Best practices in assessing cardiac protein O-GlcNAcylation by immunoblot.

The modification of serine and threonine amino acids of proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates the activity, stability, function, and subcellular localization of proteins. Dysregulation of O-GlcNAc homeostasis is well established as a hallmark of various cardiac diseases, including cardiac hypertrophy, heart failure, complications associated with diabetes, and responses to acute injuries such as oxidative stress and ischemia-reperfusion. Given the limited availability of site-specific O-GlcNAc antibodies, studies of changes in O-GlcNAcylation in the heart frequently use pan-O-GlcNAc antibodies for semiquantitative evaluation of overall O-GlcNAc levels. However, there is a high degree of variability in many published cardiac O-GlcNAc blots. For example, many blots often have regions that lack O-GlcNAc positive staining of proteins either below 50 or above 100 kDa. In some O-GlcNAc blots, only a few protein bands are detected, while in others, intense bands around 75 kDa dominate the gel due to nonspecific IgM band staining, making it difficult to visualize less intense bands. Therefore, the goal of this study was to develop a modifiable protocol that optimizes O-GlcNAc positive banding of proteins in cardiac tissue extracts. We showed that O-GlcNAc blots using CTD110.6 antibody of proteins ranging from <30 to ∼450 kDa could be obtained while also limiting nonspecific staining. We also show that some myofilament proteins are recognized by the CTD110.6 antibody. Therefore, by protocol optimization using the widely available CTD110.6 antibody, we found that it is possible to obtain pan-O-GlcNAc blots of cardiac tissue, which minimizes common limitations associated with this technique.NEW & NOTEWORTHY The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is recognized as mediating cardiac pathophysiology. However, there is considerable variability in the quality of O-GlcNAc immunoblots used to evaluate changes in cardiac O-GlcNAc levels. Here we show that with relatively minor changes to a commonly used protocol it is possible to minimize the intensity of nonspecific bands while also reproducibly generating O-GlcNAc immunoblots covering a range of molecular weights from <30 to ∼450 kDa.

Racial and socioeconomic disparity associates with differences in cardiac DNA methylation among men with end-stage heart failure.

Heart failure (HF) is a multifactorial syndrome that remains a leading cause of worldwide morbidity. Despite its high prevalence, only half of patients with HF respond to guideline-directed medical management, prompting therapeutic efforts to confront the molecular underpinnings of its heterogeneity. In the current study, we examined epigenetics as a yet unexplored source of heterogeneity among patients with end-stage HF. Specifically, a multicohort-based study was designed to quantify cardiac genome-wide cytosine-p-guanine (CpG) methylation of cardiac biopsies from male patients undergoing left ventricular assist device (LVAD) implantation. In both pilot (n = 11) and testing (n = 31) cohorts, unsupervised multidimensional scaling of genome-wide myocardial DNA methylation exhibited a bimodal distribution of CpG methylation found largely to occur in the promoter regions of metabolic genes. Among the available patient attributes, only categorical self-identified patient race could delineate this methylation signature, with African American (AA) and Caucasian American (CA) samples clustering separately. Because race is a social construct, and thus a poor proxy of human physiology, extensive review of medical records was conducted, but ultimately failed to identify covariates of race at the time of LVAD surgery. By contrast, retrospective analysis exposed a higher all-cause mortality among AA (56.3%) relative to CA (16.7%) patients at 2 yr following LVAD placement (P = 0.03). Geocoding-based approximation of patient demographics uncovered disparities in income levels among AA relative to CA patients. Although additional studies are needed, the current analysis implicates cardiac DNA methylation as a previously unrecognized indicator of socioeconomic disparity in human heart failure outcomes.NEW & NOTEWORTHY A bimodal signature of cardiac DNA methylation in heart failure corresponds with racial differences in all-cause mortality following mechanical circulatory support. Racial differences in promoter methylation disproportionately affect metabolic signaling pathways. Socioeconomic factors are associated with racial differences in the cardiac methylome among men with end-stage heart failure.

MitoQ regulates redox-related noncoding RNAs to preserve mitochondrial network integrity in pressure-overload heart failure.

