Drug evaluation based on phosphomimetic PDHA1 reveals the complexity of activity-related cell death in A549 non-small cell lung cancer cells.

Cancer cells predominantly generate energy via glycolysis, even in the presence of oxygen, to support abnormal cell proliferation. Suppression of PDHA1 by PDK1 prevents the conversion of cytoplasmic pyruvate into Acetyl-CoA. Several PDK inhibitors have been identified, but their clinical applications have not been successful for unclear reasons. In this study, endogenous PDHA1 in A549 cells was silenced by the CRISPR/Cas9 system, and PDHA1WT and PDHA13SD were transduced. Since PDHA13SD cannot be phosphorylated by PDKs, it was used to evaluate the specific activity of PDK inhibitors. This study highlights that PDHA1WT and PDHA13SD A549 cells can be used as a cell-based PDK inhibitor-distinction system to examine the relationship between PDH activity and cell death by established PDK inhibitors. Leelamine, huzhangoside A and otobaphenol induced PDH activity-dependent apoptosis, whereas AZD7545, VER-246608 and DCA effectively enhanced PDHA1 activity but little toxic to cancer cells. Furthermore, the activity of phosphomimetic PDHA1 revealed the complexity of its regulation, which requires further in-depth investigation. [BMB Reports 2021; 54(11): 563-568].

Fursultiamine Alleviates Choroidal Neovascularization by Suppressing Inflammation and Metabolic Reprogramming.

Purpose: To assess the therapeutic effects of fursultiamine on choroidal neovascularization (CNV) through its modulation of inflammation and metabolic reprogramming in the retinal pigment epithelium (RPE). Methods: The anti-angiogenic effects of fursultiamine were assessed by measuring vascular leakage and CNV lesion size in the laser-induced CNV mouse model. Inflammatory responses were evaluated by quantitative polymerase chain reaction, western blot, and ELISA in both CNV eye tissues and in vitro cell cultures using ARPE-19 cells or primary human RPE (hRPE) cells under lipopolysaccharide (LPS) treatment or hypoxia. Mitochondrial respiration was assessed by measuring oxygen consumption in ARPE-19 cells treated with LPS with or without fursultiamine, and lactate production was measured in ARPE-19 cells subjected to hypoxia with or without fursultiamine. Results: In laser-induced CNV, fursultiamine significantly decreased vascular leakage and lesion size, as well as the numbers of both choroidal and retinal inflammatory cytokines, including IL-1ß, IL-6, IL-8, and TNF-α. In LPS-treated ARPE-19 cells, fursultiamine decreased proinflammatory cytokine secretion and nuclear factor kappa B phosphorylation. Furthermore, fursultiamine suppressed LPS-induced upregulation of IL-6, IL-8, and monocyte chemoattractant protein-1 in a dose-dependent and time-dependent manner in primary hRPE cells. Interestingly, fursultiamine significantly enhanced mitochondrial respiration in the LPS-treated ARPE-19 cells. Additionally, fursultiamine attenuated hypoxia-induced aberrations, including lactate production and inhibitory phosphorylation of pyruvate dehydrogenase. Furthermore, fursultiamine attenuated hypoxia-induced VEGF secretion and mitochondrial fission in primary hRPE cells that were replicated in ARPE-19 cells. Conclusions: Our findings show that fursultiamine is a viable putative therapeutic for neovascular age-related macular degeneration by modulating the inflammatory response and metabolic reprogramming by enhancing mitochondrial respiration in the RPE.

Identification of Novel Resorcinol Amide Derivatives as Potent and Specific Pyruvate Dehydrogenase Kinase (PDHK) Inhibitors.

