Pyruvate Dehydrogenase Kinase 4 Inhibition Research Articles

Reducing Oxidative Stress and Inflammation by Pyruvate Dehydrogenase Kinase 4 Inhibition Is Important in Prevention of Renal Ischemia-Reperfusion Injury in Diabetic Mice.

Reactive oxygen species (ROS) and inflammation are reported to have a fundamental role in the pathogenesis of ischemia-reperfusion (IR) injury, a leading cause of acute kidney injury. The present study investigated the role of pyruvate dehydrogenase kinase 4 (PDK4) in ROS production and inflammation following IR injury. We used a streptozotocin-induced diabetic C57BL6/J mouse model, which was subjected to IR by clamping both renal pedicles. Cellular apoptosis and inflammatory markers were evaluated in NRK-52E cells and mouse primary tubular cells after hypoxia and reoxygenation using a hypoxia work station. Following IR injury in diabetic mice, the expression of PDK4, rather than the other PDK isoforms, was induced with a marked increase in pyruvate dehydrogenase E1α (PDHE1α) phosphorylation. This was accompanied by a pronounced ROS activation, as well as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) production. Notably, sodium dichloroacetate (DCA) attenuated renal IR injury-induced apoptosis which can be attributed to reducing PDK4 expression and PDHE1α phosphorylation levels. DCA or shPdk4 treatment reduced oxidative stress and decreased TNF-α, IL-6, IL-1β, and MCP-1 production after IR or hypoxia-reoxygenation injury. PDK4 inhibition alleviated renal injury with decreased ROS production and inflammation, supporting a critical role for PDK4 in IR mediated damage. This result indicates another potential target for reno-protection during IR injury; accordingly, the role of PDK4 inhibition needs to be comprehensively elucidated in terms of mitochondrial function during renal IR injury.

PDK4-dependent hypercatabolism and lactate production of senescent cells promotes cancer malignancy

Senescent cells remain metabolically active, but their metabolic landscape and resulting implications remain underexplored. Here, we report upregulation of pyruvate dehydrogenase kinase 4 (PDK4) upon senescence, particularly in some stromal cell lines. Senescent cells display a PDK4-dependent increase in aerobic glycolysis and enhanced lactate production but maintain mitochondrial respiration and redox activity, thus adopting a special form of metabolic reprogramming. Medium from PDK4+ stromal cells promotes the malignancy of recipient cancer cells in vitro, whereas inhibition of PDK4 causes tumor regression in vivo. We find that lactate promotes reactive oxygen species production via NOX1 to drive the senescence-associated secretory phenotype, whereas PDK4 suppression reduces DNA damage severity and restrains the senescence-associated secretory phenotype. In preclinical trials, PDK4 inhibition alleviates physical dysfunction and prevents age-associated frailty. Together, our study confirms the hypercatabolic nature of senescent cells and reveals a metabolic link between cellular senescence, lactate production, and possibly, age-related pathologies, including but not limited to cancer.

Suppressing succinate accumulation during ischemia protects the kidney from IRI

Previous studies have indicated that succinate accumulation during kidney ischemia, and its oxidation during reperfusion, results in the production of excessive reactive oxygen species, mitochondrial dysfunction, and kidney injury. In this issue, Oh etal. have reported that pyruvate dehydrogenase kinase 4 (PDK4) inhibition in proximal tubules ameliorates kidney ischemia/reperfusion injury via suppressed succinate accumulation. This study suggests that PDK4 inhibition is a promising new treatment strategy for ischemic acute kidney injury.

Inhibition of Pyruvate Dehydrogenase Kinase 4 Attenuates Myocardial and Mitochondrial Injury in Sepsis-Induced Cardiomyopathy.

