A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction.
ABSTRACT: OBJECTIVE:Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo actions of ACSL4 are unknown. The purpose of our studies was to determine the in vivo role of adipocyte ACSL4 in regulating obesity-associated adipocyte dysfunction. METHODS:We developed a novel mouse model with adipocyte-specific ablation of ACSL4 (Ad-KO) using loxP Cre recombinase technology. Metabolic phenotyping of Ad-KO mice relative to their floxed littermates (ACSL4floxed) was performed, including body weight and body composition over time; insulin and glucose tolerance tests; and energy expenditure, activity, and food intake in metabolic cages. Adipocytes were isolated for ex vivo adipocyte oxygen consumption by Clark electrode and lipidomics analysis. In vitro adipocyte analysis including oxygen consumption by Seahorse and real-time PCR analysis were performed to confirm our in vivo findings. RESULTS:Ad-KO mice were protected against DIO, adipocyte death, and metabolic dysfunction. Adipocytes from Ad-KO mice fed high-fat diet (HFD) had reduced incorporation of AA into phospholipids (PL), free AA, and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). Additionally, adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR), which we demonstrated are direct effects of 4-HNE on adipocytes in vitro. CONCLUSION:These studies are the first to elucidate ACSL4's in vivo actions to regulate the incorporation of AA into PL and downstream effects on DIO-associated adipocyte dysfunction. By reducing the incorporation of AA into PL and free fatty acid pools in adipocytes, Ad-KO mice were significantly protected against HFD-induced increases in adipose and liver fat accumulation, adipocyte death, gonadal white adipose tissue (gWAT) inflammation, and insulin resistance (IR). Additionally, deficiency of adipocyte ACSL4 expression in mice fed a HFD resulted in increased gWAT adipocyte OCR and whole body energy expenditure (EE).
Project description:Cellular plasticity in adipose tissue involves adipocyte death, its clearance, and de novo adipogenesis, enabling homeostatic turnover and adaptation to metabolic challenges; however, mechanisms regulating these serial events are not fully understood. The present study investigated the roles of arachidonate 15-lipoxygenase (Alox15) in the clearance of dying adipocytes by adipose tissue macrophages. First, upregulation of Alox15 expression and apoptotic adipocyte death in gonadal white adipose tissue (gWAT) were characterized during adipose tissue remodeling induced by ?3-adrenergic receptor stimulation. Next, an in vitro reconstruction of adipose tissue macrophages and apoptotic adipocytes recapitulated adipocyte clearance by macrophages and demonstrated that macrophages co-cultured with apoptotic adipocytes increased the expression of efferocytosis-related genes. Genetic deletion and pharmacological inhibition of Alox15 diminished the levels of adipocyte clearance by macrophages in a co-culture system. Gene expression profiling of macrophages isolated from gWAT of Alox15 knockout (KO) mice demonstrated distinct phenotypes, especially downregulation of genes involved in lipid uptake and metabolism compared to wild-type mice. Finally, in vivo ?3-adrenergic stimulation in Alox15 KO mice failed to recruit crown-like structures, a macrophage network clearing dying adipocytes in gWAT. Consequently, in Alox15 KO mice, proliferation/differentiation of adipocyte progenitors and ?3-adrenergic remodeling of gWAT were impaired compared to wild-type control mice. Collectively, our data established a pivotal role of Alox15 in the resolution of adipocyte death and in adipose tissue remodeling.
Project description:<h4>Objective</h4>Expansion of adipose tissue during obesity through the recruitment of newly generated adipocytes (hyperplasia) is metabolically healthy, whereas that through the enlargement of pre-existing adipocytes (hypertrophy) leads to metabolic complications. Accumulating evidence from genetic fate mapping studies suggests that in animal models receiving a high-fat diet (HFD), only adipocyte progenitors (APs) in gonadal white adipose tissue (gWAT) have proliferative potential. However, the proliferative potential and differentiating capacity of APs in the inguinal WAT (iWAT) of male mice remains controversial. The objective of this study was to investigate the proliferative and adipogenic potential of APs in the iWAT of HFD-fed male mice.<h4>Methods</h4>We generated PDGFRα-GFP-Cre-ER<sup>T2</sup>/tdTomato (KI/td) mice and traced PDGFRα-positive APs in male mice fed HFD for 8 weeks. We performed a comprehensive phenotypic analysis, including the histology, immunohistochemistry, flow cytometry, and gene expression analysis, of KI/td mice fed HFD.<h4>Results</h4>Contrary to the findings of others, we found an increased number of newly generated tdTomato<sup>+</sup> adipocytes in the iWAT of male mice, which was smaller than that observed in the gWAT. We found that in male mice, the iWAT has more proliferating tdTomato<sup>+</sup> APs than the gWAT. We also found that tdTomato<sup>+</sup> APs showed a higher expression of Dpp4 and Pi16 than tdTomato<sup>-</sup> APs, and the expression of these genes was significantly higher in the iWAT than in the gWAT of mice fed HFD for 8 weeks. Collectively, our results reveal that HFD feeding induces the proliferation of tdTomato<sup>+</sup> APs in the iWAT of male mice.<h4>Conclusion</h4>In male mice, compared with gWAT, iWAT undergoes hyperplasia in response to 8 weeks of HFD feeding through the recruitment of newly generated adipocytes due to an abundance of APs with a high potential for proliferation and differentiation.
