Pyruvate Dehydrogenase Kinase Research Articles

A metabolic switch to the pentose-phosphate pathway induces radiation resistance in pancreatic cancer

PurposePancreatic ductal adenocarcinoma (PDAC) is remarkably resistant to standard modalities, including radiotherapy. We hypothesized that metabolic reprogramming may underlie PDAC radioresistance, and moreover, that it would be possible to exploit these metabolic changes for therapeutic intent. Methods and materialsWe established two matched models of radioresistant PDAC cells by exposing the AsPC-1 and MIAPaCa-2 human pancreatic cancer cells to incremental doses of radiation. The metabolic profile of parental and radioresistant cells was investigated using Nanostring technology, labeled-glucose tracing by liquid chromatography-mass spectrometry, Seahorse analysis and exposure to metabolic inhibitors. The synergistic effect of radiation combined with a pentose-phosphate pathway inhibitor, 6-aminonicotinamide (6-AN) was evaluated in a xenograft model established by subcutaneous injection of radioresistant-AsPC-1 cells into nude mice. ResultsThe radioresistant cells overexpressed pyruvate dehydrogenase kinase (PDK) and consistently, displayed increased glycolysis and downregulated the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Metabolic flux through the pentose-phosphate pathway (PPP) was increased, as were levels of reduced glutathione; pharmacological inhibition of the PPP dramatically potentiated radiation-induced cell death. Furthermore, the combined treatment of radiation with the PPP inhibitor 6-AN synergistically inhibited tumor growth in-vivo. ConclusionsWe provide a mechanistic understanding of the metabolic changes that underlie radioresistance in PDAC. Furthermore, we demonstrate that pancreatic cancer cells can be re-sensitized to radiation via metabolic manipulation, in particular, inhibition of the PPP. Exploitation of the metabolic vulnerabilities of radioresistant pancreatic cancer cells constitutes a new approach to pancreatic cancer, with a potential to improve clinical outcomes.

Fenbendazole and Diisopropylamine Dichloroacetate Exert Synergistic Anti-cancer Effects by Inducing Apoptosis and Arresting the Cell Cycle in A549 Lung Cancer Cells.

Lung cancer is the leading cause of cancer-related mortality worldwide, accounting for approximately 2 million new cases and 1.8 million deaths annually. Standard treatment options include surgery, radiation therapy, chemotherapy, and targeted therapies. Despite advancements over the past 25 years, the prognosis of patients with lung cancer remains poor. This study evaluated the synergistic anticancer effects of fenbendazole (FZ) and diisopropylamine dichloroacetate (DADA) on A549 lung cancer cells. Fenbendazole (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl) carbamate) is a broad-spectrum benzimidazole anthelmintic commonly used in veterinary medicine. Diisopropylamine Dichloroacetate (DADA), an over-the-counter treatment for chronic liver disease, has demonstrated anti-tumor properties as an inhibitor of pyruvate dehydrogenase kinase. The combination of FZ and DADA exhibited a synergistic effect on inhibiting the proliferation of A549 lung cancer cells. After 48 h of treatment, the FZ-DADA combination produced reactive oxygen species (ROS) and promoted apoptosis by down-regulating Bcl2 and up-regulating BAX protein expression. The combination activated caspase-3, caspase-7, and PARP, further driving apoptosis in A549 cells. The FZ-DADA treatment also induced cell cycle arrest, as evidenced by the inhibition of Cyclin A and Cyclin E proteins. The synergistic anticancer effects of the FZ-DADA combination were confirmed at both cellular and protein levels in A549 lung cancer cells. The combination modulates key apoptotic proteins, induces cell cycle arrest, and increases mitochondrial ROS production, suggesting a promising approach for lung cancer treatment that warrants further investigation and development.

Hypoxia-Induced Differences in the Expression of Pyruvate Dehydrogenase Kinase 1-Related Factors in the Renal Tissues and Renal Interstitial Fibroblast-like Cells of Yak (Bos Grunniens)

