Effects Of Fish Oil Diet On Serum Glucose, Triacylglycerol And Insulin Resistance In High Fat Diet Induced Rat:An Experimental Study

BackgroudWith the accelerating rise of obese morbidity in children,we will challenge a series of severe social problems.Because obesity,always accompanied by high serum glucose, high lipids and insulin resistance, consided as the prelude to type 2 diabetes millitus and hypertension. In particular,the insulin resistance accompanied is one of the most foundmental physiopathologic factors for diabetes millitus.For decades of years,researchers have been trying to disclose the relationship between obesity and insulin resistance and trying to find ways which are expected to ameliorate insulin resistance. The biochemical basis of insulin resistance in obesity has been the subject of many studies.Earlier studies have indicated that quantitative regulation of the insulin sensitive glucose transporters and insulin receptors themselves may contribute to this disorder,however,these two factors are probably inadequate to explain the extent of insulin resistance.Studies on postreceptor defects has recently focused on the intrinsic catalytic activity of the insulin receptor and downstream signaling events.A reduction in tyrosine phosphorylation of both the insulin receptor and the insulin receptor substrates has been verified in both animal and human.Importantly,this occurs in all of the major insulin-sensitive tissues,namely the muscle,fat and liver.It is now clear that decreased signal transduction capacity of the insulin receptor is the most important component of insulin resistance. The signal transduction capacity isdetermined by effective tyrosine phosphorylation of both insulin receptor and its downstream substrates.It has been testified that an insulin receptor comprises two α subunits and two β subunits.Two α subunits construct the joining area of insulin receptor,two β subunits contain protein tyrosine kinase. When insulin joins to the area of α subunits, the β subunits are activated characterized by tyrosine phosphorylation, then the activated β subunits join to the insulin substrate-1 and catalyze its tyrosine phosphorylation ,the tyrosine phosphorylated insulin substrate-1 which acts as the second message activates various protein kinases including phosphatidylinositol-3 kinase,and this promotes transcription and translocation of glucose transporter 4 which positively regulates glucose uptakes of cells. The insulin substrate-2 has the same function as insulin substrate-1 except that it needs much more insulin to be activated.The mechanism of insulin resistance in obesity is poorly understood. Generally,it is suggested that obese subjects characterized by accumulated much more fat produce much more or less adipocytokines including unesterified fatty acid,leptin,tumor necrosis factor, interleukin-6 and adiponectin,which in turn differently affect insulin sensitivity by the local or systemic way. Studies on these adipocytokines in obesity have been finding more and more evidences to emphasize this suggestion.High fatty acids diets usually result in obesity,but this depends on the types of fatty acids contained in diets.It has been reported that different types of fatty acids have different effects on body weight gain and insulin resistance.N-3 polyunsaturated fatty acids which positively modulate the oxidation of fatty acids produce less weight gain and insulin resistance than saturated fatty acids.A high fish oil(n-3PUFA) diet was unable to induce insulin resistance supposedly,partial replacement of the saturated fatty acids with fish oil also prevented onset of high fat diet-induced insulin resistance.However,the effect of fish oil on preexisting insulin resistance is unclear.Therefore,the aims of the present study were about to evaluate the effects of fish oil on insulin resistance, glucose,triacylglycerol,and weight gain in a model of preexistent insulin resistance.Meanwhile,we also investigated the changes ofinterleukin-6,tumor necrosis factor, and leptin which might involve in the development of insulin resistance in obesity.Part l:Establishment of obese rat and effects of fish oil diet on its serum glucose,triacylglycerol,and insulin resistance一, Establishment of obese rat and comparisions of serum glucose,leptin,and insulin sensitivity between obese and obese-resistant ratsObjective:It is well known that high fat-diet can induce obesity,but the result will be adiverse when there is too much fat in diet,because studies on establishment of obese models have revealed that too much fat down-regulates the appetite of subjects.Thus,we induced obesity by feeding rats a high fat diet containing about 20% fat in order to provide a solid foundation for the next work.However,obesity does not occur in every subject consuming a high fat diet,some rats remain to be leans which are defined as obese-resistant rats.Therefore,several physiological factors such as serum glucose, leptin,triacylglycerol,and insulin were compared ,which might help us to comprehend the mechanisms of obesity .Methods:Forty-five male Wistar rats weighing about 105-135g were divided into two groups on the basis of body weight at random.Thirty-five rats recevied high fat diet were designated as high fat diet group,The remaining ten rats received normal diet designated as control group. All rats were maintained in cages under appropriate temperature,humidity,and light-dark cycle conditions.Weekly body weight and daily food intake were measured for 8 weeks. At the end of the eighth week,the rats in high fat diet group were divided into two categories according to the body weight.The rats which were 30g heavier than the controls were used as obese rats,the remains as the obese-resistant rats.Blood was