Pyruvate Dehydrogenase Protein Research Articles

Conserved moonlighting protein pyruvate dehydrogenase induces robust protection against Staphylococcus aureus infection

Developing an effective Staphylococcus aureus (S. aureus) vaccine has been a challenging endeavor, as demonstrated by numerous failed clinical trials over the years. In this study, we formulated a vaccine containing a highly conserved moonlighting protein, the pyruvate dehydrogenase complex E2 subunit (PDHC), and showed that it induced strong protective immunity against epidemiologically relevant staphylococcal strains in various murine disease models. While antibody responses contributed to bacterial control, they were not essential for protective immunity in the bloodstream infection model. Conversely, vaccine-induced systemic immunity relied on γδ T cells. It has been suggested that prior S. aureus exposure may contribute to the reduction of vaccine efficacy. However, PDHC-induced protective immunity still facilitated bacterial clearance in mice previously exposed to S. aureus. Collectively, our findings indicate that PDHC is a promising serotype-independent vaccine candidate effective against both methicillin-sensitive and methicillin-resistant S. aureus isolates.

Sperm proteome and sperm parameters of captive Amazon squirrel monkeys (Saimiri collinsi Osgood, 1916) during the non-fatted and fatted condition

The aims of this work were (1) to characterize the proteome profile of Saimiri collinsi (Amazon squirrel monkey) sperm, and (2) to evaluate if there are meaningful differences in sperm quality and sperm protein landscape during the non-fatted and fatted conditions of the animals. Semen of adult males (n=4) was collected by electroejaculation and diluted in ACP-118® extender. Sperm concentration, motility, vigor, plasma membrane integrity and functionality, and sperm morphology were evaluated. Sperm proteome was characterized by mass spectrometry. The non-fatted and fatted conditions were defined according to the fattening index. There were increases in seminal volume, pH, and sperm motility of S. collinisi during the fatted in comparison with the non-fatted condition (p < 0.05). Animals in the fatted condition had greater body mass (909 ± 23.98 g) in comparison with the non-fatted condition (818.8 ± 5.97 g, p = 0.0001). There were 1161 proteins identified in the sperm of the monkeys. Thirty-seven proteins were upregulated (p < 0.05) in non-fatted condition (such as coiled-coil domain-containing protein 105, protein kinase C alpha type isoform X1, pyruvate dehydrogenase protein X component – mitochondrial and heat shock 70 kDa protein 1-like) and they were involved in cellular and metabolic processes, catalytic activity and binding. Twenty-eight sperm proteins proteins were upregulated (p < 0.05) in fatted condition (such as Cytochrome b-c1 complex subunit 2, mitochondrial; calcium-binding mitochondrial carrier protein SCaMC-1-like; heat shock 70 kDa protein 13, and izumo sperm-egg fusion protein 1), participating in cellular processes, metabolic processes, and localization, binding, catalytic activity, ATP-dependent activity, and transport activity. Functional annotations of sperm proteins upregulated in non-fatted condition were described as hydrogen ion transport, chaperone, cilium, flagellum, and cell projection. In in fatted conditions, such annotations were described as fertilization, spermatogenesis and transport, chaperone, and motor protein. The present study brings an unprecedent contribution to our understanding about the reproductive biology of adult Saimiri collinsi. It also provides support for conservation and development of assisted reproductive technologies for Neotropical primates.

Thermoneutral housing increases the obesogenic phenotype of mice, while some effects of exercise are reduced

Introduction: Mice are extensively used to investigate the molecular foundations of obesity and the beneficial effect of exercise training (ET). Mice are most often housed at ambient temperature, ~22°C, which is below their thermoneutral environment (~30°C). Mild cold stress at ambient temperature accelerates mouse metabolic rate and may confound translation of results to humans. Aiming to determine the metabolic adaptations of ET in an obesogenic environment, we hypothesized that thermoneutral housing would increase adiposity during high-fat feeding. Moreover, thermoneutral housing would blunt the effects of ET by relieving the added cold stress. Methods: After temperature acclimatization (3 weeks, 22°C vs. 30°C), C57BL/6J mice (n=68, ♀, Age: 11 weeks) were dual-housed for 8 weeks receiving a chow diet or a 60%E high-fat diet (HFD). HFD mice were then separated into two additional groups with access to voluntary wheel running for another 8 weeks. Metabolic parameters were measured using magnetic resonance imaging, indirect calorimetry, glucose tolerance tests, and maximal running capacity on treadmill. Brown and white adipose tissues and gastrocnemius muscle mRNA and protein content were analyzed using qPCR and immunoblotting analyses, respectively. Results: In chow-fed mice, thermoneutral housing decreased metabolic rate (VO2), fasting plasma glucose, and thermogenic genes in adipose tissue depots, without changes in skeletal muscle mitochondrial protein content compared to 22°C. In HFD mice, thermoneutral housing increased fat mass, induced glucose intolerance, and fasting plasma insulin levels compared to 22°C housing. This was despite decreased food intake and metabolic rate compared to HFD mice housed at 22°C. ET mice improved the exercise capacity and glucose tolerance independent of housing temperature. Interestingly, while ET at 30°C prevented further fat gain during the intervention, ET at 22°C decreased fat mass. Moreover, only ET at 22°C increased the metabolic rate during the active phase. Gene expression responded similarly to ET independent of housing temperature in adipose tissue depots. ET induced GLUT4, peroxiredoxin 2 and pyruvate dehydrogenase protein content to a lesser extent in muscle at 30°C housing. Conclusion: Adaptations to diet-induced obesity and ET in mice critically depend upon housing temperature. Our findings underscore housing temperature as a key consideration in research related the beneficial effect of exercise in an obesogenic environment. Disclosure of funding: SHR is supported by the Lundbeck Foundation and Independent Research Fund Denmark. LS and EAR are supported by the Novo Nordisk Foundation and Independent Research Fund Denmark. IK is supported by the Novo Nordisk Foundation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comparison Between Dichloroacetate and Phenylbutyrate Treatment for Pyruvate Dehydrogenase Deficiency.

