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Growth hormone (GH) is a counter-regulatory hormone that plays an important role in preventing hypoglycemia during fasting. Because inhibition of the pyruvate dehydrogenase complex (PDC) by pyruvate dehydrogenase kinase 4 (PDK4) conserves substrates for gluconeogenesis, we tested whether GH increases PDK4 expression in liver by a signaling pathway sensitive to inhibition by metformin. The effects of GH and metformin were determined in the liver of wild-type, small heterodimer partner (SHP)-, PDK4-, and signal transducer and activator of transcription 5 (STAT5)-null mice. Administration of GH in vivo increased PDK4 expression via a pathway dependent on STAT5 phosphorylation. Metformin inhibited the induction of PDK4 expression by GH via a pathway dependent on AMP-activated protein kinase (AMPK) and SHP induction. The increase in PDK4 expression and PDC phosphorylation by GH was reduced in STAT5-null mice. Metformin decreased GH-mediated induction of PDK4 expression and metabolites in wild-type but not in SHP-null mice. In primary hepatocytes, dominant-negative mutant-AMPK and SHP knockdown prevented the inhibitory effect of metformin on GH-stimulated PDK4 expression. SHP directly inhibited STAT5 association on the PDK4 gene promoter. Metformin inhibits GH-induced PDK4 expression and metabolites via an AMPK-SHP-dependent pathway. The metformin-AMPK-SHP network may provide a novel therapeutic approach for the treatment of hepatic metabolic disorders induced by the GH-mediated pathway. [ABSTRACT FROM AUTHOR]

Skeletal muscle plays crucial roles in locomotion, protein reservoir, and maintenance of metabolic homeostasis. Loss of muscle, known as muscle atrophy, causes the metabolic diseases such as type 2 diabetes mellitus, hypertension, and so on. Therefore, great efforts have been devoted to prevent the muscle atrophy. Policosanols are a mixture of long chain fatty alcohols extracted from various natural sources. They have long been used as functional foods to lower the level of serum lipids, including triacylglycerol and cholesterol, and to protect against inflammatory stress. In this study, we examine the protective effect and molecular mechanism of Cuban policosanol on skeletal muscle cell death and mitochondrial dysfunction using lipopolysaccharide-treated C2C12 cells. Our results demonstrated that policosanol significantly rescued cell survival (40% vs. 88%; LPS vs. LPS+policosanol) via activation of the Akt pathway, resulting in inhibition of apoptosis (p<0.05). Moreover, policosanol restored the LPS-induced repression of collagen by two fold (0.33±0.04 vs. 0.67±0.03 compared to that of control; LPS vs. LPS+policosanol) via activation of ERK-mTOR-p70S6K pathways. In addition, policosanol increased the mitochondrial fusion by regulating the activities of DRP1 and Mfn2, leading to ameliorate the mitochondrial dysfunction induced by LPS. Improved mitochondria function increased the oxygen consumption rate with glucose as fuel source, indicating that policosanol could shift the glucose metabolism from lactate fermentation, induced by lipopolysaccharide, to oxidative phosphorylation. Thus, policosanol is a promising agent for preventing the inflammation-induced muscle cell death and mitochondrial dysfunction.

Cryptotanshinone is known for its inhibitory activity against tumorigenesis in various human cancer cells. However, exact mechanisms underlying the anticancer effects of cryptotanshinone are not fully elucidated. Here, we propose a plausible molecular mechanism, wherein cryptotanshinone represses rapamycin-sensitive mTORC1/S6K1 mediated cancer cell growth and cell transformation. We investigated the various effects of cryptotanshinone on the mTORC1/S6K1 axis, which is associated with the regulation of cell growth in response to nutritional and growth factor signals. We found that cryptotanshinone specifically inhibited the mTORC1-mediated phosphorylation of S6K1, which consequently suppressed the clonogenicity of SK-Hep1 cells and the neoplastic transformation of JB6 Cl41 cells induced by insulin-like growth factor-1. Finally, we observed that cryptotanshinone prevented S6K1 from binding to the Raptor/mTOR complex, rather than regulating mTOR and its upstream pathway. Overall, our findings provide a novel mechanism underlying anti-cancer effects cryptotanshinone targeting mTORC1 signaling, contributing to the development of anticancer agents involving metabolic cancer treatment., Competing Interests: CONFLICTS OF INTEREST No potential conflicts of interest were disclosed., (Copyright © 2021 Korean Society of Cancer Prevention.)

Neoplastic cells secrete autocrine motility factor (AMF) to stimulate the motility of cancer cells. In this study, AMF secreted from HT-29 colorectal cancer cells selectively suppressed liver cancer cells by downregulating pAKT and β-catenin. In addition, HT-29 AMF significantly augmented the activity of methyl jasmonate against liver cancer cells and is a promising alternative for liver cancer therapy., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Autocrine motility factor (AMF) stimulates the motility of cancer cells via an autocrine route and has been implicated in tumor progression and metastasis. Overexpression of AMF is correlated with the aggressive nature of breast cancer and is negatively associated with clinical outcomes. In contrast, AMF also has the ability to suppress cancer cells. In this study, AMFs from different cancer cells were demonstrated to have suppressive activity against MCF-7 and MDA-MB-231 breast cancer cells. In a growth and colony formation assay, AMF from AsPC-1 pancreatic cancer cells (ASPC-1:AMF) was determined to be more suppressive compared to other AMFs. It was also demonstrated that AsPC-1:AMF could arrest breast cancer cells at the G0/G1 cell cycle phase. Quantified by Western blot analysis, AsPC-1:AMF lowered levels of the AMF receptor (AMFR) and G-protein-coupled estrogen receptor (GPER), concomitantly regulating the activation of the AKT and ERK signaling pathways. JAK/STAT activation was also decreased. These results were found in estrogen receptor (ER)-positive MCF-7 cells but not in triple-negative MDA-MB-231 cells, suggesting that AsPC-1:AMF could work through multiple pathways led to apoptosis. More importantly, AsPC-1:AMF and methyl jasmonate (MJ) cooperatively and synergistically acted against breast cancer cells. Thus, AMF alone or along with MJ may be a promising breast cancer treatment option., Competing Interests: Declaration of competing interest None declared., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In cell competition, a secreted death signal can determine cell fate. However, the nature of such a signal remains unclear. In this study, conditioned medium from HeLa cells (HeLa CM) inhibited growth of A549 and MCF-7 cells. Through HeLa CM fractionation, glucose 6-phosphate isomerase/autocrine motility factor (GPI/AMF) was identified as the main growth inhibitor. Previously, AMF was known for its mitogenic, motogenic, and differentiation functions and was implicated in tumor progression and metastasis. HeLa CM lost its growth inhibitory property after treatment with erythrose-4-phosphate (E4P) or anti-GPI antibody. Purified HeLa recombinant AMF (rAMF) proteins inhibited the growth of A549, MDA-MB-232, MCF-7, AsPC-1, DU145, Hep-2, Hep G2, and HT-29 cells. However, growth of HL-60, SKOV3, U-87 MG, SNU-484, U-87 MG, and 3T3-L1 cells was little affected. In a Transwell assay, HeLa rAMF effectively reduced A549 cell migration and invasion. HeLa rAMF effectively induced apoptosis in A549 cells, apparently by reducing the levels of Bcl-2, GPI, and poly(ADP-ribose) polymerase (PARP)14 and activating caspase-3 and p53. HeLa rAMF antagonized HER2 and the AMF receptor (AMFR or GP78) in relation to the AKT/EKT signaling pathway. These results suggest that HeLa AMF could act as a diffusible death signal that could induce cancer cell-selective growth inhibition and apoptosis., Competing Interests: Declaration of competing interest None declared., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In fasting or diabetes, gluconeogenic genes are transcriptionally activated by glucagon stimulation of the cAMP-protein kinase A (PKA)-CREB signaling pathway. Previous work showed pyruvate dehydrogenase kinase (PDK) inhibition in skeletal muscle increases pyruvate oxidation, which limits the availability of gluconeogenic substrates in the liver. However, this study found upregulation of hepatic PDK4 promoted glucagon-mediated expression of gluconeogenic genes, whereas knockdown or inhibition of hepatic PDK4 caused the opposite effect on gluconeogenic gene expression and decreased hepatic glucose production. Mechanistically, PDK4 deficiency decreased ATP levels, thus increasing phosphorylated AMPK (p-AMPK), which increased p-AMPK-sensitive phosphorylation of cyclic nucleotide phosphodiesterase 4B (p-PDE4B). This reduced cAMP levels and consequently p-CREB. Metabolic flux analysis showed that the reduction in ATP was a consequence of a diminished rate of fatty acid oxidation (FAO). However, overexpression of PDK4 increased FAO and increased ATP levels, which decreased p-AMPK and p-PDE4B and allowed greater accumulation of cAMP and p-CREB. The latter were abrogated by the FAO inhibitor etomoxir, suggesting a critical role for PDK4 in FAO stimulation and the regulation of cAMP levels. This finding strengthens the possibility of PDK4 as a target against diabetes., (© 2018 by the American Diabetes Association.)

