Stearoyl-CoA desaturase 2 is required for peroxisome proliferator-activated receptor gamma expression and adipogenesis in cultured 3T3-L1 cells

Based on recent evidence that fatty acid synthase and endogenously produced fatty acid derivatives are required for adipogenesis in 3T3-L1 adipocytes, we conducted a small interfering RNA-based screen to identify other fatty acid-metabolizing enzymes that may mediate this effect. Of 24 enzymes screened, stearoyl-CoA desaturase 2 (SCD2) was found to be uniquely and absolutely required for adipogenesis. Remarkably, SCD2 also controls the maintenance of adipocyte-specific gene expression in fully differentiated 3T3-L1 adipocytes, including the expression of SCD1. Despite the high sequence similarity between SCD2 and SCD1, silencing of SCD1 did not down-regulate 3T3-L1 cell differentiation or gene expression. SCD2 mRNA expression was also uniquely elevated 44-fold in adipose tissue upon feeding mice a high fat diet, whereas SCD1 showed little response. The inhibition of adipogenesis caused by SCD2 depletion was associated with a decrease in peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA and protein, whereas in mature adipocytes loss of SCD2 diminished PPARgamma protein levels, with little change in mRNA levels. In the latter case, SCD2 depletion did not change the degradation rate of PPARgamma protein but decreased the metabolic labeling of PPARgamma protein using [(35)S]methionine/cysteine, indicating protein translation was decreased. This requirement of SCD2 for optimal protein synthesis in fully differentiated adipocytes was verified by polysome profile analysis, where a shift in the mRNA to monosomes was apparent in response to SCD2 silencing. These results reveal that SCD2 is required for the induction and maintenance of PPARgamma protein levels and adipogenesis in 3T3-L1 cells

Expression of ITGB8 in Epicardial Adipose Tissue is Highly and Directly Correlated with the Severity of Coronary Atherosclerosis

Background: In patients with coronary artery disease (CAD), epicardial adipose tissue (EAT) has been shown to express increased levels of inflammatory cytokines (IL-1β, IL-6, MCP-1, TNFα) and decreased levels of anti-inflammatory and cardioprotective adipokines. However, it is not known whether or not inflammation in EAT is a primary cause or a secondary response to atherosclerosis. In order to better understand this pathophysiology, we tested the hypothesis that expression of certain genes in EAT would correlate with the degree of coronary atherosclerosis. Purpose: The purpose of this study was to determine whether there is a difference in gene expression in epicardial fat of patients with and without coronary artery disease and if there is a difference, whether these differentially expressed genes participate in the inflammatory pathways. Methods: EAT and paired subcutaneous adipose tissue (SAT) samples collected from cardiac surgery patients with and without coronary disease were fixed for microscopy and frozen for RNA extraction. RNA was hybridized to Affymetrix Human Gene 1.0 ST chips. We used an unbiased approach to identify genes highly and differentially expressed in EAT vs. SAT (FC\u3e3.0). The probe intensities for these resultant genes were then correlated with the severity of atherosclerosis in each patient as determined by the Gensini score. Results:35 genes were differentially expressed in EAT at \u3e3.0 fold change (p Conclusions: Using an unbiased whole genome approach, we identified ITGB8 and TG2 as genes whose expression is correlated with CAD severity. ITGB8 has been previously shown to be expressed by fibroblasts and functions to activate TGFβ. TGFβ signaling has also been correlated with advanced atherosclerosis. We speculate that EAT expression of ITGB8 may have pro-inflammatory effects, possibly by activating TGFβ, and stimulating recruitment of dendritic cells or T cells to secondary lymphoid organs in EAT. Whether or not this is the case is a goal of future studies

Expression of the Integrin Beta 8 Gene (ITGB8) in Epicardial Adipose Tissue is Highly and Directly Correlated with the Degree of Coronary Atherosclerosis