Hypoxia is one of the factors severely affect renal function, and, in severe cases, it can lead to renal fibrosis. Although much progress has been made in identifying the molecular mediators of fibrosis, the mechanisms that govern renal fibrosis remain unclear, and there have been no effective therapeutic anti-fibrotic strategies to date. Mammals exposed to low oxygen in the plateau environment for a long time are prone to high-altitude disease, while yaks have been living in the plateau for generations do not develop kidney fibrosis caused by low oxygen. It has been suggested that metabolic reprogramming occurs in renal fibrosis and that pyruvate dehydrogenase kinase 1 (PDK1) plays a crucial role in metabolic reprogramming as an important node between glycolysis and the tricarboxylic acid cycle. The aim of this study was to investigate the effects of hypoxia on the renal tissues and renal interstitial fibroblasts of yaks. We found that, at the tissue level, HIF-1α, PDK1, TGF-β1, Smad2, Smad3, and α-SMA were mainly distributed and expressed in tubular epithelial cells but were barely present in the renal mesenchymal fibroblasts of healthy cattle and yak kidneys. Anoptical density analysis showed that in healthy cattle kidneys, TGF-β1, Smad2, and Smad3 expression was significantly higher than in yak kidneys (p < 0.05), and HIF-1α and PDK1 expression was significantly lower than in yak kidneys (p < 0.05). The results at the protein and gene levels showed the same trend. At the cellular level, prolonged hypoxia significantly elevated PDK1 expression in the renal mesangial fibroblasts of cattle and yak kidneys compared with normoxia (p < 0.05) and was proportional to the degree of cellular fibrosis. However, PDK1 expression remained stable in yaks compared with renal interstitial fibroblast-like cells in cattle during the same hypoxic time period. At the same time, prolonged hypoxia also promoted changes in cellular phenotype, promoting the proliferation, activation, glucose consumption, lactate production, and anti-apoptosis in the both of cattle and yaks renal interstitial fibroblasts The differences in kidney structure and expression of PDK1 and HIF-1α in kidney tissue and renal interstitial fibroblasts induced by different oxygen concentrations suggest that there may be a regulatory relationship between yak kidney adaptation and hypoxic environment at high altitude. This provides strong support for the elucidation of the regulatory relationship between PDK1 and HIF-1α, as well as a new direction for the treatment or delay of hypoxic renal fibrosis; additionally, these findings provide a basis for further analysis of the molecular mechanism of hypoxia adaptation-related factors and the adaptation of yaks to plateau hypoxia.

Downregulation of CPEB3 exacerbates cardiac injury by inhibiting cardiomyocyte adaptive metabolic reprogramming after myocardial infarction

Abstract Background Adaptive metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis in hypoxia plays a protective role in cardiomyocyte survival by reducing reactive oxygen species (ROS) and increasing ATP. Cytoplasmic polyadenylation element binding protein 3 (CPEB3) influences protein translation by modulating 3' untranslated region (3'UTR) lengths through alternative polyadenylation (APA). However, the role of CPEB3 in cardiomyocyte after myocardial infarction (MI) remains unknown. Purpose This study aims to explore the function of CPEB3 in cardiomyocyte after MI. Methods Three GEO databases of mice MI (GSE110209, GSE114695 and GSE236374) were analyzed to identify CPEB3 dysregulation. Cardiomyocyte-specific CPEB3 knockout mice and CPEB3-knockdown neonatal mouse cardiomyocytes (NMCMs), RNA sequencing, flow cytometry, TUNEL staining, Seahorse, ROS and ATP measurements were used to investigate the impact of CPEB3. APA events analysis, RIP sequencing, CUT-TAG, 3’RACE, polysome profiling and dual luciferase report were performed to elucidate the mechanisms of CPEB3. Recombinant adeno-associated virus carrying cardiac troponin T promoter was used to evaluate the therapeutic efficacy of CPEB3 in mice with MI. Results CPEB3 expression was markedly reduced in the heart tissue after MI and in NMCMs after hypoxia. Cardiomyocyte-specific CPEB3 deletion aggravated cardiomyocyte apoptosis, enlarged infarct size, and exacerbated cardiac injury after MI. RNA sequencing revealed that CPEB3 deficiency predominantly inhibited glycolysis-related pathway, with a notable decrease in pyruvate dehydrogenase kinase 1 (PDK1) expression. In CPEB3-knockdown NMCMs, adaptive metabolic reprogramming from OXPHOS to glycolysis and ATP level were decreased in hypoxia, whereas ROS production was significantly enhanced, all of which resulted in apoptosis and were regulated by PDK1. Besides, PDK1 overexpression counteracted the effects of CPEB3 knockdown on metabolic pattern, ROS, ATP and apoptosis. Mechanistically, CPEB3 deficiency led to a shortened 3’UTR of the transcription factor forkhead box O3 (FOXO3), and a decline of FOXO3 protein level. CPEB3 protein was confirmed to directly bind to FOXO3 mRNA, and the translation efficiency of FOXO3 was decreased with CPEB3 knockdown. Furthermore, FOXO3 was found to activate the transcription of PDK1, and FOXO3 overexpression alleviated the decline of PDK1 and attenuated CPEB3 knockdown-induced apoptosis in hypoxia. Finally, cardiomyocyte-specific CPEB3 overexpression reduced cardiomyocyte apoptosis, suppressed myocardial fibrosis, and improved cardiac function by upregulating FOXO3 and PDK1 levels after MI. Conclusion CPEB3 could promote FOXO3 protein expression via APA of FOXO3 3’UTR, activate transcription of PDK1 and ultimately protect cardiomyocyte from apoptosis by restoring metabolic balance in hypoxia. CPEB3 may serve as a promising therapeutic target for cardiomyocyte apoptosis after MI.Schematic diagram

