Study On Relationship Of Hepatic Endoplasmic Reticulum Stress, Hepatic Steatosis And Insulin Resistance In Obese Rats Induced By High Fat Diet

With the changes of eating habits and lifestyle, the incidence of obesity has increased year by year. In2008, overweight and obesity were estimated to afflict nearly1.5billion adults worldwide. A recent analysis of new data predicted that in2030an estimated2.16billion adults worldwide will be overweight and1.12billion will be obese. Many metabolic diseases were concomitant with obesity usually, including type2diabetes, NAFLD and atherosclerosis et al. Metabolic syndrome (MetS) to be highly prevalent and contributes to a rapidly growing problem globally. About40%of adults in US population is estimated to have MetS by the time they reach the age of60years. At least one-fourth of the adult European population may have MetS, with a similar prevalence in Latin America. MetS is also considered an emerging epidemic in Asian countries, with a prevalence of8%-13%in men and2%-18%in women. Thus, obesity and related MetS have a wide range of epidemic in the worldwide and result in huge medical and economic burdens. Therefore, it is necessary to clarify the obesity and related MetS pathogenesis and identify the prevention measures.Except for erythrocyte, all eukaryotic cells contain endoplasmic reticulum (ER), which has a variety of functions and is the important place of glucose and lipids metabolism. Interaction between ER stress and immune system can give rise to inflammation and oxidative stress. Thus, endoplasmic reticulum stress is associated with the development of metabolic diseases. In addition to that, ER is sensitive to nutrients and ER stress is also affected by dietary nutrients and ingredients.Therefore, insulin resistance and hepatic steatosis was induced in obese rats by high fat diet enriched with lard oil and soybean oil. The differences of insulin resistance and hepatic steatosis were observed induced by high fat diet with different fat compositions. We discussed the possible pathological mechanism of insulin resistance and hepatic steatosis from the perspective of ER stress, inflammation and innate immune system responses.Part1Study on differences of insulin resistance and hepatic steatosis in obese rats induced by high fat diets with different fat compositionsSection one Study on differences of obesity in rats induced by high fat diets with different fat compositionsObjective Obese rats were induced by isocaloric high fat diet enrich with lard oil and soybean oil respectively. We study the differences of incidence of obesity in rats induced by different high fat diet and observe the influences on body weight and body fat in rats by low fat diet intervention.Methods One hundred male Sprague-Dawley rats (purchased from Shanghai Sippr-VK lab animal Co. Ltd.) weighing120-130g were housed individually in cages under controlled conditions (a12-h light/dark cycle;17-27℃;40-60%relative humidity). After10days acclimation, the rats were weight-matched and randomly divided into3groups:a HL group (n=45) on a high lard oil diet (rich in saturated fatty acids); the HS group (n=45) on a high soybean oil diet (rich in polyunsaturated fatty acids); and a LF group (n=10) remained on standard laboratory chow food (provided by Tongji Medical college lab animal center, Wuhan). The standard chow food contains3.29kcal/g and the high fat diet contains4.62kcal/g. Except for enriched fat, the other compositions of HL and HS are all the same. Dietary intake was recorded daily and body weight was measured weekly in the morning throughout the study. After10weeks of free access to their corresponding diet, rats in HL or HS group with body weights more than x-+1.96s of LF group were classified as diet induced obesity (DIO) and selected for further study. The DIO rats from HL and HS group were randomly subdivided into two groups respectively. One group of the HL-DIO or HS-DIO rats were switched to LF chow food (HL-DIO-LF, HS-DIO-LF) and the other group of each were kept on their original high fat diet (HL-DIO-HL, HS-DIO-HS) for the following8weeks. All rats were provided water and food ad libitum. Tail blood was collected at Ow and the end of the10th week, and serum samples were stored at-80℃. Terminally, all rats were sacrificed after12h fasting. Trunk blood was collected and centrifuged, serum was stored