Metabolic stress induces a double-positive feedback loop between AMPK and SQSTM1/p62 conferring dual activation of AMPK and NFE2L2/NRF2 to synergize antioxidant defense.

Co-occurring mutations in KEAP1 in STK11/LKB1-mutant NSCLC activate NFE2L2/NRF2 to compensate for the loss of STK11-AMPK activity during metabolic adaptation. Characterizing the regulatory crosstalk between the STK11-AMPK and KEAP1-NFE2L2 pathways during metabolic stress is crucial for understanding the implications of co-occurring mutations. Here, we found that metabolic stress increased the expression and phosphorylation of SQSTM1/p62, which is essential for the activation of NFE2L2 and AMPK, synergizing antioxidant defense and tumor growth. The SQSTM1-driven dual activation of NFE2L2 and AMPK was achieved by inducing macroautophagic/autophagic degradation of KEAP1 and facilitating the AXIN-STK11-AMPK complex formation on the lysosomal membrane, respectively. In contrast, the STK11-AMPK activity was also required for metabolic stress-induced expression and phosphorylation of SQSTM1, suggesting a double-positive feedback loop between AMPK and SQSTM1. Mechanistically, SQSTM1 expression was increased by the PPP2/PP2A-dependent dephosphorylation of TFEB and TFE3, which was induced by the lysosomal deacidification caused by low glucose metabolism and AMPK-dependent proton reduction. Furthermore, SQSTM1 phosphorylation was increased by MAP3K7/TAK1, which was activated by ROS and pH-dependent secretion of lysosomal Ca2+. Importantly, phosphorylation of SQSTM1 at S24 and S226 was critical for the activation of AMPK and NFE2L2. Notably, the effects caused by metabolic stress were abrogated by the protons provided by lactic acid. Collectively, our data reveal a novel double-positive feedback loop between AMPK and SQSTM1 leading to the dual activation of AMPK and NFE2L2, potentially explaining why co-occurring mutations in STK11 and KEAP1 happen and providing promising therapeutic strategies for lung cancer.Abbreviations: AMPK: AMP-activated protein kinase; BAF1: bafilomycin A1; ConA: concanamycin A; DOX: doxycycline; IP: immunoprecipitation; KEAP1: kelch like ECH associated protein 1; LN: low nutrient; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MCOLN1/TRPML1: mucolipin TRP cation channel 1; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NSCLC: non-small cell lung cancer; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PPP2/PP2A: protein phosphatase 2; ROS: reactive oxygen species; PPP3/calcineurin: protein phosphatase 3; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TCL: total cell lysate; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; V-ATPase: vacuolar-type H+-translocating ATPase.

Metabolic determinants of leukemia onset variability in genetically homogeneous AKR mice.

Leukemia is a complex disease shaped by the intricate interplay of genetic and environmental factors. Given our preliminary data showing different leukemia incidence in genetically homogenous AKR mice harboring the spontaneous leukemia-inducing mutation Rmcfs, we sought to unravel the role of metabolites and gut microbiota in the leukemia penetrance. Our metabolomic analysis revealed distinct serum metabolite profiles between mice that developed leukemia and those that did not. We discovered that linoleic acid (LA), an essential ω-6 polyunsaturated fatty acid, was significantly decreased in the leukemia group, with the lower levels observed starting from 25 weeks before the onset. A predictive model based on LA levels demonstrated high accuracy in predicting leukemia development (area under curve 0.82). In vitro experiment confirmed LA's cytotoxic effects against leukemia cells, and in vivo study showed that a diet enriched with LA prolonged survival in AKR mice. Furthermore, gut microbiome analysis identified specific Lachnospiraceae species, that affect host lipid metabolism, are exclusively present in the leukemia group, suggesting their potential influence on LA metabolism and leukemia development. These findings shed light on the complex relationship between metabolites, gut microbiota, and leukemia development, providing valuable insights into the role of non-genetic factors in leukemia penetrance and potential strategies for leukemia prevention.