Sepsis-induced cardiomyopathy (SIC) is a cardiac dysfunction caused by sepsis, with mitochondrial dysfunction being a critical contributor. Pyruvate dehydrogenase kinase 4 (PDK4) is a kinase of pyruvate dehydrogenase with multifaceted actions in mitochondrial metabolism. However, its role in SIC remains unknown. Serum PDK4 levels were measured and analyzed in 27 children with SIC, 30 children with sepsis, and 29 healthy children. In addition, for mice exhibiting SIC, the effects of PDK4 knockdown or inhibition on the function and structure of the myocardium and mitochondria were assessed. The findings from the analysis of children with SIC revealed that PDK4 was significantly elevated and correlated with disease severity and organ injury. Nonsurvivors displayed higher serum PDK4 levels than survivors. Furthermore, mice with SIC benefited from PDK4 knockdown or inhibition, showing improved myocardial contractile function, reduced myocardial injury, and decreased mitochondrial structural injury and dysfunction. In addition, inhibition of PDK4 decreased the inhibitory phosphorylation of PDHE1α (pyruvate dehydrogenase complex E1 subunit α) and improved abnormal pyruvate metabolism and mitochondrial dysfunction. PDK4 is a potential biomarker for the diagnosis and prognosis of SIC. In experimental SIC, PDK4 promoted mitochondrial dysfunction with increased phosphorylation of PDHE1α and abnormal pyruvate metabolism.

Inhibition of pyruvate dehydrogenase kinase 4 ameliorates kidney ischemia-reperfusion injury by reducing succinate accumulation during ischemia and preserving mitochondrial function during reperfusion

Ischemia-reperfusion (IR) injury, a leading cause of acute kidney injury (AKI), is still without effective therapies. Succinate accumulation during ischemia followed by its oxidation during reperfusion leads to excessive reactive oxygen species (ROS) and severe kidney damage. Consequently, the targeting of succinate accumulation may represent a rational approach to the prevention of IR-induced kidney injury. Since ROS are generated primarily in mitochondria, which are abundant in the proximal tubule of the kidney, we explored the role of pyruvate dehydrogenase kinase 4 (PDK4), a mitochondrial enzyme, in IR-induced kidney injury using proximal tubule cell-specific Pdk4 knockout (Pdk4ptKO) mice. Knockout or pharmacological inhibition of PDK4 ameliorated IR-induced kidney damage. Succinate accumulation during ischemia, which is responsible for mitochondrial ROS production during reperfusion, was reduced by PDK4 inhibition. PDK4 deficiency established conditions prior to ischemia resulting in less succinate accumulation, possibly because of a reduction in electron flow reversal in complex II, which provides electrons for the reduction of fumarate to succinate by succinate dehydrogenase during ischemia. The administration of dimethyl succinate a cell-permeable form of the Complex II inhibitor malonate, attenuated the beneficial effects of PDK4 deficiency, suggesting that the kidney-protective effect is succinate-dependent. Finally, genetic or pharmacological inhibition of PDK4 prevented IR-induced mitochondrial damage in mice and normalized mitochondrial function in an in vitro model of IR injury. Thus, inhibition of PDK4 represents a novel means of preventing IR-induced kidney injury, and involves the inhibition of ROS-induced kidney-toxicity through reduction in succinate accumulation and mitochondrial dysfunction.

MicroRNA-15b-5p shuttled by mesenchymal stem cell-derived extracellular vesicles protects podocytes from diabetic nephropathy via downregulation of VEGF/PDK4 axis.

Diabetic nephropathy (DN) is a severe complication of diabetes lethal for end-stage renal disease, with less treatment methodologies and uncertain pathogenesis. In the current study, we determined the role of mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) containing microRNA (miR)-15b-5p in DN. After extraction and identification of MSC-derived EVs, mouse podocyte line MPC5 was selected to establish an in vitro high-glucose (HG) cell model, where expression of miR-15b-5p, pyruvate dehydrogenase kinase 4 (PDK4) and VEGFA expression in tissues and cells were determined. The loss- and gain- function assays were conducted to determine the roles of miR-15b-5p, PDK4 and VEGFA. MPC5 cells were then co-cultured with MSC-derived EVs and their biological behaviors were detected by Western blot, CCK-8 assay, and flow cytometry. The binding relationship between miR-15b-5p and PDK43 by dual luciferase reporter gene assay. The expression of miR-15b-5p was downregulated in podocytes under HG environment, but highly expressed in mouse MSCs-derived EVs. EVs-derived miR-15b-5p could protect MPC5 cell apoptosis and inflammation. miR-15b-5p inhibited the expression of PDK4 by directly bound to the 3'UTR region of PDK4 gene. miR-15b-5p inhibits VEGF expression by binding to PDK4. Inhibition of PDK4 decreased VEGFA expression and reduced apoptosis and inflammation. Collectively, miR-15b-5p shuttled by MSC-derived EV can play protective roles in HG-induced mouse podocyte injury, possibly by targeting PDK4 and decreasing the VEGFA expression.