for further use. Perirenal and epididymal white adipose tissues were dissected and weighed.Results At the end of the10th week,19(42.2%of HL group) and22(48.9%of HS group) rats were designated as DIO rats according to the mean body weight of LF group. No statistical difference was found between HL and HS in obesity incidence. During the first10weeks, the body of DIO rats fed with HL and HS were both significantly higher than that in LF rats starting from the3rd week to the10th week, whereas no significant difference in body weight was observed between obese rats fed with the HL and HS throughout the whole experiment. During the following8weeks, shifting from high fat diet to LF chow food reduced the weight gain in HL-DIO-LF rats but not in HS-DIO-LF rats. Energy intake was calculated according to food intake recorded everyday. Cumulative energy intake during the first10weeks and the following8weeks were compared among groups. Over the first10weeks, HL-DIO and HS-DIO rats had higher energy intake than LF rats, whereas no significant difference was found between HL-DIO and HS-DIO group (P>0.05). During the following8weeks, the differences of energy intake in all groups were not significant. At the end of the18th week, HL-DIO-HL rats had the highest percentage of body fat among all groups, even though the HS-DIO-HS rats also had significantly higher percentage of body fat compared to LF rats. HL-DIO-LF and HS-DIO-LF rats had lower percentage of body fat compared to their respective counterparts remaining on high fat diet, but the percentage of body fat in HL-DIO-LF rats was still higher than that of LF rats. At the end of10th week, TG in HL-DIO rats were higher than that in HS-DIO and LF rats, TG in HS-DIO rats were higher than LF rats also. TC in HL-DIO and HS-DIO rats were higher than that in LF rats, no significant differences were observed between HL-DIO and HS-DIO rats. At the end of18th week, TG in All the other four groups were significantly higher than that in LF rats, TG in HL-DIO-HL rats were higher than HS-DIO-HS rats, TG in HL-DIO-LF rats were lower than HL-DIO-HL rats. TC in HL-DIO-HL and HS-DIO-HS rats were significantly higher than that in LF rats, TC in HL-DIO-LF and HS-DIO-LF rats were significantly reduced compared to their respective counterparts.Conlusions The incidence of obesity induced by HL and HS had no statistical difference. Although no significant differences were observed in body weights between HL-DIO-HL and HS-DIO-HL rats, the total body fat in HL-DIO-HL rats was significantly higher than that in HS-DIO-HS rats. Both HL and HL gave rise to dyslipidemia, especially HL. Body weight, body fat and dyslipidemia can be reduced by low fat diet intervention.Section two Study on differences of hepatic steatosis in obese rats induced by high fat diets with different fat compositionsObjective Obesity in rats were induced by isocaloric high fat diet enrich with lard oil and soybean oil respectively. We study the differences of hepatic steatosis in obese rats induced by different high fat diet and observe the influences on levels of hepatic steatosis in rats by low fat diet intervention.Methods The obesity model was established as part one section one. Terminally, all rats were sacrificed after12h fasting. Trunk blood was collected and centrifuged, serum was stored for further use. Livers were removed and weighed, then parts of the livers were fixed with formaldehyde for histology and the remaining parts were snap-frozen in liquid nitrogen immediately and stored at-80℃for further use.Results At the end of18th week, no significant differences in liver weight were found in all five groups of rats. Liver TG levels in HL-DIO-HL rats were significantly increased compared with any other groups of rats. The HL-DIO-HL and HS-DIO-HS rats had higher liver TG content compared with their LF intervention counterparts. The liver TG levels in HS-DIO-HS rats were increased as well when compared to LF rats but still lower than that in HL-DIO-HL rats. Comparisons in pathological section, although the incidence of hepatic steatosis induced by HL and HS were both100%, the levels of hepatic steatosis in HL-DIO-HL rats were more serious than that of HS-DIO-HS rats. The morphological characters of hepatocyte in HL-DIO-LF and HS-DIO-HS were similar to LF rats.Conclusions Hepatic steatosis can be observed in obese rats induced