Obesity and aging are associated to non-alcoholic fatty liver disease (NAFLD) development. Here, we investigate whether long-term feeding with a docosahexaenoic acid (DHA)-enriched diet and aerobic exercise, alone or in combination, are effective in ameliorating NAFLD in aged obese mice. Two-month-old female C57BL/6J mice received control or high fat diet (HFD) for 4 months. Then, the diet-induced obese (DIO) mice were distributed into four groups: DIO, DIO + DHA (15% dietary lipids replaced by a DHA-rich concentrate), DIO + EX (treadmill running), and DIO + DHA + EX up to 18 months. The DHA-rich diet reduced liver steatosis in DIO mice, decreasing lipogenic genes (Dgat2, Scd1, Srebp1c), and upregulated lipid catabolism genes (Hsl/Acox) expression. A similar pattern was observed in the DIO + EX group. The combination of DHA + exercise potentiated an increase in Cpt1a and Ppara genes, and AMPK activation, key regulators of fatty acid oxidation. Exercise, alone or in combination with DHA, significantly reversed the induction of proinflammatory genes (Mcp1, Il6, Tnf¿, Tlr4) in DIO mice. DHA supplementation was effective in preventing the alterations induced by the HFD in endoplasmic reticulum stress-related genes (Ern1/Xbp1) and autophagy markers (LC3II/I ratio, p62, Atg7). In summary, long-term DHA supplementation and/or exercise could be helpful to delay NAFLD progression during aging in obesity.

Maresin 1 (MaR1) is a DHA-derived pro-resolving lipid mediator. The present study aimed to characterize the ability of MaR1 to prevent the alterations induced by TNF-alpha on insulin actions in glucose uptake and Akt phosphorylation in cultured human adipocytes from overweight/obese subjects, as well as to investigate the effects of MaR1 acute and chronic administration on Akt phosphorylation in absence/presence of insulin in white adipose tissue (WAT) and skeletal muscle from lean and diet-induced obese (DIO) mice. MaR1 (0.1 nM) prevented the inhibitory effect of TNF-alpha on insulin-stimulated 2-Deoxy-D-glucose uptake and Akt phosphorylation in human adipocytes. Acute treatment with MaR1 (50 mu g/kg, 3 h, i.p.) induced Akt phosphorylation in WAT and skeletal muscle of lean mice. However, MaR1 did not further increase the stimulatory effect of insulin on Akt activation. Interestingly, intragastric chronic treatment with MaR1 (50 mu g/kg, 10 days) in DIO mice reduced the hyperglycemia induced by the high fat diet (HFD) and improved systemic insulin sensitivity. In parallel, MaR1 partially restored the impaired insulin response in skeletal muscle of DIO mice and reversed HFD-induced lower Akt phosphorylation in WAT in non-insulin-stimulated DIO mice while did not restore the defective Akt activation in response to acute insulin observed in DIO mice. Our results suggest that MaR1 attenuates the impaired insulin signaling and glucose uptake induced by proinflammatory cytokines. Moreover, the current data support that MaR1 treatment could be useful to reduce the hyperglycemia and the insulin resistance associated to obesity, at least in part by improving Akt signaling.

Aging usually comes associated with increased visceral fat accumulation, reaching even an obesity state, and favoring its associated comorbidities. One of the processes involved in aging is cellular senescence, which is highly dependent on the activity of the regulators of the cell cycle. The aim of this study was to analyze the changes in the expression of p27 and cdk2 in different adipose tissue depots during aging, as well as their regulation by obesity in mice. Changes in the expression of p27 and CDK2 in visceral and subcutaneous white adipose tissue (WAT) biopsies were also analyzed in a human cohort of obesity and type 2 diabetes. p27, but not cdk2, exhibits a lower expression in subcutaneous than in visceral WAT in mice and humans. p27 is drastically downregulated by aging in subcutaneous WAT (scWAT), but not in gonadal WAT, of female mice. Obesity upregulates p27 and cdk2 expression in scWAT, but not in other fat depots of aged mice. In humans, a significant upregulation of p27 was observed in visceral WAT of subjects with obesity. Taken together, these results show a differential adipose depot-dependent regulation of p27 and cdk2 in aging and obesity, suggesting that p27 and cdk2 could contribute to the adipose-tissue depot's metabolic differences. Further studies are necessary to fully corroborate this hypothesis.

