Increased adipocyte S-nitrosylation targets anti-lipolytic action of insulin: relevance to adipose tissue dysfunction in obesity

Protein S-nitrosylation is a reversible protein modification implicated in both physiological and pathophysiological regulation of protein function. In obesity, skeletal muscle insulin resistance is associated with increased S-nitrosylation of insulin-signaling proteins. However, whether adipose tissue is similarly affected in obesity and, if so, what are the causes and functional consequences of increased S-nitrosylation in this tissue are unknown. Total protein S-nitrosylation was increased in intra-abdominal adipose tissue of obese humans and in high fat-fed or leptin-deficient ob/ob mice. Both the insulin receptor β-subunit and Akt were S-nitrosylated, correlating with body weight. Elevated protein and mRNA expression of inducible NO synthase and decreased protein levels of thioredoxin reductase were associated with increased adipose tissue S-nitrosylation. Cultured differentiated pre-adipocyte cell lines exposed to the NO donors S-nitrosoglutathione (GSNO) or S-nitroso-N-acetylpenicillamine exhibited diminished insulin-stimulated phosphorylation of Akt but not of GSK3 nor of insulin-stimulated glucose uptake. Yet the anti-lipolytic action of insulin was markedly impaired in both cultured adipocytes and in mice injected with GSNO prior to administration of insulin. In cells, impaired ability of insulin to diminish phosphorylated PKA substrates in response to isoproterenol suggested impaired insulin-induced activation of PDE3B. Consistently, increased S-nitrosylation of PDE3B was detected in adipose tissue of high fat-fed obese mice. Site-directed mutagenesis revealed that Cys-768 and Cys-1040, two putative sites for S-nitrosylation adjacent to the substrate-binding site of PDE3B, accounted for ～50% of its GSNO-induced S-nitrosylation. Collectively, PDE3B and the anti-lipolytic action of insulin may constitute novel targets for increased S-nitrosylation of adipose tissue in obesity.     

Decreased sensitivity to insulin in white adipose tissue from adrenalectomised rats

The ability of insulin to increase both [14C]-glucose incorporation into fatty acids and pyruvate dehydrogenase activity in incubated rat epididymal adipose tissues was considerably lessened after adrenalectomy. Insulin antagonism of adrenaline-stimulated lipolysis in isolated fat cells was abolished after adrenalectomy. Percentage stimulation of lipolysis above basal by adrenaline was not appreciably altered by adrenalectomy.     

Hyperinsulinaemia increases insulin action in vivo in white adipose tissue but not in muscles.

The effect of 4 days of stable hyperglycaemia and resulting hyperinsulinaemia on insulin-induced glucose utilization by individual rat tissues was studied in vivo. The treatment produced a net increase in the glucose utilization index under both basal and insulin-stimulated (euglycaemic/hyperinsulinaemic clamp) conditions in white adipose tissue. On the contrary, glucose utilization was unchanged in aerobic muscles but was decreased in glycolytic skeletal muscles during the clamp.     

Hyperglycemia contributes insulin resistance in hepatic and adipose tissue but not skeletal muscle of ZDF rats

To determine the contribution of hyperglycemia to the insulin resistance in various insulin-sensitive tissues of Zucker diabetic fatty (ZDF) rats, T-1095, an oral sodium-dependent glucose transporter (SGLT) inhibitor, was administered by being mixed into food. Long-term treatment with T-1095 lowered both fed and fasting blood glucose levels to near normal ranges. A hyperinsulinemic euglycemic clamp study that was performed after 4 wk of T-1095 treatment demonstrated partial recovery of the reduced glucose infusion rate (GIR) in the T-1095-treated group. In the livers of T-1095-treated ZDF rats, hepatic glucose production rate (HGP) and glucose utilization rate (GUR) showed marked recovery, with almost complete normalization of reduced glucokinase/glucose-6-phosphatase (G-6-Pase) activities ratio. In adipose tissues, decreased GUR was also shown to be significantly improved with a normalization of insulin-induced GLUT-4 translocation. In contrast, in skeletal muscles, the reduced GUR was not significantly improved in response to amelioration of hyperglycemia by T-1095 treatment. These results suggest that the contribution of hyperglycemia to insulin resistance in ZDF rats is very high in the liver and considerably elevated in adipose tissues, although it is very low in skeletal muscle.     

