The Study Of Relationship Between Hypoglycemic Drugs And Amylin

Type 2 diabetes is characterized by insulin resistance and progressiveβ-cell dysfunction leading to insulin deficiency.Type 2 diabetes is a chronic metabolic disease,which is characterized by fasting hyperglycemia that worsens as the disease progresses.Data from the UK Prospective Diabetes Study(UK-PDS) have shown that an almost inevitable progressiveβ-cell failure occurs despite the use of various therapies aimed at ameliorating hyperglycemia.Several mechanisms may contribute to the progressiveβ-cell failure in type 2 diabetes,including loss ofβ-cell mass,β-cell exhaustion,and the cytotoxic effects of elevated glucose and lipid levels.A growing body of evidence suggests that islet amyloid deposits may play an important role in the loss ofβ-cells and the progressive decline in insulin secretion.Westermark identified the major component of islet amyloid as a 37 amino acid peptide and named it amylin or islet amyloid polypeptide.Amylin is colocalized with insulin in the isleβ-cells and is cosecreted with insulin in response toβ-cell stimulation by both glucose and non-glucose secretagogues agents, such as arginine.In type 2 diabetes,this peptide aggregates to form amyloid fibrils that are toxic toβ-cells.The mechanism responsible for islet amyloid formation in type 2 diabetes is still unclear,but it appears that an increase in the secretion and expression of amylin can result in its onset.Therefore,therapies that alter endogenous insulin secretion are likely to cause parallel changes in amylin secretion.In fact,previous studies have suggested that sulfonylurea therapy increases the post-prandial amylin concentration,but not so in insulin therapy.These changes in turn may influence the rate of the formation of islet amyloids,which may be disadvantageous in the long term.In the present study,sulfonylurea and recombined human glucagon-like peptide 1(GLP-1) were adopted to study the relationship between hypoglycemic drugs and amylin,and explore the role of amylin in type 2 diabetes therapy and the mechanism of hypoglycemic drugs.The present project was supported by a research fund for the Doctoral Program of Higher Education(№2004-024-6069). The major methods,results and conclusions were described in two parts.PartⅠ:The relationship between amylin and effectiveness of SUAIM:To investigate the effect sulfonylurea(SU) on glycometabolism andβ-cell function in normal mice treated with a large dose in a long-term.The effect of amylin on hypoglycemic effectiveness of SU was evaluated in the present study.METHODS:The diabetes mice model was established by intravenous injection alloxan The mice were treated for 5 weeks with Glibenclamide 7.2mg/kg/d tid and gliclazide 80mg/kg/d tid,respectively.The weight and blood glucose were measured during the experiment.At the 5^th week,the glucose tolerance test was performed and the blood sample was taken to measure glucose,amylin level in the fasting state and for 120min after injection glucose.When the treatment finished,the mice pancreas were taken and made section.The Histological examination was performed in light microscopy.Mice were treated with Glibenclamide 3.6 mg/kg.After 1 hour,they were injected amylin 80μg/kg by i.v,and given glucose 2g/kg by i.p at once.The blood glucose spike, area under the curve(AUC),incremental glucose,time -averaged mean incremental are determined after glucose loading.Glipizide and amylin were incubated together for 2h in the primary culture islet cell,and then insulin level was measured in the culture supernatant by ELASA.RESULTS:Effects of Sulfonylureas on the level of blood glucose in diabetic mice induced by alloxan.Alloxan-treated mice had markedly elevated level of blood glucose compared with normal mice(P＜0.001),which indicated the diabetes model induced by alloxan was available.Treatment with Glibenclamide 3.6mg/kg(b.i.d) and gliclazide 40mg/kg(b.i.d) for 5weeks did not reduce the level of blood glucose in diabetes mice. It suggests that the diabetes model induced by alloxan is not applicable to study the drugs treated type 2 diabetes,such as SU. Effect of SU on the level of blood glucose and glucose tolerance in normal miceTreatment with Glibenclamide 3.6mg/kg(b.i.d) caused a marked fasting hypoglycemia compared with untreated group.during administration for 2,3,4 weeks(P＜0.05,P＜0.01).However,the role of hypoglycemia disappeared at treatment for 5 weeks.Gliclazide 40mg/kg(b.i.d) had not effect on the level of blood glucose in normal mice.The glucose tolerance of normal mice was not changed by treatment with Glibenclamide 3.6mg/kg(b.i.d) and Gliclazide 40mg/kg(b.i.d) for 5 weeks.Effect of SU on the level of amylin in normal mice loaded glucoseAll experimental mice showed an increase in the level of amylin after administration glucose.However,there is not