Pyruvate Dehydrogenase Kinase Isoenzyme Research Articles

Stevioside Ameliorates Palmitic Acid-Induced Abnormal Glucose Uptake via the PDK4/AMPK/TBC1D1 Pathway in C2C12 Myotubes.

Stevioside (SV) with minimal calories is widely used as a natural sweetener in beverages due to its high sweetness and safety. However, the effects of SV on glucose uptake and the pyruvate dehydrogenase kinase isoenzyme (PDK4) as an important protein in the regulation of glucose metabolism, remain largely unexplored. In this study, we used C2C12 skeletal muscle cells that was induced by palmitic acid (PA) to assess the effects and mechanisms of SV on glucose uptake and PDK4. The glucose uptake of C2C12 cells was determined by 2-NBDG; expression of the Pdk4 gene was measured by quantitative real-time PCR; and expression of the proteins PDK4, p-AMPK, TBC1D1 and GLUT4 was assessed by Western blotting. In PA-induced C2C12 myotubes, SV could significantly promote cellular glucose uptake by decreasing PDK4 levels and increasing p-AMPK and TBC1D1 levels. SV could promote the translocation of GLUT4 from the cytoplasm to the cell membrane in cells. Moreover, in Pdk4-overexpressing C2C12 myotubes, SV decreased the level of PDK4 and increased the levels of p-AMPK and TBC1D1. SV was found to ameliorate PA-induced abnormal glucose uptake via the PDK4/AMPK/TBC1D1 pathway in C2C12 myotubes. Although these results warranted further investigation for validation, they may provide some evidence of SV as a safe natural sweetener for its use in sugar-free beverages to prevent and control T2DM.

Changes in the Expression of Pyruvate Dehydrogenase Kinase Isoenzymes During Early Differentiation of Mouse Embryonic Stem Cells

The embryonic stem cells (ESCs) are pluripotent, self-renewing cells that able to differentiate into any of the germ layers involved in embryogenesis. However, the molecular mechanisms that control ESC pluripotency and differentiation remain poorly understood. The family of Pyruvate dehydrogenase kinase (PDK1-4), inactivates the mitochondrial pyruvate dehydrogenase complex via phosphorylation, plays a crucial role in the control of glucose homeostasis. In the current study, gene expression levels of PDK isoenzymes were analyzed on undifferentiated mouse embryonic stem cells (mESCs) and compared to mESCs induced to differentiate by removal of leukemia inhibitory factor (LIF) for 1, 3 and 5 days. Besides, we performed gene expression analysis of several genes related to pluripotency and differentiation. In addition, we also determined glucose uptake rates by a colorimetric assay kit in early differentiated and undifferentiated mESCs. Differently expression level of PDK isoenzymes in pluripotent and LIF-depleted mESCs suggest that they may have roles in differentiation and pluripotency of ESCs. Furthermore, this study lays the foundation for detailed investigation of molecular mechanisms underlying the roles of PDKs in the pluripotency and transition to differentiated state of ESCs.

Suppression of Pyruvate Dehydrogenase Kinase by Dichloroacetate in Cancer and Skeletal Muscle Cells Is Isoform Specific and Partially Independent of HIF-1α.

Inhibition of pyruvate dehydrogenase kinase (PDK) emerged as a potential strategy for treatment of cancer and metabolic disorders. Dichloroacetate (DCA), a prototypical PDK inhibitor, reduces the abundance of some PDK isoenzymes. However, the underlying mechanisms are not fully characterized and may differ across cell types. We determined that DCA reduced the abundance of PDK1 in breast (MDA-MB-231) and prostate (PC-3) cancer cells, while it suppressed both PDK1 and PDK2 in skeletal muscle cells (L6 myotubes). The DCA-induced PDK1 suppression was partially dependent on hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of PDK1, in cancer cells but not in L6 myotubes. However, the DCA-induced alterations in the mRNA and the protein levels of PDK1 and/or PDK2 did not always occur in parallel, implicating a role for post-transcriptional mechanisms. DCA did not inhibit the mTOR signaling, while inhibitors of the proteasome or gene silencing of mitochondrial proteases CLPP and AFG3L2 did not prevent the DCA-induced reduction of the PDK1 protein levels. Collectively, our results suggest that DCA reduces the abundance of PDK in an isoform-dependent manner via transcriptional and post-transcriptional mechanisms. Differential response of PDK isoenzymes to DCA might be important for its pharmacological effects in different types of cells.

