Study On Extrapancreatic Effects Of Sulfonylureas And Its Mechanisms

BackgroundThe sulfonylurea receptor(SUR)belongs to the ATP-binding cassette(ABC)family of proteins and appears in several isoforms:SUR1 in pancreatic β-cells and neurons,SU R2A in cardiac and skeletal muscle,and SUR2B in vascular smooth muscle.SUR1 is unique and serves as an ion channel regulator,in which ATP hydrolysis modulates the gating of a separate Kir6.2 channel pore.SUR1,together with pore-forming Kir6.2,comprises the ATP-sensitive K+(KATP)channel,which serves to transduce metabolic changes into biophysical signals through the following sequence of events:elevated glucose increases the cytoplasmic[ATP]:[ADP]ratio in pancreatic β-cells,which causes closure of the KATP channels,β-cell membrane depolarization,opening of the voltage-dependent Ca2+ channels and Ca2+ influx.The resultant rise in the intracellular Ca2+ concentration triggers exocytosis of insulin granules and insulin secretion.Compared with other subtypes of SUR,SUR1 play a vital role in glucose metabolism.Hence,it is clear that research on the specific functions of SUR1 is valuable.Thanks to various animal models,we have gained a better understanding of the aetiology,pathology,and molecular mechanisms of different disorders.Over the past few decades,the adoption of molecular biology techniques for the genetic manipulation of rodents has resulted in a surge of interest in using these rodents as model systems for the investigation of almost all facets of mammalian biology.Not only does this allow the direct assessment of gene function in intact animals,it also allows the design of increasingly useful animal models of human disease.Therefore,we can apply gene knockout technology to explore the special function of the SUR1 gene in mammalian biology.Although much effort has been given in this field by using the SUR1 gene knockout mice,no direct evidence has been available for rats.When compared with mice,rats are closer to humans in cognitive behaviour and often serve as an ideal animal model of hypertension,diabetes mellitus,breast carcinoma and neurological disorders.In addition,considering that such studies require invasive procedures and large blood and tissue samples,the large body size of rats could be an advantage over the smaller mice.Therefore,we established a SUR1-/-rat model via the TALEN technique and investigated changes in glucose metabolism when knocking out the SUR1 gene,which was primarily responsible for glucose regulation under normal conditions.Objectives1.To investigate the changes in glucose metabolism in SURF1-/-rat model,so as to better understand the role of SUR1 in rat glucose metabolism.2.To explore the possible mechanisms in above processes.Materials and methods1.Animal model for SUR1 gene knockout and grouping(1)Establishment of SUR1 gene knockout rats:The SUR1 gene ID(25559)was searched for in the Entrez Gene database.The SUR1 null Sprague-Dawley rat lines were acquired by TALEN-mediated gene targeting from Cyagen Biosciences Inc.,Guangzhou,China.Rats of the 5th generation or greater were used in the experiments.Genotypes were determined by Sanger sequencing of PCR products for each rat to confirm deletion sites.The SUR1 protein expression was examined by Western blot to further corroborate the successful deletion of the SUR1 gene.(2)Grouping:All experimental rats in the study were divided into four groups as follows:SUR1-/-male rats,SUR1-/-female rats,wild-type(WT)male rats and WT female rats.The weight of the rats ranged from 60-140 g,with an age of four weeks at the beginning of the experiments.After 8 weeks of feeding with the standard diet,all rats were euthanized,and the organs were collected for multiple biochemical analyses.2.Body weight(BW)and fasting blood glucose(FBG)(1)BW:After initiation of experiments with 4-week-old rats,the rats were weighed once per week until the age of 12 weeks.