Lipids play an essential role in physiology. In addition to serving as an energy source, bioactive lipids, also known as lipokines, function as signaling molecules regulating metabolism and inflammation. A major source of lipokines, brown adipose tissue (BAT) has been shown to have beneficial effects on cardiometabolic health by increasing energy expenditure and improving glucose tolerance, thus providing a potential target for therapeutic interventions. Recent studies have identified 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME, produced by cytochrome P450-epoxide hydrolase metabolism of linoleic acid (C18:2n-6), as a lipokine produced by BAT that modulates fatty acid uptake (FAU) into brown adipocytes and skeletal muscle cells in response to cold stimulation or exercising, respectively. Here, we characterized the molecular mechanism and structure-activity relationship (SAR) of 12,13-diHOME in BAT fatty acid uptake. Conversion of the terminal carboxylic acid of 12,13-diHOME to ethyl acetate and changes in saturation results in decreased stimulated FAU indicating 12,13-diHOME's activity is structurally dependent. In addition, we provide insight into a novel protein target responsible for enhancing FAU in BAT, in part, by enhancing intracellular calcium mobilization and influencing the expression of lipolytic genes. Furthermore, we have determined that the 12,13-diHOME-stimulated FAU is, in part, dependent on Gq-mediated calcium mobilization as determined by the utilization of Gq inhibitor YM-254890. Thus, the receptor of 12,13-diHOME could represent a target to augment BAT activity by modulating fatty acid metabolism, which could contribute to increased energy expenditure and, consequently, to the prevention of cardiometabolic diseases. Disclosure J.I.Senfeld: None. M.Lynes: None. S.Kodani: None. K.Lee: None. Y.Tseng: Consultant; Cellarity, LyGenesis.
Brown adipocytes hold the potential to mitigate body fat accumulation and type 2 diabetes (T2D) via their specialized function in dissipating energy as heat, a process known as thermogenesis. To fuel their function, brown adipocytes release the bond energy of fat through lipolysis and β-oxidation; then, uncoupling cellular respiration releases this energy as physical heat. In addition to their energy-dissipating function, brown adipocytes can release factors, such as lipids, metabolites, peptides, or microRNA, to regulate metabolism. Signaling lipids have demonstrated roles in regulating thermogenesis in brown adipocytes by regulating lipid uptake and utilization. To further explore these pathways, we performed signaling lipidomics analysis in in vitro differentiated human brown adipocytes and found 15-keto-PGF2α was increased when treated with forskolin, an adenylyl cyclase activator that mimics adrenergic stimulation. Previous studies have shown PGF2α, the parent molecule of 15-keto-PGF2α, inhibits maturation, or adipogenesis, in white adipocyte cell lines by preventing the expression of key adipogenesis genes, such as PPARγ and C/EBPα. Similarly, PGF2α inhibits thermogenic gene expression in brown adipocyte cells. To explore and compare the functional activities of PGF2α and 15-keto-PGF2α, we characterized the dose-response relationship of these lipids on thermogenic gene and protein expression. We found that 15-keto-PGF2α is not as potent as PGF2α at inhibiting both thermogenic gene and protein expression. This suggests the conversion of PGF2α to 15-keto-PGF2α represents a significant signaling event to promote thermogenesis in brown adipocytes. Interestingly, carbonyl reductase 1, an enzyme that catalyzes this conversion, is regulated by treatment with the adrenergic agonist, norepinephrine, in vitro and by cold exposure in vivo. Targeting this conversion may be an attractive approach towards activating thermogenesis to mitigate obesity and T2D. Disclosure E.Fadumiye: None. S.Kodani: None. M.Kiebish: None. V.Bussberg: None. Y.Tseng: Consultant; Cellarity. Funding National Institutes of Health (R01DK122808)
White adipose tissue is characterized by substantial functional heterogeneity based on anatomic location and developmental lineages. Such differences may contribute to depot-dependent differences in adipose regulation of systemic metabolism and the relative impact of adipose distribution (e.g. subcutaneous, SQ, vs. intraabdominal, IA) on risk for type 2 diabetes. While mouse adipose depot heterogeneity at the single-cell level has recently been explored, comparative analysis of human white fat depots is still in its infancy.Hereby, we establish a single-nucleus RNA sequencing (snRNA Seq) method for isolating and sequencing intact single nuclei from frozen human WAT biopsies. Using this approach, we analyzed the transcriptome of individual nuclei from 22 WAT SQ or IA biopsies (SQ, 12 IA, 3 paired) acquired from subjects (16 females, 3 males) with or without type 2 diabetes (T2D) across a wide BMI range (Mean+/-SD = 45.4+/-10.3 kg/m2. Range: 23.6 - 60.9 kg/m2) . We show that both SQ and IA human fat depots demonstrate substantial cell-to-cell heterogeneity, and identify multiple adipose and non-adipose-related cell types resident in both tissues. Our analysis identifies several novel subclusters, including two distinct mature adipocyte subclusters, characterized by low vs. high adiponectin expression and thus potentially contributing to adipose-mediated effects on systemic metabolism. Moreover, we identify a novel cluster present in both SQ and IA depots, with a unique transcriptomic signature marked by DAPK1 and enrichment for hedgehog signaling and endocytosis KEGG terms. Comprehensive cross-sample integrative bioinformatics analysis using the software tools scVI and VISION reveals distinct molecular markers and transcriptomic signatures between depots, between clusters, and across a range of BMI, that may serve as promising targets for understanding the mechanism by which specific adipose-resident cell populations mediate metabolic risk associated with adiposity. Disclosure V.Efthymiou: None. S.Kodani: None. A.Gupta: None. F.Shamsi: None. A.Streets: None. Y.Tseng: Consultant; Cellarity. M.Patti: Consultant; AstraZeneca, Eiger BioPharmaceuticals, Hanmi Pharm. Co., Ltd., MBX Biosciences, Poxel SA, Other Relationship; Fractyl Health, Inc., Xeris Pharmaceuticals, Inc., Research Support; Dexcom, Inc. Funding Chan Zuckerberg Initiative (CZI)
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