Fish oil reverses the altered glucose transporter,phosphorylation, insulin receptor substrate-1 protein level and lipid contents in the skeletal muscle of sucrose-rich diet fed rats

The role and underlying mechanisms by which n?3 polyunsaturated fatty acids (PUFA) prevent/reverse SRD-induced insulin resistance (IR) in the muscle are not completely understood. Therefore, we examined: triglyceride, diacylglycerol, PKCθ, Glut-4, enzymatic hexokinase activity, IRS-1 protein mass level, and fatty acid composition of muscle phospholipids. Rats were fed a SRD during 6 months. Thereafter, half the animals continued with SRD up to 8 months; the other half was fed a SRD in which CO (8% wt/wt) was replaced by FO (7%+1% CO) for 2 months. Results were compared with those obtained in rats fed a control diet (CD). In SRD-fed rats, FO oil normalized/improved lipid storage and PKCθ protein mass level. Effects of insulin were comparable with those of CD-fed rats. FO reversed impaired glucose phosphorylation, IRS-1, and, under insulin stimulation, Glut-4 protein mass level. FO normalized insulin resistance and increased n?3 PUFAs in muscle phospholipids.     

Effect of capsaicin on skeletal muscle lipoprotein lipase in rats fed high fat diet

A synthetic analogue of capsaicin (0.2 mg%) fed to female Wistar rats along with a high fat diet for 11 weeks, lowered adipose tissue weight and also liver and serum triglycerides. The compound elevated total post heparin plasma lipase and skeletal muscle lipase activities. The increase in the latter indicates the possible mechanism by which capsaicin enhances serum triglyceride uptake by muscle tissue and in turn lowers triglyceride levels. A single dose of capsaicin even at a much higher level failed to lower serum triglycerides emphasizing the necessity of continuous ingestion of capsaicin for exerting its hypolipidemic effect.     

"In vitro" effects of insulin on the PDH complex of the isolated perfused heart of rats fed a sucrose-rich diet.

We have recently reported that the "in situ" myocardial concentrations of the active form of the Pyruvate Dehydrogenase Complex (PDHa) were significantly decreased in hearts obtained from normal rats fed for 3 weeks on an isocaloric sucrose rich (63%) diet (SRD) when compared to age matched controls fed on the standard laboratory chow (STD). Since, on the one hand SRD rats present glucose intolerance and impaired "in vivo" insulin action and, on the other hand the effects of insulin on the interconversion of heart PDH remains a controversial matter, we found it relevant to study the effects of insulin on the PDH complex in the "in vitro" perfused (Langendorff technique) heart preparations obtained from SRD rats. After a 35 minute perfusion period with 5.5 mM glucose as the only nutrient in the perfusate, PDHa as a percentage of total PDH was found to remain significantly lower in SRD hearts (M +/- SEM 32.6 +/- 2.3) when compared to STD hearts (68.3 +/- 4.6, P less than 0.05) in spite of comparable total PDH activities in both groups of animals. Although the addition of insulin to the perfusate (20 mu/ml) resulted in a significant increase in the percentage of PDHa (45.8 +/- 3.4) of SRD heart, values attained still remained significantly lower than those obtained in STD controls (67.5 +/- 3.6; P less than 0.05). Simultaneously, the addition of insulin to the perfusate, significantly reduced the Acetyl-CoA/CoASH ratio in SRD hearts although this ratio remained still much higher than those observed in STD controls under the same experimental conditions.     

Substituting dietary linoleic acid with alpha-linolenic acid improves insulin sensitivity in sucrose fed rats

