in adipocytes.

Abstract: Exercise increases AMPK (AMP-activated protein kinase) activity in human and rat adipocytes, but the underlying molecular mechanisms and functional consequences of this activation are not known. Since adrenaline (epinephrine) concentrations increase with exercise, in the present study we hypothesized that adrenaline activates AMPK in adipocytes. We show that a single bout of exercise increases AMPKα1 and α2 activities and ACC (acetyl-CoA carboxylase) Ser79 phosphorylation in rat adipocytes. Similarly to exercise, adrenaline treatment in vivo increased AMPK activities and ACC phosphorylation. Pre-treatment of rats with the β-blocker propranolol fully blocked exercise-induced AMPK activation. Increased AMPK activity with exercise and adrenaline treatment in vivo was accompanied by an increased AMP/ATP ratio. Adrenaline incubation of isolated adipocytes also increased the AMP/ATP ratio and AMPK activities, an effect blocked by propranolol. Adrenaline incubation increased lipolysis in isolated adipocytes, and Compound C, an AMPK inhibitor, attenuated this effect. Finally, a potential role for AMPK in the decreased adiposity associated with chronic exercise was suggested by marked increases in AMPKα1 and α2 activities in adipocytes from rats trained for 6 weeks. In conclusion, both acute and chronic exercise are significant regulators of AMPK activity in rat adipocytes. Our findings suggest that adrenaline plays a critical role in exercise-stimulated AMPKα1 and α2 activities in adipocytes, and that AMPK can function in the regulation of lipolysis.

Aberrant activation of AMP-activated protein kinase remodels metabolic network in favor of cardiac glycogen storage.

Abstract: AMP-activated protein kinase (AMPK) responds to impaired cellular energy status by stimulating substrate metabolism for ATP generation. Mutation of the γ2 regulatory subunit of AMPK in humans renders the kinase insensitive to energy status and causes glycogen storage cardiomyopathy via unknown mechanisms. Using transgenic mice expressing one of the mutant γ2 subunits (N488I) in the heart, we found that aberrant high activity of AMPK in the absence of energy deficit caused extensive remodeling of the substrate metabolism pathways to accommodate increases in both glucose uptake and fatty acid oxidation in the hearts of γ2 mutant mice via distinct, yet synergistic mechanisms resulting in selective fuel storage as glycogen. Increased glucose entry in the γ2 mutant mouse hearts was directed through the remodeled metabolic network toward glycogen synthesis and, at a substantially higher glycogen level, recycled through the glycogen pool to enter glycolysis. Thus, the metabolic consequences of chronic activation of AMPK in the absence of energy deficiency is distinct from those previously reported during stress conditions. These findings are of particular importance in considering AMPK as a target for the treatment of metabolic diseases.

Genetic model for the chronic activation of skeletal muscle AMP-activated protein kinase leads to glycogen accumulation

Abstract: The AMP-activated protein kinase (AMPK) is an important metabolic sensor/effector that coordinates many of the changes in mammalian tissues during variations in energy availability. We have sought to create an in vivo genetic model of chronic AMPK activation, selecting murine skeletal muscle as a representative tissue where AMPK plays important roles. Muscle-selective expression of a mutant noncatalytic gamma1 subunit (R70Qgamma) of AMPK activates AMPK and increases muscle glycogen content. The increase in glycogen content requires the presence of the endogenous AMPK catalytic alpha-subunit, since the offspring of cross-breeding of these mice with mice expressing a dominant negative AMPKalpha subunit have normal glycogen content. In R70Qgamma1-expressing mice, there is a small, but significant, increase in muscle glycogen synthase (GSY) activity associated with an increase in the muscle expression of the liver isoform GSY2. The increase in glycogen content is accompanied, as might be expected, by an increase in exercise capacity. Transgene expression of this mutant AMPKgamma1 subunit may provide a useful model for the chronic activation of AMPK in other tissues to clarify its multiple roles in the regulation of metabolism and other physiological processes.

Calmodulin-binding domain of AS160 regulates contraction- but not insulin-stimulated glucose uptake in skeletal muscle.

Abstract: OBJECTIVE— Insulin and contraction increase skeletal muscle glucose uptake through distinct and additive mechanisms However, recent reports have demonstrated that both signals converge on the Akt substrate of 160 kDa (AS160), a protein that regulates GLUT4 translocation Although AS160 phosphorylation is believed to be the primary factor affecting its activity, AS160 also possesses a calmodulin-binding domain (CBD) This raises the possibility that contraction-stimulated increases in Ca 2+ /calmodulin could also modulate AS160 function RESEARCH DESIGN AND METHODS— To evaluate the AS160 CBD in skeletal muscle, empty-vector, wild-type, or CBD-mutant AS160 cDNAs were injected into mouse muscles followed by in vivo electroporation One week later, AS160 was overexpressed by ∼14-fold over endogenous protein RESULTS— Immunoprecipitates of wild-type and CBD-mutant AS160 were incubated with biotinylated calmodulin in the presence of Ca 2+ Wild-type AS160, but not the CBD-mutant AS160, associated with calmodulin Next, we measured insulin- and contraction-stimulated glucose uptake in vivo Compared with empty-vector and wild-type AS160, insulin-stimulated glucose uptake was not altered in muscles expressing CBD-mutant AS160 In contrast, contraction-stimulated glucose uptake was significantly decreased in CBD-mutant–expressing muscles This inhibitory effect on glucose uptake was not associated with aberrant contraction-stimulated AS160 phosphorylation Interestingly, AS160 expressing both calmodulin-binding and Rab-GAP (GTPase-activating protein) domain point mutations (CBD + R/K) fully restored contraction-stimulated glucose uptake CONCLUSIONS— Our results suggest that the AS160 CBD directly regulates contraction-induced glucose uptake in mouse muscle and that calmodulin provides an additional means of modulating AS160 Rab-GAP function independent of phosphorylation These findings define a novel AS160 signaling component, unique to contraction and not insulin, leading to glucose uptake in skeletal muscle