Pdk Gene Research Articles

BCKDK regulates the TCA cycle through PDC in the absence of PDK family during embryonic development.

Pyruvate dehydrogenase kinases (PDK1-4) inhibit the TCA cycle by phosphorylating pyruvate dehydrogenase complex (PDC). Here, we show that PDK family is dispensable for murine embryonic development and that BCKDK serves as a compensatory mechanism by inactivating PDC. First, we knocked out all four Pdk genes one by one. Surprisingly, Pdk total KO embryos developed and were born in expected ratios but died by postnatal day 4 because of hypoglycemia or ketoacidosis. Moreover, PDC was phosphorylated in these embryos, suggesting that another kinase compensates for PDK family. Bioinformatic analysis implicated branched-chain ketoacid dehydrogenase kinase (Bckdk), a key regulator of branched-chain amino acids (BCAAs) catabolism. Indeed, knockout of Bckdk and Pdk family led to the loss of PDC phosphorylation, an increase in PDC activity and pyruvate entry into the TCA cycle, and embryonic lethality. These findings reveal a regulatory crosstalk hardwiring BCAA and glucose catabolic pathways, which feed the TCA cycle.

Metabolic plasticity mediates differential responses to spring and autumn migrations: Evidence from gene expression patterns in migratory buntings.

What is the central question of this study? What are the molecular underpinnings of seasonal metabolic plasticity during spring and autumn migrations in songbirds? What is the main finding and its importance? We report differences in mRNA levels of genes involved in the regulation of glucose and fat metabolism between photoinduced non-migratory and migratory states and between the spring and autumn migratory states. Higher expression of genes associated with fat mobilization and energy generation in the spring than in the autumn migration suggests differential activation of the metabolic pathways or alteration in the efficiency of existing functional machinery during annual journeys between nearly fixed destinations. The molecular underpinnings of metabolic plasticity underlying differential responses to spring and autumn migrations are not well understood. We investigated this by examining the differences in mRNA levels of metabolic genes in the liver, muscle and adipose tissues of night-migratory red-headed buntings between photostimulated non-migratory and migratory states and between spring and autumn migratory states. Buntings accumulated more subcutaneous fat and hepatic lipid, had higher body mass, larger adipose cells and higher circulating triglyceride and free fatty acid levels and exhibited more intense Zugunruhe in the spring migratory state than in the autumn migratory state. More importantly, we found differences in the hepatic expression of pdc and pdk genes, indicating a differential acetyl-CoA requirement, and of the mdh and ogdh genes, suggesting differential oxidative phosphorylation between the non-migratory and migratory states and between the spring and autumn migratory states. Differences in fasn, bmal1 and glut1 mRNA levels were consistent with this and suggested seasonal differences in lipogenesis and/or glucose uptake. Likewise, differences in mRNA levels of genes coding for lipases (atgl and lpl) suggested that adipose triglycerides and free fatty acids serve largely as the metabolic substrate. Furthermore, changes in mRNA levels of genes coding for the fatty acid binding protein (fabp3) and fatty acid translocases (cd36) were consistent with differential fat fuel supply (via circulating free fatty acids) to aerobically exercising flight muscles between the spring and autumn migrations. These results show seasonal adaptation of genetic pathway(s) underlying seasonal metabolic plasticity that seems to mediate differential responses to spring and autumn migrations in latitudinal migratory songbirds.

Pyruvate dehydrogenase kinase 1 is essential for transplantable mouse bone marrow hematopoietic stem cell and progenitor function.