Evidence suggests that mitochondrial network integrity is impaired in cardiomyocytes from failing hearts. While oxidative stress has been implicated in heart failure (HF)-associated mitochondrial remodeling, the effect of mitochondrial-targeted antioxidants, such as mitoquinone (MitoQ), on the mitochondrial network in a model of HF (e.g., pressure overload) has not been demonstrated. Furthermore, the mechanism of this regulation is not completely understood with an emerging role for posttranscriptional regulation via long noncoding RNAs (lncRNAs). We hypothesized that MitoQ preserves mitochondrial fusion proteins (i.e., mitofusin), likely through redox-sensitive lncRNAs, leading to improved mitochondrial network integrity in failing hearts. To test this hypothesis, 8-wk-old C57BL/6J mice were subjected to ascending aortic constriction (AAC), which caused substantial left ventricular (LV) chamber remodeling and remarkable contractile dysfunction in 1 wk. Transmission electron microscopy and immunostaining revealed defective intermitochondrial and mitochondrial-sarcoplasmic reticulum ultrastructure in AAC mice compared with sham-operated animals, which was accompanied by elevated oxidative stress and suppressed mitofusin (i.e., Mfn1 and Mfn2) expression. MitoQ (1.36 mg·day-1·mouse-1, 7 consecutive days) significantly ameliorated LV dysfunction, attenuated Mfn2 downregulation, improved interorganellar contact, and increased metabolism-related gene expression. Moreover, our data revealed that MitoQ alleviated the dysregulation of an Mfn2-associated lncRNA (i.e., Plscr4). In summary, the present study supports a unique mechanism by which MitoQ improves myocardial intermitochondrial and mitochondrial-sarcoplasmic reticulum (SR) ultrastructural remodeling in HF by maintaining Mfn2 expression via regulation by an lncRNA. These findings underscore the important role of lncRNAs in the pathogenesis of HF and the potential of targeting them for effective HF treatment.NEW & NOTEWORTHY We have shown that MitoQ improves cardiac mitochondrial network integrity and mitochondrial-SR alignment in a pressure-overload mouse heart-failure model. This may be occurring partly through preventing the dysregulation of a redox-sensitive lncRNA-microRNA pair (i.e., Plscr4-miR-214) that results in an increase in mitofusin-2 expression.

Genome-wide DNA methylation encodes cardiac transcriptional reprogramming in human ischemic heart failure.

Ischemic cardiomyopathy (ICM) is the clinical endpoint of coronary heart disease and a leading cause of heart failure. Despite growing demands to develop personalized approaches to treat ICM, progress is limited by inadequate knowledge of its pathogenesis. Since epigenetics has been implicated in the development of other chronic diseases, the current study was designed to determine whether transcriptional and/or epigenetic changes are sufficient to distinguish ICM from other etiologies of heart failure. Specifically, we hypothesize that genome-wide DNA methylation encodes transcriptional reprogramming in ICM. RNA-sequencing analysis was performed on human ischemic left ventricular tissue obtained from patients with end-stage heart failure, which enriched known targets of the polycomb methyltransferase EZH2 compared to non-ischemic hearts. Combined RNA sequencing and genome-wide DNA methylation analysis revealed a robust gene expression pattern consistent with suppression of oxidative metabolism, induced anaerobic glycolysis, and altered cellular remodeling. Lastly, KLF15 was identified as a putative upstream regulator of metabolic gene expression that was itself regulated by EZH2 in a SET domain-dependent manner. Our observations therefore define a novel role of DNA methylation in the metabolic reprogramming of ICM. Furthermore, we identify EZH2 as an epigenetic regulator of KLF15 along with DNA hypermethylation, and we propose a novel mechanism through which coronary heart disease reprograms the expression of both intermediate enzymes and upstream regulators of cardiac metabolism such as KLF15.

DNA methylation reprograms cardiac metabolic gene expression in end-stage human heart failure.

