Maresin 1 mitigates liver steatosis in ob/ob and diet-induced obese mice.

BACKGROUND/OBJECTIVES: The aim of this study was to characterize the effects of Maresin 1 (MaR1) in obesity-related liver steatosis and the mechanisms involved. METHODS: MaR1 effects on fatty liver disease were tested in ob/ob (2-10 µg kg-1 i.p., 20 days) and in diet-induced obese (DIO) mice (2 µg kg-1, i.p., or 50 µg kg-1, oral gavage for 10 days), as well as in cultured hepatocytes. RESULTS: In ob/ob mice, MaR1 reduced liver triglycerides (TG) content, fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 protein expression, while increased acetyl-CoA carboxylase (ACC) phosphorylation and LC3II protein expression, in parallel with a drop in p62 levels. Similar effects on hepatic TG, ACC phosphorylation, p62 and LC3II were observed in DIO mice after MaR1 i.p. injection. Interestingly, oral gavage of MaR1 also decreased serum transaminases, reduced liver weight and TG content. MaR1-treated mice exhibited reduced hepatic lipogenic enzymes content (FAS) or activation (by phosphorylation of ACC), accompanied by upregulation of carnitine palmitoyltransferase (Cpt1a), acyl-coenzyme A oxidase (Acox1) and autophagy-related proteins 5 and 7 (Atg5-7) gene expression, along with increased number of autophagic vacuoles and reduced p62 protein levels. MaR1 also induced AMP-activated protein kinase (AMPK) phosphorylation in DIO mice and in primary hepatocytes, and AMPK inhibition completely blocked MaR1 effects on Cpt1a, Acox1, Atg5 and Atg7 expression. CONCLUSIONS: MaR1 ameliorates liver steatosis by decreasing lipogenic enzymes, while inducing fatty acid oxidation genes and autophagy, which could be related to AMPK activation. Thus, MaR1 may be a new therapeutic candidate for reducing fatty liver in obesity.

Eicosapentaenoic acid promotes mitochondrial biogenesis and beige-like features in subcutaneous adipocytes from overweight subjects.

Eicosapentaenoic acid (EPA), a n-3 long-chain polyunsaturated fatty acid, has been reported to have beneficial effects in obesity-associated metabolic disorders. The objective of the present study was to determine the effects of EPA on the regulation of genes involved in lipid metabolism, and the ability of EPA to induce mitochondrial biogenesis and beiging in subcutaneous adipocytes from overweight subjects. Fully differentiated human subcutaneous adipocytes from overweight females (BMI: 28.1-29.8kg/m2) were treated with EPA (100-200 µM) for 24 h. Changes in mRNA expression levels of genes involved in lipogenesis, fatty acid oxidation and mitochondrial biogenesis were determined by qRT-PCR. Mitochondrial content was evaluated using MitoTracker® Green stain. The effects on peroxisome proliferator-activated receptor gamma, co-activator 1 alpha (PGC-1α) and AMP-activated protein kinase (AMPK) were also characterized. EPA down-regulated lipogenic genes expression while up-regulated genes involved in fatty acid oxidation. Moreover, EPA-treated adipocytes showed increased mitochondrial content, accompanied by an up-regulation of nuclear respiratory factor-1, mitochondrial transcription factor A and cytochrome c oxidase IV mRNA expression. EPA also promoted the activation of master regulators of mitochondrial biogenesis such as sirtuin 1, PGC1-α and AMPK. In parallel, EPA induced the expression of genes that typify beige adipocytes such as fat determination factor PR domain containing 16, uncoupling protein 1 and cell death-inducing DFFA-like effector A, T-Box protein 1 and CD137. Our results suggest that EPA induces a remodeling of adipocyte metabolism preventing fat storage and promoting fatty acid oxidation, mitochondrial biogenesis and beige-like markers in human subcutaneous adipocytes from overweight subjects.

LXR is a negative regulator of glucose uptake in human adipocytes.

