Branched-chain Alpha-ketoacid Dehydrogenase Kinase Research Articles

Dairy consumption and hepatocellular carcinoma risk.

This review provides epidemiological and translational evidence for milk and dairy intake as critical risk factors in the pathogenesis of hepatocellular carcinoma (HCC). Large epidemiological studies in the United States and Europe identified total dairy, milk and butter intake with the exception of yogurt as independent risk factors of HCC. Enhanced activity of mechanistic target of rapamycin complex 1 (mTORC1) is a hallmark of HCC promoted by hepatitis B virus (HBV) and hepatitis C virus (HCV). mTORC1 is also activated by milk protein-induced synthesis of hepatic insulin-like growth factor 1 (IGF-1) and branched-chain amino acids (BCAAs), abundant constituents of milk proteins. Over the last decades, annual milk protein-derived BCAA intake increased 3 to 5 times in Western countries. In synergy with HBV- and HCV-induced secretion of hepatocyte-derived exosomes enriched in microRNA-21 (miR-21) and miR-155, exosomes of pasteurized milk as well deliver these oncogenic miRs to the human liver. Thus, milk exosomes operate in a comparable fashion to HBV- or HCV- induced exosomes. Milk-derived miRs synergistically enhance IGF-1-AKT-mTORC1 signaling and promote mTORC1-dependent translation, a meaningful mechanism during the postnatal growth phase, but a long-term adverse effect promoting the development of HCC. Both, dietary BCAA abundance combined with oncogenic milk exosome exposure persistently overstimulate hepatic mTORC1. Chronic alcohol consumption as well as type 2 diabetes mellitus (T2DM), two HCC-related conditions, increase BCAA plasma levels. In HCC, mTORC1 is further hyperactivated due to RAB1 mutations as well as impaired hepatic BCAA catabolism, a metabolic hallmark of T2DM. The potential HCC-preventive effect of yogurt may be caused by lactobacilli-mediated degradation of BCAAs, inhibition of branched-chain α-ketoacid dehydrogenase kinase via production of intestinal medium-chain fatty acids as well as degradation of milk exosomes including their oncogenic miRs. A restriction of total animal protein intake realized by a vegetable-based diet is recommended for the prevention of HCC.

BCKDK regulates the TCA cycle through PDC in the absence of PDK family during embryonic development.

Pyruvate dehydrogenase kinases (PDK1-4) inhibit the TCA cycle by phosphorylating pyruvate dehydrogenase complex (PDC). Here, we show that PDK family is dispensable for murine embryonic development and that BCKDK serves as a compensatory mechanism by inactivating PDC. First, we knocked out all four Pdk genes one by one. Surprisingly, Pdk total KO embryos developed and were born in expected ratios but died by postnatal day 4 because of hypoglycemia or ketoacidosis. Moreover, PDC was phosphorylated in these embryos, suggesting that another kinase compensates for PDK family. Bioinformatic analysis implicated branched-chain ketoacid dehydrogenase kinase (Bckdk), a key regulator of branched-chain amino acids (BCAAs) catabolism. Indeed, knockout of Bckdk and Pdk family led to the loss of PDC phosphorylation, an increase in PDC activity and pyruvate entry into the TCA cycle, and embryonic lethality. These findings reveal a regulatory crosstalk hardwiring BCAA and glucose catabolic pathways, which feed the TCA cycle.

Branched-chain \u03b1-ketoacids are preferentially reaminated and activate protein synthesis in the heart

Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of BCAA and BCKA away from mitochondrial oxidation is likely due to low levels of expression of the mitochondrial BCAA transporter SLC25A44 in the heart, as its overexpression significantly lowers accumulation of [13C]-labeled valine from [U-13C]KIV. Finally, exposure of perfused hearts to levels of BCKA found in obese rats increases phosphorylation of the translational repressor 4E-BP1 as well as multiple proteins in the MEK-ERK pathway, leading to a doubling of total protein synthesis. These data suggest that elevated BCKA levels found in obesity may contribute to pathologic cardiac hypertrophy via chronic activation of protein synthesis.

