| Background:Insulin resistance(IR)plays a crucial role in the development of type 2 diabetes mellitus and is associated with hypertension,obesity and cardiovascular diseases(CVDs).Studies have indicated that IR precedes the clinical diagnosis of type 2 diabetes and is a major metabolic risk factor even in normoglycemic states.Several clinical trials,such as the insulin resistance atherosclerosis study(IRAS)and the San Antonio Heart Study(SAHS),have shown that IR is associated with atherosclerosis even in nondiabetic patients.These studies suggest that current clinical interventions for IR occur relatively late considering diabetes-related complications.Therefore,earlier pharmaceutical intervention for IR before the onset of diabetes is a very promising strategy for the prevention of diabetes-related complicationsAlthough the pathogenesis of IR has not yet been elucidated,elevated free fatty acid(FFA)levels are a well-established risk factor for IR.Acute exposure to FFAs leads to IR without affecting blood glucose in healthy humans and rodents.In the process of FFA induction,mitochondrial dysfunction is considered a major cause of IR formation.Mitochondrial function is regulated by Cyclophilin D(CypD),the key regulator of the mitochondrial permeability transition pore(mPTP),which is closely associated with IR.Studies have revealed that CypD-deficient mice are protected from high-fat diet(HFD)-induced IR,and the increased glucose uptake is observed in only skeletal muscle but not in liver or adipose tissue.The skeletal muscle-specific regulation of insulin sensitivity has been demonstrated by variations in the composition and function of the mPTP.These studies suggest that the inhibition of CypD might have potential therapeutic value in IR by regulating skeletal muscle mitochondrial dysfunction.Berberine(BBR),an isoquinoline alkaloid,has been used to treat intestinal infections for thousands of years.Recently,several studies have shown its chronic beneficial effects on weight loss,hyperglycemia and IR in humans and rodents with diabete.One study in db/db mice indicated that BBR reduces body weight and causes an improvement in glucose tolerance.Another study in type 2 diabetic patients showed that three months of BBR treatment reduces fasting blood glucose and was accompanied by improved insulin sensitivity and decreased body weight.However,it is hard to rule out the effect of weight loss on IR in the chronic treatment model because weight loss is sufficient to improve IR.Meanwhile,these studies focused on the hypoglycemic effect of BBR in the diabetic state.Whether BBR has a therapeutic role in the treatment of IR before the onset of hyperglycemia has not yet been elucidated.In the current study,we demonstrated that BBR alleviated FFA-induced 1R independent of glucose homeostasis by inhibiting skeletal muscle CypD,suggesting a therapeutic role of BBR in the early intervention of IR.Objectives:1.To determine the effects of berberine on intralipid-induced insulin resistant.2.To verify the effects of berberine on intralipid-induced mitochondrial swelling.3.To determine the effects of berberine on CypD protein expression in AML 12 hepatocytes and HSMCs in vitro.4.To verify whether the effect of berberine on intralipid-induced insulin resistant was associated with CypD depletion.5.To determine the target organ of berberine on insulin sensitizing effects by using different delivery routes.Methods:1.Animal experimental protocolsMale Sprague-Dawley rats weighing 250-350 g were fasted overnight before the surgery.After being anesthetized,rats were intubated to maintain a patent airway.Polyethylene cannulae(PE-50)were inserted into the carotid artery,portal vein and jugular vein for arterial blood sampling,arterial blood pressure monitoring,and various infusions.After a 30-min baseline period to assure stable anesthesia and hemodynamic stability,rats were studied with the following protocols.