Background: In patients with coronary artery disease (CAD), epicardial adipose tissue (EAT) expression of inflammatory genes is high while expression of anti-inflammatory genes is low. We hypothesized that expression of certain genes in EAT would correlate directly with the degree of adjacent CAD. Methods: EAT and paired subcutaneous adipose tissue (SAT) samples were collected from cardiac surgery patients (n=9) with and without CAD. RNA was isolated and hybridized to Affymetrix 1.0 ST chips. Genes differentially expressed in EAT vs. SAT were identified. Probe intensities were correlated with the severity of CAD in each patient using the Gensini score. Results: 35 genes were differentially expressed in EAT at \u3e3.0 fold change (p Conclusions: Expression of ITGB8 is directly correlated with CAD severity. ITGB8 has been previously shown to be expressed by fibroblasts and functions to activate TGFβ. TGFβ signaling has also been correlated with advanced atherosclerosis. We speculate that EAT expression of ITGB8 may have pro-inflammatory effects, possibly by activating TGFβ, and stimulating recruitment of dendritic cells or T cells to secondary lymphoid organs in EAT. Whether or not this is the case is a goal of future studies

G(alpha)11 signaling through ARF6 regulates F-actin mobilization and GLUT4 glucose transporter translocation to the plasma membrane

The action of insulin to recruit the intracellular GLUT4 glucose transporter to the plasma membrane of 3T3-L1 adipocytes is mimicked by endothelin 1, which signals through trimeric G(alpha)q or G(alpha)11 proteins. Here we report that murine G(alpha)11 is most abundant in fat and that expression of the constitutively active form of G(alpha)11 [G(alpha)11(Q209L)] in 3T3-L1 adipocytes causes recruitment of GLUT4 to the plasma membrane and stimulation of 2-deoxyglucose uptake. In contrast to the action of insulin on GLUT4, the effects of endothelin 1 and G(alpha)11 were not inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin at 100 nM. Signaling by insulin, endothelin 1, or G(alpha)11(Q209L) also mobilized cortical F-actin in cultured adipocytes. Importantly, GLUT4 translocation caused by all three agents was blocked upon disassembly of F-actin by latrunculin B, suggesting that the F-actin polymerization caused by these agents may be required for their effects on GLUT4. Remarkably, expression of a dominant inhibitory form of the actin-regulatory GTPase ARF6 [ARF6(T27N)] in cultured adipocytes selectively inhibited both F-actin formation and GLUT4 translocation in response to endothelin 1 but not insulin. These data indicate that ARF6 is a required downstream element in endothelin 1 signaling through G(alpha)11 to regulate cortical actin and GLUT4 translocation in cultured adipocytes, while insulin action involves different signaling pathways

Coronary disease is not associated with robust alterations in inflammatory gene expression in human epicardial fat

Epicardial adipose tissue (EAT) is the visceral fat depot of the heart. Inflammation of EAT is thought to contribute to coronary artery disease (CAD). Therefore, we hypothesized that the EAT of patients with CAD would have increased inflammatory gene expression compared with controls without CAD. Cardiac surgery patients with (n = 13) or without CAD (n = 13) were consented, and samples of EAT and subcutaneous adipose tissue (SAT) were obtained. Transcriptomic analysis was performed using Affymetrix Human Gene 1.0 ST arrays. Differential expression was defined as a 1.5-fold change (ANOVA P \u3c 0.05). Six hundred ninety-three genes were differentially expressed between SAT and EAT in controls and 805 in cases. Expression of 326 genes was different between EAT of cases and controls; expression of 14 genes was increased in cases, while 312 were increased in controls. Quantitative reverse transcription PCR confirmed that there was no difference in expression of CCL2, CCR2, TNF-alpha, IL-6, IL-8, and PAI1 between groups. Immunohistochemistry showed more macrophages in EAT than SAT, but there was no difference in their number or activation state between groups. In contrast to prior studies, we did not find increased inflammatory gene expression in the EAT of patients with CAD. We conclude that the specific adipose tissue depot, rather than CAD status, is responsible for the majority of differential gene expression

Control of Adipocyte Thermogenesis and Lipogenesis through beta3-Adrenergic and Thyroid Hormone Signal Integration

