Metabolism
Abstract
Therapeutics blocking PI3K/mTOR complex 1 (mTORC1) are commonly used for tumor treatment, and at times achieve major responses, yet minimal residual disease (MRD) persists, leading to tumor relapse. We developed multiple MRD models both in vitro (rapamycin persistent, RP) and in vivo after mTORC1 inhibition. All 11 RP/MRD cell lines showed complete growth and signaling insensitivity to rapamycin but variable sensitivity to bi-steric mTORC1 inhibitors, with MtorS2035 mutations identified in 4 of 7 RP cell lines. Multiomic analyses identified a pronounced shift toward oxidative phosphorylation and away from glycolysis with increased mitochondrial number in all RP/MRD models. MYC and SWI/SNF expression was significantly enhanced. Both the SWI/SNF inhibitor AU-15330 and the mitochondrial complex I oxidative phosphorylation inhibitor IACS-010759 showed pronounced synergy with bi-steric mTORC1 inhibitors to cause cuproptotic cell death in RP/MRD cells, suggesting these combinations as a potential patient treatment strategy for rapalog resistance.
Authors
Heng Du, Heng-Jia Liu, Magdalena Losko, Yu Chi Yang, Min Yuan, Elizabeth P. Henske, John M. Asara, Mallika Singh, David J. Kwiatkowski
Abstract
Processes that promote white adipocyte inflammatory function remain incompletely defined. Here, we demonstrated that type I interferon–dependent (IFN-I–dependent) skewing of adipocyte glycolysis, nicotinamide adenine dinucleotide (NAD+) utilization, and pyruvate kinase isozyme M2 (PKM2) function may contribute to increased systemic and tissue inflammation and disease severity in obesity. Notably, chemical and/or genetic inhibition of glycolysis, the NAD+ salvage pathway, or PKM2 restricted IFN-I–dependent increase in adipocyte inflammatory cytokine production. Further, genetic or small molecule targeting of PKM2 function in vivo was sufficient to reduce systemic and tissue inflammation and metabolic disease severity in obese mice, in an adipocyte PKM2-dependent manner. Further, white adipose tissue of individuals living with obesity and metabolic disease, compared with metabolically healthy individuals with obesity, showed an increase in expression of inflammatory and metabolic genes, while small molecule targeting of PKM2 function contributed to reduced IFN-I–driven inflammatory cytokine production by primary human adipocytes. Together, our findings invoke the IFN-I/PKM2 axis as a potential target for modulating adipocyte dysregulated inflammation.
Authors
Michelle S.M.A. Damen, Pablo C. Alarcon, Calvin C. Chan, Traci E. Stankiewicz, Hak Chung, Keisuke Sawada, Cassidy J. Ulanowicz, John Eom, Jarren R. Oates, Jennifer L. Wayland, Jessica R. Doll, Rajib Mukherjee, Miki Watanabe-Chailland, Lindsey Romick-Rosendale, Sara Szabo, Michael A. Helmrath, Joan Sanchez-Gurmaches, Maria E. Moreno-Fernandez, Senad Divanovic
Abstract
Insulin/insulin growth factor signaling is a conserved pathway that regulates lifespan. Yet, long-lived loss-of-function mutants often produce insulin-resistance, slow growth, and impair reproduction. Recently, a gain-of-function mutation in the kinase insert domain (KID) of the Drosophila insulin/IGF receptor was seen to dominantly extend lifespan without impairing insulin-sensitivity, growth and reproduction. This substitution occurs within residues conserved in mammalian insulin receptor (IR) and insulin growth factor-1 receptor (IGF-1R). We produced two knock-in mouse strains that carry the homologous KID Arg/Cys substitution in murine IR or IGF-1R, and we replicated these genotypes in human cells. Cells with heterodimer receptors of IR or IGF-1R induce receptor phosphorylation and phospho-Akt when stimulated with insulin or IGF. Heterodimer receptors of IR fully induce pERK but ERK was less phosphorylated in cells with IGF-1R heterodimers. Adults with a single KID allele (producing heterodimer receptors) have normal growth and glucose regulation. At four months, these mice variably display hormonal markers that associate with successful aging counteraction, including elevated adiponectin, FGF21, and reduced leptin and IGF-1. Livers of IGF-1R females show decreased transcriptome-based biological age, which may point toward delayed aging and warrants an actual lifespan experiment. These data suggest that KID mutants may slow mammalian aging while they avoid the complications of insulin resistance.
