Branched-chain Amino Acid Oxidation Research Articles

Abstract 6627: Boosting macrophage-specific BCAA oxidation enhances immune activation within the tumor microenvironment and diminishes tumor growth in pancreatic cancer

Abstract Treating pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge, mainly due to therapy resistance linked to the immunosuppressive tumor microenvironment (TME) within PDAC tumors. This TME is characterized by a fibrotic stroma and an abundance of immunosuppressive cells, notably tumor-associated macrophages (TAMs). TAMs crucially hinder effector T cell activity and contribute to T cell exhaustion. Redirecting TAMs toward an immunostimulatory state has emerged as a promising approach in PDAC treatment. Recent investigations, including our own studies, reveal a profound influence of metabolic pathways on the immune phenotype of TAMs. Specifically, dysregulation in branched-chain amino acid (BCAA) metabolism has been observed in PDAC patients, manifesting as elevated circulating BCAA levels correlating with a doubled risk of PDAC development. However, the extent to which BCAA metabolism influences TAM phenotype and anti-tumor immune responses remains a largely unexplored territory, presenting a challenge for PDAC treatment. Our focus revolves around understanding how heightened BCAA levels in PDAC affect TAM phenotype, the immunosuppressive tumor environment, and tumor progression. Employing an orthotopic PDAC model and genetically modified mice, specifically BCKDKfl/fl BCKDK LysMcre/+ (mimicking increased BCAA oxidation), alongside a BCKDK-targeting pharmacological inhibitor (BT-2), multicolor flow cytometry, and in vitro models, our research has revealed crucial insights. We found a diminished expression of BCAA oxidation genes in TAMs compared to normal pancreatic macrophages. Interestingly, M1 and M2 macrophages seemed to utilize BCAAs differently, reflected in varying levels of TCA cycle intermediates derived from BCAA oxidation. Notably, boosting BCAA oxidation specifically in TAMs through genetic modifications resulted in reduced PDAC growth associated with an enhanced immunostimulatory TAM phenotype and activation of CD8+ T cells in the TME. Conversely, administering BCAAs induced an immunosuppressive TAM phenotype, fostering PDAC growth, suggesting a direct influence of BCAAs on TAM phenotype. Pharmacologically enhancing BCAA oxidation via BT-2 bolstered immune activation within the tumor environment and reduced PDAC growth. Intriguingly, our analysis of publicly available data unveiled a significant correlation between high BCAA oxidation and favorable responses to anti-PD1 therapy in cancer patients. Our findings emphasize the pivotal role of BCAA metabolism in shaping the immune landscape of PDAC, offering potential avenues for therapeutic interventions targeting TAMs and mitigating TME immunosuppression. Citation Format: Gauri Mirji, Sajad Ahmad Bhat, Zachary Schug, Ben Z. Stanger, Zoltan Arany, Rahul S. Shinde. Boosting macrophage-specific BCAA oxidation enhances immune activation within the tumor microenvironment and diminishes tumor growth in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6627.

Lipodystrophy in methylmalonic acidemia associated with elevated FGF21 and abnormal methylmalonylation.

A distinct adipose tissue distribution pattern was observed in patients with methylmalonyl-CoA mutase deficiency, an inborn error of branched-chain amino acid (BCAA) metabolism, characterized by centripetal obesity with proximal upper and lower extremity fat deposition and paucity of visceral fat, that resembles familial multiple lipomatosis syndrome. To explore brown and white fat physiology in methylmalonic acidemia (MMA), body composition, adipokines, and inflammatory markers were assessed in 46 patients with MMA and 99 matched controls. Fibroblast growth factor 21 levels were associated with acyl-CoA accretion, aberrant methylmalonylation in adipose tissue, and an attenuated inflammatory cytokine profile. In parallel, brown and white fat were examined in a liver-specific transgenic MMA mouse model (Mmut-/- TgINS-Alb-Mmut). The MMA mice exhibited abnormal nonshivering thermogenesis with whitened brown fat and had an ineffective transcriptional response to cold stress. Treatment of the MMA mice with bezafibrates led to clinical improvement with beiging of subcutaneous fat depots, which resembled the distribution seen in the patients. These studies defined what we believe to be a novel lipodystrophy phenotype in patients with defects in the terminal steps of BCAA oxidation and demonstrated that beiging of subcutaneous adipose tissue in MMA could readily be induced with small molecules.

