PDK1 Expression Research Articles

Cardiomyocyte Pdk4 response is associated with metabolic maladaptation in aging.

Ischemic heart disease (IHD) is the leading cause of death, with age range being the primary factor for development. The mechanisms by which aging increases vulnerability to ischemic insult are not well understood. We aim to use single-cell RNA sequencing to discover transcriptional differences in various cell types between aged and young mice, which may contribute to aged-related vulnerability to ischemic insult. Utilizing 10× Genomics Single-Cell RNA sequencing, we were able to complete bioinformatic analysis to identity novel differential gene expression. During the analysis of our collected samples, we detected Pyruvate Dehydrogenase Kinase 4 (Pdk4) expression to be remarkably differentially expressed. Particularly in cardiomyocyte cell populations, Pdk4 was found to be significantly upregulated in the young mouse population compared to the aged mice under ischemic/reperfusion conditions. Pdk4 is responsible for inhibiting the enzyme pyruvate dehydrogenase, resulting in the regulation of glucose metabolism. Due to decreased Pdk4 expression in aged cardiomyocytes, there may be an increased reliance on glucose oxidization for energy. Through biochemical metabolomics analysis, it was observed that there is a greater abundance of pyruvate in young hearts in contrast to their aged counterparts, indicating less glycolytic activity. We believe that Pdk4 response provides valuable insight towards mechanisms that allow for the young heart to handle ischemic insult stress more effectively than the aged heart.

Analysis of a genetic region affecting mouse body weight.

Genetic factors affect an individual's risk of developing obesity, but in most cases each genetic variant has a small effect. Discovery of genes that regulate obesity may provide clues about its underlying biological processes and point to new ways the disease can be treated. Preclinical animal models facilitate genetic discovery in obesity because environmental factors can be better controlled compared with the human population. We studied inbred mouse strains to identify novel genes affecting obesity and glucose metabolism. BTBR T+ Itpr3tf/J (BTBR) mice are fatter and more glucose intolerant than C57BL/6J (B6) mice. Prior genetic studies of these strains identified an obesity locus on chromosome 2. Using congenic mice, we found that obesity was affected by a ∼316 kb region, with only two known genes, pyruvate dehydrogenase kinase 1 (Pdk1) and integrin α 6 (Itga6). Both genes had mutations affecting their amino acid sequence and reducing mRNA levels. Both genes have known functions that could modulate obesity, lipid metabolism, insulin secretion, and/or glucose homeostasis. We hypothesized that genetic variation in or near Pdk1 or Itga6 causing reduced Pdk1 and Itga6 expression would promote obesity and impaired glucose tolerance. We used knockout mice lacking Pdk1 or Itga6 fed an obesigenic diet to test this hypothesis. Under the conditions we studied, we were unable to detect an individual contribution of either Pdk1 or Itga6 to body weight. During our studies, with conditions outside our control, we were unable to reproduce some of our previous body weight data. However, we identified a previously unknown role for Pdk1 in cardiac cholesterol metabolism providing the basis for future investigations. The studies described in this paper highlight the importance and the challenge using physiological outcomes to study obesity genes in mice.

Combined Gamma Conglutin and Lupanine Treatment Exhibits In Vivo an Enhanced Antidiabetic Effect by Modulating the Liver Gene Expression Profile.

Previous studies have individually shown the antidiabetic potential of gamma conglutin (Cγ) and lupanine from lupins. Until now, the influence of combining both compounds and the effective dose of the combination have not been assessed. Moreover, the resulting gene expression profile from this novel combination remains to be explored. Therefore, we aimed to evaluate different dose combinations of Cγ and lupanine by the oral glucose tolerance test (OGTT) to identify the higher antidiabetic effect on a T2D rat model. Later, we administered the selected dose combination during a week. Lastly, we evaluated biochemical parameters and liver gene expression profile using DNA microarrays and bioinformatic analysis. We found that the combination of 28 mg/kg BW Cγ + 20 mg/kg BW lupanine significantly reduced glycemia and lipid levels. Moreover, this treatment positively influenced the expression of Pdk4, G6pc, Foxo1, Foxo3, Ppargc1a, Serpine1, Myc, Slc37a4, Irs2, and Igfbp1 genes. The biological processes associated with these genes are oxidative stress, apoptosis regulation, and glucose and fatty-acid homeostasis. For the first time, we report the beneficial in vivo effect of the combination of two functional lupin compounds. Nevertheless, further studies are needed to investigate the pharmacokinetics and pharmacodynamics of the Cγ + lupanine combined treatment.

