Metabolism
Abstract
Methylmalonic acidemia (MMA) is a severe metabolic disorder affecting multiple organs because of a distal block in branched-chain amino acid (BCAA) catabolism. Standard of care is limited to protein restriction and supportive care during metabolic decompensation. Severe cases require liver/kidney transplantation, and there is a clear need for better therapy. Here, we investigated the effects of a small molecule branched-chain amino acid transaminase (BCAT) inhibitor in human MMA hepatocytes and an MMA mouse model. Mitochondrial BCAT is the first step in BCAA catabolism, and reduction of flux through an early enzymatic step is successfully used in other amino acid metabolic disorders. Metabolic flux analyses confirmed robust BCAT inhibition, with reduction of labeling of proximal and distal BCAA-derived metabolites in MMA hepatocytes. In vivo experiments verified the BCAT inhibition, but total levels of distal BCAA catabolite disease markers and clinical symptoms were not normalized, indicating contributions of substrates other than BCAA to these distal metabolite pools. Our study demonstrates the importance of understanding the underlying pathology of metabolic disorders for identification of therapeutic targets and the use of multiple, complementary models to evaluate them.
Authors
Madeline G. Hemmingsen, Guo-Fang Zhang, Yunhan Ma, Hannah Marchuk, Kalyani R. Patel, Tong Chen, Xinning Li, Mark Chapman, Sabrina Collias, Dolores H. Lopez-Terrada, James Beasley, Ashlee R. Stiles, Randy J. Chandler, Charles P. Venditti, Sarah P. Young, Mercedes Barzi, Beatrice Bissig-Choisat, Doug Krafte, Christopher B. Newgard, Karl-Dimiter Bissig
Abstract
Impaired muscle regrowth in aging is underpinned by reduced pro-inflammatory macrophage function and subsequently impaired muscle cellular remodeling. Macrophage phenotype is metabolically controlled through TCA intermediate accumulation and activation of HIF1A. We hypothesized that transient hypoxia following disuse in old mice would enhance macrophage metabolic inflammatory function thereby improving muscle cellular remodeling and recovery. Old (20 months) and young adult mice (4 months) were exposed to acute (24h) normobaric hypoxia immediately following 14-days of hindlimb unloading and assessed during early re-ambulation (4- and 7-days) compared to age-matched controls. Treated aged mice had improved pro-inflammatory macrophage profiles, muscle cellular remodeling, and functional muscle recovery to the levels of young control mice. Likewise, young adult mice had enhanced muscle remodeling and functional recovery when treated with acute hypoxia. Treatment in aged mice restored the muscle molecular fingerprint and biochemical spectral patterns (Raman Spectroscopy) observed in young mice and strongly correlated to improved collagen remodeling. Finally, intramuscular delivery of hypoxia-treated macrophages recapitulated the muscle remodeling and recovery effects of whole-body hypoxic exposure in old mice. These results emphasize the role of pro-inflammatory macrophages during muscle regrowth in aging and highlight immunometabolic approaches as a route to improve muscle cellular dynamics and regrowth.
Authors
Zachary J. Fennel, Negar Kosari, Paul-Emile Bourrant, Elena M. Yee, Robert J. Castro, Anu S. Kurian, Jonathan Palmer, Morgan Christensen, Katsuhiko Funai, Ryan M. O'Connell, Anhong Zhou, Micah J. Drummond
Abstract
In vitro studies have implicated orphan receptor GPRC5B in β-cell survival, proliferation and insulin secretion, but its relevance for glucose homeostasis in vivo is largely unknown. Using tamoxifen-inducible, β-cell-specific GPRC5B knockout mice (Ins-G5b-KOs) we show here that loss of GPRC5B does not affect β-cell function in the lean state, but results in strongly reduced insulin secretion and disturbed glucose tolerance in mice subjected to high fat diet for 16 weeks. Flow cytometry and single-cell expression analyses in islets from obese mice show a reduced β-cell abundance and a less mature β-cell phenotype in Ins-G5b-KOs. Expression of β-cell-specific transcription factor MafA is reduced both on the RNA and protein level, as are transcripts of MafA target genes. Mechanistically, we show that phosphorylation of cAMP response element-binding protein (CREB), a major regulator of MafA expression, is reduced in islets of obese Ins-G5b-KOs, and that this phenotype precedes the downregulation of MafA and MafA target genes. Taken together, GPRC5B helps to maintain mature β-cell function in obesity through cAMP/CREB-dependent regulation of MafA expression.