This study describes the effect of substituting dietary linoleic acid (18:2 n-6) with alpha-linolenic acid (18:3 n-3) on sucrose-induced insulin resistance (IR). Wistar NIN male weanling rats were fed casein based diet containing 22 energy percent (en%) fat with approximately 6, 9 and 7 en% saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) respectively for 3 months. IR was induced by replacing starch (ST) with sucrose (SU). Blends of groundnut, palmolein, and linseed oil in different proportions furnished the following levels of 18:3 n-3 (g/100 g diet) and 18:2 n-6/18:3 n-3 ratios respectively: ST-220 (0.014, 220), SU-220 (0.014, 220), SU-50 (0.06, 50), SU-10 (0.27, 10) and SU-2 (1.1, 2). The results showed IR in the sucrose fed group (SU-220) as evidenced by increase in fasting plasma insulin and area under the curve (AUC) of insulin in response to oral glucose load. In SU-220, the increase in adipocyte plasma membrane cholesterol/phospholipid ratio was associated with a decrease in fluidity, insulin stimulated glucose transport, antilipolytic effect of insulin and increase in basal and norepinephrine stimulated lipolysis in adipocytes. In SU-50, sucrose induced alterations in adipocyte lipolysis and antilipolysis were normalized. However, in SU-2, partial corrections in plasma insulin, AUC of insulin and adipocyte insulin stimulated glucose transport were observed. Further, plasma triglycerides and cholesterol decreased in SU-2. In diaphragm phospholipids, the observed dose dependent increase in long chain (LC) n-3 PUFA was associated with a decrease in LC-n-6 PUFA but insulin stimulated glucose transport increased only in SU-2. Thus, this study shows that the substitution of one-third of dietary 18:2 n-6 with 18:3 n-3 (SU-2) results in lowered blood lipid levels and increases peripheral insulin sensitivity, possibly due to the resulting high LCn-3 PUFA levels in target tissues of insulin action. These findings suggest a role for 18:3 n-3 in the prevention of insulin resistant states. The current recommendation to increase 18:3 n-3 intake for reducing cardiovascular risk may also be beneficial for preventing IR in humans.     

Effect of dietary fish oil on the sensitivity of hepatic lipid metabolism to regulation by insulin

The contribution of dietary fat content and type to changes in the sensitivity of hepatic lipid metabolism to insulin was studied in primary hepatocyte cultures from donor rats maintained on a low-fat diet (LF), or on diets enriched in olive oil (OO) or fish oil (FO). The higher rate of fatty acid oxidation in hepatocytes from the FO-fed group was resistant to the inhibitory effects of insulin observed in hepatocytes from the other groups. Insulin stimulation of fatty acid incorporation into triglyceride (TG) was also less pronounced in hepatocytes from the FO-fed group than in those from the OO-fed group but there was no difference in the stimulatory effect of insulin on fatty acid incorporation into phospholipid (PL) in these two groups. In the case of fatty acid incorporation into both PL and TG, hepatocytes from the LF group were refractory to stimulation by insulin. At each concentration of insulin, hepatocytes from the FO-fed group secreted less very low density lipoprotein (VLDL) TG than those from the other groups. However, the absolute suppression of VLDL TG secretion by insulin was similar irrespective of the diet of the donor animals.We conclude that chronic consumption of a particular type of dietary fat does not affect the insulin sensitivity of the major pathways of hepatic lipid metabolism in a consistent manner.     

Altered estrogen receptor expression in skeletal muscle and adipose tissue of female rats fed a high-fat diet

Estrogen receptors (ERs) are expressed in adipose tissue and skeletal muscle, with potential implications for glucose metabolism and insulin signaling. Previous studies examining the role of ERs in glucose metabolism have primarily used knockout mouse models of ERα and ERβ, and it is unknown whether ER expression is altered in response to an obesity-inducing high-fat diet (HFD). The purpose of the current study was to determine whether modulation of glucose metabolism in response to a HFD in intact and ovariectomized (OVX) female rats is associated with alterations in ER expression. Our results demonstrate that a 6-wk HFD (60% calories from fat) in female rats induces whole body glucose intolerance with tissue-specific effects isolated to the adipose tissue, and no observed differences in insulin-stimulated glucose uptake, GLUT4, or ERα protein expression levels in skeletal muscle. In chow-fed rats, OVX resulted in decreased ERα with a trend toward decreased GLUT4 expression in adipose tissue. Sham-treated and OVX rats fed a HFD demonstrated a decrease in ERα and GLUT4 in adipose tissue. The HFD also increased activation of stress kinases (c-jun NH?-terminal kinase and inhibitor of κB kinase β) in the sham-treated rats and decreased expression of the protective heat shock protein 72 (HSP72) in both sham-treated and OVX rats. Our findings suggest that decreased glucose metabolism and increased inflammation in adipose tissue with a HFD in female rats could stem from a significant decrease in ERα expression.     

Regulation of muscle protein degradation,not synthesis,by dietary leucine in rats fed a protein-deficient diet

The aim of this study was to elucidate the effects of long-term intake of leucine in dietary protein malnutrition on muscle protein synthesis and degradation. A reduction in muscle mass was suppressed by leucine-supplementation (1.5% leucine) in rats fed protein-free diet for 7 days. Furthermore, the rate of muscle protein degradation was decreased without an increase in muscle protein synthesis. In addition, to elucidate the mechanism involved in the suppressive effect of leucine, we measured the activities of degradation systems in muscle. Proteinase activity (calpain and proteasome) and ubiquitin ligase mRNA (Atrogin-1 and MuRF1) expression were not suppressed in animals fed a leucine-supplemented diet, whereas the autophagy marker, protein light chain 3 active form (LC3-II), expression was significantly decreased. These results suggest that the protein-free diet supplemented with leucine suppresses muscle protein degradation through inhibition of autophagy rather than protein synthesis.     