Obesity is characterized by excessive fat accumulation and inflammation. Aging has also been characterized as an inflammatory condition, frequently accompanied by accumulation of visceral fat. Beneficial effects of exercise and 11-3 long-chain polyunsaturated fatty acids in metabolic disorders have been described. Glucose transporter 12 (GLUT12) is one of the less investigated members of the GLUT family. Glucose, insulin, and tumor necrosis factor alpha (TNF-alpha) induce GLUT12 translocation to the membrane in muscle, adipose tissue, and intestine. We aimed to investigate GLUT12 expression in obesity and aging, and under diet supplementation with docosahexaenoic acid (DHA) alone or in combination with physical exercise in mice. Aging increased GLUT12 expression in intestine, kidney, and adipose tissue, whereas obesity reduced it. No changes on the transporter occurred in skeletal muscle. In obese 18-month-old mice, DHA further decreased GLUT12 in the 4 organs. Aerobic exercise alone did not modify GLUT12, but the changes triggered by exercise were able to prevent the DHA-diminishing effect, and almost restored GLUT12 basal levels. In conclusion, the downregulation of metabolism in aging would be a stimulus to upregulate GLUT12 expression. Contrary, obesity, an excessive energy condition, would induce GLUT12 downregulation. The combination of exercise and DHA would contribute to restore basal function of GLUT12.

The aim of this study was to characterize the effects of Maresin 1 (MaR1), a DHA-derived pro-resolving lipid mediator, on obesity-related colonic inflammation and gut dysbiosis in diet-induced obese (DIO) mice. In colonic mucosa of DIO mice, the MaR1 treatment decreased the expression of inflammatory genes, such asTnf-alpha andIl-1 beta. As expected, the DIO mice exhibited significant changes in gut microbiota composition at the phylum, genus, and species levels, with a trend to a higher Firmicutes/Bacteroidetes ratio. Deferribacteres and Synergistetes also increased in the DIO animals. In contrast, these animals exhibited a significant decrease in the content of Cyanobacteria and Actinobacteria. Treatment with MaR1 was not able to reverse the dysbiosis caused by obesity on the most abundant phyla. However, the MaR1 treatment increased the content ofP. xylanivorans, which have been considered to be a promising probiotic with healthy effects on gut inflammation. Finally, a positive association was found between the Deferribacteres andIl-1 beta expression, suggesting that the increase in Deferribacteres observed in obesity could contribute to the overexpression of inflammatory cytokines in the colonic mucosa. In conclusion, MaR1 administration ameliorates the inflammatory state in the colonic mucosa and partially compensates changes on gut microbiota caused by obesity.

Chemerin is a pro-inflammatory adipokine that is increased in obesity and associated with obesity-related comorbidities. The aim of this study was to investigate the effects of omega-3 polyunsaturated fatty acids, eicosapentaenoic and docosahexaenoic acids (EPA and DHA), on basal and tumor necrosis factor-alpha (TNF-alpha)-induced chemerin production in 3T3-L1 and human subcutaneous cultured adipocytes. The potential involvement of G protein-coupled receptor 120 (GPR120), as well as the actions of DHA-derived specialized proresolving lipid mediators (SPMs), resolvin D1 and D2 (RvD1 and RvD2) and maresin 1 (MaR1), were also evaluated. DHA significantly lowered both basal and TNF-alpha-stimulated chemerin production in 3T3-L1 and human adipocytes. EPA did not modify basal chemerin production, while it attenuated the induction of chemerin by TNF-alpha. Silencing of GPR120 using siRNA blocked the ability of DHA and EPA to reduce TNF-alpha-induced chemerin secretion. Interestingly, treatment with the DHA-derived SPMs RvD1, RvD2 and MaR1 also reversed the stimulatory effect of TNF-alpha on chemerin production in human adipocytes.

Type 2 diabetes (T2D) is a complex metabolic disease, which involves a maintained hyperglycemia due to the development of an insulin resistance process. Among multiple risk factors, host intestinal microbiota has received increasing attention in T2D etiology and progression. In the present study, we have explored the effect of long-term supplementation with a non-dairy fermented food product (FFP) in Zucker Diabetic and Fatty (ZDF) rats T2D model. The supplementation with FFP induced an improvement in glucose homeostasis according to the results obtained from fasting blood glucose levels, glucose tolerance test, and pancreatic function. Importantly, a significantly reduced intestinal glucose absorption was found in the FFP-treated rats. Supplemented animals also showed a greater survival suggesting a better health status as a result of the FFP intake. Some dissimilarities have been observed in the gut microbiota population between control and FFP-treated rats, and interestingly a tendency for better cardiometabolic markers values was appreciated in this group. However, no significant differences were observed in body weight, body composition, or food intake between groups. These findings suggest that FFP induced gut microbiota modifications in ZDF rats that improved glucose metabolism and protected from T2D development.

