Metabolic inflexibility promotes mitochondrial health during liver regeneration.

Mitochondria are critical for proper organ function and mechanisms to promote mitochondrial health during regeneration would benefit tissue homeostasis. We report that during liver regeneration, proliferation is suppressed in electron transport chain (ETC)-dysfunctional hepatocytes due to an inability to generate acetyl-CoA from peripheral fatty acids through mitochondrial ß-oxidation. Alternative modes for acetyl-CoA production from pyruvate or acetate are suppressed in the setting of ETC dysfunction. This metabolic inflexibility forces a dependence on ETC-functional mitochondria and restoring acetyl-CoA production from pyruvate is sufficient to allow ETC-dysfunctional hepatocytes to proliferate. We propose that metabolic inflexibility within hepatocytes can be advantageous by limiting the expansion of ETC-dysfunctional cells.

The intrinsic substrate specificity of the human tyrosine kinome.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

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.

The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP-importin α interactions.

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin ß and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Šcrystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2-ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117-196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

Real-time hyperpolarized 13C magnetic resonance detects increased pyruvate oxidation in pyruvate dehydrogenase kinase 2/4-double knockout mouse livers.

The pyruvate dehydrogenase complex (PDH) critically regulates carbohydrate metabolism. Phosphorylation of PDH by one of the pyruvate dehydrogenase kinases 1-4 (PDK1-4) decreases the flux of carbohydrates into the TCA cycle. Inhibition of PDKs increases oxidative metabolism of carbohydrates, so targeting PDKs has emerged as an important therapeutic approach to manage various metabolic diseases. Therefore, it is highly desirable to begin to establish imaging tools for noninvasive measurements of PDH flux in rodent models. In this study, we used hyperpolarized (HP) 13C-magnetic resonance spectroscopy to study the impact of a PDK2/PDK4 double knockout (DKO) on pyruvate metabolism in perfused livers from lean and diet-induced obese (DIO) mice and validated the HP observations with high-resolution 13C-nuclear magnetic resonance (NMR) spectroscopy of tissue extracts and steady-state isotopomer analyses. We observed that PDK-deficient livers produce more HP-bicarbonate from HP-[1-13C]pyruvate than age-matched control livers. A steady-state 13C-NMR isotopomer analysis of tissue extracts confirmed that flux rates through PDH, as well as pyruvate carboxylase and pyruvate cycling activities, are significantly higher in PDK-deficient livers. Immunoblotting experiments confirmed that HP-bicarbonate production from HP-[1-13C]pyruvate parallels decreased phosphorylation of the PDH E1α subunit (pE1α) in liver tissue. Our findings indicate that combining real-time hyperpolarized 13C NMR spectroscopy and 13C isotopomer analysis provides quantitative insights into intermediary metabolism in PDK-knockout mice. We propose that this method will be useful in assessing metabolic disease states and developing therapies to improve PDH flux.

Therapeutic Effect of Targeting Branched-Chain Amino Acid Catabolic Flux in Pressure-Overload Induced Heart Failure.

Background Branched-chain amino acid (BCAA) catabolic defect is an emerging metabolic hallmark in failing hearts in human and animal models. The therapeutic impact of targeting BCAA catabolic flux under pathological conditions remains understudied. Methods and Results BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), a small-molecule inhibitor of branched-chain ketoacid dehydrogenase kinase, was used to enhance BCAA catabolism. After 2 weeks of transaortic constriction, mice with significant cardiac dysfunctions were treated with vehicle or BT2. Serial echocardiograms showed continuing pathological deterioration in left ventricle of the vehicle-treated mice, whereas the BT2-treated mice showed significantly preserved cardiac function and structure. Moreover, BT2 treatment improved systolic contractility and diastolic mechanics. These therapeutic benefits appeared to be independent of impacts on left ventricle hypertrophy but associated with increased gene expression involved in fatty acid utilization. The BT2 administration showed no signs of apparent toxicity. Conclusions Our data provide the first proof-of-concept evidence for the therapeutic efficacy of restoring BCAA catabolic flux in hearts with preexisting dysfunctions. The BCAA catabolic pathway represents a novel and potentially efficacious target for treatment of heart failure.