Reducing branched-chain amino acids improves cardiac stress response in mice by decreasing histone H3K23 propionylation.

Identification of branched-chain amino acid (BCAA) oxidation enzymes in the nucleus led us to predict that they are a source of the propionyl-CoA that is utilized for histone propionylation and, thereby, regulate gene expression. To investigate the effects of BCAAs on the development of cardiac hypertrophy and failure, we applied pressure overload on the heart in mice maintained on a diet with standard levels of BCAAs (BCAA control) versus a BCAA-free diet. The former was associated with an increase in histone H3K23-propionyl (H3K23Pr) at the promoters of upregulated genes (e.g., cell signaling and extracellular matrix genes) and a decrease at the promoters of downregulated genes (e.g., electron transfer complex [ETC I-V] and metabolic genes). Intriguingly, the BCAA-free diet tempered the increases in promoter H3K23Pr, thus reducing collagen gene expression and fibrosis during cardiac hypertrophy. Conversely, the BCAA-free diet inhibited the reductions in promoter H3K23Pr and abolished the downregulation of ETC I-V subunits, enhanced mitochondrial respiration, and curbed the progression of cardiac hypertrophy. Thus, lowering the intake of BCAAs reduced pressure overload-induced changes in histone propionylation-dependent gene expression in the heart, which retarded the development of cardiomyopathy.

Reactive nitrogen species inhibit branched chain alpha-ketoacid dehydrogenase complex and impact muscle cell metabolism

Branched chain α-ketoacid dehydrogenase complex (BCKDC) is the rate-limiting enzyme in branched chain amino acid (BCAA) catabolism, a metabolic pathway with great importance for human health. BCKDC belongs to the mitochondrial α-ketoacid dehydrogenase complex family, which also includes pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex. Here, we revealed that BCKDC can be substantially inhibited by reactive nitrogen species (RNS) via a mechanism similar to what we recently discovered with pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex-RNS can cause inactivating covalent modifications of the lipoic arm on its E2 subunit. In addition, we showed that such reaction between RNS and the lipoic arm of the E2 subunit can further promote inhibition of the E3 subunits of α-ketoacid dehydrogenase complexes. We examined the impacts of this RNS-mediated BCKDC inhibition in muscle cells, an important site of BCAA metabolism, and demonstrated that the nitric oxide production induced by cytokine stimulation leads to a strong inhibition of BCKDC activity and BCAA oxidation in myotubes and myoblasts. More broadly, nitric oxide production reduced the level of functional lipoic arms across the multiple α-ketoacid dehydrogenases and led to intracellular accumulation of their substrates (α-ketoacids), decrease of their products (acyl-CoAs), and a lower cellular energy charge. In sum, this work revealed a new mechanism for BCKDC regulation, demonstrated that RNS can generally inhibit all α-ketoacid dehydrogenases, which has broad physiological implications across multiple cell types, and elucidated the mechanistic connection between RNS-driven inhibitory modifications on the E2 and E3 subunits of α-ketoacid dehydrogenases.