Clinical prescription of cisplatin, one of the most widely used chemotherapeutic agents, is limited by its side effects, particularly tubular injury-associated nephrotoxicity. Since details of the underlying mechanisms are not fully understood, we investigated the role of pyruvate dehydrogenase kinase (PDK) in cisplatin-induced acute kidney injury. Among the PDK isoforms, PDK4 mRNA and protein levels were markedly increased in the kidneys of mice treated with cisplatin, and c-Jun N-terminal kinase activation was involved in cisplatin-induced renal PDK4 expression. Treatment with the PDK inhibitor sodium dichloroacetate (DCA) or genetic knockout of PDK4 attenuated the signs of cisplatin-induced acute kidney injury, including apoptotic morphology of the kidney tubules along with numbers of TUNEL-positive cells, cleaved caspase-3, and renal tubular injury markers. Cisplatin-induced suppression of the mitochondrial membrane potential, oxygen consumption rate, expression of electron transport chain components, cytochrome c oxidase activity, and disruption of mitochondrial morphology were noticeably improved in the kidneys of DCA-treated or PDK4 knockout mice. Additionally, levels of the oxidative stress marker 4-hydroxynonenal and mitochondrial reactive oxygen species were attenuated, whereas superoxide dismutase 2 and catalase expression and glutathione synthetase and glutathione levels were recovered in DCA-treated or PDK4 knockout mice. Interestingly, lipid accumulation was considerably attenuated in DCA-treated or PDK4 knockout mice via recovered expression of peroxisome proliferator-activated receptor-α and coactivator PGC-1α, which was accompanied by recovery of mitochondrial biogenesis. Thus, PDK4 mediates cisplatin-induced acute kidney injury, suggesting that PDK4 might be a therapeutic target for attenuating cisplatin-induced acute kidney injury., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Hepatic steatosis is associated with increased insulin resistance and tricarboxylic acid (TCA) cycle flux, but decreased ketogenesis and pyruvate dehydrogenase complex (PDC) flux. This study examined whether hepatic PDC activation by inhibition of pyruvate dehydrogenase kinase 2 (PDK2) ameliorates these metabolic abnormalities. Wild-type mice fed a high-fat diet exhibited hepatic steatosis, insulin resistance, and increased levels of pyruvate, TCA cycle intermediates, and malonyl-CoA but reduced ketogenesis and PDC activity due to PDK2 induction. Hepatic PDC activation by PDK2 inhibition attenuated hepatic steatosis, improved hepatic insulin sensitivity, reduced hepatic glucose production, increased capacity for β-oxidation and ketogenesis, and decreased the capacity for lipogenesis. These results were attributed to altered enzymatic capacities and a reduction in TCA anaplerosis that limited the availability of oxaloacetate for the TCA cycle, which promoted ketogenesis. The current study reports that increasing hepatic PDC activity by inhibition of PDK2 ameliorates hepatic steatosis and insulin sensitivity by regulating TCA cycle anaplerosis and ketogenesis. The findings suggest PDK2 is a potential therapeutic target for nonalcoholic fatty liver disease., (© 2016 by the American Diabetes Association.)

In this study, we investigated the effects of silymarin (SM) on gluconeogenesis during exercise in rats. After 4 weeks of exercise, blood samples, liver, and skeletal muscle tissues were collected, and the levels of triglycerides (TG), lactate, peroxisome proliferator activated receptor gamma (PPARγ), phosphoenol pyruvate carboxykinase (PEPCK), pyruvate dehydrogenase kinase 4 (PDK4), and phosphorylated 5-AMP activated protein kinase (AMPK) were measured. The TG and lactate level of the serum were reduced. In addition, the expression of Akt, PEPCK, and PPARγ in liver was decreased as well as the expression of AMPK in muscle. On the contrary, the level of PDK4 in muscle was increased. These results showed that that administration of SM ameliorated exerciseinduced gluconeogenesis and β-oxidation through the regulation of PPARγ, PEPCK, and PDK4. Thus, intake of SM during exercise may improve endurance by modulating of the metabolism of glucose, lipids, and lactate.

