Branched-chain Keto Acid Research Articles

The BCKDH kinase inhibitor BT2 promotes BCAA disposal and mitochondrial proton leak in both insulin-sensitive and insulin-resistant C2C12 myotubes.

Elevated circulating branched-chain amino acids (BCAAs) have been correlated with the severity of insulin resistance, leading to recent investigations that stimulate BCAA metabolism for the potential benefit of metabolic diseases. BT2(3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), an inhibitor of branched-chain ketoacid dehydrogenase kinase, promotes BCAA metabolism by enhancing BCKDH complex activity. The purpose of this report was to investigate the effects of BT2 on mitochondrial and glycolytic metabolism, insulin sensitivity, and de novo lipogenesis both with and without insulin resistance. C2C12 myotubes were treated with or without low or moderate levels of BT2 with or without insulin resistance. Western blot and quantitative real-time polymerase chain reactionwere used to assess protein and gene expression, respectively. Mitochondrial, nuclei, and lipid content were measured using fluorescent staining and microscopy. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Liquid chromatography-mass spectrometry was used to quantify BCAA media content. BT2 treatment consistently promoted mitochondrial uncoupling following 24-h treatment, which occurred largely independent of changes in expressional profiles associated with mitochondrial biogenesis, mitochondrial dynamics, BCAA catabolism, insulin sensitivity, or lipogenesis. Acute metabolic studies revealed a significant and dose-dependent effect of BT2 on mitochondrial proton leak, suggesting BT2 functions as a small-molecule uncoupler. Additionally, BT2 treatment consistently and dose-dependently reduced extracellular BCAA levels without altering expression of BCAA catabolic enzymes or pBCKDHa activation. BT2 appears to act as a small-molecule mitochondrial uncoupler that promotes BCAA utilization, though the interplay between these two observations requires further investigation.

Reduced Branched-Chain Amino Acid Intake Improved High-Fat Diet-Induced Nonalcoholic Fatty Pancreas Disease in Mice.

To explore the effects of branched-chain amino acids (BCAAs) on nonalcoholic fatty pancreas disease (NAFPD) and its possible mechanism in high-fat diet (HFD) induced mice. Pancreatic morphology and lipid infiltration was assessed by hematoxylin-eosin staining and immunohistochemistry, and lipid levels in the pancreas were determined using colorimetric enzymatic method. Relevant mechanism was investigated using western blotting and biochemical test. In HFD-fed mice, dietary BCAAs restriction could attenuate body weight increase, improve glucose metabolism, and reduce excessive lipid accumulation in the pancreas. Furthermore, expression of AMPKα and downstream uncoupling protein 1 were upregulated, while genes related to mammalian target of rapamycin complex 1 (mTORC1) signal pathway and lipid de novo synthesis were suppressed in HFD-BCAA restriction group compared with HFD and HFD-high BCAAs fed mice. In addition, BCAA restriction upregulated expression of BCAAs related metabolic enzymes including PPM1K and BCKDHA, and decreased the levels of BCAAs and branched chain keto acid in the pancreas. However, there was no difference in levels of lipid content in the pancreas and gene expression of AMPKα and mTORC1 between HFD and HFD-high BCAAs groups. Branched-chain amino acid restriction ameliorated HFD-induced NAFPD in mice by activation of AMPKα pathway and suppression of mTORC1 pathway.

Elevated BCAA catabolism reverses the effect of branched-chain ketoacids on glucose transport in mTORC1-dependent manner in L6 myotubes.

Plasma levels of branched-chain amino acids (BCAA) and their metabolites, branched-chain ketoacids (BCKA), are increased in insulin resistance. We previously showed that ketoisocaproic acid (KIC) suppressed insulin-stimulated glucose transport in L6 myotubes, especially in myotubes depleted of branched-chain ketoacid dehydrogenase (BCKD), the enzyme that decarboxylates BCKA. This suggests that upregulating BCKD activity might improve insulin sensitivity. We hypothesised that increasing BCAA catabolism would upregulate insulin-stimulated glucose transport and attenuate insulin resistance induced by BCKA. L6 myotubes were either depleted of BCKD kinase (BDK), the enzyme that inhibits BCKD activity, or treated with BT2, a BDK inhibitor. Myotubes were then treated with KIC (200 μM), leucine (150 μM), BCKA (200 μM), or BCAA (400 μM) and then treated with or without insulin (100 nM). BDK depletion/inhibition rescued the suppression of insulin-stimulated glucose transport by KIC/BCKA. This was consistent with the attenuation of IRS-1 (Ser612) and S6K1 (Thr389) phosphorylation but there was no effect on Akt (Ser473) phosphorylation. The effect of leucine or BCAA on these measures was not as pronounced and BT2 did not influence the effect. Induction of the mTORC1/IRS-1 (Ser612) axis abolished the attenuating effect of BT2 treatment on glucose transport in cells treated with KIC. Surprisingly, rapamycin co-treatment with BT2 and KIC further reduced glucose transport. Our data suggests that the suppression of insulin-stimulated glucose transport by KIC/BCKA in muscle is mediated by mTORC1/S6K1 signalling. This was attenuated by upregulating BCAA catabolic flux. Thus, interventions targeting BCAA metabolism may provide benefits against insulin resistance and its sequelae.