(1)Euglycemic hyperinsulinemic clamps.Rats received systemic infusions with regular insulin(3 mU/kg/min)for 2 h.The arterial blood glucose level was determined every 10 min using an Accu-Chek Advantage glucometer(Roche Diagnostics,Indianapolis,IN,USA),and 30%dextrose(30%wt/vol)was infused into the jugular vein at a variable rate to maintain blood glucose levels within 10%of basal levels.The time course and steady-state whole-body glucose infusion rate(GIR)were calculated.At the end of the study,rats were sacrificed,and the gastrocnemius muscle was obtained to evaluate mitochondrial swelling,Akt,AMP-activated protein kinase(AMPK)phosphorylation and CypD protein expression using Western blotting.(2)Insulin tolerance test.After fasting for 8 h,mice received an i.p.injection of insulin(1.0 U/kg,human insulin;Lily,Indianapolis,IN,USA).The blood glucose levels were determined at 0,15,30,60,90 and 120 min after the injection of insulin.The insulin sensitivity index(Kitt)was calculated as(0.693/t1/2)× 100.(3)Protocol 1.Rats received an intragastric(i.g.)administration of berberine(10 mg/kg,Sigma-Aldrich,St.Louis,MO,USA)1 h before or after the insulin clamp in the presence or absence of an intralipid(6.6%,Sigma-Aldrich)plus heparin(60 U/ml)infusion(5 ?l/min).(4)Protocol 2.Male wild-type mice aged 19-20 weeks were divided into three groups.Each group received saline or intralipid(4 ?l/g,20%,Sigma-Aldrich)plus heparin(60 U/ml)injection through the caudal vein with or without BBR(14mg/kg)i.g.delivery 60 min before the insulin tolerance test(ITT).(5)Protocol 3.Mice were divided into three Ppif+/+(wild type)groups and three Ppif-/-(CypD deficiency)groups.Each group received saline or intralipid(4?l/g,20%,Sigma-Aldrich)plus heparin(60 U/ml)injection through the caudal vein with or without a BBR(14 mg/kg)i.g.delivery 60 min before the ITT.(6)Protocol 4.Rats received BBR via 3 routes[intragastric(i.g.,10 mg/kg),intraportal(p.v.,20 ?g/kg)or intravenous(i.v.,15?g/kg)],1 h before the insulin clamp was performed in the presence of intralipid(6.6%,Sigma-Aldrich)plus heparin(60 U/ml)infusion.The dose we used for the p.v.delivery(20?g/kg)was calculated according to BBR pharmacokinetics,which could make the intraportal BBR concentration similar to that after BBR(10 mg/kg)i.g.delivery.The dose we used for the i.v.delivery(15 ?g/kg)was calculated according to BBR pharmacokinetics,which could make the plasma BBR concentration similar to that after BBR(20 ?g/kg)p.v.delivery(7)Protocol 5.Rats received an intralipid(6.6%,Sigma-Aldrich)plus heparin(60 U/ml)infusion with or without BBR(10 mg/kg)i.g.delivery 1 h before the insulin clamp in the presence or absence of NG-nitro-L-arginine methyl ester(L-NAME;50 ?g/kg/min)via i.v.infusion.2.Cell cultureAML 12 hepatocytes,human skeletal muscle cells,human umbilical vein endothelial cells were cultured in a humidified atmosphere with 5%CO2 at 37?Cells received the indicated treatment and were then collected for further analysis AML12 hepatocytes and HSMCs were pretreated with 0.4 mM palmitate acid(PA)for 2 h before coincubation with 5 ?M BBR for 24 h.DMSO and BBR(5 ?M)were treated as control.HSMCs were treated with BBR 1,2.5,5 ?M for 24 h,DMSO were treated as control.HUVECs were treated with BBR 1,5 ?M for 24 h,DMSO were treated as control.3.Western blottingSkeletal muscle tissue and cells p-Ser1177 eNOS,p-Thr172 AMPK,p-Ser473 Akt,AMPK,eNOS,?-actin,CypD,and GAPDH were measured by western blotting.Results:1.BBR improves intralipid-induced IR and skeletal muscle mitochondrial swellingInsulin clamp experiments were performed for 2 h with or without BBR i.g.administration at-60 min in rats receiving a 3-h systemic intralipid infusion(protocol 1).Intralipid infusion inhibited insulin-stimulated glucose disposal by?70%.BBR i.g.delivery partially improved IR,as indicated by an?2.2-fold increase in the glucose infusion rate(GIR)at steady-state.BBR was i.g.delivered 2 h after intralipid infusion.The glucose disposal rate was acutely increased 30 min after BBR i.g.administration,and the steady-state GIR was similar to that after BBR i.g.delivery at-60 min.The basal fasting blood glucose levels of rats were within the normal range and were not affected by either intralipid or BBR treatment.Similar experiments were also performed in C56BL/6 mice(protocol 2),and insulin sensitivity was measured by the ITT.The insulin sensitivity index(kitt)decreased in the intralipid