(2)FBG:All experimental rats of the four groups were fasted for 12 hours with access to drinking water.The next day,the FBG level of each rat was tested.Tail vein blood was collected sequentially,and glucose level was measured by the One-Touch Glucometer.FBG was determined once per week for 8 weeks.3.Glucose/insulin tolerance(IPGTT and IPITT)IPGTT:An intraperitoneal glucose tolerance test(IPGTT)was performed to evaluate glucose tolerance after the rats were fasted for 12 hours.The following day,rats were injected with a bolus of glucose(1 g/kg i.p.),and blood from the tail vein was tested at 0,15,30,60,and 120 min from injection by a One-Touch Glucometer.The curves were drawed and the mean areas under the curve(AUC)were obtained.IPITT:Intraperitoneal insulin tolerance test(IPITT)was carried out after they were fasted for 4 hours.A bolus of insulin(1 unit/kg i.p.)was administered to the animals,and blood samples were collected as described above.AUC was calculated as the primary result of the insulin tolerance test.IPGTT and IPITT experiments were performed every four weeks.4.PAS staining and immunofluorescence staining(1)The glycogen contents in liver and muscle were determined by periodic acid-Schiff(PAS)staining.(2)Pancreas tissues from the four groups were extracted for immune fluorescence staining to evaluate the expression of insulin and glucagon.(3)Muscle sections were extracted for immunofluorescence staining to evaluate the expression of GLUT4.5.Western blotting analysisAfter euthanasia,tissues including liver and muscle were removed and homogenized with RIPA using a homogenizer,and the tissue proteins were isolated.Expressions of p-GSK and GSK in liver and the expression of GLUT4 in muscle were detected by western blotting6.Statistical analysisAll statistical analyses were performed using Statistical Product and Service Solutions(SPSS)20.0 software(IBM).A Student’s t-test was used to assess significance for data within two groups.All data are presented as the means ± SEM,and significance was set at P<0.05.Results1.Identification of SUR1 gene knockoutSanger sequencing of the PCR products from each rat showed a 16 bp deletion in the SUR1 gene in the SUR1-/-rats corresponding to CCTCACGGGGCTTCTG compared with WT rats.Western blot showed that SUR1 knockout rats had no SUR1 protein expression compared with WT rats,further confirming that the SUR1 gene knockout model was successfully constructed.2.BW and FBG(1)BW:The body weights of SUR1-/-rats at the age of 4 weeks were 35-40%less than that of the WT group,and they remained significantly smaller than the WT rats throughout adulthood.Compared with WT rats,SUR1-/-rats weighed less(P<0.05).Body weight gain curves showed that the body weight of SUR1-/-male rats gained significantly slower than that of WT male rats(P<0.05).(2)FBG:SUR1-/-rats were significantly more hypoglycaemic than WT rats when fasted for 12 hours(4.10±0.75 vs 7.07±1.22 mmol/L for male,P<0.05;4.77±0.67 vs 6.03±0.49 mmol/L for female,P<0.05)at the age of 6 weeks.In addition,among the SUR1-/-rats,the males showed a lower FBG than the females(4.40±1.41 vs.6.57±0.91mmol/L,P<0.05)at the age of 8 weeks.3.IPGTT and IPITTIPGTT:Plasma glucose levels were significantly elevated in SUR1-/-rats at 15 min,and peak glucose values were significantly higher for SUR1-/-female rats than WT females.The glucose clearance time was longer than that of the WT rats.In addition,SUR1-/-rats failed to release insulin in response to the glucose challenge1 compared with WT rats.The IPGTT results showed that SUR1-/-female rats had a more severely impaired glucose tolerance compared to SUR1-/-males.Additionally,IPGTT indicated a significantly impaired glucose tolerance of SUR1-/-rats,especially for female rats.IPITT:Following intraperitoneal injection of insulin,WT rats showed a typical decrease and rebound in serum glucose after insulin challenge,whereas SUR1-/-ats showed a rapid and sustained reduction in serum glucose.SUR1-/-rats exhibited a stronger reduction of the AUC for blood glucose levels in response to insulin compared with WT rats,which demonstrated a significantly enhanced insulin sensitivity.In addition,a better insulin sensitivity was observed in SUR1-/-male than that in SUR1-/-females.4.Expression of insulin and glucagon in pancreas in SUR1-/-ratsResults showed that a reduced fluorescence staining for insulin was observed in both SUR1-/-male rats and SUR1-/-female rats,without changes of glucagon expression,compared with WT rats.Reduced fluorescent staining for insulin further confirmed that the SUR1 gene knockout induced decreased insulin secretion.5.Glucose metabolism in liver and muscle in SUR1-/-ratsLiver:(1)PAS staining:The level of hepatic glycogen was significantly higher in the SUR1-/-rats than in the WT rats.