Regulation of myocardial fatty acid oxidation by substrate supply.
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We tested the hypothesis that myocardial substrate supply regulates fatty acid oxidation independent of changes in acetyl-CoA carboxylase (ACC) and 5'-AMP-activated protein kinase (AMPK) activities. Fatty acid oxidation was measured in isolated working rat hearts exposed to different concentrations of exogenous long-chain (0.4 or 1.2 mM palmitate) or medium-chain (0.6 or 2.4 mM octanoate) fatty acids. Fatty acid oxidation was increased with increasing exogenous substrate concentration in both palmitate and octanoate groups. Malonyl-CoA content only rose as acetyl-CoA supply from octanoate oxidation increased. The increases in octanoate oxidation and malonyl-CoA content were independent of changes in ACC and AMPK activity, except that ACC activity increased with very high acetyl-CoA supply levels. Our data suggest that myocardial substrate supply is the primary mechanism responsible for alterations in fatty acid oxidation rates under nonstressful conditions and when substrates are present at physiological concentrations. More extreme variations in substrate supply lead to changes in fatty acid oxidation by the additional involvement of intracellular regulatory pathways. (+info)
5'-AMP-activated protein kinase phosphorylates IRS-1 on Ser-789 in mouse C2C12 myotubes in response to 5-aminoimidazole-4-carboxamide riboside.
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Exercise is known to increase insulin sensitivity and is an effective form of treatment for the hyperglycemia observed in type 2 diabetes. Activation of 5'-AMP-activated protein kinase (AMPK) by 5-aminoimidazole-4-carboxamide riboside (AICAR), exercise, or electrically stimulated contraction leads to increased glucose transport in skeletal muscle. Here we report the first evidence of a direct interaction between AMPK and the most upstream component of the insulin-signaling cascade, insulin receptor substrate-1 (IRS-1). We find that AMPK rapidly phosphorylates IRS-1 on Ser-789 in cell-free assays as well as in mouse C2C12 myotubes incubated with AICAR. In the C2C12 myotubes activation of AMPK by AICAR matched the phosphorylation of IRS-1 on Ser-789. This phosphorylation correlates with a 65% increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity in C2C12 myotubes preincubated with AICAR. The binding of phosphatidylinositol 3-kinase to IRS-1 was not affected by AICAR. These results demonstrate the existence of an interaction between AMPK and early insulin signaling that could be of importance to our understanding of the potentiating effects of exercise on insulin signaling. (+info)
Role of AMP-activated protein kinase in mechanism of metformin action.
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Metformin is a widely used drug for treatment of type 2 diabetes with no defined cellular mechanism of action. Its glucose-lowering effect results from decreased hepatic glucose production and increased glucose utilization. Metformin's beneficial effects on circulating lipids have been linked to reduced fatty liver. AMP-activated protein kinase (AMPK) is a major cellular regulator of lipid and glucose metabolism. Here we report that metformin activates AMPK in hepatocytes; as a result, acetyl-CoA carboxylase (ACC) activity is reduced, fatty acid oxidation is induced, and expression of lipogenic enzymes is suppressed. Activation of AMPK by metformin or an adenosine analogue suppresses expression of SREBP-1, a key lipogenic transcription factor. In metformin-treated rats, hepatic expression of SREBP-1 (and other lipogenic) mRNAs and protein is reduced; activity of the AMPK target, ACC, is also reduced. Using a novel AMPK inhibitor, we find that AMPK activation is required for metformin's inhibitory effect on glucose production by hepatocytes. In isolated rat skeletal muscles, metformin stimulates glucose uptake coincident with AMPK activation. Activation of AMPK provides a unified explanation for the pleiotropic beneficial effects of this drug; these results also suggest that alternative means of modulating AMPK should be useful for the treatment of metabolic disorders. (+info)
AMP-activated protein kinase activation prevents denervation-induced decline in gastrocnemius GLUT-4.
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This study was designed to determine whether the reductions in GLUT-4 seen in 3-day-denervated muscles can be prevented through chemical activation of 5'-AMP-activated protein kinase (AMPK). Muscle AMPK can be chemically activated in rats using subcutaneous injections with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). In this study, the tibial nerve was sectioned on one side; the other was sham operated but without nerve section. Acute injections of AICAR resulted in significantly increased AMPK activity in denervated gastrocnemius but not soleus muscles. Acetyl-CoA carboxylase activity, a reporter of AMPK activation, declined in both gastrocnemius and soleus in both denervated and contralateral muscles. Three days after denervation, GLUT-4 levels were significantly decreased by approximately 40% in gastrocnemius muscles and by approximately 30% in soleus muscles. When rats were injected with AICAR (1 mg/g body wt) for 3 days, the decline in GLUT-4 levels was prevented in denervated gastrocnemius muscles but not in denervated soleus muscles. The extent of denervation-induced muscle atrophy was similar in AICAR-treated vs. saline-treated rats. These studies provide evidence that some effects of denervation may be prevented by chemical activation of the appropriate signaling pathways. (+info)
Yeast AMP pathway genes respond to adenine through regulated synthesis of a metabolic intermediate.