Pyruvate dehydrogenase (PDH) deficiency is caused by a number of pathogenic variants and the most common are found in the PDHA1 gene. The PDHA1 gene encodes one of the subunits of the PDH enzyme found in a carbohydrate metabolism pathway involved in energy production. Pathogenic variants of PDHA1 gene usually impact the α-subunit of PDH causing energy reduction. It potentially leads to increased mortality in sufferers. Potential treatments for this disease include dichloroacetate and phenylbutyrate, previously used for other diseases such as cancer and maple syrup urine disease. However, not much is known about their efficacy in treating PDH deficiency. Effective treatment for PDH deficiency is crucial as carbohydrate is needed in a healthy diet and rice is the staple food for a large portion of the Asian population. This review analysed the efficacy of dichloroacetate and phenylbutyrate as potential treatments for PDH deficiency caused by PDHA1 pathogenic variants. Based on the findings of this review, dichloroacetate will have an effect on most PDHA1 pathogenic variant and can act as a temporary treatment to reduce the lactic acidosis, a common symptom of PDH deficiency. Phenylbutyrate can only be used on patients with certain pathogenic variants (p.P221L, p.R234G, p.G249R, p.R349C, p.R349H) on the PDH protein. It is hoped that the review would provide an insight into these treatments and improve the quality of lives for patients with PDH deficiency.

Proteomic analysis of the mitochondrial glucocorticoid receptor interacting proteins reveals pyruvate dehydrogenase and mitochondrial 60 kDa heat shock protein as potent binding partners

Glucocorticoids are steroid hormones that regulate plethora biological actions such as growth and metabolism, immune response, and apoptosis. Glucocorticoids actions are mediated via glucocorticoid receptors which act mainly as transcription factors, but it is also found to be localized in mitochondria. Mitochondrial localization of the receptor indicates novel functions of the receptor. Characterization of the mitochondrial glucocorticoid receptor (mtGR) interacting proteins will shed light on these actions and the biochemical mechanisms that underlie mitochondrial glucocorticoid receptor import and functions. In this study, applying immunoprecipitation, mass spectrometry and Western blot analysis of the GR interacting proteins in total or mitochondrial extracts of HepG2 cells and of HepG2 cells overexpressing a mitochondrial targeted GR we found pyruvate dehydrogenase (PDH), chaperones such as and heat shock protein (HSP) −60, −70, −75 and −90, and 78 kDa glucose-regulated protein, mitochondrial transcription factors and enzymes involved in the regulation of the mitochondrial protein biosynthesis, lipid metabolism, ATP production and apoptosis as glucocorticoid receptor interacting proteins. Our results uncover potential novel mitochondrial partners of the receptor, suggesting possible new regulatory roles of mtGR in the control of mitochondrial-associated functions of the cell. SignificanceIn this study the mitochondrial GR interacting proteins were characterized highlighting novel regulatory roles of the receptor in mitochondria. Detection of the mtGR/PDH and mtGR/HSP60 interaction in almost all the analyses performed uncovered PDH and HSP60 proteins as potent mtGR binding partners. The interesting finding of the PDH/mtGR interaction possibly indicates involvement of mtGR in the regulation of the balance between glycolytic and oxidative phosphorylation energy production. Characterization of the mitochondrial heat shock −60, −70, −75 and 78 proteins as mtGR binding partners contribute to the characterization of the biochemical mechanisms of the mitochondrial import of the receptor. Moreover, identification of mitochondrial heat shock proteins, metabolic enzymes, transcription factors, OXPHOS, and regulatory molecules in mitochondrial protein biosynthesis as mtGR binding partners indicates possible new regulatory roles of mtGR in the glucocorticoids-induced regulation and orchestration of nuclear and mitochondrial functions, the exact biochemical mechanism of which remain to be established. The study discloses potential new regulatory roles of the receptor in mitochondria, pointing out its importance as a promising target molecule for the control of the mitochondria-associated pathophysiology of the cell.