Definition of the Neurotoxicity-Associated Metabolic Signature Triggered by Berberine and Other Respiratory Chain Inhibitors.

To characterize the hits from a phenotypic neurotoxicity screen, we obtained transcriptomics data for valinomycin, diethylstilbestrol, colchicine, rotenone, 1-methyl-4-phenylpyridinium (MPP), carbaryl and berberine (Ber). For all compounds, the concentration triggering neurite degeneration correlated with the onset of gene expression changes. The mechanistically diverse toxicants caused similar patterns of gene regulation: the responses were dominated by cell de-differentiation and a triggering of canonical stress response pathways driven by ATF4 and NRF2. To obtain more detailed and specific information on the modes-of-action, the effects on energy metabolism (respiration and glycolysis) were measured. Ber, rotenone and MPP inhibited the mitochondrial respiratory chain and they shared complex I as the target. This group of toxicants was further evaluated by metabolomics under experimental conditions that did not deplete ATP. Ber (204 changed metabolites) showed similar effects as MPP and rotenone. The overall metabolic situation was characterized by oxidative stress, an over-abundance of NADH (>1000% increase) and a re-routing of metabolism in order to dispose of the nitrogen resulting from increased amino acid turnover. This unique overall pattern led to the accumulation of metabolites known as biomarkers of neurodegeneration (saccharopine, aminoadipate and branched-chain ketoacids). These findings suggest that neurotoxicity of mitochondrial inhibitors may result from an ensemble of metabolic changes rather than from a simple ATP depletion. The combi-omics approach used here provided richer and more specific MoA data than the more common transcriptomics analysis alone. As Ber, a human drug and food supplement, mimicked closely the mode-of-action of known neurotoxicants, its potential hazard requires further investigation.

Sex Differences in Branched-chain Amino Acid and Tryptophan Metabolism and Pathogenesis of Youth-onset Type 2 Diabetes.

Insulin resistance is associated with elevations in plasma branched-chain amino acids (BCAAs). BCAAs compete with aromatic amino acids including tryptophan for uptake into β cells. To explore relationships between BCAAs and tryptophan metabolism, adiposity, and glucose tolerance, we compared urine metabolites in overweight/obese youth with type 2 diabetes (T2D) with those in nondiabetic overweight/obese and lean youth. Metabolites were measured in 24-hour and first-morning urine samples of 56 nondiabetic adolescents with overweight/obesity, 42 adolescents with T2D, and 43 lean controls, aged 12 to 21 years. Group differences were assessed by Kruskal Wallis or ANOVA. Groups were comparable for age, pubertal status, and ethnicity. Youth with T2D were predominantly female and had highest percent body fat. BCAAs, branched-chain ketoacids (BCKAs), tryptophan, and kynurenine were higher in urine of subjects with T2D. There were no differences between lean controls and nondiabetic youth with overweight/obesity. T2D was associated with diversion of tryptophan from the serotonin to the kynurenine pathway, with higher urinary kynurenine/serotonin ratio and lower serotonin/tryptophan and 5-HIAA/kynurenine ratios. Urinary BCAAs, BCKAs, tryptophan, and ratios reflecting diversion to the kynurenine pathway correlated positively with metrics of body fat and hemoglobin A1c. Increases in these metabolites in the obese T2D group were more pronounced and statistically significant only in adolescent girls. Increases in urinary BCAAs and BCKAs in adolescent females with T2D are accompanied by diversion of tryptophan metabolism from the serotonin to the kynurenine pathway. These adaptations associate with higher risks of T2D in obese adolescent females than adolescent males.

Abstract 16786: Integrative Omics in Failing Myocardium Highlights Metabolic Substrate Utilization Defects in Human Heart Failure