PPM1K mediates metabolic disorder of branched-chain amino acid and regulates cerebral ischemia-reperfusion injury by activating ferroptosis in neurons

Ischemic stroke is a neurological disorder caused by vascular stenosis or occlusion, accounting for approximately 87% of strokes. Clinically, the most effective therapy for ischemic stroke is vascular recanalization, which aims to rescue neurons undergoing ischemic insults. Although reperfusion therapy is the most effective treatment for ischemic stroke, it still has limited benefits for many patients, and ischemia-reperfusion (I/R) injury is a widely recognized cause of poor prognosis. Here, we aim to investigate the mechanism of protein phosphatase Mg2+/Mn2+ dependent 1 K (PPM1K) mediates metabolic disorder of branched-chain amino acids (BCAA) by promoting fatty acid oxidation led to ferroptosis after cerebral I/R injury. We established the I/R model in mice and used BT2, a highly specific BCAA dehydrogenase (BCKD) kinase inhibitor to promote BCAA metabolism. It was further verified by lentivirus knocking down PPM1K in neurons. We found that BCAA levels were elevated after I/R injury due to dysfunctional oxidative degradation caused by phosphorylated BCKD E1α subunit (BCKDHA). Additionally, the level of phosphorylated BCKDHA was determined by decreased PPM1K in neurons. We next demonstrated that BCAA could induce oxidative stress, lipid peroxidation, and ferroptosis in primary cultured cortical neurons in vitro. Our results further showed that BT2 could reduce neuronal ferroptosis by enhancing BCAA oxidation through inhibition of BCKDHA phosphorylation. We further found that defective BCAA catabolism could induce neuronal ferroptosis by PPM1K knockdown. Furthermore, BT2 was found to alleviate neurological behavior disorders after I/R injury in mice, and the effect was similar to ferroptosis inhibitor ferrostatin-1. Our findings reveal a novel role of BCAA in neuronal ferroptosis after cerebral ischemia and provide a new potential target for the treatment of ischemic stroke.

Abstract P3202: The Branched Chain α-Ketoacid Dehydrogenase Kinase (BCKDK) Inhibitor BT2 Is A Mitochondrial Uncoupler

Elevated levels of branched-chain amino acids (BCAAs) are associated with cardiovascular and metabolic disease including heart failure, diabetes, and obesity. BCAAs are first transaminated into their respective branched-chain α-ketoacids (BCKAs) by the mitochondrial branched-chain amino-transaminase (BCAT m ). The pharmacological agent BT2 (dichloropheno[b]thiophene-2-carboxylic-acid) was designed to induce catabolism of BCAAs by inhibiting the branched-chain α-ketoacid dehydrogenase kinase (BCKDK). The function of BCKDK is to phosphorylate and therefore deactivate the branched-chain α-ketoacid dehydrogenase (BCKDH), an enzymatic complex that converts the BCKAs into their respective R-CoAs. Although it has been shown that BT2 protects from heart failure and metabolic disease in animal models, recent studies have suggested that its protective effects may originate from a mechanism independent of targeting BCAA oxidation. The objective of this study was to investigate alternative molecular mechanisms by which BT2 may confer cardioprotection and protect from metabolic disease. We hypothesized that BT2 is a mitochondrial uncoupler based on its chemical structure as a lipophilic weak acid. We demonstrate BT2 is a mitochondrial uncoupler using a combination of respirometry, membrane potential, and patch-clamp electrophysiology. BT2 has 20% of the potency of the chemical uncoupler 2,4-dinitrophenol (DNP). The data suggest BT2 may exert some of its cardioprotective effects and protection from metabolic disease via mitochondrial uncoupling.

Branched-chain amino acid catabolism in muscle affects systemic BCAA levels but not insulin resistance.

Elevated levels of plasma branched-chain amino acids (BCAAs) have been associated with insulin resistance and type 2 diabetes since the 1960s. Pharmacological activation of branched-chain α-ketoacid dehydrogenase (BCKDH), the rate-limiting enzyme of BCAA oxidation, lowers plasma BCAAs and improves insulin sensitivity. Here we show that modulation of BCKDH in skeletal muscle, but not liver, affects fasting plasma BCAAs in male mice. However, despite lowering BCAAs, increased BCAA oxidation in skeletal muscle does not improve insulin sensitivity. Our data indicate that skeletal muscle controls plasma BCAAs, that lowering fasting plasma BCAAs is insufficient to improve insulin sensitivity and that neither skeletal muscle nor liver account for the improved insulin sensitivity seen with pharmacological activation of BCKDH. These findings suggest potential concerted contributions of multiple tissues in the modulation of BCAA metabolism to alter insulin sensitivity.

