Chinese Medicine Jinlida Ameliorates High-fat Diet Induced Insulin Resistance In Rats Through Alleviating Lipid Accumulation In Skeletal Muscle

Insulin resistance (IR) is an important risk factor for the development ofT2DM, indicating a reduced response to insulin in the peripheral tissue andtarget organs. Skeletal muscle, the major tissue contributing to whole-bodyenergy metabolism, is the main site for insulin-stimulated glucoseuptake(about70%of blood glucose) and fatty acid intake; therefore,responsiveness to insulin in skeletal muscle greatly influence whole-bodyglucose homeostasis. Several studies have implicated that insulin resistance ischaracterized by excessive lipid accumulation in skeletal muscle due to areduction in fatty acid oxidation, such as triglyceride (TG), free fatty acid(FFA) and long-chain fatty acyl-CoA (LCACoA).The impairment of mitochondrial function has been suggested to be a crucialfactor in the pathogenesis of insulin resistance and lipid accumulation inskeletal muscle. Mitochondrial function has been implicated to be impaired insubjects with T2DM, resulting in reduced fatty acid oxidation and insulinresistance in skeletal muscle.Jinlida (JLD) superfine powder (also known as JLD Recipe) is a Chineseherbal compound that has been widely used in the treatment of insulinresistance and T2DM in China over recent years. However, the underlyingmechanisms are poorly understood. Since JLD can lower blood glucose andcirculating serum lipid levels through clinical experience, a randomizedcontrolled experiment to centre on lipid accumulation and mitochondrialfunction in skeletal muscle was conducted to explore the mechanisms bywhich JLD ameliorate insulin resistance.Objective: To establish insulin resistance model in rats induced byhigh-fat diet, and to explore the effects of Jinlida on ameliorating insulin resistance by examining AMPK signal pathway and mitochondrial function inskeletal muscle in insulin resistant rats.Methods: Totally48SD rats were randomly divided into two groups:normal diet group (n=12) and high-fat diet group (n=36). Rats in normal dietgroup were given a normal diet, and the energy contents were as follows:65.5%of carbohydrate,24.2%of protein and10.3%of fat; Rats in high-fatdiet group were provided with a high-fat diet, and the energy contents were asfollows:20.1%of carbohydrate,20.1%of protein and59.8%of fat. Six rats ofeach group were subjected to a euglycemic-hyperinsulinemic clamp after6weeks of diet to confirm the onset of insulin resistance in the high-fat dietgroup. Then six rats in each group were killed randomly by abdominal aortableeding. Plasma insulin (FINS) and blood glucose (FBG) were determined atthe end of treatment. The remaining6rats administered with normal feedingwere given normal diet continuously (ND group); The remaining30rats givenhigh-fat diet were further subdivided into five subgroups: high-fat diet group(HFD group), high-fat diet with pioglitazone and high-fat diet with3doses ofJLD (n=6per group). Body weight was monitored weekly. Animals weresacrificed on week14after starved for12h overnight. Blood samples of ratswere obtained from the abdominal aorta. Skeletal muscle samples werecollected and preserved at-80℃after being immediately frozen in liquidnitrogen. Rats of all groups were fasted for12hours before they were killed,then hyperinsulinemic-euglycemic clamp test and intraperitoneal glucosetolerance test (IPGTT) were performed. Glucose infusion rate (GIR) and areaunder curve of blood glucose (AUCglu) were calculated respectively. SerumFINS, adiponectin (APN), fasting blood glucose (FBG), triglyceride(TG), totalcholesterol(TC) and free fatty acids(FFA) were detected. TG, FFA andlong-chain fatty acyl-CoA (LCACoA) in skeletal muscle were exzamined bykits. ADIPOR1and ADIPOR2for adiponectin receptor1and2in skeletalmuscle were determined by quantitative real-time polymerase chain reaction.Proteins of AMPK signal pathway were assayed by RT-PCR andWestern-blotting. Expression of genes involved in mitochondrial oxidation were performed by RT-PCR. Transmission electron microscope (TEM) wasperformed to observe the changes of mitochondrial morphology.Lipid dropletsin skeletal muscle were observed by Oil red O staining.Results：1The change after6weeks of high-fat feeding: rats in high-fatdiet group had gained more weight than normal diet group (P<0.05),bloodglucose (FBG)(6.38±0.23mmol/L vs4.05±0.14mmol/L) and plasma insulin(FINS)(19.64±1.50mU/Lvs11.70±0.86mU/L) were significantly higher inhigh-fat diet group in comparison with normal diet group (P<0.05); GIR wassignificantly lower in high-fat diet group in contrast with normal diet group(14.74±0.42mg/kg/min vs27.61±0.56mg/kg/min)(P<0.05).2The change after treatment with