Brown and white adipose tissue lipid composition in three successive progenies of rats: Effects of ethanol exposure

The effect of ethanol exposure on the fatty acid composition of brown and white adipose tissue in three successive rat progenies at the end of an experimental period (24 weeks) was studied. Ethanol‐treated rats received a standard rat chow diet and 5, 10 and 15% ethanol in the ad libitum drinking fluid over 3 successive weeks. Then a concentration of 20% ethanol was maintained for 5 additional weeks up to the end of the experimental period. The males and females in the ethanol treated group were mated to obtain the 1st generation of offspring. Then female and male rats from the 1st generation were mated to obtain the 2nd generation. Finally, males and females from the 2nd generation were mated to obtain the 3rd generation of ethanol treated rats. Another group served as control and received only water and a standard rat chow diet. The control group was handled in the same way as the other experimental groups. In the 1st and 2nd generations the percentage of stearic acid (18:0) decreased and palmitoleic (16:ln7) and oleic acid (18:ln9) increased in both adipose tissues of ethanol‐treated rats with respect to control. Additionally, n‐3 and n‐6 series were reduced both in brown and white adipose tissues. In the 3rd generation the fatty acid composition of the white adipose tissue was similar to that of control rats. Thus, no significant difference in essential fatty acids and oleic acid (18:ln9) were found. However, the fatty acid composition of the brown adipose tissue, in the 3rd generation, was similar to that observed in the 1st and 2nd generation. Thus, a decrease in essential fatty acids and an increase in oleic acid (18:ln9) was found. This suggests adaptation to ethanol consumption during successive progenies in white adipose tissue. However, in brown adipose tissue the values indicate a triglyceride storing during the thermogenesis, which is more important to newborns.     

Reconstitution of insulin action in muscle, white adipose tissue, and brain of insulin receptor knock-out mice fails to rescue diabetes

Type 2 diabetes results from an impairment of insulin action. The first demonstrable abnormality of insulin signaling is a decrease of insulin-dependent glucose disposal followed by an increase in hepatic glucose production. In an attempt to dissect the relative importance of these two changes in disease progression, we have employed genetic knock-outs/knock-ins of the insulin receptor. Previously, we demonstrated that insulin receptor knock-out mice (Insr(-/-)) could be rescued from diabetes by reconstitution of insulin signaling in liver, brain, and pancreatic β cells (L1 mice). In this study, we used a similar approach to reconstitute insulin signaling in tissues that display insulin-dependent glucose uptake. Using GLUT4-Cre mice, we restored InsR expression in muscle, fat, and brain of Insr(-/-) mice (GIRKI (Glut4-insulin receptor knock-in line 1) mice). Unlike L1 mice, GIRKI mice failed to thrive and developed diabetes, although their survival was modestly extended when compared with Insr(-/-). The data underscore the role of developmental factors in the presentation of murine diabetes. The broader implication of our findings is that diabetes treatment should not necessarily target the same tissues that are responsible for disease pathogenesis.     

The role of insulin in norepinephrine turnover and thermogenesis in brown adipose tissue after acute cold-exposure

The role of insulin in norepinephrine turnover (NE) and thermogenesis in brown adipose tissue (BAT) after acute cold-exposure was studied using streptozocin (STZ)-induced diabetic rats. NE turnover was estimated by the NE synthesis inhibition technique with alpha-methyl-p-tyrosine. BAT thermogenesis was estimated by measuring mitochondrial guanosine-5'-diphosphate (GDP), cytochrome oxidase activity and mitochondrial oxygen consumption in BAT at an ambient temperature of 22 degrees C and during a six-hour cold-exposure at 4 degrees C. In insulin-deficient diabetic rats, the NE turnover, mitochondrial GDP binding, cytochrome oxidase activity and mitochondrial oxygen consumption in BAT at 22 degrees C were significantly reduced, compared with those of control rats. Treatment of STZ-induced diabetic rats with insulin prevented a decrease in NE turnover and BAT thermogenesis. Acute cold-exposure increased the NE turnover of BAT in insulin-deficient diabetic rats. The BAT thermogenic response to acute cold-exposure, however, did not occur in insulin-deficient diabetic rats. These results suggest that insulin is not essential in potentiating NE turnover in BAT after acute cold-exposure, but is required for cold-induced thermogenesis.     

