AMPK (AMP-activated protein kinase) is activated allosterically by AMP and by phosphorylation of Thr172 within the catalytic alpha subunit. Here we show that mutations in the regulatory gamma subunit reduce allosteric activation of the kinase by AMP. In addition to its allosteric effect, AMP significantly reduces the dephosphorylation of Thr172 by PP (protein phosphatase)2Calpha. Moreover, a mutation in the gamma subunit almost completely abolishes the inhibitory effect of AMP on dephosphorylation. We were unable to detect any effect of AMP on Thr172 phosphorylation by either LKB1 or CaMKKbeta (Ca2+/calmodulin-dependent protein kinase kinase beta) using recombinant preparations of the proteins. However, using partially purified AMPK from rat liver, there was an apparent AMP-stimulation of Thr172 phosphorylation by LKB1, but this was blocked by the addition of NaF, a PP inhibitor. Western blotting of partially purified rat liver AMPK and LKB1 revealed the presence of PP2Calpha in the preparations. We suggest that previous studies reporting that AMP promotes phosphorylation of Thr172 were misinterpreted. A plausible explanation for this effect of AMP is inhibition of dephosphorylation by PP2Calpha, present in the preparations of the kinases used in the earlier studies. Taken together, our results demonstrate that AMP activates AMPK via two mechanisms: by direct allosteric activation and by protecting Thr172 from dephosphorylation. On the basis of our new findings, we propose a simple model for the regulation of AMPK in mammalian cells by LKB1 and CaMKKbeta. This model accounts for activation of AMPK by two distinct signals: a Ca2+-dependent pathway, mediated by CaMKKbeta and an AMP-dependent pathway, mediated by LKB1.
Maintaining energy balance is a key process at both the level of the individual cell and the whole body. In mammals, defects in energy homeostasis underlie the development of metabolic diseases, including type 2 diabetes and obesity, the incidence of which is increasing at a significant rate in humans. Understanding the molecular basis for energy balance is a prerequisite for developing new strategies, including pharmacological intervention, for combating the rise in these metabolic diseases. An important component in the regulation of energy homeostasis that has emerged over the last few years is the AMP-activated protein kinase (AMPK) 6 pathway. AMPK is a heterotrimeric protein kinase complex that acts as an energy sensor, responding to a rise in AMP levels by increasing ATP-generating pathways and reducing ATP-consuming pathways (1-3). Initially, AMPK was considered primarily as a gauge of energy status at the cellular level (4), and consistent with this idea, orthologues of AMPK have been identified in single cell eukaryotes, such as Saccharomyces cerevisiae (1). Accumulating evidence indicates, however, that in mammals AMPK regulates whole body energy homeostasis acting in metabolic tissues in response to nutrient and hormonal signals. For instance, the adipokines leptin and adiponectin activate AMPK stimulating fatty acid oxidation in liver and muscle (5, 6), while suppressing hepatic glucose production (6, 7). In addition to its peripheral effects, AMPK has been implicated in the central control of energy balance. Activation of AMPK in the hypothalamus has been reported to stimulate food intake, whereas inhibition leads to reduced food intake (8 -11). However, a recent study has reported that in mice a genetic deletion of AMPK in pro-opiomelanocortin neurons leads to an obese phenotype (12), conflicting with the results of previous studies.Many of the downstream effects of AMPK are predicted to be beneficial in treating, and potentially preventing, aspects of metabolic diseases. Consistent with this hypothesis, 5-aminoimidazole-4-carboxamide riboside, a compound that results in activation of AMPK in cells and in vivo, improved insulin sensitivity in animal models of insulin resistance (13-15). Furthermore, metformin, which has been used for nearly 50 years as an anti-diabetic drug and is currently estimated to be used by over 120 million people, activates AMPK via an indirect mechanism (16). It was reported recently that the glucose lowering effect of metformin requires hepatic expression of LKB1, an upstream kinase in the AMPK pathway, providing further evidence that in liver metformin acts via activation of AMPK (17). Taken together, these results indicate that activation of AMPK may provide an effective means for treatment of metabolic disorders.