We have previously demonstrated in Caco-2 cells that tumor necrosis factor-alpha (TNF-alpha) inhibits sugar uptake, acting from the apical membrane, by decreasing the expression of the Na+-glucose cotransporter SGLT1 in the brush border membrane. The goal was to investigate the hypothesis that TNF-alpha from abdominal adipose tissue (adipocytes and macrophages) would decrease sugar and amino acid transport acting from the basolateral membrane of the enterocytes. TNF-alpha placed in the basal compartment of Caco-2 cells decreased alpha-methyl- d-glucose (alphaMG) and glutamine uptake. The apical medium derived from these Caco-2 cells apically placed in another set of cells, also reduced sugar and glutamine transport. Reverse-transcription polymerase chain reaction analysis demonstrated upregulation of TNF-alpha, IL-1beta, and MCP1 expression in Caco-2 cells exposed to basal TNF-alpha. Similarly, MG uptake was inhibited after Caco-2 cells were incubated, in the basal compartment, with medium from visceral human mesenchymal stem cells-derived adipocytes of overweight individuals. The apical medium collected from those Caco-2 cells, and placed in the upper side of other set of cells, also decreased sugar uptake. Basal presence of medium derived from lipopolysaccharide-activated macrophages and nonactivated macrophages decreased MG uptake as well. Diet-induced obese mice showed an increase in the visceral adipose tissue surrounding the intestine.

A potential role of marine n-3 polyunsaturated fatty acids (-3 PUFAs) has been suggested in memory, learning, and cognitive processes. Therefore, -3 PUFAs might be a promising treatment option, albeit controversial, for Alzheimer's disease (AD). Among the different mechanisms that have been proposed as responsible for the beneficial effects of -3 PUFAs, inhibition of JNK stands as a particularly interesting candidate. In the present work, it has been studied whether the administration of two different PUFAs (docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)) and a DHA-derived specialized pro-resolving lipid mediator (MaR1) is able to reverse cognitive deficits in the senescence-accelerated mouse prone 8 (SAMP8) mouse model of sporadic AD. The novel object recognition test (NORT) test showed that recognition memory was significantly impaired in SAMP8 mice, as shown by a significantly decreased discrimination index that was reversed by MaR1 and DHA. In the retention phase of the Morris water maze (MWM) task, SAMP8 mice showed memory deficit that only DHA treatment was able to reverse. pJNK levels were significantly increased in the hippocampus of SAMP8 mice compared to SAMR1 mice, and only DHA treatment was able to significantly reverse these increased pJNK levels. Similar results were found when measuring c-Jun, the main JNK substrate. Consequently to the increases in tau phosphorylation after increased pJNK, it was checked that tau phosphorylation (PHF-1) was increased in SAMP mice, and this effect was reversed after DHA treatment. Altogether, DHA could represent a new approach for the treatment of AD through JNK inhibition.

SCOPE: To study the effects of Maresin 1 (MaR1), a docosahexaenoic-acid-derived lipid mediator, on fibroblast growth factor 21 (FGF21) production and to characterize the tissue-specific regulation of Fgf21 and its signaling pathway in liver, skeletal muscle, and white adipose tissue (WAT). METHODS AND RESULTS: Diet-induced obese (DIO) mice are treated with MaR1 (50g kg-1 , 10 days, oral gavage) and serum FGF21 levels and liver, muscle and WAT Fgf21, beta-Klotho, Fgfr1, Egr1, and cFos mRNA expression are evaluated. Additionally, MaR1 effects are tested in mouse primary hepatocytes, HepG2 human hepatocytes, C2C12 myotubes, and 3T3-L1 adipocytes. In DIO mice, MaR1 decreases circulating FGF21 levels and HFD-induced hepatic Fgf21 mRNA expression. MaR1 increases hepatic beta-Klotho, Egr1, and cFos in DIO mice. In WAT, MaR1 counteracts the HFD-induced downregulation of Fgf21, Fgfr1, and beta-Klotho. In muscle, MaR1 does not modify Fgf21 but promoted Fgfr1 expression. In mouse primary hepatocytes, MaR1 decreases Fgf21 expression and downregulated Pparalpha mRNA levels. In HepG2 cells, MaR1 reverses the increased production of FGF21 and the downregulation of FGFR1, Beta-KLOTHO, EGR1, and cFOS induced by palmitate. Preincubation with a PPARalpha antagonist prevents MaR1 effects on FGF21 secretion. CONCLUSION: The ability of MaR1 to modulate FGF21 can contribute to its beneficial metabolic effects.