Identification of the osteoarthritis signature gene PDK1 by machine learning and its regulatory mechanisms on chondrocyte autophagy and apoptosis.

Osteoarthritis (OA) is a degenerative joint disease frequently diagnosed in the elderly and middle-aged population. However, its specific pathogenesis has not been clarified. This study aimed to identify biomarkers for OA diagnosis and elucidate their potential mechanisms for restoring OA-dysregulated autophagy and inhibiting chondrocyte apoptosis in vitro. Two publicly available transcriptomic mRNA OA-related datasets (GSE10575 and GSE51588) were explored for biomarker identification by least absolute shrinkage and selection operator (LASSO) regression, weighted gene co-expression network analysis (WGCNA), and support vector machine recursive feature elimination (SVM-RFE). We applied the GSE32317 and GSE55457 cohorts to validate the markers' efficacy for diagnosis. The connections of markers to chondrocyte autophagy and apoptosis in OA were also comprehensively explored in vitro using molecular biology approaches, including qRT-PCR and Western blot. We identified 286 differentially expressed genes (DEGs). These DEGs were enriched in the ECM-receptor interaction and PI3K/AKT signaling pathway. After external cohort validation and protein-protein interaction (PPI) network construction, PDK1 was finally identified as a diagnostic marker for OA. The pharmacological properties of BX795-downregulated PDK1 expression inhibited LPS-induced chondrocyte inflammation and apoptosis and rescued OA-dysregulated autophagy. Additionally, the phosphorylation of the mediators associated with the MAPK and PI3K/AKT pathways was significantly downregulated, indicating the regulatory function of PDK1 in apoptosis and autophagy via MAPK and PI3K/AKT-associated signaling pathways in chondrocytes. A significantly positive association between the PDK1 expression and Neutrophils, Eosinophils, Plasma cells, and activated CD4 memory T cells, as well as an evident negative correlation between T cells follicular helper and CD4 naive T cells, were detected in the immune cell infiltration analysis. PDK1 can be used as a diagnostic marker for OA. Inhibition of its expression can rescue OA-dysregulated autophagy and inhibit apoptosis by reducing the phosphorylation of PI3K/AKT and MAPK signaling pathways.

Identification of PDK4 as Hub Gene for Diabetic Nephropathy Using Co-Expression Network Analysis

Introduction: Diabetic nephropathy (DN) is related to type 1 and type 2 diabetes. They are the leading cause of end-stage renal disease, but the underling specific pathogenesis of DN is not yet clear. Our study was conducted to explore how DN changed the transcriptome profiles in the kidney. Methods: The gene expression profile of microdissected glomeruli of 41 type 2 DN patients and 20 healthy controls were included. The sample dataset GSE96804 was obtained from the GEO database. Differentially expressed genes (DEGs) were analyzed in R with the limma package and the important modules were found by weighted gene co-expression network analysis (WGCNA) clustering. The modules were then analyzed based on Gene Ontology (GO) gene set enrichment analysis, and the hub genes were found out. We next validated the hub gene, PDK4, in a cell model of DN. We also constructed the PDK4-related PPI network to investigate the correlation between PDK4 expression and other genes. Results: Heatmap and volcano map were drawn to illustrate the mRNA expression profile of 1,204 DEGs in both samples of DN patients and the control group. Using WGCNA, we selected the blue module in which genes showed the strongest correlation with the phenotype and the smallest p value. We also identified PDK4 as a hub gene. PDK4 expression was upregulated in human diabetic kidney tissue. Moreover, PDK4 was speculated to play a role in glomerular basement membrane development and kidney development according to the enrichment of functions and signaling pathways. Furthermore, PDK4 and two key genes GSTA2 and G6PC protein expression were verified highly expressed in the cell model of DN. Conclusion: During the pathogenesis of DN, many genes may change expression in a coordinated manner. The discovery of PDK4 as key gene using WGCNA is of great significance for the development of new treatment strategies to block the development of DN.

Determination of PDK1, SLC2A1, EGFR, PTEN, CD276 Gene Expression Levels and IDH1 Gene R132H Polymorphism in Brain Tumor Tissues.