significant difference among untreated group,Glibenclamide treated group and Gliclazide treated group.That is to say, Glibenclamide 3.6mg/kg(b.i.d)and Gliclazide 40mg/kg(b.i.d) had no effect on the secretion of amylin.Effect of amylin on the role of SU hypoglycemia in normal mice.Treatment with amylin 80μg/kg didn't cause the reduction of fasting glucose and glucose area under the curve(AUC).However,the glucose peak and the incremental glucose after injection glucose were markedly decreased by amylin 80μg/kg compared with untreated group(P＜0.01).The hypoglycemic effect of Glibenclamide was significantly augmented by amylin 80μg/kg intravenously compared with untreated group(P＜0.001),which resulted in a reduction of glucose AUC and a improvement on glucose tolerance compared with untreated group(P＜0.05).These findings indicated that amyin was in coordination with Glibenclamide in hypoglycemic action.The reduction of the incremental glucose in treatment with amylin group was much more than that of amylin combination with Glibenclamide group.Effect of amylin on the nsulinotropin of SU in primary culture islet cell.The islet cells were incubated in 16.7mmol/L glucose,and then stimulated for 2h in a series of concentration Glipizide.Glipizide 5.36μmol/L stimulated a marked increase in insulin level of in culture supernatants(P＜0.01).When 1μM amylin was added into culture medium,no significant difference was detectable in insulin level compared with Glipizide-stimulated insulin release.However,5 and 10μM amylin significantly inhibited the release of insulin stimulated by Glipizide compared with Glipizide group (P＜0.05).Conclusion:The effectiveness of Glibenclamide on fasting hypoglycemia was weaken by a long-term treatment with a large dose in normal mice.However,it doesn't significantly correlate with the level of amylin.The present data indicate that amylin increase or restrain the insulinotropin of sulfonylurea(SU) in vivo and in vitro,respectively.PartⅡ:Effect of GLP-1 on the secretion and expression of amylin in GK ratsAIM:To observe the effect of recombined human glucagon-like peptide 1(GLP-1) on the secretion and expression of amylin in Goto-Kakizaki(GK) rats(type 2 diabetes model).To determine whether amylin had disadvantaged effects on the role of GLP-1 and explore the mechanism of GLP-1 in the molecular level.METHODS:The GK rats were treated with rhGLP-1(7-36) 56 and 133μg/kg subcutaneously for 12 weeks.The body weight and fasting blood glucose were monitored at 0,1,3,5,7, and 11 weeks.In the 11th week,the post-prandial blood glucose level was measured at 30 and 60 min after feeding by the Roche Glucotrend-2 glucometer.In the 12th week of treatment,the rats were subjected to an intraperitoneal glucose tolerance test(IPGTT). The blood samples were collected from the ophthalmic vein after the glucose injection. Serum amylin was determined using an ELISA kit based on the standard curve.Directly after the glucose tolerance test,the rats were killed by dislocation of the cervical vertebra,and the pancreatic tissues were taken and made section.The Histological examination and islet number counting was assayed by light microscopy. For the immunohistochemical demonstration of amylin,the streptavidin-biotin-peroxidase complex(SABC) technique was employed to detect the amylin protein.The transcription levels of amylin and insulin mRNA were evaluated by fluorescent-quantitative-PCR.Effect of GLP-1 on the level of blood glucose and glucose tolerance in GK ratsBoth the fasting and post-prandial blood glucose levels were significantly higher in the untreated GK rats than the Wistar rats(P＜0.01).Treatment with 56 and 133μg/kg GLP-1 showed significantly lower blood glucose levels at 30 and 60 min after feeding compared with the untreated GK group(P＜0.05).There was no significant difference in the fasting blood glucose level(P＞0.05).The similar manifestation was observed in intraperitoneal glucose tolerance and GLP-1 showed significantly lower blood glucose levels after glucose loading compared with the untreated GK group (P＜0.05).Effect of GLP-1 on the level of amylin in serumThe plasma amylin levels were lower in the untreated GK rats than in the Wistar controls,both during basal conditions(P＜0.05) and after the glucose administration (P＜0.01).The basal plasma amylin levels in the rats treated by 56 and 133μg/kg GLP-lwere found to show a descending trend compared to those in the untreated GK rats(P＞0.05).In response to the intraperitoneal glucose administration,the plasma amylin levels of the rats treated by 133μg/kg GLP-1 displayed a marked increase at 30 min after the injection compared with the untreated GK rats(P＜0.05),whereas the increase in the rats treated by 56μg/kg GLP-1 did not reach significance(P=0.09).Effect of GLP-1 on the level of amylin in pancreatic isletsImmunostaining for amylin showed