Pyruvate dehydrogenase kinases (PDHKs) promote abnormal respiration in cancer cells. Studies with novel resorcinol amide derivatives based on VER-246608 (6) led to the identification of 19n and 19t containing five-membered heteroaromatic rings as unique structural features. These substances possess single-digit nanomolar activities against PDHKs. 19t exhibits higher potencies against PDHK1/2/4 than does 6 and inhibits only PDHKs among 366 kinases. Moreover, 19g, 19l, and 19s were found to be isotype-selective PDHK inhibitors. Molecular dynamics simulations provide a better understanding of how the heteroaromatic rings affect the activities of 19n and 19t on PDHK1/2/3/4. Moreover, 19n possesses a much higher antiproliferative activity against cancer cells than does 6. We demonstrated that the results of PDH assays better correlate with cellular activities than do those of PDHK kinase assays. Furthermore, 19n induces apoptosis of cancer cells via mitochondrial dysfunction, suppresses tumorigenesis, and displays a synergistic effect on satraplatin suppression of cancer cell proliferation.

Pyruvate Dehydrogenase Kinase Is a Metabolic Checkpoint for Polarization of Macrophages to the M1 Phenotype.

Metabolic reprogramming during macrophage polarization supports the effector functions of these cells in health and disease. Here, we demonstrate that pyruvate dehydrogenase kinase (PDK), which inhibits the pyruvate dehydrogenase-mediated conversion of cytosolic pyruvate to mitochondrial acetyl-CoA, functions as a metabolic checkpoint in M1 macrophages. Polarization was not prevented by PDK2 or PDK4 deletion but was fully prevented by the combined deletion of PDK2 and PDK4; this lack of polarization was correlated with improved mitochondrial respiration and rewiring of metabolic breaks that are characterized by increased glycolytic intermediates and reduced metabolites in the TCA cycle. Genetic deletion or pharmacological inhibition of PDK2/4 prevents polarization of macrophages to the M1 phenotype in response to inflammatory stimuli (lipopolysaccharide plus IFN-γ). Transplantation of PDK2/4-deficient bone marrow into irradiated wild-type mice to produce mice with PDK2/4-deficient myeloid cells prevented M1 polarization, reduced obesity-associated insulin resistance, and ameliorated adipose tissue inflammation. A novel, pharmacological PDK inhibitor, KPLH1130, improved high-fat diet-induced insulin resistance; this was correlated with a reduction in the levels of pro-inflammatory markers and improved mitochondrial function. These studies identify PDK2/4 as a metabolic checkpoint for M1 phenotype polarization of macrophages, which could potentially be exploited as a novel therapeutic target for obesity-associated metabolic disorders and other inflammatory conditions.

Discovery of Novel Pyruvate Dehydrogenase Kinase 4 Inhibitors for Potential Oral Treatment of Metabolic Diseases.

Pyruvate dehydrogenase kinase 4 (PDK4) activation is associated with metabolic diseases including hyperglycemia, insulin resistance, allergies, and cancer. Structural modifications of hit anthraquinone led to the identification of a new series of allosteric PDK4 inhibitors. Among this series, compound 8c showed promising in vitro activity with an IC50 value of 84 nM. Good metabolic stability, pharmacokinetic profiles, and possible metabolites were suggested. Compound 8c improved glucose tolerance in diet-induced obese mice and ameliorated allergic reactions in a passive cutaneous anaphylaxis mouse model. Additionally, compound 8c exhibited anticancer activity by controlling cell proliferation, transformation, and apoptosis. From the molecular docking studies, compound 8c displayed optimal fitting in the lipoamide binding site (allosteric) with a full fitness, providing a new scaffold for drug development toward PDK4 inhibitors.

Progression of Multifaceted Immune Cells in Atherosclerotic Development.