Differences in gene expression of enzymes involved in branched-chain amino acid metabolism of abdominal subcutaneous adipose tissue between pregnant women with and without PCOS

ObjectivePolycystic ovary syndrome (PCOS) appears to be a common endocrine disorder of women in reproductive age. Adipose tissue (AT) is known as an active tissue in the metabolism of branched-chain amino acids (BCAA; Valine, Leucine, and Isoleucine) that they have associated with blood BCAA levels is a prognostic factor for insulin-resistant. Although the crucial roles of AT in women suffering from PCOS was reported, little information exists on the BCAA metabolism in AT of PCOS women. The aim was to assess and compare the expression of BCAAs metabolism pathway genes in abdominal subcutaneous AT of pregnant women with PCOS and non-PCOS pregnant women. Materials and methodsAT samples from 13 PCOS were compared with samples collected from 6 non-PCOS women, all of whom underwent caesarean. Quantitative real-time PCR technique was used for gene expression of branched chain aminotransferase 2 mitochondrial (BCAT2), branched chain ketoacid dehydrogenase E1-alpha (BCKDHA), branched chain ketoacid dehydrogenase E1-Beta (BCKDHB), dihydrolipoamide branched chain transacylase E2 (DBT), dihydrolipoamide dehydrogenase E3 (DLD), branched chain ketoacid dehydrogenase kinase (BCKDK), Data were analyzed using t-test or U-test. ResultsNo significant differences were found in age and body mass index (BMI) between non-PCOS and PCOS women. The mRNA level of BCAT2 and DLD in PCOS group was not significantly different from non-PCOS group whereas mRNA level of BCKDHB and DBT was significantly increased in PCOS group (P < 0.0001). In contrast, mRNA level of BCKDHA (P = 0.0001) and BCKDK (P < 0.0001) was significantly decreased in PCOS group. ConclusionThe alterations in gene expressions involved BCAA metabolism in age-matched and BMI- matched non-PCOS and PCOS pregnant women at delivery day was shown which warrants further studies regards functional activity. More attention should be given to AT of PCOS mothers that was previously ignored.

Antihypertensive drug valsartan as a novel BDK inhibitor

Catabolism of branched-chain amino acids (BCAAs) is affected by various physiological conditions and its abnormality is associated with glucose metabolism, heart disease, and neurological dysfunction. The first two steps of the BCAA metabolic pathway are common to the three BCAAs (leucine, isoleucine, and valine). The second step is an irreversible rate-limited reaction catalyzed by branched-chain α-keto acid dehydrogenase (BCKDH), which is bound to a specific kinase, BCKDH kinase (BDK), and inactivated by phosphorylation. Here, we investigated potential new BDK inhibitors and discovered valsartan, an angiotensin II type 1 receptor (AT1R) blocker, as a new BDK inhibitor. BCKDH phosphorylation and the BCKDH-BDK interaction were inhibited by valsartan in vitro. Valsartan administration in rats resulted in increased BCKDH activity by decreasing the dephosphorylated level of BCKDH complex, bound forms of BDK from BCKDH complex as well as decreased plasma BCAA concentrations. Valsartan is a novel BDK inhibitor that competes with ATP, via a different mechanism from allosteric inhibitors. The BDK inhibitor has been shown to preserve cardiac function in pressure overload-induced heart failure mice and to attenuate insulin resistance in obese mice. Our findings suggest that valsartan is a potent seed compound for developing a powerful BDK inhibitor and useful medication for treating heart failure and metabolic diseases with suppressed BCAA catabolism.

Dichloroacetate and Pyruvate Metabolism: Pyruvate Dehydrogenase Kinases as Targets Worth Investigating for Effective Therapy of Toxoplasmosis.