Introduction: Changes in substrate utilization may be a biomarker of, or a contributor to, heart failure with reduced ejection fraction (HFrEF). Previous work has described circulating metabolites, proteins and transcripts that reflect myocardial bioenergetics, but direct assessment in relevant tissues is lacking. Here, we leverage integrative omics to identify tissue-level biomarkers and pathways of HFrEF that may play a role in pathogenesis. Methods: Myocardial tissue was obtained from 27 HFrEF donors and 21 non-failing hearts (noHF) from Duke University and the University of Colorado Medical Centers. Targeted (n=139 metabolites) and nontargeted (Metabolon, n=817) metabolic profiling was performed; in addition, 496 proteins were assayed (Olink) and ~31,000 transcripts were quantified via RNA sequencing. Individual metabolites were tested for association with HFrEF in models adjusted for age, sex and diabetes. Metabolomics, proteomics and RNA-seq data were integrated using block sparse partial least squares discriminant analysis (sPLS-DA) to identify correlated biomarkers that discriminate HFrEF from noHF hearts. Results: The targeted metabolomics platform primarily assayed metabolic fuel substrate pathways; 71 analytes (51%) were associated with HFrEF after controlling the false discovery rate (FDR) at 5%. Medium- and long-chain acylcarnitines (C10s-C18s) were significantly lower in tissue from failing hearts, while branched-chain amino acids (BCAA) and ketoacids were higher. In the nontargeted set, 293 metabolites (36%) differed between HFrEF and noHF. BCAA and acylcarnitine associations were recapitulated along with a decrease in malonylcarnitine. The sPLS-DA model selected 25 targeted and 50 nontargeted metabolites, 35 proteins and 35 transcripts, and successfully discriminated HFrEF from no HF (balanced error rate = 3.2%). 39/186 tested KEGG pathways were overrepresented in these analytes, including fatty acid metabolism (FDR q=0.002). Conclusions: Analysis of tissue-level metabolites suggests both decreased use of fatty acids as a fuel and disrupted enzymatic breakdown of BCAAs in the failing heart, while integrative omics pathway analysis also supports the association of disrupted fatty acid metabolism with HF.

Divergent Skeletal Muscle Metabolomic Signatures of Different Exercise Training Modes Independently Predict Cardiometabolic Risk Factors.

We aimed to understand the link between skeletal muscle metabolites and cardiometabolic health after exercise training. Although aerobic, resistance, and combined exercise training each enhance muscle insulin sensitivity as well as other cardiometabolic parameters, they disparately alter amino and citric acid metabolites as well as the lipidome, linking these metabolomic changes independently to the improvement of cardiometabolic risks with each exercise training mode. These findings reveal an important layer of the unique exercise mode-dependent changes in muscle metabolism, which may eventually lead to more informed exercise prescription for improving SI.

Mitophagy in Astrocytes Is Required for the Health of Optic Nerve.

Mitochondrial dysfunction in astrocytes has been implicated in the development of various neurological disorders. Mitophagy, mitochondrial autophagy, is required for proper mitochondrial function by preventing the accumulation of damaged mitochondria. The importance of mitophagy, specifically in the astrocytes of the optic nerve (ON), has been little studied. We introduce an animal model in which two separate mutations act synergistically to produce severe ON degeneration. The first mutation is in Cryba1, which encodes βA3/A1-crystallin, a lens protein also expressed in astrocytes, where it regulates lysosomal pH. The second mutation is in Bckdk, which encodes branched-chain ketoacid dehydrogenase kinase, which is ubiquitously expressed in the mitochondrial matrix and involved in the catabolism of the branched-chain amino acids. BCKDK is essential for mitochondrial function and the amelioration of oxidative stress. Neither of the mutations in isolation has a significant effect on the ON, but animals homozygous for both mutations (DM) exhibit very serious ON degeneration. ON astrocytes from these double-mutant (DM) animals have lysosomal defects, including impaired mitophagy, and dysfunctional mitochondria. Urolithin A can rescue the mitophagy impairment in DM astrocytes and reduce ON degeneration. These data demonstrate that efficient mitophagy in astrocytes is required for ON health and functional integrity.

THU136 Insulin Resistance, Insulin Secretion, And Body Fat In Youth: Associations With Branched-chain Amino-acids (BCAA), Branched-chain Keto-acids (BCKAs), Glutamate/Glutamine (Glx), And Kynurenine (KYN)