Chronic inflammation augments the deleterious effects of several diseases, particularly diabetes, cancer, and sepsis. It is also involved in the process of metabolic shift from glucose oxidation to lactate production. Although several studies suggest that the change in activity of the pyruvate dehydrogenase complex (PDC) is a major factor causing this metabolic change, the exact mechanism of the inflammatory state remains unclear. In this study, we investigated the effect of lipopolysaccharide (LPS) on the expression of pyruvate dehydrogenase kinase 4 (PDK4), which is strongly associated with inactivation of the PDC in C2C12 myoblasts. In C2C12 myoblasts, LPS exposure led to increased PDK4 mRNA and protein expression levels as well as lactate production in culture medium. However, the expression levels of other PDK isoenzymes (PDK1 - 3) remained unchanged. Additionally, we observed that LPS treatment induced phosphorylation of Jun N-Terminal Kinases (JNK). To confirm the role of JNK, we inhibited the JNK pathway and observed that PDK4 expression and lactate production were decreased, but p38 and ERK were not significantly changed. Taken together, our results suggest that LPS induces PDK4 expression and alters glucose metabolism via the JNK pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pyruvate dehydrogenase kinases (PDK1-4) are mitochondrial metabolic regulators that serve as decision makers via modulation of pyruvate dehydrogenase (PDH) activity to convert pyruvate either aerobically to acetyl-CoA or anaerobically to lactate. Metabolic dysregulation and inflammatory processes are two sides of the same coin in several pathophysiological conditions. The lactic acid surge associated with the metabolic shift has been implicated in diverse painful states. In this study, we investigated the role of PDK-PDH-lactic acid axis in the pathogenesis of chronic inflammatory pain. Deficiency of Pdk2 and/or Pdk4 in mice attenuated complete Freund's adjuvant (CFA)-induced pain hypersensitivities. Likewise, Pdk2/4 deficiency attenuated the localized lactic acid surge along with hallmarks of peripheral and central inflammation following intraplantar administration of CFA. In vitro studies supported the role of PDK2/4 as promoters of classical proinflammatory activation of macrophages. Moreover, the pharmacological inhibition of PDKs or lactic acid production diminished CFA-induced inflammation and pain hypersensitivities. Thus, a PDK-PDH-lactic acid axis seems to mediate inflammation-driven chronic pain, establishing a connection between metabolism and inflammatory pain., Significance Statement: The mitochondrial pyruvate dehydrogenase (PDH) kinases (PDKs) and their substrate PDH orchestrate the conversion of pyruvate either aerobically to acetyl-CoA or anaerobically to lactate. Lactate, the predominant end product of glycolysis, has recently been identified as a signaling molecule for neuron-glia interactions and neuronal plasticity. Pathological metabolic shift and subsequent lactic acid production are thought to play an important role in diverse painful states; however, their contribution to inflammation-driven pain is still to be comprehended. Here, we report that the PDK-PDH-lactic acid axis constitutes a key component of inflammatory pain pathogenesis. Our findings establish an unanticipated link between metabolism and inflammatory pain. This study unlocks a previously ill-explored research avenue for the metabolic control of inflammatory pain pathogenesis., (Copyright © 2015 the authors 0270-6474/15/3514354-17$15.00/0.)

Impaired glucose homeostasis is one of the risk factors for causing metabolic diseases including obesity, type 2 diabetes, and cancers. In glucose metabolism, pyruvate dehydrogenase complex (PDC) mediates a major regulatory step, an irreversible reaction of oxidative decarboxylation of pyruvate to acetyl-CoA. Tight control of PDC is critical because it plays a key role in glucose disposal. PDC activity is tightly regulated using phosphorylation by pyruvate dehydrogenase kinases (PDK1 to 4) and pyruvate dehydrogenase phosphatases (PDP1 and 2). PDKs and PDPs exhibit unique tissue expression patterns, kinetic properties, and sensitivities to regulatory molecules. During the last decades, the up-regulation of PDKs has been observed in the tissues of patients and mammals with metabolic diseases, which suggests that the inhibition of these kinases may have beneficial effects for treating metabolic diseases. This review summarizes the recent advances in the role of specific PDK isoenzymes on the induction of metabolic diseases and describes the effects of PDK inhibition on the prevention of metabolic diseases using pharmacological inhibitors. Based on these reports, PDK isoenzymes are strong therapeutic targets for preventing and treating metabolic diseases.

Excessive proliferation of vascular smooth muscle cells (VSMCs) and incomplete re-endothelialization is a major clinical problem limiting the long-term efficacy of percutaneous coronary angioplasty. We tested if dimethylfumarate (DMF), an anti-psoriasis drug, could inhibit abnormal vascular remodeling via NF-E2-related factor 2 (Nrf2)-NAD(P)H quinone oxidoreductase 1 (NQO1) activity. DMF significantly attenuated neointimal hyperplasia induced by balloon injury in rat carotid arteries via suppression of the G1 to S phase transition resulting from induction of p21 protein in VSMCs. Initially, DMF increased p21 protein stability through an enhancement in Nrf2 activity without an increase in p21 mRNA. Later on, DMF stimulated p21 mRNA expression through a process dependent on p53 activity. However, heme oxygenase-1 (HO-1) or NQO1 activity, well-known target genes induced by Nrf2, were dispensable for the DMF induction of p21 protein and the effect on the VSMC proliferation. Likewise, DMF protected endothelial cells from TNF-α-induced apoptosis and the dysfunction characterized by decreased eNOS expression. With knock-down of Nrf2 or NQO1, DMF failed to prevent TNF-α-induced cell apoptosis and decreased eNOS expression. Also, CD31 expression, an endothelial specific marker, was restored in vivo by DMF. In conclusion, DMF prevented abnormal proliferation in VSMCs by G1 cell cycle arrest via p21 upregulation driven by Nrf2 and p53 activity, and had a beneficial effect on TNF-α-induced apoptosis and dysfunction in endothelial cells through Nrf2-NQO1 activity suggesting that DMF might be a therapeutic drug for patients with vascular disease.

Pyruvate is an obligatory intermediate in the oxidative disposal of glucose and a major precursor for the synthesis of glucose, glycerol, fatty acids, and non-essential amino acids. Stringent control of the fate of pyruvate is critically important for cellular homeostasis. The regulatory mechanisms for its metabolism are therefore of great interest. Recent advances include the findings that (a) the mitochondrial pyruvate carrier is sensitive to inhibition by thiazolidinediones; (b) pyruvate dehydrogenase kinases induce the Warburg effect in many disease states; and (c) pyruvate carboxylase is an important determinate of the rates of gluconeogenesis in humans with type 2 diabetes. These enzymes are potential therapeutic targets for several diseases.

During recovery from glycogen-depleting exercise, there is a shift from carbohydrate oxidation to glycogen resynthesis. The activity of the pyruvate dehydrogenase (PDH) complex may decrease to reduce oxidation of carbohydrates in favor of increasing gluconeogenic recycling of carbohydrate-derived substrates for this process. The precise mechanism behind this has yet to be elucidated; however, research examining mRNA content has suggested that the less-abundant pyruvate dehydrogenase kinase-4 (PDK4) may reduce PDH activation during exercise recovery. To investigate this, skeletal muscle and liver of wild-type (WT) and PDK4-knockout (PDK4-KO) mice were analyzed at rest (Rest), after exercise to exhaustion (Exh), and after 2 h of recovery with ad libitum feeding (Rec). Although there were no differences in exercise tolerance between genotypes, caloric consumption was doubled by PDK4-KO mice during Rec. Because of this, PDK4-KO mice at Rec supercompensated muscle glycogen to 120% of resting stores. Therefore, an extra group of PDK4-KO mice were pair-fed (PF) with WT mice during Rec for comparison. PF mice fully replenished muscle glycogen but recovered only 50% of liver glycogen stores. Concentrations of muscle lactate and alanine were also lower in PF than in WT mice, indicating that this decrease may lead to a potential reduction of recycled gluconeogenic substrates, due to oxidation of their carbohydrate precursors in skeletal muscle, leading to observed reductions in hepatic glucose and glycogen concentrations. Because of the impairments seen in PF PDK4-KO mice, these results suggest a role for PDK4 in regulating the PDH complex in muscle and promoting gluconeogenic precursor recirculation during recovery from exhaustive exercise.