conclusive positivity in many cells of the pancreatic islets in the Wistar rats.Few scattered cells were immunopositive for amylin in the untreated GK rats.However,the GLP-1-treated GK rats showed more rich amylin-positive cells compared to the untreated GK rats,although the positively-stained cells were still less than those of the Wistar rats.This result was coincidence with the change of amylin in serum.Effect of GLP-1 on the histology and number of isletsIn contrast to the findings in the Wistar control rats,the islets of the GK rats usually had a very irregular shape,and the islets sometimes had a broken appearance. The boundary between the islets and exocrine pancreas was irregular.Some islet cells seemed degenerate and swollen.In many sections,a few islets were also found that displayed a rounded,clear-cut shape like the normal ones seen in the Wistar control rats. The number of islets was markedly decreased in the GK rats compared to the Wistar control rats(P＜0.01).No differences were found in the number of islets between the GLP-1 treated rats and untreated GK rats.However,the treatment with GLP-1 showed slight histological amelioration.Effect of GLP-1 on the amylin and insulin mRNA levelsIn the untreated GK rats,the levels of amylin and insulin mRNA were significantly reduced(P＜0.01).However,there was a more pronounced reduction in the levels of insulin mRNA than amylin mRNA.The GLP-1 56,133μg/kg treated rats both showed marked increases of amylin and insulin mRNA compared with the untreated GK rats.The amylin to insulin mRNA ratio of the untreated GK rats was significantly higher than that of the Wistar rats(P＜0.05,164.51%±43.86%vs 63.25%±13.76%).The ratio in the GK/GL and GK/GH rats was elevated with the GLP-1 treatment compared to the untreated GK rats(P＜0.05).Conclusion:GLP-1 stimulates the secretion and expression of amylin,which doesn't change the effectiveness of GLP-1 on hypoglycemia and improvement in islet histology.It may be contributed to a beneficial effect on the ratio of amylin to insulin mRNA.These findings suggest that GLP-1 and GLP-1 analogs are ideal candidates for the treatment of type 2 diabetes. (一) The whole conclusions1.The effectiveness of Glibenclamide on fasting hypoglycemia was weaken by a 5 weeks long-term treatment with 3.6mg/kg b.i.d dose in normal mice.However,it doesn't significantly correlate with the level of amylin.2.Amylin 80μg/kg had a hypoglycemic role in normal mice,which was in coordination with Glibenclamide in hypoglycemic action.However,amylin significantly inhibited restrain the insulinotropin of sulfonylurea(SU).These findings indicated that hypoglycemia of amylin was independence on the release of insulin.3.GLP-1 showed significantly lower postprandial blood glucose levels and improvement the glucose tolerance in GK rats.Toleration of GLP-1 was not observed during 8 weeks treatment.4.The treatment with GLP-1 showed slight histological amelioration and an increase of amylin secretion and content in islet cell.These findings suggeste that increase of amylin secretion stimulated by GLP-1 doesn't change the effectiveness of GLP-1 on hypoglycemia and improvement in islet histology.5.GLP-1 increased the levels of amylin and insulin mRNA and reduced the ratio of amylin to insulin mRNA.The data showed that the amylin and insulin mRNA was negative correlation on the blood glucose level,nevertheless the ratio of amylin to insulin mRNA was positive correlation on the blood glucose level.It may be contributed to a beneficial effect on the ratio of amylin to insulin mRNA.(二) New ideas1.That is the first study that the effectiveness of Glibenclamide on fasting hypoglycemia was weaken by a 5 weeks long-term treatment with 3.6mg/kg b.i.d dose in normal mice and it doesn't significantly correlate with the level of amylin.2.Amylin was in coordination with Glibenclamide in hypoglycemic action,which indicated amylin hypoglycemia is independence on SU hypoglycemic action.3.GLP-1 increased amylin secretion and did not induce toleration in a long-term therapy in the GK rat,a type 2 diabetes model.4.GLP-1 increased the levels of amylin and insulin mRNA simultaneously and reduced the ratio of amylin to insulin mRNA.GLP-1 reverse the changes of the amylin and insulin mRNA in diabetes model,which provided theory evidence for clinical application of GLP-1.5.The study showed that the ratio of amylin to insulin mRNA was positive correlation on the blood glucose level.The result suggest that the reduction in the ratio of amylin to insulin mRNA possibly is an important mechanism of some hypoglycemia,which provided a new route to exploit and screen a new pattern hypoglycemic drugs.6.The difference effect of SU and GLP-1 on amylin secretion indicated that amylin possibly was concerned with toleration of SU.At the same time,our result supported the academic concept.