BackgroundAccumulating evidence suggests that hypoxic areas in the bone marrow are crucial for maintenance of hematopoietic stem cells (HSCs) by supporting a quiescent state of cell cycle and regulating the transplantation capacity of long-term (LT)-HSCs. In addition, HSCs seem to express a metabolic profile of energy production away from mitochondrial oxidative phosphorylation in favor of glycolysis. At oxygen deprivation, hypoxia inducible factor 1α (HIF-1α) is known to induce glycolytic enzymes as well as suppressing mitochondrial energy production by inducing pyruvate dehydrogenase kinase 1 (Pdk1) in most cell types. It has not been established whether PDK1 is essential for HSC function and mediates hypoxia-adapting functions in HSCs. While the Pdk gene family contains four members (Pdk1-4), it was recently shown that Pdk2 and Pdk4 have an important role in regulating LT-HSCs.Principle findingsHere we demonstrate that PDK1 activity is crucial for transplantable HSC function. Whereas Pdkl, Pdk2, and Pdk3 transcripts were expressed at higher levels in different subtypes of HSCs compared to differentiated cells, we could not detect any major differences in expression between LT-HSCs and more short-term HSCs and multipotent progenitors. When studying HIF-1α-mediated regulation of Pdk activity in vitro, Pdk1 was the most robust target regulated by hypoxia, whereas Pdk2, Pdk3, and Pdk4 were not affected. Contrary, genetic ablation in a cre-inducible Hif-1α knockout mouse did not support a link between HIF-1α and Pdk1. Silencing of Pdk1 by shRNA lentiviral gene transfer partially impaired progenitor colony formation in vitro and had a strong negative effect on both long-term and short-term engraftment in mice.ConclusionsOur study demonstrates that PDK1 has broad effects in hematopoiesis and is a critical factor for engraftment of both HSCs and multipotent progenitors upon transplantation to recipient mice. While Pdk1 was a robust hypoxia-inducible gene mediated by HIF-1α in vitro, we could not find evidence of any in vivo links between Pdk1 and HIF-1α.

Genetic Inactivation of Pyruvate Dehydrogenase Kinases Improves Hepatic Insulin Resistance Induced Diabetes

Pyruvate dehydrogenase kinases (PDK1-4) play a critical role in the inhibition of the mitochondrial pyruvate dehydrogenase complex especially when blood glucose levels are low and pyruvate can be conserved for gluconeogenesis. Under diabetic conditions, the Pdk genes, particularly Pdk4, are often induced, and the elevation of the Pdk4 gene expression has been implicated in the increased gluconeogenesis in the liver and the decreased glucose utilization in the peripheral tissues. However, there is no direct evidence yet to show to what extent that the dysregulation of hepatic Pdk genes attributes to hyperglycemia and insulin resistance in vivo. To address this question, we crossed Pdk2 or Pdk4 null mice with a diabetic model that is deficient in hepatic insulin receptor substrates 1 and 2 (Irs1/2). Metabolic analyses reveal that deletion of the Pdk4 gene had better improvement in hyperglycemia and glucose tolerance than knockout of the Pdk2 gene whereas the Pdk2 gene deletion showed better insulin tolerance as compared to the Pdk4 gene inactivation on the Irs1/2 knockout genetic background. To examine the specific hepatic effects of Pdks on diabetes, we also knocked down the Pdk2 or Pdk4 gene using specific shRNAs. The data also indicate that the Pdk4 gene knockdown led to better glucose tolerance than the Pdk2 gene knockdown. In conclusion, our data suggest that hepatic Pdk4 may be critically involved in the pathogenesis of diabetes.

Expression Characteristic of TaPDC-E1α Gene and Its regulatory Enzymes Gene in Male Sterile Line of Wheat

To understand the mechanism of male sterility induced by gametocide SQ-1 in wheat ( Triticum aestivum L.), TaPDC-E1α gene was isolated using silcon cloning technique. The open reading frame of this gene is 1401 bp in length, putatively encoding 388 amino acid residues. This gene possesses the conserved TPP domains. Two potential phosphorylation sites of serine residues might be present in the TaPDC-E1α protein of wheat. According to semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis, the expression levels of TaPDC-E1α in the physiological and genetic male sterile lines were lower than those in the fertile lines. Compared with the fertile lines, the expression of PDK was obviously down-regulated in the physiological male sterile line induced by SQ-1. However, PDK gene was highly expressed in the genetic male sterile lines. The expression levels of PDP gene were similar in the fertile and the male-sterile lines. These results suggest that the pathway of energy metabolism in the SQ-1 induced sterile line is more susceptible than that of the genetic male-sterile line. The upstream signal mechanism of mediating PDK gene may be inconsistent between the male-sterile line induced by SQ-1 and the genetic male-sterile line.

