Silvia Lorente-Cebrián

Role of omega-3 fatty acids in obesity, metabolic syndrome, and cardiovascular diseases: a review of the evidence

The present review aims to illustrate current knowledge about the efficacy of omega-3 long-chain polyunsaturated fatty acids (n−3 LC-PUFAs) in treating/preventing several metabolic pathologies. We reviewed systematically the published evidence on the effectiveness of n−3 LC-PUFAs fish consumption or n−3 LC-PUFAs supplementation on prevention/treatment of obesity, metabolic syndrome, and cardiovascular diseases. Most of the reviewed studies were randomized-controlled interventional trials, although some relevant prospective and cross-sectional studies as well as some meta-analysis were also reviewed. Supplementation with n−3 LC-PUFAs might improve some obesity-associated metabolic syndrome features such as insulin resistance, hypertension and dyslipidemia by decreasing plasma triglycerides. Moreover, the blood pressure-lowering and anti-inflammatory properties of these fatty acids and their benefits in vascular function might confer cardioprotection. However, the efficacy of n−3 LC-PUFA on reducing myocardial infarction, arrhythmia, cardiac and sudden death, or stroke is controversial. Due to the beneficial actions of n−3 LC-PUFAs, several worldwide government and health organizations have established some recommendations of n−3 LC-PUFAs intake for groups of population. In general, the recommended levels for diseases prevention are lower than those advised for particular treatments. However, more clinical trials are necessary to recommend the most effective dosages and formulas (type of n−3 LC-PUFA, EPA/DHA ratio) for specific pathologies.

Regulation of adipokine secretion by n-3 fatty acids.

Obesity leads to several chronic morbidities including type 2 diabetes, dyslipidaemia, atherosclerosis and hypertension, which are major components of the metabolic syndrome. White adipose tissue (WAT) metabolism and WAT-derived factors (fatty acids and adipokines) play an important role in the development of these metabolic disturbances. In fact, dysregulated adipokine secretion from the expanded WAT of obese individuals contributes to the development of systemic low-grade inflammation, insulin resistance and metabolic syndrome. The n-3 PUFA EPA and DHA have been widely reported to have protective effects in a range of chronic inflammatory conditions including obesity. In fact, n-3 PUFA have been shown to ameliorate low-grade inflammation in adipose tissue associated with obesity and up-regulate mitochondrial biogenesis and induce beta-oxidation in WAT in mice. Moreover, the ability of n-3 PUFA to regulate adipokine gene expression and secretion has been observed both in vitro and in vivo in rodents and human subjects. The present article reviews: (1) the physiological role of adiponectin, leptin and pre-B cell colony-enhancer factor/visfatin, three adipokines with immune-modulatory properties involved in the regulation of metabolism and insulin sensitivity and (2) the actions of n-3 PUFA on these adipokines focusing on the underlying mechanisms and the potential relationship with the beneficial effects of these fatty acids on obesity-associated metabolic disorders. It can be concluded that the ability of n-3 PUFA to improve obesity and insulin resistance conditions partially results from the modulation of WAT metabolism and the secretion of bioactive adipokines including leptin, adiponectin and visfatin.

Eicosapentaenoic acid stimulates AMP-activated protein kinase and increases visfatin secretion in cultured murine adipocytes

