Discordant Dose-Dependent Metabolic Effects of Eicosapentanoic Acid in Diet-Induced Obese Mice.

Obesity is a widespread epidemic that increases the risk for several metabolic diseases. Despite several beneficial health effects of eicosapentaenoic acid (C20:5n-3, EPA), previous studies have used very high doses of EPA. In this study, dose-dependent effects of EPA on metabolic outcomes were determined in diet-induced obese mice. We used B6 male mice, fed high-fat diet (HF, 45% kcal fat) or HF diet supplemented with 9, 18, and 36 g/kg of EPA-enriched fish oil for 14 weeks. We conducted metabolic phenotyping during the feeding period, and harvested tissues and blood at termination. Only mice fed 36 g/kg of EPA significantly (p < 0.05) lowered body weight, fat content and epididymal fat pad weight, compared to HF. Both 18 and 36 g/kg doses of EPA significantly increased glucose clearance and insulin sensitivity, compared to HF or 9 g/kg of EPA. Locomotor activity was significantly increased with both 18 and 36 g/kg doses of EPA. Interestingly, all doses of EPA compared to HF, significantly increased energy expenditure and oxygen consumption and significantly reduced serum insulin, leptin, and triglycerides levels. These results demonstrate weight- and adiposity-independent metabolic benefits of EPA, at doses comparable to those currently used to treat hypertriglyceridemia.

Angiotensin II Increases Endoplasmic Reticulum Stress in Adipose Tissue and Adipocytes.

The Renin Angiotensin System (RAS), a key regulator of blood pressure has been linked to metabolic disorders. We have previously reported that adipose overexpression of angiotensinogen in mice (Agt-Tg) induces obesity, in part mediated by adipose tissue inflammation, through yet unidentified mechanisms. Hence, we hypothesize that adipose tissue enrichment of angiotensinogen leads to activation of inflammatory cascades and endoplasmic reticulum (ER) stress, thereby, contributing to obesity. We used wild type (Wt), Agt-Tg and Agt-knockout (KO) mice along with 3T3-L1 and human adipocytes treated with RAS, ER stress and inflammation inhibitors. ER stress and pro-inflammation markers were significantly higher in Agt-Tg compared to Wt mice and captopril significantly reduced their expression. Furthermore, in vitro treatment with Ang II significantly induced ER stress and inflammation, whereas angiotensin II receptor inhibitor, telmisartan reduced RAS effects. Moreover, miR-30 family had significantly lower expression in Agt-Tg group. MiR-708-5p and -143-3p were upregulated when RAS was overexpressed, and RAS antagonists reduced miR-143-3p and -708-5p in both mouse adipose tissue and adipocytes. Activation of RAS by Ang II treatment, increased inflammation and ER stress in adipocytes mainly via AT1 receptor, possibly mediated by miR-30 family, -708-5p and/or -143-3p. Hence, RAS and mediating microRNAs could be used as potential targets to reduce RAS induced obesity and related comorbid diseases.

Eicosapentaenoic Acid Reduces Adiposity, Glucose Intolerance and Increases Oxygen Consumption Independently of Uncoupling Protein 1.

SCOPE: Brown adipose tissue (BAT) dissipates energy through uncoupling protein 1 (UCP1) and has been proposed as an anti-obesity target. It was reported previously that a high-fat (HF) diet enriched in eicosapentaenoic acid (EPA) significantly increased UCP1 and other thermogenic markers in BAT. It is hypothesized that these effects are mediated through UCP1-dependent regulation. METHODS AND RESULTS: Wild-type (WT) and UCP1 knockout (KO) B6 male mice were housed at thermoneutrality and fed a HF diet, without or with eicosapentaenoic acid (EPA)-enriched fish oil. HF-fed KO mice were heavier and had higher BAT lipid content than other groups. Protective effects of EPA in WT, previously observed at 22 °C (reduced adiposity, improved glucose tolerance, and increased UCP1), disappeared at thermoneutrality. Mitochondrial proteins, cytochrome c oxidase subunit 1 (COX I), COX I, II, and IV were reduced in the KO mice compared to WT. Unexpectedly, EPA attenuated weight and fat mass gain and improved glucose tolerance in the KO mice. Finally, EPA increased BAT peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) protein and gene expression, and whole-body oxygen consumption in KO mice, consistent with increased mitochondria DNA (mtDNA)/nuclear DNA (nucDNA) ratio. CONCLUSIONS: EPA rescued the weight gain and glucose intolerance in UCP1 KO mice at thermoneutrality, independent of UCP1; these effects may be mediated in part via increased oxygen consumption and BAT PGC1α.