To determine IDH1 R132H codon and the mRNA levels of PDK1, SLC2A1, EGFR, PTEN, and CD276 genes in brain tumors. This study included 15 brain tumor tissues [pituitary adenoma (1), pilocytic astrocytoma (1), mixed meningioma (2), mesothelial meningioma (2), atypical meningioma (1), immature teratoma (1), glioblastoma (4), meningioma (2), and bladder cancer metastasis (1)]. The expression levels of genes in brain tumor tissues were analyzed using real-time PCR. Sanger sequencing was performed to identify the IDH1 gene R132H codon. All cases were wild-type in terms of IDH1 R132H: nucleotide 395 G > A; codon CGT > CAT. The mRNA level of PDK1 was lower in grade I tumor tissues (0.675-fold) and increased in grades II-III-IV (7.135, 16.912, and 7.081-fold, respectively) (p < 0.001). The mRNA level of SLC2A1 decreased in all grades I-II-III-IV [(0.424-, 0.093-, 0.234 (p < 0.001), and 0.141-fold (p < 0.005), respectively)]. The mRNA level of EGFR increased in all grades I-II-III-IV [1.388, 5.452 (p < 0.017), 4.624-, and 4.137-fold, respectively]. The mRNA level of PTEN increased in grades I-II-III [1.802-, 1.702-, and 1.5-fold, respectively] and decreased in grade IV (0.176-fold). The mRNA level of CD276 increased in all grades I-II-III-IV [1.8-, 5.756-(p < 0.001), 10.303 (p < 0.001), and 2.5-fold, respectively]. We obtained similar findings for previously reported PDK1, EGFR, PTEN, and CD276 gene expression levels. In contrast, SLC2A1 expression was markedly downregulated, as reported in other tumor studies. These findings may be due to the unique nature of brain tumor tissues. Additionally, a decrease in PTEN gene expression has been observed in grade IV brain tumors, including glioblastoma and meningioma. Although the size of the analyzed study group was limited, the gene expression results showed similarities in the behavior of genes during cancer staging.

PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer

BackgroundResistance to chemotherapeutic drugs is a key factor for cancer recurrence and metastases in head and neck cancer (HNC). Cancer stem cells (CSCs) in tumors have self-renewal, differentiation, and higher drug resistance capabilities, resulting in a poor prognosis for patients. In glucose metabolism, pyruvate dehydrogenase kinase (PDK) inhibits pyruvate dehydrogenase and impedes pyruvate from being metabolized into acetyl-CoA and entering the tricarboxylic acid cycle to generate energy. Studies have reported that PDK1 and PDK2 inhibition suppresses the growth, motility, and drug resistance of cancer cells. Furthermore, while TGFβ1 levels are persistently elevated in HNC patients with poor prognosis, the role of PDK isoforms in the TGFβ1-promoted progression and stem-like properties of HNC is unclear.MethodsLevels of PDK1 and PDK2 were evaluated in HNC tissue microarrays by immunohistochemistry to explore potential clinical relevance. PDK1 and PDK2 were knocked down by the lentivirus shRNA system to investigate their role in TGFβ1-promoted tumor progression in vitro.ResultsWe found that PDK2 levels were increased in the later stage of HNC tissues compared to constant PDK1 expression. After PDK1 and PDK2 knockdown, we discovered increased ATP production and decreased lactate production in TGFβ1-treated and untreated HNC cells. However, only PDK2 silencing significantly inhibited the clonogenic ability of HNC cells. We subsequently found that TGFβ1-promoted migration and invasion capabilities were decreased in PDK1 and PDK2 knockdown cells. The tumor spheroid-forming capability, motility, CSC genes, and multidrug-resistant genes were downregulated in PDK1 and PDK2 silencing CSCs. PDK1 and PDK2 inhibition reversed cisplatin and gemcitabine resistance of CSCs, but not paclitaxel resistance.ConclusionThe results demonstrated that the PDK1- and PDK2-mediated Warburg effect contributes to the TGFβ1-enhanced stemness properties of HNC. Therefore, PDK1 and PDK2 may serve as molecular targets for the combination therapy of HNC.

