Mechanisms underlying impaired GLUT-4 translocation in glycogen-supercompensated muscles of exercised rats. (33/2280)

Exercise training induces an increase in GLUT-4 in muscle. We previously found that feeding rats a high-carbohydrate diet after exercise, with muscle glycogen supercompensation, results in a decrease in insulin responsiveness so severe that it masks the effect of a training-induced twofold increase in GLUT-4 on insulin-stimulated muscle glucose transport. One purpose of this study was to determine whether insulin signaling is impaired. Maximally insulin-stimulated phosphatidylinositol (PI) 3-kinase activity was not significantly reduced, whereas protein kinase B (PKB) phosphorylation was approximately 50% lower (P < 0.01) in muscles of chow-fed, than in those of fasted, exercise-trained rats. Our second purpose was to determine whether contraction-stimulated glucose transport is also impaired. The stimulation of glucose transport and the increase in cell surface GLUT-4 induced by contractions were both decreased by approximately 65% in glycogen-supercompensated muscles of trained rats. The contraction-stimulated increase in AMP kinase activity, which has been implicated in the activation of glucose transport by contractions, was approximately 80% lower in the muscles of the fed compared with the fasted rats 18 h after exercise. These results show that both the insulin- and contraction-stimulated pathways for muscle glucose transport activation are impaired in glycogen-supercompensated muscles and provide insight regarding possible mechanisms.  (+info)

The AMP-activated protein kinase prevents ceramide synthesis de novo and apoptosis in astrocytes. (34/2280)

Fatty acids induce apoptosis in primary astrocytes by enhancing ceramide synthesis de novo. The possible role of the AMP-activated protein kinase (AMPK) in the control of apoptosis was studied in this model. Long-term stimulation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevented apoptosis. AICAR blunted fatty acid-mediated induction of serine palmitoyltransferase and ceramide synthesis de novo, without affecting fatty acid synthesis and oxidation. Prevention of ceramide accumulation by AICAR led to a concomitant blockade of the Raf-1/extracellular signal-regulated kinase cascade, which selectively mediates fatty acid-induced apoptosis. Data indicate that AMPK may protect cells from apoptosis induced by stress stimuli.  (+info)

Post-translational modifications of the beta-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization. (35/2280)

The AMP-activated protein kinase (AMPK) is a ubiquitous mammalian protein kinase important in the adaptation of cells to metabolic stress. The enzyme is a heterotrimer, consisting of a catalytic alpha subunit and regulatory beta and gamma subunits, each of which is a member of a larger isoform family. The enzyme is allosterically regulated by AMP and by phosphorylation of the alpha subunit. The beta subunit is post-translationally modified by myristoylation and multi-site phosphorylation. In the present study, we have examined the impact of post-translational modification of the beta-1 subunit on enzyme activity, heterotrimer assembly and subcellular localization, using site-directed mutagenesis and expression of subunits in mammalian cells. Removal of the myristoylation site (G2A mutant) results in a 4-fold activation of the enzyme and relocalization of the beta subunit from a particulate extranuclear distribution to a more homogenous cell distribution. Mutation of the serine-108 phosphorylation site to alanine is associated with enzyme inhibition, but no change in cell localization. In contrast, the phosphorylation site mutations, SS24, 25AA and S182A, while having no effects on enzyme activity, are associated with nuclear redistribution of the subunit. Taken together, these results indicate that both myristoylation and phosphorylation of the beta subunit of AMPK modulate enzyme activity and subunit cellular localization, increasing the complexity of AMPK regulation.  (+info)

Domain fusion between SNF1-related kinase subunits during plant evolution. (36/2280)

Members of the conserved SNF1/AMP-activated protein kinase (AMPK) family regulate cellular responses to environmental and nutritional stress in eukaryotes. Yeast SNF1 and animal AMPKs form a complex with regulatory SNF4/AMPKgamma and SIP1/SIP2/GAL83/AMPKbeta subunits. The beta-subunits function as target selective adaptors that anchor the catalytic kinase and regulator SNF4/gamma-subunits to their kinase association (KIS) and association with the SNF1 complex (ASC) domains. Here we demonstrate that plant SNF1-related protein kinases (SnRKs) interact with an adaptor-regulator protein, AKINbetagamma, in which an N-terminal KIS domain characteristic of beta-subunits is fused with a C-terminal region related to the SNF4/AMPKgamma proteins. AKINbetagamma is constitutively expressed in plants, suppresses the yeast delta snf4 mutation, and shows glucose-regulated interaction with the Arabidopsis SnRK, AKIN11. Our results suggest that evolution of AKINbetagamma reflects a unique function of SNF1-related protein kinases in plant glucose and stress signalling.  (+info)

Effects of stimulation of AMP-activated protein kinase on insulin-like growth factor 1- and epidermal growth factor-dependent extracellular signal-regulated kinase pathway. (37/2280)