Dietary essential amino acids for the treatment of heart failure with reduced ejection fraction.

Heart failure with reduced ejection fraction (HFrEF) is a leading cause of mortality worldwide, requiring novel therapeutic and lifestyle interventions. Metabolic alterations and energy production deficit are hallmarks and thereby promising therapeutic targets for this complex clinical syndrome. We aim to study the molecular mechanisms and effects on cardiac function in rodents with HFrEF of a designer diet in which free essential amino acids-in specifically designed percentages-substituted for protein. Wild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricle (LV) pressure overload or sham surgery. Whole-body glucose homeostasis was studied with glucose tolerance test, while myocardial dysfunction and fibrosis were measured with echocardiogram and histological analysis. Mitochondrial bioenergetics and morphology were investigated with oxygen consumption rate measurement and electron microscopy evaluation. Circulating and cardiac non-targeted metabolite profiles were analyzed by ultrahigh performance liquid chromatography-tandem mass spectroscopy, while RNA-sequencing was used to identify signalling pathways mainly affected. The amino acid-substituted diet shows remarkable preventive and therapeutic effects. This dietary approach corrects the whole-body glucose metabolism and restores the unbalanced metabolic substrate usage-by improving mitochondrial fuel oxidation-in the failing heart. In particular, biochemical, molecular, and genetic approaches suggest that renormalization of branched-chain amino acid oxidation in cardiac tissue, which is suppressed in HFrEF, plays a relevant role. Beyond the changes of systemic metabolism, cell-autonomous processes may explain at least in part the diet's cardioprotective impact. Collectively, these results suggest that manipulation of dietary amino acids, and especially essential amino acids, is a potential adjuvant therapeutic strategy to treat systolic dysfunction and HFrEF in humans.

Inhibition of branched-chain amino acid oxidation aggravates adverse remodeling in the failing heart

Inhibition of branched-chain amino acid oxidation aggravates adverse remodeling in the failing heart

Metabolon formation regulates branched-chain amino acid oxidation and homeostasis.

The branched-chain aminotransferase isozymes BCAT1 and BCAT2, segregated into distinct subcellular compartments and tissues, initiate the catabolism of branched-chain amino acids (BCAAs). However, whether and how BCAT isozymes cooperate with downstream enzymes to control BCAA homeostasis in an intact organism remains largely unknown. Here, we analyse system-wide metabolomic changes in BCAT1- and BCAT2-deficient mouse models. Loss of BCAT2 but not BCAT1 leads to accumulation of BCAAs and branched-chain α-keto acids (BCKAs), causing morbidity and mortality that can be ameliorated by dietary BCAA restriction. Through proximity labelling, isotope tracing and enzymatic assays, we provide evidence for the formation of a mitochondrial BCAA metabolon involving BCAT2 and branched-chain α-keto acid dehydrogenase. Disabling the metabolon contributes to BCAT2 deficiency-induced phenotypes, which can be reversed by BCAT1-mediated BCKA reamination. These findings establish a role for metabolon formation in BCAA metabolism in vivo and suggest a new strategy to modulate this pathway in diseases involving dysfunctional BCAA metabolism.

Muscle Amino Acid and Adenine Nucleotide Metabolism during Exercise and in Liver Cirrhosis: Speculations on How to Reduce the Harmful Effects of Ammonia.