Authors
Ulalume Hernández-Arciga, Jun Kyoung Kim, Jacob L. Fisher, Alexander Tyshkovskiy, Alibek Moldakozhayev, Catherine Hall, Souvik Ghosh, Yashvandhini Govindaraj, Ian J. Sipula, Jake Kastroll, Diana Cooke, Jinping Luo, Jonathan K. Alder, Stacey J. Sukoff Rizzo, Gene P. Ables, Eunhee Choi, Vadim N. Gladyshev, Michael J. Jurczak, Marc Tatar, Andrey A. Parkhitko
Abstract
The present study aims to explore the role and possible underlying mechanisms of histone lactylation modifications in diabetes-associated cognitive impairment (DACD). In this study, behavioral tests, Hematoxylin & Eosin (HE) staining, and immunohistochemistry were used to evaluate cognitive function and the extent of cerebral tissue injury. We quantified the levels of lactic acid and Pan-lysine lactylation (Pan Kla) in the brains of type 2 diabetes mellitus (T2DM) mice and in high glucose–treated microglia. We also identified all Kla sites in isolated microglia. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were subsequently conducted to identify the functions and pathways that were enriched at the differentially expressed modification sites. cleavage under targets and tagmentation (CUT&Tag) technology was used to identify candidate genes that are regulated by H3K18la. Small interfering RNA (siRNA) and H3K18R mutant sequences were used to knock down crucial components in key signaling pathways to assess the effects of histone lactylation on microglial polarization. We found that lactic acid levels were significantly greater in the brains of T2DM mice and high glucose-treated microglia than in those of their corresponding controls, which increased the level of Pan-Kla. We discovered that lactate can directly stimulate an increase in H3K18la. The global landscape of the lactylome reveals information about modification sites, indicating a correlation between the upregulation of H3K18la and protein lactylation and Toll-like receptor signaling. CUT&Tag demonstrated that enhanced H3K18la directly stimulates the nuclear factor kappa-B (NF-κB) signaling pathway by increasing binding to the promoter of Toll Like Receptor 4 (TLR4), thereby promoting M1 microglial polarization. The present study demonstrated that enhanced H3K18la directly stimulates TLR4 signaling to promote M1 microglial polarization, thereby facilitating DACD phenotypes. Targeting such loop may be a potential therapeutic approach for the treatment of DACD.
Authors
Ying Yang, Fei Chen, Lulu Song, Liping Yu, Jinping Zhang, Bo Zhang
Abstract
X-linked myotubular myopathy (XLMTM) due to MTM1 mutations is a rare and often lethal congenital myopathy. Its downstream molecular and cellular mechanisms are currently incompletely understood. The most abundant protein in muscle, myosin, has been implicated in the pathophysiology of other congenital myopathies. Hence, in the present study, we aimed to define whether myosin is also dysfunctional in XLMTM and whether it thus may constitute a potential drug target. To this end, we used skeletal muscle tissue from human patients and canine/mouse models; we performed Mant-ATP chase experiments coupled with X-ray diffraction analyses and LC/MS-based proteomics studies. In XLMTM humans, we found that myosin molecules are structurally disordered and preferably adopt their ATP-consuming biochemical state. This phosphorylation-related (mal)adaptation was mirrored by a striking remodelling of the myofibre energetic proteome in XLMTM dogs. In line with these, we confirmed an accrued myosin ATP consumption in mice lacking MTM1. Hence, we treated these, with a myosin ATPase inhibitor, mavacamten. After a four-week treatment period, we observed a partial restoration of the myofibre proteome, especially proteins involved in cytoskeletal, sarcomeric and energetic pathways. Altogether, our study highlights myosin inhibition as a new potential drug mechanism for the complex XLMTM muscle phenotype.