Here, we show that beta adrenergic signaling coordinately upregulates de novo lipogenesis (DNL) and thermogenesis in subcutaneous white adipose tissue (sWAT), and both effects are blocked in mice lacking the cAMP-generating G protein-coupled receptor Gs (Adipo-GsalphaKO) in adipocytes. However, UCP1 expression but not DNL activation requires rapamycin-sensitive mTORC1. Furthermore, beta3-adrenergic agonist CL316243 readily upregulates thermogenic but not lipogenic genes in cultured adipocytes, indicating that additional regulators must operate on DNL in sWAT in vivo. We identify one such factor as thyroid hormone T3, which is elevated locally by adrenergic signaling. T3 administration to wild-type mice enhances both thermogenesis and DNL in sWAT. Mechanistically, T3 action on UCP1 expression in sWAT depends upon cAMP and is blocked in Adipo-GsalphaKO mice even as elevated DNL persists. Thus, T3 enhances sWAT thermogenesis by amplifying cAMP signaling, while its control of adipocyte DNL can be mediated independently of both cAMP and rapamycin-sensitive mTORC1

CRISPR-delivery particles targeting nuclear receptor-interacting protein 1 (Nrip1) in adipose cells to enhance energy expenditure

RNA-guided, engineered nucleases derived from the prokaryotic adaptive immune system CRISPR-Cas represent a powerful platform for gene deletion and editing. When used as a therapeutic approach, direct delivery of Cas9 protein and single-guide RNA (sgRNA) could circumvent the safety issues associated with plasmid delivery and therefore represents an attractive tool for precision genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fatty acid oxidation, and secretion of bioactive factors through a browning process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed CRISPR-delivery particles, denoted CriPs, composed of nano-size complexes of Cas9 protein and sgRNA that are coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9-mediated gene deletion of ectopically expressed GFP by CriPs was achieved in multiple cell types, including a macrophage cell line, primary macrophages, and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP-expressing mice following intraperitoneal injection of CriPs containing Gfp-targeting sgRNA. Furthermore, disruption of a nuclear co-repressor of catabolism, the Nrip1 gene, in white adipocytes by CriPs enhanced adipocyte browning with a marked increase of uncoupling protein 1 (UCP1) expression. Of note, the CriP-mediated Nrip1 deletion did not produce detectable off-target effects. We conclude that CriPs offer an effective Cas9 and sgRNA delivery system for ablating targeted gene products in cultured cells and in vivo, providing a potential therapeutic strategy for metabolic disease

Adipocyte-specific Hypoxia-inducible gene 2 promotes fat deposition and diet-induced insulin resistance

OBJECTIVE: Adipose tissue relies on lipid droplet (LD) proteins in its role as a lipid-storing endocrine organ that controls whole body metabolism. Hypoxia-inducible Gene 2 (Hig2) is a recently identified LD-associated protein in hepatocytes that promotes hepatic lipid storage, but its role in the adipocyte had not been investigated. Here we tested the hypothesis that Hig2 localization to LDs in adipocytes promotes adipose tissue lipid deposition and systemic glucose homeostasis. METHOD: White and brown adipocyte-deficient (Hig2fl/fl x Adiponection cre+) and selective brown/beige adipocyte-deficient (Hig2fl/fl x Ucp1 cre+) mice were generated to investigate the role of Hig2 in adipose depots. Additionally, we used multiple housing temperatures to investigate the role of active brown/beige adipocytes in this process. RESULTS: Hig2 localized to LDs in SGBS cells, a human adipocyte cell strain. Mice with adipocyte-specific Hig2 deficiency in all adipose depots demonstrated reduced visceral adipose tissue weight and increased glucose tolerance. This metabolic effect could be attributed to brown/beige adipocyte-specific Hig2 deficiency since Hig2fl/fl x Ucp1 cre+ mice displayed the same phenotype. Furthermore, when adipocyte-deficient Hig2 mice were moved to thermoneutral conditions in which non-shivering thermogenesis is deactivated, these improvements were abrogated and glucose intolerance ensued. Adipocyte-specific Hig2 deficient animals displayed no detectable changes in adipocyte lipolysis or energy expenditure, suggesting that Hig2 may not mediate these metabolic effects by restraining lipolysis in adipocytes. CONCLUSIONS: We conclude that Hig2 localizes to LDs in adipocytes, promoting adipose tissue lipid deposition and that its selective deficiency in active brown/beige adipose tissue mediates improved glucose tolerance at 23 degrees C. Reversal of this phenotype at thermoneutrality in the absence of detectable changes in energy expenditure, adipose mass, or liver triglyceride suggests that Hig2 deficiency triggers a deleterious endocrine or neuroendocrine pathway emanating from brown/beige fat cells

