Fish oil-derived n-3 polyunsaturated fatty acids downregulate aquaporin 9 protein expression of liver and white adipose tissues in diabetic KK mice and 3T3-L1 adipocytes.

Aquaporin 9 (AQP9) is an integral membrane protein that facilitates glycerol transport in hepatocytes and adipocytes. Glycerol is necessary as a substrate for gluconeogenesis in the physiological fasted state, suggesting that inhibiting AQP9 function may be beneficial for treating type 2 diabetes associated with fasting hyperglycemia. The n-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are rich in fish oil and lower the risk of metabolic syndrome; however, the effects of EPA and DHA on AQP9 expression in obese and type 2 diabetes are unclear. The KK mouse is an animal model of obesity and type 2 diabetes because of the polymorphisms on leptin receptor gene, which results in a part of cause for obese and diabetic conditions. In this study, we determined the effect of fish oil-derived n-3 PUFA on AQP9 protein expression in the liver and white adipose tissue (WAT) of KK mice and mouse 3T3-L1 adipocytes. The expression of AQP9 protein in the liver, epididymal WAT, and inguinal WAT were markedly decreased following fish oil administration. We also demonstrated that n-3 PUFAs, such as DHA, and to a lesser extent EPA, downregulated AQP9 protein expression in 3T3-L1 adipocytes. Our results suggest that fish oil-derived n-3 PUFAs may regulate the protein expressions of AQP9 in glycerol metabolism-related organs in KK mice and 3T3-L1 adipocytes.

Impact of discontinuation of fish oil after pioglitazone-fish oil combination therapy in diabetic KK mice.

Pioglitazone is one of the thiazolidinediones (TZDs) and an insulin-sensitive drug for type 2 diabetes. In our previous study, a combination of pioglitazone and fish oil rich in n-3 polyunsaturated fatty acids (PUFAs) was shown to inhibit pioglitazone-induced side effects, such as accumulation of subcutaneous fat and body weight gain. However, the effects of the discontinuation of fish oil after combination treatment with TZD and fish oil are not clear. In this study, discontinuation of fish oil for 4 weeks showed several unfavorable effects: (1) return of plasma adiponectin level, (2) reversal of the inhibition of lipogenesis and activation of fatty acid ß-oxidation in liver, (3) increase in hypertrophic adipocytes in epidydimal white adipose tissue (WAT) and (4) accumulation of lipids in brown adipose tissue (BAT). However, insulin resistance was ameliorated by pioglitazone with or without fish oil treatment and the discontinuation of fish oil. These findings indicate that discontinuation of n-3 PUFA after combination therapy with TZDs adversely affects lipid metabolism and energy homeostasis in liver, epididymal WAT and BAT.

Benefits of combination low-dose pioglitazone plus fish oil on aged type 2 diabetes mice.

The elderly patients with type 2 diabetes suffer more adverse drug events than young adults due to pharmacokinetic and pharmacodynamic changes associated with aging. Reducing the risks of these medication-related problems are equally important for the clinical care of older type 2 diabetes patients. Pioglitazone is used for treating type 2 diabetes as an oral antidiabetic drug. Despite pioglitazone is used helpful insulin sensitizers, the accumulation of subcutaneous fat is considered a major adverse effect of pioglitazone therapy. We investigated to reduce the adverse effect of pioglitazone by combination with fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in aged diabetic KK mice. The accumulation of subcutaneous fat associated with high-dose pioglitazone is reduced by fish oil, suppressing lipogenesis and stimulating fatty acid ß-oxidation in the liver. Our data suggest that adding fish oil to low-dose pioglitazone results in antidiabetic efficacy similar to that of the high-dose without concomitant body weight gain.

Fish oil prevents excessive accumulation of subcutaneous fat caused by an adverse effect of pioglitazone treatment and positively changes adipocytes in KK mice.

Pioglitazone, a thiazolidinedione (TZD), is widely used as an insulin sensitizer in the treatment of type 2 diabetes. However, body weight gain is frequently observed in TZD-treated patients. Fish oil improves lipid metabolism dysfunction and obesity. In this study, we demonstrated suppression of body weight gain in response to pioglitazone administration by combination therapy of pioglitazone and fish oil in type 2 diabetic KK mice. Male KK mice were fed experimental diets for 8 weeks. In safflower oil (SO), safflower oil/low-dose pioglitazone (S/PL), and safflower oil/high-dose pioglitazone (S/PH) diets, 20% of calories were provided by safflower oil containing 0%, 0.006%, or 0.012% (wt/wt) pioglitazone, respectively. In fish oil (FO), fish oil/low-dose pioglitazone (F/PL), and fish oil/high-dose pioglitazone (F/PH) diets, 20% of calories were provided by a mixture of fish oil and safflower oil. Increased body weight and subcutaneous fat mass were observed in the S/PL and S/PH groups; however, diets containing fish oil were found to ameliorate these changes. Hepatic mRNA levels of lipogenic enzymes were significantly decreased in fish oil-fed groups. These findings demonstrate that the combination of pioglitazone and fish oil decreases subcutaneous fat accumulation, ameliorating pioglitazone-induced body weight gain, through fish oil-mediated inhibition of hepatic de novo lipogenesis.