Maternal and Postnatal Supplementation of Fish Oil Improves Metabolic Health of Mouse Male Offspring.

OBJECTIVE: Over half of American women of childbearing age have either obesity or overweight. Hence, maternal programming through diet is critical for prevention of diseases in the offspring. Clinical trials with fish oil (FO) report various health benefits; however, it remains unclear whether maternal and postnatal consumption of FO protects offspring from adverse effects of consuming a high-fat (HF) diet. METHODS: Female mice were fed HF diets supplemented without (HF) or with FO from 8 weeks before pregnancy through lactation. A low-fat (LF) diet was included as a control diet. After weaning, male offspring from HF or FO dams were either continued on their respective diet (HF-HF and FO-FO) or switched to the other diet (HF-FO and FO-HF) and compared with LF. Phenotypic and mechanistic studies were performed. RESULTS: FO-FO offspring demonstrated significantly higher glucose clearance and insulin sensitivity compared with other pups fed the HF diet (P < 0.05). Furthermore, FO-FO pups had lower adiposity, inflammation, and fat deposition in the liver, consistent with reduced markers of hepatic lipogenesis and increased hepatic lipid oxidation. CONCLUSIONS: Supplementation of FO during pregnancy and early life is more beneficial than treating with FO either during pregnancy or in pups.

Effects of delta-tocotrienol on obesity-related adipocyte hypertrophy, inflammation and hepatic steatosis in high-fat-fed mice.

Inflammation is a major underlying cause for obesity-associated metabolic diseases. Hence, anti-inflammatory dietary components may improve obesity-related disorders. We hypothesized that delta-tocotrienol (δT3), a member of the vitamin E family, reduces adiposity, insulin resistance and hepatic triglycerides through its anti-inflammatory properties. To test this hypothesis, C57BL/6J male mice were fed a high-fat diet (HF) with or without supplementation of δT3 (HF+δT3) at 400 mg/kg and 1600 mg/kg for 14 weeks, and they were compared to mice fed a low-fat diet (LF) or HF supplemented with metformin as an antidiabetic control. Glucose tolerance tests were administered 2 weeks prior to the end of treatments. Histology, quantitative polymerase chain reaction and protein analyses were performed to assess inflammation and fatty acid metabolism in adipose and liver tissues. Significant improvements in glucose tolerance, and reduced hepatic steatosis and serum triglycerides were observed in δT3-supplemented groups compared to the HF group. Body and fat pad weights were not significantly reduced in HF+δT3 groups; however, we observed smaller fat cell size and reduced macrophage infiltration in their adipose tissues compared to other groups. These changes were at least in part mechanistically explained by a reduction of mRNA and protein expression of proinflammatory adipokines and increased expression of anti-inflammatory adipokines in HF+δT3 mice. Moreover, δT3 dose-dependently increased markers of fatty acid oxidation and reduced markers of fatty acid synthesis in adipose tissue and liver. In conclusion, our studies suggest that δT3 may promote metabolically healthy obesity by reducing fat cell hypertrophy and decreasing inflammation in both liver and adipose tissue.

Eicosapentaenoic acid regulates brown adipose tissue metabolism in high-fat-fed mice and in clonal brown adipocytes.