Heart failure (HF) is a leading cause of morbidity and mortality in the United States and worldwide. As a multifactorial syndrome with unpredictable clinical outcomes, identifying the common molecular underpinnings that drive HF pathogenesis remains a major focus of investigation. Disruption of cardiac gene expression has been shown to mediate a common final cascade of pathological hallmarks wherein the heart reactivates numerous developmental pathways. Although the central regulatory mechanisms that drive this cardiac transcriptional reprogramming remain unknown, epigenetic contributions are likely. In the current study, we examined whether the epigenome, specifically DNA methylation, is reprogrammed in HF to potentiate a pathological shift in cardiac gene expression. To accomplish this, we used paired-end whole genome bisulfite sequencing and next-generation RNA sequencing of left ventricle tissue obtained from seven patients with end-stage HF and three nonfailing donor hearts. We found that differential methylation was localized to promoter-associated cytosine-phosphate-guanine islands, which are established regulatory regions of downstream genes. Hypermethylated promoters were associated with genes involved in oxidative metabolism, whereas promoter hypomethylation enriched glycolytic pathways. Overexpression of plasmid-derived DNA methyltransferase 3A in vitro was sufficient to lower the expression of numerous oxidative metabolic genes in H9c2 rat cardiomyoblasts, further supporting the importance of epigenetic factors in the regulation of cardiac metabolism. Last, we identified binding-site competition via hypermethylation of the nuclear respiratory factor 1 (NRF1) motif, an established upstream regulator of mitochondrial biogenesis. These preliminary observations are the first to uncover an etiology-independent shift in cardiac DNA methylation that corresponds with altered metabolic gene expression in HF.NEW & NOTEWORTHY The failing heart undergoes profound metabolic changes because of alterations in cardiac gene expression, reactivating glycolytic genes and suppressing oxidative metabolic genes. In the current study, we discover that alterations to cardiac DNA methylation encode this fetal-like metabolic gene reprogramming. We also identify novel epigenetic interference of nuclear respiratory factor 1 via hypermethylation of its downstream promoter targets, further supporting a novel contribution of DNA methylation in the metabolic remodeling of heart failure.

Pyruvate dehydrogenase kinase 4 deficiency attenuates cisplatin-induced acute kidney injury.

Clinical prescription of cisplatin, one of the most widely used chemotherapeutic agents, is limited by its side effects, particularly tubular injury-associated nephrotoxicity. Since details of the underlying mechanisms are not fully understood, we investigated the role of pyruvate dehydrogenase kinase (PDK) in cisplatin-induced acute kidney injury. Among the PDK isoforms, PDK4 mRNA and protein levels were markedly increased in the kidneys of mice treated with cisplatin, and c-Jun N-terminal kinase activation was involved in cisplatin-induced renal PDK4 expression. Treatment with the PDK inhibitor sodium dichloroacetate (DCA) or genetic knockout of PDK4 attenuated the signs of cisplatin-induced acute kidney injury, including apoptotic morphology of the kidney tubules along with numbers of TUNEL-positive cells, cleaved caspase-3, and renal tubular injury markers. Cisplatin-induced suppression of the mitochondrial membrane potential, oxygen consumption rate, expression of electron transport chain components, cytochrome c oxidase activity, and disruption of mitochondrial morphology were noticeably improved in the kidneys of DCA-treated or PDK4 knockout mice. Additionally, levels of the oxidative stress marker 4-hydroxynonenal and mitochondrial reactive oxygen species were attenuated, whereas superoxide dismutase 2 and catalase expression and glutathione synthetase and glutathione levels were recovered in DCA-treated or PDK4 knockout mice. Interestingly, lipid accumulation was considerably attenuated in DCA-treated or PDK4 knockout mice via recovered expression of peroxisome proliferator-activated receptor-α and coactivator PGC-1α, which was accompanied by recovery of mitochondrial biogenesis. Thus, PDK4 mediates cisplatin-induced acute kidney injury, suggesting that PDK4 might be a therapeutic target for attenuating cisplatin-induced acute kidney injury.

Estrogen-Related Receptor γ Plays a Key Role in Vascular Calcification Through the Upregulation of BMP2 Expression.

