Adipose tissue-specific ablation of PGC-1ß impairs thermogenesis in brown fat.

Impaired thermogenesis observed in mice with whole-body ablation of peroxisome proliferator-activated receptor-γ coactivator-1ß (PGC-1ß; officially known as PPARGC1B) may result from impaired brown fat (brown adipose tissue; BAT) function, but other mechanism(s) could be involved. Here, using adipose-specific PGC-1ß knockout mice (PGC-1ß-AT-KO mice) we aimed to learn whether specific PGC-1ß ablation in adipocytes is sufficient to drive cold sensitivity. Indeed, we found that warm-adapted (30°C) mutant mice were relatively sensitive to acute cold exposure (6°C). When these mice were subjected to cold exposure for 7 days (7-day-CE), adrenergic stimulation of their metabolism was impaired, despite similar levels of thermogenic uncoupling protein 1 in BAT in PGC-1ß-AT-KO and wild-type mice. Gene expression in BAT of mutant mice suggested a compensatory increase in lipid metabolism to counteract the thermogenic defect. Interestingly, a reduced number of contacts between mitochondria and lipid droplets associated with low levels of L-form of optic atrophy 1 was found in BAT of PGC-1ß-AT-KO mice. These genotypic differences were observed in warm-adapted mutant mice, but they were partially masked by 7-day-CE. Collectively, our results suggest a role for PGC-1ß in controlling BAT lipid metabolism and thermogenesis. This article has an associated First Person interview with the first author of the paper.

Additive Effects of Omega-3 Fatty Acids and Thiazolidinediones in Mice Fed a High-Fat Diet: Triacylglycerol/Fatty Acid Cycling in Adipose Tissue.

Long-chain n-3 polyunsaturated fatty acids (Omega-3) and anti-diabetic drugs thiazolidinediones (TZDs) exhibit additive effects in counteraction of dietary obesity and associated metabolic dysfunctions in mice. The underlying mechanisms need to be clarified. Here, we aimed to learn whether the futile cycle based on the hydrolysis of triacylglycerol and re-esterification of fatty acids (TAG/FA cycling) in white adipose tissue (WAT) could be involved. We compared Omega-3 (30 mg/g diet) and two different TZDs-pioglitazone (50 mg/g diet) and a second-generation TZD, MSDC-0602K (330 mg/g diet)-regarding their effects in C57BL/6N mice fed an obesogenic high-fat (HF) diet for 8 weeks. The diet was supplemented or not by the tested compound alone or with the two TZDs combined individually with Omega-3. Activity of TAG/FA cycle in WAT was suppressed by the obesogenic HF diet. Additive effects in partial rescue of TAG/FA cycling in WAT were observed with both combined interventions, with a stronger effect of Omega-3 and MSDC-0602K. Our results (i) supported the role of TAG/FA cycling in WAT in the beneficial additive effects of Omega-3 and TZDs on metabolism of diet-induced obese mice, and (ii) showed differential modulation of WAT gene expression and metabolism by the two TZDs, depending also on Omega-3.

Omega-3 index in the Czech Republic: No difference between urban and rural populations.

Naturally occurring long-chain omega-3 PUFA such as eicosapentaenoic acid (EPA; 20:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3) exert multiple effects on health, which are related to the intake of these lipids in the diet and correlate with the levels of omega-3 PUFA in the body. These levels are reflected by the omega-3 PUFA index, i.e. the EPA and DHA content as % of all fatty acids in red blood cells. The aim of this study was to evaluate omega-3 index in the Czech Republic, using blood samples collected from the capital city (n = 476) and the rural region (n = 388). The mean omega-3 index was 3.56 mol % with a maximal value of 8.10% and a minimal value of 1.12%. There was no difference in the index value between rural and urban / industrial regions, but this value was higher in subjects who reported eating fish or omega-3 PUFA supplements. In conclusion, the results indicated suboptimal values of the omega-3 index in the Czech population independent of the sampling region.

Cell type-specific modulation of lipid mediator's formation in murine adipose tissue by omega-3 fatty acids.