SIRT6 inhibits hypoxia-induced pulmonary arterial smooth muscle cells proliferation via HIF-1α/PDK4 signaling

SIRT6 is an NAD+-dependent protein that plays a vital role in regulating the cell proliferation, differentiation and apoptosis. Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) in peripheral vascular is one of the major pathological findings of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). However, whether SIRT6 is involved in hypoxia-induced proliferation of PASMCs and its possible mechanisms remain unknown. In the present study, we found that the expression of SIRT6 was decreased in both hypoxia-induced PAH rats model and HPASMCs. Hypoxia promoted the proliferation of HPASMCs in a time-dependent manner, inhibited the activity of caspase-3 and the production of PDH, increased the activity of LDH, ROS level, mitochondrial membrane potential(MMP) and the expression of HIF-1α and PDK4, which induced glycolysis. SIRT6 over-expression could inhibit the proliferation of HPASMCs and increase the apoptosis rate, impelled the retardation of cell cycle in phase G1. Meanwhile, SIRT6 over-expression reduced LDH activity, the levels of ROS and MMP, which simultaneously increased the production of PDH, the expression of HIF-1α, PDK4, Cyclin D1 and PCNA in hypoxia-induced HPASMCs. Moreover, SIRT6 over-expression inhibited the transcriptional activation of HIF-1α/PDK4 signaling. In addition, SIRT6 knockdown with SIRT6 siRNA exhibited the same effect as hypoxia. Together, our results indicated that SIRT6 was participant in regulating hypoxia-induced imbalance of proliferation and apoptosis of HPASMCs, which was associated with the activation of HIF-1α/PDK4 signaling pathway. Targeting at SIRT6 gene and regulating the downstream metabolism signaling pathway may be a novel strategy for the treatment of hypoxia-induced PAH.

Abstract 13180: Trans-Valvular Unloading Reduces Anaerobic Glycolysis Before Reperfusion and Preserves Energy Substrate Utilization After Reperfusion in Models of Acute Myocardial Infarction

Background: The impact of mechanical left ventricular (LV) unloading before reperfusion on myocardial energetics in acute myocardial infarction (AMI) is unknown. We hypothesized that LV unloading reduces anaerobic glycolysis in models of ischemia reperfusion injury (IRI). Methods: Myocardial ischemia was induced by percutaneous occlusion of the left anterior descending (LAD) artery for 90 minutes in adult swine. In Groups A and B, an additional 120 minutes of LAD occlusion without reperfusion was performed in the presence or absence of a trans-valvular pump (TVP). In Group C and D, an additional 120 minutes of reperfusion was performed in the presence or absence TVP (Fig. 1A). Untargeted Metabolomics was performed on the tissues from the infarct zones. Results: Compared to Group A, infarct size normalized to the area at risk (IS/AAR) was significantly smaller with LV unloading after ischemia alone (Group B; Fig 1B) and reperfusion (Group D; Fig 1B). Principal component analysis analysis of 800 metabolites identified 5 distinct clusters corresponding to the 4 study groups and sham controls (Fig 1C). LV unloading without reperfusion reduced lactate by 30±4% (p&lt;0.006), glucose by 25±5% (p&lt;0.02), and intermediates of glucose metabolism, Fructose 1,6-diphosphate by 9.3%, p&lt;0.008) and ATP by 14±5% (p&lt;0.04) consistent with decreased anaerobic glycolysis and reduced myocardial oxygen demand (Fig 1D Groups A and B). LV unloading followed by reperfusion increased lactate and glucose levels by 30±5% and 75±30% (p&lt;0.05). Consistent with reduced glycolytic metabolites during ischemia with unloading, we observed significantly reduced levels of genes and proteins associated with glycolysis including mRNA expression of Glut1 and PDK1 within the infarct zone.We report for the first time that LV unloading during coronary occlusion reduces anaerobic glycolysis, energy substrate utilization and ATP production before reperfusion.

Abstract 10461: Single-Cell Transcriptional Analysis of Altered Cardiomyocyte Pdk4 as Indicator of Ischemic Stress Vulnerability in Aging

