Experimental Study On The Mechanism Of Insulin Resistance In Nonalcoholic Fatty Liver Disease

ObjectiveWith the changes of diet compositions, nonalcoholic fatty liver disease(NAFLD) is highly common in many countries including China, and up to date it has become the second prevalent liver disease after viral hepatitis in China. NASH is the key stage in the spectrum of NAFLD which ranges from simple fatty liver disease to liver cirrhosis. The pathogenesis of NAFLD remains unclear. IR, as "second hit to liver", is a pivotal step in pathogenisis of NAFLD, which has been widely accepted for investigators. In this study, our aims are to study the metabolic state of insulin sensitive organs and tissues and the features of biochemistry in the established experimental NAFLD model of rats. Through exploring the mechanism and the effect of IR in NAFLD, we try to provide new potential therapeutic strategy for NAFLD. MethodThe experimental NAFLD model of rat was established with the high-fat diet and was evaluated by histopathologic and biochemical test. The IR was assessed by HOMA_IR, HOMA_β, DI. The insulin transduction signal proteins and metabolic regulation proteins including SREBP-1c, PPARγ, GLUT-4, SOCS-3 were detected by immunostaining in pancreas, liver and abdominal fat tissues to explain the molecular mechanism of IR. Meanwhile, the hyperplasia and apoptosis of pancreas islet cells were estimated through PCNA and Fas-L immunostaining. The expression of ISR and GLUT-4 at mRNA levels was detected by using in situ hybridization.Result1. According to the histological evaluation of liver, the model was highly similar to human NAFLD. The typical changes of NASH which blonged to pivotal pathological stage of the NAFLD spectrum were present at the period of 6-8 week in the livers of the experimental rats, while liver fibrosis occurred at 12^th week.2. The serological biochemical examination showed that HOMA_IR, HOMA_βstarted to increase at 2^th week and reached the peaks at 12^th week and 8^th week respectively, while the levels of DI decreased in the NAFLD model of rats. IRI was significantly correlated with the levels of TC, ALT, GGT in sera (P<0.05, P<0.05, P<0.01) .3. The qualitative and quantitative observtions of IR related organs and tissues were as follows:(1) The observation of pancreas: 1) Compared with control rats, the production of insulin inβcells presented up-regulated and significantly increased at 6^th week. 2) With the development of NAFLD, the expression of PCNA and Fas-L was markedly increased at 2^th week and 8^th week respectively, so the hyperplasia and apoptosis ofβcell co-existed in the early and late stages of the NAFLD model. 3) There were no obvious changes of the expression of ISR,IRS at the early stage of NAFLD, but decreased markedly at 6^th week, which were negatively correlated with the levels of TNFαand leptin(P<0.05,respectively). However, the expression of AKT in the panceas tissues significantly increased at 2^th week. 4) SOCS-3 was diffusely stained in the whole of islets cells, and PPARγwas positively stained in the center of the islets. The quantitative analysis of SOCS-3 and PPARγexpression in the pancreas tissues were negatively correlated with ISR and IRS(P<0.05, respectively).(2) The observation of liver: 1) In the experimental NAFLD model of rats, ISR and IRS positive cells were mainly located in the centrilobular area, and markly decreased at 6^th week and 8^th week respectively. The up-regulated expression of ISR mRNA occured at 2^th week and reached the peak at 6^th week. 2) GLUT-4 and GLUT-4 mRNA positive cells were decreased obviously at 4^th week, 6^th week respectively, which was closely correlated with leptin expression(P<0.05) 3) .SOCS-3 positive cells were mainly located in the centrilobular area which started to increase at 2^th week, and reach the peak at 6^th week, it was inversely related to IRS(P<0.05). 4)SREBP-1c and PPARγpositive cells were predominant in the centrilobular area, and they increased obviously at 2^th week and 8^th week respectively.(3) The observation of abdominal fat tissues: 1) In the experimental NAFLD model of rats, the expression of ISR, IRS, GLUT-4 in the abdominal fat tissues were decreased obviously at 6^th week, 6^th week, 8^th week respectively and negatively correlated with TNFαexpression(P<0.05). The expression of SOCS-3, SREBP-1c, PPARγenhanced notablly at 2^th week, 2^th week, 6^th week respectively. Furthermore, The quantitative analysis of immunostaining detection showed that SOCS-3 was negatively correlated with IRS (P=0.05), while SREBP-1c was correlated with apidonectin, HDL and TC respectively(P<0.05).Conclusions:1. We have successfully established experimental NAFLD model of rat whosepathological characteristic was very similar to the human NAFLD and exerted IR at any stage, which provided a rational experimental model for the exploration of pathogenesis and novel treatment of NAFLD.2. The results showed that the enhanced secretion and hyperplasia ofβcell were co-exsited in the whole process of NAFLD, which suggested that the pancreas islets occurred an adaptive reaction in the pathogenesis of NAFLD. Meanwhile, we deduced that the apoptosis ofβcells in the progression of NAFLD might be a regressive change after over-secretion and proliferation of pancreas islets.3. TNFα, leptin and SOCS-3 might take part in down-regulating the expression of ISR and IRS, and up-regulating expression of PPARγin pancreas, which means the negative feedback mechanism might play an important role in the process of NAFLD. 4. The results show that IR was happened in liver and abdominal fat tissues, followed by the change of insulin transduction signal proteins. Among the insulin transduction signal proteins, the change of ISR expression was more predominant and earlier than others, while PI3K was no obvious change. Among the metabolic regulation proteins, the early up-expressed SOCS-3 was positively correlated with TNFα. The expression of GLUT-4 was decreased in liver and fat, and the former changed more significant and earlier. These results suggested that insulin transduction signal proteins and regulatory molecules paiticipated in the initiation and maintenance of IR in the beginning and developing of NAFLD, Therefore, we could deduce that the intervention of the insulin transduction passageway and regulatory proteins might be a key step for improving insulin sensitivity, which provide a new strategy for NAFLD therapy.5. According to our results, since the serological level of GGT was significantly correlated with IRI, TNFα, leptin, HDL, examination of GGT might be a potential useful predictor for estimation of IR in NAFLD.