Reactive oxygen species and reactive nitrogen species promote endothelial dysfunction in old age and contribute to the development of cardiovascular diseases such as atherosclerosis, diabetes, and hypertension. α-Lipoic acid was identified as a catalytic agent for oxidative decarboxylation of pyruvate and α-ketoglutarate in 1951, and it has been studied intensively by chemists, biologists, and clinicians who have been interested in its role in energetic metabolism and protection from reactive oxygen species-induced mitochondrial dysfunction. Consequently, many biological effects of α-lipoic acid supplementation can be attributed to the potent antioxidant properties of α-lipoic acid and dihydro α-lipoic acid. The reducing environments inside the cell help to protect from oxidative damage and the reduction-oxidation status of α-lipoic acid is dependent upon the degree to which the cellular components are found in the oxidized state. Although healthy young humans can synthesize enough α-lipoic acid to scavenge reactive oxygen species and enhance endogenous antioxidants like glutathione and vitamins C and E, the level of α-lipoic acid significantly declines with age and this may lead to endothelial dysfunction. Furthermore, many studies have reported α-lipoic acid can regulate the transcription of genes associated with anti-oxidant and anti-inflammatory pathways. In this review, we will discuss recent clinical studies that have investigated the beneficial effects of α-lipoic acid on endothelial dysfunction and propose possible mechanisms involved.

The excessive accumulation of adipocytes contributes to the development of obesity and obesity-related diseases. The interactions of several transcription factors, such as C/EBPβ, PPARγ, C/EBPα, Nrf2, and STAT3, are required for adipogenic differentiation. Dimethylfumarate (DMF), an immune modulator and antioxidant, may function as an inhibitor of STAT3 and an activator of Nrf2. This study examined whether DMF inhibits adipogenic differentiation of 3T3-L1 preadipocytes by inhibiting STAT3 or activating Nrf2. DMF suppressed 3T3-L1 preadipocyte differentiation to mature adipocytes in a dose-dependent manner as determined by Oil Red O staining. The mRNA and protein levels of adipogenic genes, including C/EBPβ, C/EBPα, PPARγ, SREBP-1c, FAS, and aP2, were significantly lower in DMF-treated 3T3-L1 preadipocytes. Suppression of adipogenic differentiation by DMF treatment resulted primarily from inhibition of the early stages of differentiation. DMF inhibits clonal expansion during adipogenic differentiation through induction of a G1 cell cycle arrest. Additionally, DMF regulates cell cycle-related proteins, such as p21, pRb, and cyclin D. DMF treatment markedly inhibited differentiation medium-induced STAT3 phosphorylation and inhibited STAT3 transcriptional activation of a reporter construct composed of four synthetic STAT3-response elements. Moreover, inhibition of endogenous Nrf2 activity using a dominant negative Nrf2 did not abolish the DMF-induced inhibition of adipogenic differentiation of 3T3-L1 preadipocytes. In summary, DMF is a negative regulator of adipogenic differentiation based on its regulation of adipogenic transcription factors and cell cycle proteins. This negative regulation by DMF is mediated by STAT3 inhibition, but is unlikely to involve Nrf2 activation.

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.

The importance of PDHK (pyruvate dehydrogenase kinase) 2 and 4 in regulation of the PDH complex (pyruvate dehydrogenase complex) was assessed in single- and double-knockout mice. PDHK2 deficiency caused higher PDH complex activity and lower blood glucose levels in the fed, but not the fasted, state. PDHK4 deficiency caused similar effects, but only after fasting. Double deficiency intensified these effects in both the fed and fasted states. PDHK2 deficiency had no effect on glucose tolerance, PDHK4 deficiency produced only a modest effect, but double deficiency caused a marked improvement and also induced lower insulin levels and increased insulin sensitivity. In spite of these beneficial effects, the double-knockout mice were more sensitive than wild-type and single-knockout mice to long-term fasting, succumbing to hypoglycaemia, ketoacidosis and hypothermia. Stable isotope flux analysis indicated that hypoglycaemia was due to a reduced rate of gluconeogenesis and that slightly more glucose was converted into ketone bodies in the double-knockout mice. The findings establish that PDHK2 is more important in the fed state, PDHK4 is more important in the fasted state, and survival during long-term fasting depends upon regulation of the PDH complex by both PDHK2 and PDHK4.

NADPH oxidase (NOX) is a predominant source of reactive oxygen species (ROS), and the activity of NOX, which uses NADPH as a common rate-limiting substrate, is upregulated by prolonged dietary salt intake. β-Lapachone (βL), a well-known substrate of NAD(P)H:quinone oxidoreductase 1 (NQO1), decreases the cellular NAD(P)H/NAD(P)(+) ratio via activation of NQO1. In this study, we evaluated whether NQO1 activation by βL modulates salt-induced renal injury associated with NOX-derived ROS regulation in an animal model. Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet were used to investigate the renoprotective effect of NQO1 activation. βL treatment significantly lowered the cellular NAD(P)H:NAD(P)(+) ratio and dramatically reduced NOX activity in the kidneys of HS diet-fed DS rats. In accordance with this, total ROS production and expression of oxidative adducts also decreased in the βL-treated group. Furthermore, HS diet-induced proteinuria and glomerular damage were markedly suppressed, and inflammation, fibrosis, and apoptotic cell death were significantly diminished by βL treatment. This study is the first to demonstrate that activation of NQO1 has a renoprotective effect that is mediated by NOX activity via modulation of the cellular NAD(P)H:NAD(P)(+) ratio. These results provide strong evidence that NQO1 might be a new therapeutic target for the prevention of salt-induced renal injury., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aims: Pyruvate dehydrogenase kinase 4 (PDK4) plays a crucial role in glucose utilization and lipid metabolism by regulating the pyruvate dehydrogenase complex (PDC) and is an emerging therapeutic target for type 2 diabetes. To date, no study has specifically examined the relationship between PDK4 gene polymorphisms and type 2 diabetes or metabolic syndrome., Methods: The association of common single nucleotide polymorphisms (SNPs) was examined in PDK4 [-208A/G (rs10085637), IVS3+192C/T (rs3779478), IVS6+31A/G (rs2301630), IVS7+514A/G (rs12668651), IVS10+75C/T (rs10247649)] with type 2 diabetes and metabolic syndrome in 651 Korean subjects with type 2 diabetes and 350 nondiabetic Korean subjects. The association of these SNPs with clinical parameters related to metabolic syndromes including obesity, hyperglycemia, hypertension, and dyslipidemia was also examined., Results: No significant association was found between the studied SNPs and type 2 diabetes, metabolic syndrome, or clinical parameters. The PDK4 gene haplotype ACAGC showed a modest association with type 2 diabetes. However, the significance of this association was lost after considering for multiple comparisons., Conclusions: PDK4 polymorphisms may not be associated with type 2 diabetes or metabolic syndrome. Further studies utilizing a larger study population are required to confirm these results., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The oxidation of carbohydrates in mammals is regulated by the pyruvate dehydrogenase (PDH) complex, which is covalently regulated by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) unique to the PDH complex. To investigate the role that PDK4 plays in regulating PDH activation (PDHa) during muscle contraction, mouse extensor digitorum muscle was removed from wild type (WT) and PDK4-knockout (PDK4-KO) mice after a 24 h fast and stimulated for 3 min either at 10 Hz (low-intensity contraction), 40 Hz (moderate-intensity contraction), or allowed to rest. Force was recorded and muscle PDHa activity and metabolite concentrations were measured. PDHa activity was ∼2.5-fold higher at rest in PDK4-KO mice than WT mice (P = 0.009) and ∼2-fold higher in PDK4-KO mice at both 10 Hz (P < 0.001) and 40 Hz (P < 0.001). Force relative to muscle weight was similar at 10 Hz, but was 5.8 ± 0.7 mN·g(-1) in PDK4-KO mice and 3.5 ± 0.7 mN·g(-1) in WT mice at 40 Hz (P < 0.001), with a similar rate of fatigue in both genotypes. From these results it was concluded that PDK4 plays a role in reducing PDHa activity during low to moderate-intensity muscle stimulation, and that absence of PDK4 and the subsequent changes in carbohydrate utilization may alter force production.