Authors
Tianpeng Wang, Remy Bonnavion, Janett Piesker, Stefan Günther, Nina Wettschureck
Abstract
Adipose inflammation plays a key role in obesity-induced metabolic abnormalities. Epigenetic regulation, including DNA methylation, is a molecular link between environmental factors and complex diseases. Here we found that high fat diet (HFD) feeding induced a dynamic change of DNA methylome in mouse white adipose tissue (WAT) analyzed by reduced representative bisulfite sequencing. Interestingly, DNA methylation at the promoter of estrogen receptor α (Esr1) was significantly increased by HFD, concomitant with a down-regulation of Esr1 expression. HFD feeding in mice increased the expression of DNA methyltransferase 1 (Dnmt1) and Dnmt3a, and binding of DNMT1 and DNMT3a to Esr1 promoter in WAT. Mice with adipocyte-specific Dnmt1 deficiency displayed increased Esr1 expression, decreased adipose inflammation and improved insulin sensitivity upon HFD challenge; while mice with adipocyte-specific Dnmt3a deficiency showed a mild metabolic phenotype. Using a modified CRISPR/RNA-guided system to specifically target DNA methylation at the Esr1 promoter in WAT, we found that reducing DNA methylation at Esr1 promoter increased Esr1 expression, decreased adipose inflammation and improved insulin sensitivity in HFD-challenged mice. Our study demonstrated that DNA methylation at Esr1 promoter played an important role in regulating adipose inflammation, which may contribute to obesity-induced insulin resistance.
Authors
Rui Wu, Fenfen Li, Shirong Wang, Jia Jing, Xin Cui, Yifei Huang, Xucheng Zhang, Jose A. Carrillo, Zufeng Ding, Jiuzhou Song, Liqing Yu, Huidong Shi, Bingzhong Xue, Hang Shi
Abstract
T cells rely on different metabolic pathways to differentiate into effector or memory cells, and metabolic intervention is a promising strategy to optimize T cell function for immunotherapy. Pyruvate dehydrogenase (PDH) is a nexus between glycolytic and mitochondrial metabolism, regulating pyruvate conversion to either lactate or acetyl-CoA. Here, we retrovirally transduced pyruvate dehydrogenase kinase 1 (PDK1) or pyruvate dehydrogenase phosphatase 1 (PDP1), which control PDH activity, into CD8+ T cells to test effects on T cell function. Although PDK1 and PDP1 were expected to influence PDH in opposing directions, by several criteria they induced similar changes relative to control T cells. Seahorse metabolic flux assays showed both groups exhibited increased glycolysis and oxidative phosphorylation. Both groups had improved primary and memory recall responses following infection with murine gammaherpesvirus-68. However, metabolomics using labeled fuels indicated differential usage of key fuels by metabolic pathways. Importantly, CD8+ T cell populations after B cell lymphoma challenge were smaller in both groups, resulting in poorer protection, which was rescued by glutamine and acetate supplementation. Overall, this study indicates that PDK1 and PDP1 both enhance metabolic capacity, but the context of the antigenic challenge significantly influences the consequences for T cell function.
