HIF prolyl hydroxylases (PHD1-3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparalpha pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2alpha and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.

High lactate generation and low glucose oxidation, despite normal oxygen conditions, are commonly seen in cancer cells and tumors. Historically known as the Warburg effect, this altered metabolic phenotype has long been correlated with malignant progression and poor clinical outcome. However, the mechanistic relationship between altered glucose metabolism and malignancy remains poorly understood. Here we show that inhibition of pyruvate dehydrogenase complex (PDC) activity contributes to the Warburg metabolic and malignant phenotype in human head and neck squamous cell carcinoma. PDC inhibition occurs via enhanced expression of pyruvate dehydrogenase kinase-1 (PDK-1), which results in inhibitory phosphorylation of the pyruvate dehydrogenase ␣ (PDH␣) subunit. We also demonstrate that PDC inhibition in cancer cells is associated with normoxic stabilization of the malignancy-promoting transcription factor hypoxia-inducible factor-1␣ (HIF-1␣) by glycolytic metabolites. Knockdown of PDK-1 via short hairpin RNA lowers PDH␣ phosphorylation, restores PDC activity, reverts the Warburg metabolic phenotype, decreases normoxic HIF-1␣ expression, lowers hypoxic cell survival, decreases invasiveness, and inhibits tumor growth. PDK-1 is an HIF-1-regulated gene, and these data suggest that the buildup of glycolytic metabolites, resulting from high PDK-1 expression, may in turn promote HIF-1 activation, thus sustaining a feed-forward loop for malignant progression. In addition to providing anabolic support for cancer cells, altered fuel metabolism thus supports a malignant phenotype. Correction of metabolic abnormalities offers unique opportunities for cancer treatment and may potentially synergize with other cancer therapies.Cancer is a disease whereby genetic mutation results in uncontrolled cell growth combined with malignancy. High lactate accumulation, despite adequate oxygen availability, is a metabolic pattern commonly associated with malignant transformation of the uncontrolled dividing cell. This metabolic phenotype, termed aerobic glycolysis and historically known as the Warburg effect, is characterized by high glycolytic rates and reduced mitochondrial oxidation (1, 2), features that may favor cell survival in the hypoxic microenvironments found in tumors. This phenotype also favors the routing of key metabolic intermediates away from oxidative destruction and toward anabolic processes required by rapidly dividing cells (2). Hypoxia and growth factors may select for this phenotype by activating hypoxia-inducible transcription factor-1 (HIF-1), 3 which induces transcription of glucose transporters, glycolytic enzymes, and many other genes associated with hypoxic survival, angiogenesis, and tissue invasion (3). Hypoxia, HIF-1 activation, and high lactate levels in tumors are all independently correlated with poor clinical outcome for many human cancers (3-5). A causative role for hypoxia and HIF-1 stabilization in tumor formation and progression has been demonstrated (reviewed in Ref. 3). However, the buildup of glycoly...

The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4-/- mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4-/- mice. Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4-/- mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4-/- mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4-/- mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis. BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4-/- mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4-/- mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity. Up-regulation of PDHK4 in tissues other than the liver is clearly important during starvation for regulation of PDC activity and glucose homoeostasis.

Jeoung NH, Harris RA. Pyruvate dehydrogenase kinase-4 deficiency lowers blood glucose and improves glucose tolerance in diet-induced obese mice. Am J Physiol Endocrinol Metab 295: E46 -E54, 2008. First published April 22, 2008 doi:10.1152/ajpendo.00536.2007.-The effect of pyruvate dehydrogenase kinase-4 (PDK4) deficiency on glucose homeostasis was studied in mice fed a high-fat diet. Expression of PDK4 was greatly increased in skeletal muscle and diaphragm but not liver and kidney of wild-type mice fed the high-fat diet. Wild-type and PDK4 Ϫ/Ϫ mice consumed similar amounts of the diet and became equally obese. Insulin resistance developed in both groups. Nevertheless, fasting blood glucose levels were lower, glucose tolerance was slightly improved, and insulin sensitivity was slightly greater in the PDK4 Ϫ/Ϫ mice compared with wild-type mice. When the mice were killed in the fed state, the actual activity of the pyruvate dehydrogenase complex (PDC) was higher in the skeletal muscle and diaphragm but not in the liver and kidney of PDK4 Ϫ/Ϫ mice compared with wild-type mice. When the mice were killed after overnight fasting, the actual PDC activity was higher only in the kidney of PDK4 Ϫ/Ϫ mice compared with wild-type mice. The concentrations of gluconeogenic substrates were lower in the blood of PDK4 Ϫ/Ϫ mice compared with wild-type mice, consistent with reduced formation in peripheral tissues. Diaphragms isolated from PDK4 Ϫ/Ϫ mice oxidized glucose faster and fatty acids slower than diaphragms from wild-type mice. Fatty acid oxidation inhibited glucose oxidation by diaphragms from wild-type but not PDK4 Ϫ/Ϫ mice. NEFA, ketone bodies, and branched-chain amino acids were elevated more in PDK4 Ϫ/Ϫ mice, consistent with slower rates of oxidation. These findings show that PDK4 deficiency lowers blood glucose and slightly improves glucose tolerance and insulin sensitivity in mice with diet-induced obesity.pyruvate dehydrogenase kinase-4-knockout mice; diet-induced obesity; glucose oxidation; fatty acid oxidation; branched-chain amino acids THE PYRUVATE DEHYDROGENASE COMPLEX (PDC) is required for the oxidative disposal of dietary carbohydrate. Decarboxylation of pyruvate produces acetyl-CoA for oxidation by the citric acid cycle or conversion to fat. Since acetyl-CoA cannot be converted back to glucose, tight control of flux through PDC is required for maintenance of euglycemia during starvation. PDC activity has to be increased after meals but reduced during fasting. PDC activity is regulated by feedback inhibition by its products acetyl-CoA and NADH and by a balance between inactivation by pyruvate dehydrogenase kinases (PDKs) and activation by pyruvate dehydrogenase phosphatases (PDPs). In the well-fed state, PDC is relatively dephosphorylated and active for disposal of excess glucose. In the fasted state, PDC is highly phosphorylated and inactive so that three-carbon compounds can be converted back to glucose (50, 51). Four PDK isoenzymes (PDK1-4) and two PDP isoenzymes (PDP1 and -2) are expressed in mammalian tis...