Abnormal mitochondrial structure and function are retained in gingival tissues and human gingival fibroblasts from patients with chronic periodontitis.

The abnormal structure and function of mitochondria in cells is closely associated with inflammatory diseases. However, the physiology of mitochondria within gingival tissues and human gingival fibroblasts (HGFs) in patients with chronic periodontitis (CP) remains unclear. The objective of this study was to investigate the structure profile and function of mitochondria in gingival tissues and in HGFs derived from patients with or without CP. These features of mitochondria in HGFs were further analyzed when HGFs were induced by lipopolysaccharide (LPS) from Porphyromonas gingivalis (P.g). Gingival tissues and HGFs were collected from CP and healthy patients. Mitochondrial structure was assessed by transmission electron microscopy. Tissues or cells lysis was performed for mitochondrial DNA (mtDNA) quantification, and real-time polymerase chain reaction (RT-PCR) tests were used to determine mtDNA copy numbers. Western blot analysis was used to evaluate autophagy-related protein (ATG)-5, microtubule-associated protein light chain 3 (LC3), and mitochondrial matrix protein pyruvate dehydrogenase kinase isozyme 2 (PDK2) levels in tissues and HGFs from CP and healthy individuals. Tissues and HGFs from CP showed a significant greater mitochondrial structure destruction, lower mtDNA level, increased ATG5, LC3-II, and lower PDK2 protein levels than those of healthy individuals. In addition, LPS from P.g also triggered the same results in HGFs from healthy donors. Moreover, the challenge of HGFs from CP with LPS worsened these parameters. Mitochondrial structure and function within gingival tissues and HGFs from CP individuals were abnormal compared to those from healthy donors, and LPS could promote mitochondrial destruction.

Evaluation of immune effect of Streptococcus suis biofilm-associated protein PDH

As a zoonotic pathogen, Streptococcus suis(S. suis) takes pigs as the main host and is mainly colonizes in the upper respiratory tract and tonsil of pigs, causing septicemia, endocarditis and meningitis in pigs. Pyruvate dehydrogenase (PDH) is an enzyme that catalyzes the conversion of pyruvate to acetyl-CoA. As an immunogenic membrane-associated protein in S. suis, it has been found to be closely related to the formation of biofilm. In this study, the recombinant PDH (rPDH) of S. suis ZY05719 (serotype 2) was expressed and purified in E. coli by His affinity chromatography. Western blotting analysis showed that there was a strong specific reaction between PDH protein and PDH antiserum. Mice were immunized with recombinant PDH and inactivated bacteria, and the relative survival rates were 70 % and 60 %, respectively. In addition, mice immunized with PDH caused high levels of antibodies and high expression of immune-related genes in the spleen, which significantly protected the liver, brain and spleen from pathological damage. In addition, PDH antiserum could significantly inhibit the growth of S. suis and the formation of S. suis biofilm in vitro. These results further suggest that PDH is a promising candidate for S. suis biofilm-related subunit vaccine.

MiR-26a enhances colorectal cancer cell growth by targeting RREB1 deacetylation to activate AKT-mediated glycolysis.

We previously reported the inhibitory effects of microRNA-26a (miR-26a) on the conversion of pyruvate to acetyl coenzyme A in glucose metabolism by directly targeting pyruvate dehydrogenase protein X component in colorectal cancer (CRC) cells (Chen B et al., BMC Cancer 2014). Here, using microRNA in situ hybridization, we confirmed that miR-26a levels were elevated in 77 human CRC tissue samples and further investigated the key miR-26a-mediated metabolic regulation elements and signaling pathways in CRC cells through quantitative proteomic dissection combined with cancer cell biology and biochemical loss-of-function analysis. We found that AKT transcription signaling was a target pathway via miR-26a-mediated deacetylation modification of Ras-responsive element-binding protein 1 (RREB1) at the Lys-60 residue. miR-26a improved the deacetylation level of RREB1, thus contributing to RREB1 binding to the AKT1 promoter to activate AKT transcription and its related signaling pathway in glycolysis. Moreover, miR-26a promoted CRC tumorigenesis in CRC cells and subcutaneous xenograft mice. Thus, miR-26a is a key regulator of CRC tumorigenesis that mediates the deacetylation modification of RREB1 to enhance AKT1 transcription and downstream target gene expression in glycolysis for CRC growth.