(2)Western blot showed that a significant reduction in GSK3 expression,whereas the p-GSK3/GSK3 ratio was significantly increased in the SUR1-/-rats(P<0.05),which indirectly reflected the improvement of hepatic insulin sensitivity Muscle:(1)PAS staining:The level of muscle glycogen was significantly higher in the SUR1-/-rats than in the WT rats.(2)GLUT4:Glucose uptake in the peripheral tissues is primarily mediated by GLUT4,which is a crucial pathway to mediate glucose transport.The expression of GLUT4 in the muscle of SUR1-/-rats was signifficantly increased by 2-fold(P<0.01)compared to that of the WT group.In accordance with the above western blotting results,increased GLUT4 fluorescence staining was observed in SUR1-/-rat muscle.Conclusions1.SUR1-/-rats showed glucose intolerance for KATP channel dysfunction to secret sufficient insulin,enhanced peripheral insulin responsiveness can provide a direct account for survival and protection from hyperglycaemia.2.Enhancement of hepatic and muscle glycogen levels,GSK3 inactivation and increased GLUT4 expression levels might contribute to the improvement of insulin sensitivity in SUR1-/-rats.3.SUR1-/-male rats showed better insulin sensitivity compared to females,possibly due to excess androgen secretion in adulthood.BackgroundThe hypoglycaemic effect of sulfonylureas(SUs)is mainly due to a drug-induced release of insulin from pancreatic β cells.Whereas,studies suggested that SUs might decrease blood glucose via improving insulin resistance,which is known as extrapancreatic glucose-lowering activity of SUs.However,whether above effects of SUs exist in vivo has not yet been fully determined.SUs-induced blood glucose control without adequately elevated mean plasma insulin level was interpreted as a drug-induced postprandial kick of insulin release resulting in lower mean blood glucose values and thereby also reduced basal insulin levels.Moreover,the improvement of insulin resistance has also been attributed to an alleviation of glucotoxicity derived from a SU-induced increase in insulin secretion.Although in vitro insulin-sensitizing effects of SUs have been suggested,in vivo induced effects are unknown.Thus,sufficient in vivo studies are urgently needed to clarify the occurrence of these effects of SUs,such as glimepiride and gliclazide.Glimepiride,a widely used SU,harbors the typical SU moiety and shares the wide spectrum of activities attributed to conventional SUs.In pancreatic beta cells,the glimepiride-binding affinity is lower than that of other SUs,leading to a lower effect on insulin secretion.However,glimepiride effect upon glycemic homeostasis is similar to that observed for other SUs,which hints that glimepiride possesses extrapancreatic blood glucose-improving activity.Similar activity may also exist in gliclazide for its unique molecular structure.SUR1,the main functional receptor of SUs on islet beta cell,could stimulate insulin secretion via combining with SUs.In this study,SUR1 gene knockout(SUR1-/-)rat model was used to determine the extrapancreatic glucose-lowering activity of glimepiride and gliclazide,in which these SUs could not stimulate insulin secretion by combining with SUR1,thus excluding its intrinsic action in decreasing glucose levels.In the present study,we aim to investigate whether glimepiride and gliclazide possess extra-pancreatic glucose-lowering effects in vivo and their mechanisms and difference in this process.Objectives1.To clarify the existence of extrapancreatic glucose-lowering effect of glimepiride