Hesperidin prevents androgen deficiency-induced bone loss in male mice.

The purpose of this study was to examine whether hesperidin inhibits bone loss in androgen-deficient male mice. Male ddY mice aged 7 weeks underwent either a sham operation or orchidectomy (ORX) and were divided into five groups: a sham-operated group fed a control diet (Sham) based on AIN-93G formulation with corn oil instead of soy bean oil, an ORX group fed the control diet (ORX), a group fed the control diet containing 0.5% hesperidin (ORX + H), a group fed the control diet containing 0.7% α-glucosylhesperidin (ORX + αG), and a group fed the control diet containing 0.013% simvastatin (ORX + St). Four weeks after intervention, ORX mice showed a striking decrease in seminal vesicle weight, which was not affected by the administration of hesperidin, α-glucosylhesperidin, or simvastatin. Femoral BMD was significantly reduced by ORX, and bone loss was inhibited by the administration of hesperidin, α-glucosylhesperidin or simvastatin. Histomorphometric analysis showed that the bone volume and trabecular thickness were significantly lower, and the osteoclast number was higher in the distal femoral cancellous bone in the ORX group than in the Sham group, and these were normalized in the ORX + H, ORX + αG and ORX + St groups. These results indicate that hesperidin inhibited bone resorption and hyperlipidemia, in ORX mice, and the preventive effect was stronger than that observed in ovariectomized mice in our previous study.

Effects of dietary fat energy restriction and fish oil feeding on hepatic metabolic abnormalities and insulin resistance in KK mice with high-fat diet-induced obesity.

We investigated the effects of dietary fat energy restriction and fish oil intake on glucose and lipid metabolism in female KK mice with high-fat (HF) diet-induced obesity. Mice were fed a lard/safflower oil (LSO50) diet consisting of 50 energy% (en%) lard/safflower oil as the fat source for 12 weeks. Then, the mice were fed various fat energy restriction (25 en% fat) diets - LSO, FO2.5, FO12.5 or FO25 - containing 0, 2.5, 12.5, or 25 en% fish oil, respectively, for 9 weeks. Conversion from a HF diet to each fat energy restriction diet significantly decreased final body weights and visceral and subcutaneous fat mass in all fat energy restriction groups, regardless of fish oil contents. Hepatic triglyceride and cholesterol levels markedly decreased in the FO12.5 and FO25 groups, but not in the LSO group. Although plasma insulin levels did not differ among groups, the blood glucose areas under the curve in the oral glucose tolerance test were significantly lower in the FO12.5 and FO25 groups. Real-time polymerase chain reaction analysis showed fatty acid synthase mRNA levels significantly decreased in the FO25 group, and stearoyl-CoA desaturase 1 mRNA levels markedly decreased in the FO12.5 and FO25 groups. These results demonstrate that body weight gains were suppressed by dietary fat energy restriction even in KK mice with HF diet-induced obesity. We also suggested that the combination of fat energy restriction and fish oil feeding decreased fat droplets and ameliorated hepatic hypertrophy and insulin resistance with suppression of de novo lipogenesis in these mice.

Combined effects of soy isoflavone and fish oil on ovariectomy-induced bone loss in mice.

Both soy isoflavone and n-3 polyunsaturated fatty acids are known to reduce the levels of bone-resorbing cytokines; however, the synergistic effects of these food ingredients have not been examined yet. This study was performed to elucidate the effect of concomitant intake of soy isoflavone and fish oil on bone mass in ovariectomized mice. Eight-week-old ddY female mice were subjected to ovariectomy (OVX) or sham surgery, and then fed an AIN-93G with safflower oil (So) as a control lipid source, isoflavone-supplemented safflower oil (So + I), fish oil instead of safflower oil (Fo) or isoflavone-supplemented fish oil (Fo + I) for 4 weeks. Femoral bone mineral density was significantly decreased by OVX; however, this decrease was inhibited by the intake of isoflavone and/or fish oil. Histomorphometric analyses showed that bone volume and trabecular thickness in the distal femoral trabecular bone were significantly lower in the So group than in the sham group, but those were restored in the Fo + I groups. The number of osteoclasts was significantly decreased by isoflavone intake. The increased rate of bone resorption after OVX was inhibited by isoflavone and/or fish oil. The serum concentration of tumor necrosis factor alpha was increased after OVX, but was significantly lower with the combination of isoflavone with fish oil than isoflavone or fish oil alone. The results of this study indicated that the intakes of soy isoflavone and/or fish oil might have ameliorating effects on bone loss due to OVX. Further, the concomitant intake of soy isoflavone and fish oil at a low dose showed better effects on cytokines related with bone resorption.