There is interest in how altered lipid metabolism could contribute to muscle insulin resistance. Many animal and human states of insulin resistance have increased muscle triglyceride content, and there are now plausible mechanistic links between muscle lipid accumulation and insulin resistance, which go beyond the classic glucose-fatty acid cycle. We postulate that muscle cytosolic accumulation of the metabolically active long-chain fatty acyl CoAs (LCACoA) is involved, leading to insulin resistance and impaired insulin signalling or impaired enzyme activity (e.g. glycogen synthase or hexokinase) either directly or via chronic translocation/activation of mediators such as a protein kinase C (particularly PKC theta and epsilon ). Ceramides and diacylglycerols (DAGs) have also been implicated in forms of lipid-induced muscle insulin resistance. Dietary lipid-induced muscle insulin resistance in rodents is relatively easily reversed by manipulations that lessen cytosolic lipid accumulation (e.g. diet change, exercise or fasting). PPAR agonists (both gamma and alpha) also lower muscle LCACoA and enhance insulin sensitivity. Activation of AMP-activated protein kinase (AMPK) by AICAR leads to muscle enhancement (especially glycolytic muscle) of insulin sensitivity, but involvement of altered lipid metabolism is less clear cut. In rodents there are similarities in the pattern of muscle lipid accumulation/PKC translocation/altered insulin signalling/insulin resistance inducible by 3-5-h acute free fatty acid elevation, 1-4 days intravenous glucose infusion or several weeks of high-fat feeding. Recent studies extend findings and show relevance to humans. Muscle cytosolic lipids may accumulate either by increased fatty acid flux into muscle, or by reduced fatty acid oxidation. In some circumstances muscle insulin resistance may be an adaptation to optimize use of fatty acids when they are the predominant available energy fuel. The interactions described here are fundamental to optimizing therapy of insulin resistance based on alterations in muscle lipid metabolism.
The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10⌬2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10⌬2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10⌬2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health.Insulin controls glucose homeostasis by regulating protein, lipid, and carbohydrate metabolism. Cellular responses to insulin in target tissues, such as skeletal muscle, adipose tissue, and liver, are mediated via the insulin receptor (Insr) (reviewed in reference 51). Activation of the Insr results in tyrosine phosphorylation of intracellular docking proteins such as Shc and IRS-1 through IRS-4, which then bind specific Src homology 2 (SH2) domain-containing enzymes and adapters, leading to the activation of downstream signaling cascades. A critical event mediating insulin regulation of metabolic endpoints is the activation of phosphatidylinositol 3-kinase (PI3K). This stimulates the synthesis of phosphatidylinositol 3,4,5-triphosphate, which induces plasma membrane recruitment and subsequent phosphorylation of protein kinase B (also known as Akt), a key player in the regulation of glucose uptake and glycogen synthesis. Activation of the Insr and downstream signaling results in increased glucose uptake, utilization, and storage in adipose tissue and skeletal muscle, while decreased gluconeogenesis and glycogenolysis and increased glycogen synthesis occur in the liver (reviewed in reference 51). Resistance to these effects of insulin is a defining feature of type 2 diabetes, a polygenic disease afflicting over 110 million people worldwide. Impaired insulin action is also a feature of obesity and predisposes people to arteriosclerosis and cardiovascular diseases, facts which highlight its importance in human health. The fundamental role of the Insr in insulin action was demonstrated following targeted disruption of the receptor (1, 29). To dissect the contribution of individual tissues to gl...
SUMMARY Activation of AMP-activated protein kinase (AMPK) is thought to convey many of the beneficial effects of exercise via its inhibitory effect on acetyl-CoA carboxylase 2 (ACC2) and promotion of fatty acid oxidation. Hence, AMPK and ACC have become major drug targets for weight loss and improved insulin action. However, it remains unclear if or how activation of the fatty acid oxidation pathway without a concomitant increase in energy expenditure could be beneficial. In this study we have used either pharmacological (administration of the AMPK agonist 5′ aminoimidazole-4-carboxamide-riboside (AICAR)) or genetic means (mutation of the ACC2 gene in mice) to manipulate fatty acid oxidation to determine if this is sufficient to promote leanness. Both of these strategies increased whole body fatty acid oxidation without altering energy expenditure or adiposity. We conclude that negative energy balance is a pre-requisite for weight reduction and increased fatty acid oxidation per se has little, if any, effect to reduce adiposity.