P–341 Epigenetic role of the H2BK5ac histone modification on the expression of ALX1 and PDHX genes in the endometriotic tissues and normal endometrium

Abstract Study question Evolution of the ALX1 and PDHX genes expression and incorporation of H2BK5ac mark on the promoter of these genes in endometriotic tissues versus normal endometrium Summary answer Lower incorporation of H2BK5ac mark in ALX1 and PDHX promoters can because to downregulation of these genes in endometriotic tissues compared to normal endometrium. What is known already Endometriosis is considered as multifactorial disease affected by genetic, hormonal, and environmental factors. Recent evidences suggest the role of epigenetic mechanisms in this disease. Aristaless-like homeobox1 (ALX1) and Pyruvate Dehydrogenase Protein X (PDHX) genes are considered in this study. Studies show that upregulation of the ALX1 gene cause cell proliferation, migration, and invasion in cancer cells. PDHX is involved in cellular metabolism and acts as a tumor suppressor gene while maintaining normal homeostasis. It is Hypothesized that H2BK5ac which is known as a dynamic marker in promoter regions of active genes, may be involved in regulation of these gene expression. Study design, size, duration Ten eutopic and ectopic endometrium tissue, as well as ten normal endometrium, were collected. Ectopic biopsies were obtained using diagnostic laparoscopy, while the endometrial control samples and eutopic samples were collected via pipelle. Participants/materials, setting, methods RNA extraction and cDNA synthesis were done then the expression of ALX1 and PDHX genes evaluated by quantitative real-time PCR. Promoter regions of mentioned genes investigated for the incorporation of the epigenetic mark of H2BK5ac using Chromatin immunoprecipitation (ChIP) followed by real-time PCR. Data analysis performed using One-way ANOVA analysis (SPSS software) considered the significant level of P &amp;lt; 0.05. Main results and the role of chance Results showed that the expression of ALX1 was significantly decreased in eutopic endometrial samples compared to normal endometrium (p = 0.007). Also, there was a significant reduction in PDHX mRNA level in the eutopic and ectopic samples vs. normal endometrium (p = .017 and p =.021, respectively). The chromatin immunoprecipitation real-time PCR (ChIP PCR) analyses showed significantly lower incorporation of H2BK5ac epigenetic mark in ALX1 promoter in eutopic endometrial samples compared to normal endometrium (p = 0.007). Also, reduced incorporation of H2BK5ac at the PDHX promoter region was observed in both eutopic and ectopic endometrial samples compared to normal endometrium (p = 0.004 and p = 0.003, respectively). Limitations, reasons for caution The main limitation of our study is the low number of samples. Wider implications of the findings: Our results suggest that the marked lower levels of H2BK5ac in regulatory regions of ALX1and PDHX might lead to deregulation of these genes in tissue endometriotic samples. Trial registration number ‘not applicable’ for non-clinical trials

Abstract 2824: SHP2-PDHA metabolic axis is critical for adipocyte function by mitigating ER stress-induced adipocytes dysfunction: Implication for obesity-driven pancreatic ductal adenocarcinoma progression

Abstract Obesity remains a major risk factor for the development of pancreatic cancer. Animal studies demonstrated that obese mice develop larger and more metastatic pancreatic cancer compared to their lean counterpart. Within the tumor environment are adipocytes that secrete adipokines, which promote inflammation and directly stimulate pancreatic cancer cells. Understanding the mechanisms that alter adipocyte function and adipokine secretion is critical for circumventing adipocyte-mediated influence on pancreatic cancer progression. Targeting of the Src homology region 2 containing protein tyrosine phosphatase (SHP-2) in the cancer cells has recently shown promising potential, yet the effect of SHP2 inhibition on stromal components has not been addressed. Here we demonstrate that SHP2 and the mitochondrial protein pyruvate dehydrogenase (PDHA) are critical mediators of adipocyte function, through mitigation of ER stress in adipocytes. In order to understand the role of SHP2 and PDHA in adipocytes, we performed Oil Red O staining and western blot assays on cultured adipocytes. We found that the transition from pre-adipocytes to fully developed adipocytes is marked by significant accumulation of phospho-SHP2, PDHA and the ER stress marker Bip. Chemical inhibition of SHP2 or PDHA augmented ER stress and impaired lipid production in the adipocytes; these effects were reversed by the ER stress inhibitor, 4-Phenylbyturic acid (4-PBA). Moreover, pancreatic cancer cells exposed to conditioned media from differentiated adipocytes, in which either SHP2 or PDHA was inhibited, are less aggressive and invasive compared to PDAC cells grown in conditioned media from control adipocytes with no inhibitor treatment. Therefore, our studies indicate that SHP2 and PDHA, in adipocytes, are key mediators of obesity-driven pancreatic cancer progression. Future studies will focus on understanding the SHP2-PDHA axis in adipocytes and further to elucidate how cancer cell extrinsic effects of SHP2 inhibition could alter PDAC progression. Citation Format: Appolinaire A. Olou, Jarrid Jack, Sharon Manley, Joseph Ambrose, McKinnon Walsh, Austin Eades, Michael N. VanSaun. SHP2-PDHA metabolic axis is critical for adipocyte function by mitigating ER stress-induced adipocytes dysfunction: Implication for obesity-driven pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2824.

