The Mechanism Of Improving The Insulin Resistance Of Db/db Diabetic Mice By Generational Syndrome

Diabetes is one of the most urgent health problems worldwide.Up to 425 million adults suffer from diabetes,among which 90%belong to type 2 diabetes(T2DM).Insulin resistance(IR)runs through T2DM,and is a critical pathophysiological basis of T2DM.The improvement of IR is of great importance for the control of T2DM.IR is the result of the combined action of different tissues,cell types,as well as multiple mechanisms.From a traditional Chinese medicine(TCM)point of view,the mechanism of insulin resistance could mainly be as follows.Over intake of high fat diets and sugar and sedentary life style etc.induce "abdominal distension and internal heat".On the one side,due to congestion of middle-jiao associated with liver qi stagnation,phlegm accumulates in the liver(hepatic lipid deposits).On the other hand,because of the food essence being transformed abnormally and overflowing to four extremities,lipid deposits in skeletal muscle.And there exists energy deficiency in tissue cells such as myocytes due to defection in transportation(low energy metabolism).Dai-Zong-Fang(DZF)is a compound herbal formula created by my supervisor.It is based on over two decades of clinical experience in the treatment of MetS and IR.DZF is mainly composed of Rhizoma Coptidis(Huanglian)and Fructus Aurantii Immaturus(Zhishi).Though DZF has been clinically used for treatment of IR and metabolic syndrome for decades,its mechanism in vivo remains unknown.Based on the combination of modern medicine and TCM understandings of IR,the present study aims to observe the effects and mechanisms of DZF treatment on insulin resistant and diabetic db/db mice.OBJECTIVE:1,To observe the effects of DZF exerts on insulin resistance in spontaneous T2DM db/db mice.2.To investigate the mechanism of DZF effect on insulin resistance in db/db mice.METHODS:1.Animals:6-week-old male db/db mice and 10 male wild-type C57BL/Ksj mice were housed.After acclimation,db/db mice were randomly divided into 4 groups(n=10)and were treated daily via gavage with vehicle,metformin(0,25 g·kg-1)and DZF(0.5 g·kg-1,-g·kg-1).The vehicle group and ten wild-type mice(normal control,WT)were treated with distilled water.After 12 weeks of administration,mice were starved for 12 h and sacrificed under anesthesia by chloral hydrate.Blood samples were collected for serum assessment.Main tissues such as liver were removed,weighed and frozen at-80 ? until analyzed or fixed in paraformaldehyde(PFA).Parts of the skeletal muscle were cut into small cubes and fixed in 2.5%glutaraldehyde for electron microscopy.2.Glucose and lipid metabolism and insulin resistance evaluation:Fasting blood glucose was detected every 2 weeks.ITT and OGTT were performed.The mouse insulin ELISA kit was used to detect serum insulin concentration,after which HOMA-IR was calculated according to formula.Serum TG,CHO,HDL,LDL were assessed by an automatic biochemical analyzer.Serum NEFA was assayed by an assay kit of colorimetric method.H&E staining was used to observe the micromorphology of the pancreases.3.Histological analysis,glucose and lipid metabolism in liver:The assay kit of colorimetric method and PAS staining were used to detect hepatic glycogen.H&E and Oil red O staining were used to observe micromorphology and lipid accumulation in the hepatic tissue.Hepatic TG was assayed by an assay kit of enzymatic method.Hepatic SOD was measured by assay kits of WST-1 method.Serum ALT and AST levels were detected by an automatic biochemical analyzer.4.Protein and gene expressions in liver:Western blot were used to detect IRS-1,Akt,AMPK and their phosphorylated proteins,as well as NICD,Hesl in the hepatic tissue.RT-PCR were used to detected Fasn?Ppara?Cpt1a relative expressions in liver.5.Ultrastructral alteration,protein and gene expressions of the skeletal muscle:Ultrastructral alteration of the skeletal muscle in db/db mice were observed under a transmission electron microscope.AMPK,plasma membrane GLUT4 were detected by western blot in the skeletal muscle.RT-PCR was used to detected CdS6,Cpt1b in the skeletal muscle.RESULTS:1.Effects of DZF on glucose and lipid metabolism in db/db mice:FBG in the vehicle db/db mice was 2.3-5.3 folds high of that in WT mice.Metformin si'gnificantly reduced FBG compared with the vehicle group.And a reduction of 47.1%was seen at the 4th week(p<0.001).DZF lowered FBG from 2th to 8th weeks(p<0.05),with a reduction of 23.2%and 27.8%in DZF-0.5 and DZF-1 groups respectively at the 4th week(p<0.01).Compared with the WT mice,all lipid profiles were significantly higher in db/db mice(p<0.001 or p<0.05).Metformin lowered LDL-C and TG levels(p<0.01 and p<0.05,respectively),and raised HDL-C levels(p<0.05)in db/db mice.Compared with