Abstract Disclosure: K. Blew: None. Q. Wang: None. H. Hong: None. O. Ilkayeva: None. M. Muehlbauer: None. M. Crawford: Employee; Self; Laboratory Corporations of America. Stock Owner; Self; Laboratory Corporations of America. R.P. Grant: Employee; Self; Laboratory Corporations of America. Stock Owner; Self; Laboratory Corporations of America. D.S. Hsia: None. J.R. Bain: None. M. Freemark: None. P. Gumus Balikcioglu: None. Objective: We recently showed that insulin resistance (IR) and Type 2 diabetes (T2D) in youth with obesity are associated with disordered metabolism of BCAA and diversion of tryptophan (TRP) metabolism from the serotonin to the kynurenine (KYN) pathway. In this study we determined if BCAAs, BCKAs, Glx and KYN/TRP correlate with metrics of obesity, insulin sensitivity, and insulin secretion. Methods: We performed frequently sampled oral glucose tolerance tests (OGTT) in 16 youth with normal weight and 16 non-diabetic youth with overweight/obesity and mild-moderate IR ages 18-21 yo. After an overnight fast of 8-12 hours, subjects underwent a 3-hour (1.75 g/kg, max 75 g) frequently sampled OGTT. Glucose (G) and insulin (I) were measured at times -15, 0, 10, 15, 30, 45, 60, 90, 120, and 180 minutes. Plasma metabolomic profiles were obtained at time 0. HOMA-S, HOMA-B, HOMA-IR, Matsuda Index, AUC glucose, AUC insulin, insulinogenic index (IGI) and disposition index (DI) at 10, 15 and 30 min were calculated. % Body fat was measured by TANITA. Results: Groups were comparable in age (19±1.2 vs 19.6±1.1 years), sex, race, ethnicity and pubertal status. Overweight/obese participants had higher BMI (22±2.3 vs 33.7±8.1), BF% (19.6±7.2 vs 37±9.2), leptin, and CRP and were more insulin resistant as evidenced by higher HOMA-IR and HOMA-B and lower HOMA-S and Matsuda Index. Glucose at 120’, insulin at -15’,0’,15’ and 120’, and IGI at 15’ were higher among overweight/obese subjects. AUC glucose, AUC insulin and DI were comparable across groups, as were fasting BCAA, BCAA/BCKA ratio, TRP, KYN, Kynurenic acid, and the KYN/TRP ratio. BCKA levels trended lower (p=0.05) in the overweight/obese group. Glx levels were higher among overweight/obese subjects (p=0.03) and correlated positively with BMI, % body fat, and metrics of hepatic IR including HOMA IR and ALT. BCAA levels correlated positively with fasting KYN and negatively with DI at 15’ and HMW-adiponectin, a metric of insulin sensitivity, but did not correlate with BMI or % body fat. The ratio of BCAA to BCKA correlated positively with KYN, KYN/TRP ratio and BMI. In contrast, BCKAs correlated negatively with BMI and % body fat but did not correlate with metrics of insulin sensitivity. KIV, the α-keto-acid of valine, associated negatively with metrics of insulin secretion including DI and IGI at 15’ and AUC glucose. Conclusions: In this cohort of non-diabetic youth, the levels of Glx correlated strongly with body fat mass and metrics of hepatic insulin resistance. The levels of BCAA and the ratio of BCAA/BCKA associated positively with KYN and negatively with HMW-adiponectin, a metric of insulin sensitivity, but did not correlate with % body fat. In contrast, BCKAs correlated negatively with % body fat and insulin secretion but did not correlate with metrics of IR. These findings suggest complex effects of fat deposition and insulin sensitivity on BCAA and glutamate metabolism. Presentation: Thursday, June 15, 2023

SAT415 Development Of A Novel, Preclinical Swine Model Of Transgender Men To Assess Testosterone Impact On Insulin Sensitivity And Whole-Body Metabolism

Abstract Disclosure: N. Mahabir: None. I. Ahmad: None. T. Wright: None. G. Ten Have: None. N. Deutz: None. A.E. Newell-Fugate: None. The effects oftestosterone therapy on peripheral insulin sensitivity in transgender men areequivocal due to heterogeneity of the population, short term patientfollow-up, and small cohort sizes. Most research has found testosterone therapyto decrease or have no effect on insulin sensitivity, but in one studytransgender men had increased insulin sensitivity. We have found thatshort-term, virilizing doses of testosterone in female pigs suppress circulatinginsulin levels. The objective of the current study was to develop a long-term,preclinical model of transgender men to assess insulin sensitivity and whole-bodymetabolism. A swine model of transgender men is superior to other models,because pigs closely mimic human cardiometabolic physiology and their largebody size permits longitudinal sampling and interventional procedures. Togenerate stable, long-term serum testosterone (TEST) concentrations in femalepigs, we fabricated testosterone silastic implants at the following doses:placebo (30 mm, n=1) or 15 mm (n=2), 30 mm (n=2), 60 mm (n=2), or 120 mm (n=1)TEST. Eight sexually mature female pigs were administered 14 days of progestin(Altrenogest) which was withdrawn on day 15. On day 16, 400 I.U. of pregnantmare serum gonadotropin and 200 I.U of human chorionic gonadotropin (PG600®,Merck Animal Health) were administered intramuscularly to stimulatefolliculogenesis and ovulation. Pigs had estrus detection performed dailythroughout the study and came into estrus 3-7 days post PG600® injection.Twenty-four hours post-estrus, implants were surgically placed subcutaneously,blood was taken, and biopsies of subcutaneous white adipose tissue werecollected from the incision site for baseline assessment of mitochondrialoxygen flux (Oroboros) and proteomic measurement of mitochondrial and insulinpathway abundance. After implant placement, fasted blood was collected at 24,48, 72 hours and every three days thereafter for six weeks. During weeks 2-6post-implantation, continuous glucose monitoring was performed (Freestyle Libre3). Twenty-four hours prior to study completion, a catheter was placed in thecranial vena cava and an intravenous glucose tolerance test was performed (0,5, 10, 20, 30, 40, 50, 60 min). The next morning fasted blood was collected (time0) and a stable isotopic tracer bolus was administered to measure whole bodyproduction of glycerol, branched chain amino acids, branched chain keto acids, and β-hydroxyβ-methylbutyric acid. After tracer bolus, blood was collected at 10, 20, 40, 60and 120 minutes. At 120 minutes post-bolus, tissues were collected for isotopiccompound measurement, mitochondrial function assays, and insulin signalingpathway assessment. This translational swine model will help uncover themechanistic effects of testosterone therapy on insulin sensitivity and whole-bodymetabolism in female to male transitioning patients. Presentation: Saturday, June 17, 2023