Obesity is associated with high levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-¿), which promotes inflammation in adipose tissue. The omega-3 PUFAs, and their derived lipid mediators, such as Maresin 1 (MaR1) have anti-inflammatory effects on adipose tissue. This study aimed to analyze if MaR1 may counteract alterations induced by TNF-¿ on lipolysis and autophagy in mature 3T3-L1 adipocytes. Our data revealed that MaR1 (1-100¿nM) inhibited the TNF-¿-induced glycerol release after 48¿hr, which may be related to MaR1 ability of preventing the decrease in lipid droplet-coating protein perilipin and G0/G1 Switch 2 protein expression. MaR1 also reversed the decrease in total hormone sensitive lipase (total HSL), and the ratio of phosphoHSL at Ser-565/total HSL, while preventing the increased ratio of phosphoHSL at Ser-660/total HSL and phosphorylation of extracellular signal-regulated kinase 1/2 induced by TNF-¿. Moreover, MaR1 counteracted the cytokine-induced decrease of p62 protein, a key autophagy indicator, and also prevented the induction of LC3II/LC3I, an important autophagosome formation marker. Current data suggest that MaR1 may ameliorate TNF-¿-induced alterations on lipolysis and autophagy in adipocytes. This may also contribute to the beneficial actions of MaR1 on adipose tissue and insulin sensitivity in obesity.

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 mu g kg(-1) i.p., 20 days) and in diet-induced obese (DIO) mice (2 mu g kg(-1), i.p., or 50 mu g 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.

Background/Objectives: Obesity is related to a dynamic extracellular matrix (ECM) remodeling, which involves the synthesis and degradation of different proteins, such as tenascin C (TNC) in the adipose tissue (AT). Given the functional relationship between leptin and inducible nitric oxide synthase (iNOS), our aim was to analyze the impact of the absence of the iNOS gene in AT inflammation and ECM remodeling in ob/ob mice. Subjects/Methods: The expression of genes involved in inflammation and ECM remodeling was evaluated in 10-week-old male double knockout (DBKO) mice simultaneously lacking the ob and iNOS genes as well as in ob/ob mice classified into three groups [control, leptin-treated (1 mg kg(-1) day(-1)) and pair-fed]. Results: Leptin deficiency increased inflammation and fibrosis in AT. As expected, leptin treatment improved the obesity phenotype. iNOS deficiency in ob/ob mice improved insulin sensitivity, AT inflammation, and ECM remodeling, as evidenced by lower AT macrophage infiltration and collagen deposition, a downregulation of proinflammatory and profibrogenic genes Tnf, Emr1, Hif1a, Col6a1, Col6a3, and Tnc, as well as lower circulating TNC levels. Interestingly, leptin upregulated TNC expression and release in 3T3-L1 adipocytes, and iNOS knockdown in 3T3-L1 fat cells produced a significant decrease in basal and leptin-induced Tnc expression. Conclusions: Ablation of iNOS in leptin-deficient mice improved AT inflammation and ECM remodeling-related genes, attenuating fibrosis, and metabolic dysfunction. The activation of iNOS by leptin is necessary for the synthesis and secretion of TNC in adipocytes, suggesting an important role of this alarmin in the development of AT inflammation and fibrosis.

Cardiotrophin-1 (CT-1) belongs to the IL-6 family of cytokines. Previous studies of our group revealed that CT-1 is a key regulator of glucose and lipid metabolism. The aim of the present study was to analyze the in vitro and in vivo effects of CT-1 on the production of several adipokines involved in body weight regulation, nutrient metabolism, and inflammation. For this purpose, 3T3-L1 adipocytes were incubated with recombinant protein CT-1 (rCT-1) (1-40 ng/ml) for 1 and 18 h. Moreover, the acute effects of rCT-1 administration (0.2 mg/kg, i.v.) for 30 min and 3 h on adipokines levels were also evaluated in high-fat fed obese mice. In 3T3-L1 adipocytes, rCT-1 treatment downregulated the expression and secretion of leptin, resistin, and visfatin. However, rCT-1 significantly stimulated apelin mRNA and secretion. rCT-1 (18 h) also promoted the activation by phosphorylation of AKT, ERK 1/2, and STAT3. Interestingly, pretreatment with the PI3K inhibitor LY294002 reversed the stimulatory effects of rCT-1 on apelin expression, suggesting that this pathway could be mediating the effects of rCT-1 on apelin production. In contrast, acute administration of rCT-1 (30 min and 3 h) to diet-induced obese mice downregulated leptin and resistin, without significantly modifying apelin or visfatin mRNA in adipose tissue. Furthermore, CT-1 null mice exhibited altered expression of adipokines in adipose tissue. The present study demonstrates that rCT-1 modulates the production of adipokines in vitro and in vivo, suggesting that the regulation of the secretory function of adipocytes could be involved in the metabolic actions of this cytokine. (C) 2016 Wiley Periodicals, Inc.