Authors
Taewook Kang, Young-Kwang Usherwood, Julie A. Reisz, Sukrut C. Kamerkar, Rachel Culp-Hill, Owen M. Wilkins, Andreia F. Verissimo, Fred W. Kolling IV, Anton M. Hung, Shawn C. Musial, Pamela C. Rosato, Angelo D’Alessandro, Henry N. Higgs, Edward J. Usherwood
Abstract
Transforming growth factor beta (TGF-β) signaling is the master modulator of renal fibrosis. However, targeting drugs have failed to prevent the progression of chronic kidney disease (CKD) in clinical trials due to the extensive biological regulation of TGF-β signaling. It is necessary to investigate the precise downstream mechanisms of TGF-β signaling that regulate renal fibrosis. In this study, we found that PR-domain containing 16 (PRDM16) expression in human renal tubular epithelial cells was markedly reduced by TGF-β. Mechanistically, activated Smad3 induced by TGF-β interacted with the cofactor H-Ras and bound to the promoter of PRDM16, downregulating its transcription. Tubular-specific knockout of PRDM16 promoted renal fibrosis in models of unilateral ureteral occlusion (UUO) and unilateral ischemia-reperfusion injury (UIRI) by exacerbating mitochondrial dysfunction. In vitro, PRDM16 blocked TGF-β-induced mitochondrial injury and lipid deposition by upregulating Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1α (PGC-1α). Delivery of the exogenous PRDM16 gene preserved renal function and ameliorated the progression of renal fibrosis by protecting mitochondrial function. We report PRDM16 as a novel downstream target of TGF-β signaling that attenuates renal fibrosis by safeguarding tubular mitochondrial function.
Authors
Qian Yuan, Ben Tang, Yuting Zhu, Chao Wan, Yaru Xie, Yajuan Xie, Cheng Wan, Hua Su, Youhua Liu, Chun Zhang
Abstract
Cellular metabolism plays a key role in T cell biology. Increased glycolysis and mitochondrial respiration have been identified in CD4+ helper T cells from both patients with systemic lupus erythematosus (SLE) and lupus mouse models. Inhibiting this metabolic activity can reduce T cell activation and ameliorate disease symptoms in lupus mice. However, the metabolic differences among circulating follicular helper T (cTfh) cell subsets in patients with SLE versus healthy controls (HCs) have not been thoroughly studied. While the frequencies of cTfh cells and their subsets were similar between patients with SLE and HCs, patients exhibited a higher proportion of activated ICOS+ programmed cell death 1–positive cells, which correlated with disease activity. cTfh17 cells from both patients with SLE and HCs demonstrated heightened glycolytic activity and expression of glycolysis-related genes compared with cTfh1 and cTfh2. Glucose deprivation significantly diminished costimulatory molecule expression and cytokine production, including IL-17A, IL-10, IL-2, and TNF-α. Glycolysis inhibition reduced the B cell activation capacity of cTfh17 cells. This glucose dependence was more pronounced in cTfh17 than cTfh2 from patients with SLE, but it similarly affected both cTfh2 and cTfh17 cells from HCs. These findings highlight distinct metabolic dependencies among cTfh subsets and the critical role of glycolysis in cTfh17-mediated B cell activation in SLE.
Authors
Vera Kim, Takaya Misao, Hong Tian, Meggan Mackay, Cynthia Aranow, Sun Jung Kim
Abstract
Psoriasis is a chronic autoimmune skin disease characterized by abnormal keratinocyte proliferation and immune dysregulation. Altered lipid metabolism has been implicated in its pathogenesis, but the underlying mechanisms remain unclear. In this study, we generated an keratinocyte-specific Sprouty RTK signaling antagonist 1 (SPRY1) knockout (Spry1ΔEpi) mouse model, which exhibits psoriasis-like symptoms. Using both psoriasis patient samples and Spry1ΔEpi mice, we investigated the role of diacylglycerol acyltransferase 2 (DGAT2) in psoriasis. Our results show that DGAT2 expression is reduced, and glycerides metabolism is disrupted in psoriatic lesions in both psoriasis patients and Spry1ΔEpi mice. Lipidomic analysis reveals significant alterations in glycerides, glycerophospholipids, sphingolipids, and fatty acids in Spry1ΔEpi mice. At the cellular level, DGAT2 downregulation and lipid dysregulation enhance Toll-like receptor 3 (TLR3)-mediated inflammatory signaling in keratinocytes. Furthermore, increased DGAT2 secretion from keratinocytes promotes CD8⁺ T cell activation, proliferation and survival, amplifying psoriatic inflammation. These findings highlight the role of DGAT2 and lipid metabolism in the pathogenesis of psoriasis and reveal their interaction with immune responses in psoriasis.