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In Saccharomyces cerevisiae, AMP biosynthesis genes (ADE genes) are transcriptionally activated in the absence of extracellular purines by the Bas1p and Bas2p (Pho2p) transcription factors. We now show that expression of the ADE genes is low in mutant strains affected in the first seven steps of the pathway, while it is constitutively derepressed in mutant strains affected in later steps. Combined with epistasy studies, these results show that 5'-phosphoribosyl-4-succinocarboxamide-5-aminoimidazole (SAICAR), an intermediate metabolite of the pathway, is needed for optimal activation of the ADE genes. Two-hybrid studies establish that SAICAR is required to promote interaction between Bas1p and Bas2p in vivo, while in vitro experiments suggest that the effect of SAICAR on Bas1p-Bas2p interaction could be indirect. Importantly, feedback inhibition by ATP of Ade4p, catalyzing the first step of the pathway, appears to regulate SAICAR synthesis in response to adenine availability. Consistently, both ADE4 dominant mutations and overexpression of wild-type ADE4 lead to deregulation of ADE gene expression. We conclude that efficient transcription of yeast AMP biosynthesis genes requires interaction between Bas1p and Bas2p which is promoted in the presence of a metabolic intermediate whose synthesis is controlled by feedback inhibition of Ade4p acting as the purine nucleotide sensor within the cell. (+info)
Mechanism for fatty acid "sparing" effect on glucose-induced transcription: regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase.
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Carbohydrate-responsive element-binding protein (ChREBP) is a new transcription factor that binds to the carbohydrate-responsive element of the l-type pyruvate kinase gene (l-PK). The aim of this study was to investigate the mechanism by which feeding high fat diets results in decreased activity of ChREBP in the liver (Yamashita, H., Takenoshita, M., Sakurai, M., Bruick, R. K., Henzel, W. J., Shillinglaw, W., Arnot, D., and Uyeda, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 9116-9121). We cloned the rat liver ChREBP gene for use throughout this study. Acetate, octanoate, and palmitate inhibited the glucose-induced activation of l-PK transcription in ChREBP-overexpressed hepatocytes. In these hepatocytes, the cytosolic AMP concentration increased 30-fold and AMP-activated protein kinase activity was activated 2-fold. Similarly to the fatty acids, 5-amino-4-imidazolecarboxamide ribotide, a specific activator of AMP-activated protein kinase (AMPK) also inhibited the l-PK transcription activity in ChREBP-overexpressed hepatocytes. Using as a substrate a truncated ChREBP consisting of the C-terminal region, we demonstrated that phosphorylation by AMPK resulted in inactivation of the DNA binding activity. AMPK specifically phosphorylated Ser(568) of ChREBP. A S568A mutant of the ChREBP gene showed tight DNA binding and lost its fatty acid sensitivity, whereas a S568D mutant showed weak DNA binding and inhibited l-PK transcription activity even in the absence of fatty acid. These results strongly suggested that the fatty acid inhibition of glucose-induced l-PK transcription resulted from AMPK phosphorylation of ChREBP at Ser(568), which inactivated the DNA binding activity. AMPK was activated by the increased AMP that was generated by the fatty acid activation. (+info)
Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation.
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Apoptosis has been observed in vascular cells, nerve, and myocardium of diabetic humans and experimental animals, although whether it contributes to or is a marker of complications in these tissues is unclear. Previous studies have shown that incubation of human umbilical vein endothelial cells (HUVECs) with 30 vs. 5 mmol/l glucose for 72 h causes a significant increase in apoptosis, possibly related to an increase in oxidative stress. We report here that this increase in apoptosis (assessed morphologically by TdT-mediated dUTP nick- end labeling staining) is preceded (24 h of incubation) by inhibition of fatty acid oxidation, by increases in diacylglycerol synthesis, the concentration of malonyl CoA, and caspase-3 activity, and by decreases in mitochondrial membrane potential and cellular ATP content. In addition, the phosphorylation of Akt in the presence of 150 microU/ml insulin was impaired. No increases in ceramide content or its de novo synthesis were observed. AMP-activated protein kinase (AMPK) activity was not diminished; however, incubation with the AMPK activator 5-aminoimidazole-4-carboxamide-riboside increased AMPK activity twofold and completely prevented all of these changes. Likewise, expression of a constitutively active AMPK in HUVEC prevented the increase in caspase-3 activity. The results indicate that alterations in fatty-acid metabolism, impaired Akt activation by insulin, and increased caspase-3 activity precede visible evidence of apoptosis in HUVEC incubated in a hyperglycemic medium. They also suggest that AMPK could play an important role in protecting the endothelial cell against the adverse effects of sustained hyperglycemia. (+info)
An mRNA splice variant of the AFX gene with altered transcriptional activity.
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Several studies indicate that FKHR and AFX, mammalian homologues of the Caenorhabditis elegans forkhead transcription factor DAF-16, function in the insulin signaling pathway. Here we describe the discovery of a novel AFX isoform, which we designated AFX zeta, in which the first 16 amino acids of the forkhead domain are not present. PCR analysis showed that this isoform is most abundant in the liver, kidney, and pancreas. In HepG2 cells, overexpressed AFX zeta induced reporter gene activity through the insulin-responsive sequences of the phosphoenolpyruvate carboxykinase (PEPCK), IGFBP-1, and G6Pase promoters. AFX zeta-mediated stimulation was repressed by insulin treatment, by bisperoxovanadate treatment, and by overexpression of constitutively active protein kinase B (PKB). Insulin treatment and PKB overexpression resulted in phosphorylation of AFX zeta. Furthermore, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), an AMP-activated protein kinase activator, repressed AFX zeta-dependent reporter activation. Taken together, these findings suggest that AFX zeta is a downstream target of both the phosphatidylinositol 3-kinase/PKB insulin signaling pathway and an AMP-activated protein kinase-dependent pathway. (+info)