delivery group but was restored by BBR supplementation.we examined Ca2+-overload-induced mitochondrial swelling in the skeletal muscle of SD rats in protocol 1.Mitochondria from intralipid-treated skeletal muscle were more sensitive to Ca2+ overload,indicating that more mPTP opened in skeletal muscle mitochondria after intralipid treatment than saline treatment(P<0.05,ANOVA).BBR i.g.administration before intralipid infusion alleviated intralipid-induced mitochondrial swelling.2.BBR inhibits intralipid-induced CypD expressionBBR treatment strongly inhibited palmitate acid(PA)-induced CypD protein expression in AML12 hepatocytes and HSMCs.BBR treatment decreased CypD protein expression and increased AMPK phosphorylation levels in a dose-dependent manner without influencing Akt phosphorylation in HSMCs.3.The improvement of IR in response to BBR is associated with CypD inhibitionAll mice were divided into wild-type(Ppif+/+)and CypD-deficient(Ppif-/-)groups(protocol 3).Each group was divided into three subgroups that received saline i.v.,intralipid i.v.or intralipid i.v.+ BBR i.g.60 min before the ITT.The absence of CypD protein expression in skeletal muscle validated the CypD deficiency model.The insulin sensitivity index(kitt)was lower in the intralipid-treated group than in the saline-treated group,while kitt was not different in the two subgroups of CypD-deficient mice.These results suggest that intralipid-induced IR was alleviated due to CypD deficiency.BBR cotreatment with intralipid partially restored Kitt in wild-type mice(compared with the intralipid-treated subgroup,P<0.05,ANOVA)but not in CypD-deficient mice.Collectively,these results revealed that CypD participated in the protective effect of BBR on intralipid-induced IR.4.The insulin-sensitizing effect of BBR is independent of the gut and liverTo examine the effect on the intestine,we injected a much lower dose of BBR directly into the portal veins of rats(protocol 4),which could make the intraportal BBR concentration similar to that after BBR i.g.administration while avoiding high intestinal concentrations.Then,we performed insulin clamp experiments on the rats.Compared with i.g.delivery,the p.v.delivery of BBR did not weaken the effect of BBR on IR,indicating that the insulin-sensitizing effect of BBR was independent of the intestine.Similarly,we injected a lower dose of BBR directly into the jugular veins of the rats,which could make the plasma BBR concentration similar to that after BBR p.v.administration while avoiding high liver concentrations.Compared with p.v.delivery,the i.v.delivery of BBR did not weaken the effect of BBR on IR,indicating that the insulin-sensitizing effect of BBR was independent of the liver.5.Endothelial-dependent vasodilation is not associated with BBR's insulin-sensitizing effectBBR treatment increased eNOS phosphorylation at Ser1177 in HUVECs in a dose-dependent manner.To examine the role of NO-dependent vasodilation on the insulin-sensitizing effect of BBR,L-NAME was used to inhibit NO-dependent vasodilation in rats receiving BBR and intralipid administration.However,L-NAME coinfusion with BBR failed to abolish the effect of BBR on IR,indicating that endothelial-dependent vasodilation was not involved in the insulin-sensitizing effect of BBR.6.BBR inhibits skeletal muscle CypD expressionCypD protein expression was upregulated in the intralipid-treated group and was dramatically inhibited by BBR cotreatment in skeletal muscle.AMPK phosphorylation levels were increased by BBR treatment in skeletal muscle.However,this effect was strongly inhibited by intralipid administration.The phosphorylation of Akt was not influenced by BBR in the skeletal muscle of SD rats.Conclusions:1.Berberine improved intralipid-induced insulin resistant,which was accompanied by decreased skeletal muscle mitochondrial swelling.2.Berberine inhibited CypD protein expression in HSMCs and AML hepatocytes.3.Berberine improved intralipid-induced insulin resistant,which was associated with the inhibition of CypD protein expression4.The targart organ of berberine on insulin sensitizing effect was skeletal muscle. |
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