Reversing tozasertib resistance in glioma through inhibition of pyruvate dehydrogenase kinases.

Acquired resistance to conventional chemotherapeutic agents limits their effectiveness and can cause cancer treatment to fail. Because enzymes in the aurora kinase family are vital regulators of several mitotic events, we reasoned that targeting these kinases with tozasertib, a pan‐aurora kinase inhibitor, would not only cause cytokinesis defects, but also induce cell death in high‐grade pediatric and adult glioma cell lines. We found that tozasertib induced cell cycle arrest, increased mitochondrial permeability and reactive oxygen species generation, inhibited cell growth and migration, and promoted cellular senescence and pro‐apoptotic activity. However, sustained exposure to tozasertib at clinically relevant concentrations conferred resistance, which led us to examine the mechanistic basis for the emergence of drug resistance. RNA‐sequence analysis revealed a significant upregulation of the gene encoding pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), a pyruvate dehydrogenase (PDH) inhibitory kinase that plays a crucial role in the control of metabolic flexibility under various physiological conditions. Upregulation of PDK1, PDK2, PDK3, or PDK4 protein levels was positively correlated with tozasertib‐induced resistance through inhibition of PDH activity. Tozasertib‐resistant cells exhibited increased mitochondrial mass as measured by 10‐N‐nonyl‐Acridine Orange. Inhibition of PDK with dichloroacetate resulted in increased mitochondrial permeability and cell death in tozasertib‐resistant glioma cell lines. Based on these results, we believe that PDK is a selective target for the tozasertib resistance phenotype and should be considered for further preclinical evaluations.

Glavonoid-rich oil supplementation reduces stearoyl-coenzyme A desaturase 1 expression and improves systemic metabolism in diabetic, obese KK-Ay mice

AimsGlavonoid-rich oil (GRO) derived from ethanol extraction of licorice (Glycyrrhiza glabra Linne) root has been reported to have beneficial effects on health. In this study, we aimed to determine the effect of long-term administration of GRO on metabolic disorders and to elucidate the molecular mechanism. Main methodsFemale obese, type 2 diabetic KK-Ay mice were fed diets supplemented with 0.3% or 0.8% GRO (w/w) for 4–12 weeks. Mice were euthanized and autopsied at 20 weeks old. The effects of GRO on lipid and glucose metabolism were evaluated by measuring physiological and biochemical markers using mRNA sequencing, quantitative reverse-transcription PCR, and western blot analyses. Key findingsCompared to mice fed the control diet, GRO-supplemented mice had reduced body and white adipose tissue weights, serum levels of triglycerides and cholesterol, and improved glucose tolerance, while food intake was not affected. We found remarkable reductions in the gene expression levels of stearoyl-coenzyme A desaturase 1 (Scd1) and pyruvate dehydrogenase kinase isoenzyme 4 (Pdk4) in the liver, in addition to decreased expression of fatty acid synthase (Fasn) in inguinal white adipose tissue (iWAT). These results suggest that GRO supplementation improves lipid profiles via reduced de novo lipogenesis in the liver and white adipose tissue. Glucose metabolism may also be improved by increased glycolysis in the liver. SignificanceOur analysis of long-term supplementation of GRO in obese and diabetic mice should provide novel insight into preventing insulin resistance and metabolic syndromes.

Skeletal Response to Insulin in the Naturally Occurring Type 1 Diabetes Mellitus Mouse Model.