Atherosclerosis is a major cause of morbidity and mortality due to cardiovascular diseases, such as coronary artery disease, stroke, and peripheral vascular disease, that are associated with thrombosis-induced organ infarction. In Westernized countries, the high prevalence of obesity-induced insulin resistance is predicted to be a major factor leading to atherosclerotic vascular disease. Both genetic and environmental factors interfere with immune responses in atherosclerosis development with chronic and non-resolving states. The most known autoimmune disease therapy is cytokine-targeted therapy, which targets tumor necrosis factor-α and interleukin (IL)-17 antagonists. Recently, a clinical trial with the anti-IL-1ß antibody (canakinumab) had shown that the anti-inflammatory effects in canakinumab-treated subjects play a critical role in reducing cardiovascular disease prevalence. Recent emerging data have suggested effective therapeutics involving anti-obesity and anti-diabetic agents, as well as statin and anti-platelet drugs, for atherothrombosis prevention. It is well-known that specialized immune differentiation and activation completely depends on metabolic reprogramming mediated by mitochondrial dynamics in distinct immune cells. Therefore, there is a strong mechanistic link between metabolism and immune function mediated by mitochondrial function. In this review, we describe that cellular metabolism in immune cells is strongly interconnected with systemic metabolism in terms of diverse phenotypes and activation.

Lifestyle of Patients with Alcoholic Liver Disease and Factors Leading to Hospital Readmission: A Prospective Observational Study.

The objective of this study was to clarify the lifestyle characteristics of patients with alcoholic liver disease (ALD) who were readmitted to the hospital, and to identify the background factors associated with these characteristics. This was a prospective observational study. Over a period of 3 months following hospital discharge, we conducted structured interviews to investigate the following five lifestyle characteristics based on our previous research: dietary intake, alcohol consumption or abstinence, psycho-emotional status, regularity of life habits, adherence to treatment. We also collected data on background factors from medical records and questionnaires. The analysis was performed using conceptual cluster matrices, with participants divided into two groups (at-home recovery and readmission). Lifestyle, health status, and background factors were compared between the two groups. Of the 34 patients with ALD recruited, 21 completed the one-month follow-up and were included in the analysis-14 patients were in the at-home recovery group and 7 in the readmission group. The at-home group's lifestyle was characterized by controlled alcohol consumption, but with maintenance of regular life and eating habits and adherence to treatment. In contrast, irregular eating habits (p=0.006) and the development of irregular life habits or the discontinuation of treatment very quickly after hospital discharge characterized the readmission group's lifestyle. Experiences of loss were a lifestyle-related background factor that was associated with readmission (p=0.017). Based on these findings, supporting patients with ALD in maintaining regular eating habits and taking experiences of loss into consideration would be important in avoiding readmission over the short-term.

Role of the Pyruvate Dehydrogenase Complex in Metabolic Remodeling: Differential Pyruvate Dehydrogenase Complex Functions in Metabolism.

Mitochondrial dysfunction is a hallmark of metabolic diseases such as obesity, type 2 diabetes mellitus, neurodegenerative diseases, and cancers. Dysfunction occurs in part because of altered regulation of the mitochondrial pyruvate dehydrogenase complex (PDC), which acts as a central metabolic node that mediates pyruvate oxidation after glycolysis and fuels the Krebs cycle to meet energy demands. Fine-tuning of PDC activity has been mainly attributed to post-translational modifications of its subunits, including the extensively studied phosphorylation and de-phosphorylation of the E1α subunit of pyruvate dehydrogenase (PDH), modulated by kinases (pyruvate dehydrogenase kinase [PDK] 1-4) and phosphatases (pyruvate dehydrogenase phosphatase [PDP] 1-2), respectively. In addition to phosphorylation, other covalent modifications, including acetylation and succinylation, and changes in metabolite levels via metabolic pathways linked to utilization of glucose, fatty acids, and amino acids, have been identified. In this review, we will summarize the roles of PDC in diverse tissues and how regulation of its activity is affected in various metabolic disorders.

Dipeptidyl peptidase-4 inhibition by gemigliptin prevents abnormal vascular remodeling via NF-E2-related factor 2 activation.