Studies from the last decades indicate that increased levels of ammonia contribute to muscle wasting in critically ill patients. The aim of the article is to examine the effects of two different causes of hyperammonemia—increased ATP degradation in muscles during strenuous exercise and impaired ammonia detoxification to urea due to liver cirrhosis. During exercise, glycolysis, citric acid cycle (CAC) activity, and ATP synthesis in muscles increase. In cirrhosis, due to insulin resistance and mitochondrial dysfunction, glycolysis, CAC activity, and ATP synthesis in muscles are impaired. Both during exercise and in liver cirrhosis, there is increased ammonia detoxification to glutamine (Glu + NH3 + ATP → Gln + ADP + Pi), increased drain of ketoglutarate (α-KG) from CAC for glutamate synthesis by α-KG-linked aminotransferases, glutamate, aspartate, and α-KG deficiency, increased oxidation of branched-chain amino acids (BCAA; valine, leucine, and isoleucine), and protein-energy wasting in muscles. It is concluded that ammonia can contribute to muscle wasting regardless of the cause of its increased levels and that similar strategies can be designed to increase muscle performance in athletes and reduce muscle loss in patients with hyperammonemia. The pros and cons of glutamate, α-KG, aspartate, BCAA, and branched-chain keto acid supplementation are discussed.

Extra-cardiac BCAA catabolism lowers blood pressure and protects from heart failure

Pharmacologic activation of branched-chain amino acid (BCAA) catabolism is protective in models of heart failure (HF). How protection occurs remains unclear, although a causative block in cardiac BCAA oxidation is widely assumed. Here, we use invivo isotope infusions to show that cardiac BCAA oxidation in fact increases, rather than decreases, in HF. Moreover, cardiac-specific activation of BCAA oxidation does not protect from HF even though systemic activation does. Lowering plasma and cardiac BCAAs also fails to confer significant protection, suggesting alternative mechanisms of protection. Surprisingly, activation of BCAA catabolism lowers blood pressure (BP), a known cardioprotective mechanism. BP lowering occurred independently of nitric oxide and reflected vascular resistance to adrenergic constriction. Mendelian randomization studies revealed that elevated plasma BCAAs portend higher BP in humans. Together, these data indicate that BCAA oxidation lowers vascular resistance, perhaps in part explaining cardioprotection in HF that is not mediated directly in cardiomyocytes.

Obese Skeletal Muscle-Expressed Interferon Regulatory Factor 4 Transcriptionally Regulates Mitochondrial Branched-Chain Aminotransferase Reprogramming Metabolome.

In addition to the significant role in physical activity, skeletal muscle also contributes to health through the storage and use of macronutrients associated with energy homeostasis. However, the mechanisms of regulating integrated metabolism in skeletal muscle are not well-defined. Here, we compared the skeletal muscle transcriptome from obese and lean control subjects in different species (human and mouse) and found that interferon regulatory factor 4 (IRF4), an inflammation-immune transcription factor, conservatively increased in obese subjects. Thus, we investigated whether IRF4 gain of function in the skeletal muscle predisposed to obesity and insulin resistance. Conversely, mice with specific IRF4 loss in skeletal muscle showed protection against the metabolic effects of high-fat diet, increased branched-chain amino acids (BCAA) level of serum and muscle, and reprogrammed metabolome in serum. Mechanistically, IRF4 could transcriptionally upregulate mitochondrial branched-chain aminotransferase (BCATm) expression; subsequently, the enhanced BCATm could counteract the effects caused by IRF4 deletion. Furthermore, we demonstrated that IRF4 ablation in skeletal muscle enhanced mitochondrial activity, BCAA, and fatty acid oxidation in a BCATm-dependent manner. Taken together, these studies, for the first time, established IRF4 as a novel metabolic driver of macronutrients via BCATm in skeletal muscle in terms of diet-induced obesity.