by both HL and HS, especially by HL. The severity of hepatic steatosis can be alleviated by low fat diet intervention.Section three Study on differences of insulin resistance in obese rats induced by high fat diets with different fat compositionsObjective Obesity in rats was induced by isocaloric high fat diet enrich with lard oil and soybean oil respectively. We study the differences of insulin resistance in obese rats induced by different high fat diet and observe the influences on severity of insulin resistance in rats by low fat diet intervention.Methods The obesity model was established as part one section one. Tail blood was collected at Ow and the end of the10th week, and serum samples were stored at-80℃. Terminally, all rats were sacrificed after12h fasting. Trunk blood was collected and centrifuged, serum was stored for further use. Livers were removed and weighed, then snap-frozen in liquid nitrogen immediately and stored at-80℃for further use.Recults Cumulative body weight gains over18weeks in HL-DIO-HL, HS-DIO-HS and HS-DIO-LF rats were higher than that of LF rats, HL-DIO-LF rats were lower than that of HL-DIO-HL rats. Cumulative energy intake over18weeks in HL-DIO-HL and HS-DIO-HS rats were higher than that of their respective high fat diet counterparts and LF rats. At the end of18th week, no significant differences of fasting serum glucose levels were found among all five groups. Levels of fasting serum insulin and HOMA-IR in HL-DIO-HL rats were significantly higher than that of HS-DIO-HS and LF rats; HL-DIO-LF were decreased comparing to their HL counterparts, but still higher than that of LF rats; no significant differences were found between HS-DIO-HS and HS-DIO-LF rats in levels of fasting serum insulin and HOMA-IR.Conclusions Although no significant differences were observed in cumulative body weight gain and energy intake between HL-DIO-HL and HS-DIO-HS rats, better insulin sensitivity was found in HS-DIO-HS rats comparing to HL-DIO-HL rats. Sensitivity of insulin was improved by low fat diet intervention.Part2Study on mechanisms of differences in insulin resistance and hepatic steatosis in obese rats induced by high fat diets with different fat compositionsSection one Study on relationship between hepatic endoplasmic reticulum stress and hepatic steatosis in obese rats induced by high fat diets with different fat compositions and the impact of low fat diet interventionObjective To explore the impact of high fat diets with different fat compositions on hepatic endoplasmic reticulum stress and the mechanism of hepatic endoplasmic reticulum stress on the development of hepatic steatosis through detecting the expression of hepatic steatosis and endoplasmic reticulum stress related molecules.Methods The obesity model was established as part one section one. At the end of18th week, all rats were sacrificed after12h fasting. Trunk blood was collected and centrifuged, serum was stored for further use. Livers were removed and weighed, then snap-frozen in liquid nitrogen immediately and stored at-80℃for further use. Content of hepatic GSH and MDA was assayed by GSH and MDA kits. Hepatic mRNA expression of GRP78, CHOP, PPARy, PPARa and SREBPlc were detected by Real-time PCR. Protein expression of CHOP in liver was determinged by Western Blot.Results Hepatic content of GSH in HL-DIO-HL and HS-DIO-HS rats was lower than that of LF rats,; there was no significant difference between HL-DIO-HL and HS- DIO-HS rats; hepatic GSH content in HL-DIO-LF rats was increased significantly comparing to HL-DIO-HL rats, but not in HS-DIO-LF rats. Hepatic content of MDA in HL-DIO-HL rats was higher than that of HS-DIO-HS and LF rats; there was no significant difference between HL-DIO-HL and HS-DIO-HS rats; HL-DIO-LF and HS-DIO-LF was decreased significantly comparing to their respective HL or HS counterparts. Hepatic mRNA expression of GRP78and CHOP in HL-DIO-HL rats were both higher than that of HS-DIO-HS and LF rats; HL-DIO-LF and HS-DIO-LF was decreased significantly comparing to their respective HL or HS counterparts. CHOP protein expression was similar to its mRNA expression. PPARγ mRNA expression in all the other four groups were increased significantly comparing to LF rats; there was no significant difference among the four groups. PPARα mRNA expression in HL-DIO-HL and HL-DIO-LF rats were higher than that of LF rats; PPARα