Dexmedetomidine Ameliorates Myocardial Ischemia-Reperfusion Injury by Inhibiting MDH2 Lactylation via Regulating Metabolic Reprogramming.

Myocardial ischemia-reperfusion injury (MIRI) significantly worsens the outcomes of patients with cardiovascular diseases. Dexmedetomidine (Dex) is recognized for its cardioprotective properties, but the related mechanisms, especially regarding metabolic reprogramming, have not been fully clarified. A total of 60 patients with heart valve disease are randomly assigned to Dex or control group. Blood samples are collected to analyze cardiac injury biomarkers and metabolomics. In vivo and vitro rat models of MIRI are utilized to assess the effects of Dex on cardiac function, lactate production, and mitochondrial function. It is found that postoperative CK-MB and cTNT levels are significantly lower in the Dex group. Metabolomics reveals that Dex regulates metabolic reprogramming and reduces lactate level. In Dex-treated rats, the myocardial infarction area is reduced, and myocardial contractility is improved. Dex inhibits glycolysis, reduces lactate, and improves mitochondrial function following MIRI. Lactylation proteomics identifies that Dex reduces the lactylation of Malate Dehydrogenase 2（MDH2), thus alleviating myocardial injury. Further studies reveal that MDH2 lactylation induces ferroptosis, leading to MIRI by impairing mitochondrial function. Mechanistic analyses reveal that Dex upregulates Nuclear Receptor Subfamily 3 Group C Member 1（NR3C1)phosphorylation, downregulates Pyruvate Dehydrogenase Kinase 4 (PDK4), and reduces lactate production and MDH2 lactylation. These findings provide new therapeutic targets and mechanisms for the treatment for MIRI.

Dichloroacetate reduces cisplatin-induced apoptosis by inhibiting the JNK/14-3-3/Bax/caspase-9 pathway and suppressing caspase-8 activation via cFLIP in murine tubular cells

Cisplatin-induced injury to renal proximal tubular cells stems from mitochondrial damage-induced apoptosis and inflammation. Dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, a potential generator of ROS and ATP, protects against cisplatin-induced nephrotoxicity by promoting the TCA cycle. However, its effects on apoptotic pathways and ROS production in renal tubular cells remain unclear. Here, we investigated the detailed molecular mechanisms of the DCA’s effects by immunoblot, RT-PCR, RNA-sequencing, and RNA-silencing in a murine renal proximal tubular (mProx) cell line and mouse kidneys. In mProx cells, DCA suppressed cisplatin-induced apoptosis by attenuating the JNK/14-3-3/Bax/caspase-9 and death receptor/ligand/caspase-8 pathways without impeding inflammatory signaling. RNA-sequencing demonstrated that DCA increased the cisplatin-reduced expression of cFLIP, a caspase-8 inactivator, and decreased the expression of almost all oxidative phosphorylation (OXPHOS) genes. DCA also increased NF-kB activation and ROS production, probably enhancing the cFLIP induction and OXPHOS gene reduction, respectively. Furthermore, cFLIP silencing weakened the DCA’s anti-apoptotic effects. Finally, in mouse kidneys, DCA attenuated cisplatin-caused injuries such as functional and histological damages, caspase activation, JNK/14-3-3 activation, and cFLIP reduction. Conclusively, DCA mitigates cisplatin-induced nephrotoxicity by attenuating the JNK/14-3-3/Bax/caspase-9 pathway and inhibiting the caspase-8 pathways via cFLIP induction, probably outweighing the cisplatin plus DCA-derived cytotoxic effects including ROS.

Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.