Loss of prion protein control of glucose metabolism promotes neurodegeneration in model of prion diseases.

Corruption of cellular prion protein (PrPC) function(s) at the plasma membrane of neurons is at the root of prion diseases, such as Creutzfeldt-Jakob disease and its variant in humans, and Bovine Spongiform Encephalopathies, better known as mad cow disease, in cattle. The roles exerted by PrPC, however, remain poorly elucidated. With the perspective to grasp the molecular pathways of neurodegeneration occurring in prion diseases, and to identify therapeutic targets, achieving a better understanding of PrPC roles is a priority. Based on global approaches that compare the proteome and metabolome of the PrPC expressing 1C11 neuronal stem cell line to those of PrPnull-1C11 cells stably repressed for PrPC expression, we here unravel that PrPC contributes to the regulation of the energetic metabolism by orienting cells towards mitochondrial oxidative degradation of glucose. Through its coupling to cAMP/protein kinase A signaling, PrPC tones down the expression of the pyruvate dehydrogenase kinase 4 (PDK4). Such an event favors the transfer of pyruvate into mitochondria and its conversion into acetyl-CoA by the pyruvate dehydrogenase complex and, thereby, limits fatty acids β-oxidation and subsequent onset of oxidative stress conditions. The corruption of PrPC metabolic role by pathogenic prions PrPSc causes in the mouse hippocampus an imbalance between glucose oxidative degradation and fatty acids β-oxidation in a PDK4-dependent manner. The inhibition of PDK4 extends the survival of prion-infected mice, supporting that PrPSc-induced deregulation of PDK4 activity and subsequent metabolic derangements contribute to prion diseases. Our study posits PDK4 as a potential therapeutic target to fight against prion diseases.

MicroRNA -148 alleviates cardiac dysfunction, immune disorders and myocardial apoptosis in myocardial ischemia-reperfusion (MI/R) injury by targeting pyruvate dehydrogenase kinase (PDK4)

ABSTRACT Ischemic heart disease in children may be induced by varied factors, and there is no corresponding systematic treatment up to now. This study aims to investigate the effects of microRNA (miR)-148 on myocardial injury in immature rats with myocardial ischemia-reperfusion (MI/R) injury. In this study, MI/R model was established by ligating the coronary artery of heart. The results showed that miR-148 alleviated myocardial injury and rescued relevant parameters (mean ventricular systolic blood pressure (MAP), left ventricular systolic blood pressure (LVSP), heart rate (HR), creatine kinase-MB (CK-MB), cTn1 and Mb in immature rats with MI/R injury. Besides, miR-148 improved the immune dysfunction induced by MI/R through increasing the number of interleukin (IL)-10+ cells and reducing the number of inducible nitric oxide synthase (iNOS)+ cells. In addition, miR-148 relieved the apoptosis of cardiomyocytes induced by MI/R through inhibiting the expression of Bax and elevating the expression of Bcl-2. Further molecular mechanism indicated that pyruvate dehydrogenase kinase 4 (PDK4) was the downstream target of miR-148, which was further confirmed by dual luciferase reporter assay and related expression detection. Accordingly, silenced PDK4 attenuated cardiac dysfunction, immune disorder and myocardial apoptosis in immature rats and enhanced the ability of antioxidant enzymes. What is more, activated SMAD pathway induced by MI/R injury was then blocked by silenced PDK4. Taken together, our study demonstrated that overexpressed miR-148 relieved cardiac dysfunction, immune disorder and cardiomyocyte apoptosis in immature MI/R rats by PDK4 inhibition, which provided novel targets for MI/R injury treatment.