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.

In vivo assessment of increased oxidation of branched-chain amino acids in glioblastoma.

Altered branched-chain amino acids (BCAAs) metabolism is a distinctive feature of various cancers and plays an important role in sustaining tumor proliferation and aggressiveness. Despite the therapeutic and diagnostic potentials, the role of BCAA metabolism in cancer and the activities of associated enzymes remain unclear. Due to its pivotal role in BCAA metabolism and rapid cellular transport, hyperpolarized 13C-labeled α-ketoisocaproate (KIC), the α-keto acid corresponding to leucine, can assess both BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase complex (BCKDC) activities via production of [1-13C]leucine or 13CO2 (and thus H13CO3-), respectively. Here, we investigated BCAA metabolism of F98 rat glioma model in vivo using hyperpolarized 13C-KIC. In tumor regions, we observed a decrease in 13C-leucine production from injected hyperpolarized 13C-KIC via BCAT compared to the contralateral normal-appearing brain, and an increase in H13CO3-, a catabolic product of KIC through the mitochondrial BCKDC. A parallel ex vivo 13C NMR isotopomer analysis following steady-state infusion of [U-13C]leucine to glioma-bearing rats verified the increased oxidation of leucine in glioma tissue. Both the in vivo hyperpolarized KIC imaging and the leucine infusion study indicate that KIC catabolism is upregulated through BCAT/BCKDC and further oxidized via the citric acid cycle in F98 glioma.

The BCKDH Kinase and Phosphatase Integrate BCAA and Lipid Metabolism via Regulation of ATP-Citrate Lyase.

Branched-chain amino acids (BCAA) are strongly associated with dysregulated glucose and lipid metabolism, but the underlying mechanisms are poorly understood. We report that inhibition of the kinase (BDK) or overexpression of the phosphatase (PPM1K) that regulates branched-chain ketoacid dehydrogenase (BCKDH), the committed step of BCAA catabolism, lowers circulating BCAA, reduces hepatic steatosis, and improves glucose tolerance in the absence of weight loss in Zucker fatty rats. Phosphoproteomics analysis identified ATP-citrate lyase (ACL) as an alternate substrate of BDK and PPM1K. Hepatic overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis. BDK and PPM1K transcript levels were increased and repressed, respectively, in response to fructose feeding or expression of the ChREBP-ß transcription factor. These studies identify BDK and PPM1K as a ChREBP-regulated node that integrates BCAA and lipid metabolism. Moreover, manipulation of the BDK:PPM1K ratio relieves key metabolic disease phenotypes in a genetic model of severe obesity.

A novel inhibitor of pyruvate dehydrogenase kinase stimulates myocardial carbohydrate oxidation in diet-induced obesity.

The pyruvate dehydrogenase complex (PDC) is a key control point of energy metabolism and is subject to regulation by multiple mechanisms, including posttranslational phosphorylation by pyruvate dehydrogenase kinase (PDK). Pharmacological modulation of PDC activity could provide a new treatment for diabetic cardiomyopathy, as dysregulated substrate selection is concomitant with decreased heart function. Dichloroacetate (DCA), a classic PDK inhibitor, has been used to treat diabetic cardiomyopathy, but the lack of specificity and side effects of DCA indicate a more specific inhibitor of PDK is needed. This study was designed to determine the effects of a novel and highly selective PDK inhibitor, 2((2,4-dihydroxyphenyl)sulfonyl) isoindoline-4,6-diol (designated PS10), on pyruvate oxidation in diet-induced obese (DIO) mouse hearts compared with DCA-treated hearts. Four groups of mice were studied: lean control, DIO, DIO + DCA, and DIO + PS10. Both DCA and PS10 improved glucose tolerance in the intact animal. Pyruvate metabolism was studied in perfused hearts supplied with physiological mixtures of long chain fatty acids, lactate, and pyruvate. Analysis was performed using conventional 1H and 13C isotopomer methods in combination with hyperpolarized [1-13C]pyruvate in the same hearts. PS10 and DCA both stimulated flux through PDC as measured by the appearance of hyperpolarized [13C]bicarbonate. DCA but not PS10 increased hyperpolarized [1-13C]lactate production. Total carbohydrate oxidation was reduced in DIO mouse hearts but increased by DCA and PS10, the latter doing so without increasing lactate production. The present results suggest that PS10 is a more suitable PDK inhibitor for treatment of diabetic cardiomyopathy.

