Nishan S. Kalupahana

(n-3) Fatty Acids Alleviate Adipose Tissue Inflammation and Insulin Resistance: Mechanistic Insights

Obesity is associated with the metabolic syndrome, a significant risk factor for developing type 2 diabetes and cardiovascular diseases. Chronic low-grade inflammation occurring in the adipose tissue of obese individuals is causally linked to the pathogenesis of insulin resistance and the metabolic syndrome. Although the exact trigger of this inflammatory process is unknown, adipose tissue hypoxia, endoplasmic reticular stress, and saturated fatty acid-mediated activation of innate immune processes have been identified as important processes in these disorders. Furthermore, macrophages and T lymphocytes have important roles in orchestrating this immune process. Although energy restriction leading to weight loss is the primary dietary intervention to reverse these obesity-associated metabolic disorders, other interventions targeted at alleviating adipose tissue inflammation have not been explored in detail. In this regard, (n-3) PUFA of marine origin both prevent and reverse high-fat-diet-induced adipose tissue inflammation and insulin resistance in rodents. We provide an update on the pathogenesis of adipose tissue inflammation and insulin resistance in obesity and discuss potential mechanisms by which (n-3) PUFA prevent and reverse these changes and the implications in human health.

Eicosapentaenoic Acid Prevents and Reverses Insulin Resistance in High-Fat Diet-Induced Obese Mice via Modulation of Adipose Tissue Inflammation

We investigated the effects of eicosapentaenoic acid (EPA) on prevention (P) and reversal (R) of high saturated-fat (HF) diet-induced obesity and glucose-insulin homeostasis. Male C57BL/6J mice were fed low-fat (LF; 10% energy from fat), HF (45% energy from fat), or a HF-EPA-P (45% energy from fat; 36 g/kg EPA) diet for 11 wk. A 4th group was initially fed HF for 6 wk followed by the HF-EPA-R diet for 5 wk. As expected, mice fed the HF diet developed obesity and glucose intolerance. In contrast, mice fed the HF-EPA-P diet maintained normal glucose tolerance despite weight gain compared with the LF group. Whereas the HF group developed hyperglycemia and hyperinsulinemia, both HF-EPA groups (P and R) exhibited normal glycemia and insulinemia. Further, plasma adiponectin concentration was lower in the HF group but was comparable in the LF and HF-EPA groups, suggesting a role of EPA in preventing and improving insulin resistance induced by HF feeding. Further analysis of adipose tissue adipokine levels and proteomic studies in cultured adipocytes indicated that dietary EPA supplementation of HF diets was associated with reduced adipose inflammation and lipogenesis and elevated markers of fatty acid oxidation. In C57BL/6J mice, EPA minimized saturated fat-induced insulin resistance and this is in part mediated by its effects on fatty acid oxidation and inflammation.

Immunity as a link between obesity and insulin resistance.

Obesity is a major public health problem in the United States and worldwide. Further, obesity is causally linked to the pathogenesis of insulin resistance, metabolic syndrome and type-2 diabetes (T2D). A chronic low-grade inflammation occurring in adipose tissue is at least in part responsible for the obesity-induced insulin resistance. This adipose tissue inflammation is characterized by changes in immune cell populations giving rise to altered adipo/cytokine profiles, which in turn induces skeletal muscle and hepatic insulin resistance. Detailed molecular mechanisms of insulin resistance, adipose tissue inflammation and the implications of these findings on therapeutic strategies are discussed in this review.

Health benefits of n-3 polyunsaturated fatty acids: eicosapentaenoic acid and docosahexaenoic acid.

Marine-based fish and fish oil are the most popular and well-known sources of n-3 polyunsaturated fatty acids (PUFAs), namely, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These n-3 PUFAs are known to have variety of health benefits against cardiovascular diseases (CVDs) including well-established hypotriglyceridemic and anti-inflammatory effects. Also, various studies indicate promising antihypertensive, anticancer, antioxidant, antidepression, antiaging, and antiarthritis effects. Moreover, recent studies also indicate anti-inflammatory and insulin-sensitizing effects of these fatty acids in metabolic disorders. Classically, n-3 PUFAs mediate some of these effects by antagonizing n-6 PUFA (arachidonic acid)-induced proinflammatory prostaglandin E₂ (PGE₂) formation. Another well-known mechanism by which n-3 PUFAs impart their anti-inflammatory effects is via reduction of nuclear factor-κB activation. This transcription factor is a potent inducer of proinflammatory cytokine production, including interleukin 6 and tumor necrosis factor-α, both of which are decreased by EPA and DHA. Other evidence also demonstrates that n-3 PUFAs repress lipogenesis and increase resolvins and protectin generation, ultimately leading to reduced inflammation. Finally, beneficial effects of EPA and DHA in insulin resistance include their ability to increase secretion of adiponectin, an anti-inflammatory adipokine. In summary, n-3 PUFAs have multiple health benefits mediated at least in part by their anti-inflammatory actions; thus their consumption, especially from dietary sources, should be encouraged.