PDK4 promotes vascular calcification by interfering with autophagic activity and metabolic reprogramming

Pyruvate dehydrogenase kinase 4 (PDK4) is an important mitochondrial matrix enzyme in cellular energy regulation. Previous studies suggested that PDK4 is increased in the calcified vessels of patients with atherosclerosis and is closely associated with mitochondrial function, but the precise regulatory mechanisms remain largely unknown. This study aims to investigate the role of PDK4 in vascular calcification and the molecular mechanisms involved. Using a variety of complementary techniques, we found impaired autophagic activity in the process of vascular smooth muscle cells (VSMCs) calcification, whereas knocking down PDK4 had the opposite effect. PDK4 drives the metabolic reprogramming of VSMCs towards a Warburg effect, and the inhibition of PDK4 abrogates VSMCs calcification. Mechanistically, PDK4 disturbs the integrity of the mitochondria-associated endoplasmic reticulum membrane, concomitantly impairing mitochondrial respiratory capacity, which contributes to a decrease in lysosomal degradation by inhibiting the V-ATPase and lactate dehydrogenase B interaction. PDK4 also inhibits the nuclear translocation of the transcription factor EB, thus inhibiting lysosomal function. These changes result in the interruption of autophagic flux, which accelerates calcium deposition in VSMCs. In addition, glycolysis serves as a metabolic adaptation to improve VSMCs oxidative stress resistance, whereas inhibition of glycolysis by 2-deoxy-D-glucose induces the apoptosis of VSMCs and increases the calcium deposition in VSMCs. Our results suggest that PDK4 plays a key role in vascular calcification through autophagy inhibition and metabolic reprogramming.

PDK4-Deficiency Reprograms Intrahepatic Glucose and Lipid Metabolism to Facilitate Liver Regeneration in Mice.

Liver regeneration requires intrahepatic and extrahepatic metabolic reprogramming to meet the high hepatic bioenergy demand for liver cell repopulation. This study aims to elucidate how pyruvate dehydrogenase kinase 4 (PDK4), a critical regulator of glucose and lipid metabolism, coordinates metabolic regulation with efficient liver growth. We found that hepatic Pdk4 expression was elevated after two‐thirds partial hepatectomy (PHx). In Pdk4 −/− PHx mice, the liver/body weight ratio was more rapidly restored, accompanied by more aggressive hepatic DNA replication; however, Pdk4 −/− mice developed more severe hypoglycemia. In Pdk4 −/− PHx livers, the pro‐regenerative insulin signaling was potentiated, as demonstrated by early peaking of the phosphorylation of insulin receptor, more remarkable induction of the insulin receptor substrate proteins, IRS1 and IRS2, and more striking activation of Akt. The hepatic up‐regulation of CD36 contributed to the enhanced transient regeneration‐associated steatosis in Pdk4 −/− PHx mice. Notably, CD36 overexpression in mice promoted the recovery of liver/body weight ratio and elevated intrahepatic adenosine triphosphate after PHx. CD36 expression was transcriptionally suppressed by FOXO1 (forkhead box protein O1), which was stabilized and translocated to the nucleus following AMPK (adenosine monophosphate–activated protein kinase) activation. PHx remarkably induced AMPK activation, which became incompetent to respond in Pdk4 −/− livers. Moreover, we defined that PDK4‐regulated AMPK activation directly depended on intracellular adenosine monophosphate in vitro and in regenerative livers. Conclusion: PDK4 inhibition reprograms glucose and lipid metabolism to promote liver regeneration by enhancing hepatic insulin/Akt signaling and activating an AMPK/FOXO1/CD36 regulatory axis of lipid. These findings may lead to potential therapeutic strategies to prevent hepatic insufficiency and liver failure.