Mutual interactions between adipocytes and immune cells in white adipose tissue (WAT) are involved in modulation of lipid metabolism in the tissue and also in response to omega-3 polyunsaturated fatty acids (PUFA), which counteract adverse effects of obesity. This complex interplay depends in part on in situ formed anti- as well as pro-inflammatory lipid mediators, but cell types engaged in the synthesis of the specific mediators need to be better characterized. We used tissue fractionation and metabolipidomic analysis to identify cells producing lipid mediators in epididymal WAT of mice fed for 5 weeks obesogenic high-fat diet (lipid content 35% wt/wt), which was supplemented or not by omega-3 PUFA (4.3 mg eicosapentaenoic acid and 14.7 mg docosahexaenoic acid per g of diet). Our results demonstrate selective increase in levels of anti-inflammatory lipid mediators in WAT in response to omega-3, reflecting either their association with adipocytes (endocannabinoid-related N-docosahexaenoylethanolamine) or with stromal vascular cells (pro-resolving lipid mediator protectin D1). In parallel, tissue levels of obesity-associated pro-inflammatory endocannabinoids were suppressed. Moreover, we show that adipose tissue macrophages (ATMs), which could be isolated using magnetic force from the stromal vascular fraction, are not the major producers of protectin D1 and that omega-3 PUFA lowered lipid load in ATMs while promoting their less-inflammatory phenotype. Taken together, these results further document specific roles of various cell types in WAT in control of WAT inflammation and metabolism and they suggest that also other cells but ATMs are engaged in production of pro-resolving lipid mediators in response to omega-3 PUFA.

Combined intervention with pioglitazone and n-3 fatty acids in metformin-treated type 2 diabetic patients: improvement of lipid metabolism.

BACKGROUND: The marine n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exert numerous beneficial effects on health, but their potency to improve treatment of type 2 diabetic (T2D) patients remains poorly characterized. We aimed to evaluate the effect of a combination intervention using EPA + DHA and the insulin-sensitizing drug pioglitazone in overweight/obese T2D patients already treated with metformin. METHODS: In a parallel-group, four-arm, randomized trial, 69 patients (66 % men) were assigned to 24-week-intervention using: (i) corn oil (5 g/day; Placebo), (ii) pioglitazone (15 mg/day; Pio), (iii) EPA + DHA concentrate (5 g/day, containing ~2.8 g EPA + DHA; Omega-3), or (iv) pioglitazone and EPA + DHA concentrate (Pio& Omega-3). Data from 60 patients were used for the final evaluation. At baseline and after intervention, various metabolic markers, adiponectin and cytokines were evaluated in serum using standard procedures, EPA + DHA content in serum phospholipids was evaluated using shotgun lipidomics and mass spectrometry, and hyperinsulinemic-euglycemic clamp and meal test were also performed. Indirect calorimetry was conducted after the intervention. Primary endpoints were changes from baseline in insulin sensitivity evaluated using hyperinsulinemic-euglycemic clamp and in serum triacylglycerol concentrations in fasting state. Secondary endpoints included changes in fasting glycemia and glycated hemoglobin (HbA1c), changes in postprandial glucose, free fatty acid and triacylglycerol concentrations, metabolic flexibility assessed by indirect calorimetry, and inflammatory markers. RESULTS: Omega-3 and Pio& Omega-3 increased EPA + DHA content in serum phospholipids. Pio and Pio& Omega-3 increased body weight and adiponectin levels. Both fasting glycemia and HbA1c were increased by Omega-3, but were unchanged by Pio& Omega-3. Insulin sensitivity was not affected by Omega-3, while it was improved by Pio& Omega-3. Fasting triacylglycerol concentrations and inflammatory markers were not significantly affected by any of the interventions. Lipid metabolism in the meal test and metabolic flexibility were additively improved by Pio& Omega-3. CONCLUSION: Besides preventing a modest negative effect of n-3 fatty acids on glycemic control, the combination of pioglitazone and EPA + DHA can be used to improve lipid metabolism in T2D patients on stable metformin therapy. TRIAL REGISTRATION: EudraCT number 2009-011106-42.

Lipid signaling in adipose tissue: Connecting inflammation & metabolism.