Augmenting oxidation of branched-chain amino acids (BCAAs) treats murine chronic Graft-Versus-Host Disease (cGVHD)

Abstract Aberrant immune responses, such as high Tfollicular helper (Tfh) and germinal center B cells (GCB) in cGVHD inducing antibody (Ab) secretion with tissue deposition and fibrosis, require distinct metabolic reprogramming to sustain pathogenesis. Leucine, isoleucine, and valine are essential BCAAs that signal via mTORC1 to support glycolysis and protein synthesis. Alternatively, BCAAs can be oxidized to metabolites that directly enter the tricarboxylic acid cycle or become substrates for gluconeogenesis and ketogenesis. Branched chain ketoacid dehydrogenase kinase (BDK) is a negative regulator of BCAA metabolism, decreasing downstream oxidation and increasing mTORC1 and glycolysis. We sought to determine whether increasing BCAA oxidation could be exploited to inhibit cGVHD. BDK is inhibitable by 3,6-Dichlorobenzo[β] thiophene-2-carboxylic acid (BT2), increasing BCAA oxidation and decreasing T effector (Teff) activation. In a multiorgan cGVHD model with bronchiolitis obliterans, mice that received BDK knockout vs wildtype donor T cells had improved survival and pulmonary function with decreased GCB frequencies (p-value 0.005) and a trend toward decreased Tfh/Tfollicular regulatory ratios. An in vitro assay measuring Tfh-mediated B cell Ab production showed that BT2 downregulates IgG class switching, which is key for cGVHD pathogenesis. BT2-treated T effectors but not T regulatory cells (Treg) exhibited increased oxidative phosphorylation (OXPHOS). Therefore, limiting BCAAs inhibits Teff switch from a highly glycolytic state to OXPHOS. In contrast, BCAAs are not required for Treg energy production or survival. These data suggest that augmenting BCAA oxidation could be a new therapeutic approach for treating cGVHD. Supported by NIH P01 AI056299

An amino acid-defined diet impairs tumour growth in mice by promoting endoplasmic reticulum stress and mTOR inhibition

ObjectiveProfound metabolic alterations characterize cancer development and, beyond glucose addiction, amino acid (AA) dependency is now recognized as a hallmark of tumour growth. Therefore, targeting the metabolic addiction of tumours by reprogramming their substrate utilization is an attractive therapeutic strategy. We hypothesized that a dietary approach targeted to stimulate oxidative metabolism could reverse the metabolic inflexibility of tumours and represent a proper adjuvant therapy. MethodsWe measured tumour development in xenografted mice fed with a designer, casein-deprived diet enriched in free essential amino acids (EAAs; SFA-EAA diet), or two control isocaloric, isolipidic, and isonitrogenous diets, identical to the SFA-EAA diet except for casein presence (SFA diet), or casein replacement by the free AA mixture designed on the AA profile of casein (SFA-CAA diet). Moreover, we investigated the metabolic, biochemical, and molecular effects of two mixtures that reproduce the AA composition of the SFA-EAA diet (i.e., EAAm) and SFA-CAA diet (i.e., CAAm) in diverse cancer and non-cancer cells. ResultsThe SFA-EAA diet reduced tumour growth in vivo, promoted endoplasmic reticulum (ER) stress, and inhibited mechanistic/mammalian target of rapamycin (mTOR) activity in the tumours. Accordingly, in culture, the EAAm, but not the CAAm, activated apoptotic cell death in cancer cells without affecting the survival and proliferation of non-cancer cells. The EAAm increased branched-chain amino acid (BCAA) oxidation and decreased glycolysis, ATP levels, redox potential, and intracellular content of selective non-essential amino acids (NEAA) in cancer cells. The EAAm-induced NEAA starvation activated the GCN2-ATF4 stress pathway, leading to ER stress, mTOR inactivation, and apoptosis in cancer cells, unlike non-cancer cells. ConclusionTogether, these results confirm the efficacy of specific EAA mixtures in promoting cancer cells’ death and suggest that manipulation of dietary EAA content and profile could be a valuable support to the standard chemotherapy for specific cancers.

Branched-Chain Amino Acid Metabolism in the Failing Heart.