Toxoplasmosis, a protozoan infection caused by Toxoplasma gondii, is estimated to affect around 2.5 billion people worldwide. Nevertheless, the side effects of drugs combined with the long period of therapy usually result in discontinuation of the treatment. New therapies should be developed by exploring peculiarities of the parasite's metabolic pathways, similarly to what has been well described in cancer cell metabolism. An example is the switch in the metabolism of cancer that blocks the conversion of pyruvate into acetyl coenzyme A in mitochondria. In this context, dichloroacetate (DCA) is an anticancer drug that reverts the tumor proliferation by inhibiting the enzymes responsible for this switch: the pyruvate dehydrogenase kinases (PDKs). DCA has also been used in the treatment of certain symptoms of malaria; however, there is no evidence of how this drug affects apicomplexan species. In this paper, we studied the metabolism of T. gondii and demonstrate that DCA also inhibits T. gondii's in vitro infection with no toxic effects on host cells. DCA caused an increase in the activity of pyruvate dehydrogenase followed by an unbalanced mitochondrial activity. We also observed morphological alterations frequently in mitochondria and in a few apicoplasts, essential organelles for parasite survival. To date, the kinases that potentially regulate the activity of pyruvate metabolism in both organelles have never been described. Here, we confirmed the presence in the genome of two putative kinases (T. gondii PDK [TgPDK] and T. gondii branched-chain α-keto acid dehydrogenase kinase [TgBCKDK]), verified their cellular localization in the mitochondrion, and provided in silico data suggesting that they are potential targets of DCA.IMPORTANCE Currently, the drugs used for toxoplasmosis have severe toxicity to human cells, and the treatment still lacks effective and safer alternatives. The search for novel drug targets is timely. We report here that the treatment of T. gondii with an anticancer drug, dichloroacetate (DCA), was effective in decreasing in vitro infection without toxicity to human cells. It is known that PDK is the main target of DCA in mammals, and this inactivation increases the conversion of pyruvate into acetyl coenzyme A and reverts the proliferation of tumor cells. Moreover, we verified the mitochondrial localization of two kinases that possibly regulate the activity of pyruvate metabolism in T. gondii, which has never been studied. DCA increased pyruvate dehydrogenase (PDH) activity in T. gondii, followed by an unbalanced mitochondrial activity, in a manner similar to what was previously observed in cancer cells. Thus, we propose the conserved kinases as potential regulators of pyruvate metabolism and interesting targets for new therapies.

BDK Deficiency in Cerebral Cortex Neurons Causes Neurological Abnormalities and Affects Endurance Capacity.

Branched-chain amino acid (BCAA) catabolism is regulated by its rate-limiting enzyme, branched-chain α-keto acid dehydrogenase (BCKDH), which is negatively regulated by BCKDH kinase (BDK). Loss of BDK function in mice and humans leads to dysregulated BCAA catabolism accompanied by neurological symptoms such as autism; however, which tissues or cell types are responsible for the phenotype has not been determined. Since BDK is highly expressed in neurons compared to astrocytes, we hypothesized that neurons are the cell type responsible for determining the neurological features of BDK deficiency. To test this hypothesis, we generated mice in which BDK deletion is restricted to neurons of the cerebral cortex (BDKEmx1-KO mice). Although BDKEmx1-KO mice were born and grew up normally, they showed clasped hind limbs when held by the tail and lower brain BCAA concentrations compared to control mice. Furthermore, these mice showed a marked increase in endurance capacity after training compared to control mice. We conclude that BDK in neurons of the cerebral cortex is essential for maintaining normal neurological functions in mice, and that accelerated BCAA catabolism in that region may enhance performance in running endurance following training.

Viral myocarditis involves the generation of autoreactive T cells with multiple antigen specificities that localize in lymphoid and non-lymphoid organs in the mouse model of CVB3 infection

Autoreactive T cells may contribute to post-viral myocarditis induced with Coxsackievirus B3 (CVB3), but the underlying mechanisms of their generation are unclear. Here, we have comprehensively analyzed the generation of antigen-specific, autoreactive T cells in the mouse model of CVB3 infection for antigens implicated in patients with myocarditis/dilated cardiomyopathy. First, comparative analysis of CVB3 proteome with five autoantigens led us to identify three mimicry epitopes, one each from adenine nucleotide translocator 1 (ANT), sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and cardiac troponin I. None of these induced cross-reactive T cell responses. Next, we generated major histocompatibility complex (MHC) class II dextramers to enumerate the frequencies of antigen-specific T cells to determine whether T cells with multiple antigen specificities are generated by CVB3 infection. These analyses revealed appearance of CD4 T cells positive for SERCA2a 971−990, and cardiac myosin heavy chain-α (Myhc) 334−352 dextramers, both in the periphery and also in the hearts of CVB3-infected animals. While ANT 21−40 dextramer+ T cells were inconsistently detected, the β1-adrenergic receptor 181−200/211−230 or branched chain α-ketoacid dehydrogenase kinase 111−130 dextramer+ cells were absent. Interestingly, SERCA2a 971−990, Myhc 334−352 and ANT 21−40 dextramer+ cells were also detected in the liver indicating that they may have a pathogenic role. Finally, we demonstrate that the SERCA2a 971−990-reactive T cells generated in CVB3 infection could transfer disease to naïve mice. The data suggest that CVB3 infection can lead to the generation of autoreactive T cells for multiple antigens indicating a possibility that the autoreactive T cells localized in the liver can potentially circulate and contribute to the development of viral myocarditis.