Brown adipose tissue (BAT) plays a key role in energy expenditure through its specialized thermogenic function. Therefore, BAT activation may help prevent and/or treat obesity. Interestingly, subcutaneous white adipose tissue (WAT) also has the ability to differentiate into brown-like adipocytes and may potentially contribute to increased thermogenesis. We have previously reported that eicosapentaenoic acid (EPA) reduces high-fat (HF)-diet-induced obesity and insulin resistance in mice. Whether BAT mediates some of these beneficial effects of EPA has not been determined. We hypothesized that EPA activates BAT thermogenic program, contributing to its antiobesity effects. BAT and WAT were harvested from B6 male mice fed HF diets supplemented with or without EPA. HIB 1B clonal brown adipocytes treated with or without EPA were also used. Gene and protein expressions were measured in adipose tissues and H1B 1B cells by quantitative polymerase chain reaction and immunoblotting, respectively. Our results show that BAT from EPA-supplemented mice expressed significantly higher levels of thermogenic genes such as PRDM16 and PGC1α and higher levels of uncoupling protein 1 compared to HF-fed mice. By contrast, both WATs (subcutaneous and visceral) had undetectable levels of these markers with no up regulation by EPA. HIB 1B cells treated with EPA showed significantly higher mRNA expression of PGC1α and SIRT2. EPA treatment significantly increased maximum oxidative and peak glycolytic metabolism in H1B 1B cells. Our results demonstrate a novel and promising role for EPA in preventing obesity via activation of BAT, adding to its known beneficial anti-inflammatory effects.

Eicosapentaenoic acid reduces adipocyte hypertrophy and inflammation in diet-induced obese mice in an adiposity-independent manner.

BACKGROUND: Obesity is associated with an overexpansion of adipose tissue, along with increases in blood pressure, glycemia, inflammation, and thrombosis. Research to develop nutritional interventions to prevent or treat obesity and its associated diseases is greatly needed. Previously, we demonstrated the ability of eicosapentaenoic acid (EPA) to prevent high-fat (HF) diet-induced obesity, insulin resistance, and inflammation in mice. OBJECTIVE: The objective of the current study was to determine the mechanisms mediating the anti-inflammatory and antilipogenic actions of EPA. METHODS: In a previous study, male C57BL/6J mice were fed a low-fat diet (10% of energy from fat), an HF diet (45% of energy from fat), or an HF diet supplemented with EPA (45% of energy from fat; 36 g/kg EPA; HF+EPA) for 11 wk or an HF diet for 6 wk and then switched to the HF+EPA diet for 5 wk. In this study, we used histology/immunohistochemistry, gene expression, and metabolomic analyses of white adipose tissue from these mice. In addition, cultured mouse 3T3-L1 adipocytes were treated with 100 µM EPA for 48 h and then used for extracellular flux assays with untreated 3T3-L1 adipocytes used as a control. RESULTS: Compared with the HF diet, the HF+EPA diet significantly reduced body weight, adiposity, adipocyte size, and macrophage infiltration into adipose tissue. No significant differences in overall body weight or fat pad weights were observed between HF-fed mice vs. those fed the HF+EPA diet for a short time after first inducing obesity with the HF diet. Interestingly, both histology and immunohistochemistry results showed a significantly lower mean adipocyte size and macrophage infiltration in mice fed the HF diet and then switched to the HF+EPA diet vs. those fed HF diets only. This indicated that EPA was able to prevent as well as reverse HF-diet-induced adipocyte inflammation and hypertrophy and that some of the metabolic effects of EPA were independent of body weight or adiposity. In addition, adipose tissue metabolomic data and cultured adipocyte extracellular flux bioenergetic assays indicated that EPA also regulated mitochondrial function by increasing fatty acid oxidation and oxygen consumption, respectively. CONCLUSION: With the use of mice and cultured adipocytes, we showed that EPA ameliorates HF-diet effects at least in part by increasing oxygen consumption and fatty acid oxidation and reducing adipocyte size, adipogenesis, and adipose tissue inflammation, independent of obesity.