| Background:Withthe development of the living standard of the people,diabetes mellitus is a serious metabolic disease which is becoming an important health problem.The total number of diabetic patients will be over 110 million in China.Recent studies have indicated that more than 90%of the total number of diabetic patients are type 2 diabetes mellitus.Although numerous factors are implicated in the pathogenesis of type 2 diabetes,insulin resistance is the major causes of type 2 diabetes mellitus.In particular,the defect of GLUT4 function is an important factor leading to increase in insulin resistance and decrease in glucose uptake in type 2 diabetes.The 85%of insulin-induced glucose uptake take place in skeletal muscle.Therefore,the quantity and activity of GLUT4 on the cell membrane of skeletal muscle determine the glucose uptake and insulin sensitivity.Our previous studies found that intracerebroventricular and abdominal injection of galanin can elevate insulin sensitivity in skeletal muscles of rats.To date,three galanin subtype receptors have been characterized,GALR1,GALR2 and GALR3,which have different function in different tissues.As yet it is unknown whether the GALR2 is an effective receptor to mediate the effect of galanin on increased insulin sensitivity.Chinese Medicine does not have the name "diabetes mellitus".It can be attributed to "lung elimination" or "GE Consumer".The pathophysiology of collateral is "Yin deficiency and dryness heat" in traditional Chinese medicine.Insulin resistance of type 2 diabetes is similar to the symptom of "Nei Jing" of "Pi Dan"."Dryness heat" is an important role in the development of the disease as described in "Nei Jing".The clinical treatment of insulin resistance of type 2 diabetes mellitus emphasizes on the maintenance of Yin and qi,especially the removing heat treatment.A lot of traditional herbals have been used to treat type 2 diabetic insulin resistance and its complications.Among them,Scutellaria baicalensis Georgi(known as huang qin)is a traditional Chinese herb,exhibiting antidiabetic effect.Scutellaria baicalensis contains several bioactive chemical compounds,baicalin is one of the most potent and abundant polyphenolic compounds extracted from it.Recent studies indicated that administration of baicalin could alleviate hyperglycemia and insulin resistance.However,the ameliorative effect of baicalin on insulin resistance has not been sufficiently documented as yet.Besides,it is unknown whether baicalin could attenuate insulin resistance through activation of GALR2 pathway.These are significant subjects for further exploration.Objectives:1.To explore the mechanism of GALR2 regulating insulin resistance in skeletal muscles of insulin-resistant mice and GALR2 knockout mice as well as GALR2 silencing myotubes.2.To investigate whether baicalin could increase the expression and translocation of GLUT4 and its signal pathways in skeletal muscles of insulin-resistant mice and myotubes.3.It is unknown whether baicalin could attenuate insulin resistance through activation of GALR2 pathway in skeletal muscles of insulin-resistant mice and GALR2 knockout mice as well as GALR2 silencing myotubes.Methods:1.We investigated the changes of insulin resistance after i.p.administration of agonist or antagonist in skeletal muscles of insulin-resistant mice and myotubes as well as skeletal muscles of GALR2 knockout mice and GALR2 silencing myotubes.Glucose tolerance test and insulin tolerance test were used to determine insulin sensitivity.The insulin levels were quantified using competitive insulin ELISA kits according to the manufacturer’s specification.The 2-NBDG uptake of cells was detected by flow cytometric measurement.The GLUT4 contents in plasma membranes of myotubes and skeletal muscle and GLUT4 mRNA expression in myotubes and skeletal muscle were detected by Western Blot and real-time PCR respectively.The PGC-la mRNA in myotubes and skeletal muscle was detected by real-time PCR.The PGC-1α,pAKT,pAS160 and pP38MAPK contents were detected by Western Blot.The GLUT4 contents in plasma membranes of myotubes in p38MAPK and AKT inhibition experiments were detected by Western Blot.The morphological changes of GLUT4 contents in myotubes and skeletal muscle were detected by immunohistochemical and immunofluorescence staining respectively.2.We investigated the changes of insulin resistance after i.p.administration of baicalin in skeletal muscles of insulin-resistant mice and myotubes.Glucose tolerance test and insulin tolerance test were used to determine insulin sensitivity.The insulin levels were quantified using competitive insulin ELISA kits according to the manufacturer’s specification.The 2-NBDG uptake of cells was detected by flow cytometric measurement.The GLUT4 contents in plasma membranes of myotubes and skeletal muscle and GLUT4 mRNA expression in myotubes and skeletal muscle were detected by Western Blot and real-time PCR respectively.The PGC-la mRNA in myotubes and skeletal muscle was detected by real-time PCR.The PGC-la,pAKT,pAS160 and pP38MAPK contents were detected by Western Blot.The GLUT4 contents in plasma membranes of myotubes in p38MAPK and AKT inhibition experiments were detected by Western Blot.The morphological changes of GLUT4 contents in myotubes and skeletal muscle were detected by immunohistochemical and immunofluorescence staining respectively.3.We investigated the changes of insulin resistance after i.p.administration of baicalin in skeletal muscles of GALR2 knockout mice and GALR2 silencing myotubes.Glucose tolerance test and insulin tolerance test were used to determine insulin sensitivity.The insulin