Effects Of Diet (Ethanol Consumption And High-Fat Consumption) On Levels Of Adipokines In Visceral Adipose Tissues And Sera Of Rats

Background:Both white and brown adipose tissue (WAT and BAT, respectively) can be distinguished histologically and functionally. Whereas BAT is specialized in the production of heat, WAT stores excess energy as triacylglycerols. Recent studies demonstrate that WAT is an important endocrine organ that produces numerous peptides and proteins with broad biological activity. Secretory products of WAT are collectively called adipokines. Among these adipokines, adiponectin, leptin, resistin and visfatin are implicated in the development of metabolic syndrome. Leptin is considered one of the main peripheral endocrine signals involved in the regulation of appetite and body weight. Adiponectin is a potent anti-inflammatory and anti-diabetic protein. In contrast, resistin causes—at least in the murine system-insulin resistance. Visfatin is highly enriched in the visceral adipose tissue of both humans and rodents. It mimics the effects of insulin by lowering plasma glucose levels. Cartonectin is an adipocyte secretory protein that was identified in 2001. It is an adiponectin paralog and a member of the C1q/TNF-a molecular superfamily, which not only has anti-inflammatory properties but also can effectively stimulate adiponectin and resistin secretion from murine adipocytes in vitro. This stimulatory property of cartonectin demonstrates that it is a potentially important adipokine in the diabetes research field.At present, ethanol is the most widely abused addictive substance. In addition to decreasing glucose uptake in rat adipocytes, chronic ethanol exposure in rats has been shown to increase the rate of triglyceride degradation in adipose tissue, resulting in elevated circulating free fatty acids (FFAs). Our prior results measured depressed insulin-stimulated glucose uptake and increased insulin resistance in isolated skeletal muscle of rats exposed to chronic excess ethanol. We also observed down-regulated expression of the glucose transporter, GLUT4, in rat cardiac muscle tissue. However, how is the effect of chronic ethanol feeding on adipokines?Previous studies investigating the effects of ethanol on adipokines have concentrated mainly on their detection levels in serum. Due to different experimental conditions, no consistent conclusions can be drawn concerning the effects of ethanol on adipokines. Most studies report that ethanol elevated serum levels of leptin and resistin. Other studies indicate that serum leptin levels in rodents and humans were decreased or unchanged with ethanol intake and that resistin levels in human serum were not affected by moderate ethanol intake. Serum adiponectin levels decreased in rats chronically treated with high doses of ethanol, while rats treated with moderate doses of ethanol had elevated adiponectin levels. Nevertheless, data are sparse regarding the effects of ethanol on adipokine levels in adipose tissue. In addition, to the best of our knowledge, no previous studies have described the effects of ethanol on visfatin and cartonectin levels.As for unhealthy lifestyles of excess ethanol and/or fat intake, now it has been fairly general with the gradually elevated living standard. Many studies reported alcohol and high-fat diet are risk factors for the development of type 2 diabetes. Our previous studies demonstrated that both long-time ethanol intake and high-fat diet impaired glucose uptake in rat skeletal muscles and cardiac muscles. Up to date, literature on effects of ethanol intake or high-fat diet on adipokines present the following characters:(i) although detailed observations exist on the effects of either ethanol or high-fat alone on adipokines, no data are available on the comparison of effects between ethanol and high-fat diet on adipokines under the same study conditions; (ii) most studies pay close attention to serum adipokine levels and lack, even completely, attention to adipokine expression in their origin, adipose tissue, and so it is not entirely clear that whether the consistency between adipose tissue and serum levels of adipokines exists or not.Objective:1 To determine VAT and sera levels of leptin, adiponectin, resistin, visfatin and cartonectin in rats exposed to ethanol treatments using enzyme-linked immunosorbent assays (ELISAs)2 To explore correlations between these VAT and serum adipokine levels.3 To compare the changes of adiponectin, resistin and cartonectin in both VAT and sera between long-term ethanol intake and high-fat diet feeding.Materials and methods:1 AnimalsGrouping 1:Forty-eight SPF male Wistar rats (weight,180-200 g; age,8 wk,) were randomly allocated into four experimental groups (n=12 in each group): control group (distilled water:5.0 g·kg-1·d-1), low-, middle-, high-dose group (corresponding ethanol:0.5,2.5,5.0 g·kg-1·d-1). Distilled water or edible ethanol was given once daily between 8:00 AM and 9:00 AM through a gastric tube. All the treatments lasted for 22 weeks.Grouping 2:Thirty-six SPF male Wistar rats (weight,180-200 g; age,8 wk) were randomly allocated into three experimental groups (n=12 in each group): normal diet group (N), high-dose ethanol group (HE) and high fat group (HF). Rats in group HE received edible ethanol (50%, v/v) once at a total daily dosage of 5 g-kg-1 between 8:00 AM and 9:00 AM through a gastric tube and rats in other group received a corresponding volume of distilled water. All the treatments lasted for 22 weeks.2 Specimen collection and storage After a 22-wk-feeding period, Blood samples were obtained from inferior vena cava, centrifuged, allocated to distinct EP tubes, and stored in-80℃.The epididymal and perirenal fat pads were quickly remove. The tissues were frozen in liquid nitrogen for adipokines analyses.3 Biochemical analysis and evaluation of insulin sensitivity Fasting blood glucose levels (FBG) were measured by glucose oxidase method and fasting serum insulin levels (FINS) were measured by radioimmunoassay, respectively. HOMA-IR was calculated by the following formula:FBG (mmol/1)×FINS (μmol/ml)/22.5.4 Extraction of total proteins in adipose tissues The adipose tissues from liquid nitrogen were placed on ice and 200 mg of each sample was excised. The excised tissues were dissolved in 200μl PBS (0.01M PH 7.4) and homogenized with VCX450 Ultrasonic Cell Disruption System. The adipose tissue homogenate was frozen in-80℃overnight and thawed on ice the next day. After two freeze-thaw cycles, the homogenate was centrifuged thrice and the middle layer was the protein-required liquid.5 Enzyme-linked immunosorbent assay (ELISA) Concentrations of leptin, adiponectin, resistin, visfatin and cartonectin in both adipose tissues and sera were respectively measured by ELISA kits.Results1 Chronic ethanol consumption and high fat diet feeding raised insulin resistance index.2 Chronic ethanol consumption elevated leptin contents in VAT and sera.3 Chronic ethanol consumption decreased adiponectin contents in VAT and sera.4 Chronic ethanol consumption increased resistin contents in VAT and sera.5 Chronic ethanol consumption raised visfatin contents in VAT and sera.6 Chronic ethanol consumption decreased cartonectin contents in VAT. 7 Chronic ethanol consumption elevated TNF-a levels in serum.8 Positive correlation between VAT contents and serum levels of adiponectin, leptin, resistin or visfatin existed.9 Tthe adiponectin changed more largely in Group HE than Group HE10 Long-term ethanol intake increased resistin contents in VAT and serum significantly; whereas HF diet, under the same treatment period, significantly decreased the resistin contents in VAT and did not yet alter the serum resistin levels remarkably.11 Long-term ethanol intake had decreased significantly the cartonectin contents in VAT and did not yet change the serum cartonectin levels; whereas HF diet feeding notably raised the serum cartonectin levels and did not alter significantly the VAT cartonectin contents.Conclusions:1 Chronic ethanol consumption affects, in a dose-response manner, adipokines contents in both VAT and sera except serum cartonectin.2 There exists positive correlation between VAT contents and serum levels of adiponectin, leptin, resistin or visfatin.3 Adiponectin, resistin, cartonectin are more susceptible to chronic ethanol intake relative to HF diet.