Comparative Analysis of Skeleton Muscle Proteome Profile between Yak and Cattle Provides Insight into High-Altitude Adaptation

Background::Mechanisms underlying yak adaptation to high-altitude environments have been investigated at the levels of morphology, anatomy, physiology, genome and transcriptome, but have not been explored at the proteome level.Objective:The protein profiles were compared between yak and cattle to explore molecular mechanisms underlying yak adaptation to high altitude conditions.Methods:In the present study, an antibody microarray chip was developed, which included 6,500 mouse monoclonal antibodies. Immunoprecipitation and mass spectrometry were performed on 12 selected antibodies which showed that the chip was highly specific. Using this chip, muscle tissue proteome was compared between yak and cattle, and 12 significantly Differentially Expressed Proteins (DEPs) between yak and cattle were identified. Their expression levels were validated using Western blot.Results:ompared with cattle, higher levels of Rieske Iron-Sulfur Protein (RISP), Cytochrome C oxidase subunit 4 isoform 1, mitochondrial (COX4I1), ATP synthase F1 subunit beta (ATP5F1B), Sarcoplasmic/ Endoplasmic Reticulum Calcium ATPase1 (SERCA1) and Adenosine Monophosphate Deaminase1 (AMPD1) in yak might improve oxygen utilization and energy metabolism. Pyruvate Dehydrogenase protein X component (PDHX) and Acetyltransferase component of pyruvate dehydrogenase complex (DLAT) showed higher expression levels and L-lactate dehydrogenase A chain (LDHA) showed lower expression level in yak, which might help yak reduce the accumulation of lactic acid. In addition, higher expression levels of Filamin C (FLNC) and low levels of AHNAK and Four and a half LIM domains 1 (FHL1) in yak might reduce the risks of pulmonary arteries vasoconstriction, remodeling and hypertension.Conclusion:Overall, the present study reported the differences in protein profile between yak and cattle, which might be helpful to further understand molecular mechanisms underlying yak adaptation to high altitude environments.

Sevoflurane preconditioning attenuates hypoxia/reoxygenation injury of H9c2 cardiomyocytes by activation of the HIF-1/PDK-1 pathway.

BackgroundSevoflurane preconditioning (SPC) can provide myocardial protective effects similar to ischemic preconditioning (IPC). However, the underlying molecular mechanism of SPC remains unclear. Studies confirm that hypoxia-inducible factor-1 (HIF-1) can transform cells from aerobic oxidation to anaerobic glycolysis by activating the switch protein pyruvate dehydrogenase kinase-1 (PDK-1), thus providing energy for the normal life activities of cells under hypoxic conditions. The purpose of this study was to investigate whether the cardioprotective effects of SPC are associated with activation of the HIF-1a/PDK-1 signal pathway.MethodsThe H9c2 cardiomyocytes hypoxia/reoxygenation model was established and treated with 2.4% sevoflurane at the end of equilibration. Lactate dehydrogenase (LDH) level, cell viability, cell apoptosis, mitochondrial membrane potential, key enzymes of glycolysis, ATP concentration of glycolysis were assessed after the intervention. Apoptosis related protein(Bcl-2, Bax), HIF-1a protein, and PDK-1 protein were assessed by western blot.ResultsCompared with the H/R group, SPC significantly increased the expression of HIF-1a, PDK-1, and Bcl-2 and reduced the protein expression of Bax, which markedly decreased the apoptosis ratio and Lactate dehydrogenase (LDH) level, increasing the cell viability, content of key enzymes of glycolysis, ATP concentration of glycolysis and stabilizing the mitochondrial membrane potential. However, the cardioprotective effects of SPC were disappeared by treatment with a HIF-1a selective inhibitor.ConclusionThis study demonstrates that the cardioprotective effects of SPC are associated with the activation of the HIF-1a/PDK-1 signaling pathway. The mechanism may be related to increasing the content of key enzymes and ATP of glycolysis in the early stage of hypoxia.