Introduction: The mechanisms by which aging increases vulnerability to ischemic insult are not well understood. Single-Cell RNA sequencing (scRNA seq) was employed to characterize transcriptional differences in various cell types between aged and young mice which may contribute to aged-related vulnerability to ischemic insult. Pyruvate Dehydrogenase Kinase 4 (Pdk4) has been shown to impact capacity for fatty-acid oxidation (FAO) and consequently adaption to metabolic alterations of ischemic insult. Hypothesis: Alteration of Pdk4 expression levels is an adaptive response transcriptionally under pathological stress to maintain metabolic homeostasis during ischemia and reperfusion (I/R). Methods: Young (3-5 months) and aged (24-26 months) C57BL/6J mice were subjected to in vivo regional 45 min of ischemia and 24 hr of reperfusion (I/R) or sham operations. Bioinformatic analysis was done using Seurat integration vignettes allowing for cell type identification and differential expression testing. Observing genetic expression profiles by cell types. Substrate metabolism was determined with working heart system and Seahorse XF Analyzer. Results: Pdk4 was profoundly found in cardiomyocytes versus fibroblasts, endothelial cells and macrophages of both young and aged hearts. Interestingly, I/R stress triggered Pdk4 mRNA expression of young but not aged cardiomyocytes. Moreover, there is a relative higher Pdk4 level in aged versus young cardiomyocytes under sham operations, indicating Pdk4 mRNA respond to pathological stress. The ex vivo working heart perfusion data showed that young versus aged hearts exhibited higher FAO in response to I/R. Moreover, Seahorse XF Analyzer showed a higher glycolysis rate in young versus aged cardiomyocytes during I/R. It suggests that an impaired cardiomyocyte Pdk4 mRNA response occurred in aging under I/R stress conditions, this could result in cardiac maladaptive metabolic alterations in aging during pathological stress. Conclusions: Alterations in Pdk4 levels of cardiomyocytes regulates cardiac adaptive metabolism in response to pathological stress. This adaptive regulation of cardiomyocyte Pdk4 is impaired in aging that leads to more vulnerability of aged versus young hearts to ischemic insult.

Incarvillea compacta Maxim ameliorates inflammatory response via inhibiting PI3K/AKT pathway and NLRP3 activation.

Incarvillea compacta Maxim is a traditional Tibetan medicine used to treat inflammation-related diseases, such as pneumonia, fever, jaundice, and otitis media. However, no studies have examined its anti-inflammatory mechanism. To validate the anti-inflammatory activity of I. compacta extract (ICE) and its protective effect on acute alcoholic gastritis, Phytochemicals of I. compacta were identified using Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Lipopolysaccharide (LPS)-induced RAW 264.7 macrophages were used in vitro along with an in vivo a mouse acute gastritis model. Pro-inflammatory mediators and cytokines were measured using the Griess reagent and Cytometric bead array (CBA) assay. Furthermore, inflammation-related molecules were analysed by Western blotting, RNA-Seq, and real-time quantitative PCR (RT-qPCR). The experimental results revealed that ICE decreased the nitric oxide (NO), IL-6, MCP-1, and TNF-α levels in LPS-stimulated RAW 264.7 cells, and downregulated the expression and phosphorylation of PDK1, AKT, and GSK3β. Moreover, ICE also downregulated the activation of NLRP3. The RNA-Seq analysis revealed that 340 differentially expressed genes (DEGs) response to ICE treatment was enriched in several inflammation-related biological processes. The results of the in vivo mouse acute gastritis model showed that ICE significantly reduced inflammatory lesions in the gastric mucosa and remarkably downregulated the expression of iNOS, TNF-α, IL-1β, and IL-6 mRNA in gastric tissue. Therefore, the results of this study obtained scientific evidence supporting the use of I. compacta.

Pyruvate Dehydrogenase Kinase 2 Accelerates Endotoxin Shock by Promoting Mitogen-Activated Protein Kinase Activation.

Endotoxin shock remains one of the major causes of mortality worldwide. Pyruvate dehydrogenase kinase (PDK) 2 is an important regulatory enzyme involved in glucose metabolism. The purpose of this study was to determine the regulatory effect of PDK2 on LPS-induced endotoxin shock and explore the mechanisms in vivo and in vitro. Here, we showed that PDK2 contributed to Toll-like receptor (TLR)-mediated inflammation. Lipopolysaccharide (LPS) activation of TLR4 pathways resulted in PDK2 upregulation in macrophages and dendritic cells (DCs). PDK2 overexpression enhanced TLR4 signaling pathway activation, whereas downregulating PDK2 expression inhibited TLR4 signaling pathway activation. Pharmacological inhibition of PDK2 significantly decreased the mortality rate and alleviated pathological injury in the lungs and livers of LPS-challenged mice, while significantly suppressing proinflammatory cytokine production. Thus, we confirmed that PDK2 is involved in LPS-induced endotoxin shock by modulating TLR4-mitogen-activated protein kinase signaling and inducing the production of proinflammatory cytokines in macrophages and DCs. Our findings highlight the importance of PDK2 as a novel target to treat septic shock.

A novel organic arsenic derivative MZ2 remodels metabolism and triggers mtROS-mediated apoptosis in acute myeloid leukemia.