Anti-obesity effect of fish oil and fish oil-fenofibrate combination in female KK mice.

AIM: The aim of our study is to elucidate the effects of EPA- or DHA-rich fish oil, and of the latter plus fenofibrate, on lipid metabolism in female KK mice. METHODS: Female KK mice were fed purified experimental diets containing lard/safflower oil (4:6, Lard/SO), EPA-rich fish oil (EPA), DHA-rich fish oil (DHA), or DHA-rich fish oil plus 0.2% (w/w) fenofibrate (DHA+FF) for 8 weeks. At the end of the experiments, we measured levels of plasma lipids, hepatic triglycerides, and cholesterol, as well as the hepatic mRNA expression of lipogenic and lipidolytic genes. RESULTS: The final body weight of EPA- and DHA-fed groups was significantly lower than that of the Lard/SO-fed group, and that of the DHA+FF-fed group was the lowest. All three fish oil treatments significantly reduced plasma insulin levels. Hepatic lipid levels significantly decreased in all three of these groups compared with the Lard/SO-fed group. Plasma adiponectin increased in both the EPA-and DHA-fed groups, but the increase was suppressed in the DHA+FF-fed group. Hepatocytes of Lard/SO-fed mice were filled with numerous fat droplets, but fat accumulation was inhibited in both EPA- and DHA-fed mice and was significantly prevented by fenofibrate treatment. SREBP-1c mRNA levels were decreased by about half in EPA- and DHA-fed mice compared with Lard/SO-fed mice. FAS, Insig-1, HMG-CoA reductase, and LDL-receptor mRNA levels also markedly decreased in both EPA- and DHA-fed mice, but there was no additional decrease in DHA+FF fed mice. Fenofibrate treatment significantly induced mRNA expression of AOX and UCP-2, but not of PPARalpha. CONCLUSION: These data suggest that fish oil inhibited body weight gain and exhibited an anti-obesity effect through the inhibition of lipid synthesis in female KK mice. Furthermore, fenofibrate treatment markedly inhibited body weight gain by the induction of fatty acid oxidation. Plasma adiponectin levels did not increase in mice fed DHA-rich fish oil with fenofibrate, although white adipose tissue (WAT) weight significantly decreased. We considered that adiponectin sensitivity increased more in mice fed DHA-rich fish oil with fenofibrate than in mice fed DHA-rich fish oil alone.

Interaction of fenofibrate and fish oil in relation to lipid metabolism in mice.

AIM: The aim of our study is to elucidate the interactive effects on lipid metabolism of fenofibrate and two fish oils with EPA and DHA contents in mice. METHODS: Female C57BL/6J mice were fed purified experimental diets containing safflower oil (SO), EPA-rich menhaden oil (MO) or DHA-rich tuna oil (TO) with or without 0.1% fenofibrate for 8 weeks. At the end of the experiments, we measured plasma lipids and hepatic triglycerides and cholesterol, and the hepatic mRNA expression of lipogenic and lipidolytic genes. RESULTS: Plasma TG levels fell in the group fed MO or TO alone and fell significantly in all fenofibrate-treated groups. Although plasma total cholesterol levels fell significantly in fish oil-fed groups, fenofibrate treatments increased significantly plasma total cholesterol levels in these fish oil groups, but not in the group fed SO alone; however, hepatic triglyceride and total cholesterol levels markedly decreased in MO-or TO-fed mice. In lipid synthesis, the hepatic mRNA level of SREBP-1c was not reduced in either fish oil group; however, Insig-1 mRNA decreased in MO and TO feeding groups by about half and FAS or SCD-1 mRNA decreased significantly in MO and TO feeding groups, compared with the SO feeding group. In both fish oil groups, SREBP-2 mRNA decreased significantly and HMG-CoA reductase mRNA also decreased with/without fenofibrate. On the other hand, fenofibrate supplementation significantly induced the mRNA expression of AOX and UCP-2, which play a role in lipid catabolism, in all diets. CYP7A1 mRNA increased markedly in mice fed MO diet with fenofibrate, compared with TO diet with fenofibrate. CONCLUSION: These data suggest that differences in dietary contents of EPA and DHA do not influence the inhibition of lipogenesis, and that fenofibrate supplementation stimulates fatty acid oxidation, regardless of the oil type; however, cholesterol catabolism was induced by a combination of EPA-rich fish oil and fenofibrate, which suggests that EPA has a greater synergistic ability for cholesterol catabolism induction by fenofibrate than DHA.