Sterol regulatory element-binding proteins (SREBPs) are transcription factors central to the regulation of lipid metabolism. The SREBPs are synthesized as precursor proteins that require proteolytic processing to become transcriptionally active. Whereas the regulation of SREBP-1a and -2 cleavage by cellular sterol content is well defined, much less is known about the regulation of SREBP-1c, the predominant SREBP isoform in the liver. Both insulin and liver X receptor ␣ (LXR␣) induce SREBP-1c transcription; however, the respective roles of these factors and the mechanism responsible for proteolytic cleavage of this SREBP isoform are not known. In this study, we compare the effects of insulin and LXR agonist TO-901317 on SREBP-1c expression and transcriptional activity in isolated rat hepatocytes. We report that full induction of the mature and transcriptionally active form of SREBP-1c protein requires insulin. Although activation of LXR leads to the induction of SREBP-1c gene expression and precursor protein, it has a very poor effect in inducing the mature nuclear form of the transcription factor. This may be due to the induction of insulin-induced gene-2a mRNA and protein by LXR activation. The LXR-induced SREBP-1c precursor, however, is rapidly cleaved on acute exposure to insulin via a phosphatidylinositol 3-kinase-dependent mechanism. Finally, we show through experiments in suckling mice that this acute action of insulin to stimulate the proteolytic processing of SREBP-1c is functional in vivo.glucose homeostasis ͉ cholesterol ͉ lipogenesis ͉ hepatocytes T he sterol regulatory element-binding proteins (SREBPs) are transcription factors integral to the maintenance of lipid homeostasis. The three SREBP isoforms (SREBP-1a, -1c, and -2) have overlapping target genes and show differential expression across tissues (1). SREBP-1c is the major isoform expressed in the liver and tissues involved in energy homeostasis (2). It regulates fatty acid synthesis through selective induction of hepatic glucokinase (GK) and an array of lipogenic genes (3-6). SREBP-2 is widely expressed and primarily regulates genes involved in cholesterol biosynthesis (7). The SREBP-1a isoform, which can transactivate both lipogenic and cholesterogenic genes, is highly expressed in cell lines but has very low expression in most organs in vivo (2).The SREBPs are synthesized in the endoplasmic reticulum (ER) in the form of a precursor protein. To become transcriptionally active, the SREBP precursor must undergo proteolytic cleavage in the Golgi apparatus to liberate its N-terminal domain, which constitutes the mature transcription factor (1). Two proteins are essential to this cleavage process: SREBP cleavage-activating protein (SCAP) and insulin-induced gene (Insig). SCAP is a large integral membrane protein of the ER that interacts with newly synthesized SREBP precursor and escorts it to the Golgi apparatus (8, 9). However, SCAP can also interact with Insig, another ER protein that is deeply embedded in the membranes. Insig functions to retain...
In humans and animal models, increased lipid content of skeletal muscle is strongly associated with insulin resistance. However, it is unclear whether this accumulation is due to increased uptake or reduced utilization of fatty acids (FAs). We used 3 H-R-bromopalmitate tracer to assess the contribution of tissue-specific changes in FA uptake to the lipid accumulation observed in tissues of insulin-resistant, high fat-fed rats (HFF) compared with control rats (CON) fed a standard diet. To study FA metabolism under different metabolic states, tracer was infused under basal conditions, during hyperinsulinemic-euglycemic clamp (low FA availability) or during the infusion of intralipid and heparin (high FA availability). FA clearance was significantly increased in the red gastrocnemius muscle of HFF under conditions of low (HFF ؍ 10.4 ؎ 1.1; CON ؍ 7.4 ؎ 0.5 ml ⅐ min ؊1 ⅐ 100 g ؊1 ; P < 0.05), basal (HFF ؍ 8.3 ؎ 1.4; CON ؍ 4.5 ؎ 0.7 ml ⅐ min ؊1 ⅐ 100 g ؊1 ; P < 0.01), and high (HFF ؍ 7.0 ؎ 0.8; CON ؍ 4.3 ؎ 0.5 ml ⅐ min ؊1 ⅐ 100 g ؊1 ; P < 0.05) FA levels. This indicates an adaptation by muscle for more efficient uptake of lipid. Associated with the enhanced efficiency of FA uptake, we observed increases in CD36/FA translocase mRNA expression (P < 0.01) and acyl-CoA synthetase activity (P < 0.02) in the same muscle. FA clearance into white adipose tissue was also increased in HFF when circulating FA were elevated, but there was little effect of the high-fat diet on hepatic FA uptake. In conclusion, insulin resistance induced by feeding rats a high-fat diet is associated with tissue-specific adaptations that enhance utilization of increased dietary lipid but could also contribute to the accumulation of intramuscular lipid with a detrimental effect on insulin action.
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