Study On Effects Of Adipocyte Differentiation In Improvement By Sports On Insulin Resistance In High-fat-diet Rats

Insulin resistance(IR)refers to as the amount of insulin secretion in the normal levels, the physiological effects to stimulate the target cells, uptake and use of glucose reduces, or the maintenance of the target cell uptake of glucose in the normal physiological effects needs extraordinary amount of insulin. IR is not only an important feature in the development process of type 2 diabetes, but also the key program to metabolic syndrome, closely linked with obesity, hyperlipidemia, hypertension and cardiovascular disease. Studies have shown that adipose tissue and insulin resistance are closely linked. Obesity can lead to abnormal adipocyte differentiation, cell hypertrophy, leading to the occurrence of insulin resistance. Abnormal differentiation also activate the insulin-inhibited lipolysis process, which induced a large number of FFA from adipose tissue secreted into the bloodstream and affect the insulin sensitivity in other tissues. Adipose tissue can secrete resistin, inflammatory cytokines and other substances to regulate the body's energy metabolism and play an important role in insulin resistance. It can be concluded that the normalization of adipocyte differentiation is the key to improving insulin resistance. From the research of exercise treatment on diabetes, sports really improves insulin resistance and as a prevention and treatment of diabetes. Some experiments have confirmed that adipose tissue may be the origin parts of insulin resistance, then how to connect the function of insulin resistance and adipocyte differentiation? What changes in the differentiation to impact insulin resistance? It can be better understood through the changes of adipoctye differentiation in insulin resistance improved by exercise. Therefore, the purpose of this study is to explore the molecular mechanism of adipocyte differentiation and analyze adipocyte differentiation and involved morphological and functional changes in the influence of insulin resistance in order to learn the mechanism of exercise improve the abnormal adipocyte differentiation and insulin resistance and provide the theory basis for prevention and treatment of obesity and some metabolic syndrome.Animal experiments was designed to explore the effects of high-fat diet and exercise training on rat adipocyte differentiation and insulin resistance and analyze the mechanism of changes in adipocyte differentiation in the formation of insulin resistance in rats. In the one hand, treadmill training intervention was used in high-fat diet rat model of insulin resistance. Some indicators including fasting blood glucose, blood insulin levels, insulin sensitivity index, resistance index of model group and intervention group were measured and analyzed to determine the improvement of treadmill training. while some differentiation indicators were also detected to analyze the mechanism of adipocyte differentiation in insulin resistance improved by exercise. In the other hand, different stages were selected to observe the changes of indicators involved adipocyte differentiation to learn the effects of treadmill training and high-fat diet intervention to provide a basis to clarify the relationship of adipocyte differentiation and insulin resistance.PartⅠThe effects of treadmill exercise of eight weeks on adipocyte differentiation and insulin sensitivity in high-fat diet ratFourty Sprague-Dawley (SD) rats were randomly assigned into four groups: rats in group C were fed by standard diet without exercise training, rats in group H were fed by high-fat diet without exercise training, rats in group HE were exercised by treadmill training (60min/d, 20 m/min, 5d/w) and fed by high-fat diet, rats in group E were exercised by same training but fed by standard diet. By the end of 8th week, all rats were killed and serum and adipocyte parameters were evaluated.1. Serum indicators: Blood glucose concentration among the groups no significant difference (P>0.05); The serum insulin concentrations of H wasand higher compared with C group and the HE group, but did not reach significant difference, E group were lower than C, H and HE group, the differences were significant (respectively P<0.05, P <0.01, P<0.05); ISI and HOMA-IR of H group has significant differences with the C group (P<0.01 and P <0.05). In the comparison of the groups, there is no difference.2. Different protein expression in adipose tissuePPARγ: E group was higher than C group, the difference was significant (P<0.01), showed that exercise can up-regulate PPARγ. H was higher than C, HE was lower than H, but no statistical significance. C/EBPα: H group was higher than the other groups, the difference is highly significant (P<0.01); E was higher than C, HE was lower than H, indicating that exercise enhances C / EBPαincreasesPI3K: E group was higher than C group, the difference was significant (P<0.05); H was the most higher than the other groups, the difference was significant compared with the C and HE(respevtively P<0.05 and P<0.05), E was higher than HE, but no statistical significance, indicating exercise can promote the expression of PI3K.PKB: H group was the highest, C was lower than E, but in all groups there was no significant difference.The trend was simple to PI3K.3. Differentiation and proliferation index in adipose tissuesFat cell differentiation: E group was the highest and the difference compared with the other groups was significant (P<0.05), HE was higher than H, H slightly higher than C, but there was no significant difference.Proliferation index: C group was the highest, there were significant differences compared with H and E (respectively, P<0.01 and P<0.05); HE was lower than H and E (P <0.05).4. Histological analysis and morphometry: Size of adipose cell in E group was smaller than C, the difference was significant (P<0.01), number of cells were more than C, suggesting that exercise may increase the amount of adipose tissue of small cells; size of H was smaller than C (P <0.01), and the number increased; HE has larger size and decreased number than H.5. Correlation analysis between ISI and the other index: r value of ISI and HOMA-IR reached -0.97. In insulin resistance-related indicators, r value of PI3K and ISI was 0.50, while the PKB was only -0.04. In adipocyte differentiation-related