The Role Of Exendin-4 On Hepatocytes Edoplasmic Reticulum Stress Pathway Of Rats With IGT Complicated NAFLD

Objective To compare the changes of CHOP mRNA、CHOP protein、SREBP-1c mRNAexpression in rats with impair glucose tolerance (IGT) complicated nonalcoholic fattyliver(NAFLD) with GLP-1 agonists intervention, and to do liver tissues HE dying which observeby light microscope，compare the ratio changes of the number of hepatocytes steatosis in thehepatic lobule to the total liver cell number, to explore protective effect and possible mechanismof GLP-1 agonists on the hepatocytes endoplasmic reticulum stress pathway of NAFLD.Methods Sixty-eight male Wistar rats in 4-5 weeks (150g-180g) of clean grade werepurchased in institute of animal central of Shanxi Medical Shool. After one week adaptivefeeding (five/cage), they were divided into two groups randomly, the normal glucose tolerancegroup rats(NGT group, n=18) were fed by normal diet (13.4kJ/g, which accounted for 10.2% ofcalories, fat and protein accounted for 23.3% respectively, carbohydrate accounted for 66.5%);the IGT group rats(n=50) were given high-sugar and high-fat feed (21.8kJ/g, which accountedfor 56.0% of calories, fat and protein accounted for 7.0% respectively, carbohydrates accountedfor 37.0%) and free access to water, at room temperature controlled at 18-22℃, relative humidity30%-70%. Two groups of rats twice a day morning and evening feeding, daily food in take foreach rat to vote for 3% of body weight. When 12 weeks, do oral glucose tolerance test (OGTT):after fasting 8h, cut the tail blood and rapid blood glucose meters measure fasting blood glucose(FBG), intragastric administration with 50% glucose injection 2g/kg body weight, after 2h cutthe tail blood again and measure 2h postprandial blood glucose (2hPG). Building a suceessfulIGT model was 7.8mmol/L≤2hPG＜1l.lmmol/L and continued for more than a week. Fouty rats inIGT group were into models，it has the making model sueeess rate of over 80%. Then randomlyextracted 8 mouse to do liver tissue HE dyeing which observed by light microscope, more thanone third hepatocytes appeared with diffuse large bubble steatosis and ballooning degenerationwere determined successful IGT merger NAFLD model. It has the making NAFLD modelsueeess rate of over 100%, and making IGT merger NAFLD model sueeess rate of over 80%.Then half rats were selected to establish IGT/NAFLDExgroup(n=16)，another half number is setto IGT/NAFLDNSgroup(n=16). Select randomly half rats in three groups put to executionrespectively. After fasting 12h, weighing, and then do oral glucose tolerance test measure FBGand 2hPG. The next day, after fasting 12 hours, anesthetizd rats with 5% chloral hydrate byintraperitoneal injection 0.6ml/100g body weight, taken abdominal blood, rapid centrifugation of blood samples of serum specimens, measure blood fat and liver function.Then quickly removethe liver and adipose tissue after taking the blood, and with ice PBS wash clean and dry withfilter paper, weighing, measure visceral fat content(VF/W), obtain three 1.5cm×1.5cm×0.5cmliver tissues rapidly in liquid nitrogen, after thoroughly frozen, stored in -70℃refrigerator.Measured the expression of CHOP mRNA、SREBP-1c mRNA by real time quantitativepolymerase chain reaction (RT-PCR) and measured the expression of CHOP protein by WesternBlotting. And then obtain a 1cm×1cm×0.5cm of liver tissues, fixed with 10% formaldehydesolution, dehydrated, embedded in paraffin, regular slices to observe by the light microscope.IGT/NAFLDExgroup received Exendin-4 subcutaneously twice daily (5ug/kg) interventing 4weeks. NGT group and IGT/NAFLDNSgroup were given equal volume of saline injection. Wheninterventing 4 weeks，all put to execution and detect above index. All results were measured by( x±s), using SPSS 13.0 software package, multiple groups means were compared with singlefactor analysis of variance, LSD test. P≤0.05 was considered statistically significant. ThePearson correlation were used to do correlation analysis of the expression of CHOP mRNA、CHOP protein、SREBP-1c mRNA and hepatocytes steatosis.Results1. The changes of clinical characteristics between rats in each groups Before intervented,compared with the NGT group, the body weigh of rats in the IGT/ NAFLDNSgroup andIGT/NAFLDExgroup were higher（464.78 33.67 vs. 554.88 19.06）（464.78 33.67 vs.557.25 31.92）and the differences were also significant (both P<0.05); the liver wet weight ofrats were higher（13.11 1.05 vs. 16.38 1.06）（13.11 1.05 vs. 16.62 1.06）, the differenceswere also significant (both P<0.05); the VF/W of rats were higher (1.83 0.34 vs. 8.61 1.34)(1.83 0.34 vs. 8.80 1.52) , the differences were also significant (both P<0.05). Thedifferences of each parameter of IGT/NAFLDExgroup compared to IGT/NAFLDNSgroup werenot significant (P＞0.05),（554.88 19.06 vs 557.25 31.92.）（16.38 1.06 vs. 16.62 1.06）（8.61 1.34 vs. 8.80 1.52）. After intervented with Exendin-4 4 weeks, compared with theIGT/NAFLDNSgroup, the body weigh，the liver wet weight and VF/W of IGT/NAFLDExgroupwere decreased (614.00 25.11 vs. 487.44 24.56) (17.25 1.04 vs. 13.38 0.92) (11.28 1.69vs. 4.03 1.10) of rats in the IGT/NAFLDExgroup were decreased , the differences weresignificant (both P<0.05). Compared with the IGT/NAFLDExgroup before intervented, the levelswere decreased（557.25 31.92 vs. 487.44 24.56）（16.62 1.06 vs. 13.38 0.92）（8.801.52 vs. 4.03 1.10），the differences were significant (P<0.05).2. The changes of of blood sugar between rats in each groups Before intervented, compared with the NGT group, the 2hPG of rats in the IGT/ NAFLDNSgroup and IGT/NAFLDExgroupwere higher (5.96±0.77 vs. 9.69±0.33)(5.96±0.77 vs. 9.26±0.38), the differences were alsosignificant (both P<0.05). The FBG of rats were higher(4.79±0.55 vs. 5.05±0.72)(4.79±0.55vs. 5.23±0.39), but the differences were not significant (both P>0.05). The differences of eachparameter of IGT/NAFLDExgroup compared to IGT/NAFLDNSgroup were not significant (P＞0.05), (9.69±0.33 vs. 9.28±0.38) (5.05±0.72 vs. 5.23±0.39). After intervented withExendin-4 4 weeks, compared with the IGT/NAFLDNSgroup, 2hPG of IGT/NAFLDExgroupwere decreased（10.28±0.38 vs. 7.19±0.34）of rats in the IGT/NAFLDExgroup weredecreased , the differences were significant (both P<0.05). The FBG in the IGT/NAFLDExgroupwere decreased (4.98±0.36 vs. 4.89±0.31), but the difference was not significant (P>0.05).Compared with the IGT/NAFLDExgroup before intervented, the levels were decreased（9.28±0.38 vs. 7.19±0.34），the differences were significant (P<0.05). The FBG was also decreased(5.05±0.7 2 vs. 4.89±0.31), but the difference was not significant (P>0.05).3. The changes of blood lipid between rats in each groups Before intervented, compared withthe NGT group, the TG of rats in the IGT/NAFLDNSgroup and IGT/NAFLDExgroup werehigher（0.87±0.22 vs. 2.08±0.21）（0.87±0.22 vs. 2.12±0.21）and the differences were alsosignificant (both P<0.05); the TC of rats were higher（0.84±0.19 vs. 1.91±0.16）（0.84±0.19 vs.2.00±0.17）, the differences were also significant (both P<0.05); the LDL of rats were higher（0.21±0.06 vs. 0.92±0.05）（0.21±0.06 vs. 0.93±0.04）, the differences were also significant(both P<0.05), the HDL of rats were decreased（1.09±0.06 vs. 0.86±0.07）（1.09±0.06 vs.0.84±0.08）. The differences of each parameter of IGT/NAFLDExgroup compared toIGT/NAFLDNSgroup were not significant (P＞0.05),（2.08±0.21 vs. 2.12±0.21）（1.91±0.16 vs.2.00±0.17）（0.92±0.05 vs. 0.93±0.04）（0.86±0.07 vs. 0.84±0.08）. After intervented withExendin-4 4 weeks, compared with the IGT/NAFLDNSgroup, TG ,TC, LDL, were decreased ofrats in the IGT/NAFLDExgroup（2.22±0.14 vs. 1.06±0.13）（2.09±0.18 vs. 1.02±0.09）（1.04±0.11vs. 0.25±0.04）, HDL is higher（0.82±0.06 vs. 1.05±0.04）, the differences were significant(P<0.05). Compared with the IGT/NAFLDExgroup before intervented, TG ,TC, LDL weredecreased of rats in the IGT/NAFLDExgroup（2.12±0.21 vs. 1.06±0.13）（2.00±0.17 vs.1.02±0.09）（0.93±0.04 vs. 0.25±0.04）, HDL is highe（r0.84±0.08 vs. 1.05±0.04）, the differenceswere significant (both P<0.05).4. The changes of liver function between rats in each groups Before intervented, comparedwith the NGT group, the AST of rats were higher（29.56±8.20 vs. 81.75±8.60）（29.56±8.20 vs.85.25±5.06）, the differences were also significant (both P<0.05); the ALT of rats were higher （30.22±7.87 vs. 81.75±8.46）（30.22±7.87 vs. 82.38±7.85）, the differences were alsosignificant (both P<0.05). The differences of each parameter of IGT/NAFLDExgroupcompared to IGT/NAFLDNSgroup were not significant (P＞0.05) ,（81.75±8.60 vs. 85.25±5.06）（81.75±8.46vs.82.38±7.85）. After intervented with Exendin-4 4 weeks, compared with theIGT/NAFLDNSgroup, AST and ALT were decreased of rats in the IGT/NAFLDExgroup（85.25±7.63 vs. 33.25±6.63）（85.88±4.76 vs. 32.75±6.98）the differences were significant(P<0.05). Compared with the IGT/NAFLDExgroup before intervented, AST and ALT weredecreased of rats in the IGT/NAFLDExgroup（85.25±5.06 vs. 33.25±6.63）（82.38±7.85 vs.32.75±6.98）, the differences were significant (both P<0.05).5. The expression of CHOP mRNA and CHOP protein in liver cells with eath groupsBefore intervented, compared with the NGT group, the expression of CHOP mRNA with rats inthe IGT/NAFLDNSgroup and IGT/NAFLDExgroup were increased（0.15±0.04 vs. 1.06±0.15）（0.15±0.04 vs. 1.09±0.15），and the differences were significant (both P<0.05); CHOP proteinof rats were higher（214.59±2.76 vs. 221.11±2.15）（214.59±2.76 vs. 221.25±2.09）, thedifferences were also significant (both P<0.05). The differences of each parameter ofIGT/NAFLDExgroup compared to IGT/NAFLDNSgroup were not significant (P＞0.05) ,（1.06±0.15 vs. 1.09±0.15）（221.11±2.15 vs. 221.25±2.09）.After intervented with Exendin-4 4weeks, compared with the IGT/NAFLDNSgroup and IGT/NAFLDExgroup before intervented,the expression of CHOP mRNA and CHOP protein in the IGT/NAFLDExgroup was decreased（1.23±0.12 vs. 0.49±0.12）（222.65±1.28 vs. 216.40±5.44），the differences was significant(both P<0.05); compared with the IGT/NAFLDExgroup before intervented the level wasdecreased（1.09±0.15vs. 0.49±0.12）（221.25±2.09 vs. 216.40±5.44），the differences wassignificant (both P<0.05). Compared with the NGT group the level was incresed（0.18±0.02 vs.0.49±0.12）（214.57±2.32 vs. 216.40±5.44），but the difference was significant ( P<0.05).6. The expression of SREBP-1c mRNA in liver cells with eath groups Before intervented,compared with the NGT group, the expression of SREBP-1c mRNA with rats in theIGT/NAFLDNSgroup and IGT/NAFLDExgroup were increased（0.17±0.05vs. 4.79±1.37）（0.17±0.05 vs. 5.00±1.23），and the differences were significant (both P<0.05). Compared withthe IGT/NAFLDNSgroup, the expression of SREBP-1c mRNA with rats in the IGT/NAFLDExgroup was higher（4.79±1.37 vs. 5.00±1.23），but the difference was not significant ( P＞0.05).After intervented with Exendin-4 4 weeks, compared with the IGT/NAFLDNSgroup andIGT/NAFLDExgroup before intervented, the expression of SREBP-1c mRNA in theIGT/NAFLDExgroup was decreased（5.47±1.23 vs. 0.26±0.10）（5.00±1.23vs. 0.26±0.10）, the differences was significant (both P<0.05).7. The histological analyses of liver cells in each groups The liver appearance of NGT group isreddish brown, soft and elastic. The liver volume in IGT/NAFLD group is larger, its appearanceis yellow, many yellowish gray particles scattered on liver surfaces, greasy feeling; afterintervented with physiological saline 4 weeks, liver morphology was not change. Afterintervented with Exendin-4 4 weeks, the liver appearance of IGT/ NAFLDExgroup is yellowish-brown, a few yellowish gray particles scattered on liver surface. HE dyeing : before intervented,the liver tissue structure in NGT group is complete and clear, the liver lobular structure is normal,the central venous big and thin, liver cells in cable around the central vein radiate out indistribution. Liver cells volume in IGT/ NAFLDExgroup is increased, varying sizes lipid dropsempty bubble can be seen in the cytoplasm, the nucleus is pulled to the peripheral. Afterintervented with physiological saline 4 weeks, liver cells volume is larger , and contain a largelipid drops empty bubble. After intervented with Exendin-4 4 weeks, liver lobules and liver sinusstructure is clear, the cells are arranged in order, and only contain several lipid drops emptybubble.8. The expression of the ratio of the number of hepatocytes steatosis in the hepatic lobule tothe total liver cell number with eath groups Before intervented, compared with the NGTgroup, the ratio of the number of hepatocytes steatosis in the hepatic lobule to the total liver cellnumber with rats in the IGT/NAFLDNSgroup and IGT/NAFLDExgroup were increased（0.00±0.00 vs. 50.00±11.54）（0.00±0.00 vs. 51.25±11.14），and the differences were significant(both P<0.05). Compared with the IGT/NAFLDNSgroup, the ratio of the number of hepatocytessteatosis in the hepatic lobule to the total liver cell number with rats in the IGT/NAFLDExgroupwas higher（50.00±11.54 vs. 51.25±11.14），but the difference was not significant (P＞0.05).After intervented with Exendin-4 4 weeks, compared with the IGT/NAFLDNSgroup andIGT/NAFLDExgroup before intervented, the ratio of the number of hepatocytes steatosis in thehepatic lobule to the total liver cell number with rats in the IGT/NAFLDExgroup was decreased（88.50±6.72 vs. 40.38±6.84）、（51.25±11.14 vs. 40.38±6.84），the differences was significant(both P<0.05). Compared with the NGT group the level was incresed（0.00±0.00 vs.40.38±6.84），but the difference was significant ( both P<0.05).9. The expression of CHOP mRNA、CHOP protein、SREBP-1c mRNA and hepatocytessteatosis was significantly correlated in rats with impaired glucose tolorence complicatedNAFLD（r =0.933、0.946、0.978，P＜0.01）. 10. The expression of CHOP mRNA and the expression of SREBP-1c mRNA in liver wassignificantly correlated in rats with IGT complicated NAFLD（r =0.923，P＜0.01）.Conclusions1. Given high-sugar and high-fat feed 12 weeks, IGT complicated NAFLD model weresuccessful induced. It has the making IGT complicated NAFLD model sueeess rate over 80%.2. Exendin-4 can reduce the endoplasmic reticulum stress in hepatocytes of IGT complicatedNAFLD rats, decrease hepatic steatosis. Exendin-4 could ameliorate the endoplasmic reticulumstress which is one of the risk factors of liver steatosis.