Joint analysis of left ventricular expression and circulating plasma levels of Omentin after myocardial ischemia

BACKGROUND: Omentin-1, also known as Intelectin-1 (ITLN1), is an adipokine with plasma levels associated with diabetes, obesity, and coronary artery disease. Recent studies suggest that ITLN1 can mitigate myocardial ischemic injury but the expression of ITLN1 in the heart itself has not been well characterized. The purpose of this study is to discern the relationship between the expression pattern of ITLN1 RNA in the human heart and the level of circulating ITLN1 protein in plasma from the same patients following myocardial ischemia. METHODS: A large cohort of patients (n = 140) undergoing elective cardiac surgery for aortic valve replacement were enrolled in this study. Plasma and left ventricular biopsy samples were taken at the beginning of cardiopulmonary bypass and after an average of 82 min of ischemic cross clamp time. The localization of ITLN1 in epicardial adipose tissue (EAT) was also further characterized with immunoassays and cell fate transition studies. RESULTS: mRNA expression of ITLN1 decreases in left ventricular tissue after acute ischemia in human patients (mean difference 280.48, p = 0.001) whereas plasma protein levels of ITLN1 increase (mean difference 5.24, p \u3c 0.001). Immunohistochemistry localized ITLN1 to the mesothelium or visceral pericardium of EAT. Epithelial to mesenchymal transition in mesothelial cells leads to a downregulation of ITLN1 expression. CONCLUSIONS: Myocardial injury leads to a decrease in ITLN1 expression in the heart and a corresponding increase in plasma levels. These changes may in part be due to an epithelial to mesenchymal transition of the cells that express ITLN1 following ischemia. Trial Registration Clinicaltrials.gov ID: NCT00985049

CRISPR-enhanced human adipocyte \u27browning\u27 as cell therapy for metabolic disease [preprint]

Obesity and type 2 diabetes (T2D) are associated with poor tissue responses to insulin [1,2], disturbances in glucose and lipid fluxes [3-5] and comorbidities including steatohepatitis [6] and cardiovascular disease [7,8]. Despite extensive efforts at prevention and treatment [9,10], diabetes afflicts over 400 million people worldwide [11]. Whole body metabolism is regulated by adipose tissue depots [12-14], which include both lipid-storing white adipocytes and less abundant \u27brown\u27 and \u27brite/beige\u27 adipocytes that express thermogenic uncoupling protein UCP1 and secrete factors favorable to metabolic health [15-18]. Application of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing [19,20] to enhance \u27browning\u27 of white adipose tissue is an attractive therapeutic approach to T2D. However, the problems of cell-selective delivery, immunogenicity of CRISPR reagents and long term stability of the modified adipocytes are formidable. To overcome these issues, we developed methods that deliver complexes of SpyCas9 protein and sgRNA ex vivo to disrupt the thermogenesis suppressor gene NRIP1 [21,22] with near 100% efficiency in human or mouse adipocytes. NRIP1 gene disruption at discrete loci strongly ablated NRIP1 protein and upregulated expression of UCP1 and beneficial secreted factors, while residual Cas9 protein and sgRNA were rapidly degraded. Implantation of the CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreased adiposity and liver triglycerides while enhancing glucose tolerance compared to mice implanted with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic modification of human adipocytes without exposure of the recipient to immunogenic Cas9 or delivery vectors