drawn by tail vein puncture with anaesthetizing sodium pentobarbital (40mg/kg) ,following 12h food deprivation at a fixed time between8PM-8AM.Serum was isolated and serum glucose and triacylglycerol levels were measured with automatic biochemical apparatus,serum leptin and insulin levels were measured by radioimmunoassay.The ratio of serum insulin to glucose was as an indicator for insulin sensitivity in the present study.Results:From the sixth week on,the weights of rats in high fat diet group were heavier than those in control group.By the end of the eighth week, 18 out of 35 rats reached the criterion of obesity .The morbidity of obesity in high fat diet group was 51% that coincided with literature.In addition,serum glucose, triacylglycerol, leptin,and insulin levels were significantly higher in obese models than those in controls. Serum leptin,insulin levels were also significantly increased in obese rats compared to those in obese-resistant rats ,however, there were no significant differences in serum glucose and triacylglycerol between them.Conclusion:Obese rats induced by high fat diet have similar characteristics with human obese subjects and are fit for the study of obesity in human. Meanwhile, obese-resistant rats induced by the same high fat diet and feeding period as obese rats are also fit for the study of obese-resistance in human.The results of the control study on obese and obese-resistant rats manifest that they have the similar energy disorder except that the obese-resistant rats have less tendency to develop leptin and insulin resistance.二, Effects offish oil diet on serum glucose,triacylglycerol,and insulin resistance in obese ratObjective:Fish oil involves in and regulates the metabolism of glucose and fat. Studies have reported that fish oil compared to saturated fat can ameliorate the development of serum glucose,triacylglycerol levels as well as insulin resistance in normal subjects,but effects of fish oil on these parameters in obese subjects are not very clear,In particular,the effect of fish oil on preexisting insulin resistance isobscure.Therefore,the aims of the present part were about to observe the changes of these parameters in obese rats after a 4-week fish oil diet feeding period.Methods:The obese rats from above were randomly divided into two groups by body weighthigh fat diet group(HFD) and fish oil diet group(FOD).The rats in HFD continued to be fed with the same high fat diet as above, and the rats in FOD were fed with the same high fat diet except that the saturated fatty acids were completely replaced by fish oil. The normal diet rats were remained and continued to be fed with the same diet as the base controls(CON).All rats were maintained in cages under appropriate temperature,humidity,and light-dark cycle conditions. Weekly body weight and daily food intake were measured for 4 weeks. At the end of the fourth week, Blood was drawn by heart puncture with anaesthetizing sodium pentobarbital (40mg/kg) , following 12h food deprivation at a fixed time between 8PM-8AM. Serum was isolated and divided into two parts which either was used for the present assays or stored in a -85 ± freezer for assays later. Retroperitoneal fat mass and liver were collected carefully ,rinsed with physiological saline, weighed and then divided into two parts which either was used for pathophysiological examination or stored in a -85 ± freezer for later assays.Serum glucose and triacylglycerol levels were measured immediately with automatic biochemical apparatus by the same methods as above,serum insulin level was measured by radioimmunoassay.The ratio of insulin to glucose was still an indicator for insulin resistance.Results:Body weight and weekly food intake in response to dietary fat types were not significant differences between HFD and FOD ,but body weight gain in FOD and CON was significantly lower than that in HFD .There was no significant difference in liver weight between HFD and FOD ,but liver weight in CON was much more lower than that in HFD or FOD.Retroperitoneal fat mass and serum triacylglycerol were significantly reduced in FOD compared to those in HFD.Serum glucose level in FOD was reduced by 15.2% compared to that in HFD despite it did not reach statistic significance(p=0.0666). However,the preexisting insulin resistance in FOD was not ameliorated.There existed severe liver steatosis in HFD and fish oil diet did not improve it.But fish oil diet reduced adipocyte diameter enlarged by highfat diet.Conclusion:Fish oil diet can reduce body weight gain,retroperitoneal fat mass ,diameter of adipocyte,and serum triacylglycerol level in obese rat after a four-week feeding period, and can reduce serum glucose level to some extent ,but it can not reverse insulin resistance.Part 2:Effects of fish oil diet on serum TNF- α and TNF- α mRNA expression in retroperitoneal fat in obese rat: relationships to insulin resistanceObjective:Adipose tissue is a significant source of endogeneous TNF- α ,with its expression in fat tissue being elevated in obesity.It is a candidate for insulin resistance in obese subject.However,evidence for significant circulating levels of this cytokine is variable,and mechanisms for it in insulin resistance in obesity are not very clear,although it is thought to operate mainly via autocrine/paracrine mechanisms. Therefore,the aims of the present part were to investigate the roles of serum TNF- α and TNF- α mRNA expression for insulin resistance in obese rat.Importantly,we investigated the potential changes of serum TNF- α and TNF- α mRNA expression 　as well as their relationships to insulin resistance before and after a fish oil diet feeding period,which may help us to evaluate the effect of fish oil on insulin resistance in obesity.Methods:Serum TNF- α