ABSTRACTPatients with type 1 diabetes mellitus (T1DM) exhibit reduced BMD and significant increases in fracture risk. Changes in BMD are attributed to blunted osteoblast activity and inhibited bone remodeling, but these cannot fully explain the impaired bone integrity in T1DM. The goal of this study was to determine the cellular mechanisms that contribute to impaired bone morphology and composition in T1DM. Nonobese diabetic (NOD) mice were used, along with μCT, histomorphometry, histology, Raman spectroscopy, and RNAseq analyses of several skeletal sites in response to naturally occurring hyperglycemia and insulin treatment. The bone volume in the axial skeleton was found to be severely reduced in diabetic NOD mice and was not completely resolved with insulin treatment. Decreased bone volume in diabetic mice was associated with increased sclerostin expression in osteocytes and attenuation of bone formation indices without changes in bone resorption. In the face of blunted bone remodeling, decreases in the mineral:matrix ratio were found in cortical bones of diabetic mice by Raman microspectroscopy, suggesting that T1DM did not affect the bone mineralization process per se, but rather resulted in microenvironmental alterations that favored mineral loss. Bone transcriptome analysis indicated metabolic shifts in response to T1DM. Dysregulation of genes involved in fatty acid oxidation, transport, and synthesis was found in diabetic NOD mice. Specifically, pyruvate dehydrogenase kinase isoenzyme 4 and glucose transporter 1 levels were increased, whereas phosphorylated‐AKT levels were significantly reduced in diabetic NOD mice. In conclusion, in addition to the blunted bone formation, osteoblasts and osteocytes undergo metabolic shifts in response to T1DM that may alter the microenvironment and contribute to mineral loss from the bone matrix. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

PDK2 Deficiency Prevents Ovariectomy-Induced Bone Loss in Mice by Regulating the RANKL-NFATc1 Pathway During Osteoclastogenesis.

Estrogen deficiency leads to osteoporosis as a result of an imbalance in bone remodeling due to greater bone resorption. Estrogen deficiency increases the osteoclastic resorption of bone, and many of the FDA-approved therapies for osteoporosis are antiresorptive drugs that mainly act by reducing osteoclast activity. The mitochondrial enzyme pyruvate dehydrogenase kinase (PDK) is a critical regulator of aerobic glycolysis that exerts its effects by phosphorylating the pyruvate dehydrogenase complex (PDC), which is responsible for oxidative phosphorylation. In the present study, we found that during osteoclast differentiation, PDK2 expression increased more than that of the other PDK isoenzymes. Bone loss was delayed and the number of osteoclasts was lower in ovariectomized (OVX) Pdk2-/- mice than in OVX wild-type mice. The differentiation of osteoclasts was suppressed in Pdk2-/- bone marrow-derived monocyte/macrophage lineage cells, which was associated with lower phosphorylation of cAMP response element-binding protein (CREB) and c-FOS, and a consequent reduction in NFATc1 transcription. Administration of AZD7545, a specific inhibitor of PDK2, prevented the OVX-induced bone loss and reduced the phosphorylation of CREB and c-FOS, and the protein expression of NFATc1, in osteoclasts. Collectively, these results indicate that the inhibition of PDK2 prevents osteoporosis in estrogen-deficient mice by reducing aberrant osteoclast activation, probably via inhibition of the RANKL-CREB-cFOS-NFATc1 pathway. These findings imply that PDK2 inhibitors might be repurposed for the therapy of estrogen deficiency-induced osteoporosis. © 2020 American Society for Bone and Mineral Research (ASBMR).