Visfatin is an adipokine highly expressed in visceral AT (adipose tissue) of humans and rodents, the production of which seems to be dysregulated in excessive fat accumulation and conditions of insulin resistance. EPA (eicosapentaenoic acid), an n-3 PUFA (polyunsaturated fatty acid), has been demonstrated to exert beneficial effects in obesity and insulin resistance conditions, which have been further linked to its reported ability to modulate adipokine production by adipocytes. TNF-alpha (tumour necrosis factor-alpha) is a pro-inflammatory cytokine whose production is increased in obesity and is involved in the development of insulin resistance. Control of adipokine production by some insulin-sensitizing compounds has been associated with the stimulation of AMPK (AMP-activated protein kinase). The aim of the present study was to examine in vitro the effects of EPA on visfatin production and the potential involvement of AMPK both in the absence or presence of TNF-alpha. Treatment with the pro-inflammatory cytokine TNF-alpha (1 ng/ml) did not modify visfatin gene expression and protein secretion in primary cultured rat adipocytes. However, treatment of these primary adipocytes with EPA (200 mumol/l) for 24 h significantly increased visfatin secretion (P<0.001) and mRNA gene expression (P<0.05). Moreover, the stimulatory effect of EPA on visfatin secretion was prevented by treatment with the AMPK inhibitor Compound C, but not with the PI3K (phosphoinositide 3-kinase) inhibitor LY294002. Similar results were observed in 3T3-L1 adipocytes. Moreover, EPA strongly stimulated AMPK phosphorylation alone or in combination with TNF-alpha in 3T3-L1 adipocytes and pre-adipocytes. The results of the present study suggest that the stimulatory action of EPA on visfatin production involves AMPK activation in adipocytes.

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

Adiponectin, a hormone produced by adipocytes, is involved in glucose metabolism and insulin sensitivity. The production of this adipokine is impaired in obesity and insulin resistance. Eicosapentaenoic acid (EPA) is a dietary n-3 polyunsaturated fatty acid that improves insulin sensitivity in several models of obesity and diabetes, which has been suggested to be related to adiponectin induction. An increase in adiponectin production has been also associated with an up-regulation of the transcriptional factor PPARγ. The aim of this trial was to evaluate the direct effects of EPA on adiponectin gene expression and protein secretion in isolated rat adipocytes as well as to explore the potential mechanisms involved. A comparative study with troglitazone, a PPARγ agonist, was also performed. For these purposes, primary rat adipocytes were cultured with EPA (100 and 200 μM) and with troglitazone (10 μM) for 96 hours. Both EPA and troglitazone improved glucose utilization by adipocytes. As expected, troglitazone enhanced adiponectin secretion and increased PPARγ gene expression. However, EPA significantly decreased adiponectin gene expression and protein secretion and reduced PPARγ mRNA levels, suggesting that the inhibition of adiponectin by EPA is likely to be secondary to the down-regulation of this adipogenic transcription factor. Moreover, these results suggest that other mechanisms different from the direct stimulation of adiponectin by the fatty acid are underlying the insulin-sensitizing properties observed after EPA treatment in vivo.ResumenLa adiponectina es una hormona producida por los adipocitos que esta implicada en el metabolismo de la glucosa y la sensibilidad a insulina. Situaciones de obesidad y resistencia insulínica se han asociado con hipoadiponectinemia. El ácido eicosapentaenoico (EPA) es un acido graso poliinsaturado omega-3 que mejora la sensibilidad a la insulina en diversos modelos de obesidad y diabetes, y se ha sugerido que esto podría estar relacionado con un incremento en la producción de adiponectina. Por otra parte, se ha observado que la activación del factor de transcripción PPARγ induce aumento en la producción de adiponectina. El objetivo del presente trabajo fue evaluar los efectos directos del EPA sobre la expresión génica y la secreción de adiponectina en adipocitos primarios de rata, así como investigar los potenciales mecanismos implicados. Además, se realizó un estudio comparativo con las acciones de la troglitazona, un agonista de PPARγ. Los adipocitos primarios de rata se cultivaron con EPA (100 y 200 μM) o con troglitazona (10 μM) durante 96 horas. Ambos tratamientos mejoraron la utilización de glucosa y como era de esperar, la troglitazona incrementó la secreción de adiponectina y la expresión de PPARγ. Sin embargo, el EPA disminuyó significativamente la secreción y expresión génica de adiponectina, además de reducir los niveles de ARNm de PPARγ, sugiriendo que la inhibición de la adiponectina causada por el EPA podría ser secundaria a la reducción del factor de transcripción PPARγ. Estos hallazgos sugieren que las propiedades insulino-sensibilizadoras observadas tras los tratamientos con EPA in vivo no derivan del incremento directo de la adiponectina.

