p27Kip1 Deficiency Impairs Brown Adipose Tissue Function Favouring Fat Accumulation in Mice.

The aim of this work was to investigate the effect of the whole-body deletion of p27 on the activity of brown adipose tissue and the susceptibility to develop obesity and glucose homeostasis disturbances in mice, especially when subjected to a high fat diet. p27 knockout (p27-/-) and wild type (WT) mice were fed a normal chow diet or a high fat diet (HFD) for 10-weeks. Body weight and composition were assessed. Insulin and glucose tolerance tests and indirect calorimetry assays were performed. Histological analysis of interscapular BAT (iBAT) was carried out, and expression of key genes/proteins involved in BAT function were characterized by qPCR and Western blot. iBAT activity was estimated by 18F-fluorodeoxyglucose (18FDG) uptake with microPET. p27-/- mice were more prone to develop obesity and insulin resistance, exhibiting increased size of all fat depots. p27-/- mice displayed a higher respiratory exchange ratio. iBAT presented larger adipocytes in p27-/- HFD mice, accompanied by downregulation of both Glut1 and uncoupling protein 1 (UCP1) in parallel with defective insulin signalling. Moreover, p27-/- HFD mice exhibited impaired response to cold exposure, characterized by a reduced iBAT 18FDG uptake and difficulty to maintain body temperature when exposed to cold compared to WT HFD mice, suggesting reduced thermogenic capacity. These data suggest that p27 could play a role in BAT activation and in the susceptibility to develop obesity and insulin resistance.

Changes in brown adipose tissue lipid mediator signatures with aging, obesity, and DHA supplementation in female mice.

Brown adipose tissue (BAT) dysfunction in aging and obesity has been related to chronic unresolved inflammation, which could be mediated by an impaired production of specialized proresolving lipid mediators (SPMs), such as Lipoxins-LXs, Resolvins-Rvs, Protectins-PDs, and Maresins-MaRs. Our aim was to characterize the changes in BAT SPMs signatures and their association with BAT dysfunction during aging, especially under obesogenic conditions, and their modulation by a docosahexaenoic acid (DHA)-rich diet. Lipidomic, functional, and molecular studies were performed in BAT of 2- and 18-month-old lean (CT) female mice and in 18-month-old diet-induced obese (DIO) mice fed with a high-fat diet (HFD), or a DHA-enriched HFD. Aging downregulated Prdm16 and UCP1 levels, especially in DIO mice, while DHA partially restored them. Arachidonic acid (AA)-derived LXs and DHA-derived MaRs and PDs were the most abundant SPMs in BAT of young CT mice. Interestingly, the sum of LXs and of PDs were significantly lower in aged DIO mice compared to young CT mice. Some of the SPMs most significantly reduced in obese-aged mice included LXB4 , MaR2, 4S,14S-diHDHA, 10S,17S-diHDHA (a.k.a. PDX), and RvD6. In contrast, DHA increased DHA-derived SPMs, without modifying LXs. However, MicroPET studies showed that DHA was not able to counteract the impaired cold exposure response in BAT of obese-aged mice. Our data suggest that a defective SPMs production could underlie the decrease of BAT activity observed in obese-aged mice, and highlight the relevance to further characterize the physiological role and therapeutic potential of specific SPMs on BAT development and function.

Regulation of p27 and Cdk2 Expression in Different Adipose Tissue Depots in Aging and Obesity.

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.

Basolateral presence of the proinflammatory cytokine tumor necrosis factor -α and secretions from adipocytes and macrophages reduce intestinal sugar transport.

We have previously demonstrated in Caco-2 cells that tumor necrosis factor-α (TNF-α) 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-α from abdominal adipose tissue (adipocytes and macrophages) would decrease sugar and amino acid transport acting from the basolateral membrane of the enterocytes. TNF-α placed in the basal compartment of Caco-2 cells decreased α-methyl- d-glucose (αMG) 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-α, IL-1ß, and MCP1 expression in Caco-2 cells exposed to basal TNF-α. Similarly, αMG 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 αMG uptake as well. Diet-induced obese mice showed an increase in the visceral adipose tissue surrounding the intestine. In this physiological condition, there was a reduction on αMG uptake in jejunal everted rings. Altogether, these results suggest that basolateral TNF-α, which can be produced by adipocytes and macrophages during obesity, would be able to activate TNF-α and other proinflammatory proteins expression in the small intestine and diminish intestinal sugar and amino acids transport.