Authors
Elise Gerlach Melhedegaard, Fanny Rostedt, Charlotte Gineste, Robert A.E. Seaborne, Hannah F. Dugdale, Vladimir Belhac, Edmar Zanoteli, Michael W. Lawlor, David L. Mack, Carina Wallgren-Pettersson, Anthony L. Hessel, Heinz Jungbluth, Jocelyn Laporte, Yoshihiko Saito, Ichizo Nishino, Julien Ochala, Jenni Laitila
Abstract
Impaired cardiac lipid metabolism has been reported to cause heart failure. Lipin1, a multifunctional protein, is a phosphatidate phosphatase that generates diacylglycerol from phosphatidic acid and a transcriptional cofactor that regulates lipid metabolism-related gene expression. Here, we investigated the roles of lipin1 in cardiac remodeling after myocardial infarction (MI). The expression levels of lipin1 significantly decreased in cardiomyocytes of the human failing heart and murine ischemic myocardium. Cardiomyocyte-specific Lpin1 knockout (cKO) mice showed left ventricle enlargement and reduced fractional shortening after MI, compared to control mice. This was accompanied by elevated cardiac fibrosis, accumulation of reactive oxygen species, and increased expression of inflammatory cytokines. In contrast, cardiomyocyte-specific Lpin1 overexpression (cOE) mice showed reduced fibrosis and inflammation and improved cardiac function compared to control mice. Cardiac lipid droplets (LDs) were reduced after MI in wild-type (WT) mice hearts and were further downregulated in the hearts of cKO mice with a decrease in triglyceride and free fatty acid content, while cOE mice hearts exhibited increased LDs and lipid content. Expression levels of genes involved in fatty acid oxidation, such as Ppargc1a (PGC1A) and Acaa2, were decreased and increased in the MI hearts of cKO mice and cOE mice, respectively. These results suggest the protective role of lipin1 against ischemic injury by maintaining lipid metabolism in ischemic cardiomyocytes.
Authors
Jiaxi Guo, Kohei Karasaki, Kazutaka Ueda, Manami Katoh, Masaki Hashimoto, Toshiyuki Ko, Masato Ishizuka, Satoshi Bujo, Chunxia Zhao, Risa Kishikawa, Haruka Yanagisawa-Murakami, Hiroyuki Sowa, Bowen Zhai, Mutsuo Harada, Seitaro Nomura, Norihiko Takeda, Brian N. Finck, Haruhiro Toko, Issei Komuro
Abstract
Clear cell renal cell carcinomas (ccRCC) are largely driven by HIF2α and are avid consumers of glutamine. However, inhibitors of glutaminase1 (GLS1), the first step in glutaminolysis, have not shown benefit in phase III trials, and HIF2α inhibition, recently FDA-approved for treatment of ccRCC, shows significant but incomplete benefits. This highlights the need to better understand the interplay between glutamine metabolism and HIF2α in ccRCC. Here, we report that glutamine deprivation rapidly redistributes GLS1 into isolated clusters within mitochondria in diverse cell types, but not in ccRCC. GLS1 clustering occurs rapidly within 1 to 3 hours, is reversible, is specifically triggered by reduced intracellular glutamate, and is dependent on mitochondrial fission. Clustered GLS1 markedly enhances glutaminase activity and promotes cell death under glutamine-deprived conditions. HIF2α prevents GLS1 clustering, independently of its transcriptional activity, thereby maintaining low GLS activity and protecting ccRCC cells from glutamine deprivation-induced cell death. Forced clustering of GLS1, using constitutively clustering mutants, restores high GLS activity, promotes apoptosis, and suppresses ccRCC tumor growth in vivo. These findings reveal multiple insights into cellular glutamine handling, including a previously unrecognized process by which HIF2α promotes ccRCC: by suppressing GLS1 clustering and maintaining low GLS activity. This mechanism provides a potential explanation for the lack of clinical efficacy of GLS inhibitors in ccRCC and suggests a therapeutic avenue to combine HIF2α inhibition with strategies that restore GLS1 clustering.
Authors
Wencao Zhao, Sara M. Demczyszyn, Nathan J. Coffey, Yanqing Jiang, Boyoung Kim, Schuyler Bowers, Caitlyn E. Bowman, Michael C. Noji, Cholsoon Jang, M. Celeste Simon, Zoltan Arany, Boa Kim
Abstract
Angiopoietin-like 3 (ANGPTL3) is a major regulator of lipoprotein metabolism. ANGPTL3 deficiency results in lower levels of triglycerides, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C), and may protect from cardiovascular disease. ANGPTL3 oligomerizes with ANGPTL8 to inhibit lipoprotein lipase (LPL), the enzyme responsible for plasma triglyceride hydrolysis. Independent of ANGPTL8, oligomers of ANGPTL3 can inhibit endothelial lipase (EL), which regulates circulating HDL-C and LDL-C levels through the hydrolysis of lipoprotein phospholipids. The N-terminal region of ANGPTL3 is necessary for both oligomerization and lipase inhibition. However, our understanding of the specific residues that contribute to these functions is incomplete. In this study, we performed mutagenesis of the N-terminal region to identify residues important for EL inhibition and oligomerization. We also assessed the presence of different ANGPTL3 species in human plasma. We identified a motif important for lipase inhibition, and protein structure prediction suggested that this region interacted directly with EL. We also found that recombinant ANGPTL3 formed a homotrimer and was unable to inhibit EL activity when trimerization was disrupted. Surprisingly, we observed that human plasma contained more monomeric ANGPTL3 than trimeric ANGPTL3. An important implication of these findings is that previous correlations between circulating ANGPTL3 and circulating triglyceride-rich lipoproteins need to be revisited.