Pyruvate dehydrogenase kinase (PDK) 1 is one of four isozymes that inhibit the oxidative decarboxylation of pyruvate to acetyl-CoA via pyruvate dehydrogenase. PDK activity is elevated in fasting or starvation conditions to conserve carbohydrate reserves. PDK has also been shown to increase mitochondrial fatty acid utilization. In cardiomyocytes, metabolic flexibility is crucial for the fulfillment of high energy requirements. The PDK1 isoform is abundant in cardiomyocytes, but its specific contribution to cardiomyocyte metabolism is unclear. Here we show that PDK1 regulates cardiomyocyte fuel preference by mediating triacylglycerol turnover in differentiated H9c2 myoblasts using lentiviral shRNA to knockdown Pdk1 . Somewhat surprisingly, PDK1 loss did not affect overall PDH activity, basal glycolysis, or glucose oxidation revealed by oxygen consumption rate experiments and 13 C 6 glucose labelling. On the other hand, we observed decreased triacylglycerol turnover in H9c2 cells with PDK1 knockdown, which was accompanied by decreased mitochondrial fatty acid utilization following nutrient deprivation. 13 C 16 palmitate tracing of uniformly labelled acyl chains revealed minimal acyl chain shuffling within triacylglycerol, indicating that the triacylglycerol hydrolysis, and not re-esterification, was dysfunctional in PDK1 suppressed cells. Importantly, PDK1 loss did not significantly impact the cellular lipidome or triacylglycerol accumulation following palmitic acid treatment, suggesting that effects of PDK1 on lipid metabolism were specific to the nutrient-deprived state. We validated that PDK1 loss decreased triacylglycerol turnover in Pdk1 knockout mice. Together, these findings implicate a novel role for PDK1 in lipid metabolism in cardiomyocytes, independent of its canonical roles in glucose metabolism.

Pdk3's role in RANKL-induced osteoclast differentiation: insights from a bone marrow macrophage model.

Osteoporosis (OP) is a chronic disease characterized by decreased bone mass, loss of skeletal structural integrity and increased susceptibility to fracture. Available studies have shown that the pyruvate dehydrogenase kinase (PDK) family is associated with osteoclastogenesis and bone loss, but the specific role of Pdk3 in bone pathology has not been systematically investigated. A cell OP model was established in receptor activator for nuclear factor-κB Ligand (RANKL)-induced bone marrow macrophages (BMMs). Hereafter, the expression levels of Pdk3 and osteoclastogenesis feature genes including nuclear factor of activated T cells 1 (Nfatc1), Cathepsin K (Ctsk), osteoclast associated Ig-like receptor (Oscar) in BMMs-derived osteoclasts were examined based on real-time quantitative PCR and western blotting methods. Further, the phosphorylation of ERK, P65 and JAK/STAT and their correlation was Pdk3 was gauged. In particular, changes in the activity of these signaling pathways were observed by silencing experiments of the Pdk3 gene (using small interfering RNA). Finally, the effects of Pdk3 gene silencing on signaling pathway activity, osteoclastogenesis, and related inflammatory and apoptotic indicators were observed by transfection with PDK3-specific siRNA. Following RANKL exposure, the levels of Pdk3 and osteoclastogenesis feature genes were all elevated, and a positive correlation between Pdk3 and osteoclastogenesis feature genes was seen. Meanwhile, ERK, P65 and JAK/STAT phosphorylation was increased by RANKL, and Pdk3 was confirmed to be positively correlated with the phosphorylation of ERK, P65 and JAK/STAT. Additionally, in RANKL-exposed osteoclasts, Pdk3 knockdown diminished the phosphorylation of ERK, P65 and JAK/STAT, reduced the expressions of osteoclastogenesis feature genes. Importantly, knockdown of Pdk3 also reduced the expression of inflammatory cytokines and resulted in elevated levels of Bax and Casp3 expression, as well as downregulation of Bcl2 expression. This study reveals for the first time the role of Pdk3 in RANKL-induced osteoclastogenesis and OP. These findings provide a foundation for future studies on the role of Pdk3 in other bone diseases and provide new ideas for the development of OP therapeutics targeting Pdk3.