Acute myeloid leukemia (AML) is one of the most common neoplasms in adults, and it is difficult to achieve satisfactory results with conventional drugs. Here, we synthesized a novel organic arsenic derivative MZ2 and evaluated its ability to remodel energy metabolism to achieve anti-leukemia. MZ2 was characterized by the average 1-min full mass spectra analysis. Biological methods such as Western blot, qPCR, flow cytometry and confocal microscopy were used to assess the mode and mechanism of MZ2-induced death. The in vivo efficacy of MZ2 was assessed by constructing a patient-derived xenograft (PDX) AML model. Unlike the precursor organic arsenical Z2, MZ2 can effectively reduce the level of aerobic glycolysis. Our in-depth found that MZ2 inhibited the expression of PDK2 in a dose-dependent manner and did not affect the expression of LDHA, another key enzyme of the glycolytic pathway. MZ2 reconstituted energy metabolism to induce the generation of mitochondrial ROS (mtROS) and then triggerd intrinsic apoptosis pathway. We also assessed whether MZ2 generates autophagy and results showed that MZ2 can induce autophagy of AML cells, which may be associated with the precursor organic arsenic drug. In vivo, MZ2 effectively attenuated leukemia progression in mice, and immunohistochemical results suggested its PDK2 inhibitory effect. In summary, the novel organic arsine derivative MZ2 exhibited excellent anti-tumor effects in acute myeloid leukemia, which may provide a potential strategy for the treatment of acute myeloid leukemia.

The alignment of the substrate nanofibers directing cellular energy metabolism

Electrospinning technology is critical in tissue engineering due to its ability to mimic the extracellular matrix. Cells sense their surroundings and perform a series of adaptive behaviors, including proliferation, differentiation, and migration. Here, we prepared two electrospinning fibers with different alignments to investigate the effect of the fibrous network on cell metabolism. We found that cells spread along the fiber orientation. Compared to random fibers, cells on aligned fibers were more elongated and expressed less phosphorylated myosin II. These morphological changes were associated with altered cellular metabolism. HK2, PDK1, and LDHA proteins expression and glycolytic capacity were enhanced in cells on random fibers. These results tentatively demonstrated the influence of substrate morphology on the metabolic state of cells.

Retinoic acid regulates pyruvate dehydrogenase kinase 4 (Pdk4) to modulate fuel utilization in the adult heart: Insights from wild-type and β-carotene 9',10' oxygenase knockout mice.

Regulation of the pyruvate dehydrogenase (PDH) complex by the pyruvate dehydrogenase kinase PDK4 enables the heart to respond to fluctuations in energy demands and substrate availability. Retinoic acid, the transcriptionally active form of vitamin A, is known to be involved in the regulation of cardiac function and growth during embryogenesis as well as under pathological conditions. Whether retinoic acid also maintains cardiac health under physiological conditions is unknown. However, vitamin A status and intake of its carotenoid precursor β‐carotene have been linked to the prevention of heart diseases. Here, we provide in vitro and in vivo evidence that retinoic acid regulates cardiac Pdk4 expression and thus PDH activity. Furthermore, we show that mice lacking β‐carotene 9′,10′‐oxygenase (BCO2), the only enzyme of the adult heart that cleaves β‐carotene to generate retinoids (vitamin A and its derivatives), displayed cardiac retinoic acid insufficiency and impaired metabolic flexibility linked to a compromised PDK4/PDH pathway. These findings provide novel insights into the functions of retinoic acid in regulating energy metabolism in adult tissues, especially the heart.

RNA-seq transcriptome profiling of liver regeneration in mice identifies the miR-34b-5p/phosphoinositide-dependent protein kinase 1 axis as a potential target for hepatocyte proliferation