Glycerol 3-phosphate dehydrogenases (1 and 2) in cancer and other diseases.

The glycerol 3-phosphate shuttle (GPS) is composed of two different enzymes: cytosolic NAD+-linked glycerol 3-phosphate dehydrogenase 1 (GPD1) and mitochondrial FAD-linked glycerol 3-phosphate dehydrogenase 2 (GPD2). These two enzymes work together to act as an NADH shuttle for mitochondrial bioenergetics and function as an important bridge between glucose and lipid metabolism. Since these genes were discovered in the 1960s, their abnormal expression has been described in various metabolic diseases and tumors. Nevertheless, it took a long time until scientists could investigate the causal relationship of these enzymes in those pathophysiological conditions. To date, numerous studies have explored the involvement and mechanisms of GPD1 and GPD2 in cancer and other diseases, encompassing reports of controversial and non-conventional mechanisms. In this review, we summarize and update current knowledge regarding the functions and effects of GPS to provide an overview of how the enzymes influence disease conditions. The potential and challenges of developing therapeutic strategies targeting these enzymes are also discussed.

Acetyl-CoA synthetase 2 contributes to a better prognosis for liver cancer by switching acetate-glucose metabolism.

Acetyl-CoA synthetase 2 (ACSS2)-dependent acetate usage has generally been associated with tumorigenesis and increased malignancy in cancers under nutrient-depleted conditions. However, the nutrient usage and metabolic characteristics of the liver differ from those of other organs; therefore, the mechanism of ACSS2-mediated acetate metabolism may also differ in liver cancer. To elucidate the underlying mechanisms of ACSS2 in liver cancer and acetate metabolism, the relationships between patient acetate uptake and metabolic characteristics and between ACSS2 and tumor malignancies were comprehensively studied in vitro, in vivo and in humans. Clinically, we initially found that ACSS2 expression was decreased in liver cancer patients. Moreover, PET-CT imaging confirmed that lower-grade cancer cells take up more 11C-acetate but less 18F-fluorodeoxyglucose (18F-FDG); however, this trend was reversed in higher-grade cancer. Among liver cancer cells, those with high ACSS2 expression avidly absorbed acetate even in a glucose-sufficient environment, whereas those with low ACSS2 expression did not, thereby showing correlations with their respective ACSS2 expression. Metabolomic isotope tracing in vitro and in vivo revealed greater acetate incorporation, greater lipid anabolic metabolism, and less malignancy in high-ACSS2 tumors. Notably, ACSS2 downregulation in liver cancer cells was associated with increased tumor occurrence in vivo. In human patient cohorts, patients in the low-ACSS2 subgroup exhibited reduced anabolism, increased glycolysis/hypoxia, and poorer prognosis. We demonstrated that acetate uptake by ACSS2 in liver cancer is independent of glucose depletion and contributes to lipid anabolic metabolism and reduced malignancy, thereby leading to a better prognosis for liver cancer patients.

Visualizing Cancer-Originating Acetate Uptake Through MCT1 in Reactive Astrocytes in the Glioblastoma Tumor Microenvironment.