Branched-chain amino acid transaminase 1 regulates glioblastoma cell plasticity and contributes to immunosuppression.

Glioblastoma is the most common malignant brain tumor in adults. Cellular plasticity and the poorly differentiated features result in a fast relapse of the tumors following treatment. Moreover, the immunosuppressive microenvironment proved to be a major obstacle to immunotherapeutic approaches. Branched-chain amino acid transaminase 1 (BCAT1) was shown to drive the growth of glioblastoma and other cancers;however, its oncogenic mechanism remains poorly understood. Using human tumor data, cell line models and orthotopic immuno-competent and -deficient mouse models, we investigated the phenotypic and mechanistic effects of BCAT1 on glioblastoma cell state and immunomodulation. Here, we show that BCAT1 is crucial for maintaining the poorly differentiated state of glioblastoma cells and that its low expression correlates with a more differentiated glioblastoma phenotype. Furthermore, orthotopic tumor injection into immunocompetent mice demonstrated that the brain microenvironment is sufficient to induce differentiation of Bcat1-KO tumors in vivo. We link the transition to a differentiated cell state to the increased activity of ten-eleven translocation demethylases and the hypomethylation and activation of neuronal differentiation genes. In addition, the knockout of Bcat1 attenuated immunosuppression, allowing for an extensive infiltration of CD8+ cytotoxic T-cells and complete abrogation of tumor growth. Further analysis in immunodeficient mice revealed that both tumor cell differentiation and immunomodulation following BCAT1-KO contribute to the long-term suppression of tumor growth. Our study unveils BCAT1's pivotal role in promoting glioblastoma growth by inhibiting tumor cell differentiation and sustaining an immunosuppressive milieu. These findings offer a novel therapeutic avenue for targeting glioblastoma through the inhibition of BCAT1.

Branched-chain keto-acid dehydrogenase kinase regulates vascular permeability and angiogenesis to facilitate tumor metastasis in renal cell carcinoma.

Branched-chain keto-acid dehydrogenase kinase (BCKDK) is the rate-limiting enzyme of branched-chain amino acid (BCAA) metabolism. In the last six years, BCKDK has been used as a kinase to promote tumor proliferation and metastasis. Renal cell carcinoma (RCC) is a highly vascularized tumor. A high degree of vascularization promotes tumor metastasis. Our objective is to explore the relationship between BCKDK and RCC metastasis and its specific mechanism. In our study, BCKDK is highly expressed in renal clear cell carcinoma and promotes the migration of clear cell renal cell carcinoma (ccRCC). Exosomes from ccRCC cells can promote vascular permeability and angiogenesis, especially when BCKDK is overexpressed in ccRCC cells. BCKDK can also augment the miR-125a-5p expression in ccRCC cells and derived exosomes, thereby decreasing the downstream target protein VE-cadherin level, weakening adhesion junction expression, increasing vascular permeability, and promoting angiogenesis in HUVECs. The novel BCKDK/Exosome-miR-125a-5p/VE-cadherin axis regulates intercellular communication between ccRCC cells and HUVECs. BCKDK plays a critical role in renal cancer metastasis, may be used as a molecular marker of metastatic ccRCC, and even may become a potential target of clinical anti-vascular therapy for ccRCC.

Abstract B003: Increasing branched-chain amino acid metabolism reduces clear cell renal cell carcinoma growth