Pioglitazone corrects dysregulation of skeletal muscle mitochondrial proteins involved in ATP synthesis in type 2 diabetes

ContextIn this study, we aimed to identify the determinants of mitochondrial dysfunction in skeletal muscle (SKLM) of subjects with type 2 diabetes (T2DM), and to evaluate the effect of pioglitazone (PIO) on SKLM mitochondrial proteome. MethodsTwo different groups of adults were studied. Group I consisted of 8 individuals with normal glucose tolerance (NGT) and 8 with T2DM, subjected to SKLM mitochondrial proteome analysis by 2D-gel electrophoresis followed by mass spectrometry-based protein identification. Group II included 24 individuals with NGT and 24 with T2DM, whose SKLM biopsies were subjected to immunoblot analysis. Of the 24 subjects with T2DM, 20 were randomized to receive placebo or PIO (15 mg daily) for 6 months. After 6 months of treatment, SKLM biopsy was repeated. ResultsMitochondrial proteomic analysis on Group I revealed that several mitochondrial proteins involved in oxidative metabolism were differentially expressed between T2DM and NGT groups, with a downregulation of ATP synthase alpha chain (ATP5A), electron transfer flavoprotein alpha-subunit (ETFA), cytochrome c oxidase subunit VIb isoform 1 (CX6B1), pyruvate dehydrogenase protein X component (ODPX), dihydrolipoamide dehydrogenase (DLDH), dihydrolipoamide-S-succinyltransferase (DLST), and mitofilin, and an up-regulation of hydroxyacyl-CoA-dehydrogenase (HCDH), 3,2-trans-enoyl-CoA-isomerase (D3D2) and delta3,5-delta2,4-dienoyl-CoA-isomerase (ECH1) in T2DM as compared to NGT subjects. By immunoblot analysis on SKLM lysates obtained from Group II we confirmed that, in comparison to NGT subjects, those with T2DM exhibited lower protein levels of ATP5A (−30%, P = 0.006), ETFA (−50%, P = 0.02), CX6B1 (−30%, P = 0.03), key factors for ATP biosynthesis, and of the structural protein mitofilin (−30%, P = 0.01). T2DM was associated with a reduced abundance of the enzymes involved in the Krebs cycle DLST and ODPX (−20%, P ≤ 0.05) and increased levels of HCDH and ECH1, enzymes implicated in the fatty acid catabolism (+30%, P ≤ 0.05).In subjects with type 2 diabetes treated with PIO for 6 months we found a restored SKLM protein abundance of ATP5A, ETFA, CX6B1, and mitofilin. Moreover, protein levels of HCDH and ECH1 were reduced by −10% and − 15% respectively (P ≤ 0.05 for both) after PIO treatment. ConclusionType 2 diabetes is associated with reduced levels of mitochondrial proteins involved in oxidative phosphorylation and an increased abundance of enzymes implicated in fatty acid catabolism in SKLM. PIO treatment is able to improve SKLM mitochondrial proteomic profile in subjects with T2DM.

Draft genome sequence of the termite, Coptotermes formosanus: Genetic insights into the pyruvate dehydrogenase complex of the termite

Abstract Termites are generally deficient in pyruvate dehydrogenase (PDH), which links glycolysis to the Krebs cycle; however, one termite species, Coptotermes formosanus, has some PDH activity, though it is not enough to maintain the respiration of the termite by itself. We obtained a high quality annotated draft genome of C. formosanus. We found that all genes constituting the PDH complex and controlling PDH activity are present in the genome of C. formosanus, except for the PDH protein X component that is essential for a functional PDH complex. Additionally, we found that C. formosanus has three endo-s-1,4-glucanases (EGs), for which the amino acid sequences differ from those of previously reported EGs. Despite the ability of termites to convert cellulose to glucose and the resulting glucose to pyruvate, PDH is likely to function poorly due to a missing X component of the PDH complex.

Chronic Sustained Hypoxic Exposure Can Improve Glucose Metabolism in Mice

PurposeHypoxic therapy has been reported to improve glucose metabolism. In previous studies, there were many studies that determined whether hypoxia regulate glucose metabolism. However, it is still unclear whether hypoxia would only affect whole‐body energy expenditure and energy utilization. In addition, along with energy metabolism, it needs to confirm the gene expression associated with glucose metabolism. Therefore, the purpose of the present study was to investigate the effects of hypoxia exposure on resting metabolic rate, changes of protein expression related to glucose metabolism and mitochondrial biogenesis in muscle tissue.MethodsSeven‐week‐old ICR male mice (n=16) were divided into two groups: normoxia pair‐fed to hypoxia (Nor), chronic hypoxia (Chr). Normoxia group was exposed to 21% O2 and chronic hypoxia group was 12% O2 for 3 weeks. After 3 weeks, we confirmed resting metabolic rate (RMR), body composition, and protein expression related to glucose metabolism for hexokinase 2 (HXK2), pyruvate dehydrogenase (PDH) and mitochondrial biogenesis for peroxisome proliferator‐activated receptor gamma coactivator‐1 alpha (PGC‐1α). RMR was measured using an open circuit calorimetry system.ResultsIn resting metabolic rate, carbohydrate oxidation and energy expenditure were significantly higher in the Chr group than in the Nor group. In soleus muscle, PDH protein level was higher in the Chr group than Nor (p=0.015) group. HXK 2 protein level tended to be higher about 21% in the Chr group (p=0.09). PGC1α protein level was higher in the Chr group than Nor group (p=0.032).ConclusionOur data shows that chronic sustained hypoxia significantly improved energy metabolism. Especially, carbohydrate oxidation and protein levels related to glucose metabolism and mitochondrial biogenesis were higher than normoxia group. These observations suggest that chronic exposure for 3 weeks could serve as an effective therapy for glucose metabolic disorder. In the future study, it would be necessary to elucidate whether hypoxia exposure could improve glucose metabolism through diabetic mice model.