Data on the proliferation and differentiation of C2C12 myoblast treated with branched-chain ketoacid dehydrogenase kinase inhibitor.

The catabolism of branched chain amino acids (BCAAs) is mainly carried out in skeletal muscle myofibers. It is mediated by branched chain aminotransferase 2 and branched chain alpha ketoacid dehydrogenase (BCKDH) in mitochondria for energy supply, especially during exercise. BCKDH kinase (BCKDK) is a negative regulator of BCAAs catabolism by its inhibitory phosphorylation of the BCKDH E1a subunit. The data presented in this article are related to the research article that we previously have reported entitled “Energy metabolism profile of the effects of amino acid treatment on skeletal muscle cells: Leucine inhibits glycolysis of myotubes” (Suzuki et al., 2020)[1]. In this report, we have demonstrated that 1hour treatment of BT2, an inhibitor of BCKDK, decreased the glycolysis of C2C12 differentiated myotubes compared to the control. Although BCAAs metabolism is basically assumed to be carried out in differentiated myofibers, BCKDK is expressed in both undifferentiated myoblasts and differentiated myotubes, and the biological and physiological significance of BCAAs metabolism in myoblasts is still unclear. Present data demonstrate an in vitro assessment of BT2 on C2C12 myoblasts proliferation and differentiation. The data suggest that activation of BCAAs catabolism by the BCKDK inhibitor BT2 impairs C2C12 myoblasts proliferation and differentiation.

Depletion of branched-chain aminotransferase 2 (BCAT2) enzyme impairs myoblast survival and myotube formation.

Much is known about the positive effects of branched‐chain amino acids (BCAA) in regulating muscle protein metabolism. Comparatively much less is known about the effects of these amino acids and their metabolites in regulating myotube formation. Using cultured myoblasts, we showed that although leucine is required for myotube formation, this requirement is easily met by α‐ketoisocaproic acid, the ketoacid of leucine. We then demonstrated increases in the expression of the first two enzymes in the catabolism of the three BCAA, branched‐chain amino transferase (BCAT2) and branched‐chain α‐ketoacid dehydrogenase (BCKD), with ~3× increase in BCKD protein expression (p < .05) during differentiation. Furthermore, depletion of BCAT2 abolished myoblast differentiation, as indicated by reduction in the levels of myosin heavy chain‐1, troponin and myogenin. Supplementation of incubation medium with branched‐chain α‐ketoacids or related metabolites derivable from BCAT2 functions did not rescue the defects. However, co‐depletion of BCKD kinase partially rescued the defects. Collectively, our data indicate a requirement for BCAA catabolism during myotube formation and that this requirement for BCAT2 likely goes beyond the need for this enzyme to generate the α‐ketoacids of the BCAA.

Targeting BCAA Catabolism to Treat Obesity-Associated Insulin Resistance.