Obesity-associated low-grade inflammation of white adipose tissue (WAT) contributes to development of insulin resistance and other disorders. Accumulation of immune cells, especially macrophages, and macrophage polarization from M2 to M1 state, affect intrinsic WAT signaling, namely anti-inflammatory and proinflammatory cytokines, fatty acids (FA), and lipid mediators derived from both n-6 and n-3 long-chain PUFA such as (i) arachidonic acid (AA)-derived eicosanoids and endocannabinoids, and (ii) specialized pro-resolving lipid mediators including resolvins derived from both eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), lipoxins (AA metabolites), protectins and maresins (DHA metabolites). In this respect, potential differences in modulating adipocyte metabolism by various lipid mediators formed by inflammatory M1 macrophages typical of obese state, and non-inflammatory M2 macrophages typical of lean state remain to be established. Studies in mice suggest that (i) transient accumulation of M2 macrophages could be essential for the control of tissue FA levels during activation of lipolysis, (ii) currently unidentified M2 macrophage-borne signaling molecule(s) could inhibit lipolysis and re-esterification of lipolyzed FA back to triacylglycerols (TAG/FA cycle), and (iii) the egress of M2 macrophages from rebuilt WAT and removal of the negative feedback regulation could allow for a full unmasking of metabolic activities of adipocytes. Thus, M2 macrophages could support remodeling of WAT to a tissue containing metabolically flexible adipocytes endowed with a high capacity of both TAG/FA cycling and oxidative phosphorylation. This situation could be exemplified by a combined intervention using mild calorie restriction and dietary supplementation with EPA/DHA, which enhances the formation of "healthy" adipocytes. This article is part of a Special Issue entitled Oxygenated metabolism of PUFA: analysis and biological relevance."

Fibroblast growth factor-21 and the beneficial effects of long-chain n-3 polyunsaturated fatty acids.

Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) in the diet protect against insulin resistance and obesity. Fibroblast growth factor-21 (Fgf21) is a hormonal factor released mainly by the liver that has powerful anti-diabetic effects. Here, we tested whether the beneficial metabolic effects of LC n-3 PUFA involve the induction of Fgf21. C57BL/6 J mice were exposed to an obesogenic, corn-oil-based, high-fat diet (cHF), or a diet in which corn oil was replaced with a fish-derived LC n-3 PUFA concentrate (cHF + F) using two experimental settings: short-term (3 weeks) and long-term treatment (8 weeks). CHF + F reduced body weight gain, insulinemia, and triglyceridemia compared to cHF. cHF increased plasma Fgf21 levels and hepatic Fgf21 gene expression compared with controls, but these effects were less pronounced or absent in cHF + F-fed mice. In contrast, hepatic expression of peroxisome proliferator-activated receptor (PPAR)-α target genes were more strongly induced by cHF + F than cHF, especially in the short-term treatment setting. The expression of genes encoding Fgf21, its receptors, and Fgf21 targets was unaltered by short-term LC n-3 PUFA treatment, with the exception of Ucp1 (uncoupling protein 1) and adiponectin genes, which were specifically up-regulated in white fat. In the long-term treatment setting, the expression of Fgf21 target genes and receptors was not differentially affected by LC n-3 PUFA. Collectively, our findings indicate that increased Fgf21 levels do not appear to be a major mechanism through which LC n-3 PUFA ameliorates high-fat-diet-associated metabolic disorders.

Oleuropein as an inhibitor of peroxisome proliferator-activated receptor gamma.

Oleuropein, the major phenolic compound found in olive leaves and oil, exerts antioxidant, anti-inflammatory and anti-atherogenic effects and suppresses the adipocyte differentiation in vitro. Herein, we characterized molecular mechanisms underlying the anti-adipogenic effects of oleuropein on 3T3-L1 cells and adipocytes derived from stromal-vascular fraction of dorsolumbar and gonadal fat dissected from mice. We found that oleuropein (>100 µM) decreased viability of preadipocytes proliferating in vitro and did not exerted any cytotoxic effects in post-confluent cells after induction of differentiation. Oleuropein (>100 µM) inhibited adipocyte differentiation, suppressed gene expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-/enhancer-binding protein α, sterol regulatory element-binding transcription factor 1c and fatty acid synthase. Furthermore, we tested ability of oleuropein to regulate of PPARγ-, PPARα- or PPARß-/PPARδ-mediated ß-lactamase expression in appropriate reporter gene assays. Oleuropein between 10 and 400 µM concentrations did not affect activity of PPARα or PPARß/δ. Contrary, PPARγ activity, either basal or rosiglitazone activated, was inhibited by oleuropein. Our data suggest that oleuropein exerts anti-adipogenic effect through direct inhibition of PPARγ transcriptional activity.

Omega-3 phospholipids from fish suppress hepatic steatosis by integrated inhibition of biosynthetic pathways in dietary obese mice.