The renin-angiotensin system: a link between obesity, inflammation and insulin resistance

The renin‐angiotensin system (RAS) is classically known for its role in regulation of blood pressure, fluid and electrolyte balance. Recently, several local RASs in organs such as brain, heart, pancreas and adipose tissue have also been identified. Evidence from clinical trials suggests that in addition to anti‐hypertensive effects, pharmacological inhibition of RAS also provides protection against the development of type‐2 diabetes. Moreover, animal models with targeted inactivation of RAS genes exhibit improved insulin sensitivity and are protected from high‐fat diet‐induced obesity and insulin resistance. Because there is evidence for RAS overactivation in obesity, it is possible that RAS is a link between obesity and insulin resistance. This review summarizes the evidence and mechanistic insights on the associations between RAS, obesity and insulin resistance, with special emphasis on the role of adipose tissue RAS in the pathogenesis of metabolic derangements in obesity.

Overproduction of angiotensinogen from adipose Tissue Induces adipose Inflammation, Glucose Intolerance, and Insulin Resistance

Although obesity is associated with overactivation of the white adipose tissue (WAT) renin‐angiotensin system (RAS), a causal link between the latter and systemic insulin resistance is not established. We tested the hypothesis that overexpression of angiotensinogen (Agt) from WAT causes systemic insulin resistance via modulation of adipose inflammation. Glucose tolerance, systemic insulin sensitivity, and WAT inflammatory markers were analyzed in mice overexpressing Agt in the WAT (aP2‐Agt mice). Proteomic studies and in vitro studies using 3T3‐L1 adipocytes were performed to build a mechanistic framework. Male aP2‐Agt mice exhibited glucose intolerance, insulin resistance, and lower insulin‐stimulated glucose uptake by the skeletal muscle. The difference in glucose tolerance between genotypes was normalized by high‐fat (HF) feeding, and was significantly improved by treatment with angiotensin‐converting enzyme (ACE) inhibitor captopril. aP2‐Agt mice also had higher monocyte chemotactic protein‐1 (MCP‐1) and lower interleukin‐10 (IL‐10) in the WAT, indicating adipose inflammation. Proteomic studies in WAT showed that they also had higher monoglyceride lipase (MGL) and glycerol‐3‐phosphate dehydrogenase levels. Treatment with angiotensin II (Ang II) increased MCP‐1 and resistin secretion from adipocytes, which was prevented by cotreating with inhibitors of the nuclear factor‐κB (NF‐κB) pathway or nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In conclusion, we show for the first time that adipose RAS overactivation causes glucose intolerance and systemic insulin resistance. The mechanisms appear to be via reduced skeletal muscle glucose uptake, at least in part due to Ang II‐induced, NADPH oxidase and NFκB‐dependent increases in WAT inflammation.

n-3 and n-6 polyunsaturated fatty acids differentially regulate adipose angiotensinogen and other inflammatory adipokines in part via NF-κB-dependent mechanisms

Excessive secretion of proinflammatory adipokines has been linked to metabolic disorders. We have previously documented anti-inflammatory effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) in adipose tissue; however, the mechanisms by which these fatty acids regulate adipokine secretion remain unclear. Here, we determined differential effects of eicosapentaenoic acid (EPA, n-3 PUFA) vs. arachidonic acid (AA, n-6 PUFA) on expression and secretion of angiotensinogen (Agt), interleukin 6 (IL-6) and monocyte chemotactic protein (MCP-1) in 3T3-L1 adipocytes. While both PUFAs increased intracellular Agt protein and mRNA expression, Agt secretion into culture media was increased only by AA treatment, which in turn was prevented by co-treatment with EPA. At various AA/EPA ratios, increasing AA concentrations significantly increased secretion of the above three adipokines, whereas increasing EPA dose-dependently, while lowering AA, decreased their secretion. Moreover, IL-6 and MCP-1 were more significantly reduced by EPA treatment compared to Agt (IL-6>MCP>Agt). Next, we tested whether nuclear factor-κB (NF-κB), a major proinflammatory transcription factor, was involved in regulation of these adipokines by PUFAs. EPA significantly inhibited NF-κB activation compared to control or AA treatments. Moreover, EPA attenuated tumor necrosis factor-α-induced MCP-1 and further reduced its secretion in the presence of an NF-κB inhibitor. Taken together, we reported here novel beneficial effects of EPA in adipocytes. We demonstrated direct anti-inflammatory effects of EPA, which are at least in part due to the inhibitory effects of this n-3 PUFA on the NF-κB pathway in adipocytes. In conclusion, these studies further support beneficial effects of n-3 PUFAs in adipocyte inflammation and metabolic disorders.