Recent studies implicate a strong association between elevated plasma branched-chain amino acids (BCAAs) and insulin resistance (IR). However, a causal relationship and whether interrupted BCAA homeostasis can serve as a therapeutic target for diabetes remain to be established experimentally. In this study, unbiased integrative pathway analyses identified a unique genetic link between obesity-associated IR and BCAA catabolic gene expression at the pathway level in human and mouse populations. In genetically obese (ob/ob) mice, rate-limiting branched-chain α-keto acid (BCKA) dehydrogenase deficiency (i.e., BCAA and BCKA accumulation), a metabolic feature, accompanied the systemic suppression of BCAA catabolic genes. Restoring BCAA catabolic flux with a pharmacological inhibitor of BCKA dehydrogenase kinase (BCKDK) ( a suppressor of BCKA dehydrogenase) reduced the abundance of BCAA and BCKA and markedly attenuated IR in ob/ob mice. Similar outcomes were achieved by reducing protein (and thus BCAA) intake, whereas increasing BCAA intake did the opposite; this corroborates the pathogenic roles of BCAAs and BCKAs in IR in ob/ob mice. Like BCAAs, BCKAs also suppressed insulin signaling via activation of mammalian target of rapamycin complex 1. Finally, the small-molecule BCKDK inhibitor significantly attenuated IR in high-fat diet–induced obese mice. Collectively, these data demonstrate a pivotal causal role of a BCAA catabolic defect and elevated abundance of BCAAs and BCKAs in obesity-associated IR and provide proof-of-concept evidence for the therapeutic validity of manipulating BCAA metabolism for treating diabetes.

Abstract 566: Branched Chain Ketoacid Dehydrogenase Kinase Inhibition Alters Substrate Utilization and Gene Expression in Myocytes

Circulating increases in branched chain amino acid (BCAA) levels have long been associated with type II diabetes and metabolic syndrome. Emerging data also suggest that impaired BCAA catabolism may play a role in heart failure progression. BCAA are catabolized via the branched chain ketoacid (BCKA) dehydrogenase enzyme complex (BCKDH). BCKD kinase (BCKDK) is a negative regulator of BCAA catabolism through its inhibitory phosphorylation of the BCKDHE1a subunit, and the phosphatase PPM1k dephosphorylates this same site to activate BCAA catabolism. Using an inhibitor of BCKDK (BT2), BCAA catabolism is increased in vivo. Here, we utilized metabolomics to evaluate the contribution of BCAA catabolism to substrate preference in heart and skeletal muscle. Surprisingly, BCKDK inhibition with BT2 had no effect on incorporation of glucose into TCA cycle intermediates in heart or skeletal muscle. Because others have recently shown that the primary site of BCAA catabolism is skeletal muscle, we knocked down BCKDK and PPM1k in human skeletal myocytes to further investigate how BCKDK loss or inhibition affects substrate utilization. Similar to our in vivo observations, knockdown of BCKDK and PPM1k had no effect on glucose and pyruvate utilization in a mitochondrial function assay. However, an increase in maximal respiration was observed after BCKDK knockdown when fatty acids were used. To evaluate the mechanisms underlying this increase we then performed RNAseq in these cells after BCKDK and PPM1K knockdown and observed changes in a number of genes that may explain these alterations in substrate utilization. Finally, we performed C13 BCAA metabolomics in human skeletal myocytes after BT2 treatment or knockdown of BCKDK and PPM1k. Using BT2, we observed a dose-responsive reduction in BCKA production from C13 BCAA by the muscle cells as expected; however, though BCKA production was increased after PPM1k was knocked down, we surprisingly did not observe a decrease in BCKA production after BCKDK knockdown. Collectively these data suggest that BCKDK inhibition may improve metabolism and cardiac function by altering substrate preference in skeletal myocytes.