Non-alcoholic fatty liver disease (NAFLD) accompanies obesity and insulin resistance. Recent meta-analysis suggested omega-3 polyunsaturated fatty acids DHA and EPA to decrease liver fat in NAFLD patients. Antiinflammatory, hypolipidemic, and insulin-sensitizing effects ofDHA/EPA depend on their lipid form, with marine phospholipids showing better efficacy than fish oils. We characterized the mechanisms underlying beneficial effects of DHA/EPA phospholipids, alone or combined with an antidiabetic drug, on hepatosteatosis. C57BL/6N mice were fed for 7 weeks an obesogenic high-fat diet (cHF) or cHF-based interventions: (i) cHF supplemented with phosphatidylcholine-rich concentrate from herring (replacing 10% of dietary lipids; PC), (ii) cHF containing rosiglitazone (10 mg/kg diet; R), or (iii) PC + R. Metabolic analyses, hepatic gene expression and lipidome profiling were performed. Results showed that PC and PC + R prevented cHlF-induced weight gain and glucose intolerance, while all interventions reduced abdominal fat and plasma triacylglycerols. PC and PC + R also lowered hepatic and plasma cholesterol and reduced hepatosteatosis. Microarray analysis revealed integrated downregulation of hepatic lipogenic and cholesterol biosynthesis pathways by PC, while R-induced lipogenesis was fully counteracted in PC + R Gene expression changes in PC and PC + R were associated with preferential enrichment of hepatic phosphatidylcholine and phosphatidylethanolamine fractions by DHA/EPA. The complex downregulation of hepatic lipogenic and cholesterol biosynthesis genes and the antisteatotic effects were unique to DHA/EPA-containing phospholipids, since they were absent in mice fed soy-derived phosphatidylcholine. Thus, inhibition of lipid and cholesterol biosynthesis associated with potent antisteatotic effects in the liver in response to DHA/EPA-containing phospholipids support their use in NAFLD prevention and treatment.

Fibroblast growth factor-21 is expressed in neonatal and pheochromocytoma-induced adult human brown adipose tissue.

OBJECTIVE: In rodents, brown (BAT) and white (WAT) adipose tissues are targets and expression sites for fibroblast growth factor-21 (FGF21). In contrast, human WAT expresses negligible levels of FGF21. We examined FGF21 expression in human BAT samples, including the induced BAT found in adult patients with pheochromocytoma, and interscapular and visceral BAT from newborns. METHODS: The expression of FGF21 and uncoupling protein-1 (UCP1, a brown adipocyte marker), was determined by quantitative real-time-PCR and immunoblotting. The transcript levels of marker genes for developmentally-programmed BAT (zinc-finger-protein of the cerebellum-1, ZIC1) and inducible-BAT (cluster of differentiation-137, CD137) were also determined. RESULTS: FGF21 and UCP1 are significantly expressed in visceral adipose tissue from pheochromocytoma patients, but not in visceral fat from healthy individuals. In neonates, FGF21 and UCP1 are both expressed in visceral and interscapular fat, and their expression levels show a significant positive correlation. Marker gene expression profiles suggest that inducible BAT is present in visceral fat from pheochromocytoma patients and neonates, whereas developmentally-programmed BAT is present in neonatal interscapular fat. CONCLUSIONS: Human BAT, but not WAT, expresses FGF21. The expression of FGF21 is especially high in inducible, also called beige/brite, neonatal BAT, but it is also found in the interscapular, developmentally-programmed, BAT of neonates.

Stimulation of mitochondrial oxidative capacity in white fat independent of UCP1: a key to lean phenotype.