Dipeptidyl peptidase-4 (DPP-4) inhibitors exert a potent anti-hyperglycemic effect and reduce cardiovascular risk in type 2 diabetic patients. Several studies have shown that DPP-4 inhibitors including sitagliptin have beneficial effects in atherosclerosis and cardiac infarction involving reactive oxygen species. Here, we show that gemigliptin can directly attenuate the abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) via enhanced NF-E2-related factor 2 (Nrf2) activity. Gemigliptin dramatically prevented ligation injury-induced neointimal hyperplasia in mouse carotid arteries. Likewise, the proliferation of primary VSMCs was significantly attenuated by gemigliptin in a dose-dependent manner consistent with a decrease in phospho-Rb, resulting in G1 cell cycle arrest. We found that gemigliptin enhanced Nrf2 activity not only by mRNA expression, but also by increasing Keap1 proteosomal degradation by p62, leading to the induction of Nrf2 target genes such as HO-1 and NQO1. The anti-proliferative role of gemigliptin disappeared with DPP-4 siRNA knockdown, indicating that the endogenous DPP-4 in VSMCs contributed to the effect of gemigliptin. In addition, gemigliptin diminished TNF-α-mediated cell adhesion molecules such as MCP-1 and VCAM-1 and reduced MMP2 activity in VSMCs. Taken together, our data indicate that gemigliptin exerts a preventative effect on the proliferation and migration of VSMCs via Nrf2.

Scoparone interferes with STAT3-induced proliferation of vascular smooth muscle cells.

Scoparone, which is a major constituent of Artemisia capillaries, has been identified as an anticoagulant, hypolipidemic, vasorelaxant, anti-oxidant and anti-inflammatory drug, and it is used for the traditional treatment of neonatal jaundice. Therefore, we hypothesized that scoparone could suppress the proliferation of VSMCs by interfering with STAT3 signaling. We found that the proliferation of these cells was significantly attenuated by scoparone in a dose-dependent manner. Scoparone markedly reduced the serum-stimulated accumulation of cells in the S phase and concomitantly increased the proportion of cells in the G0/G1 phase, which was consistent with the reduced expression of cyclin D1, phosphorylated Rb and survivin in the VSMCs. Cell adhesion markers, such as MCP-1 and ICAM-1, were significantly reduced by scoparone. Interestingly, this compound attenuated the increase in cyclin D promoter activity by inhibiting the activities of both the WT and active forms of STAT3. Similarly, the expression of a cell proliferation marker induced by PDGF was decreased by scoparone with no change in the phosphorylation of JAK2 or Src. On the basis of the immunofluorescence staining results, STAT3 proteins phosphorylated by PDGF were predominantly localized to the nucleus and were markedly reduced in the scoparone-treated cells. In summary, scoparone blocks the accumulation of STAT3 transported from the cytosol to the nucleus, leading to the suppression of VSMC proliferation through G1 phase arrest and the inhibition of Rb phosphorylation. This activity occurs independent of the form of STAT3 and upstream of kinases, such as Jak and Src, which are correlated with abnormal vascular remodeling due to the presence of an excess of growth factors following vascular injury. These data provide convincing evidence that scoparone may be a new preventative agent for the treatment of cardiovascular diseases.

Dimethylfumarate attenuates restenosis after acute vascular injury by cell-specific and Nrf2-dependent mechanisms.