AMP-activated protein kinase (AMPK) is tightly regulated by the cellular AMP:ATP ratio and plays a central role in the regulation of energy homeostasis. Previously, AMPK was reported to phosphorylate serine 621 of Raf-1 in vitro. In the present study, we investigated a possible role of AMPK in extracellular signal-regulated kinase (Erk) cascades, using 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), a cell-permeable activator of AMPK and antisense RNA experiments. Activation of AMPK by AICAR in NIH-3T3 cells resulted in drastic inhibitions of Ras, Raf-1, and Erk activation induced by insulin-like growth factor 1 (IGF-1). Expression of an antisense RNA for the AMPK catalytic subunit decreased the AMPK activity and significantly diminished the AICAR effect on IGF-1-induced Ras activation and the subsequent Erk activation, indicating that its effect is indeed mediated by AMPK. Phosphorylation of Raf-1 serine 621, however, was not involved in AMPK-mediated inhibition of Erk cascades. In contrast to IGF-1, AICAR did not block epidermal growth factor (EGF)-dependent Raf-1 and Erk activation, but our results demonstrated that multiple Raf-1 upstream pathways induced by EGF were differentially affected by AICAR: inhibition of Ras activation and simultaneous induction of Ras-independent Raf activation. The activities of IGF-1 and EGF receptor were not affected by AICAR. Taken together, our results suggest that AMPK differentially regulate Erk cascades by inhibiting Ras activation or stimulating the Ras-independent pathway in response to the varying energy status of the cell.  (+info)

AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle. (38/2280)

The AMP-activated protein kinase (AMPK) has been hypothesized to mediate contraction and 5-aminoimidazole-4-carboxamide 1-beta-D-ribonucleoside (AICAR)-induced increases in glucose uptake in skeletal muscle. The purpose of the current study was to determine whether treadmill exercise and isolated muscle contractions in rat skeletal muscle increase the activity of the AMPK alpha 1 and AMPK alpha 2 catalytic subunits in a dose-dependent manner and to evaluate the effects of the putative AMPK inhibitors adenine 9-beta-D-arabinofuranoside (ara-A), 8-bromo-AMP, and iodotubercidin on AMPK activity and 3-O-methyl-D-glucose (3-MG) uptake. There were dose-dependent increases in AMPK alpha 2 activity and 3-MG uptake in rat epitrochlearis muscles with treadmill running exercise but no effect of exercise on AMPK alpha1 activity. Tetanic contractions of isolated epitrochlearis muscles in vitro significantly increased the activity of both AMPK isoforms in a dose-dependent manner and at a similar rate compared with increases in 3-MG uptake. In isolated muscles, the putative AMPK inhibitors ara-A, 8-bromo-AMP, and iodotubercidin fully inhibited AICAR-stimulated AMPK alpha 2 activity and 3-MG uptake but had little effect on AMPK alpha 1 activity. In contrast, these compounds had absent or minimal effects on contraction-stimulated AMPK alpha 1 and -alpha 2 activity and 3-MG uptake. Although the AMPK alpha 1 and -alpha 2 isoforms are activated during tetanic muscle contractions in vitro, in fast-glycolytic fibers, the activation of AMPK alpha 2-containing complexes may be more important in regulating exercise-mediated skeletal muscle metabolism in vivo. Development of new compounds will be required to study contraction regulation of AMPK by pharmacological inhibition.  (+info)

Subcellular localization of the Snf1 kinase is regulated by specific beta subunits and a novel glucose signaling mechanism. (39/2280)

The Snf1/AMP-activated protein kinase family has broad roles in transcriptional, metabolic, and developmental regulation in response to stress. In Saccharomyces cerevisiae, Snf1 is required for the response to glucose limitation. Snf1 kinase complexes contain the alpha (catalytic) subunit Snf1, one of the three related beta subunits Gal83, Sip1, or Sip2, and the gamma subunit Snf4. We present evidence that the beta subunits regulate the subcellular localization of the Snf1 kinase. Green fluorescent protein fusions to Gal83, Sip1, and Sip2 show different patterns of localization to the nucleus, vacuole, and/or cytoplasm. We show that Gal83 directs Snf1 to the nucleus in a glucose-regulated manner. We further identify a novel signaling pathway that controls this nuclear localization in response to glucose phosphorylation. This pathway is distinct from the glucose signaling pathway that inhibits Snf1 kinase activity and responds not only to glucose but also to galactose and sucrose. Such independent regulation of the localization and the activity of the Snf1 kinase, combined with the distinct localization of kinases containing different beta subunits, affords versatility in regulating physiological responses.  (+info)

AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise. (40/2280)

Insulin-stimulated GLUT4 translocation is impaired in people with type 2 diabetes. In contrast, exercise results in a normal increase in GLUT4 translocation and glucose uptake in these patients. Several groups have recently hypothesized that exercise increases glucose uptake via an insulin-independent mechanism mediated by the activation of AMP-activated protein kinase (AMPK). If this hypothesis is correct, people with type 2 diabetes should have normal AMPK activation in response to exercise. Seven subjects with type 2 diabetes and eight matched control subjects exercised on a cycle ergometer for 45 min at 70% of maximum workload. Biopsies of vastus lateralis muscle were taken before exercise, after 20 and 45 min of exercise, and at 30 min postexercise. Blood glucose concentrations decreased from 7.6 to 4.77 mmol/l with 45 min of exercise in the diabetic group and did not change in the control group. Exercise significantly increased AMPK alpha2 activity 2.7-fold over basal at 20 min in both groups and remained elevated throughout the protocol, but there was no effect of exercise on AMPK alpha1 activity. Subjects with type 2 diabetes had similar protein expression of AMPK alpha1, alpha2, and beta1 in muscle compared with control subjects. AMPK alpha2 was shown to represent approximately two-thirds of the total alpha mRNA in the muscle from both groups. In conclusion, people with type 2 diabetes have normal exercise-induced AMPK alpha2 activity and normal expression of the alpha1, alpha2 and beta1 isoforms. Pharmacological activation of AMPK may be an attractive target for the treatment of type 2 diabetes.  (+info)