Targeting hepatic pyruvate dehydrogenase kinases restores insulin signaling and mitigates ChREBP-mediated lipogenesis in diet-induced obese mice.

OBJECTIVE: Mitochondrial pyruvate dehydrogenase kinases 1-4 (PDKs1-4) negatively regulate activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation. PDKs play a pivotal role in maintaining energy homeostasis and contribute to metabolic flexibility by attenuating PDC activity in various mammalian tissues. Cumulative evidence has shown that the up-regulation of PDK4 expression is tightly associated with obesity and diabetes. In this investigation, we test the central hypothesis that PDKs1-4 are a pharmacological target for lowering glucose levels and restoring insulin sensitivity in obesity and type 2 diabetes (T2D). METHODS: Diet-induced obese (DIO) mice were treated with a liver-specific pan-PDK inhibitor 2-[(2,4-dihydroxyphenyl) sulfonyl]isoindoline-4,6-diol (PS10) for four weeks, and results compared with PDK2/PDK4 double knockout (DKO) mice on the same high fat diet (HFD). RESULTS: Both PS10-treated DIO mice and HFD-fed DKO mice showed significantly improved glucose, insulin and pyruvate tolerance, compared to DIO controls, with lower plasma insulin levels and increased insulin signaling in liver. In response to lower glucose levels, phosphorylated AMPK in PS10-treated DIO and HFD-fed DKO mice is upregulated, accompanied by decreased nuclear carbohydrate-responsive element binding protein (ChREBP). The reduced ChREBP signaling correlates with down-regulation of hepatic lipogenic enzymes (ACC1, FAS, and SCD1), leading to markedly diminished hepatic steatosis in both study groups, with lower circulating cholesterol and triacylglyceride levels as well as reduced fat mass. PS10-treated DIO as well as DKO mice showed predominant fatty acid over glucose oxidation. However, unlike systemic DKO mice, increased hepatic PDC activity alone in PS10-treated DIO mice does not raise the plasma total ketone body level. CONCLUSION: Our findings establish that specific targeting of hepatic PDKs with the PDK inhibitor PS10 is an effective therapeutic approach to maintaining glucose and lipid homeostasis in obesity and T2D, without the harmful ketoacidosis associated with systemic inhibition of PDKs.

Identification of the ferredoxin interaction sites on ferredoxin-dependent glutamate synthase from Synechocystis sp. PCC 6803.

Based on in silico docking methods, five amino acids in glutamate synthase (Gln-467, His-1144, Asn-1147, Arg-1162, and Trp-676) likely constitute key binding residues in the interface of a glutamate synthase:ferredoxin complex. Although all interfacial mutants studied showed the ability to form a complex under low ionic strength, these docking mutations showed significantly less ferredoxin-dependent activities, while still retaining enzymatic activity. Furthermore, isothermal titration calorimetry showed a possible 1:2 molar ratio between the wild-type glutamate synthase and ferredoxin. However, each of our interfacial mutants showed only a 1:1 complex with ferredoxin, suggesting that the mutations directly affect the glutamate synthase:ferredoxin heterodimer interface.

Development of Dihydroxyphenyl Sulfonylisoindoline Derivatives as Liver-Targeting Pyruvate Dehydrogenase Kinase Inhibitors.