BACKGROUND: Reactive astrogliosis is a hallmark of various brain pathologies, including neurodegenerative diseases and glioblastomas. However, the specific intermediate metabolites contributing to reactive astrogliosis remain unknown. This study investigated how glioblastomas induce reactive astrogliosis in the neighboring microenvironment and explores 11C-acetate PET as an imaging technique for detecting reactive astrogliosis. METHODS: Through in vitro, mouse models, and human tissue experiments, we examined the association between elevated 11C-acetate uptake and reactive astrogliosis in gliomas. We explored acetate from glioblastoma cells, which triggers reactive astrogliosis in neighboring astrocytes by upregulating MAO-B and MCT1 expression. We evaluated the presence of cancer stem cells in the reactive astrogliosis region of glioblastomas and assessed the correlation between the volume of 11C-acetate uptake beyond MRI and prognosis. RESULTS: Elevated 11C-acetate uptake is associated with reactive astrogliosis and astrocytic MCT1 in the periphery of glioblastomas in human tissues and mouse models. Glioblastoma cells exhibit increased acetate production as a result of glucose metabolism, with subsequent secretion of acetate. Acetate derived from glioblastoma cells induces reactive astrogliosis in neighboring astrocytes by increasing the expression of MAO-B and MCT1. We found cancer stem cells within the reactive astrogliosis at the tumor periphery. Consequently, a larger volume of 11C-acetate uptake beyond contrast-enhanced MRI was associated with worse prognosis. CONCLUSION: Our results highlight the role of acetate derived from glioblastoma cells in inducing reactive astrogliosis and underscore the potential value of 11C-acetate PET as an imaging technique for detecting reactive astrogliosis, offering important implications for the diagnosis and treatment of glioblastomas.

Lactate as a major epigenetic carbon source for histone acetylation via nuclear LDH metabolism.

Histone acetylation involves the transfer of two-carbon units to the nucleus that are embedded in low-concentration metabolites. We found that lactate, a high-concentration metabolic byproduct, can be a major carbon source for histone acetylation through oxidation-dependent metabolism. Both in cells and in purified nuclei, 13C3-lactate carbons are incorporated into histone H4 (maximum incorporation: ~60%). In the purified nucleus, this process depends on nucleus-localized lactate dehydrogenase (LDHA), knockout (KO) of which abrogates incorporation. Heterologous expression of nucleus-localized LDHA reverses the KO effect. Lactate itself increases histone acetylation, whereas inhibition of LDHA reduces acetylation. In vitro and in vivo settings exhibit different lactate incorporation patterns, suggesting an influence on the microenvironment. Higher nuclear LDHA localization is observed in pancreatic cancer than in normal tissues, showing disease relevance. Overall, lactate and nuclear LDHA can be major structural and regulatory players in the metabolism-epigenetics axis controlled by the cell's own status or the environmental status.

TAK-981, a SUMOylation inhibitor, suppresses AML growth immune-independently.

Acute myeloid leukemia (AML) generally has an unsatisfactory prognosis despite the recent introduction of new regimens, including targeted agents and antibodies. To find a new druggable pathway, we performed integrated bioinformatic pathway screening on large OHSU and MILE AML databases, discovered the SUMOylation pathway, and validated it independently with an external data set (totaling 2959 AML and 642 normal sample data). The clinical relevance of SUMOylation in AML was supported by its core gene expression which is correlated with patient survival, European LeukemiaNet 2017 risk classification, and AML-relevant mutations. TAK-981, a first-in-class SUMOylation inhibitor currently under clinical trials for solid tumors, showed antileukemic effects with apoptosis induction, cell-cycle arrest, and induction of differentiation marker expression in leukemic cells. It exhibited potent nanomolar activity, often stronger than that of cytarabine, which is part of the standard of care. TAK-981's utility was further demonstrated in in vivo mouse and human leukemia models as well as patient-derived primary AML cells. Our results also indicate direct and cancer cell-inherent anti-AML effects by TAK-981, different from the type 1 interferon and immune-dependent mechanism in a previous solid tumor study. Overall, we provide a proof-of-concept for SUMOylation as a new targetable pathway in AML and propose TAK-981 as a promising direct anti-AML agent. Our data should prompt studies on optimal combination strategies and transitions to clinical trials in AML.