LKB1 Represses ATOH1 via PDK4 and Energy Metabolism and Regulates Intestinal Stem Cell Fate

In addition to the Notch and Wnt signaling pathways, energy metabolism also regulates intestinal stem cell (ISC) function. Tumor suppressor and kinase STK11 (also called LKB1) regulates stem cells and cell metabolism. We investigated whether loss of LKB1 alters ISC homeostasis in mice. We deleted LKB1 from ISCs in mice using Lgr5-regulated CRE-ERT2 (Lkb1Lgr5-KO mice) and the traced lineages by using a CRE-dependent TdTomato reporter. Intestinal tissues were collected and analyzed by immunohistochemical and immunofluorescence analyses. We purified ISCs and intestinal progenitors using flow cytometry and performed RNA-sequencing analysis. We measured organoid-forming capacity and ISC percentages using intestinal tissues from Lkb1Lgr5-KO mice. We analyzed human Ls174t cells with knockdown of LKB1 or other proteins by immunoblotting, real-time quantitative polymerase chain reaction, and the Seahorse live-cell metabolic assay. Some intestinal crypts from Lkb1Lgr5-KO mice lost ISCs compared with crypts from control mice. However, most crypts from Lkb1Lgr5-KO mice contained functional ISCs that expressed increased levels of Atoh1 messenger RNA (mRNA), acquired a gene expression signature associated with secretory cells, and generated more cells in the secretory lineage compared with control mice. Knockdown of LKB1 in Ls174t cells induced expression of Atoh1 mRNA and a phenotype of increased mucin production; knockdown of ATOH1 prevented induction of this phenotype. The increased expression of Atoh1 mRNA after LKB1 loss from ISCs or Ls174t cells did not involve Notch or Wnt signaling. Knockdown of pyruvate dehydrogenase kinase 4 (PDK4) or inhibition with dichloroacetate reduced the up-regulation of Atoh1 mRNA after LKB1 knockdown in Ls174t cells. Cells with LKB1 knockdown had a reduced rate of oxygen consumption, which was partially restored by PDK4 inhibition with dichloroacetate. ISCs with knockout of LKB1 increased the expression of PDK4 and had an altered metabolic profile. LKB1 represses transcription of ATOH1, via PDK4, in ISCs, restricting their differentiation into secretory lineages. These findings provide a connection between metabolism and the fate determination of ISCs.

Inhibition of pyruvate dehydrogenase kinase-4 by l-glutamine protects pregnant rats against fructose-induced obesity and hepatic lipid accumulation

ObjectiveAccumulation of lipids in non-adipose tissues particularly the liver is a feature of tissue insulin resistance. Hepatic glycogen depletion reflects counter glucoregulation in an insulin-resistant state and/or obesity. The effect of l-glutamine on fructose-induced increased hepatic lipid accumulation and depleted glycogen content, particularly in pregnancy, is not known. We therefore aimed at investigating the effect of glutamine on fructose-induced weight gain, hepatic lipids and glycogen contents in pregnant rats and also tested the hypothesis that hepatoprotective role of l-glutamine is through suppression of PDK-4. MethodsEleven-week-old pregnant Wistar rats were allotted into the Control, Glutamine, Fructose and Fructose plus Glutamine groups (6 rats/group). The groups received distilled water (vehicle, p.o.), 1 g/kg bwl-glutamine (p.o.), 10% Fructose (w/v) and 10% Fructose (w/v) plus 1 g/kg bwl-glutamine (p.o.) respectively, daily for 19 days. Biochemical analysis and histology of the liver were performed. ResultsData showed that fructose intake caused insulin resistance, hyperglycemia, hyperlipidemia, increased body weight gain, visceral fat mass, hepatic mass, lactate production, uric acid production, lipid peroxidation and decreased pancreatic β-cell function and hepatic glycogen synthesis. These alterations were accompanied by elevated pyruvate dehydrogenase kinase-4 (PDK-4). However, the fructose-induced dysmetabolism were improved by l-glutamine. ConclusionOur results demonstrate that obesity and hepatic lipid accumulation induced by fructose in pregnant rats is accompanied by increased PDK-4. The findings also suggest that l-glutamine would protect against obesity and hepatic lipid accumulation by suppression of PDK-4.