Abstract 5430: Evaluating a novel phytobiologic mixture against breast cancer cell lines: Effect on glycolytic enzymes and EMT gene expression profile

Abstract Background: Recently, there has been a growing evidence proposed regarding role of phytobiological compounds against various malignancies. One of the novel phytobiological mixture (PB), which is a mixture composed of the following: quercetin, resveratrol, cruciferex, epigallocatechin-3-gallate and curcumin at different proportions. To the best of our knowledge, this is the first study to evaluate the effects of a PB on breast cancer cell lines and its impact on the expression profile of these cells. Aim: Herein, we investigated the anti-proliferative effects of PB against two breast cancer cell lines: MDA-MB-231 and MCF-7, in addition, to investigating whether PB may alter the expression profile of glycolytic enzymes and epithelial mesenchymal transition (EMT) genes in these cell lines. Method: The anti-proliferative effect of PB was evaluated in vitro against MCF-7 and MDA-MB-231 cell lines using SRB assay. The effect of PB on the relative expression profile of some of the glycolytic enzymes and EMT genes was conducted using TaqMan gene expression assays via reverse transcription-quantitative PCR (RT-qPCR). Results: At 72 hrs, treatment with PB showed cytotoxic effects against MDA-MB-231 and MCF-7 cells with an IC50's of 25.9±3 µg/ml and 29.5± 0.9 µg/ml, respectively. At 24 hrs post PB treatment, there was a significant increase in the gene expression of most glycolytic enzymes such as; Phosphofructokinase muscle type (PFKM) (1.4± 0.1, p<0.01) and Pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1) (1.6± 0.2 fold, p< 0.001). PB did not seem to alter the EMT gene expression profile with both cadherin 1 and 2 showing no significant changes. Interestingly, however, a significant up-regulation was seen with ZEB2 member of the zinc finger E-box binding homeobox 2, post PB treatment. Conclusion: In conclusion PB showed a promising anti-proliferative effect against two breast cancer cell lines. However, we postulate that the drug could be modulating glycolytic enzymes expression, which ultimately can affect key molecules in cell differentiation. Further studies are needed to elucidate the exact underlying mechanism and to better understand the most beneficial drug combinations that can work synergistically with PB to interfere with different tumor hallmarks. Citation Format: Aliaa A. Alamoudi, Afnan A. Alqarni, Ghada Ajabnoor, Aleksandra Niedwiecki, Matthias Rath, Steve M. Harakeh, Ahmed M. Al-Abd. Evaluating a novel phytobiologic mixture against breast cancer cell lines: Effect on glycolytic enzymes and EMT gene expression profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5430.

Transcriptomics in the wild: Hibernation physiology in free-ranging dwarf lemurs.

Hibernation is an adaptive strategy some mammals use to survive highly seasonal or unpredictable environments. We present the first investigation on the transcriptomics of hibernation in a natural population of primate hibernators: Crossley's dwarf lemurs (Cheirogaleus crossleyi). Using capture-mark-recapture techniques to track the same animals over a period of 7months in Madagascar, we used RNA-seq to compare gene expression profiles in white adipose tissue (WAT) during three distinct physiological states. We focus on pathway analysis to assess the biological significance of transcriptional changes in dwarf lemur WAT and, by comparing and contrasting what is known in other model hibernating species, contribute to a broader understanding of genomic contributions of hibernation across Mammalia. The hibernation signature is characterized by a suppression of lipid biosynthesis, pyruvate metabolism and mitochondrial-associated functions, and an accumulation of transcripts encoding ribosomal components and iron-storage proteins. The data support a key role of pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) in regulating the shift in fuel economy during periods of severe food deprivation. This pattern of PDK4 holds true across representative hibernating species from disparate mammalian groups, suggesting that the genetic underpinnings of hibernation may be ancestral to mammals.

Pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes to the pathogenesis of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive disease and poses a high risk of severe liver damage. However, the pathogenesis of NASH is still unclear. Accumulation of lipid droplets and insulin resistance is the hallmark of NASH. Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) plays key role in glucose metabolism via regulating the activity of pyruvate dehydrogenase complex (PDC). Here, we demonstrated a novel of PDK4 in NASH by regulating hepatic steatosis and insulin signaling pathway in methionine and choline deficient (MCD) diet induced NASH model. Hepatic PDK4 levels were highly induced in human patients with NASH and MCD diet fed mice, as well as in hepatocytes treated with oleic acid. The glucose and lipid metabolism were impaired in Pdk4−/− mice. Pdk4 deficiency ameliorated the hepatic steatosis significantly in NASH mice. Pdk4−/−-MCD mice had reduced liver weights and triglyceride (TG) levels. And Pdk4 deficiency dramatically reduced the expression of genes related to fatty acid uptake, synthesis and gluconeogenesis. In addition, elevated phosphorylated AMPK (p-AMPK), p-SAPK/JNK and diminished p-ERK, p-P38, p-Akt and p-mTOR/p-4EBP1 proteins were observed. In conclusion, our data indicated that PDK4 potentially contributes to the hepatic steatosis in NASH via regulating several signaling pathway and PDK4 may be a new therapeutic strategy against NAFLD.

Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3 mutation

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. An X-linked form of CMT (CMTX6) is caused by a missense mutation (R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. PDK3 is one of 4 isoenzymes that negatively regulate the activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation of its first catalytic component pyruvate dehydrogenase (designated as E1). Mitochondrial PDC catalyses the oxidative decarboxylation of pyruvate to acetyl CoA and links glycolysis to the energy-producing Krebs cycle. We have previously shown the R158H mutation confers PDK3 enzyme hyperactivity. In this study we demonstrate that the increased PDK3 activity in patient fibroblasts (PDK3R158H) leads to the attenuation of PDC through hyper-phosphorylation of E1 at selected serine residues. This hyper-phosphorylation can be reversed by treating the PDK3R158H fibroblasts with the PDK inhibitor dichloroacetate (DCA). In the patient cells, down-regulation of PDC leads to increased lactate, decreased ATP and alteration of the mitochondrial network. Our findings highlight the potential to develop specific drug targeting of the mutant PDK3 as a therapeutic approach to treating CMTX6.

Effects of neonatal dexamethasone administration on cardiac recovery ability under ischemia-reperfusion in 24-wk-old rats.

Evaluations of stress-induced cardiac functional alterations in adults after neonatal glucocorticoid (GC) treatment have been limited. In the present study, we evaluated adult cardiac functional recovery during postischemic reperfusion and measured cardiac gene expression involved energy metabolism in rats neonatally treated with dexamethasone (DEX). Male Wistar rats were injected DEX in first 3 d after birth and controls were received saline (SAL). At 24 wk of age, insulin tolerance tests were performed, plasma lipid levels were measured, and left ventricular function and myocardial infarct size were evaluated. Expressions of genes involved in cardiac energy metabolism were measured by quantitative real-time polymerase chain reaction (PCR) and western blot. In 24-wk-old rats, neonatal DEX administration caused dyslipidemia, impaired cardiac recovery function and increased size of infarction, decreased cardiac expression of glucose transporter 4(GLUT4), peroxisome proliferative-activated receptor gamma coactivator 1α (PGC-1α) and ratios of phospho-forkhead box O1/forkhead box O1 (p-FoxO1/FoxO1) and phospho AMP-activated protein kinase/AMP-activated protein kinase (p-AMPK/AMPK) but increased pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) expression compared with controls. Neonatal DEX administration impairs cardiac functional recovery during reperfusion following ischemia in 24-wk-old rats. Reduced cardiac glucose utilization may contribute to the long-term detrimental effects caused by neonatal DEX treatment.