levels were quantified using competitive insulin ELISA kits according to the manufacturer’s specification.The 2-NBDG uptake of cells was detected by flow cytometric measurement.The GLUT4 contents in plasma membranes of myotubes and skeletal muscle were detected by Western Blot.The PGC-la mRNA and GLUT4 mRNA in myotubes and skeletal muscle were detected by real-time PCR.The GLUT4,PGC-la,pAKT,pAS160 and pP38MAPK contents were detected by Western Blot.The morphological changes of GLUT4 contents in myotubes and skeletal muscle were detected by immunohistochemical and immunofluorescence staining respectively.Results:1.Compared with normal mice,the insulin-resistant mice appeared more drinks,polyuria,sleepiness,squinting,lazy burnout,slow response,hair yellow scarce,stool stem node,red tongue and weight gain etc.The blood glucose and insulin levels as well as insulin resistance index of mice significantly increased.Besides,the GLUT4 and PGC-la expression levels,and pAKT,pAS160 and pP38MAPK as well as GLUT4 translocation levels significantly decreased in skeletal muscles of insulin-resistant mice.Furthermore,the activation of GALR2 enhanced energy expenditure,insulin sensitivity,GLUT4 expression and translocation,as well as PGC-la,pP38MAPK,pAKT and pAS160 levels,reversed high fat diet-induced glucose and insulin intolerance,hyperglycemia and insulin resistance in skeletal muscles of mice.Compared with normal mice,insulin sensitivity,GLUT4 expression and translocation,as well as PGC-1α,pP38MAPK,pAKT and pAS160 levels were significantly reduced in skeletal muscles of GALR2 knockout mice(GKO).Last,treatment with M1145 significantly elevated GLUT4 translocation and 2-NBDG levels,the GLUT4 and PGC-la expression,and PGC-1α,pP38MAPK,pAKT and pAS160 levels in myotubes.However,after treatment with p38MAPK or AKT inhibitors,the GLUT4 and 2-NBDG contents in the M1145 group were significantly decreased in myotubes.After treatment with M1145 the 2-NBDG,GLUT4,PGC-1α,pP38MAPK,pAKT and pAS 160 levels,GLUT4 and PGC-1α expression in the myotubes with silence GALR2 were insignificantly changed.2.The results of the current study revealed that administration of baicalin can decreased food intake,body weight,HOMA-IR,circulating glucose and insulin levels,but enhanced GLUT4,PGC-la,pP38MAPK,pAKT and pAS160 expression,reversed high fat diet-induced glucose and insulin intolerance,hyperglycemia and insulin resistance in skeletal muscle of insulin-resistant mice compared with controls.Also,treatment with baicalin significantly elevated the GLUT4 and PGC-1αexpression in skeletal muscles and myotubes.Furthermore,the 2-NBDG,GLUT4,PGC-1α,pP38MAPK,pAKT and pAS160 levels were significantly increased in the group of baicalin compared with control group.After treatment with p38MAPK or AKT inhibitors the GLUT4 and 2-NBDG contents in the baicalin group were significantly decreased in myotubes.3.The HOMA-IR,GLUT4,PGC-1α,pP38MAPK,pAKT and pAS160 levels as well as GLUT4 and PGC-la expression levels were significantly decreased in the M871+baicalin group compared with the baicalin group.However,the HOMA-IR,GLUT4,PGC-1α,pP38MAPK,pAKT and pAS160 levels as well as GLUT4 and PGC-1α expression levels were insignificantly changed in the GKO+baicalin group compared with the GKO group.In vitro,the 2-NBDG,GLUT4,PGC-1α,pP38MAPK,pAKT and pAS160 levels as well as GLUT4 and PGC-la expression were significantly decreased in the M871+baicalin group compared with the baicalin group too.While,after treatment with baicalin,the 2-NBDG,GLUT4,PGC-1α,pP38MAPK,pAKT and pAS160 levels as well as GLUT4 and PGC-1α expression in the myotubes with silence GALR2 were insignificantly changed.Conclusions:1.The results showed that the mice had the symptom of polydipsia and polyuria in model gr oup compare to normal group.The result of blood glucose and glucose tolerance of this model were similar to the pathogenesis of insulin resistance.These results have indicated that the insuli n-resistant mice were similar to the early onset of type 2 diabetes.Moreover,our results in this study found that activation of GALR2 could alleviate insulin resistance through P38MAPK/PGC-la/GLUT4 and AKT/AS160/GLUT4 pathway in the skeletal muscle and myotubes,indicating that activiating GALR2 represented a promising strategy against obesity-induced insulin resistance.2.The results in the current study showed that treatment with baicalin significantly elevated PGC-1α,pP38MAPK,pAKT and pAS160 levels as well as GLUT4 expression and translocation levels in the skeletal muscle and myotubes.The results suggested that baicalin alleviated insulin resistance through P38MAPK/PGC-la/GLUT4 and AKT/AS160/GLUT4 pathways.These contribute to our understanding of the target of baicalin in relieving insulin resistance,enriching the scientific connotation of the removing heat treatment of Chinese Medicine,and providing a pharmacological foundation for clinical treatment and new drug development of Scutellaria baicalensis Georgi.3.The GALR2-mediated beneficial effects of baicalin on GLUT4 expression and translocation as well as relative signal pathway have been explored in skeletal muscle and myotubes.These results suggested that baicalin alleviated insulin resistance through GALR2/P38MAPK/PGC-1α/GLUT4 pathway and GALR2/AKT/AS160/GLUT4 pathway in the skeletal muscle and myotubes.In brief,GALR2 is a novel target for baicalin against obesity-induced insulin resistance.In conclusion,this study may provide a promising strategy for modern mechanism of Scutellaria baicalensis Georgi of Chinese medicine to treat insulin resistance in clinic. |
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