The beneficial actions of n-3 fatty acids on obesity-induced insulin resistance and inflammation have been related to the synthesis of specializedproresolving lipid mediators (SPMs) like resolvins.The aimof this study was to evaluate the ability of one of these SPMs, maresin 1 (MaR1), to reverse adipose tissue inflammation and/or insulin resistance in twomodels of obesity: diet-induced obese (DIO)mice and genetic (ob/ob) obesemice. In DIO mice, MaR1 (2 mg/kg; 10 d) reduced F4/80-positive cells and expression of the proinflammatory M1 macrophage phenotype marker Cd11c in white adipose tissue (WAT). Moreover, MaR1 decreased Mcp-1, Tnf-a, and Il-1b expression, upregulated adiponectin and Glut-4, and increasedAkt phosphorylation inWAT.MaR1 administration (2 mg/kg; 20 d) to ob/ob mice did not modify macrophage recruitment but increased the M2 macrophage markers Cd163 and Il-10.MaR1 reduced Mcp-1, Tnf-a, Il-1b, andDpp-4 and increased adiponectin gene expression inWAT. MaR1treatment also improved the insulin tolerance test of ob/ob mice and increased Akt andAMPKphosphorylation in WAT. These data suggest that treatment with MaR1 can counteract the dysfunctional inflamed WAT and could be useful to improve insulin sensitivity in murine models of obesity.

Cardiotrophin (CT)-1 is a regulator of glucose and lipid homeostasis. In the present study, we analyzed whether CT-1 also acts to peripherally regulate metabolic rhythms and adipose tissue core clock genes in mice. Moreover, the circadian pattern of plasma CT-1 levels was evaluated in normal-weight and overweight subjects. The circadian rhythmicity of oxygen consumption rate (Vo(2)) was disrupted in aged obese CT-1-deficient (CT-1(-/-)) mice (12 mo). Although circadian rhythms of Vo(2) were conserved in young lean CT-1(-/-) mice (2 mo), CT-1 deficiency caused a phase shift of the acrophase. Most of the clock genes studied (Clock, Bmal1, and Per2) displayed a circadian rhythm in adipose tissue of both wild-type (WT) and CT-1(-/-) mice. However, the pattern was altered in CT-1(-/-) mice toward a lower percentage of the rhythm or lower amplitude, especially for Bmal1 and Clock. Moreover, CT-1 mRNA levels in adipose tissue showed significant circadian fluctuations in young WT mice. In humans, CT-1 plasma profile exhibited a 24-h circadian rhythm in normal-weight but not in overweight subjects. The 24-h pattern of CT-1 was characterized by a pronounced increase during the night (from 02:00 to 08:00). These observations suggest a potential role for CT-1 in the regulation of metabolic circadian rhythms.-Lopez-Yoldi, M., Stanhope, K. L., Garaulet, M., Chen, X. G., Marcos-Gomez, B., Carrasco-Benso, M. P., Santa Maria, E. M., Escote, X., Lee, V., Nunez, M. V., Medici, V., Martinez-Anso, E., Sainz, N., Huerta, A. E., Laiglesia, L. M., Prieto, J., Martinez, J. A., Bustos, M., Havel, P. J., Moreno-Aliaga, M. J. Role of cardiotrophin-1 in the regulation of metabolic circadian rhythms and adipose core clock genes in mice and characterization of 24-h circulating CT-1 profiles in normal-weight and overweight/obese 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 ¿M) 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-1¿) 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-¿ 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.

Background: The proinflammatory state induced by obesity plays an important role in obesity-related metabolic complications. Objective: Our objective was to evaluate whether dietary supplementation with ¿-lipoic acid (LA) and eicosapentaenoic acid (EPA), separately or in combination, could improve inflammatory and cardiovascular disease risk markers in healthy overweight or obese women consuming an energy-restricted diet. Methods: Within the context of the Effects of Lipoic Acid and Eicosapentaenoic Acid in Human Obesity (OBEPALIP) study, Caucasian women (n = 73) aged 20¿50 y with a BMI (in kg/m2) between 27.5 and 40 consumed an energy-restricted diet for 10 wk after being randomly assigned to 1 of 4 parallel experimental groups: a control group or groups supplemented with 1.3 g EPA/d, 0.3 g LA/d, or both. Secondary outcomes were measured at baseline and at the end of the study. These included circulating inflammatory [C-reactive protein (CRP), adiponectin, interleukin 6 (IL-6), chemerin, haptoglobin, amyloid A, and leukocytes] and cardiovascular disease risk markers (platelet count and circulating apelin, asymmetric dimethylarginine, vascular endothelial growth factor, and plasminogen activator inhibitor 1). Gene expression of IL6, adhesion G protein¿coupled receptor E1 (ADGRE1), interleukin 10 (IL10), chemokine (C¿C motif) ligand 2, and adiponectin was measured in subcutaneous abdominal adipose tissue biopsies at endpoint. Results: Supplementation with LA caused a greater reduction in some circulating inflammatory risk markers, such as CRP (¿0.13 ± 0.07 mg/dL compared with 0.06 ± 0.07 mg/dL, P < 0.05) and leukocyte count (¿0.74 ± 0.18 × 103/mm3 compared with 0.06 ± 0.18 × 103/mm3, P < 0.01), than in the groups that were not supplemented with LA. In contrast, the fall in apelin concentrations that accompanied weight loss was less pronounced in groups that were supplemented with LA (¿1.1 ± 4.9 pg/mL) than in those that were not (¿21.3 ± 4.8 pg/mL, P < 0.01). In adipose tissue, compared with those who did not receive EPA, EPA-supplemented groups exhibited a downregulation of ADGRE1 (0.7 ± 0.1¿fold compared with 1.0 ± 0.1¿fold) (P < 0.05) and an upregulation of IL10 (1.8 ± 0.2¿fold compared with 1.0 ± 0.2¿fold) (P < 0.05) gene expression. Conclusions: Dietary supplementation with LA improves some systemic inflammatory and cardiovascular disease¿related risk markers in healthy overweight or obese women independently of weight loss, whereas EPA modulates inflammation-related genes in adipose tissue. This trial was registered at clinicaltrials.gov as NCT01138774.