was assayed by enzyme linked immunosorbant assay.Total RNA was isolated from the retroperitoneal fat by the method of GIT,and reverse transcription-polymerase chain reaction was performed to quantify mRNA of TNF-α .Results: Serum TNF- α was not significantly enhanced in HFD compared to CON,and there was no significance relationship between serum TNF- α and I/G in every group.But expression of TNF- α mRNA in retroperitoneal fat had a weakpositive relationship to I/G in HFD. Expression of TNF- α mRNA in retroperitoneal fat reduced significantly after intervention of fish oil diet.However, reduced expression of TNF- α mRNA did neither reduce serum TNF- α nor improve insulin resistance.Conclusion:Serum TNF- α was neither higher nor related to insulin resistance in obese rat ,which demonstrates that serum TNF- α is not a mechanism for insulin resistance .The level of TNF- α mRNA in retroperitoneal fat had a weak but positive relationship to insulin resistance in obese rat ,demonstrating the autocrine/paracrine is a mechanism for TNF- α in insulin resistance.A 4-week fish oil diet feeding period significantly reduced TNF- α mRNA ,but it was unable to improve insulin resistance which needs to be further studied.Part 3: Effects of fish oil diet on serum leptin and leptin mRNA expression in retroperitoneal fat in obese rat:relationships to insulin resistanceObjective:Leptin is produced mainly by adipose tissue ,with its expression in adipose tissue being elevated in obesity.It involves in the regulation of body weight by the central or systemic way.Evidences in this study and the others have suggested that it is associated with insulin resistance in obesity.Leptin is modulated by a series of factors which may include the types of fatty acids .Some studies have demonstrated that n-3 polyunsaturated fatty acids influence leptin expression and so retard the onset of insulin resistance or obesity. However,effect of n-3 polyunsaturated fatty acids on preexisting leptin resistance in obesity is unknown.Therefore,the aims of the present part were about to investigate the roles of serum leptin and leptin mRNA expression for insulin resistance in obese rat.Furtherly,we investigated the possible changes of serum leptin and leptin mRNA expression as well as their relationships to insulin resistance after a fish oil diet feeding period,which may help us to evaluate the effectof fish oil diet on preexisting insulin resistance in obesity.Methods: Serum leptin was assayed by radioimmunoassay.Total RNA was isolated from the retroperitoneal fat by the method of GIT,and reverse transcription-polymerase chain reaction was performed to quantify mRNA of leptin.Results:Serum leptin in HFD was significantly higher than that in CON,and was significantly related to I/G .There was no significant relationship between I/G and expression of leptin mRNA in HFD. The expression of leptin mRNA in retroperitoneal fat reduced significantly after intervention of fish oil diet.However, reduced expression of leptin mRNA did not decrease serum leptin level ,and the relationship between serum leptin and I/G still remained in fish oil feeding rat..Conclusion: It is serum leptin but not leptin in fat tissue that may be an important mechanism for insulin resistance.Fish oil diet feeding can reduce expression of leptin mRNA in retroperitoneal fat ,with no supposedly subsquent reduction in serum leptin, which implicates the regulation of n-3 polyunsaturated fatty acids for leptin may be at post-transcriptional level. The maintenance of the relationship between serum leptin and insulin resistance demonstrates the important role of leptin for insulin resistance in fish oil diet group.Part 4: Effects of fish oil diet on serum IL-6 and IL-6 mRNA expression in retroperitoneal fat in obese rat: relationships to insulin resistanceObjective:IL-6 is another adipocytokine produced by adipose tissue,with its expression in fat tissue being elevated in obesity by recent reports.It is thought to be involved in energy metabolism and insulin resistance in obesity.But mechanisms for it in insulin resistance are poorly understood. In the present part, the roles of serum IL-6 and IL-6mRNA expression for insulin resistance in obesity were investigated. Furthermore,in this study,fish oil diet feeding decreased fat mass and fat cell sizewhich might affect the expression of IL-6 ,and thus the insulin resistance in obesity. Therefore,we investigate the potential changes of serum IL-6 and IL-6 mRNA expression as well as their relationships to insulin resistance after a fish oil diet feeding period, which might help us to understand the effect of fish oil diet on insulin resistance in obesity.Methods: Serum IL-6 was assayed by enzyme linked immunosorbant assay.Total RNA was isolated from the retroperitoneal fat by the method of GIT,and reverse transcription-polymerase chain reaction was performed to quantify mRNA of IL-6.Results:Serum IL-6 was significantly higher in HFD rat than that in CON,and positively related to the ratio of I/G.But the expression of IL-6 mRNA in retroperitoneal fat had no significant relationship to I/G.A 4-week fish oil diet feeding period significantly reduced IL-6 mRNA expression in retroperitoneal fat,and significantly reduced serum IL-6,The reduced serum IL-6 did not have significant relationship to I/G anymore.However,this did not improve the preexistent insulin resistance.Conclusion:It is serum IL-6 but not IL-6 in retroperitoneal fat that plays an important role for insulin resistance in obesity.Fish oil diet can reduce expression of IL-6 mRNA in retroperitoneal fat and reduce serum IL-6 .Reduced serum IL-6 in FOD rat may not play an important role for insulin resistance. However, insulin resistance is not ameliorated ,further study is needed.