Insulin signaling is essential for normal glucose homeostasis. Rho-kinase (ROCK) isoforms have been shown to participate in insulin signaling and glucose metabolism in cultured cell lines. To investigate the physiological role of ROCK1 in the regulation of whole body glucose homeostasis and insulin sensitivity in vivo, we studied mice with global disruption of ROCK1. Here we show that, at 16 -18 weeks of age, ROCK1-deficient mice exhibited insulin resistance, as revealed by the failure of blood glucose levels to decrease after insulin injection. However, glucose tolerance was normal in the absence of ROCK1. These effects were independent of changes in adiposity. Interestingly, ROCK1 gene ablation caused a significant increase in glucose-induced insulin secretion, leading to hyperinsulinemia. To determine the mechanism(s) by which deletion of ROCK1 causes insulin resistance, we measured the ability of insulin to activate phosphatidylinositol 3-kinase and multiple distal pathways in skeletal muscle. Insulin-stimulated phosphatidylinositol 3-kinase activity associated with IRS-1 or phospho-tyrosine was also reduced ϳ40% without any alteration in tyrosine phosphorylation of insulin receptor in skeletal muscle. Concurrently, serine phosphorylation of IRS-1 at serine 632/635, which is phosphorylated by ROCK in vitro, was also impaired in these mice. Insulin-induced phosphorylation of Akt, AS160, S6K, and S6 was also decreased in skeletal muscle. These data suggest that ROCK1 deficiency causes systemic insulin resistance by impairing insulin signaling in skeletal muscle. Thus, our results identify ROCK1 as a novel regulator of glucose homeostasis and insulin sensitivity in vivo, which could lead to new treatment approaches for obesity and type 2 diabetes.The ability of insulin to acutely stimulate glucose uptake and metabolism in peripheral tissues such as skeletal muscle and adipose tissue is critical for the regulation of normal glucose homeostasis (1). Impairments in insulin secretion and in the response of peripheral tissues to insulin (i.e. insulin resistance) are major pathogenic features of type 2 diabetes and contribute to the morbidity of obesity (1, 2). Insulin action involves a series of signaling cascades initiated by insulin binding to its receptor, eliciting receptor autophosphorylation and activation of the receptor tyrosine kinase, resulting in tyrosine phosphorylation of insulin receptor substrates (IRSs) 4 (3). Phosphorylation of IRSs leads to activation of phosphatidylinositol 3-kinase (PI3K) and subsequently to activation of Akt and its downstream mediator AS160, all of which are important steps for the stimulation of glucose transport induced by insulin (4 -6). Although the mechanism(s) underlying insulin resistance are not completely understood in peripheral tissues such as skeletal muscle, they are thought to result, at least in part, from impaired insulin-stimulated signal transduction (7).Rho-kinase (ROCK) is a Ser/Thr protein kinase identified as a GTP-Rho-binding protein (8). There are two ...

The pyruvate dehydrogenase complex (PDC) activity is crucial to maintains blood glucose and ATP levels, which largely depends on the phosphorylation status by pyruvate dehydrogenase kinase (PDK) isoenzymes. Although it has been reported that PDC is phosphorylated and inactivated by PDK2 and PDK4 in metabolically active tissues including liver, skeletal muscle, heart, and kidney during starvation and diabetes, the precise mechanisms by which expression of PDK2 and PDK4 are transcriptionally regulated still remains unclear. Insulin represses the expression of PDK2 and PDK4 via phosphorylation of FOXO through PI3K/Akt signaling pathway. Several nuclear hormone receptors activated due to fasting or increased fat supply, including peroxisome proliferator-activated receptors, glucocorticoid receptors, estrogen-related receptors, and thyroid hormone receptors, also participate in the up-regulation of PDK2 and PDK4; however, the endogenous ligands that bind those nuclear receptors have not been identified. It has been recently suggested that growth hormone, adiponectin, epinephrine, and rosiglitazone also control the expression of PDK4 in tissue-specific manners. In this review, we discuss several factors involved in the expressional regulation of PDK2 and PDK4, and introduce current studies aimed at providing a better understanding of the molecular mechanisms that underlie the development of metabolic diseases such as diabetes.

Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons vs. astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDHα). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorlyated PDHα. Dephosphorylation of astrocytic PDHα restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed.

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