OBJECTIVE: Vascular calcification which refers to ectopic mineralization in vascular cells is associated with several conditions, such as chronic kidney disease, atherosclerosis, and diabetes mellitus. Estrogen-related receptor (ERR)γ is a member of the orphan nuclear receptor superfamily, which plays diverse roles in regulating homeostatic and metabolic processes. However, the role of ERRγ in vascular calcification has not been investigated to date. The aim of the present study was to examine the role of ERRγ in vascular calcification. APPROACH AND RESULTS: Vascular calcification was induced by treating rat aortic vascular smooth muscle cells with calcification medium. ERRγ expression in vascular smooth muscle cells was induced during calcification medium-induced vascular calcification. Adenovirus-mediated overexpression of ERRγ in vascular smooth muscle cells resulted in the upregulation of the expression of osteogenic genes, including runt-related transcription factor 2, osteopontin, and Msx2, and the downregulation of α-smooth muscle actin. Adenovirus-mediated overexpression of ERRγ induced bone morphogenetic protein 2 (BMP2) expression, leading to increased phosphorylation of the intracellular BMP2 effector proteins SMAD1/5/8. Collectively, these data suggested that ERRγ promotes dedifferentiation of vascular smooth muscle cells to an osteogenic phenotype during the vascular calcification process. Inhibition of endogenous ERRγ expression or activity using a specific siRNA or the selective inverse agonist GSK5182 attenuated vascular calcification and osteogenic gene expression in vitro and in vivo. CONCLUSIONS: The present results indicate that ERRγ plays a key role in vascular calcification by upregulating the BMP2 signaling pathway, suggesting that inhibition of ERRγ is a potential therapeutic strategy for the prevention of vascular calcification.

Role of Mitochondria-Associated Endoplasmic Reticulum Membrane in Inflammation-Mediated Metabolic Diseases.

Inflammation is considered to be one of the most critical factors involved in the development of complex metabolic diseases such as type 2 diabetes, cancer, and cardiovascular disease. A few decades ago, the discovery of mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) was followed by the identification of its roles in regulating cellular homeostatic processes, ranging from cellular bioenergetics to apoptosis. MAM provides an excellent platform for numerous signaling pathways; among them, inflammatory signaling pathways associated with MAM play a critical role in cellular defense during pathogenic infections and metabolic disorders. However, induction of MAM causes deleterious effects by amplifying mitochondrial reactive oxygen species generation through increased calcium transfer from the ER to mitochondria, thereby causing mitochondrial damage and release of mitochondrial components into the cytosol as damage-associated molecular patterns (DAMPs). These mitochondrial DAMPs rapidly activate MAM-resident inflammasome components and other inflammatory factors, which promote inflammasome complex formation and release of proinflammatory cytokines in pathological conditions. Long-term stimulation of the inflammasome instigates chronic inflammation, leading to the pathogenesis of metabolic diseases. In this review, we summarize the current understanding of MAM and its association with inflammation-mediated metabolic diseases.

Enzyme-based assay for quantification of UDP-GlcNAc in cells and tissues.

In this issue of Cell Reports Methods, Sunden et al. develop an enzymatic assay to measure UDP-GlcNAc levels from cells and tissue.1 By reporting on the level of the substrate itself, this approach can potentially enhance the fields' understanding of UDP-GlcNAc concentration under a variety of conditions.

Sustained Increases in Cardiomyocyte Protein O-Linked ß-N-Acetylglucosamine Levels Lead to Cardiac Hypertrophy and Reduced Mitochondrial Function Without Systolic Contractile Impairment.

Background Lifestyle and metabolic diseases influence the severity and pathogenesis of cardiovascular disease through numerous mechanisms, including regulation via posttranslational modifications. A specific posttranslational modification, the addition of O-linked ß-N acetylglucosamine (O-GlcNAcylation), has been implicated in molecular mechanisms of both physiological and pathologic adaptations. The current study aimed to test the hypothesis that in cardiomyocytes, sustained protein O-GlcNAcylation contributes to cardiac adaptations, and its progression to pathophysiology. Methods and Results Using a naturally occurring dominant-negative O-GlcNAcase (dnOGA) inducible cardiomyocyte-specific overexpression transgenic mouse model, we induced dnOGA in 8- to 10-week-old mouse hearts. We examined the effects of 2-week and 24-week dnOGA overexpression, which progressed to a 1.8-fold increase in protein O-GlcNAcylation. Two-week increases in protein O-GlcNAc levels did not alter heart weight or function; however, 24-week increases in protein O-GlcNAcylation led to cardiac hypertrophy, mitochondrial dysfunction, fibrosis, and diastolic dysfunction. Interestingly, systolic function was maintained in 24-week dnOGA overexpression, despite several changes in gene expression associated with cardiovascular disease. Specifically, mRNA-sequencing analysis revealed several gene signatures, including reduction of mitochondrial oxidative phosphorylation, fatty acid, and glucose metabolism pathways, and antioxidant response pathways after 24-week dnOGA overexpression. Conclusions This study indicates that moderate increases in cardiomyocyte protein O-GlcNAcylation leads to a differential response with an initial reduction of metabolic pathways (2-week), which leads to cardiac remodeling (24-week). Moreover, the mouse model showed evidence of diastolic dysfunction consistent with a heart failure with preserved ejection fraction. These findings provide insight into the adaptive versus maladaptive responses to increased O-GlcNAcylation in heart.