Prevention of skeletal muscle insulin resistance by dietary cod protein in high fat-fed rats

In the present study, we tested the hypothesis that fish protein may represent a key constituent of fish with glucoregulatory activity. Three groups of rats were fed a high-fat diet in which the protein source was casein, fish (cod) protein, or soy protein; these groups were compared with a group of chow-fed controls. High-fat feeding led to severe whole body and skeletal muscle insulin resistance in casein- or soy protein-fed rats, as assessed by the euglycemic clamp technique coupled with measurements of 2-deoxy-D-[(3)H]glucose uptake rates by individual tissues. However, feeding cod protein fully prevented the development of insulin resistance in high fat-fed rats. These animals exhibited higher rates of insulin-mediated muscle glucose disposal that were comparable to those of chow-fed rats. The beneficial effects of cod protein occurred without any reductions in body weight gain, adipose tissue accretion, or expression of tumor necrosis factor-alpha in fat and muscle. Moreover, L6 myocytes exposed to cod protein-derived amino acids showed greater rates of insulin-stimulated glucose uptake compared with cells incubated with casein- or soy protein-derived amino acids. These data demonstrate that feeding cod protein prevents obesity-induced muscle insulin resistance in high fat-fed obese rats at least in part through a direct action of amino acids on insulin-stimulated glucose uptake in skeletal muscle cells.     

Reduced anorexic effects of insulin in obesity-prone rats fed a moderate-fat diet

Rats prone to develop diet-induced obesity (DIO) have reduced central sensitivity to many metabolic and hormonal signals involved in energy homeostasis. High-fat diets produce similar defects in diet-resistant (DR) rats. To test the hypothesis that genotype and diet exposure would similarly affect central insulin signaling, we assessed the anorectic effects of 8 mU third ventricular (iv3t) insulin before and after 4 wk intake of a 31% fat, high-energy (HE) diet intake in outbred (OutB) rats. Rats were retrospectively designated as DR or DIO by their low or high weight gains on HE diet. Before the HE diet, iv3t insulin reduced 4-h and 24-h chow intake by 53% and 69% in DR rats but by only 17% and 27% in DIO rats, respectively. Also, the anorectic response to iv3t insulin in OutB rats was inversely correlated (r = 0.72, P = 0.002) with subsequent 4-wk weight gain on the HE diet. Similarly, in selectively bred (SB) chow-fed DR rats, 8 mU iv3t insulin reduced 4-h and 24-h intake by 21% and 22%, respectively, but had no significant effect in SB DIO rats. Four-week HE diet intake reduced 4-h and 24-h insulin-induced anorexia by 45% in OutB DR rats and completely abolished it in SB DR rats. Reduced insulin responsiveness was unassociated with differences in arcuate nucleus insulin receptor mRNA expression between DIO and DR rats or between rats fed chow or HE diet. These data suggest that DIO rats have a preexisting reduction in central insulin signaling, which might contribute to their becoming obese on the HE diet. However, since the HE diet reduced central insulin sensitivity in DR rats but did not make them obese, it is likely that other brain areas are involved in insulin's anorectic action or that other pathways contribute to the development and maintenance of obesity.     

Protein synthesis in liver,skeletal muscle,and brown adipose tissue of rats fed a protein-deficient diet

Feeding protein-deficient diets to rats is known to stimulate diet-induced thermogenesis and activate brown adipose tissue (BAT). The fact that BAT protein content, unlike that of other tissues, is unnaffected by protein deficiency prompted us to measure tissue protein synthesis in vivo in animals maintained on normal- (18.8%) and low- (7.6%) protein (LP) diets. Protein synthesis was depressed in the liver of the LP rats due to a fall in RNA activity, with no change in RNA content, and synthesis was also reduced in skeletal muscle from the LP group, but this was due to decreased RNA content with no change in RNA activity. Conversely, protein synthesis, RNA, DNA, and protein content of interscapular BAT were all unaltered in protein-restricted animals. These data indicate that, unlike liver, skeletal muscle, and whole carcass, BAT protein synthesis is not reduced in protein-restricted rats, and this may be related to activation of thermogenesis in the tissue.     