P2‐166: BRANCHED‐CHAIN AMINO ACIDS (BCAAS): A NEW PLAYER IN ALZHEIMER'S DISEASE

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that is the third leading cause of mortality in the US. AD brain is mainly characterized by accumulated plaques and tangles and diminished neurotransmitters. AD is strongly associated with type 2 diabetes (T2D). Emerging studies suggest that branched-chain amino acids (BCAAs), the essential amino acids we need to obtain from food, are involved in the pathogenesis of insulin resistance and T2D. In support of this concept, BCAA degradation is impaired in obese and/or diabetic individuals, and BCAA supplementation leads to insulin resistance and perturbed glycemic control. It is currently unknown if similar defective BCAA metabolism exists in AD patients. Since BCAAs along with aromatic amino acids are critical for production and maintenance of brain neurotransmitters, here we tested if BCAA metabolism in liver – an organ with the highest BCAA degradation activity – is impaired in AD. Eight month-old wildtype or AD transgenic mice were fed a standard chow diet until sacrifice. Serum BCAA levels were measured by BCAA assay, and proteins and genes related to BCAA metabolism in liver were determined by western blot and RT-qPCR, respectively. Serum BCAA profile of healthy or AD individuals were assessed by metabolomics analysis. The activity of branched-chain α-keto acid dehydrogenase (BCKDH), the rate-limiting enzyme in BCAA degradation pathway, in liver was significantly suppressed in AD mice compared to wildtypes as evidenced by the protein expression and its phosphorylated, inactive, state. This is supported by increased hepatic BCKDH Kinase at both protein and gene levels in AD mice. Serum BCAAs and/or their metabolites were higher in both AD mice and humans compared to healthy controls, indicating impaired BCAA metabolism. Our findings suggest that hepatic BCAA catabolism is impaired in AD mice. This may lead to high plasma BCAAs and their metabolites that can potentially contribute to the imbalance of brain neurotransmitters and development of AD or related dementia. A longitudinal assessment of BCAA metabolism will allow us to determine if they play a predictive and/or causal role in the development of AD.

1922-P: Branched-Chain Ketoacid Dehydrogenase Kinase Inhibition Acutely Improves Glycemia and Alters the Response to Feeding in Mice

Circulating increases in branched chain amino acid (BCAA) levels have long been associated with type 2 diabetes and metabolic syndrome. Emerging data also suggest that BCAA catabolism may play a role in heart failure progression. It is hypothesized that decreased catabolism, rather than increased consumption of BCAAs, is responsible for these correlations. Branched chain ketoacid (BCKA) dehydrogenase (BCKDH) kinase (BCKDK) is a negative regulator of BCAA catabolism through its inhibitory phosphorylation of the BCKDH E1a subunit. Using the BCKDK inhibitor molecule BT2, we demonstrate here a reduction of BCAA, BCKA and tissue p-BCKDH levels concomitant with improved glucose tolerance and reduced insulin levels in diet-induced obese mice after only one day of treatment. To investigate the mechanisms underlying this protection, we assessed plasma biomarkers and tissue gene expression after fasting and re-feeding in glucose intolerant high fat diet-fed animals. Remarkably, BT2 treated animals demonstrated reduced plasma glucose levels after re-feed, which was accompanied by dramatic reductions in plasma insulin levels, reduced activation of Akt in peripheral tissues, and a failure to suppress plasma free fatty acids and lipolytic machinery in adipose tissue. RNAseq was performed in liver to assess changes in gene expression profiles, and while over 400 genes were differentially regulated in vehicle treated re-fed mice compared with vehicle treated fasted mice, there were no differentially regulated genes in BT2 re-fed animals compared with BT2 fasted animals. These data suggest that activation of BCAA catabolism with the BCKDK inhibitor BT2 impairs the systemic response to feeding. Disclosure E. Bollinger: Employee; Self; Pfizer Inc. C.P. Siddall: Employee; Self; Pfizer Inc. Employee; Spouse/Partner; Vertex Pharmaceuticals. T. Greizer: None. J.L. Libera: None. G. Hariri: None. E.E. Pashos: Employee; Self; Pfizer Inc. A. Shipstone: None. A. Hadjipanayis: None. Z. Sun: None. G. Xing: None. M.F. Clasquin: Employee; Spouse/Partner; Merck &amp; Co., Inc. Employee; Self; Pfizer Inc. B.B. Zhang: Employee; Spouse/Partner; Janssen Pharmaceuticals, Inc. Employee; Self; Pfizer Inc. Other Relationship; Self; Eli Lilly and Company. R.A. Miller: Employee; Self; Pfizer Inc. R. Roth Flach: Employee; Self; Pfizer Inc.

