MCT1-governed pyruvate metabolism is essential for antibody class-switch recombination through H3K27 acetylation.

Monocarboxylate transporter 1 (MCT1) exhibits essential roles in cellular metabolism and energy supply. Although MCT1 is highly expressed in activated B cells, it is not clear how MCT1-governed monocarboxylates transportation is functionally coupled to antibody production during the glucose metabolism. Here, we report that B cell-lineage deficiency of MCT1 significantly influences the class-switch recombination (CSR), rendering impaired IgG antibody responses in Mct1f/fMb1Cre mice after immunization. Metabolic flux reveals that glucose metabolism is significantly reprogrammed from glycolysis to oxidative phosphorylation in Mct1-deficient B cells upon activation. Consistently, activation-induced cytidine deaminase (AID), is severely suppressed in Mct1-deficient B cells due to the decreased level of pyruvate metabolite. Mechanistically, MCT1 is required to maintain the optimal concentration of pyruvate to secure the sufficient acetylation of H3K27 for the elevated transcription of AID in activated B cells. Clinically, we found that MCT1 expression levels are significantly upregulated in systemic lupus erythematosus patients, and Mct1 deficiency can alleviate the symptoms of bm12-induced murine lupus model. Collectively, these results demonstrate that MCT1-mediated pyruvate metabolism is required for IgG antibody CSR through an epigenetic dependent AID transcription, revealing MCT1 as a potential target for vaccine development and SLE disease treatment.

Hepatic mitochondrial NAD + transporter SLC25A47 activates AMPKα mediating lipid metabolism and tumorigenesis.

BACKGROUND AIMS: SLC25A47 was initially identified as a mitochondrial HCC-downregulated carrier protein, but its physiological functions and transport substrates are unknown. We aimed to investigate the physiological role of SLC25A47 in hepatic metabolism. APPROACH RESULTS: In the treatment of hepatocytes with metformin, we found that metformin can transcriptionally activate the expression of Slc25a47 , which is required for AMP-activated protein kinase α (AMPKα) phosphorylation. Slc25a47 -deficient mice had increased hepatic lipid content, triglycerides, and cholesterol levels, and we found that Slc25a47 deficiency suppressed AMPKα phosphorylation and led to an increased accumulation of nuclear SREBPs, with elevated fatty acid and cholesterol biosynthetic activities. Conversely, when Slc25a47 was overexpressed in mouse liver, AMPKα was activated and resulted in the inhibition of lipogenesis. Moreover, using a diethylnitrosamine-induced mouse HCC model, we found that the deletion of Slc25a47 promoted HCC tumorigenesis and development through the activated mammalian target of rapamycin cascade. Employing homology modeling of SLC25A47 and virtual screening of the human metabolome database, we demonstrated that NAD + was an endogenous substrate for SLC25A47, and the activity of NAD + -dependent sirtuin 3 declined in Slc25a47 -deficient mice, followed by inactivation of AMPKα. CONCLUSIONS: Our findings reveal that SLC25A47, a hepatocyte-specific mitochondrial NAD + transporter, is one of the pharmacological targets of metformin and regulates lipid homeostasis through AMPKα, and may serve as a potential drug target for treating NAFLD and HCC.

Embigin facilitates monocarboxylate transporter 1 localization to the plasma membrane and transition to a decoupling state.

Cell-surface ancillary glycoproteins basigin or embigin form heterodimeric complexes with proton-coupled monocarboxylate transporters (MCTs), facilitating the membrane trafficking of MCTs and regulating their transport activities. Here, we determine the cryoelectron microscopy (cryo-EM) structure of the human MCT1-embigin complex and observe that embigin forms extensive interactions with MCT1 to facilitate its localization to the plasma membrane. In addition, the formation of the heterodimer effectively blocks MCT1 from forming a homodimer through a steric hindrance effect, releasing the coupling between two signature motifs and driving a significant conformation change in transmembrane helix 5 (TM5) of MCTs. Consequently, the substrate-binding pocket alternates between states of homodimeric coupling and heterodimeric decoupling states and exhibits differences in substrate-binding affinity, supporting the hypothesis that the substrate-induced motion originating in one subunit of the MCT dimer could be transmitted to the adjacent subunit to alter its substrate-binding affinity.