Branched-chain amino acids (BCAAs) are essential amino acids which have critical roles in protein synthesis and energy metabolism in the body. In the heart, there is a strong correlation between impaired BCAA oxidation and contractile dysfunction in heart failure. Plasma and myocardial levels of BCAA and their metabolites, namely branched-chain keto acids (BCKAs), are also linked to cardiac insulin resistance and worsening adverse remodelling in the failing heart. This review discusses the regulation of BCAA metabolism in the heart and the impact of depressed cardiac BCAA oxidation on cardiac energy metabolism, function, and structure in heart failure. While impaired BCAA oxidation in the failing heart causes the accumulation of BCAA and BCKA in the myocardium, recent evidence suggested that the BCAAs and BCKAs have divergent effects on the insulin signalling pathway and the mammalian target of the rapamycin (mTOR) signalling pathway. Dietary and pharmacological interventions that enhance cardiac BCAA oxidation and limit the accumulation of cardiac BCAAs and BCKAs have been shown to have cardioprotective effects in the setting of ischemic heart disease and heart failure. Thus, targeting cardiac BCAA oxidation may be a promising therapeutic approach for heart failure.

Dihydrolipoamide dehydrogenase, pyruvate oxidation, and acetylation-dependent mechanisms intersecting drug iatrogenesis.

In human metabolism, pyruvate dehydrogenase complex (PDC) is one of the most intricate and large multimeric protein systems representing a central hub for cellular homeostasis. The worldwide used antiepileptic drug valproic acid (VPA) may potentially induce teratogenicity or a mild to severe hepatic toxicity, where the underlying mechanisms are not completely understood. This work aims to clarify the mechanisms that intersect VPA-related iatrogenic effects to PDC-associated dihydrolipoamide dehydrogenase (DLD; E3) activity. DLD is also a key enzyme of α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, α-ketoadipate dehydrogenase, and the glycine decarboxylase complexes. The molecular effects of VPA will be reviewed underlining the data that sustain a potential interaction with DLD. The drug-associated effects on lipoic acid-related complexes activity may induce alterations on the flux of metabolites through tricarboxylic acid cycle, branched-chain amino acid oxidation, glycine metabolism and other cellular acetyl-CoA-connected reactions. The biotransformation of VPA involves its complete β-oxidation in mitochondria causing an imbalance on energy homeostasis. The drug consequences as histone deacetylase inhibitor and thus gene expression modulator have also been recognized. The mitochondrial localization of PDC is unequivocal, but its presence and function in the nucleus were also demonstrated, generating acetyl-CoA, crucial for histone acetylation. Bridging metabolism and epigenetics, this review gathers the evidence of VPA-induced interference with DLD or PDC functions, mainly in animal and cellular models, and highlights the uncharted in human. The consequences of this interaction may have significant impact either in mitochondrial or in nuclear acetyl-CoA-dependent processes.

Abstract P388: Extra-cardiac BCAA Catabolism Lowers Blood Pressure And Protects From Heart Failure

Pharmacologic activation of branched chain amino acid (BCAA) catabolism is protective in numerous models of heart failure (HF). How this protection occurs has remained unclear, although a causative block in cardiac BCAA oxidation has been proposed. We use here in vivo heavy isotope infusion studies to show that cardiac preference for BCAA oxidation increases, rather than decreases, in multiple models of HF. We use various genetic models to show that cardiac-specific activation of BCAA oxidation does not protect from HF, even though systemic activation of BCAA oxidation does. Lowering plasma and cardiac BCAAs by genetic means is also not sufficient to confer protection comparable to that conferred by pharmacologic activation of BCAA oxidation, suggesting alternative mechanisms of protection. Surprisingly, telemetry and invasive hemodynamic studies showed that pharmacological activation of BCAA catabolism lowers blood pressure, a well-established cardioprotective mechanism. The effects on blood pressure occurred independently of nitric oxide (NO), and reflected a vascular resistance to adrenergic constriction. Finally, mendelian randomization studies revealed that elevations in plasma BCAAs portend higher blood pressure in large human cohorts. Together, these data indicate that activation of BCAA oxidation lowers blood pressure and protects from heart failure independently of any direct effects on the heart itself.