GPX8 regulates clear cell renal cell carcinoma tumorigenesis through promoting lipogenesis by NNMT.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC), with its hallmark phenotype of high cytosolic lipid content, is considered a metabolic cancer. Despite the implication of this lipid-rich phenotype in ccRCC tumorigenesis, the roles and regulators of de novo lipid synthesis (DNL) in ccRCC remain largely unexplained. METHODS: Our bioinformatic screening focused on ccRCC-lipid phenotypes identified glutathione peroxidase 8 (GPX8), as a clinically relevant upstream regulator of DNL. GPX8 genetic silencing was performed with CRISPR-Cas9 or shRNA in ccRCC cell lines to dissect its roles. Untargeted metabolomics, RNA-seq analyses, and other biochemical assays (e.g., lipid droplets staining, fatty acid uptake, cell proliferation, xenograft, etc.) were carried out to investigate the GPX8's involvement in lipid metabolism and tumorigenesis in ccRCC. The lipid metabolic function of GPX8 and its downstream were also measured by isotope-tracing-based DNL flux measurement. RESULTS: GPX8 knockout or downregulation substantially reduced lipid droplet levels (independent of lipid uptake), fatty acid de novo synthesis, triglyceride esterification in vitro, and tumor growth in vivo. The downstream regulator was identified as nicotinamide N-methyltransferase (NNMT): its knockdown phenocopied, and its expression rescued, GPX8 silencing both in vitro and in vivo. Mechanically, GPX8 regulated NNMT via IL6-STAT3 signaling, and blocking this axis suppressed ccRCC survival by activating AMPK. Notably, neither the GPX8-NNMT axis nor the DNL flux was affected by the von Hippel Lindau (VHL) status, the conventional regulator of ccRCC high lipid content. CONCLUSIONS: Taken together, our findings unravel the roles of the VHL-independent GPX8-NNMT axis in ccRCC lipid metabolism as related to the phenotypes and growth of ccRCC, which may be targeted for therapeutic purposes.

Non-bioenergetic roles of mitochondrial GPD2 promote tumor progression.

Rationale: Despite growing evidence for mitochondria's involvement in cancer, the roles of specific metabolic components outside the respiratory complex have been little explored. We conducted metabolomic studies on mitochondrial DNA (mtDNA)-deficient (ρ0) cancer cells with lower proliferation rates to clarify the undefined roles of mitochondria in cancer growth. Methods and results: Despite extensive metabolic downregulation, ρ0 cells exhibited high glycerol-3-phosphate (G3P) level, due to low activity of mitochondrial glycerol-3-phosphate dehydrogenase (GPD2). Knockout (KO) of GPD2 resulted in cell growth suppression as well as inhibition of tumor progression in vivo. Surprisingly, this was unrelated to the conventional bioenergetic function of GPD2. Instead, multi-omics results suggested major changes in ether lipid metabolism, for which GPD2 provides dihydroxyacetone phosphate (DHAP) in ether lipid biosynthesis. GPD2 KO cells exhibited significantly lower ether lipid level, and their slower growth was rescued by supplementation of a DHAP precursor or ether lipids. Mechanistically, ether lipid metabolism was associated with Akt pathway, and the downregulation of Akt/mTORC1 pathway due to GPD2 KO was rescued by DHAP supplementation. Conclusion: Overall, the GPD2-ether lipid-Akt axis is newly described for the control of cancer growth. DHAP supply, a non-bioenergetic process, may constitute an important role of mitochondria in cancer.

Chemical Biopsy for GNMT as Noninvasive and Tumorigenesis-Relevant Diagnosis of Liver Cancer.

Early diagnosis of hepatocellular carcinoma (HCC) is difficult; the lack of convenient biomarker-based diagnostic modalities renders high-risk HCC patients burdened by life-long periodical examinations. Here, a new chemical biopsy approach was developed for noninvasive diagnosis of HCC using urine samples. Bioinformatic screening for tumor suppressors yielded glycine N-methyltransferase (GNMT) as a biomarker with clinical relevance to HCC tumorigenesis. A liquid chromatography-mass spectrometry (LC-MS)-based chemical biopsy detecting nonradioactive 13C-sarcosine from 13C-glycine was designed to noninvasively assess liver GNMT activity extrahepatically. 13C-Sarcosine showed a strong correlation with GNMT in normal and cancerous liver cells. In an autochthonous animal model developing visible cancer nodules at 17 weeks, the urinary 13C-sarcosine chemical biopsy exhibited notable changes as early as 8 weeks, showing significant correlations with liver GNMT and molecular pathological changes. Our chemical biopsy approach should facilitate early and noninvasive diagnosis of HCC, with direct relevance to tumorigenesis, which can be straightforwardly applied to other diseases.