8071 Prevention Of Acute Kidney Injury By Reducing Mitochondrial Dysfunction In Diabetic Mice

Abstract Disclosure: M. Kim: None. C. Oh: None. A. Khang: None. J. Jeon: None. I. Lee: None. Background: Acute Kidney Injury (AKI) significantly contributes to the pathogenesis of diabetic kidney disease, often exacerbating chronic kidney disease (CKD) and accelerating the need for dialysis. Mitochondrial dysfunction, induced by an increase in reactive Oxygen Species (ROS) and inflammation, plays a crucial role in AKI. This study investigates the prevention of AKI by reducing mitochondrial dysfunction through pyruvate dehydrogenase kinase (PDK) inhibition in an ischemia-reperfusion (IR)-induced AKI model using streptozotocin (STZ)-induced diabetic mice. Methods: We utilized an IR-induced AKI model in STZ-induced diabetic C57BL6/J mice, subjecting them to IR by clamping both renal pedicles. Mitochondrial function tests, cellular apoptosis, and inflammatory markers were evaluated in NRK-52E cells and mouse primary tubular cells after hypoxia and reoxygenation using a hypoxia workstation. Results: In diabetic mice experiencing AKI following IR injury, there was a significant increase in pyruvate dehydrogenase E1α (PDHE1α) phosphorylation. This correlated with a notable increase in mitochondrial damage - TEM images of mice with IR-induced kidney injury revealed distension, loss of cristae, and fragmentation of the mitochondria. We also observed a decrease in oxygen consumption rate (OCR) accompanied by increased ROS, as well as increased production of inflammatory markers. Remarkably, the pan-PDK inhibitor, sodium dichloroacetate (DCA), mitigated apoptosis, attributable to the reduction of PDHE1α phosphorylation levels as well as normalized mitochondrial morphology and function. DCA treatment lessened oxidative stress and decreased the production of inflammatory markers following IR-induced AKI or hypoxia-reoxygenation injury. Conclusion: Restoration of mitochondrial function by DCA effectively alleviated renal injury by decreasing ROS production and inflammation, highlighting its critical role in IR-mediated damage. These results suggest another potential target for renal protection during AKI; therefore, the prevention of acute kidney injury through the inhibition of PDK could be a promising therapeutic approach for treating Diabetic Kidney Disease (DKD). Presentation: 6/1/2024

Novel Dichloroacetophenone-Based PDHK1 Inhibitors as Potent Anticancer Agents.

Pyruvate dehydrogenase kinases (PDHKs), important metabolic and abnormally expressed enzymes in cancer cells, are promising targets for cancer therapy, especially for non-small-cell lung cancer (NSCLC). In this study, a new hit, dichloroacetophenone (DAP) analog 9, was postulated to bind to the PDHK1 allosteric pocket, guided by molecular modeling and kinase biochemical experiments. Based on this binding mode, novel DAP analogs were designed and synthesized to confirm the importance of Phe180, Tyr411, and the hydrophobic core at the bottom of the pocket. This structure-activity relationship (SAR) study led to the discovery of a novel potent hybrid scaffold, dichloroacetophenone biphenylsulfone ether. Dichloroacetophenone biphenylsulfone ether 31 and 32 inhibited PDHK1 with IC50 values of 86 and 140 nM, respectively. Compound 32 with acceptable in vitro metabolic stability, predicted drug-likeness properties and ADME/T profiles, showed promising therapeutic efficacy in a lung cancer xenograft mouse model.

Inducible and reversible SOD2 knockdown in mouse skeletal muscle drives impaired pyruvate oxidation and reduced metabolic flexibility.

Skeletal muscle mitochondrial dysfunction is a key characteristic of aging muscle and contributes to age related diseases such as sarcopenia, frailty, and type 2 diabetes. Mitochondrial oxidative distress has been implicated as a driving factor in these age-related diseases, however whether it is a cause, or a consequence of mitochondrial dysfunction remains to be determined. The development of more flexible genetic models is an important tool to test the mechanistic role of mitochondrial oxidative stress on skeletal muscle metabolic dysfunction. We characterize a new model of inducible and reversible mitochondrial redox stress using a tetracycline controlled skeletal muscle specific short hairpin RNA targeted to superoxide dismutase 2 (iSOD2). iSOD2 KD and control (CON) animals were administered doxycycline for 3- or 12- weeks and followed for up to 24 weeks and mitochondrial respiration and muscle contraction were measured to define the time course of SOD2 KD and muscle functional changes and recovery. Maximum knockdown of SOD2 protein occurred by 6 weeks and recovered by 24 weeks after DOX treatment. Mitochondrial aconitase activity and maximum mitochondrial respiration declined in KD muscle by 12 weeks and recovered by 24 weeks. There were minimal changes in gene expression between KD and CON muscle. Twelve-week KD showed a small, but significant decrease in muscle fatigue resistance. The primary phenotype was reduced metabolic flexibility characterized by impaired pyruvate driven respiration when other substrates are present. The pyruvate dehydrogenase kinase inhibitor dichloroacetate partially restored pyruvate driven respiration, while the thiol reductant DTT did not. We use a model of inducible and reversible skeletal muscle SOD2 knockdown to demonstrate that elevated matrix superoxide reversibly impairs mitochondrial substrate flexibility characterized by impaired pyruvate oxidation. Despite the bioenergetic effect, the limited change in gene expression suggests that the elevated redox stress in this model is confined to the mitochondrial matrix.