Chronic treatment with myo-inositol reduces white adipose tissue accretion and improves insulin sensitivity in female mice

Type 2 diabetes is a complex disease characterized by a state of insulin resistance in peripheral tissues such as skeletal muscle, adipose tissue or liver. Some inositol isomers have been reported to possess insulin-mimetic activity and to be efficient in lowering blood glucose level. The aim of the present study was to assess in mice the metabolic effects of a chronic treatment with myo-inositol, the most common stereoisomer of inositol. Mice given myo-inositol treatment (0.9 or 1.2 mg g^?1 day^?1, 15 days, orally or intraperitoneally) exhibited an improved glucose tolerance due to a greater insulin sensitivity. Mice treated with myo-inositol exhibited a decreased white adipose tissue accretion (?33%, P<.005) compared with controls. The decrease in white adipose tissue deposition was due to a decrease in adipose cell volume (?33%, P<.05), while no change was noticed in total adipocyte number. In skeletal muscle, in vivo as well as ex vivo myo-inositol treatment increased protein kinase B/Akt phosphorylation under baseline and insulin-stimulated conditions, suggesting a synergistic action of myo-inositol treatment and insulin on proteins of the insulin signalling pathway. Myo-inositol could therefore constitute a viable nutritional strategy for the prevention and/or treatment of insulin resistance and type 2 diabetes.     

Reduced noradrenaline turnover in brown adipose tissue of lactating rats

Brown adipose tissue properties as well as noradrenaline turnover in the tissue were determined in 15-day lactating rats and virgin controls. Brown adipose tissue thermogenic activity was reduced in lactating rats as shown by a decrease in weight, cytochrome oxidase activity and mitochondrial GDP-binding. The noradrenaline turnover rate was lower in brown adipose tissue from lactating rats. It is suggested that diminished sympathetic activity in brown adipose tissue may be a major cause of the reduced tissue thermogenic activity during lactation.     

Effects of chronic exercise on cytokine production in white adipose tissue and skeletal muscle of rats

White adipose tissue (WAT) is a major source of production of cytokines involved in chronic diseases such as obesity, type 2 diabetes, and atherosclerosis. Long-term exercise has been proposed as a therapy to reduce chronic inflammation. We investigated here the influence of an intense exercise training (over 7 weeks) on several cytokine concentrations including interleukin 1 receptor antagonist (IL-1ra), IL-1beta, and IL-12 in serum, WAT, and skeletal muscle (SM) from non-obese rats. Two groups of 10 rats were investigated: one group was progressively trained (the two last weeks: 120min per day, 25m/min, 7% grade, 5 days per week) and the other age-matched group was used as a sedentary control. Compared to sedentary rats, weight gain was lower in the trained rats (P<0.01). In WAT, concentrations of IL-1ra, IL-1beta, and IL-12 were lower (P<0.001 for IL-1ra and IL-12, P<0.05 for IL-1beta) while they were higher in SM (P<0.01 for IL-1ra, P<0.001 for IL-1beta, P<0.05 for IL-12), and similar in serum. Significant correlations were noted between (i) body weight and WAT concentrations of IL-1ra, IL-1beta, and IL-12 (0.595, 0.450, and 0.481, respectively), (ii) body weight and IL-1beta concentration in SM (-0.526). We also observed significant negative correlations between WAT and SM concentrations of the three cytokines. We show here for the first time that intense exercise training with weight loss reduced concentrations of IL-1ra, IL-1beta, and IL-12 in WAT, while it increased them in SM. These results suggest that exercise could help reduce inflammation in WAT through mobilization of immune cells producing pro- and anti-inflammatory cytokines in SM.     

Uncoupling protein-3 mRNA levels are increased in white adipose tissue and skeletal muscle of bezafibrate-treated rats.

Fibrates are hypolipidemic drugs that are also able to improve glucose tolerance in animals and diabetic patients through an unknown mechanism. Since uncoupling proteins (UCP) seem to play an important role in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM), we examined whether treatment of rats with bezafibrate for 3, 7, or 15 days modified UCP mRNA levels. Using RT-PCR, we observed a weak ectopic expression of UCP-1 and a 2-fold increase in UCP-3 mRNA levels in white adipose tissue after 7 and 15 days of treatment. Moreover, bezafibrate administration caused a 1. 7-fold induction in UCP-3 mRNA levels in skeletal muscle on day 7. Since UCP-3 mRNA levels are reduced in skeletal muscle of diabetic patients, this effect may be involved in the improvement of insulin sensitivity caused by bezafibrate in NIDDM.     