Irisin is a myokine/adipokine with potential role in obesity and diabetes. The objectives of the present study were to analyse the relationship between irisin and glucose metabolism at baseline and during an oral glucose tolerance test (OGTT) and to determine the effects of eicosapentaenoic acid (EPA) and/or alpha-lipoic acid treatment on irisin production in cultured human adipocytes and in vivo in healthy overweight/obese women following a weight loss program. Seventy-three overweight/obese women followed a 30 % energy-restricted diet supplemented without (control) or with EPA (1.3 g/day), alpha-lipoic acid (0.3 g/day) or both EPA + alpha-lipoic acid (1.3 + 0.3 g/day) during 10 weeks. An OGTT was performed at baseline. Moreover, human adipocytes were treated with EPA (100-200 mu M) or alpha-lipoic acid (100-250 mu M) during 24 h. At baseline plasma, irisin circulating levels were positively associated with glucose levels; however, serum irisin concentrations were not affected by the increment in blood glucose or insulin during the OGTT. Treatment with alpha-lipoic acid (250 mu M) upregulated Fndc5 messenger RNA (mRNA) and irisin secretion in cultured adipocytes. In overweight/obese women, irisin circulating levels decreased significantly after weight loss in all groups, while no additional differences were induced by EPA or alpha-lipoic acid supplementation. Moreover, plasma irisin levels were positively associated with higher glucose concentrations at beginning and at endp

Taken together, our results provide evidence for a regulatory role of leptin on FNDC5/irisin, favoring muscle accretion but reducing fat browning

Matrix metalloproteinases (MMPs), a family of endopeptidases that are involved in the degradation of extracellular matrix components, have been implicated in skeletal muscle regeneration. Among the MMPs, MMP-2 and MMP-9 are upregulated in Duchenne muscular dystrophy (DMD), a fatal X-linked muscle disorder. However, inhibition or overexpression of specific MMPs in a mouse model of DMD (mdx) has yielded mixed results regarding disease progression, depending on the MMP studied. Here, we have examined the role of MMP-10 in muscle regeneration during injury and muscular dystrophy. We found that skeletal muscle increases MMP-10 protein expression in response to damage (notexin) or disease (mdx mice), suggesting its role in muscle regeneration. In addition, we found that MMP-10-deficient muscles displayed impaired recruitment of endothelial cells, reduced levels of extracellular matrix proteins, diminished collagen deposition, and decreased fiber size, which collectively contributed to delayed muscle regeneration after injury. Also, MMP-10 knockout in mdx mice led to a deteriorated dystrophic phenotype. Moreover, MMP-10 mRNA silencing in injured muscles (wild-type and mdx) reduced muscle regeneration, while addition of recombinant human MMP-10 accelerated muscle repair, suggesting that MMP-10 is required for efficient muscle regeneration. Furthermore, our data suggest that MMP-10-mediated muscle repair is associated with VEGF/Akt signaling. Thus, our findings indicate that MMP-10 is