different amounts of JLD andpioglitazone:(1) Rat body weight, serum biochemical index and APN: At theend of14th week, rats in HFD group had gained more weight than NDgroup(P<0.05),treatment with different amounts of JLD (0.75g/Kg,1.5g/Kg,3g/Kg) and pioglitazone had gained less weight than HFD group (P<0.05); Atthe end of14th week, TG and FFA in HFD group were significantly higherthan in ND group (P<0.05), treatment with different amounts of JLD (0.75g/Kg,1.5g/Kg,3g/Kg) and pioglitazone had decreased the content of TG andFFA in serum(P<0.05), JLD (0.75g/Kg,1.5g/Kg,3g/Kg) had also decreasedthe content of TC in serum; At the end of14th week, serum APN in HFDgroup was obviously less than ND group(P<0.05), treatment with differentamounts of JLD (0.75g/Kg,1.5g/Kg,3g/Kg) and pioglitazone hadsignificantly up-regulated APN in serum(P<0.05).(2)At14th week, FBG andFINS were significantly higher in HFD group in comparison with ND group(P<0.05); while glucose infusion rate (GIR) was obviously lower in HFDgroup compared with ND group (P<0.05); Treatment with different amountsof JLD (0.75g/Kg,1.5g/Kg,3g/Kg) and pioglitazone had significantlydecreased FBG and FINS, increased GIR (P<0.05).(3)Intraperitoneal glucosetolerance test (IPGTT): At the end of14th week, area under the curve ofglucose (AUCglu) was significantly higher in HFD group in contrast with NDgroup (P<0.05); Treatment with different amounts of JLD (0.75g/Kg,1.5g /Kg,3g/Kg) and pioglitazone had significantly reverted AUCglu(P<0.05).(4)Skeletal muscle lipid parameters: At the end of14th week TG, FFA andlong-chain fatty acyl-CoA (LCACoA) in skeletal muscle were markedlyhigher in HFD group compared with ND group (P<0.05); Treatment withdifferent amounts of JLD (0.75g/Kg,1.5g/Kg,3g/Kg) and pioglitazone hadsignificantly reverted the fat content of muscle tissue to that of normal muscle(P<0.05).(5)At the end of14th week, there were obvious lipid deposition inskeletal muscle tissues of HFD group; The accumulation of excess lipid in theskeletal muscle was ameliorated by JLD (0.75g/Kg,1.5g/Kg,3g/Kg) andpioglitazone.(6) The protein and gene expression of AMPK, P-AMPK, ACC,P-ACC, CPT1, GLUT4and PGC-1α:At the end of14th week, the proteinexpression of P-AMPK, P-ACC, CPT1, GLUT4, PGC-1α weredown-regulated in HFD group in comparison with the ND group(P<0.05);JLD(0.75g/Kg,1.5g/Kg) and pioglitazone significantly up-regulatedthe expression of P-AMPK, P-ACC and CPT1, JLD(1.5g/Kg) andpioglitazone increased the expression of PGC-1α;JLD (0.75g/Kg,1.5g/Kg,3g/Kg) and pioglitazone up-regulated the expression of GLUT4(P<0.05).There was no significant difference among groups in proteinexpression of AMPK and ACC(P>0.05).The mRNA expression of AMPK,ACC, GLUT4and CPT1were consistant with the protein expression ofthem.(7)The mRNA and protein expression of NRF1, COXⅣ, ACADM,PPARα,PPARγ, PGC-1α,ADIPOR1and ADIPOR2: At the end of14th week,the mRNA expression of NRF1,COXⅣ, ACADM, PPARα,PPARγ, PGC-1α,ADIPOR1and ADIPOR2were notably decreased (P<0.05).JLD (0.75g/Kg,1.5g/Kg,3g/Kg) significantly increased mRNA expression of ACADM,PPARα, PPARγ, ADIPOR1and ADIPOR2(P<0.05); JLD (1.5g/Kg) increasedPGC-1α、COXⅣmRNA expression(P<0.05); The mRNA expression of COXⅣ and NRF1were not affected by pioglitazone(P>0.05). The proteinexpression of PGC-1α was consistant with the gene expression.(8) Thechanges of mitochondria morphology：compared with those from HFD rats,mitochondria from skeletal muscle have a more clearly defined internal membrane structure after treatment with different amounts of JLD andpioglitazone.Conclusion:1High fat diet led to insulin resistance as reflected by elevated bloodglucose, serum insulin, and decreased GIR, while treatment with Jinlida andpioglitazone improved insulin sensitivity.2High fat diet led to high blood triglyceride and total cholesterol levels,as well as lipid accumulation in skeletal muscle, while treatment with Jinlidahad significant effect on blood triglyceride and total cholesterol, also reducedlipid accumulation in muscle. Jinlida was better than pioglitazone in reducingblood lipid.3High fat diet resulted in down-regulation of plasma adiponectin leveland the expression of both ADIPOR1and ADIPOR2in skeletal muscle. Theexpression of proteins involved in the AMPK signaling pathway in musclewere also down-regulated.Treatment with Jinlida and pioglitazone amelioratedthe condition described above.4High fat diet down-regulated the expression of genes involved inmitochondrial function and fat oxidation, treatment with Jinlida up-regulatedthe expression of genes described above. The mitochondria of high-fat dietgroup were less in quantities and smaller in volumn, while treatment withJinlida and pioglitazone ameliorated the condition described above.