Calorie restriction prevents the development of insulin resistance and impaired insulin signaling in skeletal muscle of ovariectomized rats

Insulin resistance of skeletal muscle glucose transport due to prolonged loss of ovarian function in ovariectomized (OVX) rats is accompanied by other features of the metabolic syndrome and may be confounded by increased calorie consumption. In this study, we investigated the role of calorie consumption in the development of insulin resistance in OVX rats. In addition, we examined the cellular mechanisms underlying skeletal muscle insulin resistance in OVX rats. Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (SHAM). OVX rats either had free access to food, pair feeding (PF) with SHAM or received a 35% reduction in food intake (calorie restriction; CR) for 12weeks. Compared with SHAM, ovariectomy induced skeletal muscle insulin resistance, which was associated with decreases (32-70%) in tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), IRS-1 associated p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), and Akt Ser(473) phosphorylation whereas insulin-stimulated phosphorylation of IRS-1 Ser(307), SAPK/JNK Thr(183)/Tyr(185), and p38 mitogen-activated protein kinase (MAPK) Thr(180)/Tyr(182) was increased (24-62%). PF improved the serum lipid profile but did not restore insulin-stimulated glucose transport, indicating that insulin resistance in OVX rats is a consequence of ovarian hormone deprivation. In contrast, impaired insulin sensitivity and defective insulin signaling were not observed in the skeletal muscle of OVX+CR rats. Therefore, we provide evidence for the first time that CR effectively prevents the development of insulin resistance and impaired insulin signaling in the skeletal muscle of OVX rats.     

Rosiglitazone and fenofibrate improve insulin sensitivity of pre-diabetic OLETF rats by reducing malonyl-CoA levels in the liver and skeletal muscle

AimsRosiglitazone and fenofibrate, specific agonists of the peroxisome proliferator activated receptors-γ (PPARγ) and -α (PPARα), respectively, improve insulin sensitivity in diabetic animals and in patients with type 2 diabetes. Here we investigated how pre-diabetic Otsuka Long–Evans Tokushima Fatty (OLETF) rats fed with normal and high-fat diets respond to these PPAR agonists.Main methodsPre-diabetic OLETF rats were subjected to high-fat or standard diets with or without rosiglitazone or fenofibrate for 2 weeks. The metabolism of the rats and the levels of malonyl-CoA and activities of malonyl-CoA decarboxylase (MCD), acetyl-CoA carboxylase (ACC), and AMP-activated protein kinase (AMPK) in metabolic tissues were assessed.Key findingsRosiglitazone and fenofibrate significantly improved insulin sensitivity and reduced the levels of plasma triglycerides and free fatty acids in OLETF rats fed with a high-fat diet. Fenofibrate particularly reduced the body weight, fat, and total cholesterol in high fat diet OLETF rats. The highly elevated malonyl-CoA levels in the skeletal muscle and liver of OLETF rat were significantly reduced by rosiglitazone or fenofibrate due to, in part, the increased MCD activities and expression. On the other hand, ACC activities were unchanged in skeletal muscle and decreased in liver in high fat diet group. AMPK activities were dramatically decreased in OLETF rats and not affected by these agonists.SignificanceThese results demonstrate that treatment of pre-diabetic OLETF rats–particularly those fed a high-fat diet–with rosiglitazone and fenofibrate significantly improves insulin sensitivity and fatty acid metabolism by increasing the activity of MCD and reducing malonyl-CoA levels in the liver and skeletal muscle.     

Alterations in the level of insulin receptor and GLUT-4 mRNA in skeletal muscle from rats fed a kidney bean (Phaseolus vulgaris) diet.

1. A decline in the level of circulating insulin was observed in rats fed a diet containing kidney bean. 2. Consumption of a diet containing kidney bean caused an increase in the level of mRNAs for the insulin receptor (327%) and GLUT-4 (185%) in the gastrocnemius muscle. In contrast there was only a small increase in the amount of actin mRNA (125%). Since the kidney bean-fed rats are euglycaemic the results suggest that insulin receptor and GLUT-4 mRNA levels are regulated in response to circulating insulin concentrations rather than glucose. 3. No increases in the level of insulin receptor and actin mRNA were evident in the soleus muscle of rats fed the diet containing kidney bean; however a decline was observed in the level of GLUT-4 mRNA. 4. It is proposed that a component of kidney beans, most likely the lectin phytohaemagglutinin, has systemic effects which lead to changes in expression of the insulin receptor and GLUT-4 genes and to the sensitivity of muscle to insulin.     