Project description:OBJECTIVE:This study investigated the role of microRNAs generated from adipose tissue macrophages (ATMs) during adipose tissue remodeling induced by pharmacological and nutritional stimuli. METHODS:Macrophage-specific Dicer knockout (KO) mice were used to determine the roles of microRNA generated in macrophages in adipose tissue remodeling induced by the ?3-adrenergic receptor agonist CL316,243 (CL). RNA-seq was performed to characterize microRNA and mRNA expression profiles in isolated macrophages and PDGFR?+ adipocyte stem cells (ASCs). The role of miR-10a-5p was further investigated in cell culture, and in adipose tissue remodeling induced by CL treatment and high fat feeding. RESULTS:Macrophage-specific deletion of Dicer elevated pro-inflammatory gene expression and prevented CL-induced de novo beige adipogenesis in gonadal white adipose tissue (gWAT). Co-culture of ASCs with ATMs of wild type mice promoted brown adipocyte gene expression upon differentiation, but co-culture with ATMs of Dicer KO mice did not. Bioinformatic analysis of RNA expression profiles identified miR-10a-5p as a potential regulator of inflammation and differentiation in ATMs and ASCs, respectively. CL treatment increased levels of miR-10a-5p in ATMs and ASCs in gWAT. Interestingly, CL treatment elevated levels of pre-mir-10a in ATMs but not in ASCs, suggesting possible transfer from ATMs to ASCs. Elevating miR-10a-5p levels inhibited proinflammatory gene expression in cultured RAW 264.7 macrophages and promoted the differentiation of C3H10T1/2 cells into brown adipocytes. Furthermore, treatment with a miR-10a-5p mimic in vivo rescued CL-induced beige adipogenesis in Dicer KO mice. High fat feeding reduced miR-10a-5p levels in ATMs of gWAT, and treatment of mice with a miR-10a-5p mimic suppressed pro-inflammatory responses, promoted the appearance of new white adipocytes in gWAT, and improved systemic glucose tolerance. CONCLUSIONS:These results demonstrate an important role of macrophage-generated microRNAs in adipogenic niches and identify miR-10a-5p as a key regulator that reduces adipose tissue inflammation and promotes therapeutic adipogenesis.
Project description:To investigate the role of TNF-like weak inducer of apoptosis (TWEAK) in pathological adipose tissue (AT) remodeling and complications of obesity.Wild type (WT) and TWEAK knockout (KO) mice were fed normal diet (ND) or a high fat diet (HFD) for up to 17 weeks. Adipocyte death was induced using an established transgenic mouse model of inducible adipocyte apoptosis (FAT-ATTAC). Metabolic, biochemical, histologic, and flow cytometric analyses were performed.TWEAK and its receptor, fibroblast growth factor-inducible molecule 14 (Fn14) were upregulated in gonadal (g)AT of WT mice after HFD week 4 and 24 h after induction of adipocyte apoptosis. Phenotypes of KO and WT mouse were indistinguishable through HFD week 8. However, at week 17 obese KO mice had ?30% larger gAT adipocytes and gAT mass than WT mice, coincident with reduced adipocyte death, enhanced insulin signaling, Th2/M2 immune skewing, fewer thick collagen fibers, and altered expression of extracellular matrix constituents and modulators that is consistent with reduced fibrosis and larger adipocytes. KO mice were less steatotic and became more insulin sensitive and glucose tolerant than WT mice after HFD week 12.TWEAK constrains "healthy" gAT expansion and promotes metabolic complications in severe obesity.