We are facing a revival of the strategy to counteract obesity and associated metabolic disorders by inducing thermogenesis mediated by mitochondrial uncoupling protein-1 (UCP1). Thus, the main focus is on the adaptive non-shivering thermogenesis occurring both in the typical depots of brown adipose tissue (BAT) and in UCP1-containing cells that could be induced in white adipose tissue (WAT). Because contribution of WAT to resting metabolic rate is relatively small, the possibility to reduce adiposity by enhancing energy expenditure in classical white adipocytes is largely neglected. However, several pieces of evidence support a notion that induction of energy expenditure based on oxidation of fatty acids (FA) in WAT may be beneficial for health, namely: (i) studies in both humans and rodents document negative association between oxidative capacity of mitochondria in WAT and obesity; (ii) pharmacological activation of AMPK in rats as well as cold-acclimation of UCP1-ablated mice results in obesity resistance associated with increased oxidative capacity in WAT; and (iii) combined intervention using long-chain n-3 polyunsaturated FA (omega 3) and mild calorie restriction exerted synergism in the prevention of obesity in mice fed a high-fat diet; this was associated with strong hypolipidemic and insulin-sensitizing effects, as well as prevention of inflammation, and synergistic induction of mitochondrial oxidative phosphorylation (OXPHOS) and FA oxidation, specifically in epididymal WAT. Importantly, these changes occurred without induction of UCP1 and suggested the involvement of: (i) futile substrate cycle in white adipocytes, which is based on lipolysis of intracellular triacylglycerols and re-esterification of FA, in association with the induction of mitochondrial OXPHOS capacity, ß-oxidation, and energy expenditure; (ii) endogenous lipid mediators (namely endocannabinoids, eicosanoids, prostanoids, resolvins, and protectins) and their cognate receptors; and (iii) AMP-activated protein kinase in WAT. Quantitatively, the strong induction of FA oxidation in WAT in response to the combined intervention is similar to that observed in the transgenic mice rendered resistant to obesity by ectopic expression of UCP1 in WAT. The induction of UCP1-independent FA oxidation and energy expenditure in WAT in response to the above physiological stimuli could underlie the amelioration of obesity and low-grade WAT inflammation, and it could reduce the release of FA from adipose tissue and counteract harmful consequences of lipid accumulation in other tissues. In this respect, new combination treatments may be designed using naturally occurring micronutrients (e.g. omega 3), reduced calorie intake or pharmaceuticals, exerting synergism in the induction of the mitochondrial OXPHOS capacity and stimulation of lipid catabolism in white adipocytes, and improving metabolic flexibility of WAT. The role of mutual interactions between adipocytes and immune cells contained in WAT in tissue metabolism should be better characterised. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.

Preservation of metabolic flexibility in skeletal muscle by a combined use of n-3 PUFA and rosiglitazone in dietary obese mice.

Insulin resistance, the key defect in type 2 diabetes (T2D), is associated with a low capacity to adapt fuel oxidation to fuel availability, i.e., metabolic inflexibility. This, in turn, contributes to a further damage of insulin signaling. Effectiveness of T2D treatment depends in large part on the improvement of insulin sensitivity and metabolic adaptability of the muscle, the main site of whole-body glucose utilization. We have shown previously in mice fed an obesogenic high-fat diet that a combined use of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and thiazolidinediones (TZDs), anti-diabetic drugs, preserved metabolic health and synergistically improved muscle insulin sensitivity. We investigated here whether n-3 LC-PUFA could elicit additive beneficial effects on metabolic flexibility when combined with a TZD drug rosiglitazone. Adult male C57BL/6N mice were fed an obesogenic corn oil-based high-fat diet (cHF) for 8 weeks, or randomly assigned to various interventions: cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids (cHF+F), cHF with 10 mg rosiglitazone/kg diet (cHF+ROSI), cHF+F+ROSI, or chow-fed. Indirect calorimetry demonstrated superior preservation of metabolic flexibility to carbohydrates in response to the combined intervention. Metabolomic and gene expression analyses in the muscle suggested distinct and complementary effects of the interventions, with n-3 LC-PUFA supporting complete oxidation of fatty acids in mitochondria and the combination with n-3 LC-PUFA and rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism. These beneficial metabolic effects were associated with the activation of the switch between glycolytic and oxidative muscle fibers, especially in the cHF+F+ROSI mice. Our results further support the idea that the combined use of n-3 LC-PUFA and TZDs could improve the efficacy of the therapy of obese and diabetic patients.

Unmasking differential effects of rosiglitazone and pioglitazone in the combination treatment with n-3 fatty acids in mice fed a high-fat diet.

Combining pharmacological treatments and life style interventions is necessary for effective therapy of major diseases associated with obesity, which are clustered in the metabolic syndrome. Acting via multiple mechanisms, combination treatments may reduce dose requirements and, therefore, lower the risk of adverse side effects, which are usually associated with long-term pharmacological interventions. Our previous study in mice fed high-fat diet indicated additivity in preservation of insulin sensitivity and in amelioration of major metabolic syndrome phenotypes by the combination treatment using n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and rosiglitazone, i.e. an anti-diabetic drug of the thiazolidinedione (TZD) family. We investigated here whether pioglitazone, a TZD-drug in clinical use, could elicit the additive beneficial effects when combined with n-3 LC-PUFA. Adult male mice (C57BL/6N) were fed an obesogenic corn oil-based high-fat diet (cHF) for 8 weeks, or randomly assigned to various dietary treatments (i) cHF+F, cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids; (ii) cHF+ROSI, cHF with 10 mg rosiglitazone/kg diet; (iii) cHF+F+ROSI; (iv) cHF+PIO, cHF with 50 mg pioglitazone/kg diet; and (v) cHF+F+PIO, or chow-fed. Plasma concentrations of 163 metabolites were evaluated using a targeted metabolomics approach. Both TZDs preserved glucose homeostasis and normal plasma lipid levels while inducing adiponectin, with pioglitazone showing better effectiveness. The beneficial effects of TZDs were further augmented by the combination treatments. cHF+F+ROSI but not cHF+F+PIO counteracted development of obesity, in correlation with inducibility of fatty acid ß-oxidation, as revealed by the metabolomic analysis. By contrast, only cHF+F+PIO eliminated hepatic steatosis and this treatment also reversed insulin resistance in dietary obese mice. Our results reveal differential effects of rosiglitazone and pioglitazone, unmasked in the combination treatment with n-3 LC-PUFA, and support the notion that n-3 LC-PUFA could be used as add-on treatment to TZDs in order to improve diabetic patient's therapy.

