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Proteome and phosphoproteome analysis of brown adipocytes reveals that RICTOR loss dampens global insulin/AKT signaling

ABSTRACT: Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 has been shown to be important for regulating BAT metabolism, yet its mechanism of activation is not known, nor are the identities of its downstream effectors. In this study, we apply proteomics to investigate the role of mTORC2 in brown adipocytes. To assess the role of mTORC2 in brown adipocytes, we compare wild-type controls to isogenic cells with an induced knockout of the mTORC2-specific subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30 minute time course, and measuring the proteomes and phosphoproteomes. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation, suggesting increased interferon-like signaling. We observe significant differences to basal phosphorylation including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells and Rictor-null mouse BAT, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. And finally, we observe a general dampening of the insulin signaling response in Rictor-iKO cells. Some sites exhibit significant dependence on RICTOR, including an AKT substrate site on ATP citrate lyase, which could partially explain the previously-observed RICTOR dependence of de novo lipogenesis from glucose in BAT.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Judit Villen

PROVIDER: MSV000084310 | MassIVE |

REPOSITORIES: MassIVE

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Transplantation of Committed Pre-adipocytes from Brown Adipose Tissue improves Whole-body Glucose Homeostasis

Project description:Obesity and its co-morbidities including type 2 diabetes are increasing at epidemic rates in the U.S. and worldwide. Brown adipose tissue (BAT) is a potential therapeutic to combat obesity and type 2 diabetes. Increasing BAT mass by transplantation improves metabolic health in rodents, but its clinical translation remains a challenge. Here, we investigated if transplantation of 2-4 million differentiated brown pre-adipocytes from mouse BAT stromal fraction (SVF) or human pluripotent stem cells (hPSCs) could improve metabolic health. Transplantation of differentiated brown pre-adipocytes, termed ‘committed pre-adipocytes’ from BAT SVF from mice or derived from hPSCs improves glucose homeostasis and insulin sensitivity in recipient mice under conditions of diet-induced obesity, and this improvement is mediated through the collaborative actions of the liver transcriptome, tissue AKT signaling and FGF21. These data demonstrate that transplantation of a small number of brown adipocytes has significant long-term translational and therapeutic potential to improve glucose metabolism.

2023-12-13 | GSE250078 | GEO

Apoptotic brown adipocytes enhance energy expenditure via SLC29A1-regulated extracellular inosine

Project description:Brown adipose tissue (BAT) dissipates energy and promotes cardio-metabolic health4. However, loss of BAT during obesity and aging is a principal hurdle for BAT-centered obesity therapies. So far not much is known about BAT apoptosis and signals released by apoptotic brown adipocytes. Here, untargeted metabolomics demonstrated that apoptotic brown adipocytes release a specific pattern of metabolites with purine metabolites being highly enriched. Interestingly, this apoptotic secretome enhances expression of the thermogenic program in healthy adipocytes to maintain tissue functionality. This effect is mediated by the purine inosine which stimulates energy expenditure (EE) in brown adipocytes. Phosphoproteomic analysis demonstrated activation of the cAMP/protein kinase A signaling pathway and of pro-thermogenic transcription factors by inosine.

2022-05-31 | PXD032153 | Pride

Uncoupling Metabolic Health from Thermogenesis via BCAA Flux in Brown Fat

Project description:Brown adipose tissue (BAT) is best known for thermogenesis. Whereas numerous studies in rodents found tight associations between the metabolic benefits of BAT and enhanced whole-body energy expenditure, emerging evidence in humans suggests that BAT is protective against Type 2 diabetes independent of body-weight. The underlying mechanism for this dissociation remained unclear. Here, we report that impaired mitochondrial flux of branched-chain amino acids (BCAA) in BAT, by deleting mitochondrial BCAA carrier (MBC, encoded by Slc25a44), was sufficient to cause systemic insulin resistance without affecting whole-body energy expenditure or body-weight. We found that brown adipocytes catabolized BCAAs in the mitochondria as essential nitrogen donors for the biosynthesis of glutamate, N-acetylated amino acids, and one of the products, glutathione. BAT-selective impairment in mitochondrial BCAA flux led to elevated oxidative stress and insulin resistance in the liver, accompanied by reduced levels of BCAA-nitrogen derived metabolites in the circulation. In turn, supplementation of glutathione restored insulin sensitivity of BAT-specific MBC knockout mice. Notably, a high-fat diet rapidly impaired BCAA catabolism and the synthesis of BCAA-nitrogen derived metabolites in the BAT, while cold-induced BAT activity is coupled with an active synthesis of these metabolites. Together, the present work uncovers a mechanism through which brown fat controls metabolic health independent of thermogenesis via BCAA-derived nitrogen carriers acting on the liver.