and gliclazide in vivo.2.To gain insight into their underlying mechanisms and to clarify the difference between these two SUs in above processes.Materials and methods1.Establishment of SUR1 knockout rats:The SUR1-/-rats(rABCC8,the approved symbol for the SUR1 gene is completely ablated)were established by TALEN-mediated gene targeting technology(Cyagen Biosciences Inc,Guangzhou,China)in the genetic background of Sprague Dawley(SD)rats.2.Establishment of type 2 diabetes modelType 2 diabetes SUR1-/-reats model:4 weeks High-fat diet(HFD,34.5%fat,17.5%protein,and 48%carbohydrate;Beijing Keaoxieli FEEDS CO.,LTD,China)combine with low dose of streptozocin(STZ,27.5 mg/kg i.p.in 0.1 mol/L citrate buffer,pH4.5;Sigma,St.Louis,MO)were used to establish type 2 diabetes model.Intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were performed before and after HFD.Blood was sampled through the tail vein to test fasting blood glucose(FBG),triglycerides(TG)and total cholesterol(TC)before and after STZ injection.The FBG>11.1 mmol/L at two consecutive tests and lower insulins resistance were regarded as diabetics.Then rats were randomly divided into four groups:(1)Glimepiride(0.5mg/kg/d);(2)Gliclazide(10mg/kg/d);(3)Metformin(212.5mg/kg/d);(4)Control(saline).3.BW and FBG:Body weight,fast blood glucose,TC and TG were measured at 0,1,2 week.4.Insulin sensitivity:(1)IPGTT and IPITTIPGTT and IPITT were measured at 0,1,2 week.The measurement method is the same as the first part.Mean area under curve(AUC)was calculated for glucose to assess the overall effect and not just at a point of time.(2)Hyperinsulinemic-euglycemic clamps testHyperinsulinemic-euglycemic clamps were performed in six rats randomly selected from each group.One week before the clamps,rats were anesthetized with isoflurane.Catheters were implanted in the left femoral artery and fixed to the back of the central neck.After overnight fasting,clamp was performed in conscious rats.A venous indwelling needle was inserted into the tail vein so as to establish a three-way channel,which was used to simultaneously infuse glucose and insulin.Insulin(10 mU/kg/min)was continuously infused to raise plasma insulin within a physiologic range,and an infusion of 20%glucose was started at variable rates to maintain the plasma glucose at 4-6 mmol/L.Blood glucose was collected through the arterial catheter implanted before and tested every 5 minutes.At the same time,blood samples were also collected,centrifuged and stored.5.Specimen retentionAt the end of the experiment,rats were fasted overnight and deeply anesthetized with an overdose of isoflurane.Blood was collected from the abdominal aorta,and then tissues were immediately dissected,weighted,and stored at-80℃ until further analysis.6.Histopathological examination and immunofluorescence staining(1)Periodic acid-Schiff(PAS)staining was used to detect hepatic glycogen deposits.(2)Immunofluorescence staining was used to evaluate expressions of total and membrane GLUT4 in muscle and fat tissues.7.Western blottingTissues were removed and homogenized with commercial RIPA lysis buffer(Beyotime,China)added with protease inhibitor(PMSF,1:100)and phosphatase inhibitor(1:100)(Beijing Solarbio Science&Technology Co.,Ltd.).Expressions of glycogen synthase kinase-3(GSK3α/β.Ser21/9),p-GSK3α/β(Ser21/9),glycogen synthase(GS),p-GS(Ser641),AKT,p-AKT,AMPK,p-AMPK,insulin receptor(IR),phospho-IGF-Ⅰ receptor β(Tyr1 135/1 136)/insulin receptor β(Tyr1 150/1 151),insulin receptor substrate(IRS-1),p-IRS-1(Thr608),glucose 6-phosphatase(G6pase),glucose transporter 4(GLUT4),Peroxisome proliferator activated receptor-gamma(PPAR-γ)and PEPCK,β-actin were tested.8.Data and Statistical analysisData are presented as mean ± SEM.Comparisons between two groups were performed by Student t test,while one-way ANOVA followed by Tukey’s post hoc test using SPSS statistics(version 20.0,IBM)were used for multi-group comparison.Post-hoc tests were only carried out if F achieved P<0.05 and there was no significant variance inhomogeneity.Significance was defined as P<0.05.Results1.Establishment