Maresin 1 inhibits TNF-alpha-induced lipolysis and autophagy in 3T3-L1 adipocytes.

Obesity is associated with high levels of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), 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-α on lipolysis and autophagy in mature 3T3-L1 adipocytes. Our data revealed that MaR1 (1-100 nM) inhibited the TNF-α-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-α. 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-α-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.

Maresin 1 improves insulin sensitivity and attenuates adipose tissue inflammation in ob/ob and diet-induced obese mice.

The beneficial actions of n-3 fatty acids on obesity-induced insulin resistance and inflammation have been related to the synthesis of specialized proresolving lipid mediators (SPMs) like resolvins. The aim of 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 two models of obesity: diet-induced obese (DIO) mice and genetic (ob/ob) obese mice. In DIO mice, MaR1 (2 µg/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-α, and Il-1ß expression, upregulated adiponectin and Glut-4, and increased Akt phosphorylation in WAT. MaR1 administration (2 µg/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-α, Il-1ß, and Dpp-4 and increased adiponectin gene expression in WAT. MaR1 treatment also improved the insulin tolerance test of ob/ob mice and increased Akt and AMPK phosphorylation 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.-Martínez-Fernández, L., González-Muniesa, P., Laiglesia, L. M., Sáinz, N., Prieto-Hontoria, P. L., Escoté, X., Odriozola, L., Corrales, F. J., Arbones-Mainar, J. M., Martínez, J. A., Moreno-Aliaga, M. J. Maresin 1 improves insulin sensitivity and attenuates adipose tissue inflammation in ob/ob and diet-induced obese mice.

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.

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 (Vo2) was disrupted in aged obese CT-1-deficient (CT-1-/-) mice (12 mo). Although circadian rhythms of Vo2 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.-López-Yoldi, M., Stanhope, K. L., Garaulet, M., Chen, X. G., Marcos-Gómez, B., Carrasco-Benso, M. P., Santa Maria, E. M., Escoté, X., Lee, V., Nunez, M. V., Medici, V., Martínez-Ansó, E., Sáinz, N., Huerta, A. E., Laiglesia, L. M., Prieto, J., Martínez, 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.

Cardiotrophin-1 Regulates Adipokine Production in 3T3-L1 Adipocytes and Adipose Tissue From Obese Mice.

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, pre-treatment 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. J. Cell. Physiol. 232: 2469-2477, 2017. © 2016 Wiley Periodicals, Inc.

MMP-10 is required for efficient muscle regeneration in mouse models of injury and muscular dystrophy.

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 critical for skeletal muscle maintenance and regeneration during injury and disease.

Supplementation with α-Lipoic Acid Alone or in Combination with Eicosapentaenoic Acid Modulates the Inflammatory Status of Healthy Overweight or Obese Women Consuming an Energy-Restricted Diet.

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 α-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.

SIRT1 and FOXO1 role on MASLD risk: effects of DHA-rich n-3 PUFA supplementation and exercise in aged obese female mice and in post-menopausal overweight/obese women.

Sirtuins 1 (SIRT1) and Forkhead box protein O1 (FOXO1) expression have been associated with obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Exercise and/or docosahexaenoic acid (DHA) supplementation have shown beneficial effects on MASLD. The current study aims to assess the relationships between Sirt1, Foxo1 mRNA levels and several MASLD biomarkers, as well as the effects of DHA-rich n-3 PUFA supplementation and/or exercise in the steatotic liver of aged obese female mice, and in peripheral blood mononuclear cells (PBMCs) of postmenopausal women with overweight/obesity. In the liver of 18-month-old mice, Sirt1 levels positively correlated with the expression of genes related to fatty acid oxidation, and negatively correlated with lipogenic and proinflammatory genes. Exercise (long-term treadmill training), especially when combined with DHA, upregulated hepatic Sirt1 mRNA levels. Liver Foxo1 mRNA levels positively associated with hepatic triglycerides (TG) content and the expression of lipogenic and pro-inflammatory genes, while negatively correlated with the lipolytic gene Hsl. In PBMCs of postmenopausal women with overweight/obesity, FOXO1 mRNA expression negatively correlated with the hepatic steatosis index (HSI) and the Zhejiang University index (ZJU). After 16-weeks of DHA-rich PUFA supplementation and/or progressive resistance training (RT), most groups exhibited reduced MASLD biomarkers and risk indexes accompanying with body fat mass reduction, but no significant changes were found between the intervention groups. However, in PBMCs n-3 supplementation upregulated FOXO1 expression, and the RT groups exhibited higher SIRT1 expression. In summary, SIRT1 and FOXO1 could be involved in the beneficial mechanisms of exercise and n-3 PUFA supplementation related to MASLD manifestation.