The inhibition of fat cell proliferation by n-3 fatty acids in dietary obese mice.

BACKGROUND: Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) of marine origin exert multiple beneficial effects on health. Our previous study in mice showed that reduction of adiposity by LC n-3 PUFA was associated with both, a shift in adipose tissue metabolism and a decrease in tissue cellularity. The aim of this study was to further characterize the effects of LC n-3 PUFA on fat cell proliferation and differentiation in obese mice. METHODS: A model of inducible and reversible lipoatrophy (aP2-Cre-ERT2 PPARγL2/L2 mice) was used, in which the death of mature adipocytes could be achieved by a selective ablation of peroxisome proliferator-activated receptor γ in response to i.p. injection of tamoxifen. Before the injection, obesity was induced in male mice by 8-week-feeding a corn oil-based high-fat diet (cHF) and, subsequently, mice were randomly assigned (day 0) to one of the following groups: (i) mice injected by corn-oil-vehicle only, i.e."control" mice, and fed cHF; (ii) mice injected by tamoxifen in corn oil, i.e. "mutant" mice, fed cHF; (iii) control mice fed cHF diet with15% of dietary lipids replaced by LC n-3 PUFA concentrate (cHF+F); and (iv) mutant mice fed cHF+F. Blood and tissue samples were collected at days 14 and 42. RESULTS: Mutant mice achieved a maximum weight loss within 10 days post-injection, followed by a compensatory body weight gain, which was significantly faster in the cHF as compared with the cHF+F mutant mice. Also in control mice, body weight gain was depressed in response to dietary LC n-3 PUFA. At day 42, body weights in all groups stabilized, with no significant differences in adipocyte size between the groups, although body weight and adiposity was lower in the cHF+F as compared with the cHF mice, with a stronger effect in the mutant than in control mice. Gene expression analysis documented depression of adipocyte maturation during the reconstitution of adipose tissue in the cHF+F mutant mice. CONCLUSION: Dietary LC n-3 PUFA could reduce both hypertrophy and hyperplasia of fat cells in vivo. Results are in agreement with the involvement of fat cell turnover in control of adiposity.

AMP-activated protein kinase α2 subunit is required for the preservation of hepatic insulin sensitivity by n-3 polyunsaturated fatty acids.

OBJECTIVE: The induction of obesity, dyslipidemia, and insulin resistance by high-fat diet in rodents can be prevented by n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). We tested a hypothesis whether AMP-activated protein kinase (AMPK) has a role in the beneficial effects of n-3 LC-PUFAs. RESEARCH DESIGN AND METHODS: Mice with a whole-body deletion of the α2 catalytic subunit of AMPK (AMPKα2(-/-)) and their wild-type littermates were fed on either a low-fat chow, or a corn oil-based high-fat diet (cHF), or a cHF diet with 15% lipids replaced by n-3 LC-PUFA concentrate (cHF+F). RESULTS: Feeding a cHF diet induced obesity, dyslipidemia, hepatic steatosis, and whole-body insulin resistance in mice of both genotypes. Although cHF+F feeding increased hepatic AMPKα2 activity, the body weight gain, dyslipidemia, and the accumulation of hepatic triglycerides were prevented by the cHF+F diet to a similar degree in both AMPKα2(-/-) and wild-type mice in ad libitum-fed state. However, preservation of hepatic insulin sensitivity by n-3 LC-PUFAs required functional AMPKα2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content. Under hyperinsulinemic-euglycemic conditions, AMPKα2 was essential for preserving low levels of both hepatic and plasma triglycerides, as well as plasma free fatty acids, in response to the n-3 LC-PUFA treatment. CONCLUSIONS: Our results show that n-3 LC-PUFAs prevent hepatic insulin resistance in an AMPKα2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity. AMPKα2 is also essential for hypolipidemic and antisteatotic effects of n-3 LC-PUFA under insulin-stimulated conditions.

n-3 PUFA: bioavailability and modulation of adipose tissue function.