Characterized by compensatory hyperplasia dependent on hepatocyte proliferation, the liver will initiate regeneration after partial hepatectomy (PH) and acute or chronic injuries. A variety of genes and noncoding RNAs play pivotal roles in these cell proliferation and growth processes. However, it is still unclear how competition endogenous RNAs (ceRNAs) modulate cellular activities during each phase of liver regeneration, and the specific mechanisms of posttranscriptional gene expression regulation in hepatocyte proliferation remain to be elucidated. To investigate the mechanism of liver regeneration through RNA-seq profiling and to determine the role of miR-34b-5p/PDK1 on hepatocyte proliferation, we established a 2/3 PH mouse model for whole transcriptome profiling based on high-throughput sequencing techniques. We subsequently constructed a lncRNA-miRNA-mRNA ceRNA regulatory network through integrative analyses of RNA interactions. Finally, plasmid transfection in NCTC 1469 cells, dual luciferase reporter gene assay, quantitative real-time PCR, western blotting, Cell Counting Kit-8, and EdU-DNA synthesis cell proliferation assay were used to demonstrate the role of the miR-34b-5p/PDK1 axis in hepatocyte proliferation in vitro. A total of 1443 mRNAs (962 up, 481 down), 48 miRNAs (35 up, 13 down), and 1955 lncRNAs (986 up, 969 down) were identified as significantly differentially expressed. We then successfully constructed a ceRNA regulatory network consisting of 7 lncRNAs, 15 miRNAs, and 347 mRNAs based on the predicted inverse interactions among ceRNAs. Additionally, miR-34b-5p/PDK1 was predicted to be closely related to hepatocyte proliferation. We further demonstrated that miR-34b-5p could bind specifically to the 3′-untranslated region (3′-UTR) of PDK1 using the dual luciferase reporter assay. Ectopic overexpression of miR-34b-5p significantly reduced the mRNA and protein expression of PDK1, while it markedly inhibited the proliferation of mouse NCTC 1469 cells in vitro. In contrast, knocking down miR-34b-5p exhibited the inverse effects on PDK1 expression and hepatocyte proliferation. Through analyzing the ceRNA network during mouse liver regeneration, this study reveals that miR-34b-5p can inhibit hepatocyte proliferation through negatively regulating PDK1 and may be a potential pharmacological intervention target.

Skeletal-Muscle-Specific Overexpression of Chrono Leads to Disruption of Glucose Metabolism and Exercise Capacity.

Disruption of circadian rhythms is related to disorders of glucose metabolism, and the molecular clock also exists in skeletal muscle. The ChIP-derived repressor of network oscillator (Chrono) and brain and muscle ARNT-like 1 (Bmal1) are core circadian components. Chrono is considered to be the repressor of Bmal1, and the Chrono–Bmal1 pathway is important in regulating the circadian rhythm; it has been speculated that this pathway could be a new mechanism for regulating glucose metabolism. The purpose of this study was to investigate the effects of Chrono on glucose metabolism in skeletal muscle and exercise capacity by using mice with skeletal-muscle-specific overexpression of Chrono (Chrono TG) and wild-type (WT) mice as the animal models. The results of this cross-sectional study indicated that the Chrono TG mice had an impaired glucose tolerance, lower exercise capacity, and higher levels of nonfasted blood glucose and glycogen content in skeletal muscle compared to WT mice. In addition, the Chrono TG mice also showed a significant increase in the amount of Chrono bound to Bmal1 according to a co-IP analysis; a remarkable decrease in mRNA expression of Tbc1d1, Glut4, Hk2, Pfkm, Pdp1, Gbe1, and Phka1, as well as in activity of Hk and protein expression of Ldhb; but higher mRNA expression of Pdk4 and protein expression of Ldha compared with those of WT mice. These data suggested the skeletal-muscle-specific overexpression of Chrono led to a greater amount of Chrono bound to Bmal1, which then could affect the glucose transporter, glucose oxidation, and glycogen utilization in skeletal muscle, as well as exercise capacity.

Mining of Differentially Expressed Genes in Diabetic Cardiomyopathy Based on GEO Database

Objective To screen out the key genes leading to diabetic cardiomyopathy by analyzing the mRNA array associated with diabetic cardiomyopathy in the GEO database. Methods The online tool GEO2R of GEO was used to mine the differentially expressed genes (DEG) in the datasets GSE4745 and GSE5606.R was used to draw the volcano map of the DEG,and the Venn diagram was established online to identify the common DEG shared by the two datasets.The clusterProfile package in R was used for gene ontology annotation and Kyoto encyclopedia of genes and genomes pathway enrichment of the DEG.GSEA was used for gene set enrichment analysis,and STRING for the construction of a protein-protein interaction network.The maximal clique centrality algorithm in the plug-in Cytohubba of Cytoscape was used to determine the top 10 key genes. The expression of key genes was studied in the primary cardiomyocytes of rats and compared between the normal control group and high glucose group. Results The expression of Pdk4,Ucp3,Hmgcs2,Asl6,and Slc2a4 was consistent with the array analysis results.The expression of Pdk4,Ucp3,and Hmgcs2 was up-regulated while that of Acsl6 and Slc2a4 was down-regulated in the cardiomyocytes stimulated by high glucose (25 mmol/L) for 72 h. Conclusion Pdk4,Ucp3,Hmgcs2,Asl6,and Slc2a4 may be associated with the occurrence and development of diabetic cardiomyopathy,and may serve as the potential biomarkers of diabetic cardiomyopathy.