ING5 inhibits aerobic glycolysis of lung cancer cells by promoting TIE1-mediated phosphorylation of pyruvate dehydrogenase kinase 1 at Y163.

Aerobic glycolysis is critical for tumor growth and metastasis. Previously, we have found that the overexpression of the inhibitor of growth 5 (ING5) inhibits lung cancer aggressiveness and epithelial-mesenchymal transition (EMT). However, whether ING5 regulates lung cancer metabolism reprogramming remains unknown. Here, by quantitative proteomics, we showed that ING5 differentially regulates protein phosphorylation and identified a new site (Y163) of the key glycolytic enzyme PDK1 whose phosphorylation was upregulated 13.847-fold. By clinical study, decreased p-PDK1Y163 was observed in lung cancer tissues and correlated with poor survival. p-PDK1Y163 represents the negative regulatory mechanism of PDK1 by causing PDHA1 dephosphorylation and activation, leading to switching from glycolysis to oxidative phosphorylation, with increasing oxygen consumption and decreasing lactate production. These effects could be impaired by PDK1Y163F mutation, which also impaired the inhibitory effects of ING5 on cancer cell EMT and invasiveness. Mouse xenograft models confirmed the indispensable role of p-PDK1Y163 in ING5-inhibited tumor growth and metastasis. By siRNA screening, ING5-upregulated TIE1 was identified as the upstream tyrosine protein kinase targeting PDK1Y163. TIE1 knockdown induced the dephosphorylation of PDK1Y163 and increased the migration and invasion of lung cancer cells. Collectively, ING5 overexpression-upregulated TIE1 phosphorylates PDK1Y163, which is critical for the inhibition of aerobic glycolysis and invasiveness of lung cancer cells.

A novel PDHK inhibitor restored cognitive dysfunction and limited neurodegeneration without affecting amyloid pathology in 5xFAD mouse, a model of Alzheimer’s disease

BackgroundAlzheimer’s disease (AD) is the most common form of dementia. Although drugs focusing on reducing amyloid β slow progression, they fail to improve cognitive function. Deficits in glucose metabolism are reflected in FDG-PET and parallel the neurodegeneration and synaptic marker loss closely preceding cognitive decline, but the role of metabolic deficits as a cause or consequence of neurodegeneration is unclear. Pyruvate dehydrogenase (PDH) is lost in AD and an important enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle by converting pyruvate into acetyl-CoA. It is negatively regulated by pyruvate dehydrogenase kinase (PDHK) through phosphorylation.MethodsIn the present study, we assessed the in vitro/ in vivo pharmacological profile of the novel PDHK inhibitor that we discovered, Compound A. We also assessed the effects of Compound A on AD-related phenotypes including neuron loss and cognitive impairment using 5xFAD model mice.ResultsCompound A inhibited human PDHK1, 2 and 3 but had no inhibitory activity on PDHK4. In primary neurons, Compound A enhanced pyruvate and lactate utilization, but did not change glucose levels. In contrast, in primary astrocytes, Compound A enhanced pyruvate and glucose utilization and enhanced lactate production. In an efficacy study using 5xFAD mice, Compound A ameliorated the cognitive dysfunction in the novel object recognition test and Morris water maze. Moreover, Compound A prevented neuron loss in the hippocampus and cerebral cortex of 5xFAD without affecting amyloid β deposits.ConclusionsThese results suggest ameliorating metabolic deficits by activating PDH by Compound A can limit neurodegeneration and is a promising therapeutic strategy for treating AD.

Effective strategies alleviate mitochondrial toxicity of perfluorooctanoic acid: Modification of functional head group and inhibition of toxic target

Minimizing the detrimental impacts of perfluorooctanoic acid (PFOA) on human health is a daunting task. Here, we aimed to propose effective strategies for reducing PFOA-induced mitochondrial toxicity in human liver and intestinal cells. PFOA could occupy the fatty acid-binding pockets of human peroxisome proliferator-activated receptor alpha (hPPARα). PFOA not only could structurally interact with hPPARα, but also substantially upregulated the expression levels of PPARα and its downstream gene (i.e., pyruvate dehydrogenase kinase (PDK4)). The increased expression of PDK4 was associated with the mitochondrial toxicity of PFOA, and inhibition of PDK4 or knock-down of PDK4 could effectively attenuate the mitochondrial toxicity of PFOA. Moreover, modification of carboxyl group via an esterification of PFOA into methyl perfluorooctanoate (MePFOA) decreased the affinity to hPPARα, resulting in the loss of upregulated expressions of PPARα and PDK4. Lower mitochondrial toxicity and cytotoxicity were found in the MePFOA-treated cells compared to PFOA exposure. Our study supported that the carboxyl group of PFOA (as functional head group) was required for inducing its mitochondrial toxicity. Two strategies, including modification of functional head group and inhibition of toxic target of PFOA, are feasible to ameliorate mitochondrial toxicity of PFOA.