Branched-chain Amino Acid Metabolism Is Impaired in Mice and Humans with Alzheimer’s Disease (OR27-04-19)

ObjectivesAlzheimer’s disease (AD) is an irreversible neurodegenerative disorder that is the third leading cause of mortality in the US. AD brain is mainly characterized by accumulated plaques and tangles and diminished neurotransmitters. AD is strongly associated with type 2 diabetes (T2D). Emerging studies suggest that branched-chain amino acids (BCAAs), the essential amino acids we need to obtain from food, are involved in the pathogenesis of insulin resistance and T2D. In support of this concept, BCAA degradation is impaired in obese and/or diabetic individuals, and BCAA supplementation leads to insulin resistance and perturbed glycemic control. It is currently unknown if similar defective BCAA metabolism exists in AD patients. Since BCAAs along with aromatic amino acids are critical for production and maintenance of brain neurotransmitters, here we tested if BCAA metabolism in liver – an organ with the highest BCAA degradation activity – is impaired in AD. MethodsEight month-old wildtype or AD transgenic mice were fed a standard chow diet until sacrifice. Serum BCAA levels were measured by BCAA assay, and proteins and genes related to BCAA metabolism in liver were determined by western blot and RT-qPCR, respectively. Serum BCAA profile of healthy or AD individuals were assessed by metabolomics analysis. ResultsThe activity of branched-chain α-keto acid dehydrogenase (BCKDH), the rate-limiting enzyme in BCAA degradation pathway, in liver was significantly suppressed in AD mice compared to wildtypes as evidenced by the protein expression and its phosphorylated, inactive, state. This is supported by increased hepatic BCKDH Kinase at both protein and gene levels in AD mice. Serum BCAAs and/or their metabolites were higher in both AD mice and humans compared to healthy controls, indicating impaired BCAA metabolism. ConclusionsOur findings suggest that hepatic BCAA catabolism is impaired in AD mice. This may lead to high plasma BCAAs and their metabolites that can potentially contribute to the imbalance of brain neurotransmitters and development of AD or related dementia. A longitudinal assessment of BCAA metabolism will allow us to determine if they play a predictive, diagnostic, and/or causal role in the development of AD. Funding SourcesNIH DK099463, Wylie Briscoe Fund, Texas Tech University.

The ubiquitin ligase UBE3B, disrupted in intellectual disability and absent speech, regulates metabolic pathways by targeting BCKDK

Kaufman oculocerebrofacial syndrome (KOS) is a recessive neurodevelopmental disorder characterized by intellectual disability and lack of speech. KOS is caused by inactivating mutations in UBE3B, but the underlying biological mechanisms are completely unknown. We found that loss of Ube3b in mice resulted in growth retardation, decreased grip strength, and loss of vocalization. The brains of Ube3b-/- mice had hypoplasia of the corpus callosum, enlarged ventricles, and decreased thickness of the somatosensory cortex. Ube3b-/- cortical neurons had abnormal dendritic morphology and synapses. We identified 22 UBE3B interactors and found that branched-chain α-ketoacid dehydrogenase kinase (BCKDK) is an in vivo UBE3B substrate. Since BCKDK targets several metabolic pathways, we profiled plasma and cortical metabolomes from Ube3b-/- mice. Nucleotide metabolism and the tricarboxylic acid cycle were among the pathways perturbed. Substrate-induced mitochondrial respiration was reduced in skeletal muscle but not in liver of Ube3b-/- mice. To assess the relevance of these findings to humans, we identified three KOS patients who had compound heterozygous UBE3B mutations. We discovered changes in metabolites from similar pathways in plasma from these patients. Collectively, our results implicate a disease mechanism in KOS, suggest that it is a metabolic encephalomyopathy, and provide an entry to targeted therapies.

Polymorphisms of ACMSD-TMEM163, MCCC1, and BCKDK-STX1B Are Not Associated with Parkinson’s Disease in Taiwan

Previous genome-wide association studies in Caucasian populations suggest that genetic loci in amino acid catabolism may be associated with Parkinson's disease (PD). However, these genetic disease associations were limitedly reported in Asian populations. Herein, we investigated the effect of top three PD-associated genetic variants related to amino acid catabolism in Caucasians listed on the top risk loci identified by meta-analysis of genome-wide association studies in PDGene database, including aminocarboxymuconate-semialdehyde decarboxylase- (ACMSD-) transmembrane protein 163 (TMEM163) rs6430538, methylcrotonyl-CoA carboxylase 1 (MCCC1) rs12637471, and branched-chain ketoacid dehydrogenase kinase- (BCKDK-) syntaxin 1B (STX1B) rs14235, by genotyping 599 Taiwanese patients with PD and 598 age-matched control subjects. PD patients demonstrate similar allelic and genotypic frequencies in all tested genetic variants. These ethnic discrepancies of genetic variants suggest a distinct genetic background of amino acid catabolism between Taiwanese and Caucasian PD patients.