Adipose tissue has a key role in the development of metabolic syndrome (MS), which includes obesity, type 2 diabetes, dyslipidaemia, hypertension and other disorders. Systemic insulin resistance represents a major factor contributing to the development of MS in obesity. The resistance is precipitated by impaired adipose tissue glucose and lipid metabolism, linked to a low-grade inflammation of adipose tissue and secretion of pro-inflammatory adipokines. Development of MS could be delayed by lifestyle modifications, while both dietary and pharmacological interventions are required for the successful therapy of MS. The n-3 long-chain (LC) PUFA, EPA and DHA, which are abundant in marine fish, act as hypolipidaemic factors, reduce cardiac events and decrease the progression of atherosclerosis. Thus, n-3 LC PUFA represent healthy constituents of diets for patients with MS. In rodents n-3 LC PUFA prevent the development of obesity and impaired glucose tolerance. The effects of n-3 LC PUFA are mediated transcriptionally by AMP-activated protein kinase and by other mechanisms. n-3 LC PUFA activate a metabolic switch toward lipid catabolism and suppression of lipogenesis, i.e. in the liver, adipose tissue and small intestine. This metabolic switch improves dyslipidaemia and reduces ectopic deposition of lipids, resulting in improved insulin signalling. Despite a relatively low accumulation of n-3 LC PUFA in adipose tissue lipids, adipose tissue is specifically linked to the beneficial effects of n-3 LC PUFA, as indicated by (1) the prevention of adipose tissue hyperplasia and hypertrophy, (2) the induction of mitochondrial biogenesis in adipocytes, (3) the induction of adiponectin and (4) the amelioration of adipose tissue inflammation by n-3 LC PUFA.

Induction of lipid oxidation by polyunsaturated fatty acids of marine origin in small intestine of mice fed a high-fat diet.

BACKGROUND: Dietary polyunsaturated fatty acids (PUFA), in particular the long chain marine fatty acids docosahexaenoic (DHA) and eicosapentaenoic (EPA), are linked to many health benefits in humans and in animal models. Little is known of the molecular response to DHA and EPA of the small intestine, and the potential contribution of this organ to the beneficial effects of these fatty acids. Here, we assessed gene expression changes induced by DHA and EPA in the wildtype C57BL/6J murine small intestine using whole genome microarrays and functionally characterized the most prominent biological process. RESULTS: The main biological process affected based on gene expression analysis was lipid metabolism. Fatty acid uptake, peroxisomal and mitochondrial beta-oxidation, and omega-oxidation of fatty acids were all increased. Quantitative real time PCR, and -- in a second animal experiment -- intestinal fatty acid oxidation measurements confirmed significant gene expression differences and showed in a dose-dependent manner significant changes at biological functional level. Furthermore, no major changes in the expression of lipid metabolism genes were observed in the colon. CONCLUSION: We show that marine n-3 fatty acids regulate small intestinal gene expression and increase fatty acid oxidation. Since this organ contributes significantly to whole organism energy use, this effect on the small intestine may well contribute to the beneficial physiological effects of marine PUFAs under conditions that will normally lead to development of obesity, insulin resistance and diabetes.

Prevention and reversal of obesity and glucose intolerance in mice by DHA derivatives.