Diabetes is a well-known independent risk factor for vascular disease. However, its underlying mechanism remains unclear. It has been reported that increased influx of the hexosamine biosynthesis pathway (HBP) induces O-GlcNAcylation of proteins, leading to insulin resistance. In this study, we determined whether or not O-GlcNAc modification of proteins could increase vessel contraction. Using an endothelium-denuded aortic ring, we observed that glucosamine induced OGlcNAcylation of proteins and augmented vessel contraction stimulated by U46619, a thromboxane A(2) agonist, via augmentation of the phosphorylation of MLC(20), MYPT1(Thr855), and CPI17, but not phenylephrine. Pretreatment with OGT inhibitor significantly ameliorated glucosamine-induced vessel constriction. Glucosamine treatment also increased RhoA activity, which was also attenuated by OGT inhibitor. In conclusion, glucosamine, a product of glucose influx via the HBP in a diabetic state, increases vascular contraction, at least in part, through activation of the RhoA/Rho kinase pathway, which may be due to O-GlcNAcylation.

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PDH is deactivated by a set of PDH kinases (PDK1, PDK2, PDK3, PDK4), with PDK2 and PDK4 being the most predominant isoforms in skeletal muscle. Although PDK2 is the most abundant isoform, few studies have examined its physiological role. The role of PDK2 on PDH activation (PDHa) at rest and during muscle stimulation at 10 and 40 Hz (eliciting low- and moderate-intensity muscle contractions, respectively) in isolated extensor digitorum longus muscles was studied in PDK2 knockout (PDK2KO) and wild-type (WT) mice (n = 5 per group). PDHa activity was unexpectedly 35 and 77% lower in PDK2KO than WT muscle (P = 0.043), while total PDK activity was nearly fourfold lower in PDK2KO muscle (P = 0.006). During 40-Hz contractions, initial force was lower in PDK2KO than WT muscle (P < 0.001) but fatigued similarly to ∼75% of initial force by 3 min. There were no differences in initial force or rate of fatigue during 10-Hz contractions. PDK1 compensated for the lack of PDK2 and was 1.8-fold higher in PDK2KO than WT muscle (P = 0.019). This likely contributed to ensuring that resting PDHa activity was similar between the groups and accounts for the lower PDH activation during muscle contraction, as PDK1 is a very potent inhibitor of the PDH complex. Increased PDK1 expression appears to be regulated by hypoxia inducible factor-1α, which was 3.5-fold higher in PDK2KO muscle. It is clear that PDK2 activity is essential, even at rest, in regulation of carbohydrate oxidation and production of reducing equivalents for the electron transport chain. In addition, these results underscore the importance of the overall kinetics of the PDK isoform population, rather than total PDK activity, in determining transformation of the PDH complex and PDHa activity during muscle contraction.

Long-term consumption of artificial sweeteners (AS) has been the recent focus of safety concerns. However, the potential risk of the AS in cardiovascular disease and lipoprotein metabolism has not been investigated sufficiently. We compared the influence of AS (aspartame, acesulfame K, and saccharin) and fructose in terms of functional and structural correlations of apolipoprotein (apo) A-I and high-density lipoproteins (HDL), which have atheroprotective effects. Long-term treatment of apoA-I with the sweetener at physiological concentration (3 mM for 168 h) resulted in loss of antioxidant and phospholipid binding activities with modification of secondary structure. The AS treated apoA-I exhibited proteolytic cleavage to produce 26 kDa-fragment. They showed pro-atherogenic properties in acetylated LDL phagocytosis of macrophages. Each sweetener alone or sweetener-treated apoA-I caused accelerated senescence in human dermal fibroblasts. These results suggest that long-term consumption of AS might accelerate atherosclerosis and senescence via impairment of function and structure of apoA-I and HDL.

Background/aims: Papillary thyroid cancer (PTC) is the most common malignancy of the thyroid gland. It involves several molecular mechanisms. The BRAF V600E mutation has been identified as the most common genetic abnormality in PTC. Moreover, it is known to be more prevalent in Korean PTC patients than in patients from other countries. We investigated distinct genetic profiles in Korean PTC through cDNA microarray analysis., Methods: Transcriptional profiles of five PTC samples and five paired normal thyroid tissue samples were generated using cDNA microarrays. The tumors were genotyped for BRAF mutations. The results of the cDNA microarray gene expression analysis were confirmed by real-time PCR and immunohistochemistry analysis of 35 PTC patients., Results: Four of the five patients whose PTC tissues were subjected to microarray analysis were found to carry the BRAF V600E mutation. Microarrays analysis of the five PTC tissue samples showed the expression of 96 genes to be increased and that of 16 genes decreased. Real-time reverse transcription-polymerase chain reaction (RT-PCR) confirmed increased expression of SLC34A2, TM7SF4, COMP, KLK7, and KCNJ2 and decreased expression of FOXA2, SLC4A4, LYVE-1, and TFCP2L1 in PTC compared with normal tissue. Of these genes, TFCP2L1, LYVE-1, and KLK7 were previously unidentified in PTC microarray analysis. Notably, Foxa2 activity in PTC was reduced, as shown by its cytoplasmic localization, in immunohistochemical analyses., Conclusions: These findings demonstrate both similarities and differences between our results and previous reports. In Korean cases of PTC, Foxa2 activity was reduced with its cytoplasmic accumulation. Further studies are needed to confirm the relationship between FOXA2 and BRAF mutations in Korean cases of PTC.