An update on the role of omega-3 fatty acids on inflammatory and degenerative diseases

Inflammation is involved in the pathophysiology of many chronic diseases, such as rheumatoid arthritis and neurodegenerative diseases. Several studies have evidenced important anti-inflammatory and immunomodulatory properties of omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs). This review illustrates current knowledge about the efficacy of n-3 LC-PUFAs (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), particularly) in preventing and/or treating several chronic inflammatory conditions (inflammatory bowel diseases and rheumatoid arthritis) as well as their potential benefits on neurodegenerative diseases. It is well established that n-3 LC-PUFAs are substrates for synthesis of novel series of lipid mediators (e.g., resolvins, protectins, and maresins) with potent anti-inflammatory and pro-resolving properties, which have been proposed to partly mediate the protective and beneficial actions of n-3 LC-PUFAs. Here, we briefly summarize current knowledge from preclinical studies analyzing the actions of EPA- and DHA-derived resolvins and protectins on pathophysiological models of rheumatoid arthritis, Alzheimer, and irritable bowel syndrome.

Eicosapentaenoic acid up-regulates apelin secretion and gene expression in 3T3-L1 adipocytes

Recent studies have shown the ability of apelin to restore glucose tolerance in obese and insulin-resistant mice. Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) from the omega-3 family that has many beneficial effects in obesity-linked disorders. The aim of this study was to examine in vitro the effects of EPA on apelin secretion and gene expression in mature 3T3-L1 adipocytes. Treatment with EPA (100 and 200 microM) significantly increased basal (p<0.01) and insulin-stimulated (p<0.001) apelin secretion and gene expression in adipocytes. EPA also stimulated Akt phosphorylation, a down-stream target of phosphatidylinositol 3-kinase (PI3K), in 3T3-L1 adipocytes. Moreover, treatment with the PI3K inhibitor LY294002 completely blocked EPA-stimulatory action on apelin mRNA gene expression (p<0.001), but not modified the stimulatory effect of EPA on basal apelin secretion. Furthermore, the stimulatory effect of EPA on basal apelin release was also observed in the presence of Actinomycin D and Cycloheximide, suggesting that EPA might also regulate apelin secretion by via post-transcriptional mechanisms. These findings suggest that the mechanisms mediating EPA-induced apelin synthesis and/or secretion are complex, involving steps that are PI3K dependent and steps that are PI3K independent.

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

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

Eicosapentaenoic acid inhibits tumour necrosis factor-α-induced lipolysis in murine cultured adipocytes

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid with beneficial effects in obesity and insulin resistance. High levels of proinflammatory cytokine tumour necrosis factor-α (TNF-α) in obesity promote lipolysis in adipocytes, leading to the development of insulin resistance. Thus, the aims of the present study were to analyze the potential antilipolytic properties of EPA on cytokine-induced lipolysis and to investigate the possible mechanisms involved. The EPA effects on basal and TNF-α-induced lipolysis were determined in both primary rat and 3T3-L1 adipocytes. Treatment of primary rat adipocytes with EPA (100 and 200 μM) significantly decreased basal glycerol release (P<.01) and prevented cytokine-induced lipolysis in a dose-dependent manner (P<.001). Moreover, EPA decreased TNF-α-induced activation of nuclear factor-κB and extracellular-related kinase 1/2 phosphorylation. In addition, the antilipolytic action of EPA was stimulated by the AMP-kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside and blocked by the AMPK-inhibitor compound C. Moreover, we found that EPA stimulated hormone-sensitive lipase (HSL) phosphorylation on serine-565, which further supports the involvement of AMPK in EPAs antilipolytic actions. Eicosapentaenoic acid treatment (24 h), alone and in the presence of TNF-α, also decreased adipose triglyceride lipase (ATGL) protein content in cultured adipocytes. However, oral supplementation with EPA for 35 days was able to partially reverse the down-regulation of HSL and ATGL messenger RNA observed in retroperitoneal adipose tissue of high-fat-diet-fed rats. These findings suggest that EPA inhibits proinflammatory cytokine-induced lipolysis in adipocytes. This effect might contribute to explain the insulin-sensitizing properties of EPA.

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

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

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.