Advanced glycation end products accelerate calcification in VSMCs through HIF-1\u03b1/PDK4 activation and suppress glucose metabolism

Arterial media calcification is associated with diabetes mellitus. Previous studies have shown that advanced glycation end products (AGEs) are responsible for vascular smooth muscle cell (VSMC) calcification, but the underlying mechanisms remain unclear. Hypoxia-inducible factor-1α (HIF-1α), one of the major factors during hypoxia, and pyruvate dehydrogenase kinase 4 (PDK4), an important mitochondrial matrix enzyme in cellular metabolism shift, have been reported in VSMC calcification. The potential link among HIF-1α, PDK4, and AGEs-induced vascular calcification was investigated in this study. We observed that AGEs elevated HIF-1α and PDK4 expression levels in a dose-dependent manner and that maximal stimulation was attained at 24 h. Two important HIF-1α-regulated genes, vascular endothelial growth factor A (VEGFA) and glucose transporter 1 (GLUT-1), were significantly increased after AGEs exposure. Stabilization or nuclear translocation of HIF-1α increased PDK4 expression. PDK4 inhibition attenuated AGEs-induced VSMC calcification, which was evaluated by measuring the calcium content, alkaline phosphatase (ALP) activity and runt-related transcription factor 2 (RUNX2) expression levels and by Alizarin red S staining. In addition, the glucose consumption, lactate production, key enzymes of glucose metabolism and oxygen consumption rate (OCR) were decreased during AGEs-induced VSMC calcification. In conclusion, this study suggests that AGEs accelerate vascular calcification partly through the HIF-1α/PDK4 pathway and suppress glucose metabolism.

The Role of Pyruvate Dehydrogenase Kinase-4 (PDK4) in Bladder Cancer and Chemoresistance.

Advanced bladder cancer remains a major source of mortality, with poor treatment options. Cisplatin-based chemotherapy is the standard treatment, however many patients are or become resistant. One potential cause of chemoresistance is the Warburg effect, a metabolic switch to aerobic glycolysis that occurs in many cancers. Upregulation of the pyruvate dehydrogenase kinase family (PDK1-PDK4) is associated with aerobic glycolysis and chemoresistance through inhibition of the pyruvate dehydrogenase complex (PDH). We have previously observed upregulation of PDK4 in high-grade compared with low-grade bladder cancers. We initiated this study to determine if inhibition of PDK4 could reduce tumor growth rates or sensitize bladder cancer cells to cisplatin. Upregulation of PDK4 in malignant bladder cancer cell lines as compared with benign transformed urothelial cells was confirmed using qPCR. Inhibition of PDK4 with dichloroacetate (DCA) resulted in increased PDH activity, reduced cell growth, and G0-G1 phase arrest in bladder cancer cells. Similarly, siRNA knockdown of PDK4 inhibited bladder cancer cell proliferation. Cotreatment of bladder cancer cells with cisplatin and DCA did not increase caspase-3 activity but did enhance overall cell death in vitro Although daily treatment with 200 mg/kg DCA alone did not reduce tumor volumes in a xenograft model, combination treatment with cisplatin resulted in dramatically reduced tumor volumes as compared with either DCA or cisplatin alone. This was attributed to substantial intratumoral necrosis. These findings indicate inhibition of PDK4 may potentiate cisplatin-induced cell death and warrant further studies investigating the mechanism through which this occurs. Mol Cancer Ther; 17(9); 2004-12. ©2018 AACR.

Glucose oxidation positively regulates glucose uptake and improves cardiac function recovery after myocardial reperfusion.