Intestinal Permeability, Gut Inflammation, and Gut Immune System Response Are Linked to Aging-Related Changes in Gut Microbiota Composition: A Study in Female Mice.

Aging entails changes at the cellular level that increase the risk of various pathologies. An association between gut microbiota and age-related diseases has also been attributed. This study aims to analyze changes in fecal microbiota composition and their association with genes related to immune response, gut inflammation, and intestinal barrier impairment. Fecal samples of female mice at different ages (2 months, 6 months, 12 months, and 18 months) and gene expression in colon tissue were analyzed. Results showed that the older mice group had a more diverse microbiota than the younger group. Additionally, the abundance of Cyanobacteria, Proteobacteria, Flavobacteriaceae, Bacteroides, Parabacteroides, Prevotellaceae_UCG-001, Akkermansia, and Parabacteroides goldsteinii increased with age. In contrast, there was a notable decline in Clostridiaceae, Lactobacillaceae, Monoglobaceae, Ligilactobacillus, Limosilactobacillus, Mucispirillum, and Bacteroides faecichinchillae. These bacteria imbalances were positively correlated with increased inflammation markers in the colon, including Tnf-α, Ccl2, and Ccl12, and negatively with the expression of tight junction genes (Jam2, Tjp1, and Tjp2), as well as immune response genes (Cd4, Cd72, Tlr7, Tlr12, and Lbp). In conclusion, high levels of diversity did not result in improved health in older mice; however, the imbalance in bacteria abundance that occurs with aging might contribute to immune senescence, inflammation, and leaky gut disease.

Chronic docosahexaenoic acid supplementation improves metabolic plasticity in subcutaneous adipose tissue of aged obese female mice.

This study aimed to characterize the potential beneficial effects of chronic docosahexaenoic acid (DHA) supplementation on restoring subcutaneous white adipose tissue (scWAT) plasticity in obese aged female mice. Two-month-old female C57BL/6J mice received a control (CT) or a high fat diet (HFD) for 4 months. Then, 6-month-old diet-induced obese (DIO) mice were distributed into the DIO and the DIOMEG group (fed with a DHA-enriched HFD) up to 18 months. In scWAT, the DHA-enriched diet reduced the mean adipocyte size and reversed the upregulation of lipogenic genes induced by the HFD, reaching values even lower than those observed in CT animals. DIO mice exhibited an up-regulation of lipolytic and fatty oxidation gene expressions that was reversed in DHA-supplemented mice except for Cpt1a mRNA levels, which were higher in DIOMEG as compared to CT mice. DHA restored the increase of proinflammatory genes observed in scWAT of DIO mice. While no changes were observed in total macrophage F4/80+/CD11b+ content, the DHA treatment switched scWAT macrophages profile by reducing the M1 marker Cd11c and increasing the M2 marker CD206. These events occurred alongside with a stimulation of beige adipocyte specific genes, the restoration of UCP1 and pAKT/AKT ratio, and a recovery of the HFD-induced Fgf21 upregulation. In summary, DHA supplementation induced a metabolic remodeling of scWAT to a healthier phenotype in aged obese mice by modulating genes controlling lipid accumulation in adipocytes, reducing the inflammatory status, and inducing beige adipocyte markers in obese aged mice.

Maresin 1 Exerts a Tissue-Specific Regulation of Adipo-Hepato-Myokines in Diet-Induced Obese Mice and Modulates Adipokine Expression in Cultured Human Adipocytes in Basal and Inflammatory Conditions.