mRNA expression in HS-DIO-HL and HS-DIO-LF rats were lower than that of LF rats; there was no impact in PPARα mRNA expression on low fat diet intervention. Hepatic SREBPlc mRNA expression in HL-DIO-HL and HS-DIO-HS were up-regulated significantly comparing to LF rats; there was no significant difference between HL-DIO-HL and HS-DIO-HS; HL-DIO-LF and HS-DIO-LF were down-regulated significantly comparing to their respective HL or HS counterparts.Conclusions Although hepatic steatosis was observed in obese rats induced by both HL and HS, the levels of hepatic endoplasmic retimulum stress and oxidative damage in HL-DIO-HL rats were more severe than that in HS-DIO-HS rats, which may involved in the development of NAFLD.Section two Study on relationship between hepatic osteopontin expression and insulin resistance in obese rats induced by high fat diets with different fat compositions and the impact of low fat diet interventionObjective To compare the differences of insulin resistance, OPN and proinflammatory factors expression in obese rats induced by high fat diet enrich with lard oil and soybean oil and explore the impact of OPN in hepatic inflammation and the development of insulin resistance. Methods The obesity model was established as part one section one. Tail blood was collected at Ow, and serum samples were stored at-80℃.At the end of18th week, all rats were sacrificed after12h fasting. Trunk blood was collected and centrifuged, serum was stored for further use. Livers were removed and weighed, then snap-frozen in liquid nitrogen immediately and stored at-80℃for further use. Levels of serum OPN were assayed by ELISA kit. Hepatic OPN and TNF-α mRNA expression were detected by Real-time PCR. Hepatic OPN protein expression was determined by Western Blot.Results No significant difference was found of serum OPN levels in all groups at the beginning and the end of the experiment. Hepatic OPN mRNA and protein expression were both increased significantly in HL-DIO-HL rats; there was no siginifcent difference between HS-DIO-HS and LF rats; HL-DIO-LF were down-regulated comparing to HL-DIO-HL rats but not in HS-DIO-LF rats. Hepatic TNF-α mRNA expression in HL-DIO-HL rats were higher than that of HS-DIO-HS and LF rats; HS-DIO-HS were higher than that of LF also; HL-DIO-LF and HS-DIO-LF were both down-regulated significantly comparing to their respective HL or HS counterpars.Conclusions The insulin sensitivity in HS-DIO-HS rats was better than that in HL-DIO-HL rats. The better insulin sensitivity in HS rats may attribute to the low expression of OPN and proinflammatory factors in livers.Section three Study on relationship among innate immune system TLRs expression, insulin resistance and hepatic steatosis in obese rats induced by high fat diets with different fat compositions and the impact of low fat diet interventionObjective To compare the differences of insulin resistance, hepatic steatosis and hepatic TLRs expression in obese rats induced by high fat diet enrich with lard oil and soybean oil and explore the impact of TLRs in hepatic inflammation and the development of insulin resistance and hepatic steatosis.Methods The obesity model was established as part one section one. At the end of18th week, all rats were sacrificed after12h fasting. Trunk blood was collected and centrifuged, serum was stored for further use. Livers were removed and weighed, then snap-frozen in liquid nitrogen immediately and stored at-80℃for further use. Hepatic mRNA expression of TLR2, TLR4, GRP78, CHOP and TNF-α were detected by Real-time PCR.Results TLR2mRNA expression in HL-DIO-HL rats were higher significantly than that of HS-DIO-HS and LF rats; there was no significant difference between HS-DIO-HS and LF rats; HL-DIO-LF were up-regulated significantly comparing to HL-DIO-HL, but not in HS-DIO-HS rats. TLR4mRNA expression in HL-DIO-HL rats were higher significantly than that of HS-DIO-HS and LF rats; there was no significant difference between HS-DIO-HS and LF rats; HL-DIO-LF and HS-DIO-LF were down-regulated significantly comparing to their respective HL or HS counterparts. The levels of GRP78, CHOP AND TNF-α mRNA expression were same as part two.Conclusion High expression of TLRs and TLRs pathway downstream inflammatory factors were induced by high lard oil diet enriched with saturated fatty acids. Immune system is closely associated with the