Myocardial reperfusion decreases glucose oxidation and uncouples glucose oxidation from glycolysis. Therapies that increase glucose oxidation lessen myocardial ischemia-reperfusion (I/R) injury. However, the regulation of glucose uptake during reperfusion remains poorly understood. We found that glucose uptake was remarkably diminished in the myocardium following reperfusion in Sprague-Dawley rats as detected by 18F-labeled and fluorescent-labeled glucose analogs, even though GLUT1 was upregulated by threefold and GLUT4 translocation remained unchanged compared with those of sham-treated rats. The decreased glucose uptake was accompanied by suppressed glucose oxidation. Interestingly, stimulating glucose oxidation by inhibition of pyruvate dehydrogenase kinase 4 (PDK4), a rate-limiting enzyme for glucose oxidation, increased glucose uptake and alleviated I/R injury. In vitro data in neonatal myocytes showed that PDK4 overexpression decreased glucose uptake, whereas its knockdown increased glucose uptake, suggesting that PDK4 has a role in regulating glucose uptake. Moreover, inhibition of PDK4 increased myocardial glucose uptake with concomitant enhancement of cardiac insulin sensitivity following myocardial I/R. These results showed that the suppressed glucose oxidation mediated by PDK4 contributes to the reduced glucose uptake in the myocardium following reperfusion, and enhancement of glucose uptake exerts cardioprotection. The findings suggest that stimulating glucose oxidation via PDK4 could be an efficient approach to improve recovery from myocardial I/R injury.

Regulation of Pyruvate Dehydrogenase Kinase 4 in the Heart through Degradation by the Lon Protease in Response to Mitochondrial Substrate Availability

Cardiac metabolic inflexibility is driven by robust up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) and phosphorylation-dependent inhibition of pyruvate dehydrogenase (PDH) within a single day of feeding mice a high fat diet. In the current study, we have discovered that PDK4 is a short lived protein (t½ ∼ 1 h) and is specifically degraded by the mitochondrial protease Lon. Lon does not rapidly degrade PDK1 and -2, indicating specificity toward the PDK isoform that is a potent modulator of metabolic flexibility. Moreover, PDK4 degradation appears regulated by dissociation from the PDH complex dependent on the respiratory state and energetic substrate availability of mouse heart mitochondria. Finally, we demonstrate that pharmacologic inhibition of PDK4 promotes PDK4 degradation in vitro and in vivo These findings reveal a novel strategy to manipulate PDH activity by selectively targeting PDK4 content through dissociation and proteolysis.

Transforming Growth Factor β Mediates Drug Resistance by Regulating the Expression of Pyruvate Dehydrogenase Kinase 4 in Colorectal Cancer

Drug resistance is one of the main causes of colon cancer recurrence. However, our understanding of the underlying mechanisms and availability of therapeutic options remains limited. Here we show that expression of pyruvate dehydrogenase kinase 4 (PDK4) is positively correlated with drug resistance of colon cancer cells and induced by 5-fluorouracil (5-FU) treatment in drug-resistant but not drug-sensitive cells. Knockdown of PDK4 expression sensitizes colon cancer cells to 5-FU or oxaliplatin-induced apoptosis in vitro and increases the effectiveness of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo In addition, we demonstrate for the first time that TGFβ mediates drug resistance by regulating PDK4 expression and that 5-FU induces PDK4 expression in a TGFβ signaling-dependent manner. Mechanistically, knockdown or inhibition of PDK4 significantly increases the inhibitory effect of 5-FU on expression of the anti-apoptotic factors Bcl-2 and survivin. Importantly, studies of patient samples indicate that expression of PDK4 and phosphorylation of Smad2, an indicator of TGFβ pathway activation, show a strong correlation and that both positively associate with chemoresistance in colorectal cancer. These findings indicate that the TGFβ/PDK4 signaling axis plays an important role in the response of colorectal cancer to chemotherapy. A major implication of our studies is that inhibition of PDK4 may have considerable therapeutic potential to overcome drug resistance in colorectal cancer patients, which warrants the development of PDK4-specific inhibitors.

Pyruvate Dehydrogenase Kinase 4 Promotes Vascular Calcification via SMAD1/5/8 Phosphorylation.