O53 RENOMETABOLIC DISORDER IN EXPERIMENTAL RAT MODEL OF PCOS IS REVERSED VIA INHIBITION OF PYRUVATE DEHYDROGENASE KINASE 4

Background and Objective: Chronic Kidney disorders is a global public health problem, including in women with polycystic ovarian syndrome (PCOS), and is characterized by renal fibrosis, nephrotoxicity and glomerulonephritis, which increases the possibility of renal failure and organ transplant. Pyruvate dehydrogenase kinase 4 (PDK4) has been implicated in mitochondria dysfunction, contributing to metabolic dysregulation in different organs, including kidney. Studies have shown the metabolic regulatory impact of probiotics in human and experimental models. Hence, the present study investigated the therapeutic potential of probiotics on renometabolic disorders associated with experimental PCOS model. The study in addition elucidates the probable involvement of PDK4 in PCOS-associated renometabolic disorders. Methods: Eight-week-old nulliparous female Wistar rats were randomly allotted into four groups (n=6). Letrozole (1 mg/kg) was used to induce PCOS for 3 weeks. Thereafter, probiotics (2 x 107 CFU) was administered for 6 weeks, uninterruptedly. Biochemical parameters, histological analysis, immunohistochemistry and gene expression were carried out with appropriate methods. Results Experimental PCOS rats were characterized with elevated circulating testosterone and the presence of multiple ovarian cysts. In addition, rat with PCOS also manifested insulin resistance, increased plasma urea and creatinine levels, increased renal Gamma glutamyltransferase (GGT), MDA, NF-kappa B, TNF-alpha, TGF-beta 1, caspase-6, HDAC2, while a decrease in G6PD, GSH, renal NO and eNOS, when compared with animals in the control rats. These were associated with elevated level of PDK4 in the renal tissue. However, administration of probiotics ameliorates these renal/metabolic abnormalities. Conclusion: Altogether, the results from the present study revealed that probiotics ameliorates renal dysfunction in PCOS via downregulation of PDK4.

Oroxylin A, a broad‑spectrum anticancer agent, relieves monocrotaline‑induced pulmonary arterial hypertension by inhibiting the Warburg effect in rats.

Pulmonary arterial hypertension (PAH) is a chronic and fatal disease characterized by pulmonary vascular remodeling, similar to the 'Warburg effect' observed in cancer, which is caused by reprogramming of glucose metabolism. Oroxylin A (OA), an active compound derived from Scutellaria baicalensis, which can inhibit glycolytic enzymes [hexokinase 2 (HK2), Lactate dehydrogenase (LDH), and pyruvate dehydrogenase kinase 1 (PDK1) by downregulating aerobic glycolysis to achieve the treatment of liver cancer. To the best of our knowledge, however, the impact of OA on PAH has not been addressed. Consequently, the present study aimed to evaluate the potential protective role and mechanism of OA against PAH induced by monocrotaline (MCT; 55 mg/kg). The mean pulmonary artery pressure (mPAP) was measured using the central venous catheter method; HE and Masson staining were used to observe pulmonary artery remodeling. Non‑targeted metabolomics was used to analyze the metabolic pathways and pathway metabolites in MCT‑PAH rats. Western Blot analysis was employed to assess the levels of glucose transporter 1 (Glut1), HK2), pyruvate kinase (PK), isocitrate dehydrogenase 2 (IDH2), pyruvate dehydrogenase kinase 1(PDK1), and lactate dehydrogenase (LDH) protein expression in both lung tissue samples from MCT‑PAH rats. The results demonstrated that intragastric administration of OA (40 and 80 mg/kg) significantly decreased mPAP from 43.61±1.88 mmHg in PAH model rats to 26.51±1.53 mmHg and relieve pulmonary artery remodeling. Untargeted metabolomic analysis and multivariate analysis indicated abnormal glucose metabolic pattern in PAH model rats, consistent with the Warburg effect. OA administration decreased this effect on the abnormal glucose metabolism. The protein levels of key enzymes involved in glucose metabolism were evaluated by western blotting, which demonstrated that OA could improve aerobic glycolysis and inhibit PAH by decreasing the protein levels of Glut1, HK2, LDH, PDK1 and increasing the protein levels of PK and IDH2. In conclusion, OA decreased MCT‑induced PAH in rats by reducing the Warburg effect.