of SUR1 knockout rats:Sequence analysis showed that the SURF1-/-rats demonstrated the lack of a 16-base pair segment and western blot showed that SUR1 did not express in SUR1/-rats,which indicated that SUR1-/-rats were successfully established.2.T2DM SUR1-/-rats and groupingAfter four weeks HFD,insulin resistance was confirmed by IPGTT and IPITT.Then,after a low dose of STZ injection,serum levels of BW,FBG,TG and TC increased significantly in diabetic SUR1-/-rats.Thirty-six of SUR1-/-rats developed insulin resistance,moderate hyperglycemia and hyperlipidemia,which were in accordance with the clinical features of type 2 diabetes and were included in our following study.Rats without those characteristics above were excluded from this study.Grouping:Thirty-six diabetic SUR1-/-rats were then randomly divided into four groups:glimepiride(0.5 mg/kg/d,Sanofi,France;n=9),gliclazide(10 mg/kg/d,Servier,France;n=9),metformin(212.5 mg/kg/d,Bristol-Myers Squibb,Shanghai;n=9)and control(normal saline,n=9).BW,FBG,TG and TCBW,TG and TC:Before treatment,BW,TG and TC levels were same in these four groups.After two weeks treatment,there were no significant changes in BW,TG and TC in each group,and no significant differences were found between therapeutic groups and control group during two weeks treatment.FBG:After two weeks treatment,the fasting glucose levels were significantly decreased in glimepiride(15.70±1.27 vs.20.71±1.58 mmol/1,P<0.05),gliclazide(16.07±1.47 vs.20.71±1.58 mmol/1,P<0.05)and metformin(12.56±1.87 vs.20.71±1.58 mmol/l,P<0.05)group compared with that of control group.Gliclazide showed a similar fasting glucose reduction as glimepiride but not as remarkable as that of metformin.3.(1)IPGTT and IPITTIPGTT:No significant difference was found among these groups before treatment.After two weeks treatment,compared with the control group,the AUCs of IPGTT in the glimepiride(P<0.05)and metformin(P<0.05)groups were significantly decreased,while no significant difference was observed between gliclazide group(P>0.05)and control group.IPITT showed that the efficiency of insulin in T2DM SUR1-/-rats treated with metfonnin,glimepiride and gliclazide were significantly enhanced,compared to that in control group(P<0.05)with the following ranking in the improvement of insulin tolerance:metformin>glimepiride≈gliclazide>control.(2)Hyperinsulinemic-euglycemic clampingGlucose concentrations were clamped to the same level to ensure that there were no significant deviations in plasma glucose over the course of the experiment.Whole body glucose disposal,reflecting insulin sensitivity in peripheral tissues,was calculated as the sum of glucose infusion rates(GIRs)in the peripheral vein.Compared with the control group,glimepiride and gliclazide significantly increased GIR in SUR1-/-T2DM rats(25.66±2.14 and 25.99±1.65 mg/kg/min vs.14.99±3.04 mg/kg/min,P<0.05),and there was no significant difference between them.Similar but stronger effect was found in metformin group(33.59±2.38 mg/kg/min,P<0.05).These results indicated that glimepiride did possess extrapancreatic glucose-lowering effect,and gliclazide showed a similar effect as glimepiride in improving insulin sensitivity in SUR1-/-T2DM rats,but both was inferior to that of metformin.4.Hepatic insulin sensitivity(1)PAS staining:Compared with the control group,hepatic glycogen storage was increased in glimepiride-treated rats.Similar effect was also found in gliclazide-treated rats,and even higher action was observed in livers of rats treated with metformin,as indicated by obviously enhanced glycogen staining.(2)Hepatic glycogen synthesis:Our result showed that,compared with control group,the phosphorylation level of GSK3(p-GSK3)were obviously increased and that of GS(p-GS)were significantly decreased in glimepiride,gliclazide and metformin groups,especially in metformin group,which was consistent with above results of PAS staining and explained the changes in glycogen content to some extent(both P<0.05).