Expression and distribution of acyl-CoA thioesterases in the white adipose tissue of rats

Acyl-CoA thioesterases (Acots) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and coenzyme A, and have the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, Acot1, is expressed and distributed in immature adipocytes located in the perivascular region of the white adipose tissue (WAT) of rats. Immunoblot analyses detected Acot1 in all of the WATs examined, while immunohistochemistry revealed positively stained layered structures surrounding the adventitia of blood vessels in the subcutaneous WAT. When the subcutaneous WAT was digested with collagenase and centrifuged, Acot1 was recovered in the stromal vascular fraction (SVF), and not in the large mature adipocytes. In the SVF, undigested cells attached to short tubular fragments of blood vessels showed positive immunostaining, as well as a proportion of the dispersed cells. These fibroblast-like cells contained fine particulate lipid droplets, stained by oil-red O dye, in their cytoplasm, or expressed fatty acid-binding protein 4, an adipocyte marker. After induction of adipocyte differentiation following a 15-day preculture without insulin, the dedifferentiated cells showed increased Acot1 expression with a diffuse distribution throughout the cytosol. These findings suggest that Acot1 expression is transiently upregulated at an early stage of adipocyte maturation, possibly to maintain cytosolic acyl-CoAs below a certain level until the cells acquire their full capability for fat storage.     

Apoptosis in the rat brown adipose tissue after insulin treatment

In this study it is demonstrated that chronically applied high dose (4 IU/kg) of insulin induced apoptosis of rat brown adipocytes in vivo. A qualitative morphological study was performed on the brown adipose tissue using light and electron microscopy, and revealed that: (1) apoptotic areas occurred in parenchyma; (2) morphological changes observed closely resembled the apoptotic body. These results indicate that apoptosis appears to be the mechanism responsible for the loss of brown adipocytes caused by hyperinsulinaemia.     

Lipoprotein lipase activity in white adipose tissue of rats subjected to exercise--rest cycles

This study was conducted to determine serum lipid levels and the activity of lipoprotein lipase in epididymal white adipose tissue of rats undergoing exercise training. During the 8-week period of treatment, one group of rats was kept sedentary and the remaining animals were exercise trained either continually (1 h of daily treadmill running) or intermittently (alternate weeks of daily running and inactivity). Exercise training, either continual or intermittent, decreased postprandial serum total and high-density lipoprotein cholesterol concentrations, which returned to sedentary levels in the intermittently trained animals following a week of rest. Lipoprotein lipase activity in whole epididymal adipose pad was lower in rats trained continually than in the sedentary group at the end of the treatment. The intermittent training program elicited large fluctuations in both the specific (per milligram of protein) and total (per tissue) activity of lipoprotein lipase in white adipose tissue. During rest periods, enzyme activity rose to levels that were higher than those of sedentary rats, whereas lipase activity was below that of sedentary animals following a week of running. In the last exercise--rest cycle, body weight gain of the intermittently trained rats was nearly abolished during the week of running, but it increased above that of sedentary animals during weeks of rest. The present results suggest that the modulation of lipoprotein lipase activity in white adipose tissue is one of the adaptations that take place to accommodate the fluctuations in the rate of energy deposition that occur in the rat during an intermittent training program.     

Regulation of triglyceride biosynthesis in adipose and intestinal tissue

The synthesis of phosphatidic acid and di- and triglycerides via the glycerol-3-phosphate pathway is markedly inhibited by 2-monooleyl ether in microsomal and whole cell preparations obtained from adipose and intestinal tissue. Monoglycerides are also inhibitors under conditions in which their hydrolysis is minimized. A correlation between inhibition by, and the hydrolysis of, monoglycerides has been demonstrated. 2-Monooleyl ether is the most effective inhibitor of the several mono- and di- ethers and esters studied. The specificity of the inhibition of glycerol-3-phosphate acylation by 2-monoethers or 2-monoesters has been demonstrated because microsomal NADH- and NADPH-cytochrome c reductase activities were not significantly inhibited. The reported control mechanism for triglyceride biosynthesis is discussed in relation to the regulation of fatty acid uptake and release in adipose tissue and the absorption and metabolism of triglycerides by the intestinal mucosa.     

Ventromedial hypothalamic stimulation accelerates norepinephrine turnover in brown adipose tissue of rats

To establish a functional link between the ventromedial hypothalamus (VMH) and brown adipose tissue (BAT), effects of electrical stimulation of the VMH and the lateral hypothalamus (LH) on norepinephrine (NE) turnover in the interscapular BAT were examined in rats. Stimulation of the VMH elicited about 3-fold increase in the rate of NE turnover in BAT, whereas stimulation of the LH had no appreciable effects. The effect of VMH stimulation was abolished after sympathetic ganglion blockade or by surgical sympathetic denervation of BAT. It was concluded that there is a sympathetic nerve-mediated connection between the VMH and BAT, and that stimulation of the VMH induces metabolic activation and heat production in BAT through an increase in sympathetic nerve activity.