X-linked Charcot-Marie-Tooth disease type 6 (CMTX6) patients with a p.R158H mutation in the pyruvate dehydrogenase kinase isoenzyme 3 gene.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. Mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene have been found to cause X-linked dominant CMT type 6 (CMTX6). This study identified the p.R158H PDK3 mutation after screening 67 probable X-linked CMT families. The mutation fully segregated with the phenotype, and genotyping the family indicated the mutation arose on a different haplotype compared with the original Australian CMTX6 family. Results of bisulphite sequencing suggest that methylated deamination of a CpG dinucleotide may cause the recurrent p.R158H mutation. The frequency of the p.R158H PDK3 mutation in Koreans is very rare. Magnetic resonance imaging revealed fatty infiltration involving distal muscles in the lower extremities. In addition, fatty infiltrations were predominantly observed in the soleus muscles, with a lesser extent in tibialis anterior muscles. This differs from demyelinating CMT1A patients and is similar to axonal CMT2A patients. The clinical, neuroimaging, and electrophysiological findings from a second CMTX6 family with the p.R158H PDK3 mutation were similar to the axonal neuropathy reported in the Australian family.

Role of adenosine 5'-monophosphate-activated protein kinase in α-linolenic acid-induced intestinal lipid metabolism.

n-3 Long-chain PUFA up-regulate intestinal lipid metabolism. However, whether these metabolic effects of PUFA on intestine are mediated by AMP-activated protein kinase (AMPK) remains to be elucidated. To determine the effects of α-linolenic acid (ALA) on intestinal fatty acid (FA) metabolism and whether these effects were affected by AMPK deletion, mice deficient in the catalytic subunit of AMPKα1 or AMPKα2 and wild-type (WT) mice were fed either a high-fat diet (HF) or HF supplemented with ALA (HF-A). The results showed that ALA supplementation decreased serum TAG content in WT mice. ALA also increased mRNA expression of genes (carnitine palmitoyltransferase 1a, acyl-CoA oxidase 1, medium-chain acyl-CoA dehydrogenase, cytochrome P450 4A10 and pyruvate dehydrogenase kinase isoenzyme 4a) involved in intestinal lipid oxidation and mRNA expression of TAG synthesis-related genes (monoacylglycerol O-acyltransferase 2, diacylglycerol O-acyltransferases 1 and 2) in WT mice. Consistent with these, expression levels of phosphorylated AMPKα1 and AMPKα2 were also increased in WT mice after ALA addition. However, in the absence of either AMPKα1 or AMPKα2, ALA supplementation failed to increase intestinal lipid oxidation. In addition, no significant effects of either diet (HF and HF-A) or genotype (WT, AMPKα1(-/-) and AMPKα2(-/-)) on FA uptake in the intestine and faecal TAG output were observed. Our results suggest that AMPK is indispensable for the effects of ALA on intestinal lipid oxidation.

PO263 VISCERAL FAT AS RISK FACTOR FOR HYPERTENSION AND DIABETES MELLITUS – AN ASIAN PERSPECTIVE

Background: Concurrent with the existing epidemic of obesity and type 2 diabetes mellitus, hepatic steatosis is rapidly evolving to be a major medical problem in the world. It involves a variety of lipid abnormalities including enhanced fatty acid influx from the adipose tissue, increased de novo lipogenesis, reduced fatty acid oxidation and ketogenesis. The pyruvate dehydrogenase kinases (PDKs) regulate pyruvate oxidation by controlling the activity of the pyruvate dehydrogenase complex (PDC). Method: We investigated whether PDKs were increased in the liver of HFD-fed mice and how they change hepatic steatosis and insulin resistance. The physiological importance of regulation of the PDC by pyruvate dehydrogenase kinase isoenzyme 2 (PDK2) was assessed by comparing PDK2 KOmice with wild-type mice fed high fat diets (HFD) and low fat diets (LFD). Result: PDK2 contributes to the regulation of hepatic PDC activity in diet induced-obese mice. Body weight and hepatic steatosis were attenuated in the HFD-fed PDK2 KO mice. Fasting blood glucose, hepatic glucose production, insulin, and liver pyruvate, lactate, oxaloacetate, citrate, diacylglycerols, and triacylglycerols were also reduced while glucose tolerance and hepatic insulin sensitivity were increased. Enzyme capacities for fatty acid oxidation and ketogenesis were increased while lipogenesis was decreased. Consistent with greater PDC activity, the respiratory exchange ratio was higher in the PDK2 KOmice. Energy expenditure was increased without a difference in physical activity. Greater hepatic insulin sensitivity and improved glucose tolerance correlated with reduced protein kinase C e phosphorylation. Conclusion:Weconfirmed that deletion of PDK2 could prevent HFD-induced hepatic steatosis and improve hepatic insulin sensitivity, most likely by augmentation of FA consumption associated with reduction of TCA cycle intermediates in liver of HFD-mice. The findings support the premise that PDK2 is a potential therapeutic target for hepatic steatosis and insulin resistance.