Free radical production in aortic rings from rats fed a fish oil-rich diet

To study the effect of the degree of unsaturation of dietary fatty acids on the production of free radicals and on the vascular smooth muscle tone in rings of the aorta, Sprague-Dawley rats were fed a semipurified diet containing 5% lipids from either corn oil (CO) or menhaden oil (MO) for 8 wk. The MO diet did not change the basal or NADPH-dependent superoxide anion (O2-*) release. There were no significant differences in phenylephrine-induced contractions between the two groups in intact rings. However, these contractions increased in endothelium-intact aortic rings from the MO group after addition of the nitric oxide (*NO) synthase inhibitor NG-nitro-L-arginine and in endothelium-denuded rings, both indicating a greater endothelial basal *NO production in rats fed with the MO diet. Endothelium-dependent relaxations in response to acetylcholine were more prominent in rings from the MO group. These differences were not due to an increased smooth muscle response to.NO, because relaxations were the same using an exogenous *NO donor. Our results indicate that dietary MO did not modify O2-* release by the vessel wall or relaxation due to the cyclooxygenase pathway, but it potentiated endothelial production of *NO.     

Development of insulin sensitivity in rat skeletal muscle. Studies of glucose transporter and insulin receptor mRNA levels.

Expression of GLUT-4 and insulin receptor mRNAs was investigated in rat skeletal muscle by Northern hybridization. GLUT-4 mRNA was barely detectable in foetal muscle, was expressed at low levels by 1-8 days and at 2-3-fold higher levels during and after weaning (18-40 days). In contrast there was little change in insulin receptor mRNA levels prior to weaning and a reduction in mRNA abundance between 18 and 40 days. Weaning rats on to a diet rich in fat prevented the increase in GLUT-4 abundance seen between 15 and 29 days in animals weaned on a high-carbohydrate diet.     

The protective effect of dietary fish oil on murine lupus

Dietary marine lipids markedly reduce the severity of glomerulonephritis and its associated mortality in inbred strains of mice developing autoimmune disease, a model for human systemic lupus erythematosus. We report here the influence of varying the dose of menhaden oil and the timing of its administration on the mortality of female (NZB x NZW) F₁ mice. After ingesting 25 wt% menhaden oil (MO) for periods of 1.5 weeks to 12 months, there was a stable content of tissue n-3 fatty acids, with total n-3 fatty acids of 28% and 35% in spleen and liver, respectively. The extent of protection from mortality was dependent on the dose of MO with marked protection at doses of 11 to 25%, marginal protection at 5.5% and no protection at 2.5% MO. Delay in the institution of MO until ages 5 or 7 months still resulted in large reductions of mortality. Conversely, institution of a MO diet from 6 weeks until ages 5 to 7 months followed by a change to beef tallow resulted in little protection. Serum levels of 4 cyclooxygenase products were reduced ranging from 26 to 76% in mice fed MO diets, compared to mice fed beef tallow, based on radioimmunoassay. The degree of reduction of mortality on different doses of MO was correlated best with tissue levels of C22:5, and levels of C20:5 and C22:6 were similar at high and low doses of MO, suggesting that levels of 22:5 may be related to the protective effects of marine lipids on autoimmune disease.     

Antihypertensive effect of quercetin in rats fed with a high-fat high-sucrose diet

The effects of different levels of quercetin on the blood pressure were studied in 6-week-old male Sprague-Dawley rats. The rats were fed with a control diet or a high-fat high-sucrose (HFS) diet containing 0, 0.02, 0.07, 0.2, or 0.5% quercetin for 4 weeks. The systolic blood pressure and the lipid peroxides in the plasma were both higher in the rats fed with the HFS diet without quercetin than in the rats fed with the control diet. The nitric oxide synthase (NOS) activity in the vascular tissues and nitric oxide (NO) metabolites in the plasma and urine were both lower in these rats. A distinct depression of the increase in blood pressure was found in the rats fed with the HFS diets containing quercetin. Each level of quercetin examined was effective, the 0.5% level being much more effective than other levels. Dietary quercetin decreased lipid peroxidation in the plasma of the rats fed with the HFS diets. Quercetin also suppressed the decrease in NO metabolites in the plasma and urine, and the NOS activity in the vascular tissues of these rats. These results suggest that the increased NO availability caused by the elevated NOS activity, and the antioxidative activity in these rats fed with quercetin may be sources of the antihypertensive effect of quercetin.