Involvement of cell shape and lipid metabolism in glioblastoma resistance to temozolomide.

Temozolomide (TMZ) has been used as standard-of-care for glioblastoma multiforme (GBM), but the resistance to TMZ develops quickly and frequently. Thus, more studies are needed to elucidate the resistance mechanisms. In the current study, we investigated the relationship among the three important phenotypes, namely TMZ-resistance, cell shape and lipid metabolism, in GBM cells. We first observed the distinct difference in cell shapes between TMZ-sensitive (U87) and resistant (U87R) GBM cells. We then conducted NMR-based lipid metabolomics, which revealed a significant increase in cholesterol and fatty acid synthesis as well as lower lipid unsaturation in U87R cells. Consistent with the lipid changes, U87R cells exhibited significantly lower membrane fluidity. The transcriptomic analysis demonstrated that lipid synthesis pathways through SREBP were upregulated in U87R cells, which was confirmed at the protein level. Fatostatin, an SREBP inhibitor, and other lipid pathway inhibitors (C75, TOFA) exhibited similar or more potent inhibition on U87R cells compared to sensitive U87 cells. The lower lipid unsaturation ratio, membrane fluidity and higher fatostatin sensitivity were all recapitulated in patient-derived TMZ-resistant primary cells. The observed ternary relationship among cell shape, lipid composition, and TMZ-resistance may be applicable to other drug-resistance cases. SREBP and fatostatin are suggested as a promising target-therapeutic agent pair for drug-resistant glioblastoma.

Ex vivo NMR metabolomics approach using cerebrospinal fluid for the diagnosis of primary CNS lymphoma: Correlation with MR imaging characteristics.

PURPOSE: Primary central nervous system lymphoma (PCNSL) is an uncommon extranodal non-Hodgkin's lymphoma. Here, the feasibility of nuclear magnetic resonance (NMR) metabolomics for the diagnosis and prognosis prediction of PCNSL, as well as its correlation with magnetic resonance imaging (MRI) characteristics, was assessed. PATIENTS AND METHODS: Cerebrospinal fluid (CSF) samples from PCNSL and normal groups (n = 41 for each) were obtained along with MRI data including pre- and postcontrast as well as T1-, T2-, and diffusion-weighted imaging for the treatment-naïve PCNSL patients (n = 24). The CSF samples were analyzed using nuclear magnetic resonance (NMR). RESULTS: The CSF NMR metabolomic exhibited clear differences with a diagnostic sensitivity of 100% and a specificity of 97.6%. The citrate level of the leptomeningeal enhancement (LE) (+) group was significantly lower than that of the LE (-) group (p = 0.018). In addition, the MRI apparent diffusion coefficient (ADC) value of the tumor was positively correlated with the glucose level (p = 0.025). However, none of the marker metabolites were significant prognosis predictors in univariate analysis. CONCLUSIONS: In conclusion, the NMR metabolomics could be helpful to diagnose PCNSL, but not for the prognosis, and MRI features (LE or ADC) can reflect the metabolic profiles of PCNSL.

Extracellular Citrate Treatment Induces HIF1α Degradation and Inhibits the Growth of Low-Glycolytic Hepatocellular Carcinoma under Hypoxia.