In the well-fed state a relatively high activity of the pyruvate dehydrogenase complex (PDC) reduces blood glucose levels by directing the carbon of pyruvate into the citric acid cycle. In the fasted state a relatively low activity of the PDC helps maintain blood glucose levels by conserving pyruvate and other three carbon compounds for gluconeogenesis. The relative activities of the pyruvate dehydrogenase kinases (PDKs) and the opposing pyruvate dehydrogenase phosphatases determine the activity of PDC in the fed and fasted states. Up regulation of PDK4 is largely responsible for inactivation of PDC in the fasted state. PDK4 knockout mice have lower fasting blood glucose levels than wild type mice, proving that up regulation of PDK4 is important for normal glucose homeostasis. In type 2 diabetes, up regulation of PDK4 also inactivates PDC, which promotes gluconeogenesis and thereby contributes to the hyperglycemia characteristic of this disease. When fed a high fat diet, wild type mice develop fasting hyperglycemia but PDK4 knockout mice remain euglycemic, proving that up regulation of PDK4 contributes to hyperglycemia in diabetes. These finding suggest PDK4 inhibitors might prove useful in the treatment of type 2 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 versus 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 (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha 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., ((c) 2010 Wiley-Liss, Inc.)

O-Linked β-N-acetyl glucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification that occurs on serine and threonine residues of cytosolic and nuclear proteins in all cell types, including those involved in the cardiovascular system. O-GlcNAcylation is thought to act in a manner analogous to protein phosphorylation. O-GlcNAcylation rapidly cycles on/off proteins in a time scale similar to that for phosphorylation/dephosphorylation of proteins. Several studies indicate that O-GlcNAc might induce nuclear localization of some transcription factors and may affect their DNA binding activities. However, at the cellular level, it has been shown that O-GlcNAc levels increase in response to stress and augmentation of this response suppresses cell survival. Increased levels of O-GlcNAc have been implicated as a pathogenic contributor to glucose toxicity and insulin resistance, which are major hallmarks of type 2 diabetes and diabetes-related cardiovascular complications. Thus, O-GlcNAc and its metabolic functions are not yet well-understood; focusing on the role of O-GlcNAc in the cardiovascular system is a viable target for biomedical investigation. In this review, we summarize our current understanding of the role of O-GlcNAc on the regulation of cell function and survival in the cardiovascular system.

Background & Aims: Liver ischemia/reperfusion (I/R) injury is a frequent cause of organ dysfunction. Loss of the oxygen sensor prolyl hydroxylase domain enzyme 1 (PHD1) causes tolerance of skeletal muscle to hypoxia. We assessed whether loss or short-term silencing of PHD1 could likewise induce hypoxia tolerance in hepatocytes and protect them against hepatic I/R damage., Methods: Hepatic ischemia was induced in mice by clamping of the portal vessels of the left lateral liver lobe; 90 minutes later livers were reperfused for 8 hours for I/R experiments. Hepatocyte damage following ischemia or I/R was investigated in PHD1-deficient (PHD1(-/-)) and wild-type mice or following short hairpin RNA-mediated short-term inhibition of PHD1 in vivo., Results: PHD1(-/-) livers were largely protected against acute ischemia or I/R injury. Among mice subjected to hepatic I/R followed by surgical resection of all nonischemic liver lobes, more than half of wild-type mice succumbed, whereas all PHD1(-/-) mice survived. Also, short-term inhibition of PHD1 through RNA interference-mediated silencing provided protection against I/R. Knockdown of PHD1 also induced hypoxia tolerance of hepatocytes in vitro. Mechanistically, loss of PHD1 decreased production of oxidative stress, which likely relates to a decrease in oxygen consumption as a result of a reprogramming of hepatocellular metabolism., Conclusions: Loss of PHD1 provided tolerance of hepatocytes to acute hypoxia and protected them against I/R-damage. Short-term inhibition of PHD1 is a novel therapeutic approach to reducing or preventing I/R-induced liver injury., (Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective: Clusterin is induced in vascular smooth muscle cells (VSMCs) during atherosclerosis and injury-induced neointimal hyperplasia. However, its functional roles in VSMCs and endothelial cells remain controversial and elusive. This study was undertaken to clarify the role of clusterin in neointimal hyperplasia and elucidate its mechanism of action., Methods and Results: Adenovirus-mediated overexpression of clusterin (Ad-Clu) repressed TNF-alpha-stimulated expression of MCP-1, fractalkine, ICAM-1, VCAM-1, and MMP-9, leading to inhibition of VSMC migration. Both Ad-Clu and secreted clusterin suppressed VSMC proliferation by inhibiting DNA synthesis, but not by inducing apoptosis. Ad-Clu upregulated p53 and p21(cip1/waf1) but downregulated cyclins D and E, leading to suppression of pRb phosphorylation and subsequent induction of G1 arrest in VSMCs. Clusterin deficiency augmented VSMC proliferation in vitro and accelerated neointimal hyperplasia in vivo, but concomitantly impaired reendothelialization in wire-injured murine femoral arteries. Moreover, Ad-Clu significantly reduced neointimal thickening in balloon-injured rat carotid arteries. Clusterin also diminished TNF-alpha-induced apoptosis of human umbilical vein endothelial cells and restored endothelial nitric oxide synthase expression suppressed by TNF-alpha., Conclusions: These results suggest that upregulation of clusterin during vascular injury may be a protective response against, rather than a causative response to, the development of neointimal hyperplasia.

The hypothesis that PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) has potential as a target for the treatment of type 2 diabetes was tested by feeding wild-type and PDHK4 knockout mice a high saturated fat diet that induces hyperglycemia, hyperinsulinaemia, glucose intolerance, hepatic steatosis and obesity. Previous studies have shown that PDHK4 deficiency lowers blood glucose by limiting the supply of three carbon gluconeogenic substrates to the liver. There is concern, however, that the increase in glucose oxidation caused by less inhibition of the pyruvate dehydrogenase complex by phosphorylation will inhibit fatty acid oxidation, promote ectopic fat accumulation and worsen insulin sensitivity. This was examined by feeding wild-type and PDHK4 knockout mice a high saturated fat diet for 8 months. Fasting blood glucose levels increased gradually in both groups but remained significantly lower in the PDHK4 knockout mice. Hyperinsulinaemia developed in both groups, but glucose tolerance was better and body weight was lower in the PDHK4 knockout mice. At termination, less fat was present in the liver and skeletal muscle of the PDHK4 knockout mice. Higher amounts of PGC-1alpha [PPARgamma (peroxisome proliferator-activated receptor gamma) coactivator 1alpha] and PPARalpha and lower amounts of fatty acid synthase and acetyl-CoA carboxylase isoenzyme 1 were present in the liver of the PDHK4 knockout mice. These findings suggest PDHK4 deficiency creates conditions that alter upstream signalling components involved in the regulation of lipid metabolism. The findings support the hypothesis that PDHK4 is a viable target for the treatment of type 2 diabetes.