Abstract North America has the highest incidence of renal cancer in the world with the most common subtype being clear cell renal cell carcinoma (ccRCC). Metabolic dysfunction is a hallmark of ccRCC based on molecular analysis and its clear cell histologic appearance due to significant cytoplasmic accumulation of lipid droplets and glycogen. Using publicly available RNA-Seq and proteomic datasets, we identified reduced expression of branched-chain amino acid (BCAA) enzymes BCAA transaminase 2 (BCAT2) and the branched-chain ketoacid dehydrogenase complex (BCKDH) in ccRCC compared to healthy kidney tissue. We validated these results by confirming that BCAT2 and the BCKDH complex are decreased at the mRNA and protein level in ccRCC tumors and cell lines compared to pair-matched normal adjacent kidney tissue. The reduced expression of the BCKDH complex was reinforced at the genome level by copy number loss of BCKDH subunits DBT and BCKDHB, underscoring the selection for suppression of this pathway. Metabolomics analysis then showed that the abundance of BCAAs were decreased in ccRCC tumors. Additionally, public datasets revealed that ccRCC patients presenting low mRNA expression of any of the four BCKDH subunits DBT, BCKDHB, BCKDHA, and DLD have a worse overall survival compared to patients with higher expression. To test if reactivating the BCAA pathway would impact ccRCC growth, I inhibited BCKDK, a kinase that inactivates the BCKDH complex, pharmacologically with BT2 and genetically with shRNA. Multiple ccRCC cell lines demonstrated reduced proliferation due to apoptosis after being treated with shRNA targeting BCKDK. Cell viability could be rescued by the antioxidant N-acetyl cysteine, suggesting loss of BCKDK increased the production of reactive oxygen species (ROS). Flow cytometry showed that knocking down BCKDK increases cellular ROS. Our data reveal that BCAA catabolism is decreased in ccRCC and reactivating the pathway reduces ccRCC growth in vitro by inducing apoptosis through increased production of ROS. This suggests that BCKDK could be a therapeutic target in ccRCC. Citation Format: Nathan J. Coffey, Romain Riscal, Nicolas Skuli, Nicholas P. Lesner, Michael Noji, Christine Jiang, Laura C. Kim, Xu Han, Megan C. Blair, Brian Keith, Zoltan Arany, M. Celeste Simon. Increasing branched-chain amino acid metabolism reduces clear cell renal cell carcinoma growth [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B003.

Small molecule branched-chain ketoacid dehydrogenase kinase (BDK) inhibitors with opposing effects on BDK protein levels

Branched chain amino acid (BCAA) catabolic impairments have been implicated in several diseases. Branched chain ketoacid dehydrogenase (BCKDH) controls the rate limiting step in BCAA degradation, the activity of which is inhibited by BCKDH kinase (BDK)-mediated phosphorylation. Screening efforts to discover BDK inhibitors led to identification of thiophene PF-07208254, which improved cardiometabolic endpoints in mice. Structure-activity relationship studies led to identification of a thiazole series of BDK inhibitors; however, these inhibitors did not improve metabolism in mice upon chronic administration. While the thiophenes demonstrated sustained branched chain ketoacid (BCKA) lowering and reduced BDK protein levels, the thiazoles increased BCKAs and BDK protein levels. Thiazoles increased BDK proximity to BCKDH-E2, whereas thiophenes reduced BDK proximity to BCKDH-E2, which may promote BDK degradation. Thus, we describe two BDK inhibitor series that possess differing attributes regarding BDK degradation or stabilization and provide a mechanistic understanding of the desirable features of an effective BDK inhibitor.

Biochemical characterization of patients with dihydrolipoamide dehydrogenase deficiency.

Dihydrolipoamide dehydrogenase (DLD; E3) oxidizes lipoic acid. Restoring the oxidized state allows lipoic acid to act as a necessary electron sink for the four mitochondrial keto-acid dehydrogenases: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, branched-chain α-keto-acid dehydrogenase, and 2-oxoadipate dehydrogenase. DLD deficiency (DLDD) is caused by biallelic pathogenic variants in DLD. Three major forms have been described: encephalopathic, hepatic, and myopathic, although DLDD patients exhibit overlapping phenotypes. Hyperlactatemia, hyperexcretion of tricarboxylic acid cycle (TCA) metabolites and branched-chain keto acids, increased plasma branched-chain amino acids and allo-isoleucine are intermittent metabolic abnormalities reported in patients with DLDD. However, the diagnostic performance of these metabolites has never been studied. Therefore, we sought to systematically evaluate the diagnostic utility of these biomarkers for DLDD. We retrospectively analyzed the results of biochemical testing of six unrelated DLDD patients, including values obtained during both well visits and acute decompensation episodes. Elevation of branched-chain amino acid concentrations was not consistently observed. We found that five of six patients in our cohort had a maximum lifetime value of allo-isoleucine of 6 μmol/L, showing that alloisoleucine elevations even during illness may be subtle. Urine organic acid analysis (UOA) during acute decompensation episodes was abnormal in all cases; however, the pattern of abnormalities had high intersubject variability. No single biomarker was universally present, even in patients experiencing metabolic decompensation. We also observed novel biochemical associations: three patients had hyperexcretion of TCA cycle metabolites during crisis; in two patients, 2-ketoadipic and 2-hydroxyadipic acids, by products of lysine degradation, were detected. We propose that these result from 2-oxoadipate dehydrogenase deficiency, an underappreciated biochemical abnormality in DLD. Given the diversity of biochemical profiles among the patients with DLDD, we conclude that accurate biochemical diagnosis relies on a high index of suspicion and multipronged biochemical analysis, including both plasma amino acid and urine organic acid quantitation during decompensation. Biochemical diagnosis during the well state is challenging. We emphasize the critical importance of multiple simultaneous biochemical tests for diagnosis and monitoring of DLDD. We also highlight the under-recognized role of DLD in the lysine degradation pathway. Larger cohorts of patients are needed to establish a correlation between the biochemical pattern and clinical outcomes, as well as a genotype-phenotype correlation.