HCC is well known for low glycolysis in the tumors, whereas hypoxia induces glycolytic phenotype and tumor progression. This study was conducted to evaluate the expression of SLCs in human HCCs and investigated whether extracellular nutrient administration related to SLCs in low-glycolytic HCC can prevent hypoxic tumor progression. SLCs expression was screened according to the level of glycolysis in HCCs. Then, whether extracellular nutrient treatment can affect hypoxic tumor progression, as well as the mechanisms, were evaluated in an in vitro cell line and an in vivo animal model. Low-glycolytic HCCs showed high SLC13A5/NaCT and SLC16A1/MCT1 but low SLC2A1/GLUT1 and HIF1α/HIF1α expression. Especially, high SLC13A5 expression was significantly associated with good overall survival in the Cancer Genome Atlas (TCGA) database. In HepG2 cells with the highest NaCT expression, extracellular citrate treatment upon hypoxia induced HIF1α degradation, which led to reduced glycolysis and cellular proliferation. Finally, in HepG2-animal models, the citrate-treated group showed smaller tumor with less hypoxic areas than the vehicle-treated group. In patients with HCC, SLC13A5/NaCT is an important SLC, which is associated with low glycolysis and good prognosis. Extracellular citrate treatment induced the failure of metabolic adaptation to hypoxia and tumor growth inhibition, which can be a potential therapeutic strategy in HCCs.

Real-Time Monitoring of Host-Gut Microbial Interspecies Interaction in Anticancer Drug Metabolism.

Gut microbiome can affect drug metabolism considerably, leading to modified drug response. However, quantitative estimation of host vs. microbial contributions in a living host-gut microbiome system has been challenging. Using the interspecies system of Caenorhabditis elegans and gut bacteria, we developed a real-time approach for monitoring their metabolic interaction in vivo during anticancer drug 5-fluorouracil (5-FU) metabolism. The fluorine NMR-based approach yielded the quantitative contributions to the host 5-FU metabolism made by human gut-microbial species of variable genetic backgrounds. It also experimentally confirmed a bacterial gene-metabolism relationship. Differential 5-FU catabolism among bacterial substrains and the contributions to the host metabolism, unobservable by conventional 16S rRNA metagenomic sequencing, were also found. The metabolic contributions could be correlated with phenotypic developmental toxicity of 5-FU to the host fed with different substrains. Our convenient platform should help to reveal heterogeneity in host-gut microbiome interactions for many drugs in a living symbiotic system.

Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney.

BACKGROUND: MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. RESULTS: Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. CONCLUSIONS: Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.

A Serum Marker for Early Pancreatic Cancer With a Possible Link to Diabetes.

BACKGROUND: Pancreatic cancer (PC) has a grim prognosis, and an early diagnostic biomarker has been highly desired. The molecular link between diabetes and PC has not been well established. METHODS: Bioinformatics screening was performed for a serum PC marker. Experiments in cell lines (5 PC and 1 normal cell lines), mouse models, and human tissue staining (37 PC and 10 normal cases) were performed to test asprosin production from PC. Asprosin's diagnostic performance was tested with serums from multi-center cohorts (347 PC, 209 normal, and 55 additional diabetic patients) and evaluated according to PC status, stages, and diabetic status, which was compared with that of CA19-9. RESULTS: Asprosin, a diabetes-related hormone, was found from the bioinformatics screening, and its production from PC was confirmed. Serum asprosin levels from multi-center cohorts yielded an age-adjusted diagnostic area under the curve (AUC) of 0.987 (95% confidence interval [CI] = 0.961 to 0.997), superior to that of CA19-9 (AUC = 0.876, 95% CI = 0.847 to 0.905), and a cut-off of 7.18 ng/mL, at which the validation set exhibited a sensitivity of 0.957 and a specificity of 0.924. Importantly, the performance was maintained in early-stage and non-metastatic PC, consistent with the tissue staining. A slightly lower performance against additional diabetic patients (n = 55) was restored by combining asprosin and CA19-9 (AUC = 0.985, 95% CI = 0.975 to 0.995). CONCLUSIONS: Asprosin is presented as an early-stage PC serum marker that may provide clues for PC-induced diabetes. Larger prospective clinical studies are warranted to solidify its utility.

Ablation of USP21 in skeletal muscle promotes oxidative fibre phenotype, inhibiting obesity and type 2 diabetes.