indicators, significant differences in indicators were visceral fat pad weight and visceral fat%, correlation coefficient for -0.42 and -0.39 respectively; C / EBPαexpressed moderate correlation with the ISI and correlation coefficient was -0.55; the number of adipose cells showed highly correlated with the ISI, the correlation coefficient was 0.95.PartⅡThe related changes of adipocyte differentiation in rats at different stages of exercise and high-fat feeding interventionThe experiment was assigned into four stages (2,4,6,8 weeks), Fourty Sprague-Dawley (SD) rats in each stage.The rats were randomly divided into four groups: rats in group C were fed by standard diet without exercise training, rats in group H were fed by high-fat diet without exercise training, rats in group HE were exercised by treadmill training (60min/d, 20 m/min, 5d/w) and fed by high-fat diet, rats in group E were exercised by same training but fed by standard diet. By the end of each stage, all rats were killed and serum and adipocyte parameters were evaluated.1. The visceral fat weight and relative weight: In 2-week groups, C group and E has no significant difference, H was higher than HE (P<0.05); In 4-week groups, H was higher than C(P<0.01), HE was lower than H; In 6-week groups, exercise group were lower than the corresponding control group (C vs E, P<0.05; H vs HE, P<0.01), C was higher than H, but no statistical significance; In 8-weeks group, exercise group were lower than the corresponding control group (P<0.01 ), H was higher than C, the difference is highly significant (P <0.01). C and E present the trends of increase, while the H group and the HE group increase and then decrease and finally rapidly rise.2. Serum indicators: TC, TG, LDL, FFA of H group were higher than C and HE, present very significant and significant difference at different stages (P<0.01 and P<0.05,respectively), HDL appears the trend of rise-- fall– rise and with significant difference in 4 weeks (P<0.01); Except HDL and FFA, the other serum indicators in E were lower than C, but LDL is only show a significant difference (P<0.01) in 2 weeks.3. Differentiation ability in adipose cell: There was no significant difference in 2,4,6 weeks among groups; in 8-week groups, E was highest(P<0.05), HE also increased, but no statistical significance. longitudinal comparisons, C and H were not changed much; HE and E increased.4. Proliferation index in adipose tissue: In 2-week groups, there were higher proportion of proliferation, but no significant difference compared with each other; In 4-week groups, E was higher than C (P<0.05), the other groups had no significant difference; In 6-week groups, it showed no significant difference among groups, but the E group was still slightly higher; In 8-week groups, E was lower than C(P<0.05), HE is higher than H(P<0.05), H was lower than C(P<0.01). From the longitudinal comparisons, there have a higher level In 2-week groups, significantly decreased in 4,6,8 weeks, the most obvious is the H group.5. Protein expression in adipose tissueChanges of PPARγ: In 2-week groups, E was higher than the other groups(P<0.01), HE group was minimum; In 4-week and 6-week groups, there was no significant differences among groups; In 8-week groups, exercise group were higher than the corresponding control group, E and C has significant difference(P<0.01). From the longitudinal comparisons, C showed a gradual upward trend until six weeks, then declined slightly; H and HE increased gradually and the greater increase in HE; E showed elevated after the reduction trend.Changes of C/EBPα: In 2-week groups, E was highest (P<0.01), HE was higher than H, the difference was significant (P<0.05); In 4-week and 6-week groups, there was no significant difference among groups; In 8-week groups, C/EBPαof H increased dramatically, there was significant difference compared with the C (P<0.05), E was higher than C, HE slightly lower than H. From the longitudinal comparisons, C group showed rising trend; H has the same trend with C, but fell back in 6 weeks, then increased in 8 weeks; E Group was in a downward trend in 2-6 weeks and rebounded in 8 weeks; HE and E has the same changes.6. Histological analysis and morphometry: In 2-week groups, adipose cells of E group has the smallest size and most number, there was significant difference compared with H and HE(P<0.01), size in H was higher and number was more than C(P<0.01), HE group has more number than H(P<0.01); In 4-week groups, there was no significant difference, E has more size and number than C group, HE and C has larger size and less number than H; In 6-week groups, size and number in H were higher than C, but the difference was not significant. Number and size of cells in exercise group were higher than their corresponding control group. E were higher than HE(P<0.01); In 8-week groups, E has smaller size (P <0.01) and more number than C, He has larger size and less number than H(P<0.01),H group was higher than C(P<0.01). In longitudinal comparisons, C increased until 8 weeks.H group decreased then steadily rise; E has downward trend in 2-6 weeks and increase in 8 weeks , but not reach the level of 2 weeks. HE and E has the same changes. Conclusion1.It can lower insulin sensitivity in rats by 8-week high-fat diet, resulting in insulin resistance and its possible mechanism as follows:①To increase body weight and visceral fat mass;②to reduce the adipocyte synthesis and increase serum TG and TC;③to increase excessively PPARγand C/EBPαexpression and lead abnormal adipocyte differentiation;④to reduce the ability of proliferation in adipose tissue, while accumulate the number of adipose to a larger base, which may make preparations for larger size;⑤to an abnormal increase in serum FFA, which worsen the insulin resistence.2.Exercise can improve insulin function caused by high-fat diet and its possible mechanism as follows:①To decrease body weight and visceral fat mass;②to promote the adipocyte synthesis and decrease serum TG and TC;③to increase PPARγand C/EBPαexpression and promote adipocyte differentiation;④to promote the ability of proliferation in adipose tissue and has more number and smaller size of adipose cells;⑤to decrease serum FFA, which promote the lipid mechanism.3. The effects of exercise and high-fat diet need time to accumulation and the index changes has turning point in 4 weeks or 6 weeks.