Astrocytic pyruvate dehydrogenase kinase-lactic acid axis involvement in glia-neuron crosstalk contributes to morphine-induced hyperalgesia in mice

The activation of spinal astrocytes accounts for opioid-induced hyperalgesia(OIH),but the underlying mechanisms remain elusive.The presence of astrocyte-neuron lactate shuttle(ANLS)makes astrocytes necessary for some neural function and communication.The aim of this study was to explore the role of ANLS in the occurrence and maintenance of OIH.After 7 days consecutive morphine injection,a mice OIH model was established and astrocytic pyruvate dehydrogenase kinase 4(PDK4),phosphorylated pyruvate dehydrogenase(p-PDH)and accumulation of L-lactate was elevated in the spinal dorsal horn.Intrathecally administration of inhibitors of PDK,lactate dehydrogenase 5 and monocarboxylate transporters to decrease the supply of L-lactate on neurons was observed to attenuate hypersensitivity behaviors induced by repeated morphine administration and downregulate the expression of markers of central sensitization in the spinal dorsal horns.The astrocyte line and the neuronal line were co-cultured to investigate the mechanisms in vitro.In this study,we demonstrated that morphine-induced hyperalgesia was sustained by lactate overload consequent upon aberrant function of spinal ANLS.In this process,PDK-p-PDH-lactate axis serves a pivotal role,which might therefore be a new target to improve long-term opioid treatment strategy in clinical practice.

Pyruvate dehydrogenase kinase is a negative regulator of interleukin-10 production in macrophages

Interleukin-10(IL-10)is the most potent anti-inflammatory cytokine in the body and plays an essential role in determining outcomes of many inflammatory diseases.Cellular metabolism is a critical determinant of immune cell function;however,it is currently unclear whether metabolic processes are specifically involved in IL-10 production.In this study,we aimed to find the central metabolic molecule regulating IL-10 production of macrophages,which are the main producers of IL-10.Transcriptomic analysis identified that metabolic changes were predominantly enriched in Kupffer cells at the early inflammatory phase of a mouse endotoxemia model.Among them,pyruvate dehydrogenase kinase(PDK)-dependent acute glycolysis was negatively involved in IL-10 production.Inhibition or knockdown of PDK selectively increased macrophage IL-10 expression.Mechanistically,PDK inhibition increased IL-10 production via profound phosphorylation of adenosine monophosphate(AMP)-activated protein kinase alpha 1(AMPKα1)by restricting glucose uptake in lipopolysaccharide-stimulated macrophages.AMPKα1 consequently activated p38 mitogen-activated protein kinase,c-Jun N-terminal kinase,and cyclic AMP-responsive element-binding protein to regulate IL-10 production.Our study uncovers a previously unknown regulatory mechanism of IL-10 in activated macrophages involving an immunometabolic function of PDK.

Deficiency of pyruvate dehydrogenase kinase 4 sensitizes mouse liver to diethylnitrosamine and arsenic toxicity through inducing apoptosis