(3)Hepatic gluconeogenesis:PEPCK and glucose-6-phosphase(G6pase),key enzymes of hepatic gluconeogenesis,were also inhibited in livers of SUR1-/-T2DM rats treated with glimepiride(P<0.05).Similar effects were found in gliclazide group(P<0.05),but both were weaker than that of metformin group(P<0.05).We found that both glimepiride and gliclazide could still reduce blood glucose without significant elevation of plasma insulin level in this model.Hyperinsulinemic-euglycemic clamp test further indicated that insulin resistance was ameliorated in these SUs treated diabetic SUR1-/-rats.(4)Activation of AKT and AMPK:AKT,the key molecule in insulin resistance modulation,is the upstream of GSK3 and GS,whose activation facilitates hepatic glycogen synthesis.Our results showed that the ratio of p-AKT to total AKT increased by 2.15-fold in glimepiride-treated SUR1-/-rats(P<0.05),1.62-fold in gliclazide-treated rats(P<0.05)and 6.45-fold in metformin-treated rats(P<0.05)compared with that of control group.These results were in agreement with the inhibition of GSK3,activation of GS in liver and upregulation of hepatic glycogen storage.AMPK is a potential target for glucose metabolism and plays a key role in the treatment of T2DM.Our results indicated that metformin significantly increased hepatic AMPK phosphorylation at Thr172 and enhanced its activity in the liver of SUR1-/-rats(P<0.05),while glimepiride and gliclazide did not,compared with the control.5.Peripheral tissues,mainly muscle and fat,whose glucose uptake is mainly mediated by GLUT4.Improvement of insulin resistance in muscle:(1)Expression of GLUT4:the expression of GLUT4 protein in muscle was increased by 8.14-fold in glimepiride group(P<0.05),4.74-fold in gliclazide group(P<0.05)and 1.36-fold in metformin group(P>0.05)compared with that of control,respectively.The effects of glimepiride and gliclazide,especially the former,on total GLUT4 in muscle were stronger than that of metformin(both P<0.05).The expressions of PM GLUT4 protein in muscle tissues were increased by 7.73-fold(P<0.05),3.5-fold(P<0.05)and 2.84-fold(P<0.05)in glimepiride,gliclazide and metformin groups,compared to that of control group,respectively.Comparisons among the three therapeutic groups indicated that glimepiride was superior to gliclazide and metformin in enhancing the expression of PM GLUT4(both P<0.05).(2)Expression of AKT:The level of p-AKT was significant higher in muscles of glimepiride-treated rats than that of the other three groups(both P<0.05).In addition,similar but weaker effects were found in gliclazide-treated rats(P<0.05),but not in the metformin group,compared with that of control group(3)Expression of IR and IRS-1:Compared with control group,the levels of phospho-IR(p-IR)in muscle were not increased by glimepiride,gliclazide and metformin,and no significant differences were found among these therapeutic groups.The level of phospho-IRS-1(p-IRS-1)was significant higher in muscles of glimepiride-treated rats than that of the other three groups(both P<0.05).(4)Expression of AMPK:p-AMPK protein was increased significantly in muscles of rats treated with metformin(P<0.05).Glimepiride and gliclazide showed no superiority over control in the activation of AMPK.(5)Expression of PPAR-γ:PPAR-γ,a member of the nuclear receptor superfamily,plays a crucial role in the regulation of glucose metabolism and insulin resistance.Compared with the control,glimepiride significantly elevated the level of PPAR-γ in muscles of T2DM SUR1-/-rats(4.98-fold,P<0.05).PPAR-γ were also increased by 2.11-fold and 1.04-fold in gliclazide(P<0.05)and metformin(P>0.05)groups,respectively.Among the three therapeutic groups,the level of PPAR-γ of glimepiride group was elevated more significant than that of gliclazide group and metformin group(both P<0.05).6.Improvement of insulin resistance in adipose(1)Expression of GLUT4:The expressions of total GLUT4 in fat tissues were consistent with that in muscle to some extent.The expression of total GLUT4 protein were increased by 5.72-fold(P<0.05),3.30-fold(P<0.05)and 1.31-fold(P>0.05)in glimepiride,gliclazide and metformin groups,compared with that of the