Excessive proliferation of vascular smooth muscle cells (VSMCs) and incomplete re-endothelialization is a major clinical problem limiting the long-term efficacy of percutaneous coronary angioplasty. We tested if dimethylfumarate (DMF), an anti-psoriasis drug, could inhibit abnormal vascular remodeling via NF-E2-related factor 2 (Nrf2)-NAD(P)H quinone oxidoreductase 1 (NQO1) activity. DMF significantly attenuated neointimal hyperplasia induced by balloon injury in rat carotid arteries via suppression of the G1 to S phase transition resulting from induction of p21 protein in VSMCs. Initially, DMF increased p21 protein stability through an enhancement in Nrf2 activity without an increase in p21 mRNA. Later on, DMF stimulated p21 mRNA expression through a process dependent on p53 activity. However, heme oxygenase-1 (HO-1) or NQO1 activity, well-known target genes induced by Nrf2, were dispensable for the DMF induction of p21 protein and the effect on the VSMC proliferation. Likewise, DMF protected endothelial cells from TNF-α-induced apoptosis and the dysfunction characterized by decreased eNOS expression. With knock-down of Nrf2 or NQO1, DMF failed to prevent TNF-α-induced cell apoptosis and decreased eNOS expression. Also, CD31 expression, an endothelial specific marker, was restored in vivo by DMF. In conclusion, DMF prevented abnormal proliferation in VSMCs by G1 cell cycle arrest via p21 upregulation driven by Nrf2 and p53 activity, and had a beneficial effect on TNF-α-induced apoptosis and dysfunction in endothelial cells through Nrf2-NQO1 activity suggesting that DMF might be a therapeutic drug for patients with vascular disease.

Allele-specific expression of angiotensinogen in human subcutaneous adipose tissue.

The angiotensinogen gene is genetically linked with hypertension, but the mechanistic basis for association of sequence variants in the promoter and coding region of the gene remains unclear. An E-box at position -20 has been hypothesized to control the level of angiotensinogen expression, but its mechanistic importance for angiotensinogen expression in human tissues is uncertain. We developed an allele-specific polymerase chain reaction-based assay to distinguish between angiotensinogen mRNA derived from variants at the -20 position (rs5050) in the angiotensinogen promoter in adipose tissues obtained during surgery. The assay takes advantage of linkage disequilibrium between the rs5050 (located in the promoter) and rs4762 (located in the coding region) single nucleotide polymorphisms. This strategy allowed us to assess the level of allele-specific expression in A-20C heterozygous subjects comparing the relative proportion of each allele with the total, thus eliminating the problem of variability in the level of total angiotensinogen mRNA among subjects. We show that angiotensinogen mRNA derived from the -20C allele is expressed significantly higher than that derived from the -20A allele in subcutaneous adipose tissue, and increased expression correlates with enriched chromatin binding of upstream stimulatory factor-2 to the -20C E-box compared with -20A. This may be depot selective because we were unable to detect these differences in omental adipose. This provides the first data directly comparing expression of angiotensinogen mRNA and differential transcription factor binding derived from 2 variant alleles in human tissue where the ratio of expression of one allele to another can be accurately determined.

Gene trapping uncovers sex-specific mechanisms for upstream stimulatory factors 1 and 2 in angiotensinogen expression.

A single-nucleotide polymorphism (C/A) located within an E-box at the -20 position of the human angiotensinogen (AGT) promoter may regulate transcriptional activation through differential recruitment of the transcription factors upstream stimulatory factor (USF) 1 and 2. To study the contribution of USF1 on AGT gene expression, mice carrying a (-20C) human AGT (hAGT) transgene were bred with mice harboring a USF1 gene trap allele designed to knock down USF1 expression. USF1 mRNA was reduced relative to controls in liver (9 ± 1%), perigenital adipose (16 ± 3%), kidney (17 ± 1%), and brain (34 ± 2%) in double-transgenic mice. This decrease was confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation analyses revealed a decrease in USF1, with retention of USF2 binding at the hAGT promoter in the liver of male mice. hAGT expression was reduced in the liver and other tissues of female but not male mice. The decrease in endogenous AGT expression was insufficient to alter systolic blood pressure at baseline but caused reduced systolic blood pressure in female USF1 gene trap mice fed a high-fat diet. Treatment of USF1 knockdown males with intravenous adenoviral short hairpin RNA targeting USF2 resulted in reduced expression of USF1, USF2, and hAGT protein. Our data from chromatin immunoprecipitation assays suggests that this decrease in hAGT is attributed to decreased USF2 binding to the hAGT promoter. In conclusion, both USF1 and USF2 are essential for AGT transcriptional regulation, and distinct sex-specific and tissue-specific mechanisms are involved in the activities of these transcription factors in vivo.