Authors
Sydney G. Walker, Yan Q. Chen, Kelli L. Sylvers-Davie, Alex Dou, Eugene Y. Zhen, Yuewei Qian, Yi Wen, Mariam E. Ehsani, Sydney A. Smith, Rakshya Thapa, Maxwell J. Mercer, Lucy Langmack, Bharat Raj Bhattarai, Michael Ploug, Robert J. Konrad, Brandon S.J. Davies
Abstract
Recent evidence suggests that cellular metabolism, including glycolysis and fatty acid synthesis in lymphatic endothelial cells (LECs), plays essential roles in developing functional lymphatic systems. Site-1 protease (S1P) proteolytically activates membrane-bound latent transcription factor sterol regulatory element-binding proteins (SREBPs), which are required to induce lipid biosynthesis. In this study, we generated mice with pan-endothelial or LEC-specific deficiency of either S1P or SREBP2. Mouse embryos with pan-endothelial deletion of S1P showed defective lymphatic vessel migration in skin and lymphedema, while their blood vasculature formation was relatively normal. Mice lacking S1P in LECs or SREBP2 in LECs exhibited chylous ascites, reduced lipogenic gene expression, and reduced VEGFR3 expression and progressively developed wasting, resulting in postnatal death by approximately 8 weeks of age. Additionally, mice with SREBP2 deletion in LECs exhibited dilated lacteal and mesenteric lymphatics and accumulation of lipids in the lacteal before weaning age, indicating apparent lymphatic malfunctioning. These data indicate that S1P-SREBP2–mediated cholesterol biosynthesis is pivotal in lymphatic vascular development. We also found that treating human dermal LECs with VEGF-C induced proteolytic activation of SREBP2 with concomitant phosphorylation of Akt and the expression of genes involved in cholesterol biosynthesis. Those effects were canceled out by treating the cells with an S1P inhibitor or SREBP inhibitor. These data demonstrate that the S1P/SREBP2 axis is critical in VEGF-C/VEGFR3 mitogenic signaling in LECs.
Authors
Yuji Kondo, Yizhi Jiang, Xin Geng, Jianhua Song, Summer Simeroth, J. Michael McDaniel, Pengchun Yu, R. Sathish Srinivasan, Lijun Xia
Abstract
Poor skeletal muscle fitness contributes to many chronic disease states including obesity, heart failure, primary muscle disorders, and age-related sarcopenia. Receptor Interacting Protein 140 (RIP140) is a striated muscle-enriched nuclear receptor coregulator known to suppress mitochondrial oxidative capacity. To investigate the role of RIP140 in skeletal muscle, striated muscle-specific RIP140-deficient (strNrip1-/-) mice were generated and characterized. strNrip1-/- mice displayed an enhanced endurance performance phenotype. RNA-sequence (RNA-seq) analysis of glycolytic fast-twitch muscle from strNrip1-/- mice identified a broad array of differentially upregulated metabolic and structural muscle genes known to be induced by endurance training, including pathways involved in mitochondrial biogenesis and respiration, fatty acid oxidation, slow muscle fiber type, and angiogenesis. In addition, muscle RIP140-deficiency induced expansive neuromuscular junction (NMJ) remodeling. Integration of RNA sequence results with CUT&RUN analysis of strNrip1-/- myotubes identified Wnt16 as a candidate effector for the NMJ biogenesis in RIP140-deficient skeletal myotubes. We conclude that RIP140 serves as a physiological “rheostat” for a broad coordinated network of metabolic and structural genes involved in skeletal muscle fitness.
Authors
Elizabeth Pruzinsky, Kirill Batmanov, Denis M. Medeiros, Sarah M. Sulon, Brian P. Sullivan, Tomoya Sakamoto, Teresa C. Leone, Tejvir S. Khurana, Daniel P. Kelly
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