Ablation of Proton/Glucose Exporter SLC45A2 Enhances Melanosomal Glycolysis to Inhibit Melanin Biosynthesis and Promote Melanoma Metastasis.

Sequence variation in SLC45A2 are responsible for oculocutaneous albinism type 4 in many species and are associated with melanoma susceptibility, but the molecular mechanism is unclear. In this study, we used Slc45a2-deficient melanocyte and mouse models to elucidate the roles of SLC45A2 in melanogenesis and melanoma metastasis. We found that the acidified cellular environment impairs the activity of key melanogenic enzyme tyrosinase in Slc45a2-deficient melanocytes. SLC45A2 is identified as a proton/glucose exporter in melanosomes, and its ablation increases the acidification of melanosomal pH through enhanced glycolysis. Intriguingly, 13C-glucose-labeled metabolic flux and biochemical assays show that melanosomes are active glucose-metabolizing organelles, indicating that elevated glycolysis mainly occurs in melanosomes owing to Slc45a2 deficiency. Moreover, Slc45a2 deficiency significantly upregulates the activities of glycolytic enzymes and phosphatidylinositol 3-kinase/protein kinase B signaling to promote glycolysis-dependent survival and metastasis of melanoma cells. Collectively, our study reveals that the proton/glucose exporter SLC45A2 mediates melanin synthesis and melanoma metastasis primarily by modulating melanosomal glucose metabolism.

Hematopoietic Progenitor Kinase1 (HPK1) Mediates T Cell Dysfunction and Is a Druggable Target for T Cell-Based Immunotherapies.

Ameliorating T cell exhaustion and enhancing effector function are promising strategies for the improvement of immunotherapies. Here, we show that the HPK1-NFκB-Blimp1 axis mediates T cell dysfunction. High expression of MAP4K1 (which encodes HPK1) correlates with increased T cell exhaustion and with worse patient survival in several cancer types. In MAP4K1KO mice, tumors grow slower than in wild-type mice and infiltrating T cells are less exhausted and more active and proliferative. We further show that genetic depletion, pharmacological inhibition, or proteolysis targeting chimera (PROTAC)-mediated degradation of HPK1 improves the efficacy of CAR-T cell-based immunotherapies in diverse preclinical mouse models of hematological and solid tumors. These strategies are more effective than genetically depleting PD-1 in CAR-T cells. Thus, we demonstrate that HPK1 is a mediator of T cell dysfunction and an attractive druggable target to improve immune therapy responses.

Cholesterol biosynthesis supports the growth of hepatocarcinoma lesions depleted of fatty acid synthase in mice and humans.

OBJECTIVE: Increased de novo fatty acid (FA) synthesis and cholesterol biosynthesis have been independently described in many tumour types, including hepatocellular carcinoma (HCC). DESIGN: We investigated the functional contribution of fatty acid synthase (Fasn)-mediated de novo FA synthesis in a murine HCC model induced by loss of Pten and overexpression of c-Met (sgPten/c-Met) using liver-specific Fasn knockout mice. Expression arrays and lipidomic analysis were performed to characterise the global gene expression and lipid profiles, respectively, of sgPten/c-Met HCC from wild-type and Fasn knockout mice. Human HCC cell lines were used for in vitro studies. RESULTS: Ablation of Fasn significantly delayed sgPten/c-Met-driven hepatocarcinogenesis in mice. However, eventually, HCC emerged in Fasn knockout mice. Comparative genomic and lipidomic analyses revealed the upregulation of genes involved in cholesterol biosynthesis, as well as decreased triglyceride levels and increased cholesterol esters, in HCC from these mice. Mechanistically, loss of Fasn promoted nuclear localisation and activation of sterol regulatory element binding protein 2 (Srebp2), which triggered cholesterogenesis. Blocking cholesterol synthesis via the dominant negative form of Srebp2 (dnSrebp2) completely prevented sgPten/c-Met-driven hepatocarcinogenesis in Fasn knockout mice. Similarly, silencing of FASN resulted in increased SREBP2 activation and hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (HMGCR) expression in human HCC cell lines. Concomitant inhibition of FASN-mediated FA synthesis and HMGCR-driven cholesterol production was highly detrimental for HCC cell growth in culture. CONCLUSION: Our study uncovers a novel functional crosstalk between aberrant lipogenesis and cholesterol biosynthesis pathways in hepatocarcinogenesis, whose concomitant inhibition might represent a therapeutic option for HCC.