α-Lipoic acid attenuates vascular calcification via reversal of mitochondrial function and restoration of Gas6/Axl/Akt survival pathway.

Vascular calcification is prevalent in patients with chronic kidney disease and leads to increased cardiovascular morbidity and mortality. Although several reports have implicated mitochondrial dysfunction in cardiovascular disease and chronic kidney disease, little is known about the potential role of mitochondrial dysfunction in the process of vascular calcification. This study investigated the effect of α-lipoic acid (ALA), a naturally occurring antioxidant that improves mitochondrial function, on vascular calcification in vitro and in vivo. Calcifying vascular smooth muscle cells (VSMCs) treated with inorganic phosphate (Pi) exhibited mitochondrial dysfunction, as demonstrated by decreased mitochondrial membrane potential and ATP production, the disruption of mitochondrial structural integrity and concurrently increased production of reactive oxygen species. These Pi-induced functional and structural mitochondrial defects were accompanied by mitochondria-dependent apoptotic events, including release of cytochrome c from the mitochondria into the cytosol, subsequent activation of caspase-9 and -3, and chromosomal DNA fragmentation. Intriguingly, ALA blocked the Pi-induced VSMC apoptosis and calcification by recovery of mitochondrial function and intracellular redox status. Moreover, ALA inhibited Pi-induced down-regulation of cell survival signals through the binding of growth arrest-specific gene 6 (Gas6) to its cognate receptor Axl and subsequent Akt activation, resulting in increased survival and decreased apoptosis. Finally, ALA significantly ameliorated vitamin D(3) -induced aortic calcification and mitochondrial damage in mice. Collectively, the findings suggest ALA attenuates vascular calcification by inhibiting VSMC apoptosis through two distinct mechanisms; preservation of mitochondrial function via its antioxidant potential and restoration of the Gas6/Axl/Akt survival pathway.

miRNA-200b-A Potential Biomarker Identified in a Porcine Model of Cardiogenic Shock and Mechanical Unloading.

Background: Cardiogenic shock (CS) alters whole body metabolism and circulating biomarkers serve as prognostic markers in CS patients. Percutaneous ventricular assist devices (pVADs) unload the left ventricle by actively ejecting blood into the aorta. The goal of the present study was to identify alterations in circulating metabolites and transcripts in a large animal model that might serve as potential prognostic biomarkers in acute CS and additional left ventricular unloading by Impella ® pVAD support. Methods: CS was induced in a preclinical large animal model by injecting microspheres into the left coronary artery system in six pigs. After the induction of CS, mechanical pVAD support was implemented for 30 min total. Serum samples were collected under basal conditions, after the onset of CS, and following additional pVAD unloading. Circulating metabolites were determined by metabolomic analysis, circulating RNA entities by RNA sequencing. Results: CS and additional pVAD support alter the abundance of circulating metabolites involved in Aminoacyl-tRNA biosynthesis and amino acid metabolism. RNA sequencing revealed decreased abundance of the hypoxia sensitive miRNA-200b following the induction of CS, which was reversed following pVAD support. Conclusion: The hypoxamir miRNA-200b is a potential circulating marker that is repressed in CS and is restored following pVAD support. The early transcriptional response with increased miRNA-200b expression following only 30 min of pVAD support suggests that mechanical unloading alters whole body metabolism. Future studies are required to delineate the impact of serum miRNA-200b levels as a prognostic marker in patients with acute CS and pVAD unloading.

Normalizing glucose levels reconfigures the mammary tumor immune and metabolic microenvironment and decreases metastatic seeding.

Obesity and diabetes cumulatively create a distinct systemic metabolic pathophysiological syndrome that predisposes patients to several diseases including breast cancer. Moreover, diabetic and obese women with breast cancer show a significant increase in mortality compared to non-obese and/or non-diabetic women. We hypothesized that these metabolic conditions incite an aggressive tumor phenotype by way of impacting tumor cell-autonomous and tumor cell non-autonomous events. In this study, we established a type 2 diabetic mouse model of triple-negative mammary carcinoma and investigated the effect of a glucose lowering therapy, metformin, on the overall tumor characteristics and immune/metabolic microenvironment. Diabetic mice exhibited larger mammary tumors that had increased adiposity with high levels of O-GlcNAc protein post-translational modification. These tumors also presented with a distinct stromal profile characterized by altered collagen architecture, increased infiltration by tumor-permissive M2 macrophages, and early metastatic seeding compared to non-diabetic/lean mice. Metformin treatment of the diabetic/obese mice effectively normalized glucose levels, reconfigured the mammary tumor milieu, and decreased metastatic seeding. Our results highlight the impact of two metabolic complications of obesity and diabetes on tumor cell attributes and showcase metformin's ability to revert tumor cell and stromal changes induced by an obese and diabetic host environment.

Increased Glucose Availability Attenuates Myocardial Ketone Body Utilization.

Background Perturbations in myocardial substrate utilization have been proposed to contribute to the pathogenesis of cardiac dysfunction in diabetic subjects. The failing heart in nondiabetics tends to decrease reliance on fatty acid and glucose oxidation, and increases reliance on ketone body oxidation. In contrast, little is known regarding the mechanisms mediating this shift among all 3 substrates in diabetes mellitus. Therefore, we tested the hypothesis that changes in myocardial glucose utilization directly influence ketone body catabolism. Methods and Results We examined ventricular-cardiac tissue from the following murine models: (1) streptozotocin-induced type 1 diabetes mellitus; (2) high-fat-diet-induced glucose intolerance; and transgenic inducible cardiac-restricted expression of (3) glucose transporter 4 (transgenic inducible cardiac restricted expression of glucose transporter 4); or (4) dominant negative O-GlcNAcase. Elevated blood glucose (type 1 diabetes mellitus and high-fat diet mice) was associated with reduced cardiac expression of ß-hydroxybutyrate-dehydrogenase and succinyl-CoA:3-oxoacid CoA transferase. Increased myocardial ß-hydroxybutyrate levels were also observed in type 1 diabetes mellitus mice, suggesting a mismatch between ketone body availability and utilization. Increased cellular glucose delivery in transgenic inducible cardiac restricted expression of glucose transporter 4 mice attenuated cardiac expression of both Bdh1 and Oxct1 and reduced rates of myocardial BDH1 activity and ß-hydroxybutyrate oxidation. Moreover, elevated cardiac protein O-GlcNAcylation (a glucose-derived posttranslational modification) by dominant negative O-GlcNAcase suppressed ß-hydroxybutyrate dehydrogenase expression. Consistent with the mouse models, transcriptomic analysis confirmed suppression of BDH1 and OXCT1 in patients with type 2 diabetes mellitus and heart failure compared with nondiabetic patients. Conclusions Our results provide evidence that increased glucose leads to suppression of cardiac ketolytic capacity through multiple mechanisms and identifies a potential crosstalk between glucose and ketone body metabolism in the diabetic myocardium.

Maintaining Myocardial Glucose Utilization in Diabetic Cardiomyopathy Accelerates Mitochondrial Dysfunction.

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear whether these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy, we generated transgenic mice with inducible cardiomyocyte-specific expression of the GLUT4. We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in nondiabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest that reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

PDK4 Augments ER-Mitochondria Contact to Dampen Skeletal Muscle Insulin Signaling During Obesity.