Authors
Ying-Ying Li, Li-Ran Ye, Ying-Zhe Cui, Fan Xu, Xi-Bei Chen, Feng-Fei Zhang, Yi Lu, Yu-Xin Zheng, Xiao-Yong Man
Chronic integrated stress response causes dysregulated cholesterol synthesis in white matter disease
Abstract
Maladaptive integrated stress response (ISR) activation is observed in human diseases of the brain. Genetic mutations of eIF2B, a critical mediator of protein synthesis, cause chronic pathway activation resulting in a leukodystrophy but the precise mechanism is unknown. We generated N208Y eIF2Bα mice and found that this metabolite binding mutation leads to destabilization of eIF2Bα, a systemic ISR, and neonatal lethality. 2BAct, an eIF2B activator, rescued lethality and significantly extended the lifespan of this severe model, underscoring its therapeutic potential in pediatric disease. Continuous treatment was required for survival, as withdrawal led to ISR induction in all tissues and rapid deterioration, thereby providing a model to assess the impact of the ISR in vivo by tuning drug availability. Single nuclei RNA-sequencing of the CNS identified astrocytes, oligodendrocytes, and ependymal cells as the cell types most susceptible to eIF2B dysfunction and revealed dysfunctional maturation of oligodendrocytes. Moreover, ISR activation decreased cholesterol biosynthesis, a process critical for myelin formation and maintenance. As such, persistent ISR engagement may contribute to pathology in other demyelinating diseases.
Authors
Karin Lin, Nina Ly, Rejani B. Kunjamma, Ngoc Vu, Bryan King, Holly M. Robb, Eric G. Mohler, Janani Sridar, Qi Hao, José Zavala-Solorio, Chunlian Zhang, Varahram Shahryari, Nick van Bruggen, Caitlin F. Connelly, Bryson D. Bennett, James J. Lee, Carmela Sidrauski
Abstract
Autophagy is a catabolic quality control pathway that has been linked to neurodegenerative disease, atherosclerosis and ageing, and can be modified by nutrient availability in preclinical models. Consequently, there is immense public interest in stimulating autophagy in people. However, progress has been hampered by the lack of techniques to measure human autophagy. As a result, several key concepts in the field, including nutritional modulation of autophagy, have yet to be validated in humans. We conducted a single arm pre-post study in 42 healthy individuals, to assess whether an acute nutritional intervention could modify autophagy in humans. Two blood samples were collected per participant: after a 12 h overnight fast and 1 h post-consumption of a high protein meal. Autophagy turnover was assessed using a physiologically relevant measure of autophagic flux in peripheral blood mononuclear cells. A lysosomal inhibitor was added directly to whole blood, with the resulting build-up of autophagy marker LC3B-II designated as flux, and measured quantitatively via ELISA. Notably, consumption of a high protein meal had no impact on autophagy, with no differences between overnight fasting and postprandial autophagic flux. We observed sexual dimorphism in autophagy, with females having higher autophagic flux compared to males (p = 0.0031). Exploratory analyses revealed sex-specific correlations between autophagy, insulin and glucose signalling. Importantly, our findings show that an acute nutritional intervention (overnight fasting followed by consumption of a protein-rich meal) does not change autophagic flux in humans, highlighting the need to conduct further autophagy studies in humans.
Authors
Sanjna Singh, Célia Fourrier, Kathryn J. Hattersley, Leanne K. Hein, Jemima Gore, Alexis Martin, Linh V.P. Dang, Barbara King, Rachael A. Protzman, Paul J. Trim, Leonie K. Heilbronn, Julien Bensalem, Timothy J. Sargeant
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