Osteopontin (OPN) is a multifunctional extracellular matrix (ECM) protein involved in multiple physiological processes. OPN expression is dramatically increased in visceral adipose tissue in obesity and the lack of OPN protects against the development of insulin resistance and inflammation in mice. We sought to unravel the potential mechanisms involved in the beneficial effects of the absence of OPN. We analyzed the effect of the lack of OPN in the development of obesity and hepatic steatosis induced by a high-fat diet (HFD) using OPN-KO mice. OPN expression was upregulated in epididymal white adipose tissue (EWAT) and liver in wild type (WT) mice with HFD. OPN-KO mice had higher insulin sensitivity, lower body weight and fat mass with reduced adipose tissue ECM remodeling and reduced adipocyte size than WT mice under a HFD. Reduced MMP2 and MMP9 activity was involved in the decreased ECM remodeling. Crown-like structure number in EWAT as well as F4/80-positive cells and Emr1 expression in EWAT and liver increased with HFD, while OPN-deficiency blunted the increase. Moreover, our data show for the first time that OPN-KO under a HFD mice display reduced fibrosis in adipose tissue and liver, as well as reduced oxidative stress in adipose tissue. Gene expression of collagens Col1a1, Col6a1 and Col6a3 in EWAT and liver, as well as the profibrotic cytokine Tgfb1 in EWAT were increased with HFD, while OPN-deficiency prevented this increase. OPN deficiency prevented hepatic steatosis via reduction in the expression of molecules involved in the onset of fat accumulation such as Pparg, Srebf1, Fasn, Mogat1, Dgat2 and Cidec. Furthermore, OPN-KO mice exhibited higher body temperature and improved BAT function. The present data reveal novel mechanisms of OPN in the development of obesity, pointing out the inhibition of OPN as a promising target for the treatment of obesity and fatty liver.

The CXCR4/SDF1 axis participates in various cellular processes, including cell migration, which is essential for skeletal muscle repair. Although increasing evidence has confirmed the role of CXCR4/SDF1 in embryonic muscle development, the function of this pathway during adult myogenesis remains to be fully elucidated. In addition, a role for CXCR4 signaling in muscle maintenance and repair has only recently emerged. Here, we have demonstrated that CXCR4 and stromal cell-derived factor-1 (SDF1) are up-regulated in injured muscle, suggesting their involvement in the repair process. In addition, we found that notexin-damaged muscles showed delayed muscle regeneration on treatment with CXCR4 agonist (AMD3100). Accordingly, small-interfering RNA-mediated silencing of SDF1 or CXCR4 in injured muscles impaired muscle regeneration, whereas the addition of SDF1 ligand accelerated repair. Furthermore, we identified that CXCR4/SDF1-regulated muscle repair was dependent on matrix metalloproteinase-10 (MMP-10) activity. Thus, our findings support a model in which MMP-10 activity modulates CXCR4/SDF1 signaling, which is essential for efficient skeletal muscle regeneration.

OBJECTIVE: alfa-Lipoic acid (alfa-LA) is a natural occurring antioxidant with beneficial effects on obesity. The aim of this study was to investigate the putative effects of alfa-LA on triglyceride accumulation and lipogenesis in subcutaneous adipocytes from overweight/obese subjects and to determine the potential mechanisms involved. METHODS: Fully differentiated human subcutaneous adipocytes were treated with alfa-LA (100 and 250 µM) during 24 h for studying triglyceride content, de novo lipogenesis, and levels of key lipogenic enzymes. The involvement of AMP-activated protein kinase (AMPK) activation was also evaluated. RESULTS: alfa-LA down-regulated triglyceride content by inhibiting fatty acid esterification and de novo lipogenesis. These effects were mediated by reduction in fatty acid synthase (FAS), stearoyl-coenzyme A desaturase 1, and diacylglycerol O-acyltransferase 1 protein levels. Interestingly, alfa-LA increased AMPK and acetyl CoA carboxylase phosphorylation, while the presence of the AMPK inhibitor Compound C reversed the inhibition observed on FAS protein levels. CONCLUSIONS: alfa-LA down-regulates key lipogenic enzymes, inhibiting lipogenesis and reducing triglyceride accumulation through the activation of AMPK signaling pathway in human subcutaneous adipocytes from overweight/obese subjects.

Leptin and nitric oxide (NO) are implicated in the control of energy homeostasis. The aim of the present study was to examine the impact of the absence of the inducible NO synthase (iNOS) gene on the regulation of energy balance in ob/ob mice analyzing the changes in gene expression levels in brown adipose tissue (BAT). Double knockout (DBKO) mice simultaneously lacking the ob and iNOS genes were generated and the expression of genes involved in energy balance including fatty acid and glucose metabolism as well as mitochondrial genes were analyzed by microarrays. DBKO mice exhibited an improvement in energy balance with a decrease in body weight (P < 0.001), total fat pads (P < 0.05) and food intake (P < 0.05) as well as an enhancement in BAT function as compared to ob/ob mice. To better understand the molecular events associated with this improvement, BAT gene expression was analyzed. Of particular interest, gene expression levels of the key subunit of the Mediator complex Med1 was upregulated (P < 0.05) in DBKO mice. Real-Time PCR and immunohistochemistry further confirmed this data. Med1 is implicated in adipogenesis, lipid metabolic and biosynthetic processes, glucose metabolism and mitochondrial metabolic pathways. Med1 plays an important role in the transcriptional control of genes implicated in energy homeostasis, suggesting that the improvement in energy balance and BAT function of the DBKO mice is mediated, at least in part, through the transcription coactivator Med1.