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 approximately 40% 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.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are important pathogenic mechanisms in atherosclerosis and restenosis after vascular injury. In this study, we investigated the effects of beta-lapachone (betaL) (3,4-Dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), which is a potent antitumor agent that stimulates NAD(P)H:quinone oxidoreductase (NQO)1 activity, on neointimal formation in animals given vascular injury and on the proliferation of VSMCs cultured in vitro. betaL significantly reduced the neointimal formation induced by balloon injury. betaL also dose-dependently inhibited the FCS- or platelet-derived growth factor-induced proliferation of VSMCs by inhibiting G(1)/S phase transition. betaL increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase 1 in rat and human VSMCs. Chemical inhibitors of AMPK or dominant-negative AMPK blocked the betaL-induced suppression of cell proliferation and the G(1) cell cycle arrest, in vitro and in vivo. The activation of AMPK in VSMCs by betaL is mediated by LKB1 in the presence of NQO1. Taken together, these results show that betaL inhibits VSMCs proliferation via the NQO1 and LKB1-dependent activation of AMPK. These observations provide the molecular basis that pharmacological stimulation of NQO1 activity is a new therapy for the treatment of vascular restenosis and/or atherosclerosis which are caused by proliferation of VSMCs.

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 alpha (PDHalpha) subunit. We also demonstrate that PDC inhibition in cancer cells is associated with normoxic stabilization of the malignancy-promoting transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) by glycolytic metabolites. Knockdown of PDK-1 via short hairpin RNA lowers PDHalpha phosphorylation, restores PDC activity, reverts the Warburg metabolic phenotype, decreases normoxic HIF-1alpha 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.

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(-/-) 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(-/-) 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(-/-) 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(-/-) mice compared with wild-type mice. The concentrations of gluconeogenic substrates were lower in the blood of PDK4(-/-) mice compared with wild-type mice, consistent with reduced formation in peripheral tissues. Diaphragms isolated from PDK4(-/-) 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(-/-) mice. NEFA, ketone bodies, and branched-chain amino acids were elevated more in PDK4(-/-) 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.

The heart adapts to changes in nutritional status and energy demands by adjusting its relative metabolism of carbohydrates and fatty acids. Loss of this metabolic flexibility such as occurs in diabetes mellitus is associated with cardiovascular disease and heart failure. To study the long-term consequences of impaired metabolic flexibility, we have generated mice that overexpress pyruvate dehydrogenase kinase (PDK)4 selectively in the heart. Hearts from PDK4 transgenic mice have a marked decrease in glucose oxidation and a corresponding increase in fatty acid catabolism. Although no overt cardiomyopathy was observed in the PDK4 transgenic mice, introduction of the PDK4 transgene into mice expressing a constitutively active form of the phosphatase calcineurin, which causes cardiac hypertrophy, caused cardiomyocyte fibrosis and a striking increase in mortality. These results demonstrate that cardiac-specific overexpression of PDK4 is sufficient to cause a loss of metabolic flexibility that exacerbates cardiomyopathy caused by the calcineurin stress-activated pathway.

Livers from mice lacking the carbohydrate-responsive element-binding protein (ChREBP) were compared with wild type (WT) mice to determine the effect of this transcription factor on hepatic energy metabolism. The pyruvate dehydrogenase complex was considerably more active in ChREBP(-/-) mice because of diminished pyruvate dehydrogenase kinase activity. Greater pyruvate dehydrogenase complex activity caused a stimulation of lactate and pyruvate oxidation, and it significantly impaired fatty acid oxidation in perfused livers from ChREBP(-/-) mice. This shift in mitochondrial substrate utilization led to a 3-fold reduction of the free cytosolic [NAD(+)]/[NADH] ratio, a 1.7-fold increase in the free mitochondrial [NAD(+)]/[NADH] ratio, and a 2-fold decrease in the free cytosolic [ATP]/[ADP][P(i)] ratio in the ChREBP(-/-) liver compared with control. Hepatic pyruvate carboxylase flux was impaired with ChREBP deletion secondary to decreased fatty acid oxidation, increased pyruvate oxidation, and limited pyruvate availability because of reduced activity of liver pyruvate kinase and malic enzyme, which replenish pyruvate via glycolysis and pyruvate cycling. Overall, the shift from fat utilization to pyruvate and lactate utilization resulted in a decrease in the energy of ATP hydrolysis and a hypo-energetic state in the livers of ChREBP(-/-) mice.

A novel phosphatase has been cloned and partially characterized. It has a mitochondrial leader sequence and its amino acid sequence places it in the PP2C family like two known mitochondrial phosphatases. Western blot analysis of subcellular fractions and confocal microscopy of 3T3L1 preadipocytes expressing the GFP-tagged protein confirm its mitochondrial localization. Western blot analysis indicates that the protein is expressed in several mouse tissues, with highest expression in brain, heart, liver, and kidney. The recombinant protein exhibits Mn(2+)-dependent phosphoserine phosphatase activity against the branched-chain alpha-keto acid dehydrogenase complex, suggesting the enzyme may play a role in regulation of branched chain amino acid catabolism. Whether there are other mitochondrial substrates for the enzyme is not known.

Changes in the expression level of the skeletal muscle LIM protein 1 (SLIM1) in cultured A10 cells were monitored in response to 25-hydroxycholesterol (25-HC), an oxidized form of cholesterol present in the oxidized low-density lipoproteins. The level of SLIM1 mRNA was elevated in a time- and concentration-dependent manner by treatment of 25-HC. Expressions of smooth muscle (SM) alpha-actin and calponin-1 (CNN-1), early markers for SMC differentiation, were also increased by the 25-HC treatments. Expressions of all three genes (SLIM1, SM alpha-actin and CNN-1) were simultaneously elevated in the cells treated with 9-cis retinoic acid (RA). On the other hand, the SLIM1 expression induced by the 25-HC or 9-cis RA (as well as SM alpha-actin and CNN-1) was decreased by the treatment of 15d-PGJ2. Since the 25-HC, 9-cis RA and 15d-PGJ2 were ligands for the LXR, RXRalpha and PPARgamma respectively, there might be a functional positive cross-talk between LXR and RXRalpha pathways and a negative cross-talk between PPARgamma and LXR and/or RXRalpha pathways in the regulation of SLIM1 expression. The cells stably transfected with the expressional vector for SLIM1 also showed an elevation in the levels of SM alpha-actin and CNN-1. In addition, an over-production of SLIM1 in the cells resulted in a change in the cell-shape into a spindle-like form, which is identical to that observed after a prolonged treatment of the cells with cholesterol.