Mitochondria-associated endoplasmic reticulum membrane (MAM) is a structural link between mitochondria and endoplasmic reticulum (ER). MAM regulates Ca2+ transport from the ER to mitochondria via an IP3R1-GRP75-VDAC1 complex-dependent mechanism. Excessive MAM formation may cause mitochondrial Ca2+ overload and mitochondrial dysfunction. However, the exact implication of MAM formation in metabolic syndromes remains debatable. Here, we demonstrate that PDK4 interacts with and stabilizes the IP3R1-GRP75-VDAC1 complex at the MAM interface. Obesity-induced increase in PDK4 activity augments MAM formation and suppresses insulin signaling. Conversely, PDK4 inhibition dampens MAM formation and improves insulin signaling by preventing MAM-induced mitochondrial Ca2+ accumulation, mitochondrial dysfunction, and ER stress. Furthermore, Pdk4-/- mice exhibit reduced MAM formation and are protected against diet-induced skeletal muscle insulin resistance. Finally, forced formation and stabilization of MAMs with synthetic ER-mitochondria linker prevented the beneficial effects of PDK4 deficiency on insulin signaling. Overall, our findings demonstrate a critical mediatory role of PDK4 in the development of skeletal muscle insulin resistance via enhancement of MAM formation.

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.

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.

Oxidative stress and calcium dysregulation by palmitate in type 2 diabetes.

Free fatty acids (FFAs) are important substrates for mitochondrial oxidative metabolism and ATP synthesis but also cause serious stress to various tissues, contributing to the development of metabolic diseases. CD36 is a major mediator of cellular FFA uptake. Inside the cell, saturated FFAs are able to induce the production of cytosolic and mitochondrial reactive oxygen species (ROS), which can be prevented by co-exposure to unsaturated FFAs. There are close connections between oxidative stress and organellar Ca2+ homeostasis. Highly oxidative conditions induced by palmitate trigger aberrant endoplasmic reticulum (ER) Ca2+ release and thereby deplete ER Ca2+ stores. The resulting ER Ca2+ deficiency impairs chaperones of the protein folding machinery, leading to the accumulation of misfolded proteins. This ER stress may further aggravate oxidative stress by augmenting ER ROS production. Secondary to ER Ca2+ release, cytosolic and mitochondrial matrix Ca2+ concentrations can also be altered. In addition, plasmalemmal ion channels operated by ER Ca2+ depletion mediate persistent Ca2+ influx, further impairing cytosolic and mitochondrial Ca2+ homeostasis. Mitochondrial Ca2+ overload causes superoxide production and functional impairment, culminating in apoptosis. This vicious cycle of lipotoxicity occurs in multiple tissues, resulting in ß-cell failure and insulin resistance in target tissues, and further aggravates diabetic complications.

Low-Dose Persistent Organic Pollutants Impair Insulin Secretory Function of Pancreatic ß-Cells: Human and In Vitro Evidence.

Low-dose persistent organic pollutants (POPs), especially organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs), have emerged as a new risk factor for type 2 diabetes. We evaluated whether chronic exposure to low-dose POPs affects insulin secretory function of ß-cells in humans and in vitro cells. Serum concentrations of OCPs and PCBs were measured in 200 adults without diabetes. Mathematical model-based insulin secretion indices were estimated by using a 2-h seven-sample oral glucose tolerance test. Insulin secretion by INS-1E ß-cells was measured after 48 h of treatment with three OCPs or one PCB mixture. Static second-phase insulin secretion significantly decreased with increasing serum concentrations of OCPs. Adjusted means were 63.2, 39.3, 44.1, 39.3, 39.7, and 22.3 across six categories of a summary measure of OCPs (Ptrend = 0.02). Dynamic first-phase insulin secretion remarkably decreased with increasing concentrations of OCPs among only insulin-sensitive individuals (Ptrend = 0.02); the insulin levels among individuals with high OCPs were ∼30% of those with low OCPs. Compared with OCPs, PCBs showed weaker associations. The decreased insulin secretion by INS-1E ß-cells was observed for even 1 pmol/L OCP. The data from human and in vitro cell experiments suggest that chronic exposure to low-dose POPs, especially OCPs, can induce pancreatic ß-cell dysfunction.