BACKGROUND: Skeletal muscle as a metabolic consumer determines systemic energy homeostasis by regulating myofibre type conversion and muscle mass control. Perturbation of the skeletal muscle metabolism elevates the risk of a variety of diseases including metabolic disorders. However, the regulatory pathways and molecules are not completely understood. The discovery of relevant responsible molecules and the associated network could be an attractive strategy to overcome diseases associated with muscle problems. METHODS: An initial screening using quantitative trait locus analysis enabled us to extract a set of genes including ubiquitin-specific proteases21 (USP21) (r = 0.738; P = 0.004) as potential targets associated with fasting blood glucose content. Given tight regulation of the ubiquitination status of proteins in muscle, we focused on USP21 and generated whole-body (KO) and skeletal muscle-specific USP21 knockout (MKO) mice. Transcriptomics, proteomics, and lipidomics assays in combination with various in vivo and in vitro experiments were performed to understand the functions of USP21 and underlying mechanisms. A high-fat diet (60%)-fed mouse model and diabetic patient-derived samples were utilized to assess the effects of USP21 on energy metabolism in skeletal muscle. RESULTS: USP21 was highly expressed in both human and mouse skeletal muscle, and controlled skeletal muscle oxidative capacity and fuel consumption. USP21-KO or USP21-MKO significantly promoted oxidative fibre type changes (Δ36.6% or Δ47.2%), muscle mass increase (Δ13.8% to Δ22.8%), and energy expenditure through mitochondrial biogenesis, fatty acid oxidation, and UCP2/3 induction (P < 0.05 or P < 0.01). Consistently, cold exposure repressed USP21 expression in mouse skeletal muscle (Δ55.3%), whereas loss of USP21 increased thermogenesis (+1.37°C or +0.84°C; P < 0.01). Mechanistically, USP21 deubiquitinated DNA-PKcs and ACLY, which led to AMPK inhibition. Consequently, USP21 ablation diminished diet-induced obesity (WT vs. USP21-KO, Δ8.02 g, 17.1%, P < 0.01; litter vs. USP21-MKO, Δ3.48 g, 7.7%, P < 0.05) and insulin resistance. These findings were corroborated in a skeletal muscle-specific gene KO mouse model. USP21 was induced in skeletal muscle of a diabetic patient (1.94-fold), which was reciprocally changed to p-AMPK (0.30-fold). CONCLUSIONS: The outcomes of this research provide novel information as to how USP21 in skeletal muscle contributes to systemic energy homeostasis, demonstrating USP21 as a key molecule in the regulation of myofibre type switch, muscle mass control, mitochondrial function, and heat generation and, thus, implicating the potential of this molecule and its downstream substrates network as targets for the treatment and/or prevention of muscle dysfunction and the associated metabolic diseases.

Metformin Decreases 2-HG Production through the MYC-PHGDH Pathway in Suppressing Breast Cancer Cell Proliferation.

The biguanide drug metformin has been widely used for the treatment of type 2 diabetes, and there is evidence supporting the anticancer effect of metformin despite some controversy. Here, we report the growth inhibitory activity of metformin in the breast cancer (MCF-7) cells, both in vitro and in vivo, and the associated metabolic changes. In particular, a decrease in a well-known oncometabolite 2-hydroxyglutarate (2-HG) was discovered by a metabolomics approach. The decrease in 2-HG by metformin was accompanied by the reduction in histone methylation, consistent with the known tumorigenic mechanism of 2-HG. The relevance of 2-HG inhibition in breast cancer was also supported by a higher level of 2-HG in human breast cancer tissues. Genetic knockdown of PHGDH identified the PHGDH pathway as the producer of 2-HG in the MCF-7 cells that do not carry isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) mutations, the conventional producer of 2-HG. We also showed that metformin's inhibitory effect on the PHGDH-2HG axis may occur through the regulation of the AMPK-MYC pathway. Overall, our results provide an explanation for the coherent pathway from complex I inhibition to epigenetic changes for metformin's anticancer effect.