Blunted Akt/FOXO signalling and activation of genes controlling atrophy and fuel use in statin myopathy

Statins are used clinically for cholesterol reduction, but statin therapy is associated with myopathic changes through a poorly defined mechanism. We used an in vivo model of statin myopathy to determine whether statins up-regulate genes associated with proteasomal- and lysosomal-mediated proteolysis and whether PDK gene expression is simultaneously up-regulated leading to the impairment of muscle carbohydrate oxidation. Animals were dosed daily with 80 mg kg(-1) day(-1) simvastatin for 4 (n = 6) and 12 days (n = 5), 88 mg kg(-1) day(-1) simvastatin for 12 days (n = 4), or vehicle (0.5% w/v hydroxypropyl-methylcellulose and 0.1% w/v polysorbate 80; Control, n = 6) for 12 days by oral gavage. We found, in biceps femoris muscle, decreased Akt(Ser473), FOXO1(Ser253) and FOXO3a(Ser253) phosphorylation in the cytosol (P < 0.05, P < 0.05, P < 0.001, respectively) and decreased phosphorylation of FOXO1 in the nucleus after 12 days simvastatin when compared to Control (P < 0.05). This was paralleled by a marked increase in the transcription of downstream targets of FOXO, i.e. MAFbx (P < 0.001), MuRF-1 (P < 0.001), cathepsin-L (P < 0.05), PDK2 (P < 0.05) and PDK4 (P < 0.05). These changes were accompanied by increased PPARalpha (P < 0.05), TNFalpha (P < 0.01), IL6 (P < 0.01), Mt1A (P < 0.01) mRNA and increased muscle glycogen (P < 0.05) compared to Control. RhoA activity decreased after 4 days simvastatin (P < 0.05); however, activity was no different from Control after 12 days. Simvastatin down-regulated PI3k/Akt signalling, independently of RhoA, and up-regulated FOXO transcription factors and downstream gene targets known to be implicated in proteasomal- and lysosomal-mediated muscle proteolysis, carbohydrate oxidation, oxidative stress and inflammation in an in vivo model of statin-induced myopathy. These changes occurred in the main before evidence of extensive myopathy or a decline in the muscle protein to DNA ratio.

Three Members of the Human Pyruvate Dehydrogenase Kinase Gene Family Are Direct Targets of the Peroxisome Proliferator-activated Receptor β/δ

The nuclear receptors peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the metabolic syndrome. Here, we show that they are direct regulators of the family of pyruvate dehydrogenase kinase ( PDK) genes, whose products act as metabolic homeostats in sensing hunger and satiety levels in key metabolic tissues by modulating the activity of the pyruvate dehydrogenase complex. Mis-regulation of this tightly controlled network may lead to hyperglycemia. In human embryonal kidney cells we found the mRNA expression of PDK2, PDK3 and PDK4 to be under direct primary control of PPAR ligands, and in normal mouse kidney tissue Pdk2 and Pdk4 are PPAR targets. Both, treatment of HEK cells with PPARβ/δ-specific siRNA and the genetic disruption of the Pparβ/δ gene in mouse fibroblasts resulted in reduced expression of Pdk genes and abolition of induction by PPARβ/δ ligands. These findings suggest that PPARβ/δ is a key regulator of PDK genes, in particular the PDK4/ Pdk4 gene. In silico analysis of the human PDK genes revealed two candidate PPAR response elements in the PDK2 gene, five in the PDK3 gene and two in the PDK4 gene, but none in the PDK1 gene. For seven of these sites we could demonstrate both PPARβ/δ ligand responsiveness in context of their chromatin region and simultaneous association of PPARβ/δ with its functional partner proteins, such as retinoid X receptor, co-activator and mediator proteins and phosphorylated RNA polymerase II. In conclusion, PDK2, PDK3 and PDK4 are primary PPARβ/δ target genes in humans underlining the importance of the receptor in the control of metabolism.