AIMS/HYPOTHESIS: Obesity increases the risk of developing type 2 diabetes mellitus, characterised by impaired insulin-mediated glucose uptake in peripheral tissues. Liver X receptor (LXR) is a positive regulator of adipocyte glucose transport in murine models and a possible target for diabetes treatment. However, the levels of LXRα are increased in obese adipose tissue in humans. We aimed to investigate the transcriptome of LXR and the role of LXR in the regulation of glucose uptake in primary human adipocytes. METHODS: The insulin responsiveness of human adipocytes differentiated in vitro was characterised, adipocytes were treated with the LXR agonist GW3965 and global transcriptome profiling was determined by microarray, followed by quantitative RT-PCR (qRT-PCR), western blot and ELISA. Basal and insulin-stimulated glucose uptake was measured and the effect on plasma membrane translocation of glucose transporter 4 (GLUT4) was assayed. RESULTS: LXR activation resulted in transcriptional suppression of several insulin signalling genes, such as AKT2, SORBS1 and CAV1, but caused only minor changes (<15%) in microRNA expression. Activation of LXR impaired the plasma membrane translocation of GLUT4, but not the expression of its gene, SLC2A4. LXR activation also diminished insulin-stimulated glucose transport and lipogenesis in adipocytes obtained from overweight individuals. Furthermore, AKT2 expression was reduced in obese adipose tissue, and AKT2 and SORBS1 expression was inversely correlated with BMI and HOMA index. CONCLUSIONS/INTERPRETATION: In contrast to murine models, LXR downregulates insulin-stimulated glucose uptake in human adipocytes from overweight individuals. This could be due to suppression of Akt2, c-Cbl-associated protein and caveolin-1. These findings challenge the idea of LXR as a drug target in the treatment of diabetes.

Sp1-mediated transcription is involved in the induction of leptin by insulin-stimulated glucose metabolism.

We have previously demonstrated that insulin-stimulated glucose metabolism, and not insulin per se, mediates the effects of insulin to increase the transcriptional activity of the leptin promoter in adipocytes. Here, we sought to identify the specific cis-acting DNA elements required for the upregulation of leptin gene transcription in response to insulin-mediated glucose metabolism. To accomplish this, 3T3-L1 cells and primary rat adipocytes were transfected with a series of luciferase reporter genes containing portions of the mouse leptin promoter. Using this method, we identified an element between -135 and -95 bp (relative to the transcriptional start site) that mediated transcription in response to insulin-stimulated glucose metabolism in adipocytes. This effect was abolished by incubation with 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism. Gel shift electrophoretic mobility shift assays confirmed that the stimulatory effect of insulin-mediated glucose metabolism on leptin transcription was mediated by a previously identified Sp1 site. Consistent with these findings, incubation of primary rat adipocytes with WP631, a specific inhibitor of specificity protein (Sp)1-dependent transcription, inhibited glucose- and insulin-stimulated, but not basal, leptin secretion. Together, these findings support a key role for Sp1 in the transcriptional activation of the leptin gene promoter by insulin-mediated glucose metabolism.

Effects of eicosapentaenoic acid (EPA) on adiponectin gene expression and secretion in primary cultured rat adipocytes.

Adiponectin, a hormone produced by adipocytes, is involved in glucose metabolism and insulin sensitivity. The production of this adipokine is impaired in obesity and insulin resistance. Eicosapentaenoic acid (EPA) is a dietary n-3 polyunsaturated fatty acid that improves insulin sensitivity in several models of obesity and diabetes, which has been suggested to be related to adiponectin induction. An increase in adiponectin production has been also associated with an up-regulation of the transcriptional factor PPARgamma. The aim of this trial was to evaluate the direct effects of EPA on adiponectin gene expression and protein secretion in isolated rat adipocytes as well as to explore the potential mechanisms involved. A comparative study with troglitazone, a PPARgamma agonist, was also performed. For these purposes, primary rat adipocytes were cultured with EPA (100 and 200 microM) and with troglitazone (10 microM) for 96 hours. Both EPA and troglitazone improved glucose utilization by adipocytes. As expected, troglitazone enhanced adiponectin secretion and increased PPARgamma gene expression. However, EPA significantly decreased adiponectin gene expression and protein secretion and reduced PPARy mRNA levels, suggesting that the inhibition of adiponectin by EPA is likely to be secondary to the down-regulation of this adipogenic transcription factor. Moreover, these results suggest that other mechanisms different from the direct stimulation of adiponectin by the fatty acid are underlying the insulin-sensitizing properties observed after EPA treatment in vivo.