This study analyses the effects of Maresin 1 (MaR1), a docosahexaenoic acid (DHA)-derived specialized proresolving lipid mediator with anti-inflammatory and insulin-sensitizing actions, on the expression of adipokines, including adiponectin, leptin, dipeptidyl peptidase 4 (DPP-4), cardiotrophin-1 (CT-1), and irisin (FNDC5), both in vitro and in in vivo models of obesity. The in vivo effects of MaR1 (50 µg/kg, 10 days, oral gavage) were evaluated in epididymal adipose tissue (eWAT), liver and muscle of diet-induced obese (DIO) mice. Moreover, two models of human differentiated primary adipocytes were incubated with MaR1 (1 and 10 nM, 24 h) or with a combination of tumor necrosis factor-α (TNF-α, 100 ng/mL) and MaR1 (1-200 nM, 24 h) and the expression and secretion of adipokines were measured in both models. MaR1-treated DIO mice exhibited an increased expression of adiponectin and Ct-1 in eWAT, increased expression of Fndc5 and Ct-1 in muscle and a decreased expression of hepatic Dpp-4. In human differentiated adipocytes, MaR1 increased the expression of ADIPONECTIN, LEPTIN, DPP4, CT-1 and FNDC5. Moreover, MaR1 counteracted the downregulation of ADIPONECTIN and the upregulation of DPP-4 and LEPTIN observed in adipocytes treated with TNF-α. Differential effects for TNF-α and MaR1 on the expression of CT-1 and FNDC5 were observed between both models of human adipocytes. In conclusion, MaR1 reverses the expression of specific adipomyokines and hepatokines altered in obese mice in a tissue-dependent manner. Moreover, MaR1 regulates the basal expression of adipokines in human adipocytes and counteracts the alterations of adipokines expression induced by TNF-α in vitro. These actions could contribute to the metabolic benefits of this lipid mediator.

Differential remodeling of subcutaneous white and interscapular brown adipose tissue by long-term exercise training in aged obese female mice.

Obesity exacerbates aging-induced adipose tissue dysfunction. This study aimed to investigate the effects of long-term exercise on inguinal white adipose tissue (iWAT) and interscapular brown adipose tissue (iBAT) of aged obese mice. Two-month-old female mice received a high-fat diet for 4 months. Then, six-month-old diet-induced obese animals were allocated to sedentarism (DIO) or to a long-term treadmill training (DIOEX) up to 18 months of age. In exercised mice, iWAT depot revealed more adaptability, with an increase in the expression of fatty acid oxidation genes (Cpt1a, Acox1), and an amelioration of the inflammatory status, with a favorable modulation of pro/antiinflammatory genes and lower macrophage infiltration. Additionally, iWAT of trained animals showed an increment in the expression of mitochondrial biogenesis (Pgc1a, Tfam, Nrf1), thermogenesis (Ucp1), and beige adipocytes genes (Cd137, Tbx1). In contrast, iBAT of aged obese mice was less responsive to exercise. Indeed, although an increase in functional brown adipocytes genes and proteins (Pgc1a, Prdm16 and UCP1) was observed, few changes were found on inflammation-related and fatty acid metabolism genes. The remodeling of iWAT and iBAT depots occurred along with an improvement in the HOMA index for insulin resistance and in glucose tolerance. In conclusion, long-term exercise effectively prevented the loss of iWAT and iBAT thermogenic properties during aging and obesity. In iWAT, the long-term exercise program also reduced the inflammatory status and stimulated a fat-oxidative gene profile. These exercise-induced adipose tissue adaptations could contribute to the beneficial effects on glucose homeostasis in aged obese mice.

Maresin 1 activates brown adipose tissue and promotes browning of white adipose tissue in mice.