the vehicle group,DZF-0.5 and DZF-1 significantly reduced LDL-C,with a reduction of 43.9%and 35.8%respectively(p<0.05).A reduction of 16.4%of serum NEFA levels in DZF-1 treated db/db mice was observed(p<0.05).2.Effects of DZF on insulin resistance in db/db mice:the area under the curve(AUC)in vehicle db/db mice was 4.3 folds of that in WT mice.The AUCs of metformin,DZF-0.5 and DZF-1 groups were significantly reduced(3.2,3.7 and3.8 folds of that in WT mice,respectively).The AUC(ITT)in db/db mice was 8 folds of that in WT mice(p<0.001).The AUCs of metformin and DZF-0.5 administered db/db mice were reduced compared with vehicle db/db mice(4.7 and 6.7 folds of that in WT mice,p<0.001 and p<0.05 respectively).Serum insulin in vehicle db/db mice was 9.2 folds with HOMA-IR 38.4 folds of that in WT mice(p<0.001).DZF-0.5 and DZF-1 significantly reduced serum insulin levels compared with vehicle db/db mice,with reduction of 79.8%and 77.0%respectively(p<0.001).HOMA-IR reduced 79.2%and 78.5%respectively(p<0.001).3.Effects of DZF on hepatic glucose and lipid metabolism in db/db mice:The glycogen assay kit assessment showed the same results with PAS staining.Reduction in glycogen was observed in vehicle db/db mice compared with the WT controls(p<0.05),whereas metformin caused a more significant reduction and much lower observed values(p<0.05).DZF presented no specific effects on glycogen compared with the vehicle db/db mice(p>0.05).Compared with WT mice,liver weight(2.74±0.27 g)and liver index(6.04±0.64%)were both much higher in db/db mice than that in WT mice(1.12±0.14 g and 4.56±0.35%,p<0.01).Both DZF-0.5 and DZF-1 remarkably reduced liver weight,the liver weight of DZF-1 administrated db/db mice decreased 18.7%compared with vehicle(p<0.05),23.2%compared with metformin(p<0.01),with liver index 20%decreased compared with vehicle(p<0.01).The hepatic TG contents in db/db mice were 4.8 folds of that in WT mice(p<0.001).Both doses of DZF remarkably reduced hepatic TG levels at 35.1%and 40.7%,respectively(p<0.01).In hematoxylin and eosin(H&E)and Oil red O staining,DZF significantly reduced lipid diffusion in db/db mice.Compared with vehicle and metformin treated db/db mice,DZF-1 reduced serum ALT(p<0.05,respectively).Compared with vehicle db/db mice,Both doses of DZF reduced serum AST(p<0.01,respectively).With DZF(1 g·kg-1)administration,hepatic SOD activity remarkably increased compared with that in vehicle db/db mice(p<0.05).DZF(0.5 g·kg-1)and metformin administration raised SOD activity with no significant difference(p>0.05).4.Effects of DZF on critical signaling pathways of insulin resistance in the liver of db/db mice:As shown by Western blot,metformin and DZF(1 g·kg-1)increased Akt and AMPK phosphorylation(p<0.01 and p<0.05,respectively),and had the tendency to increase phospho-IRS-1 relative expression.DZF and metformin significantly reduced NICD and Hesl expressions compared with those in the vehicle db/db mice(p<0.001,p<0.01,and p<0.05).By RT-PCR,we observed a notable reduction of Fasn gene expression in both metformin-and DZF-treated db/db mice(p<0.05).On the other hand,Ppara,but not Cpt1a,was significantly up-regulated in DZF-treated mice(p<0.05).5.Effects of DZF on mechanisms of energy metabolism in the skeletal muscle of db/db mice:TEM showed disarrayed,dissolved,or ruptured myofibrils,thickening and vague Z lines,and blurred H zones and M lines and the enlarged and swollen mitochondria in the skeletal muscle of vehicle db/db mice.The muscle fibers and mitochondrial morphology of DZF-treated mice were normalized compared with vehicle db/db mice.Western blot showed an increase of AMPK phosphorylation in both metformin-and DZF-treated mice(p<0.01).GLUT4 expression in the plasma membrane was up-regulated with DZF and metformin treatment(p<0.001).Both metformin and DZF can up-regulate Cd36 expression(p<0.01 and p<0.05,respectively)and Cpt1b expression(p<0.01 and p<0.05,respectively)in the skeletal muscle of db/db mice.CONCLUSIONS:1.DZF lowers FBG,LDL-C,NEFA and serum insulin concentration and significantly attenuates insulin resistance in db/db mice with improvement of insulin tolerance and glucose tolerance,and reduction of HOMA-IR level.2.DZF reduces hepatic TG concentration as well as hepatic lipid accumulation in db/db mice.DZF protects from hepatic injury with decreased serum ALT and AST and increased hepatic SOD activity.3.The mechanism of DZF effects on insulin resistance in db/db mice may be the combined actions of IRS 1/Akt insulin signaling and AMPK activation as well as regulation of lipid metabolic gene expressions in liver and skeletal muscle,inhibition of hepatic NICD/Hesl signaling pathway,and promotion of GLUT4 translocation,inducing decrease of hepatic lipid deposition and improvement of energy metabolism in skeletal muscle.