The n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exert hypolipidemic effects and prevent development of obesity and insulin resistance in animals fed high-fat diets. We sought to determine the efficacy of alpha-substituted DHA derivatives as lipid-lowering, antiobesity, and antidiabetic agents. C57BL/6 mice were given a corn oil-based high-fat (35% weight/weight) diet (cHF), or cHF with 1.5% of lipids replaced with alpha-methyl DHA ethyl ester (Substance 1), alpha-ethyl DHA ethyl ester (Substance 2), alpha,alpha-di-methyl DHA ethyl ester (Substance 3), or alpha-thioethyl DHA ethyl ester (Substance 4) for 4 months. Plasma markers of glucose and lipid metabolism, glucose tolerance, morphology, tissue lipid content, and gene regulation were characterized. The cHF induced obesity, hyperlipidemia, impairment of glucose homeostasis, and adipose tissue inflammation. Except for Substance 3, all other substances prevented weight gain and Substance 2 exerted the strongest effect (63% of cHF-controls). Glucose intolerance was significantly prevented (~67% of cHF) by both Substance 1 and Substance 2. Moreover, Substance 2 lowered fasting glycemia, plasma insulin, triacylglycerols, and nonesterified fatty acids (73, 9, 47, and 81% of cHF-controls, respectively). Substance 2 reduced accumulation of lipids in liver and skeletal muscle, as well as adipose tissue inflammation associated with obesity. Substance 2 also induced weight loss in dietary obese mice. In contrast to DHA administered either alone or as a component of the EPA/DHA concentrate (replacing 15% of dietary lipids), Substance 2 also reversed established glucose intolerance in obese mice. Thus, Substance 2 represents a novel compound with a promising potential in the treatment of obesity and associated metabolic disturbances.

Cellular and molecular effects of n-3 polyunsaturated fatty acids on adipose tissue biology and metabolism.

Adipose tissue and its secreted products, adipokines, have a major role in the development of obesity-associated metabolic derangements including Type 2 diabetes. Conversely, obesity and its metabolic sequelae may be counteracted by modulating metabolism and secretory functions of adipose tissue. LC-PUFAs (long-chain polyunsaturated fatty acids) of the n-3 series, namely DHA (docosahexaenoic acid; C(22:6n-3)) and EPA (eicosapentaenoic acid; C(20:5n-3)), exert numerous beneficial effects, such as improvements in lipid metabolism and prevention of obesity and diabetes, which partially result from the metabolic action of n-3 LC-PUFAs in adipose tissue. Recent studies highlight the importance of mitochondria in adipose tissue for the maintenance of systemic insulin sensitivity. For instance, both n-3 LC-PUFAs and the antidiabetic drugs TZDs (thiazolidinediones) induce mitochondrial biogenesis and beta-oxidation. The activation of this 'metabolic switch' in adipocytes leads to a decrease in adiposity. Both n-3 LC-PUFAs and TZDs ameliorate a low-grade inflammation of adipose tissue associated with obesity and induce changes in the pattern of secreted adipokines, resulting in improved systemic insulin sensitivity. In contrast with TZDs, which act as agonists of PPARgamma (peroxisome-proliferator-activated receptor-gamma) and promote differentiation of adipocytes and adipose tissue growth, n-3 LC-PUFAs affect fat cells by different mechanisms, including the transcription factors PPARalpha and PPARdelta. Some of the effects of n-3 LC-PUFAs on adipose tissue depend on their active metabolites, especially eicosanoids. Thus treatments affecting adipose tissue by multiple mechanisms, such as combining n-3 LC-PUFAs with either caloric restriction or antidiabetic/anti-obesity drugs, should be explored.

Induction of muscle thermogenesis by high-fat diet in mice: association with obesity-resistance.

The obesogenic effect of a high-fat (HF) diet is counterbalanced by stimulation of energy expenditure and lipid oxidation in response to a meal. The aim of this study was to reveal whether muscle nonshivering thermogenesis could be stimulated by a HF diet, especially in obesity-resistant A/J compared with obesity-prone C57BL/6J (B/6J) mice. Experiments were performed on male mice born and maintained at 30 degrees C. Four-week-old mice were randomly weaned onto a low-fat (LF) or HF diet for 2 wk. In the A/J LF mice, cold exposure (4 degrees C) resulted in hypothermia, whereas the A/J HF, B/6J LF, and B/6J HF mice were cold tolerant. Cold sensitivity of the A/J LF mice was associated with a relatively low whole body energy expenditure under resting conditions, which was normalized by the HF diet. In both strains, the HF diet induced uncoupling protein-1-mediated thermogenesis, with a stronger induction in A/J mice. Only in A/J mice: 1) the HF diet augmented activation of whole body lipid oxidation by cold; and 2) at 30 degrees C, oxygen consumption, total content, and phosphorylation of AMP-activated protein kinase (AMPK), and AICAR-stimulated palmitate oxidation in soleus muscle was increased by the HF diet in parallel with significantly increased leptinemia. Gene expression data in soleus muscle of the A/J HF mice indicated a shift from carbohydrate to fatty acid oxidation. Our results suggest a role for muscle nonshivering thermogenesis and lipid oxidation in the obesity-resistant phenotype of A/J mice and indicate that a HF diet could induce thermogenesis in oxidative muscle, possibly via the leptin-AMPK axis.