Project description:Prolonged niacin treatment elicits beneficial effects on the plasma lipid and lipoprotein profile that is associated with a protective CVD risk profile. Acute niacin treatment inhibits nonesterified fatty acid release from adipocytes and stimulates prostaglandin release from skin Langerhans cells, but the acute effects diminish upon prolonged treatment, while the beneficial effects remain. To gain insight in the prolonged effects of niacin on lipid metabolism in adipocytes, we used a mouse model with a human-like lipoprotein metabolism and drug response [female APOE*3-Leiden.CETP (apoE3 Leiden cholesteryl ester transfer protein) mice] treated with and without niacin for 15 weeks. The gene expression profile of gonadal white adipose tissue (gWAT) from niacin-treated mice showed an upregulation of the "biosynthesis of unsaturated fatty acids" pathway, which was corroborated by quantitative PCR and analysis of the FA ratios in gWAT. Also, adipocytes from niacin-treated mice secreted more of the PUFA DHA ex vivo. This resulted in an increased DHA/arachidonic acid (AA) ratio in the adipocyte FA secretion profile and in plasma of niacin-treated mice. Interestingly, the DHA metabolite 19,20-dihydroxy docosapentaenoic acid (19,20-diHDPA) was increased in plasma of niacin-treated mice. Both an increased DHA/AA ratio and increased 19,20-diHDPA are indicative for an anti-inflammatory profile and may indirectly contribute to the atheroprotective lipid and lipoprotein profile associated with prolonged niacin treatment.
Project description:ABSTRACT Expansion of visceral white adipose tissue (vWAT) occurs in response to nutrient excess, and is a risk factor for metabolic disease. SPRY1, a feedback inhibitor of receptor tyrosine kinase (RTK) signaling, is expressed in PDGFRa+ adipocyte progenitor cells (APC) in vivo. Global deficiency of Spry1 in mice results in disproportionate postnatal growth of gonadal WAT (gWAT), while iWAT and BAT were similar in size between Spry1KO and WT mice. Spry1 deficiency increased the number of PDGFRa+ stromal vascular fraction (SVF) cells in gWAT and showed increased proliferation and fibrosis. Spry1KO gWAT had increased collagen deposition and elevated expression of markers of inflammation. In vitro, SPRY1 was transiently down regulated during early adipocyte differentiation of SVF cells, with levels increasing at later stages of differentiation. SPRY1 deficiency enhances PDGF-AA and PDGF-BB induced proliferation of SVF cells. Increased proliferation of SVF from Spry1KO gWAT accompanies an increase in AKT activation. PDGF-AA stimulated a transient down regulation of SPRY1 in wild type SVF, whereas PDGF-BB stimulated a sustained down regulation of SPRY1 in wild type SVF. Collectively, our data suggest that SPRY1 is critical for regulating postnatal growth of gWAT by restraining APC proliferation and differentiation in part by regulation of PDGFRa/b-AKT signaling.
Project description:Obesity is a growing epidemic in developed countries. Obese individuals are susceptible to comorbidities, including cardiovascular disease and metabolic disorder. Increasing the ability of adipose tissue to expend excess energy could improve protection from obesity. One promising target is microRNA (miR)-155-5p. We demonstrate that deletion of miR-155 (-5p and -3p) in female mice prevents diet-induced obesity. Body weight gain did not differ between wild-type (WT) and miR-155 knockout (KO) mice fed control diet (CD); however, miR-155 KO mice fed high-fat diet (HFD) gained 56% less body weight and 74% less gonadal white adipose tissue (WAT) than WT mice. Enhanced WAT thermogenic potential, brown adipose tissue differentiation, and/or insulin sensitivity might underlie this obesity resistance. Indeed, miR-155 KO mice on HFD had 21% higher heat release than WT HFD mice. Compared to WT adipocytes, miR-155 KO adipocytes upregulated brown (Ucp1, Cidea, Pparg) and white (Fabp4, Pnpla2, AdipoQ, Fasn) adipogenic genes, and glucose metabolism genes (Glut4, Irs1). miR-155 deletion abrogated HFD-induced adipocyte hypertrophy and WAT inflammation. Therefore, miR-155 deletion increases adipogenic, insulin sensitivity, and energy uncoupling machinery, while limiting inflammation in WAT, which together could restrict HFD-induced fat accumulation. Our results identify miR-155 as a novel candidate target for improving obesity resistance.