The Transcriptional Co-Repressor Myeloid Translocation Gene 16 Inhibits Glycolysis and Stimulates Mitochondrial Respiration

The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor–containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline–dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4), and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia–stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2) oligomerization domain and the NHR3 protein–protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen–activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti–tumor effect.

A first-in-human phase I trial of AR-12, a PDK-1 inhibitor, in patients with advanced solid tumors.

2608 Background: AR-12 (OSU-03012) is an oral celecoxib analogue lacking COX-2 inhibitory activity that inhibits pyruvate dehydrogenase kinase isoenzyme 1 (PDK-1), AKT and impacts the endoplastic reticulum stress pathway. Preclinical studies indicate antitumor activity of AR-12 in various models and enhanced activity in combination. We completed a first in human clinical trial to determine its safety and tolerability, maximum tolerated dose (MTD) and recommended phase II dose (RD). Secondary objectives included assessment of tumor response, pharmacokinetics (PK) and pharmacodynamics (PD) including food effect. Methods: Patients (pts) with advanced solid tumors, ECOG PS 0-1, and adequate organ function were recruited in a modified 3+3 dose-escalation study. Pts received a run-in dose of AR-12 to analyze PK-PD and food effect, followed by continuous daily (QD) dosing in 28-day cycles. A twice daily (BID) cohort was initiated based on safety data. PD analysis was performed in platelet-rich plasma (PRP) and paired tumor biopsies when feasible. Results: 35 pts received at least one dose of study drug; 30 were evaluable for dose limiting toxicities (DLT) at dose ranges 100-3200mg QD and 800-1600mg BID. No DLT were observed in the QD cohort; DLT in the BID cohort are listed in table 1. Drug-related events (NCI-CTCAE v3) included rash (G2-2pts; G3-1pt), fatigue (G2-2pts; G3-4pts), nausea (G2-7pts; G3-1pt) and bloating (G2-1pt). Cmax after single dose was dose-proportional but high PK variability was observed, likely due to inadequate disintegration and dissolution of the formulation in the stomach. At RD, partial GSK3ß inhibition in PRP after 4 hours suggests AKT-pathway modulation. Best response (RECIST v1.0) was stable disease >6 cycles for 2 pts. Conclusions: The RD based on safety data is 800mg BID. Signs of pathway modulation were observed in concordance with the expected mechanism of action but were short-lasting. Considering limited drug absorption and PK variability, a new formulation of the drug will be developed to overcome these limitations. Clinical trial information: NCT00978523. [Table: see text]

A new locus for X-linked dominant Charcot-Marie-Tooth disease (CMTX6) is caused by mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene.