Higher levels of AKT-interacting protein in the frontal pole from people with schizophrenia are limited to a sub-group who have a marked deficit in cortical muscarinic M1 receptors

We are studying the molecular pathology of a sub-group within schizophrenia (∼ 25 %: termed Muscarinic Receptor Deficit subgroup of Schizophrenia (MRDS)) who can be separated because they have very low levels of cortical muscarinic M1 receptors (CHRM1). Based on our transcriptomic data from Brodmann's area ((BA) 9, 10 and 33 (controls, schizophrenia and mood disorders) and the cortex of the CHRM1−/− mouse (a molecular model of aberrant CHRM1 signaling), we predicted levels of AKT interacting protein (AKTIP), but not tubulin alpha 1b (TUBA1B) or AKT serine/threonine kinase 1 (AKT1) and pyruvate dehydrogenase kinase 1 (PDK1) (two AKTIP-functionally associated proteins), would be changed in MRDS. Hence, we used Western blotting to measure AKTIP (BA 10: controls, schizophrenia and mood disorders; BA 9: controls and schizophrenia) plus TUBA1B, AKT1 and PDK1 (BA 10: controls and schizophrenia) proteins. The only significant change with diagnosis was higher levels of AKTIP protein in BA 10 (Cohen's d = 0.73; p = 0.02) in schizophrenia compared to controls due to higher levels of AKTIP only in people with MRDS (Cohen's d = 0.80; p = 0.03). As AKTIP is involved in AKT1 signaling, our data suggests that signaling pathway is particularly disturbed in BA 10 in MRDS.

Dichloroacetate Prevents Sepsis Associated Encephalopathy by Inhibiting Microglia Pyroptosis through PDK4/NLRP3.

Dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, is often used to treat lactic acidosis and malignant tumors. Increasing studies have shown that DCA has neuroprotective effects. Here, we explored the role and mechanism of DCA in Sepsis associated encephalopathy (SAE). Single-cell analysis was used to determine the important role of PDK4 in SAE and identify the cell type. GO and GSEA analysis were used to determine the correlation between DCA and pyroptosis. Through LPS + ATP stimulation, a microglia pyroptosis model was established to observe the expression level of intracellular pyroptosis-related proteins under DCA intervention, and further detect the changes in intracellular ROS and JC-1. Additionally, a co-culture environment of microglia and neuron was simply constructed to evaluate the effect of DCA on activated microglia-mediated neuronal apoptosis. Finally, Novel object recognition test and the Morris water maze were used to explore the effect of DCA on cognitive function in mice from different groups after intervention. Based on the above experiments,this study concludes thatDCA can improve the ratio of peripheral and central M1 macrophages, inhibit NLRP3-mediated pyroptosis through ROS and mitochondrial membrane potential (MMP). DCA can reduce neuron death caused by SAE and improve cognitive function in LPS mice. In SAE, DCA may be a potential candidate drug for the treatment of microglia-mediated neuroinflammation.

The immunometabolic role of pyruvate dehydrogenase kinase-1 in smooth muscle cells in atherosclerosis

The immunometabolic role of pyruvate dehydrogenase kinase-1 in smooth muscle cells in atherosclerosis

Intracellular pH differentially regulates transcription of metabolic and signaling pathways in normal epithelial cells

Intracellular pH (pHi) dynamics regulate normal cell function, and dysregulated pHi dynamics is an emerging hallmark of cancer (constitutively increased pHi) and neurodegeneration (constitutively decreased pHi). However, the molecular mechanisms by which pHi dynamics regulate cell biology are poorly understood. Here, we discovered that altering pHi in normal human breast epithelial cells triggers global transcriptional changes. We identified 176 genes differentially regulated by pHi, with pHi-dependent genes clustering in signaling and glycolytic pathways. Using various normal epithelial cell models, we showed pH-dependent Notch homolog 1 protein expression, with increased protein abundance at high pHi. This resulted in pH-dependent downstream signaling, with increased Notch homolog 1 signaling at high pHi. We also found that high pHi increased the expression of glycolytic enzymes and regulators of pyruvate fate, including lactate dehydrogenase and pyruvate dehydrogenase kinase. These transcriptional changes were sufficient to alter lactate production, with high pHi shifting these normal epithelial cells toward a glycolytic metabolism and increasing lactate production. Thus, pHi dynamics transcriptionally regulate signaling and metabolic pathways in normal epithelial cells. Our data reveal new molecular regulators of pHi-dependent biology and a role for increased pHi in driving the acquisition of cancer-associated signaling and metabolic changes in normal human epithelial cells.