control,respectively.Among the three therapeutic drugs,glimepiride showed a more significant upregulation in GLUT4 expression than metformin(P<0.05),whereas there was no significant difference compared with that of gliclazide group(P>0.05).Compared with the control group,the PM GLUT4 protein in fat tissues were increased in 2.65-fold(P<0.05),2.10-fold(P<0.05)and 0.75-fold(P>0.05)in glimepiride,gliclazide and metformin groups,respectively.Among the three therapeutic groups,the increase of PM GLUT4 protein in glimepiride group was higher than that of metformin group(P<0.05),but no significant difference was found when compared with that of gliclazide group(2)Expression of AKT:Compared with the control,both glimepiride and gliclazide significantly increased the levels of p-AKT(both P<0.05).Glimepiride and gliclazide showed superiority over metformin in increasing the levels of p-AKT(P<0.05).(3)Expression of IR and IRS-1:Compared with the control,both glimepiride and gliclazide significantly increased the levels of p-IRS-1,rather than the level of p-IR(both P<0.05).Glimepiride and gliclazide showed superiority over metformin in increasing the levels of p-IRS-1(P<0.05).(4)Expression of AMPK:The changes of AMPK in fat were similar to that in muscle.Compared with control group,the expression of p-AMPK in fat was significantly increased in metformin-treated group(P<0.05),but not in glimepiride or gliclazide group(5)Expression of PPAR-y:The change of PPAR-y in fat was similar to that in muscle,except that the increase in the glimepiride group was much higher,reaching 8.67-fold compared with that in the control group(P<0.05).Compared with control,PPAR-γ were also increased by 4.61-fold(P<0.05)and 2.63-fold(P>0.05)in the gliclazide and metformin groups,respectively.Conclusions1.The data obtained from SUR1－/-rats confirmed the extrapancreatic glucose-lowering effect of glimepiride and gliclazide in vivo.2.The enhancement of glimepiride on insulin sensitivity is similar with gliclazide,however both were weaker than metformin.3.Glimepiride and gliclazide could directly improve hepatic insulin resistance,which might be attributed to the increase of hepatic glycogen synthesis and the decrease of gluconeogenesis in this process.The activation of AKT might take a part in these processes.4.Glimepiride dominance increases the expressions of total and plasma membrane(PM)GLUT4 in muscle and fat,which might be attributed to the activation of IRS/AKT in IR-independent manner,and the augmentation of PPAR-y in muscle and adipose tissues.The above effects of glimepiride were stronger than that of gliclazide.BackgroundSulfonylureas(SUs)is one of the most commonly prescribed class of drugs for treatment of type 2 diabetes mellitus(T2DM).SUs binds to their receptors(sulfonylurea receptor,SUR),which are subunit of the ATP-dependent potassium(KATP)channel,thus closing the KATP channel in pancreatic β-cells,resulting in insulin secretion and decreases blood glucose.Diabetic kidney disease(DKD)is one of the major microvascular complications of diabetes.Glomerulopathy associated with diffuse or nodular glomerulosclerosis was originally deemed as the main pathologic change.Researchers have recently come to appreciate the key role played by proximal renal tubules in DKD.Studies show that one-third of diabetic patients with microalbuminuria having no or minimal glomerular changes,only proximal tubular lesions.Tubulopathy,especially the apoptosis of proximal tubular epithelial cells(PTECs),is shown to play an important role in DKD,which occurs earlier than glomerulopathy.The effects of SUs on DKD have been thought to be due to their indirect effects via their ability to decrease blood glucose.Few studies have examined whether SUs have direct effects on the kidney.SURs are present in a wide variety of extra-pancreatic tissues.Investigations indicated that SUR2,one common subtype of SUR,is located in PTECs.Hence,it is conceivable that SUs could act directly on the PTECs.As a KATP channel blocker,SUs close the KATP channels,leading to membrane depolarization and opening of voltage-operated Ca2+ channels,which further cause Ca2+ influx,and intracellular rise of Ca2+.This process may subsequently induce Ca2+-dependent-apoptosis.Several studies have shown that the opening of the KATP channel is renoprotective.So,SUs might have detrimental effects on kidney,which is a concern in clinical practice.Objectives(1)To explore the effects of three widely prescribed SUs(glibenclamide,glimepiride and gliclazide)on the apoptosis of PTECs,an important progress in the development of DKD.