Subunit composition of mitochondrial dehydrogenase complexes in diplonemid flagellates

In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a tripartite mitochondrial pyruvate dehydrogenase (PDH) complex to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes with analogous composition catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) complex and the 2-oxoglutarate dehydrogenase (OGDH) complex. Comparative transcriptome analyses of diplonemids, one of the most abundant and diverse groups of oceanic protists, indicate that the conventional E1, E2, and E3 subunits of the PDH complex are lacking. E1 was apparently replaced in the euglenozoan ancestor of diplonemids by an AceE protein of archaeal type, a substitution that we also document in dinoflagellates. Here, we demonstrate that the mitochondrion of the model diplonemid Paradiplonema papillatum displays pyruvate and 2-oxoglutarate dehydrogenase activities. Protein mass spectrometry of mitochondria reveal that the AceE protein is as abundant as the E1 subunit of BCKDH. This corroborates the view that the AceE subunit is a functional component of the PDH complex. We hypothesize that by acquiring AceE, the diplonemid ancestor not only lost the eukaryotic-type E1, but also the E2 and E3 subunits of the PDH complex, which are present in other euglenozoans. We posit that the PDH activity in diplonemids seems to be carried out by a complex, in which the AceE protein partners with the E2 and E3 subunits from BCKDH and/or OGDH.

Branched-chain ketoacid co-ingestion with protein lowers amino acid oxidation during hemodialysis: A randomized controlled cross-over trial

Hemodialysis removes amino acids from the circulation, thereby stimulating muscle proteolysis. Protein ingestion during hemodialysis can compensate for amino acid removal but may also increase uremic toxin production. Branched-chain ketoacid (BCKA) co-ingestion may provide an additional anabolic stimulus without adding to uremic toxin accumulation. In the present study we assessed the impact of BCKA co-ingestion with protein on forearm amino acid balance and amino acid oxidation during hemodialysis. Nine patients (age: 73±10y) on chronic hemodialysis participated in this crossover trial. During two 4-h hemodialysis sessions, patients ingested 18g protein with (PRO+BCKA) or without (PRO) 9g BCKAs in a randomized order. Test beverages were labeled with L-[ring-13C6]-phenylalanine and provided throughout the last 3h of hemodialysis as 18 equal sips consumed with 10-min intervals. Arterial and venous plasma as well as breath samples were collected frequently throughout hemodialysis. Arterial plasma total amino acid (TAA) concentrations during PRO and PRO+BCKA treatments were significantly lower after 1h of hemodialysis (2.6±0.3 and 2.6±0.3mmol/L, respectively) when compared to pre-hemodialysis concentrations (4.2±1.0 and 4.0±0.5mmol/L, respectively; time effect: P<0.001). Arterial plasma TAA concentrations increased throughout test beverage ingestion (time effect: P=0.027) without differences between treatments (time∗treatment: P=0.62). Forearm arteriovenous TAA balance during test beverage ingestion did not differ between timepoints (time effect: P=0.31) or treatments (time∗treatment: P=0.34). Whole-body phenylalanine oxidation was 33±16% lower during PRO+BCKA when compared to PRO treatments (P<0.001). BCKA co-ingestion with protein during hemodialysis does not improve forearm net protein balance but lowers amino acid oxidation.

Branched-chain keto acids inhibit mitochondrial pyruvate carrier and suppress gluconeogenesis in hepatocytes

Branched-chain amino acid (BCAA) metabolism is linked to glucose homeostasis, but the underlying signaling mechanisms are unclear. We find that gluconeogenesis is reduced in mice deficient of Ppm1k, apositive regulator of BCAA catabolism, which protects against obesity-induced glucose intolerance. Accumulation of branched-chain keto acids (BCKAs) inhibits glucose production in hepatocytes. BCKAs suppress liver mitochondrial pyruvate carrier (MPC) activity and pyruvate-supported respiration. Pyruvate-supported gluconeogenesis is selectively suppressed in Ppm1k-deficient mice and can be restored with pharmacological activation of BCKA catabolism by BT2. Finally, hepatocytes lack branched-chain aminotransferase that alleviates BCKA accumulation via reversible conversion between BCAAs and BCKAs. This renders liver MPC most susceptible to circulating BCKA levels hence a sensor of BCAA catabolism.