Objective The gut-derived hormone, ghrelin, improves cardiac function in healthy individuals and patients with chronic heart failure. The aim of this study was to investigate whether the major isoforms of the hormone, acylated and desacyl ghrelin, are related to inappropriate left ventricular mass in patients with the metabolic syndrome (MetS). Methods and results Plasma concentrations of ghrelin forms were measured in 180 white participants (65 normal weight, 60 obese without MetS and 55 obese with MetS; 56% men). MetS was defined according to Adult Treatment Panel III criteria. The presence of left ventricular hypertrophy (LVH) was diagnosed by sex-specific left ventricular mass/height(2.7) cut-off values (> 49.2 g/m(2.7) for men and > 46.7 g/m(2.7) for women). Circulating concentrations of acylated ghrelin were increased in obesity and MetS, whereas desacyl ghrelin levels were decreased. Compared with participants in the lowest tertiles, the age-adjusted and sex-adjusted odds of having MetS were lower in the highest category of desacyl ghrelin (odds ratio 0.1, 95% confidence interval 0.1-0.4, P < 0.001). The prevalence of LVH was increased in the highest tertile of acylated ghrelin (odds ratio 3.4, 95% confidence interval 1.7-5.6, P < 0.05). Plasma acylated ghrelin was increased (P < 0.05) in patients with MetS exhibiting LVH compared with those with appropriate left ventricular mass, whereas plasma desacyl ghrelin was not changed (P = 0.490). Conclusion Acylated ghrelin was positively associated with SBP and left ventricular mass indices, even after correction for BMI. These results suggest that the increased acylated ghrelin concentrations may represent a compensatory mechanism to overcome the development of hypertension and LVH in patients with MetS.

BACKGROUND: Leptin and nitric oxide (NO) on their own participate in the control of non-shivering thermogenesis. However, the functional interplay between both factors in this process has not been explored so far. Therefore, the aim of the present study was to analyze the impact of the absence of the inducible NO synthase (iNOS) gene in the regulation of energy balance in ob/ob mice. METHODS AND FINDINGS: Double knockout (DBKO) mice simultaneously lacking the ob and iNOS genes were generated, and the expression of molecules involved in the control of brown fat cell function was analyzed by real-time PCR, western-blot and immunohistochemistry. Twelve week-old DBKO mice exhibited reduced body weight (p<0.05), decreased amounts of total fat pads (p<0.05), lower food efficiency rates (p<0.05) and higher rectal temperature (p<0.05) than ob/ob mice. Ablation of iNOS also improved the carbohydrate and lipid metabolism of ob/ob mice. DBKO showed a marked reduction in the size of brown adipocytes compared to ob/ob mutants. In this sense, in comparison to ob/ob mice, DBKO rodents showed an increase in the expression of PR domain containing 16 (Prdm16), a transcriptional regulator of brown adipogenesis. Moreover, iNOS deletion enhanced the expression of mitochondria-related proteins, such as peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc-1alpha), sirtuin-1 (Sirt-1) and sirtuin-3 (Sirt-3). Accordingly, mitochondrial uncoupling proteins 1 and 3 (Ucp-1 and Ucp-3) were upregulated in brown adipose tissue (BAT) of DBKO mice as compared to ob/ob rodents. CONCLUSION: Ablation of iNOS improved the energy balance of ob/ob mice by decreasing food efficiency through an increase in thermogenesis. These effects may be mediated, in part, through the recovery of the BAT phenotype and brown fat cell function improvement.

Obese leptin-deficient ob/ob mice exhibit a low-grade chronic inflammation together with a low muscle mass. Our aim was to analyze the changes in muscle expression levels of genes related to oxidative stress and inflammatory responses in leptin deficiency and to identify the effect of in vivo leptin administration. Ob/ob mice were divided in three groups as follows: control ob/ob, leptin-treated ob/ob (1 mg/kg/d) and leptin pair-fed ob/ob mice. Gastrocnemius weight was lower in control ob/ob than in wild type mice (P < .01) exhibiting an increase after leptin treatment compared to control and pair-fed (P < .01) ob/ob animals. Thiobarbituric acid reactive substances, markers of oxidative stress, were higher in serum (P < .01) and gastrocnemius (P = .05) of control ob/ob than in wild type mice and were significantly decreased (P < .01) by leptin treatment. Leptin deficiency altered the expression of 1,546 genes, while leptin treatment modified the regulation of 1,127 genes with 86 of them being involved in oxidative stress, immune defense and inflammatory response. Leptin administration decreased the high expression of Crybb1, Hspb3, Hspb7, Mt4, Cat, Rbm9, Serpinc1 and Serpinb1a observed in control ob/ob mice, indicating that it improves inflammation and muscle loss