Peroxisome Proliferator-Activated Receptor Activation in Precision-Cut Bovine Liver Slices Reveals Novel Putative PPAR Targets in Periparturient Dairy Cows.

Metabolic challenges experienced by dairy cows during the transition between pregnancy and lactation (also known as peripartum), are of considerable interest from a nutrigenomic perspective. The mobilization of large amounts of non-esterified fatty acids (NEFA) leads to an increase in NEFA uptake in the liver, the excess of which can cause hepatic accumulation of lipids and ultimately fatty liver. Interestingly, peripartum NEFA activate the Peroxisome Proliferator-activated Receptor (PPAR), a transcriptional regulator with known nutrigenomic properties. The study of PPAR activation in the liver of periparturient dairy cows is thus crucial; however, current in vitro models of the bovine liver are inadequate, and the isolation of primary hepatocytes is time consuming, resource intensive, and prone to errors, with the resulting cells losing characteristic phenotypical traits within hours. The objective of the current study was to evaluate the use of precision-cut liver slices (PCLS) from liver biopsies as a model for PPAR activation in periparturient dairy cows. Three primiparous Jersey cows were enrolled in the experiment, and PCLS from each were prepared prepartum (−8.0 ± 3.6 DIM) and postpartum (+7.7± 1.2 DIM) and treated independently with a variety of PPAR agonists and antagonists: the PPARα agonist WY-14643 and antagonist GW-6471; the PPARδ agonist GW-50156 and antagonist GSK-3787; and the PPARγ agonist rosiglitazone and antagonist GW-9662. Gene expression was assayed through RT-qPCR and RNAseq, and intracellular triacylglycerol (TAG) concentration was measured. PCLS obtained from postpartum cows and treated with a PPARγ agonist displayed upregulation of ACADVL and LIPC while those treated with PPARδ agonist had increased expression of LIPC, PPARD, and PDK4. In PCLS from prepartum cows, transcription of LIPC was increased by all PPAR agonists and NEFA. TAG concentration tended to be larger in tissue slices treated with PPARδ agonist compared to CTR. Use of PPAR isotype-specific antagonists in PCLS cultivated in autologous blood serum failed to decrease expression of PPAR targets, except for PDK4, which was confirmed to be a PPARδ target. Transcriptome sequencing revealed considerable differences in response to PPAR agonists at a false discovery rate-adjusted p-value of 0.2, with the most notable effects exerted by the PPARδ and PPARγ agonists. Differentially expressed genes were mainly related to pathways involved with lipid metabolism and the immune response. Among differentially expressed genes, a subset of 91 genes were identified as novel putative PPAR targets in the bovine liver, by cross-referencing our results with a publicly available dataset of predicted PPAR target genes, and supplementing our findings with prior literature. Our results provide important insights on the use of PCLS as a model for assaying PPAR activation in the periparturient dairy cow.

High Expression of PDK4 Could Play a Potentially Protective Role by Attenuating Oxidative Stress after Subarachnoid Hemorrhage.

Pyruvate dehydrogenase (PDH), a key enzyme on the mitochondrial outer membrane, has been found to decrease activity notably in early brain injury (EBI) after subarachnoid hemorrhage (SAH). It has been demonstrated that PDH is associated with the production of reactive oxygen species (ROS) and apoptosis. Hence, in this study, we aimed to determine the cause of the decreased PDH activity and explore the potential role of PDH in EBI. We investigated the expression changes of PDH and pyruvate dehydrogenase kinase (PDK) in vivo and in vitro. Then, we explored the possible effects of PDH and ROS after SAH. The results showed that early overexpression of PDK4 promoted the phosphorylation of PDH, inhibited PDH activity, and may play a protective role after SAH in vivo and in vitro. Finally, we investigated the levels of PDK4 and pyruvate, which accumulated due to decreased PDH activity, in the cerebrospinal fluid (CSF) of 34 patients with SAH. Statistical analysis revealed that PDK4 and pyruvate expression was elevated in the CSF of SAH patients compared with that of controls, and this high expression correlated with the degree of neurological impairment and long-term outcome. Taken together, the results show that PDK4 has the potential to serve as a new therapeutic target and biomarker for assisting in the diagnosis of SAH severity and prediction of recovery.