The adipose tissue renin-angiotensin system and metabolic disorders: a review of molecular mechanisms

The renin-angiotensin system (RAS) is classically known for its role in regulation of blood pressure, fluid and electrolyte balance. In this system, angiotensinogen (Agt), the obligate precursor of all bioactive angiotensin peptides, undergoes two enzymatic cleavages by renin and angiotensin converting enzyme (ACE) to produce angiotensin I (Ang I) and angiotensin II (Ang II), respectively. The contemporary view of RAS has become more complex with the discovery of additional angiotensin degradation pathways such as ACE2. All components of the RAS are expressed in and have independent regulation of adipose tissue. This local adipose RAS exerts important auto/paracrine functions in modulating lipogenesis, lipolysis, adipogenesis as well as systemic and adipose tissue inflammation. Mice with adipose-specific Agt overproduction have a 30% increase in plasma Agt levels and develop hypertension and insulin resistance, while mice with adipose-specific Agt knockout have a 25% reduction in Agt plasma levels, demonstrating endocrine actions of adipose RAS. Emerging evidence also points towards a role of RAS in regulation of energy balance. Because adipose RAS is overactivated in many obesity conditions, it is considered a potential candidate linking obesity to hypertension, insulin resistance and other metabolic derangements.

n3 and n6 polyunsaturated fatty acids differentially modulate prostaglandin E secretion but not markers of lipogenesis in adipocytes

A dramatic rise in the incidence of obesity in the U.S. has accelerated the search for interventions that may impact this epidemic. One recently recognized target for such intervention is adipose tissue, which secretes a variety of bioactive substances including prostaglandins. Prostaglandin E2 (PGE2) has been shown to decrease lipolysis in adipocytes, but limited studies have explored alternative mechanisms by which PGE2 might impact obesity, such as adipogenesis or lipogenesis. Studies conducted on ApcMin/+ mice indicated that selective inhibition of the cyclooxygenase (COX)-2 enzyme led to significant reductions in fatty acid synthase (FAS) activity in adipose tissue suggesting lipogenic effects of PGE2. To further investigate whether these lipid mediators directly regulate lipogenesis, we used 3T3-L1 adipocytes to determine the impact of eicosapentaenoic acid (EPA) and celecoxib on PGE2 formation and FAS used as a lipogenic marker. Both arachidonic acid (AA) and EPA dose-dependently increased PGE secretion from adipocytes. AA was expectedly more potent and exhibiting at 150 uM dose a 5-fold increase in PGE2 secretion over EPA. Despite higher secretion of PGE by EPA and AA compared to control, neither PUFA significantly altered FAS activity. By contrast both AA and EPA significantly decreased FAS mRNA levels. Addition of celecoxib, a selective COX-2 inhibitor, significantly decreased PGE2 secretion (p < 0.05) versus control, and also significantly decreased FAS activity (p < 0.05). Unexpectedly, the combination of exogenous PGE2 and celecoxib further decreased the FAS activity compared to PGE2 alone or untreated controls. In conclusion, EPA-mediated inhibition of AA metabolism did not significantly alter FAS activity while both AA and EPA significantly decreased FAS mRNA expression. COX-2 inhibition significantly decreased PGE2 production resulting in a decrease in FAS activity and expression that was not reversed with the addition of exogenous PGE2, suggesting an additional mechanism that is independent of COX-2.

Energy-restricted high-fat diets only partially improve markers of systemic and adipose tissue inflammation.

This study aimed at investigating whether the weight loss due to energy‐restricted high‐fat diets is accompanied with parallel improvements in metabolic markers and adipose tissue inflammation. Eight‐week‐old C57BL/6J mice were given free access to a low‐fat (LF) or a high‐fat (45% of energy from fat—HF) diet for 6 months. Restricting intake of the HF diet by 30% (HFR) during the last 2 months of the HF feeding trial decreased fasting plasma insulin, homeostasis model assessment of insulin resistance (HOMAIR), and plasma triglyceride levels and improved hepatic steatosis compared to ad libitum HF feeding, indicating an improved metabolic profile. Further, analysis of gonadal white adipose tissue (GWAT) gene expression by microarray and quantitative PCR analyses demonstrated that HFR downregulated expression of genes linked to cell and focal adhesion, cytokine‐cytokine receptor interaction, and endoplasmic reticulum (ER)–associated degradation pathway. However, HFR had no effect on circulating plasminogen activator inhibitor‐1 (PAI‐1) and nonesterified fatty acid levels, which were persistently higher in both HF and HFR groups compared to the LF group. Furthermore, HFR had a negative effect on plasma total adiponectin level. Finally, while HFR decreased GWAT monocyte chemotactic protein‐1 (MCP‐1), interleukin‐2 (IL‐2), and PAI‐1 levels, it did not affect several other cytokines including granulocyte‐macrophage colony‐stimulating factor, interferon‐γ, IL‐1β, IL‐6, and IL‐10. In summary, energy‐restricted high‐fat diets improve insulin sensitivity, while only partially improving markers of systemic and adipose tissue inflammation. In conclusion, our study supports the recommended low‐fat intake for overall cardiovascular health.