Estrogen-related Receptors Stimulate Pyruvate Dehydrogenase Kinase Isoform 4 Gene Expression

The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK2 and PDK4) inhibits PDC activity. Expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA. In these studies we have investigated the transcriptional regulation of the PDK4 gene by the estrogen-related receptors (ERRalpha and ERRgamma). The ERRs are orphan nuclear receptors whose physiological roles include the induction of fatty acid oxidation in heart and muscle. Previously, we found that the peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of PDK4. Here we report that ERRalpha and ERRgamma stimulate the PDK4 gene in hepatoma cells, suggesting a novel role for ERRs in controlling pyruvate metabolism. In addition, both ERR isoforms recruit PGC-1alpha to the PDK4 promoter. Insulin, which decreases the expression of the PDK4 gene, inhibits the induction of PDK4 by ERRalpha and ERRgamma. The forkhead transcription factor (FoxO1) binds the PDK4 gene and contributes to the induction of PDK4 by ERRs and PGC-1alpha. Insulin suppresses PDK4 expression in part through the dissociation of FoxO1 and PGC-1alpha from the PDK4 promoter. Our data demonstrate a key role for the ERRs in the induction of hepatic PDK4 gene expression.

The evolution of C4 plants: acquisition of cis-regulatory sequences in the promoter of C4-type pyruvate, orthophosphate dikinase gene.

In a previous study, we identified the C4-like pyruvate, orthophosphate dikinase gene (Pdk) in the C3 plant rice, with a similar structure to the C4-type Pdk in the C4 plant maize. In order to elucidate the differences between C4-type and C4-like Pdk genes in C4 and C3 plants, we have produced chimeric constructs with the beta-glucuronidase (GUS) reporter gene under the control of the Pdk promoters. In transgenic rice, both rice and maize promoters directed GUS expression in photosynthetic organs in a light-dependent manner. However, the maize promoter exhibited a much higher transcriptional activity than the rice promoter did. These results indicate that the rice C4-like Pdk gene resembles the maize C4-type Pdk gene in terms of regulation of expression. We also tested the activity of the rice promoter in transgenic maize. GUS activity was seen in both photosynthetic and non-photosynthetic organs. Thus, the rice promoter does not confer a strict organ-specific gene expression, as the maize promoter does. Moreover, the rice promoter directed GUS expression not only in mesophyll cells but also in bundle sheath cells, whereas the maize promoter directed expression only in mesophyll cells. Taken together, the results obtained from both transgenic maize and rice demonstrate that the rice and maize promoters differ not only quantitatively, but also qualitatively, in terms of their cell- and organ-specificity. Experiments with swapped promoters using the rice and maize promoters further demonstrated that a limited sequence region from -330 to -76 of the maize promoter confers light-regulated, high-level expression to the rice promoter in maize mesophyll protoplasts. We conclude the gain of cis-acting elements conferring high-level expression and mesophyll cell specificity was necessary for establishment of a C4-type Pdk gene during the course of evolution from C3 to C4 plants.

Expression patterns of cytoplasmic pyruvate, orthophosphate dikinase of rice (C3) and maize (C4) in a C3 plant, rice

Two types of mRNAs are transcribed from the C4-type pyruvate, orthophosphate dikinase gene (Pdk) with different sizes, which encode chloroplastic and cytoplasmic forms of the enzyme. The two transcripts are pro duced by two independent promoters and this unusual dual promoter system is also found in the C4-like Pdk gene of the C3 plant, rice. In order to elucidate the expression pattern of the cytoplasmic transcript from the maize C4-type and rice C4-like Pdk genes, we have produced chimeric constructs with the ß-glucuronidase (GUS) reporter gene under the control of the cytoplasmic promoters and introduced the constructs into rice. Both cytoplasmic promoters directed GUS expression in non-photosynthetic organs, such as endosperm and roots, in transgenic rice plants, while expression was low in photosynthetic organs. These results indicate that the organ-specific localization of the cyto-plasmic enzyme is similar in C3 and C4 plants. The results also suggest the possibility that the cytoplasmic enzyme has a similar function(s) in non-photosynthetic organs both in C3 and C4 plants.