Background and Aim:Pyruvate dehydrogenase kinase 4(PDK4)is a metabolism switch that regulates glucose oxidation and the tricarboxylic acid cycle(TCA cycle)in the mitochondria.Liver detoxifies xenobiotics and is constantly challenged by various injuries.This study aims at understanding how the loss of the metabolism regulator PDK4 contributes to liver injuries.Methods:Wild-type(WT)and Pdk4 knockout(Pdk4-/-)mice of different ages were examined for spontaneous hepatic apoptosis.Juvenile or adult mice of two genotypes were insulted by diethylnitrosamine(DEN),arsenic,galactosamine(GalN)/lipopolysaccharide(LPS),anti-CD95(Jo2)antibody or carbon tetrachloride(CCl4).Liver injury was monitored by blood biochemistry test.Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)staining,poly(ADP-ribose)polymerase(PARP)cleavage,and caspase activity assay.Inflammatory response was determined by nuclear factor(NF)-kB activation and the activation of NF-kB target genes.Primary hepatocytes were isolated and cell viability was evaluated by MTS assay.Results:We showed that systematic Pdk4-/-in mice resulted in age-dependent spontaneous hepatic apoptosis.PDK4-deficiency increased the toxicity of DEN in juvenile mice,which correlated with a lethal consequence and massive hepatic apoptosis.Similarly,chronic arsenic administration induced more severe hepatic apoptosis in Pdk4-/-mice compared to WT control mice.An aggravated hepatic NF-kB mediated-inflammatory response was observed in Pdk4-/-mice livers.In vitro,Pdk4-deficient primary hepatocytes were more vulnerable to DEN and arsenic challenges and displayed higher caspase activity than wild type cells.Notably,hepatic PDK4 mRNA level was remarkably reduced during acute liver failure induced by GalN/LPS or Jo2 antibody.The diminished PDK4 expression was also observed in CCl4-induced acute liver injury.Conclusions:PDK4 may contribute to the protection from apoptotic injury in mouse liver.

The pyruvate dehydrogenase kinase 2(PDK2)is associated with conidiation,mycelial growth,and pathogenicity in Fusarium graminearum

Pyruvate dehydrogenase kinase(PDK)is a mitochondrial enzyme in a variety of eukaryotes,including the plant pathogen Fusarium graminearum.This enzyme can reduce the oxidation of glucose to acetyl-coA by phosphorylation and selectively inhibits the activity of pyruvate dehydrogenase(PDH),which is a kind of pyruvate dehydrogenase complex(PDC).In this study,we investigated the F.graminearum pyruvate dehydrogenase kinase encoded by FgPDK2,which is a homologue of Neurospora crassa PDK2.The disruption of the FgPDK2 gene led to several phenotypic defects including effects on mycelial growth,conidiation,pigmentation,and pathogenicity.The mutants also showed decreased resistance to osmotic stress and cell membrane/wall-damaging agents.The FgPDK2 deletion mutant exhibited reduced virulence.All of these defects were restored by genetic complementation of the mutant with the complete FgPDK2 gene.Overall,the results demonstrated that FgPDK2 is crucial for the growth of F.graminearum and can be exploited as a potential molecular target for novel fungicides to control Fusarium head blight caused by F.graminearum.

Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat

To further research the regulatory network of pyruvate dehydrogenase kinase （designated as TaPDK） in physiological male-sterility （PHYMS） of wheat induced by chemical hybridizing agent （CHA） SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium （SD/-Ade/-His/-Leu/-Trp） （QDO）, and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor （TanLTP）, polyubiquitin （TaPUbi）, glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen （TaPCNA）, CBS domain containing protein （TaCBS）, actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.

AAZ2 induces mitochondrial-dependent apoptosis by targeting PDK1 in gastric cancer

Drastic surges in intracellular reactive oxygen species(ROS)induce cell apoptosis,while most chemotherapy drugs lead to the accumulation of ROS.Here,we constructed an organic compound,arsenical N-(4-(1,3,2-dithiarsinan-2-yl)phenyl)acrylamide(AAZ2),which could prompt the ROS to trigger mitochondrial-dependent apoptosis in gastric cancer(GC).Mechanistically,by targeting pyruvate dehydrogenase kinase 1(PDK1),AAZ2 caused metabolism alteration and the imbalance of redox homeostasis,followed by the inhibition of phosphoinositide-3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway and leading to the activation of B-cell lymphoma 2(Bcl2)/Bcl2-associated X(Bax)/caspase-9(Cas9)/Cas3 cascades.Importantly,our in vivo data demonstrated that AAZ2 could inhibit the growth of GC xenograft.Overall,our data suggested that AAZ2 could contribute to metabolic abnormalities,leading to mitochondrial-dependent apoptosis by targeting PDK1 in GC.