OBJECTIVE: Maresin 1 (MaR1) is a docosahexaenoic acid-derived proresolving lipid mediator with insulin-sensitizing and anti-steatosis properties. Here, we aim to unravel MaR1 actions on brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning. METHODS: MaR1 actions were tested in cultured murine brown adipocytes and in human mesenchymal stem cells (hMSC)-derived adipocytes. In vivo effects of MaR1 were tested in diet-induced obese (DIO) mice and lean WT and Il6 knockout (Il6-/-) mice. RESULTS: In cultured differentiated murine brown adipocytes, MaR1 reduces the expression of inflammatory genes, while stimulates glucose uptake, fatty acid utilization and oxygen consumption rate, along with the upregulation of mitochondrial mass and genes involved in mitochondrial biogenesis and function and the thermogenic program. In Leucine Rich Repeat Containing G Protein-Coupled Receptor 6 (LGR6)-depleted brown adipocytes using siRNA, the stimulatory effect of MaR1 on thermogenic genes was abrogated. In DIO mice, MaR1 promotes BAT remodeling, characterized by higher expression of genes encoding for master regulators of mitochondrial biogenesis and function and iBAT thermogenic activation, together with increased M2 macrophage markers. In addition, MaR1-treated DIO mice exhibit a better response to cold-induced BAT activation. Moreover, MaR1 induces a beige adipocyte signature in inguinal WAT of DIO mice and in hMSC-derived adipocytes. MaR1 potentiates Il6 expression in brown adipocytes and BAT of cold exposed lean WT mice. Interestingly, the thermogenic properties of MaR1 were abrogated in Il6-/- mice. CONCLUSIONS: These data reveal MaR1 as a novel agent that promotes BAT activation and WAT browning by regulating thermogenic program in adipocytes and M2 polarization of macrophages. Moreover, our data suggest that LGR6 receptor is mediating MaR1 actions on brown adipocytes, and that IL-6 is required for the thermogenic effects of MaR1.

Transcriptional analysis of brown adipose tissue in leptin-deficient mice lacking inducible nitric oxide synthase: evidence of the role of Med1 in energy balance.

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 compared with 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.

Changes Induced by Aging and Long-Term Exercise and/or DHA Supplementation in Muscle of Obese Female Mice.

Obesity and aging promote chronic low-grade systemic inflammation. The aim of the study was to analyze the effects of long-term physical exercise and/or omega-3 fatty acid Docosahexaenoic acid (DHA) supplementation on genes or proteins related to muscle metabolism, inflammation, muscle damage/regeneration and myokine expression in aged and obese mice. Two-month-old C57BL/6J female mice received a control or a high-fat diet for 4 months. Then, the diet-induced obese (DIO) mice were distributed into four groups: DIO, DIO + DHA, DIO + EX (treadmill training) and DIO + DHA + EX up to 18 months. Mice fed a control diet were sacrificed at 2, 6 and 18 months. Aging increased the mRNA expression of Tnf-α and decreased the expression of genes related to glucose uptake (Glut1, Glut4), muscle atrophy (Murf1, Atrogin-1, Cas-9) and myokines (Metrnl, Il-6). In aged DIO mice, exercise restored several of these changes. It increased the expression of genes related to glucose uptake (Glut1, Glut4), fatty acid oxidation (Cpt1b, Acox), myokine expression (Fndc5, Il-6) and protein turnover, decreased Tnf-α expression and increased p-AKT/AKT ratio. No additional effects were observed when combining exercise and DHA. These data suggest the effectiveness of long-term training to prevent the deleterious effects of aging and obesity on muscle dysfunction.

Deficiency of MMP-10 Aggravates the Diseased Phenotype of Aged Dystrophic Mice.

Matrix metalloproteinases (MMPs) have been implicated in the progression of muscular dystrophy, and recent studies have reported the role of MMP-10 in skeletal muscle pathology of young dystrophic mice. Nevertheless, its involvement in dystrophin-deficient hearts remains unexplored. Here, we aimed to investigate the involvement of MMP-10 in the progression of severe muscular dystrophy and to characterize MMP-10 loss in skeletal and cardiac muscles of aged dystrophic mice. We examined the histopathological effect of MMP-10 ablation in aged mdx mice, both in the hind limb muscles and heart tissues. We found that MMP-10 loss compromises survival rates of aged mdx mice, with skeletal and cardiac muscles developing a chronic inflammatory response. Our findings indicate that MMP-10 is implicated in severe muscular dystrophy progression, thus identifying a new area of research that could lead to future therapies for dystrophic muscles.

Effects of Long-Term DHA Supplementation and Physical Exercise on Non-Alcoholic Fatty Liver Development in Obese Aged Female Mice.

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, Tnfα, 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.