Hepatic proteome analysis reveals altered mitochondrial metabolism and suppressed acyl-CoA synthetase-1 in colon-26 tumor-induced cachexia.

Cachexia is a life-threatening complication of cancer traditionally characterized by weight loss and muscle dysfunction. Cachexia, however, is a systemic disease that also involves remodeling of nonmuscle organs. The liver exerts major control over systemic metabolism, yet its role in cancer cachexia is not well understood. To advance the understanding of how the liver contributes to cancer cachexia, we used quantitative proteomics and bioinformatics to identify hepatic pathways and cellular processes dysregulated in mice with moderate and severe colon-26 tumor-induced cachexia; ~300 differentially expressed proteins identified during the induction of moderate cachexia were also differentially regulated in the transition to severe cachexia. KEGG pathway enrichment revealed representation by oxidative phosphorylation, indicating altered hepatic mitochondrial function as a common feature across cachexia severity. Glycogen catabolism was also observed in cachexic livers along with decreased pyruvate dehydrogenase protein X component (Pdhx), increased lactate dehydrogenase A chain (Ldha), and increased lactate transporter Mct1. Together this suggests altered lactate metabolism and transport in cachexic livers, which may contribute to energetically inefficient interorgan lactate cycling. Acyl-CoA synthetase-1 (ACSL1), known for activating long-chain fatty acids, was decreased in moderate and severe cachexia based on LC-MS/MS analysis and immunoblotting. ACSL1 showed strong linear relationships with percent body weight change and muscle fiber size (R2 = 0.73-0.76, P < 0.01). Mitochondrial coupling efficiency, which is compromised in cachexic livers to potentially increase energy expenditure and weight loss, also showed a linear relationship with ACSL1. Findings suggest altered mitochondrial and substrate metabolism of the liver in cancer cachexia, and possible hepatic targets for intervention.

Increased pyruvate dehydrogenase activity in skeletal muscle of growth-restricted ovine fetuses.

Fetal sheep with placental insufficiency-induced intrauterine growth restriction (IUGR) have lower fractional rates of glucose oxidation and greater gluconeogenesis, indicating lactate shuttling between skeletal muscle and liver. Suppression of pyruvate dehydrogenase (PDH) activity was proposed because of greater pyruvate dehydrogenase kinase (PDK) 4 and PDK1 mRNA concentrations in IUGR muscle. Although PDK1 and PDK4 inhibit PDH activity to reduce pyruvate metabolism, PDH protein concentrations and activity have not been examined in skeletal muscle from IUGR fetuses. Therefore, we evaluated the protein concentrations and activity of PDH and the kinases and phosphatases that regulate PDH phosphorylation status in the semitendinosus muscle from placenta insufficiency-induced IUGR sheep fetuses and control fetuses. Immunoblots were performed for PDH, phosphorylated PDH (E1α), PDK1, PDK4, and pyruvate dehydrogenase phosphatase 1 and 2 (PDP1 and PDP2, respectively). Additionally, the PDH, lactate dehydrogenase (LDH), and citrate synthase (CS) enzymatic activities were measured. Phosphorylated PDH concentrations were 28% lower (P < 0.01) and PDH activity was 67% greater (P < 0.01) in IUGR fetal muscle compared with control. PDK1, PDK4, PDP1, PDP2, and PDH concentrations were not different between groups. CS and LDH activities were also unaffected. Contrary to the previous speculation, PDH activity was greater in skeletal muscle from IUGR fetuses, which parallels lower phosphorylated PDH. Therefore, greater expression of PDK1 and PDK4 mRNA did not translate to greater PDK1 or PDK4 protein concentrations or inhibition of PDH as proposed. Instead, these findings show greater PDH activity in IUGR fetal muscle, which indicates that alternative regulatory mechanisms are responsible for lower pyruvate catabolism.

Key Glycolytic Metabolites in Paralyzed Skeletal Muscle Are Altered Seven Days after Spinal Cord Injury in Mice.