2024-02-15 | PXD044020 | Pride

Uncoupling Metabolic Health from Thermogenesis via BCAA Flux in Brown Fat

2024-02-15 | PXD043992 | Pride

Bulk RNAseq of Mus musculus Protein Kinase D1 (Prkd1) knock-out adipose tissue in response to thermoneutral and cold exposure stress

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2023-09-01 | E-MTAB-12170 | biostudies-arrayexpress

Next generation sequencing analysis of microRNAs in brown preadipocytes and in vitro differentiated mature brown fat cells

Project description:We performed a genome-wide deep sequencing analysis of the microRNAs abundant in mesenchymal stem cells (MSCs) derived from murine brown adipose tissue and in in vitro differentiated mature brown adipocytes. Several microRNAs were identified as differentially regulated when comparing datasets from MSCs vs. mature fat cells. These microRNAs may have an implication in the regulation of adipogenesis as well as thermogenesis in brown adipose tissue (BAT). Examination of BAT-derived MSCs (BAT-MSC; 1 sample) and in vitro differentiated mature brown fat cells (BAT-DIFF; 1 sample) vertis biotechnologie AG, D-85354 Freising, Germany (library construction and sequencing)

2013-05-28 | E-GEOD-45499 | biostudies-arrayexpress

Coenzyme Q regulates UCP1 expression and thermogenesis through the integrated stress responses

Project description:Coenzyme Q is an essential component of mitochondrial function and required for thermogenic activity in brown adipose tissues (BAT). BAT CoQ deficiency (50-75%) by genetic or pharmacological means does not interfere with basal or maximal mitochondrial respiration in brown adipocytes but increases mitochondrial oxidants and induces UPRmt, ISR, and repression of UCP1 expression. ATF4, the master regulator of ISR, is required for UCP1 suppression in BAT CoQ deficiency. In animals, BAT CoQ deficiency causes cold intolerance but activates compensatory thermogenic mechanisms in BAT and other tissues via greatly induced BAT FGF21 expression resulting in paradoxically upregulated whole-body respiration rates and protection from obesity at room temperature and thermoneutrality. BAT-specific loss of either ATF4 or FGF21 abolishes these metabolic benefits demonstrating a central role for CoQ in the modulation of whole-body energy expenditure and thus for the etiology of primary and secondary CoQ deficiencies.

2022-02-01 | GSE165940 | GEO

PKGI WT vs KO BAT

Project description:Brown adipose tissue (BAT) and brown adipocytes differentiated in vitro from preadipocytes of PKGI-/- mice vs. WT were compared on a whole genome DNA array

2008-12-31 | GSE11890 | GEO

Gene expression data of brown-like adipocytes from Lgr4 homozygous mutant SVF

Project description:Obesity has become a global health problem. Brown adipose tissue (BAT), specialized for energy expenditure through thermogenesis, potently counteracts obesity. Recently, BAT is also identified in human adults. We found that Lgr4 homozygous mutant (Lgr4m/m) mice display reduced adiposity and exhibit brown-like adipocytes in their WAT depots with higher expression of uncoupling protein 1 (Ucp1). Furthermore, Lgr4 ablation potentiates brown adipocyte differentiation from stromal vascular fraction (SVF) of epididymal WAT (eWAT) in vitro. We used microarrays to emxamine the gene expression profiles of the brown-like adipocytes differentiated from SVF of wild-type and Lgr4 mutant mice. We identified distinct gene expression profiles of these two groups. To demonstrate Lgr4 ablation can potentiate the differentiation of SVF from eWAT toward brown-like adipocytes in vitro, we isolated SVF from epididymal white adipose tissue (eWAT) of wild-type (WT) and Lgr4 mutant mice. We then plated SVF cells in 12-well plate, and differentiated them to brown adipocytes, followed by RNA extraction and hybridization with Affymetrix microarrays.

2013-11-07 | E-GEOD-47760 | biostudies-arrayexpress

EPAC1 enhances energy expenditure by selectively inducing brown and beige adipose tissue growth

Project description:Brown adipose tissue (BAT) is a central thermogenic organ that enhances energy expenditure (EE) and cardiometabolic health. However, regulators that specifically increase the number of thermogenic adipocytes are still an unmet need. Here, we show by phosphoproteomics that cAMP activates distinct signaling pathways in brown progenitors, with the cAMP-EPAC1 axis enhancing proliferation and differentiation of thermogenic but not white adipocytes. Further analysis revealed that a specific subpopulation of preadipocytes that are PDGFRα-positive express EPAC1. In vivo, pharmacological activation of EPAC1 enhances BAT growth and browning of white fat, leading to increased EE and reduced diet-induced adiposity. In contrast, mice lacking EPAC1 in PDGFRα-positive preadipocytes show the opposite phenotype. Importantly, EPAC1 activation enhances proliferation and differentiation of human brown adipocytes and human brown fat organoids. Interestingly, a coding variant in EPAC1 that positively correlates with BMI abolishes norepinephrine-induced proliferation of brown adipocytes. Thus, EPAC1 might be an attractive target to enhance thermogenic adipocyte number and EE to combat metabolic diseases.

2023-11-11 | PXD046709 | Pride

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