(2)To determine whether these SUs could be differentiated with regard to their effects on the apoptosis of PTECs and to analyze their possible underlying molecular mechanisms.Materials and methods(1)Cell culture and groupingCulture:Human proximal tubular epithelial cells(HK-2,American Type Cell Collection,Rockville,MD)were cultured in the RPMI 1640 medium containing 10%fetal bovine serum(Gibco,USA),11.1 mM glucose,100 U/ml penicillin and 100 ug/ml streptomycin(Sigma,St.Louis,MO)at 37℃,5%CO2 and 95%humidity.The culture medium was replaced with fresh medium every 2-3 days and expanded to new culture plates when the cell reach approximately 80%confluence.Grouping:1)Control group;2)Gliclazide group;3)Glimepiride group;4)Glibenclamide group.(2)The expression of SUR2 in PTECs was tested by immunofluorescence staining.(3)SUs dose selectionCCK-8 assay was employed to assess the effects of three SUs on the viability of the cells.Inhibition rate of cell vitality in all groups was calculated.Appropriate doses that these SUs have the same dynamic effects on PTECs were selected for our following study.(3)Apoptosis determinationFlow cytometry was performed to examine the apoptosis of PTECs treated with different SUs and the effects of diazoxide on the promotion or suppression effect of apoptosis of PTECs treated with different SUs.The cells were incubated with 5μl of Annexin V and 5 al of propidium iodide(PI)for 15 min at room temperature in dark,according to the manufacturer’s instruction(BD Biosciences,SanJose,CA),and then subjected to flow cytometry to measure the apoptosis rate(%).(5)Western blot analysisWestern blotting was used to examine expressions of apoptotic-related proteins,such as Bcl-2 and Bax,and autophagy-associated proteins,such as LC3I and LC3H.(6)Statistical analysisAll statistical analyses were performed using Statistical Product and Service Solutions(SPSS)20.0 software(from IBM).A Student’s t-test was used to assess significance for data within two groups.All data are presented as the means± SEM,and significance was set at P<0.05.Results(1)Expression of SUR2 in PTECs:results showed that,compared with negative control,SUR was widely expressed in PTECs.(2)Effect of SUs on human proximal tubule epithelial cell viability;Our results revealed that SUs exposure led to a dose-dependent inhibition of cell viability of the HK-2 cells by CCK-8 method.HK-2 cells treated with glibenclamide at a concentration of 45umol/L,gliclazide 1058umol/L,glimepiride 130 umol/L did not significantly affect cell viability.So,the above concentrations were used for our following experiments.(3)Effects of SUs on apoptosis of PTECsGlibenclamide promoted the apoptosis of PTECs significantly(P<0.05);the apoptosis rate(Q2+Q4)in glimepiride-treated group was not different from the control group(P>0.05).However,the effect was opposite in gliclazide group,where the apoptosis rate was reduced(P<0.05).What’s more,the above effects of glibenclamide and gliclazide were alleviated by DZ.In contrast,exposure to glimepiride with or without DZ did not induce a significant change in the number of apoptotic cells.(4)Expression of SUs on apoptosis-related proteinsBcl-2 is a crucial inhibitor and Bax is the promoter of apoptosis.Our results of western blotting showed that Bcl-2 was downregulated significantly(P<0.01)and Bax was upregulated(P<0.05)in glibenclamide group compared with that of control group.The expression of Bax was upregulated significantly in glimepiride group compared to control group(P<0.01),even though there was no significant differ...