Loss of BCAA catabolism enhances Rab1A-mTORC1 signaling activity and promotes tumor proliferation in NSCLC

BackgroundNon-small cell lung cancer (NSCLC) is a leading cause of cancer death. Branched-chain amino acid (BCAA) homeostasis is important for normal physiological metabolism. Branched-chain keto acid dehydrogenase kinase (BCKDK) is a rate-limiting enzyme involved in BCAA degradation. BCAA metabolism has been highlighted in human cancers. The aberrant activation of mTORC1 has been implicated in tumor progression. Rab1A is a small GTPase, an activator of mTORC1, and an oncogene. This study aimed to reveal the specific role of BCKDK-BCAA-Rab1A-mTORC1 signaling in NSCLC. MethodsWe analyzed a cohort of 79 patients with NSCLC and 79 healthy controls. Plasma BCAA assays, immunohistochemistry, and network and pathway analyses were performed. The stable cell lines BCKDK-KD, BCKDK-OV A549, and H1299 were constructed. BCKDK, Rab1A, p-S6 and S6 were detected using western blotting to explore their molecular mechanisms of action in NSCLC. The effects of BCAA and BCKDK on the apoptosis and proliferation of H1299 cells were detected by cell function assays. ResultsWe demonstrated that NSCLC was primarily involved in BCAA degradation. Therefore, combining BCAA, CEA, and Cyfra21-1 is clinically useful for treating NSCLC. We observed a significant increase in BCAA levels, downregulation of BCKDHA expression, and upregulation of BCKDK expression in NSCLC cells. BCKDK promotes proliferation and inhibits apoptosis in NSCLC cells, and we observed that BCKDK affected Rab1A and p-S6 in A549 and H1299 cells via BCAA modulation. Leucine affected Rab1A and p-S6 in A549 and H1299 cells and affected the apoptosis rate of H1299 cells.In conclusion, BCKDK enhances Rab1A-mTORC1 signaling and promotes tumor proliferation by suppressing BCAA catabolism in NSCLC, suggesting a new biomarker for the early diagnosis and identification of metabolism-based targeted approaches for patients with NSCLC.

High doses of rosuvastatin induce impaired branched-chain amino acid catabolism and lead to insulin resistance.

What is the central question of this study? Is there a risk of developing diabetes associated with statin treatment? What is the underlying mechanism of the increased incidence rate of new-onset diabetes in patients treated with rosuvastatin? What is the main finding and its importance? Rosuvastatin therapy reduced intraperitoneal glucose tolerance and changed the catabolism of branched-chain amino acid (BCAAs) in white adipose tissue and skeletal muscle. Protein phosphatase 2Cm knockdown completely abolished the effects of insulin and rosuvastatin on glucose absorption. This study provides mechanistic support for recent clinical data on rosuvastatin-related new-onset diabetes and underscores the logic for intervening in BCAA catabolism to prevent the harmful effects of rosuvastatin. Accumulating evidence indicates that patients treated with rosuvastatin have an increased risk of developing new-onset diabetes. However, the underlying mechanism remains unclear. In this study, we administered rosuvastatin (10mg/kg body weight) to male C57BL/6J mice for 12weeks and found that oral rosuvastatin dramatically reduced intraperitoneal glucose tolerance. Rosuvastatin-treated mice showed considerably higher serum levels of branched-chain amino acids (BCAAs) than control mice. They also showed dramatically altered expression of BCAA catabolism-related enzymes in white adipose tissue and skeletal muscle, including downregulated mRNA expression of BCAT2 and protein phosphatase 2Cm (PP2Cm) and upregulated mRNA expression of branched-chain ketoacid dehydrogenase kinase (BCKDK). The levels of BCKD in the skeletal muscle were reduced in rosuvastatin-treated mice, which was associated with lower PP2Cm protein levels and increased BCKDK levels. We also investigated the effects of rosuvastatin and insulin administration on glucose metabolism and BCAA catabolism in C2C12 myoblasts. We observed that incubation with insulin enhanced glucose uptake and facilitated BCAA catabolism in C2C12 cells, which was accompanied by elevated Akt and glycogen synthase kinase 3 β (GSK3β) phosphorylation. These effects of insulin were prevented by co-incubation of the cells with 25μM rosuvastatin. Moreover, the effects of insulin and rosuvastatin administration on glucose uptake and Akt and GSK3β signaling in C2C12 cells were abolished when PP2Cm was knocked down. Although the relevance of these data, obtained with high doses of rosuvastatin in mice, to therapeutic doses in humans remains to be elucidated, this study highlights a potential mechanism for the diabetogenic effects of rosuvastatin, and suggests that BCAA catabolism could be a pharmacological target for preventing the adverse effects of rosuvastatin.