202-OR: Cell-Autonomous Impairment of BCAA Catabolism in White Adipocytes Leads to Systemic Metabolic Dysregulation

Increased levels of serum branched-chain amino acids (BCAA) is associated with obesity, insulin resistance (IR), and type 2 diabetes (T2D). However, the mechanisms that trigger perturbations in BCAA homeostasis, and whether elevated BCAA causes obesity and other metabolic impairments or is a consequence of them is not clear. Young knock-in mice bearing the human T2D-linked R1788W variant in the cytoskeletal protein ankyrin-B (AnkB) show normal body weight and composition. These mice display increased glucose uptake by white adipose tissue (WAT) due to deficient internalization of the glucose transporter GLUT4. We reported that sustained elevation in glucose disposal in WAT leads to the onset of increased adiposity, lipotoxicity, systemic inflammation, and IR in AnkB mutant mice with age or in young animals fed Western diets. Non-obese R1788W AnkB mice also exhibit transcriptional changes consistent with lower BCAA catabolism in WAT. Here, we show that AnkB deficits impair BCAA catabolism in WAT prior to the onset of obesity. Lean R1788W AnkB mice show reduction in surface levels of the BCAA transporter Asct2 and BCAA uptake together with downregulation of BCAA catabolic enzymes in WAT. These deficits are concomitant with elevations in serum levels of BCAA and related acylcarnitines. Reduced expression of BCAA catabolic enzymes was also observed in WAT of lean WAT-specific AnkB KO mice. In contrast, AnkB deficiency did not alter BCAA oxidation in skeletal muscle (SKM), brown adipose tissue, or liver. Studies in control and R1788W AnkB mice fed isocaloric low and high fat (HFD) diets enriched in BCAA suggest that BCAA elevation triggers mitochondrial dysfunction, impairments in insulin secretion by the pancreas and in insulin signaling in SKM. Our data indicates that elevated circulating BCAA impair glucose tolerance and insulin sensitivity independently of obesity, and that these deleterious effects are potentiated by HFD. Disclosure J. Tzeng: None. A. Aguillard: None. D. Lorenzo: None. Funding American Diabetes Association (1-19-JDF-081 to D.L.)

PPARγ-induced upregulation of subcutaneous fat adiponectin secretion, glyceroneogenesis and BCAA oxidation requires mTORC1 activity

The nutrient sensors peroxisome proliferator-activated receptor γ (PPARγ) and mechanistic target of rapamycin complex 1 (mTORC1) closely interact in the regulation of adipocyte lipid storage. The precise mechanisms underlying this interaction and whether this extends to other metabolic processes and the endocrine function of adipocytes are still unknown. We investigated herein the involvement of mTORC1 as a mediator of the actions of the PPARγ ligand rosiglitazone in subcutaneous inguinal white adipose tissue (iWAT) mass, endocrine function, lipidome, transcriptome and branched-chain amino acid (BCAA) metabolism. Mice bearing regulatory associated protein of mTOR (Raptor) deletion and therefore mTORC1 deficiency exclusively in adipocytes and littermate controls were fed a high-fat diet supplemented or not with the PPARγ agonist rosiglitazone (30 mg/kg/day) for 8 weeks and evaluated for iWAT mass, lipidome, transcriptome (Rnaseq), respiration and BCAA metabolism. Adipocyte mTORC1 deficiency not only impaired iWAT adiponectin transcription, synthesis and secretion, PEPCK mRNA levels, triacylglycerol synthesis and BCAA oxidation and mRNA levels of related proteins but also completely blocked the upregulation in these processes induced by pharmacological PPARγ activation with rosiglitazone. Mechanistically, adipocyte mTORC1 deficiency impairs PPARγ transcriptional activity by reducing PPARγ protein content, as well as by downregulating C/EBPα, a co-partner and facilitator of PPARγ. In conclusion, mTORC1 and PPARγ are essential partners involved in the regulation of subcutaneous adipose tissue adiponectin production and secretion and BCAA oxidative metabolism.