Analysis of promoter activity for the gene encoding pyruvate orthophosphate dikinase in stably transformed C4 flaveria species

The C4 enzyme pyruvate orthophosphate dikinase is encoded by a single gene, Pdk, in the C4 plant Flaveria trinervia. This gene also encodes enzyme isoforms located in the chloroplast and in the cytosol that do not have a function in C4 photosynthesis. Our goal is to identify cis-acting DNA sequences that regulate the expression of the gene that is active in the C4 cycle. We fused 1.5 kb of a 5' flanking region from the Pdk gene, including the entire 5' untranslated region, to the uidA reporter gene and stably transformed the closely related C4 species Flaveria bidentis. beta-Glucuronidase (GUS) activity was detected at high levels in leaf mesophyll cells. GUS activity was detected at lower levels in bundle-sheath cells and stems and at very low levels in roots. This lower-level GUS expression was similar to the distribution of mRNA encoding the nonphotosynthetic form of the enzyme. We conclude that cis-acting DNA sequences controlling the expression of the C4 form in mesophyll cells and the chloroplast form in other cells and organs are co-located within the same 5' region of the Pdk gene.

Diverse Mechanisms Regulate the Expression of Genes Coding for C4 Enzymes

Most of the enzymes of the C4 pathway of photosynthesis are compartmentalised in either mesophyll or bundle sheath cells. We have begun to dissect the mechanisms controlling the cell-specific expression of genes coding for C4 enzymes by locating the regulatory DNA sequences from two genes in the transformable C4 dicot, Flaveria bidentis. We show that chloroplast and cytosolic forms of pyruvate,orthophosphate dikinase are encoded by a single gene, Pdk. By fusing selected regions of the Pdk gene, to the gusA reporter gene we were able to infer promoter activities from measurements of GUS enzyme activity in transgenic F. bidentis plants. We show that the 5´ end of the Pdk gene contains sequences controlling high-level, mesophyll-specific expression of the C4 form of the enzyme. The promoter controlling low-level expression of the cytosolic form was located in the large intron. The C4 isoform of NADP-malic enzyme is encoded by the Me1 gene. Analyses of a series of reporter gene fusions showed that sequences at the 5´ end of the gene control bundle sheath expression but that sequences located near the 3´ end are necessary for high-level expression. The mechanisms regulating the expression of both genes are clearly different and suggest that there is no universal mechanism controlling the expression of genes coding for C4 enzymes.

Genomic structure and expression of the pyruvate, orthophosphate dikinase gene of the dicotyledonous C4 plant Flaveria trinervia (Asteraceae)

Pyruvate orthophosphate dikinase (PPDK) is a key enzyme of C4 photosynthesis providing the acceptor molecule for the primary CO2 fixation in the mesophyll cells. Here we present the isolation and characterisation of the corresponding gene (termed pdk) from the C4 plant Flaveria trinervia (Asteraceae). Southern analysis indicates that in contrast to maize pdk sequences in F. trinervia are present as single copy. Sequence analysis of the entire gene reveals that its coding sequence is identical to the previous isolated PPDK-cDNA from this species. The gene spans about 13 kb and consists of 21 exons, it thus contains two additional exons compared to the maize gene. As in maize, a long intervening sequence of 6.1 kb is positioned at the boundary of the transit peptide segment and the mature protein region. Pdk transcripts accumulate abundantly in leaves, but are also detectable in stems and roots. While the leaf and stem transcripts are 3.4 kb in size and encode the chloroplastic PPDK isoform, a 3.0 kb transcript lacking the region encoding the plastidic transit peptide accumulates in roots. Thus two different transcripts can be produced from a single pdk gene most likely by use of alternative promoters and not by alternative splicing. The accumulation of the 3.4 kb transcript is under light control. Darkening leads to a drastic depletion of this transcript in both leaves and stems. Instead, the 3.0 kb transit peptide-lacking pdk transcript accumulates, but only in stems and roots, not in leaves.