Hereditary motor and sensory disorders of the peripheral nerve form one of the most common groups of human genetic diseases collectively called Charcot-Marie-Tooth (CMT) neuropathy. Using linkage analysis in a three generation kindred, we have mapped a new locus for X-linked dominant CMT to chromosome Xp22.11. A microsatellite scan of the X chromosome established significant linkage to several markers including DXS993 (Zmax = 3.16; θ = 0.05). Extended haplotype analysis refined the linkage region to a 1.43-Mb interval flanked by markers DXS7110 and DXS8027. Whole exome sequencing identified a missense mutation c.G473A (p.R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. The change localized within the 1.43-Mb linkage interval, segregated with the affected phenotype and was excluded in ethnically matched control chromosomes. PDK3 is one of the four isoenzymes regulating the pyruvate dehydrogenase complex (PDC), by reversible phosphorylation, and is a nuclear-coded protein located in the mitochondrial matrix. PDC catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA and is a key enzyme linking glycolysis to the energy-producing Krebs cycle and lipogenic pathways. We found that the R158H mutation confers enzyme hyperactivity and binds with stronger affinity than the wild-type to the inner-lipoyl (L2) domain of the E2p chain of PDC. Our findings suggest a reduced pyruvate flux due to R158H mutant PDK3-mediated hyper-phosphorylation of the PDC as the underlying pathogenic cause of peripheral neuropathy. The results highlight an important causative link between peripheral nerve degeneration and an essential bioenergetic or biosynthetic pathway required for the maintenance of peripheral nerves.

DNA microarray analysis identified molecular pathways mediating the effects of supplementation of branched‐chain amino acids on CCl4‐induced cirrhosis in rats

This study addresses the effects of branched-chain amino acids (BCAA) on global gene expression in liver and skeletal muscle and the molecular mechanisms underlying the improvement in liver cirrhosis using DNA microarray analysis combined with RNase protection assay. Male Wistar rats administered carbon tetrachloride (CCl(4) ) repeatedly for 19 weeks as a decompensated cirrhosis model were thereafter given BCAA-enriched diet (AL) or normal diet (LC) for 5 weeks. The control-diet rats without CCl(4) administration were used as a normal control group. Gene expression in AL was reversed by twofold greater than in LC in the microarray were selected to elucidate the improvements in nutritional and metabolic disorders. Downregulation of fatty acid translocase (FAT)/Cd36, glutamine synthetase, and pyruvate dehydrogenase kinase isoenzyme 4 is believed to promote lower uptake of fatty acids, lower ammonia incorporation, and higher uptake of glucose, and thus to provide an energy source without using BCAA. Ultimately, the catabolism of BCAA and skeletal muscle protein would be slowed, maintaining BCAA concentrations in blood. We established, for the first time, the regulatory gene pathways of processes involved in hepatic fibrosis and energy metabolism (hypoalbuminemia, hyperammonemia, and carbohydrate catabolism, and their relationships) under BCAA supplementation.

Additive effects of clofibric acid and pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) deficiency on hepatic steatosis in mice fed a high saturated fat diet

Although improving glucose metabolism by inhibition of pyruvate dehydrogenase kinase 4 (PDK4) may prove beneficial in the treatment of type 2 diabetes or diet-induced obesity, it may have detrimental effects by inhibiting fatty acid oxidation. Peroxisome proliferator-activated receptor α (PPARα) agonists are often used to treat dyslipidemia in patients, especially in type 2 diabetes. Combinational treatment using a PDK4 inhibitor and PPARα agonists may prove beneficial. However, PPARα agonists may be less effective in the presence of a PDK4 inhibitor because PPARα agonists induce PDK4 expression. In the present study, the effects of clofibric acid, a PPARα agonist, on blood and liver lipids were determined in wild-type and PDK4 knockout mice fed a high-fat diet. As expected, treatment of wild-type mice with clofibric acid resulted in less body weight gain, smaller epididymal fat pads, greater insulin sensitivity, and lower levels of serum and liver triacylglycerol. Surprisingly, rather than decreasing the effectiveness of clofibric acid, PDK4 deficiency enhanced the beneficial effects of clofibric acid on hepatic steatosis, reduced blood glucose levels, and did not prevent the positive effects of clofibric acid on serum triacylglycerols and free fatty acids. The metabolic effects of clofibric acid are therefore independent of the induction of PDK4 expression. The additive beneficial effects on hepatic steatosis may be due to induction of increased capacity for fatty acid oxidation and partial uncoupling of oxidative phosphorylation by clofibric acid, and a reduction in the capacity for fatty acid synthesis as a result of PDK4 deficiency.