The BCKDH (branched-chain alpha-keto acid dehydrogenase complex) catalyses the rate-limiting step in the oxidation of BCAAs (branched-chain amino acids). Activity of the complex is regulated by a specific kinase, BDK (BCKDH kinase), which causes inactivation, and a phosphatase, BDP (BCKDH phosphatase), which causes activation. In the present study, the effect of the disruption of the BDK gene on growth and development of mice was investigated. BCKDH activity was much greater in most tissues of BDK-/- mice. This occurred in part because the E1 component of the complex cannot be phosphorylated due to the absence of BDK and also because greater than normal amounts of the E1 component were present in tissues of BDK-/- mice. Lack of control of BCKDH activity resulted in markedly lower blood and tissue levels of the BCAAs in BDK-/- mice. At 12 weeks of age, BDK-/- mice were 15% smaller than wild-type mice and their fur lacked normal lustre. Brain, muscle and adipose tissue weights were reduced, whereas weights of the liver and kidney were greater. Neurological abnormalities were apparent by hind limb flexion throughout life and epileptic seizures after 6-7 months of age. Inhibition of protein synthesis in the brain due to hyperphosphorylation of eIF2alpha (eukaryotic translation initiation factor 2alpha) might contribute to the neurological abnormalities seen in BDK-/- mice. BDK-/- mice show significant improvement in growth and appearance when fed a high protein diet, suggesting that higher amounts of dietary BCAA can partially compensate for increased oxidation in BDK-/- mice. Disruption of the BDK gene establishes that regulation of BCKDH by phosphorylation is critically important for the regulation of oxidative disposal of BCAAs. The phenotype of the BDK-/- mice demonstrates the importance of tight regulation of oxidative disposal of BCAAs for normal growth and neurological function.

The activity of the pyruvate dehydrogenase complex has long been determined in some laboratories by coupling the production of acetyl-coenzyme A (acetyl-CoA) to the acetylation of 4-aminoazobenzene-4'-sulfonic acid by arylamine N-acetyltransferase. The assay has some advantages, but its use has been limited by the need for large amounts of arylamine N-acetyltransferase. Here we report production of recombinant chicken liver arylamine N-acetyltransferase and optimization of its use in miniaturized assays for the pyruvate dehydrogenase complex and its kinase.

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.

The branched-chain amino acids (BCAAs) are required for protein synthesis and neurotransmitter synthesis. The branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is the most important regulatory enzyme in the catabolic pathways of the BCAAs. Activity of the complex is controlled by covalent modification with phosphorylation of its branched-chain alpha-ketoacid dehydrogenase subunits by a specific kinase [branched-chain kinase (BDK)] causing inactivation and dephosphorylation by a specific phosphatase [branched-chain phosphatase (BDP)] causing activation. Tight control of BCKDC activity is important for conserving as well as disposing of BCAAs. Phosphorylation of the complex occurs when there is a need to conserve BCAAs for protein synthesis; dephosphorylation occurs when BCAAs are present in excess. The relative activities of BDK and BDP set the activity state of BCKDC. BDK activity is regulated by alpha-ketoisocaproate inhibition and altered level of expression. Less is known about BDP but a novel mitochondrial phosphatase was identified recently that may contribute to the regulation of BCKDC. Reduced capacity to oxidize BCAAs, as in maple syrup urine disease, results in excess BCAAs in the blood and profound neurological dysfunction and brain damage. In contrast, loss of control of BCAA oxidation results in growth impairment and epileptic-like seizures. These findings emphasize the importance of control of BCAA catabolism for normal neurological function. It is proposed that the safe upper limit of dietary BCAA intake could be established with a BCAA tolerance test and clamp protocol.

Genes induced or suppressed by oxidized low-density lipoproteins (oxLDL) in human monocytic THP-1 cells were searched using the differential display reverse transcriptase polymerase chain reaction. One of the differentially expressed (up-regulated) cDNA fragments was found to contain sequences corresponding to monocyte chemotactic protein-3 (MCP-3). The stimulatory effect of the oxLDL on the expression of MCP-3 mRNA was both time- and dose-dependent. Treatment with GF109203X and genistein, inhibitors of protein kinase C and tyrosine kinase, respectively, had no effect on the induction of MCP-3 mRNA by oxLDL, while treatment with cycloheximide inhibited the induction. The induction was reproduced by the lipid components in oxLDL such as 9-HODE and 13-HODE, which are known to activate the peroxisome proliferator-activated receptor gamma (PPARgamma). Introduction of an endogenous PPARgamma ligand, 15d-PGJ2, in the culture of THP-1 cells resulted in the induction of MCP-3 gene expression. Furthermore, analyses of human atherosclerotic plaques revealed that the expressional pattern of MCP-3 in the regions of neointimal and necrotic core overlapped with that of PPARgamma. These results suggest that oxLDL delivers its signal for MCP-3 expression via PPARgamma, which may be further related to the atherogenesis.

A diurnal rhythm occurs in the activity state of branched-chain alpha-keto acid dehydrogenase complex (BCKDC) in female but not male rats. We attempted to determine the role played by ovarian hormones in this difference in enzyme regulation. A series of experiments examined the effects of the 4-d estrous cycle, ovariectomy, and replacement of female sex steroids on the catabolism of BCAAs. A proestrous decrease in the activity state of the complex corresponded to an increase in the plasma 17beta-estradiol level. Withdrawal of gonadal steroids by ovariectomy resulted in an increase in the activity state of BCKDC and a decrease in the activity of the branched-chain alpha-keto acid dehydrogenase kinase (BDK). However, 17beta-estradiol reversed these effects, resulting in an increase in the BDK activity, thereby decreasing the activity of the complex. Progesterone administration was ineffective. The changes in the percentage of active BCKDC caused by 17beta-estradiol withdrawal and replacement resulted from changes in the amount of BDK protein associated with the complex and therefore its activity. Thus, the marked diurnal variation in the activity state of BCKDC exhibited by female rats involves estrogenic control of BDK activity. We hypothesize that the 17beta-estradiol-controlled feeding pattern produces these variations in BCKDC activity. This may function in female rats to conserve essential amino acids for protein synthesis.

The branched-chain amino acids (BCAAs) are essential amino acids and therefore must be continuously available for protein synthesis. However, BCAAs are toxic at high concentrations as evidenced by maple syrup urine disease (MSUD), which explains why animals have such an efficient oxidative mechanism for their disposal. Nevertheless, it is clear that leucine is special among the BCAAs. Leucine promotes global protein synthesis by signaling an increase in translation, promotes insulin release, and inhibits autophagic protein degradation. However, leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway, thereby terminating its positive effects on body protein accretion. A strong case can therefore be made that the proper leucine concentration in the various compartments of the body is critically important for maintaining body protein levels beyond simply the need of this essential amino acid for protein synthesis. The goal of the work of this laboratory is to establish the importance of regulation of the branched chain alpha-ketoacid dehydrogenase complex (BCKDC) to growth and maintenance of body protein. We hypothesize that proper regulation of the activity state of BCKDC by way of its kinase (BDK) and its phosphatase (BDP) is critically important for body growth, tissue repair, and maintenance of body protein. We believe that growth and protection of body protein during illness and stress will be improved by therapeutic control of BCKDC activity. We also believe that it is possible that the negative effects of some drugs (PPAR alpha ligands) and dietary supplements (medium chain fatty acids) on growth and body protein maintenance can be countered by therapeutic control of BCDKC activity.