development of metabolic diseases, such as insulin resistance and hepatic steatosis.Part3Study on differences of hepatic ER stress in high fat diet induced obese and obese resistance ratsSection one Animal model of obesity susceptible and obesity resistanceObjective To establish the animal model of obesity susceptible and obesity resistance and observe the differences of body weight, food intake, levels of serum lipids and insulin sensitivity in obesity susceptible and obesity resistance rats and provide animal model for further study.Methods Fifty-five male Sprague-Dawley rats weighing120-130g were housed individually in cages under controlled conditions. After10days acclimation, the rats were weight-matched and randomly divided into2groups:a high fat group (n=45) on a high lard oil diet (rich in saturated fatty acids) and a LF group (n=10) remained on standard laboratory chow food. The standard chow food contains3.29kcal/g and the high fat diet contains4.62kcal/g. Dietary intake was recorded daily and body weight was measured weekly in the morning throughout the study. After10weeks of free access to their corresponding diet, rats in HF group with body weights more than x-+1.96s of LF group were classified as diet induced obesity (DIO); rats in HF group with body weights less than x-+s of LF group were classified as diet induced obesity resistance (DIO-R), DIO and DIO-R rats were selected for further study. Terminally, all rats were sacrificed after12h fasting. Trunk blood was collected and centrifuged, serum was stored for further use.Results The incidence of obesity and obesity resistance in rats induced by high fat diet were42.2%and26.6%respectively. From the3rd to the10th week, body weights in DIO rats were higher than that in DIO-R and LF rats; there was no significant difference between DIO-R and LF rats. Food intake in LF rats were higher than that in DIO and DIO-R rats over the10weeks; food intake in DIO-R rats were less than that in DIO rats from the2nd to the10th week. Cumulative food intake over10weeks in LF rats higher significantly than that in DIO and DIO-R rats; DIO-R were less significantly than that in DIO rats. Cumulative energy intake over10weeks in DIO rats were higher than that in DIO-R and LF rats; there was no significant difference between LF and DIO-R rats. Levels of serum TG in DIO rats were higher than that in DIO-R and LF rats, there was no significant difference between LF and DIO-R rats. Levels of serum TC in DIO and DIO-R rats were both higher than that in LF rats, DIO-R were decreased significantly comparing to DIO rats. No significant difference was found of levels of serum HDL-C among all groups. Levels of fasting serum glucose, insulin and HOMA-IR in DIO rats were all higher than that of DIO-R and LF rats, DIO-R were lower than DIO but still higher than that in LF rats.Conclusions Body weight, cumulative body weight gain, food intake, cumulative energy intake, levels of serum lipids and severity of insulin resistanc in DIO rats were all increased significantly comparing those to DIO-R rats.Section two Study on differences of hepatic endoplasmic reticulum stress between obesity susceptible and obesity resistance ratsObjective To expore the differences of hepatic endoplasmic reticulum stress between obesity susceptible and obesity resistance ratsMethods The animal model was the same as part three section one. After establishing the model, all groups continued the original diet on8weeks. At the end of18th week, all rats were sacrificed after12h fasting. Livers were removed and snap-frozen in liquid nitrogen immediately and stored at-80℃for further use.Results Levels of hepatic GSH content in DIO and DIO-R rats were significantly less than that in LF rats; there was no significant difference between DIO and DIO-R rass. Levels of hepatic MDA content in DIO rats were higher significantly than that in DIO-R and LF rats; there was no significant difference between DIO-R and LF rats. Hepatic GRP78and CHOP mRNA expression of DIO rats were up-regulated comparing to DIO-R and LF rats; there was no significant difference between DIO-R and LF rats. TNF-a mRNA expression of liver in DIO rats were higher than that in DIO-R and LF rats; DIO-R were higher than that in LF rats too.Conclusions The secerity of hepatic ER stress and oxidative injury were aggrevated in DIO rats comparing those to DIO-R rats.