Spinal cord injury (SCI) results in rapid muscle atrophy and an oxidative-to-glycolytic fiber-type shift. Those with chronic SCI are more at risk for developing insulin resistance and reductions in glucose clearance than able-bodied individuals, but how glucose metabolism is affected after SCI is not well known. An untargeted metabolomics approach was utilized to investigate changes in whole-muscle metabolites at an acute (7-day) and subacute (28-day) time frame after a complete T9 spinal cord transection in 20-week-old female C57BL/6 mice. Two hundred one metabolites were detected in all samples, and 83 had BinBase IDs. A principal components analysis showed the 7-day group as a unique cluster. Further, 36 metabolites were altered after 7- and/or 28-day post-SCI (p values <0.05), with 12 passing further false discovery rate exclusion criteria; of those 12 metabolites, three important glycolytic molecules-glucose and downstream metabolites pyruvic acid and lactic acid-were reduced at 7 days compared to those values in sham and/or 28-day animals. These changes were associated with altered expression of proteins associated with glycolysis, as well as monocarboxylate transporter 4 gene expression. Taken together, our data suggest an acute disruption of skeletal muscle glucose uptake at 7 days post-SCI, which leads to reduced pyruvate and lactate levels. These levels recover by 28 days post-SCI, but a reduction in pyruvate dehydrogenase protein expression at 28 days post-SCI implies disruption in downstream oxidation of glucose.

Adaptations in Protein Expression and Regulated Activity of Pyruvate Dehydrogenase Multienzyme Complex in Human Systolic Heart Failure.

Pyruvate dehydrogenase (PDH) complex, a multienzyme complex at the nexus of glycolytic and Krebs cycles, provides acetyl-CoA to the Krebs cycle and NADH to complex I thus supporting a critical role in mitochondrial energy production and cellular survival. PDH activity is regulated by pyruvate dehydrogenase phosphatases (PDP1, PDP2), pyruvate dehydrogenase kinases (PDK 1-4), and mitochondrial pyruvate carriers (MPC1, MPC2). As NADH-dependent oxidative phosphorylation is diminished in systolic heart failure, we tested whether the left ventricular myocardium (LV) from end-stage systolic adult heart failure patients (n = 26) exhibits altered expression of PDH complex subunits, PDK, MPC, PDP, and PDH complex activity, compared to LV from nonfailing donor hearts (n = 21). Compared to nonfailing LV, PDH activity and relative expression levels of E2, E3bp, E1α, and E1β subunits were greater in LV failure. PDK4, MPC1, and MPC2 expressions were decreased in failing LV, whereas PDP1, PDP2, PDK1, and PDK2 expressions did not differ between nonfailing and failing LV. In order to examine PDK4 further, donor human LV cardiomyocytes were induced in culture to hypertrophy with 0.1 μM angiotensin II and treated with PDK inhibitors (0.2 mM dichloroacetate, or 5 mM pyruvate) or activators (0.6 mM NADH plus 50 μM acetyl CoA). In isolated hypertrophic cardiomyocytes in vitro, PDK activators and inhibitors increased and decreased PDK4, respectively. In conclusion, in end-stage failing hearts, greater expression of PDH proteins and decreased expression of PDK4, MPC1, and MPC2 were evident with higher rates of PDH activity. These adaptations support sustained capacity for PDH to facilitate glucose metabolism in the face of other failing bioenergetic pathways.

Supranutritional supplementation of vitamin E influences mitochondrial proteome profile of post-mortem &lt;i&gt;longissimus lumborum&lt;/i&gt; from feedlot heifers

Supplementation of vitamin E improves beef colour stability by minimizing lipid oxidation-induced myoglobin oxidation. Mitochondria affect myoglobin redox stability, and dietary vitamin E influences mitochondrial functionality in skeletal muscles. Nonetheless, the influence of vitamin E on the mitochondrial proteome of beef skeletal muscles has yet to be investigated. Therefore, the objective of this study was to examine the effect of dietary vitamin E on mitochondrial proteome of post-mortem beef longissimus lumborum (LL) muscle. Beef LL muscle samples (24 hours post-mortem) were obtained from the carcasses of nine (n = 9) vitamin E-fed (VITE) (1000 IU vitamin E for 89 days) and nine (n = 9) control (CONT) (diet without supplemental vitamin E) heifers. The mitochondrial proteome was analysed using two-dimensional gel electrophoresis and mass spectrometry, and nine differentially abundant spots were identified. All the differentially abundant spots were over-abundant in CONT, and the proteins were electron transport chain enzymes (NADH dehydrogenase iron-sulphur protein 8, NADH dehydrogenase flavoprotein 2, and cytochrome c oxidase subunit 5B), metabolic enzymes (succinate-CoA ligase (ADP-forming) subunit beta; dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex; pyruvate dehydrogenase protein X component), and enzymes involved in ATP regeneration (creatine kinase S-type). The low abundance of these proteins in VITE may decrease mitochondrial activity, resulting in low oxidative activity. These findings suggest that the strong antioxidant activity of vitamin E leads to less expression of mitochondrial oxidative enzymes in beef skeletal muscles. Keywords : Beef colour, lipid oxidation, mitochondrial enzymes