Project description:<b>Background:</b> Salvianolic acid A (Sal A), a natural polyphenolic compound extracted from <i>Radix Salvia miltiorrhiza</i> (Danshen), exhibits exceptional pharmacological activities against cardiovascular diseases. While a few studies have reported anti-obesity properties of Sal A, the underlying mechanisms are largely unknown. Given the prevalence of obesity and promising potential of browning of white adipose tissue to combat obesity, recent research has focused on herbal ingredients that may promote browning and increase energy expenditure. <b>Purpose:</b> The present study was designed to investigate the protective antiobesity mechanisms of Sal A, in part through white adipose browning. <b>Methods:</b> Both high-fat diet (HFD)-induced obese (DIO) male mice model and fully differentiated C3H10T1/2 adipocytes from mouse embryo fibroblasts were employed in this study. Sal A (20 and 40 mg/kg) was administrated to DIO mice by intraperitoneal injection for 13-weeks. Molecular mechanisms mediating effects of Sal A were evaluated. <b>Resluts:</b> Sal A treatment significantly attenuated HFD-induced weight gain and lipid accumulation in epididymal fat pad. Uncoupling protein 1 (UCP-1), a specialized thermogenic protein and marker for white adipocyte browning, was significantly induced by Sal A treatment in both white adipose tissues and cultured adipocytes. Further mechanistic investigations revealed that Sal A robustly reversed HFD-decreased AMP-activated protein kinase (AMPK) phosphorylation and sirtuin 1 (SIRT1) expression in mice. Genetically silencing either AMPK or SIRT1 using siRNA abolished UCP-1 upregulation by Sal A. AMPK silencing significantly blocked Sal A-increased SIRT1 expression, while SIRT1 silencing did not affect Sal A-upregulated phosphorylated-AMPK. These findings indicate that AMPK was involved in Sal A-increased SIRT1. <b>Conclusion:</b> Sal A increases white adipose tissue browning in HFD-fed male mice and in cultured adipocytes. Thus, Sal is a potential natural therapeutic compound for treating and/or preventing obesity.
Project description:<h4>Background</h4>Prostate cancer (CaP) preferentially metastasises to the bone, and we have previously shown that the poly-unsaturated fatty acid (PUFA) arachidonic acid (AA) is a potent stimulator of CaP invasion. Here we present that AA promotes CaP invasion by inducing bone marrow adipocyte formation.<h4>Methods</h4>Boyden invasion-chamber assays assessed the ability of dietary oils, their PUFA components, and specific PUFA-loaded adipocytes to induce PC-3 invasion. Lipid transfer and metabolism was followed using deuterated AA and Fourier Transform Infrared spectroscopy (FTIR).<h4>Results</h4>Poly-unsaturated fatty acid constituents, but not their corresponding dietary oils, induced PC-3 invasion. PUFAs induce bone marrow adipocyte (BM-Ad) differentiation with AA inducing higher levels of BM-Ad differentiation, as compared with other PUFAs (3998+/-514.4 vs 932+/-265.8; P=0.00002), which stimulated greater PC-3 invasion than free AA (22 408.5+/-607.4 vs 16 236+/-313.9; P=0.01111) or adipocytes generated in the presence of other PUFAs. In bone marrow co-culture PC-3 and BM-Ad interactions result in direct uptake and metabolism of AA by PC-3 cells, destruction of the adipocyte and subsequent formation of a bone metastasis.<h4>Conclusion</h4>The data supports the hypothesis that AA not only promotes CaP invasion, it also prepares the 'soil', making it more supportive for implantation and propagation of the migrating metastatic cell.
Project description:<h4>Objective</h4>IP6 kinases (IP6Ks) regulate cell metabolism and survival. Mice with global (IP6K1-KO) or adipocyte-specific (AdKO) deletion of IP6K1 are protected from diet induced obesity (DIO) at ambient (23 °C) temperature. AdKO mice are lean primarily due to increased AMPK mediated thermogenic energy expenditure (EE). Thus, at thermoneutral (30 °C) temperature, high fat diet (HFD)-fed AdKO mice expend energy and gain body weight, similar to control mice. IP6K1 is ubiquitously expressed; thus, it is critical to determine to what extent the lean phenotype of global IP6K1-KO mice depends on environmental temperature. Furthermore, it is not known whether IP6K1 regulates AMPK mediated EE in cells, which do not express UCP1.<h4>Methods</h4>Q-NMR, GTT, food intake, EE, QRT-PCR, histology, mitochondrial oxygen consumption rate (OCR), fatty acid metabolism assays, and immunoblot studies were conducted in IP6K1-KO and WT mice or cells.<h4>Results</h4>Global IP6K1 deletion mediated enhancement in EE is impaired albeit not abolished at 30 °C. As a result, IP6K1-KO mice are protected from DIO, insulin resistance, and fatty liver even at 30 °C. Like AdKO, IP6K1-KO mice display enhanced adipose tissue browning. However, unlike AdKO mice, thermoneutrality only partly abolishes browning in IP6K1-KO mice. Cold (5 °C) exposure enhances carbohydrate expenditure, whereas 23 °C and 30 °C promote fat oxidation in HFD-KO mice. Furthermore, IP6K1 deletion diminishes cellular fat accumulation via activation of the AMPK signaling pathway.<h4>Conclusions</h4>Global deletion of IP6K1 ameliorates obesity and insulin resistance irrespective of the environmental temperature conditions, which strengthens its validity as an anti-obesity target.