Pyruvate dehydrogenase kinases 1-4 (PDK1-4) negatively control activity of the pyruvate dehydrogenase complex (PDC) and are up-regulated in obesity, diabetes, heart failure, and cancer. We reported earlier two novel pan-PDK inhibitors PS8 [4-((5-hydroxyisoindolin-2-yl)sulfonyl)benzene-1,3-diol] (1) and PS10 [2-((2,4-dihydroxyphenyl)sulfonyl)isoindoline-4,6-diol] (2) that targeted the ATP-binding pocket in PDKs. Here, we developed a new generation of PDK inhibitors by extending the dihydroxyphenyl sulfonylisoindoline scaffold in 1 and 2 to the entrance region of the ATP-binding pocket in PDK2. The lead inhibitor (S)-3-amino-4-(4-((2-((2,4-dihydroxyphenyl)sulfonyl)isoindolin-5-yl)amino)piperidin-1-yl)-4-oxobutanamide (17) shows a ∼8-fold lower IC50 (58 nM) than 2 (456 nM). In the crystal structure, the asparagine moiety in 17 provides additional interactions with Glu-262 from PDK2. Treatment of diet-induced obese mice with 17 resulted in significant liver-specific augmentation of PDC activity, accompanied by improved glucose tolerance and drastically reduced hepatic steatosis. These findings support 17 as a potential glucose-lowering therapeutic targeting liver for obesity and type 2 diabetes.

Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure.

BACKGROUND: Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied. METHODS AND RESULTS: Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload. CONCLUSIONS: BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15-mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.

GM130 Regulates Golgi-Derived Spindle Assembly by Activating TPX2 and Capturing Microtubules.

Spindle assembly requires the coordinated action of multiple cellular structures to nucleate and organize microtubules in a precise spatiotemporal manner. Among them, the contributions of centrosomes, chromosomes, and microtubules have been well studied, yet the involvement of membrane-bound organelles remains largely elusive. Here, we provide mechanistic evidence for a membrane-based, Golgi-derived microtubule assembly pathway in mitosis. Upon mitotic entry, the Golgi matrix protein GM130 interacts with importin α via a classical nuclear localization signal that recruits importin α to the Golgi membranes. Sequestration of importin α by GM130 liberates the spindle assembly factor TPX2, which activates Aurora-A kinase and stimulates local microtubule nucleation. Upon filament assembly, nascent microtubules are further captured by GM130, thus linking Golgi membranes to the spindle. Our results reveal an active role for the Golgi in regulating spindle formation to ensure faithful organelle inheritance.

Benzothiophene carboxylate derivatives as novel allosteric inhibitors of branched-chain α-ketoacid dehydrogenase kinase.

The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation.BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6- dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC(50) = 3.19 µM). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-α-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T(1/2) = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[ b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[ b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations.

Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket.

Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 µM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes.

Metabolite regulation of nucleo-cytosolic trafficking of carbohydrate response element-binding protein (ChREBP): role of ketone bodies.

The carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in converting excess carbohydrate to storage fat in liver. In response to changing glucose levels, ChREBP activity is regulated by nucleo-cytoplasmic shuttling of ChREBP via interactions with 14-3-3 proteins and importins. The nuclear/cytosol trafficking is regulated partly by phosphorylation/dephosphorylation of serine 196 mediated by cAMP-dependent protein kinase and protein phosphatase. We show here that protein-free extracts of starved and high fat-fed livers contain metabolites that activate interaction of ChREBP·14-3-3 and inhibit the ChREBP/importin α interaction, resulting in cytosolic localization. These metabolites were identified as ß-hydroxybutyrate and acetoacetate. Nuclear localization of GFP-ChREBP is rapidly inhibited in hepatocytes incubated in ß-hydroxybutyrate or fatty acids, and the observed inhibition is closely correlated with the production of ketone bodies. These observations show that ketone bodies play an important role in the regulation of ChREBP activity by restricting ChREBP localization to the cytoplasm, thus inhibiting fat synthesis during periods of ketosis.

Structure-based design and mechanisms of allosteric inhibitors for mitochondrial branched-chain α-ketoacid dehydrogenase kinase.

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, (S)-α-chloro-phenylpropionic acid [(S)-CPP]. Crystal structures of the BDK-(S)-CPP complex show that (S)-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of (S)-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure.