Effect Of Jinlida On Insulin Resistance And Hepatic Oxidative Stress In High Fat Fed Rats

Insulin resistance, defined as a diminished ability of insulin-sensitivetissues in respond to insulin, plays a central role in the development of T2DMand other metabolic diseases, such as obesity and metabolic syndrome. Amyriad of factors participated in the development of insulin resistance,including free fatty acid, inflammation, cytokines, and oxidative stress.Oxidative stress is proposed to be a primary factor in the etiology ofinsulin resistance. It is reported that consumption of an adipogenic diet, suchas a high-fat diet, can significantly increase oxidative stress. Excessive energyintake leads to ectopic lipid accumulation in the liver and muscle, resulting inexcess generation of ROS. In fact, T2DM is associated with a drastic rise inoxidative damage to all cellular macromolecules including nucleic acids,lipids and proteins. Recent evidence also suggests that increased oxidativestress has a significantly inhibitory effect on insulin signaling in both in vitroand in vivo experiments. The results above suggest that oxidative stress mayplay a critical role in high-fat induced insulin resistance, and that anti-oxidanttherapy may be effective in improving insulin sensitivity.Jinlida (JLD), a compound of traditional Chinese medicine, is mainlycomposed of Ginseng, Rhizoma Polygonatum, Atractylodes lancea, Sophoraflavescens Ait and so on. JLD displays definite effects in improving thesymptoms of diabetes and reducing insulin resistance. Pharmacologicalresearch has revealed that treatment with JLD has a variety of therapeuticeffects such as improving insulin resistance, increasing insulin secretion,anti-inflammation and lowering blood lipid levels. However, the specificmechanism of JLD in reducing insulin resistance remains elusive. RhizomaPolygonatum, the primary effective compound of JLD, shows antioxidantproperties. Consequently, the current study aimed to test the hypothesis that treatment with JLD could improve insulin action by its anti-oxidant potential.In the present study, we first established insulin resistance rat model by6-week high-fat feeding. After8-week treatment of JLD, we evaluated thewhole-body insulin sensitivity by hyperinsulin-euglycemic clamp test. Theoxidative stress of liver was accessed. In addition, by detecting the protein andgene expression of insulin signaling pathway and stress-sensitive signalingpathways, the association of oxidative stress and insulin resistance wasobserved. We aimed to explore the molecular mechanism by which JLDimproves insulin resistance, thus to provide evidence on clinical practice.Objective: To investigate the effects of traditional Chinese medicineJinlida (JLD) on insulin sensitivity and liver oxidative stress in high-fatfeeding rats and to elucidate the molecular mechanism of JLD on amelioratinginsulin resistance.Methods: Male SD rats weighted125g-145g were used in the study.After a week of acclimation feeding, the rats were divided into2groups:control group (Con)(n=12) and high-fat group (HF)(n=36). Con group werefed with standard chow diet with the energy contents as follows:65.5%calories from carbohydrate,10.3%calories from fat, and24.2%calories fromprotein with total energy of348kcal/100g; HF group were fed on ahigh-fat-diet with the energy contents as follows:20.1%calories fromcarbohydrate,59.8%calories from fat and20.1%calories from protein withtotal energy of501kcal/100g. Six rats were randomly chosen from each groupafter6week of feeding. Euglycemic hyperinsulinemic clamp test andintraperitoneal glucose test (IPGTT) were performed in the6rats to evaluateinsulin sensitivity; glucose infusion rate (GIR) and the glucose area under thecurve (AUCglu) were calculated. Then the rats were sacrificed, blood samplesand liver tissues were collected for the measurement of fasting blood glucose(FBG), glycohemoglobin (HbA1c), and fasting serum insulin (FINS). Oil redO staining were carried out to observe morphological changes of live. So afterwe successfully estabolish insulin resistance models by6-week high-fatfeeding, the insulin-resistant rats (n=30) were subdivided randomly into five groups (n=6each group): high-fat fed insulin resistance group (HF); low,middle and high dose of JLD group (JLD-L, JLD-M, JLD-H)(0.75g/kg,1.5g/kg,3.0g/kg) and metformin group (Met), the control group (Con) wascontinually fed with a standard diet for the remaining8weeks. At the end of8th week, Euglycemic hyperinsulinemic clamp test and IPGTT wereperformed to reevaluate insulin sensitivity; AUCglu and GIR were recacluatedas well. After the tests the rats were sacrificed, blood samples and liver tissueswere collected for detection of FBG, FINS, HbA1c, TC, TG, free fatty acid(FFA), alanine aminotransferase (ALT), aspartate aminotransferase (AST),total bilirubin (TBIL); HOMA-IR index were calculated; liver peroxidation(LPO), reduced glutathione (GSH), antioxidant enzymes such as totalsuperoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase(CAT) were measured; reactive oxygen radicals (ROS) was detected bydihydroethidium (DHE) staining; Gene expression of the major signalingpathway molecules that regulate glucose uptake, including insulin receptor(INSR), insulin receptor substrate-1(IRS-1), phosphoinositide-3-kinase(PI3K), protein kinase beta (AKT), and glucose transporter type2(GLUT2),were assessed by quantitative RT-PCR. The protein expression of JNK,p38MAPK, IRS-1and AKT were determined by Western blot. The changes ofliver morphology were observed by HE staining and transmission electronmicroscope (TEM).Results:1Changes after6weeks high-fat feeding(1) Body weight and average food intake: There was no significantdifference between two groups in body weight at the beginning of theexperiment. After6-week of high-fat feeding, the body weight of HF groupwas significantly higher compared with Con group (P<0.05). But there was nostatistically difference in average food intake per day between two groups(P>0.05).The results above indicate that the increase of body weight as aresult of high-fat feeding was independent of changes in daily food intake.(2) Blood biochemical assays: Compared with Con group, FBG, FINS, HbA1c, HOMA-IR and TG were significantly increased in HF group. Thedifferences were all statistically significant (P<0.05). However, there was nosignificant difference in TG level between two groups (P>0.05).(3) Intraperitoneal glucose tolerance test (IPGTT): Compared with Congroup, blood glucose at0’,5’,60’ and120’ was significantly higher in HFgroup. The difference was statistically significant (P<0.05). Compared withCon group, the area under curves in HF group was significantly increasedcompared with Con group. The difference was statistically meaningful(P<0.05). The results above indicate that the high-peak of postprandial bloodglucose was delayed in high-fat fed rats.(4) Euglycemic hyperinsulinemic clamp: The glucose infusion rate ofeach group was calculated. The GIR was significantly lower in HF groupcompared with Con group (47%decreased). The difference was statisticallysignificant (P<0.05). The results above indicate that high-fat fed rats weredefinitely insulin resistance.(5) Oil red O staining of liver: There were amount of lipid droplets inliver tissues of HF group, indicating hepatic lipid deposition andhepaticsteatosis of high-fat fed rats.2Changes after drug administration for8weeks(1) Body weight and average food intake: At the end of8th week, thebody weight in HF group was significantly higher than Con group (P<0.05).The body weight of metformin and JLD groups was significantly lowercompared with HF group (P<0.05), but higher compared with Con group(P<0.05). The average food intake per day showed a significant increase in HFgroup compared with Con group (P<0.05). However, there was no statisticallydifference among metformin group, JLD groups, HF group and Con group(P>0.05). The results above indicate that JLD treatment could reduce bodyweight independent of changes in daily food intake.(2) Blood biochemical assays: The FBG, HbA1c, TG, ALT and ASTwere significantly increased in HF group compared with Con group (P<0.05).After JLD and metformin treatment, the FBG, HbA1c, TG, ALT and AST were significantly lower compared with HF group (P<0.05); but still highercompared with Con group (P<0.05). The resuts above indicated that JLDshowed more effectlly in lowering lipid than metformin. The level of FINSand FFA in HF group was significantly elevated compared with HF group(P<0.05). JLD and metformin treatment reduced FINS as well as FFAcompared with HF group (P<0.05) and showed no significantly differencewith Con group (P>0.05). As to serum TC level, there was no differencebetween HF group and Con group (P>0.05). After JLD and metforminadministration, TC was significantly decreased than HF group (P<0.05). Therewas an increasing trend in TBIL of HF group compared with Con group, butfailed to reaceh statistical difference (P>0.05).(3) The evaluation of whole-body insulin sensitivity: GIR wassignificantly decreased in HF group compared with Con group (P<0.05), JLDand metformin treatment significantly lowered GIR levels. The result ofIPGTT showed that blood glucose at0’,5’,30’,60’ and120’ weresignificantly higher in HF group than Con group. JLD administration reducedblood glucose at30’ and60’; metformin administration reduced blood glucoseat5’,30’60’ and120’. The differences were statistically significant (P<0.05).(4) Hepatic insulin sensitivity: Compared with Con group, the rats of HFgroup exhibited significantly decreased mRNA expression of INSR, IRS-1,PI3K, AKT and GLUT2. The differences were statistically significant(P<0.05). Compared with HF group, JLD treatment resulted in a modestup-regulation of INSR, IRS-1, AKT and GLUT2(P<0.05), but failed to showany change in PI3K gene expression (P>0.05). While metformin treatmentcould up-regulation the mRNA expression of INSR, IRS-1, AKT, PI3K andGLUT2. Compared with Con group, the ratio of p-IRS-1/IRS-1wassignificantly increased in HF group, while the ratio of p-Akt/t-Akt wassignificantly decreased. The differences were statistically significant (P<0.05).Compared with HF group, the ratio of p-IRS-1/IRS-1was significantlydecreased in metformin and JLD groups, while the ratio of p-Akt/t-Akt wassignificantly increased. The differences were statistically significant (P<0.05). The results indicate high-fat fed rats showed reduced liver insulin sensitivityand insulin signaling pathway, which could be improved by JLD treatment.(5) Hepatic oxidative stress: Compared with Con group, the ROSgeneration of hepatocyte and LPO were significantly increased in HF group,while the activities of anti-oxidative enzymes such as T-SOD and GSH-Pxwere significantly decreased. The differences were statistically significant(P<0.05). Howerer, there were no changes in CAT activity between the twogroups (P>0.05). Treatment with JLD and metformin significantly reducedROS and LPO generation, while increased T-SOD, GSH-Px, CAT activity andGSH level when compared with HF group (P<0.05).The results indicate thatJLD can reduce liver oxidative stress.(6) JNK and p38MAPK signaling pathways: Compared with Con group,the ratios of p-JNK/JNK and p-p38MAPK/p38MAPK were significantlyincreased in HF group. The differences were statistically significant (P<0.05).Compared with HF group, the ratios of p-JNK/JNK andp-p38MAPK/p38MAPK were significantly decreased in metformin and JLDgroups. The differences were statistically significant (P<0.05). The resultsindicate that JLD could inhibit JNK and p38MAPK signaling pathway.(7) Red O oil staining and transmission electron microscope (TEM): RedO oil staining: The liver lipid deposition and hepaticsteatosis in HF groupwere remarkable. JLD and metformin treatment reduced liver lipid depositionand hepaticsteatosis to different extent. TEM：The lipid droplets were seeneverywhere in hepatocyte’s cytoplasm in HF group, accompany by the cristaeof mitochondria fractured or disappeared and rough endoplasmic reticulum(GER) expanded. Metformn and JLD groups showed decreased number andvolum of lipid droplets and alleviated mitochondria and GER ultrastructuredamage.Conclusions:1JLD treatment can improve impaired glucose tolerance, ameliorateinsulin resistance, and correct metabolism disorders such as hyperglycemia,hyperinsulinemia and hyperlipidemia. Compared with metformin, JLD shows a stronger pharmacological effect in lowering lipid.2JLD treatment can improve abnormal liver function, reduce lipiddeposition and hepaticsteatosis.3JLD treatment can reduce oxidative stress and decrease ROS and LPOgeneration in the liver, while increase anti-oxidatant such as SOD, GSH-Px,CAT activity and GSH level.4JLD treatment can ameliorate the inhibited liver insulin signalingpathway blocked by activation of the JNK and p38MAPK pathways. Theimproved insulin sensitivity as a result of JLD treatment may be attributed tothe antioxidant properties of JLD.