Quantitative Analysis of the Whole-Body Metabolic Fate of Branched-Chain Amino Acids

Elevations in branched-chain amino acids (BCAAs) associate with numerous systemic diseases, including cancer, diabetes, and heart failure. However, an integrated understanding of whole-body BCAA metabolism remains lacking. Here, we employ invivo isotopic tracing to systemically quantify BCAA oxidation in healthy and insulin-resistant mice. We find that most tissues rapidly oxidize BCAAs into the tricarboxylic acid (TCA) cycle, with the greatest quantity occurring in muscle, brown fat, liver, kidneys, and heart. Notably, pancreas supplies 20% of its TCA carbons from BCAAs. Genetic and pharmacologic suppression of branched-chain alpha-ketoacid dehydrogenase kinase, a clinically targeted regulatory kinase, induces BCAA oxidation primarily in skeletal muscle of healthy mice. While insulin acutely increases BCAA oxidation in cardiac and skeletal muscle, chronically insulin-resistant mice show blunted BCAA oxidation in adipose tissues and liver, shifting BCAA oxidation toward muscle. Together, this work provides a quantitative framework for understanding systemic BCAA oxidation in health and insulin resistance.

Berberine alleviates insulin resistance by reducing peripheral branched-chain amino acids.

Increased circulating branched-chain amino acids (BCAAs) have been involved in the pathogenesis of obesity and insulin resistance (IR). However, evidence relating berberine (BBR), gut microbiota, BCAAs, and IR is limited. Here, we showed that BBR could effectively rectify steatohepatitis and glucose intolerance in high-fat diet (HFD)-fed mice. BBR reorganized gut microbiota populations under both the normal chow diet (NCD) and HFD. Particularly, BBR noticeably decreased the relative abundance of BCAA-producing bacteria, including order Clostridiales; families Streptococcaceae, Clostridiaceae, and Prevotellaceae; and genera Streptococcus and Prevotella. Compared with the HFD group, predictive metagenomics indicated a reduction in the proportion of gut microbiota genes involved in BCAA biosynthesis but the enrichment genes for BCAA degradation and transport by BBR treatment. Accordingly, the elevated serum BCAAs of HFD group were significantly decreased by BBR. Furthermore, the Western blotting results implied that BBR could promote the BCAA catabolism in the liver and epididymal white adipose tissues of HFD-fed mice by activation of the multienzyme branched-chain α-ketoacid dehydrogenase complex (BCKDC), whereas by inhibition of the phosphorylation state of BCKDHA (E1α subunit) and branched-chain α-ketoacid dehydrogenase kinase (BCKDK). The ex vivo assay further confirmed that BBR could increase BCAA catabolism in both AML12 hepatocytes and 3T3-L1 adipocytes. Finally, data from healthy subjects and diabetics confirmed that BBR could improve glycemic control and modulate circulating BCAAs. Together, our findings clarified BBR improving IR associated not only with gut microbiota alteration in BCAA biosynthesis but also with BCAA catabolism in liver and adipose tissues.

Ca2+-dependent inhibition of branched-chain α-ketoacid dehydrogenase kinase by thiamine pyrophosphate

Catabolism of the branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) is regulated by the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which in turn is regulated by phosphorylation catalyzed by BCKDH kinase (BDK). Thiamine pyrophosphate (TPP) is required as a coenzyme for the E1 component of the BCKDH complex and can also bring about activation of the complex by inhibiting BDK. The present study shows that free Ca2+ in the physiological range greatly increases the sensitivity of BDK to inhibition by TPP (IC50 of 2.5 μM in the presence of 1 μM free Ca2+). This novel mechanism may be responsible for the stimulation of BCAA oxidation by conditions that increase mitochondrial free Ca2+ levels, e.g. in skeletal muscle during exercise.