Vascular calcification, a pathologic response to defective calcium and phosphate homeostasis, is strongly associated with cardiovascular mortality and morbidity. In this study, we have observed that pyruvate dehydrogenase kinase 4 (PDK4) is upregulated and pyruvate dehydrogenase complex phosphorylation is increased in calcifying vascular smooth muscle cells (VSMCs) and in calcified vessels of patients with atherosclerosis, suggesting that PDK4 plays an important role in vascular calcification. Both genetic and pharmacological inhibition of PDK4 ameliorated the calcification in phosphate-treated VSMCs and aortic rings and in vitamin D3-treated mice. PDK4 augmented the osteogenic differentiation of VSMCs by phosphorylating SMAD1/5/8 via direct interaction, which enhances BMP2 signaling. Furthermore, increased expression of PDK4 in phosphate-treated VSMCs induced mitochondrial dysfunction followed by apoptosis. Taken together, our results show that upregulation of PDK4 promotes vascular calcification by increasing osteogenic markers with no adverse effect on bone formation, demonstrating that PDK4 is a therapeutic target for vascular calcification.

Metabolic and transcriptional profiling reveals pyruvate dehydrogenase kinase 4 as a mediator of epithelial-mesenchymal transition and drug resistance in tumor cells.

BackgroundAccumulating preclinical and clinical evidence implicates epithelial-mesenchymal transition (EMT) in acquired resistance to anticancer drugs; however, mechanisms by which the mesenchymal state determines drug resistance remain unknown.ResultsTo explore a potential role for altered cellular metabolism in EMT and associated drug resistance, we analyzed the metabolome and transcriptome of three lung cancer cell lines that were rendered drug resistant following experimental induction of EMT. This analysis revealed evidence of metabolic rewiring during EMT that diverts glucose to the TCA cycle. Such rewiring was at least partially mediated by the reduced expression of pyruvate dehydrogenase kinase 4 (PDK4), which serves as a gatekeeper of the TCA cycle by inactivating pyruvate dehydrogenase (PDH). Overexpression of PDK4 partially blocked TGFβ-induced EMT; conversely, PDK4 inhibition via RNAi-mediated knockdown was sufficient to drive EMT and promoted erlotinib resistance in EGFR mutant lung cancer cells. We identified a novel interaction between PDK4 and apoptosis-inducing factor (AIF), an inner mitochondrial protein that appears to play a role in mediating this resistance. In addition, analysis of human tumor samples revealed PDK4-low as a predictor of poor prognosis in lung cancer and that PDK4 expression is dramatically downregulated in most tumor types.ConclusionsTogether, these findings implicate PDK4 as a critical metabolic regulator of EMT and associated drug resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/2049-3002-2-20) contains supplementary material, which is available to authorized users.

Diisopropylamine dichloroacetate, a novel pyruvate dehydrogenase kinase 4 inhibitor, as a potential therapeutic agent for metabolic disorders and multiorgan failure in severe influenza.

Severe influenza is characterized by cytokine storm and multiorgan failure with metabolic energy disorders and vascular hyperpermeability. In the regulation of energy homeostasis, the pyruvate dehydrogenase (PDH) complex plays an important role by catalyzing oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle and fatty acid synthesis, and thus its activity is linked to energy homeostasis. The present study tested the effects of diisopropylamine dichloroacetate (DADA), a new PDH kinase 4 (PDK4) inhibitor, in mice with severe influenza. Infection of mice with influenza A PR/8/34(H1N1) virus resulted in marked down-regulation of PDH activity and ATP level, with selective up-regulation of PDK4 in the skeletal muscles, heart, liver and lungs. Oral administration of DADA at 12-h intervals for 14 days starting immediately after infection significantly restored PDH activity and ATP level in various organs, and ameliorated disorders of glucose and lipid metabolism in the blood, together with marked improvement of survival and suppression of cytokine storm, trypsin up-regulation and viral replication. These results indicate that through PDK4 inhibition, DADA effectively suppresses the host metabolic disorder-cytokine cycle, which is closely linked to the influenza virus-cytokine-trypsin cycle, resulting in prevention of multiorgan failure in severe influenza.