Zoledronate dysregulates fatty acid metabolism in renal tubular epithelial cells to induce nephrotoxicity.

Zoledronate is a bisphosphonate that is widely used in the treatment of metabolic bone diseases. However, zoledronate induces significant nephrotoxicity associated with acute tubular necrosis and renal fibrosis when administered intravenously. There is speculation that zoledronate-induced nephrotoxicity may result from its pharmacological activity as an inhibitor of the mevalonate pathway but the molecular mechanisms are not fully understood. In this report, human proximal tubular HK-2 cells and mouse models were combined to dissect the molecular pathways underlying nephropathy caused by zoledronate treatments. Metabolomic and proteomic assays revealed that multiple cellular processes were significantly disrupted, including the TGFß pathway, fatty acid metabolism and small GTPase signaling in zoledronate-treated HK-2 cells (50 µM) as compared with those in controls. Zoledronate treatments in cells (50 µM) and mice (3 mg/kg) increased TGFß/Smad3 pathway activation to induce fibrosis and kidney injury, and specifically elevated lipid accumulation and expression of fibrotic proteins. Conversely, fatty acid transport protein Slc27a2 deficiency or co-administration of PPARA agonist fenofibrate (20 mg/kg) prevented zoledronate-induced lipid accumulation and kidney fibrosis in mice, indicating that over-expression of fatty acid transporter SLC27A2 and defective fatty acid ß-oxidation following zoledronate treatments were significant factors contributing to its nephrotoxicity. These pharmacological and genetic studies provide an important mechanistic insight into zoledronate-associated kidney toxicity that will aid in development of therapeutic prevention and treatment options for this nephropathy.

Tenofovir and adefovir down-regulate mitochondrial chaperone TRAP1 and succinate dehydrogenase subunit B to metabolically reprogram glucose metabolism and induce nephrotoxicity.

Despite the therapeutic success of tenofovir (TFV) for treatment of HIV-1 infection, numerous cases of nephrotoxicity have been reported. Mitochondrial toxicity has been purported as the major target of TFV-associated renal tubulopathy but the underlying molecular mechanism remains unclear. In this report, we use metabolomics and proteomics with HK-2 cells and animal models to dissect the molecular pathways underlying nephropathy caused by TFV and its more toxic analog, adefovir (ADV). Proteomic analysis shows that mitochondrial chaperone TRAP1 and mtDNA replicating protein SSBP1 were significantly down-regulated in TFV and ADV treated HK-2 cells compared with controls. Transmission electron microscopy (TEM) revealed that TFV and ADV-treated HK-2 cells had accumulated glycogen, a phenotype that was also observed in mice treated with TFV and ADV. Analysis of the proteins in TCA cycle showed succinate dehydrogenase subunit B (SDHB) was nearly depleted in glucose oxidative phosphorylation pathway however certain enzymes in the glycolysis and glycogen synthesis pathway had elevated expression in TFV and ADV-treated HK-2 cells. These results suggest that TFV and ADV may cause mitochondrial dysfunction in renal tubular cells and reprogramming of glucose metabolism. The resulting glycogen accumulation may partially contribute to TFV and ADV induced renal dysfunction.