| [Objective] Incretin hormones, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), both of which play an important role in mediating postprandial insulin release. And the hypoglycemic effect of GLP-1 is particularly important for diabetic patients. Recent studies have shown that glutamine may regulate the GLP-1 secretion and improve blood sugar, but its exact impact on blood sugar and blood lipids of patients with impaired glucose tolerance(IGT) and varying degrees of type 2 diabetes and possible adjustment mechanisms are not yet clear. Glutamine(Gln) is a kind of abundant free amino acid in the human body. Although it is considered as a non-essential amino acid, it is now recognized as a " conditionally essential " amino acid. Gin lowers blood sugar levels and contributes to increased insulin secretion in type 2 diabetes patients. Supplementation of Gin in high fat diet can reduce hyperglycemia and hyperinsulinemia in a T2DM rodent model. It also can increase the antioxidant capacity of type 1 diabetes in mice and reduced leukocyte adhesion molecule expression and oxidative stress when added to their chow. In order to clarify whether Gln could ameliorate postprandial glucose and pancreatic islet function of early T2DM and IGT patients by adjusting the incretin system, we conducted the following studies.[Methods] Human experiment:we selected nine type 2 diabetes patients with good glycemic control and a duration of less than three years, and all cases treated herein were as follows:200ml of warm water and low-fat meal (as self-control) were given when fasting in the morning on the first visit; After 1 week,200ml warm water with 100mg sitagliptin was taken prior to a low-fat meal; After another 1 week,30g Gin dissolved in 200ml warm water was served prior to a low-fat meal. Blood glucose, insulin, C-peptide, GLP-1 and other parameters along with area under the curve (AUC) were observed and analyzed.Animal experiment I:Altogether 66 Sprague Dawley(SD) rats were purchased, after 1 week of adaptive feeding 6 of them were killed as a baseline group. Then 18 rats were randomly selected as a normal control group on a normal chow. The rest of them (42 rats) served as the high-fat group and were fed a high-fat diet(HFD). Rats in the HFD group were intraperitoneally injected STZ to induce T2DM after 7weeks of high fat diet feeding. After successful induction of T2DM models in HFD group, the HFD group were randomly divided into Gin, sitagliptin, diabetic control group, respectively, i.e.12 rats in each groups. According to their group, each rat was gavaged with Gln(lg/kg body weight daily), sitagliptin(100mg/kg body weight daily) or solution(2ml/per rat/d) for 8 weeks. Fasting blood glucose, body weight of each rat were measured every week, and oral glucose tolerance test(OGTT) every 4 weeks. Serum Insulin, C-peptide, GLP-1, GIP at all time points during OGTT were assayed. Fasting blood samples were collected to assay uric acid(UA), free fatty acid(FFA), lipids before the rats were sacrificed. Pancreas of rats were obtained and treated by HE and VG staining after they were killed. The expression of connective tissue growth factor(CTGF), transforming growth factor betal(TGF-?1), Tumor-necrosis factor-a(TNF-a),3-nitrotyrosine(3-NT), nuclear factor E2-related factor-2 (Nrf-2) and heme oxygenase-1(HO-1) was detected by immunohistochemistry. TUNEL staining was used to detect apoptosis of pancreatic islet ? cells. RT-qPCR was used to quantify the mRNA expression of PTEN, Nrf2, HO-1, SOD-1 and so forth. Western blot was used to quantify the protein expression of Nrf2, p-Akt, HO-1, SOD-1 and so forth. Statistical Methods:all data were processed using SPSS 19.0 statistical package, and AUC was calculated using the sigmaplot12.5 program.Animal experiment 2:Altogether 66 SD rats were purchased, after 1 week of adaptive feeding 6 of them were killed and the remaining rats were randomly divided into the normal control group(n=18, on a normal chow) and high-fat diet group(42 rats on a high fat diet). OGTT test was performed after 8 weeks of raising, and 6 rats in the HFD group and 6 rats in normal control group were killed before the remaining HFD rats were randomly divided into Gin, sitagliptin and high-fat control(HC) group. After 4 weeks of drug intervention, OGTT test was performed before 6 rats in each group were killed. HFD rats were intraperitoneally injected with STZ to significantly elevate their blood glucose. Drug intervention was continued for another 4 weeks and OGTT was performed again before all the remaining rats were killed. The following procedures and observation indexes were the same as animal experiment ?.[Results] Human experiment:Compared with control, both Gin and sitagliptin reduced the AUC of postprandial glycemia(both P<0.05), and the hypoglycemic effect of Gin was more potent in early(t=0-60min) postprandial glycemic response(P=0.005). Compared with control, Gln significantly augmented the AUC level of insulin and C-peptide (P=0.019 and 0.016, respectively) and its AUC level of C peptide within 60 minutes after the meal was significantly more than sitagliptin(P=0.016). The whole and early-stage(t=0-60min) postprandial AUC level of C-peptide with Gin treatment both exceeded that with sitagliptin treatment(P=0.016 and 0.012, respectively). Compared with control, sitagliptin significantly raised postprandial AUC level of both GLP-1 and GIP(P=0.003 and 0.019). Gin treatment led to a significantly higher GLP-1 level at the 45th and 90lh minute than its own baseline value(P<0.05). The AUC level of GIP at the late stage(60-180min) increased significantly with Gin than control(P=0.016).Animal experiment 1:Diabetic group:after drug intervention for 4 weeks, rats in Gin group weighed significantly lower than the diabetic control group, and the difference was statistically significant (P= 0.027). After 8 weeks of drug intervention, rats in Gin group weighed close to those in normal control group(P= 0.96). But the mean body weight in Gin group was significantly lower than that in the diabetic control group rats(P= 0.004). After 4 weeks of drug intervention, fasting glucose in both Gin and sitagliptin group was significantly lower than that in the diabetic control group (P<0.001), but higher than that in the normal control group (P= 0.032 and 0.004, respectively). After 8 weeks of intervention, fasting blood glucose in both Gin and sitagliptin group was still significantly lower than that in the diabetic control group (all P<0.01), but were significantly higher than that in the normal control group (P<0.01). There was no statistically significant difference in fasting blood glucose between Gin and sitagliptin group at the time(P= 0.87). 4 weeks after the intervention, OGTT test was performed to calculate AUC. We found AUC values of insulin, C-peptide and GLP-1 in Gin group were significantly higher than those in the diabetic control group, and the difference was statistically significant (P= 0.048, P= 0.003 and P= 0.005, respectively). After 8 weeks of intervention, AUC values of insulin, C-peptide and GLP-1 in sitagliptin group were significantly higher than those in the diabetic control group (P= 0.033, P= 0.003 and P<0.001, respectively). After the intervention for 8 weeks, the AUC level of GIP in Gin group was significantly lower than that in the diabetic control group, and the difference was statistically significant (P= 0.026).Animal experiment 2:IGT rats:body weight of rats in each group within the initial 4 weeks of drug intervention increased gradually. After 4 weeks of intervention, body weight in Gin group was significantly lower than that in high-fat control(HC) group, and the difference was statistically significant (P= 0.04). Body weight of rats on sitagliptin was also lower than HC group, although the difference was not statistically significant (P= 0.102). After a total of eight weeks of intervention, rats on Gin weighed close to normal control with no significant difference (P= 0.74) between the two groups. Body weight of rats in Gin group was significantly lower than that in the HC group (P= 0.001) while the body weight of rats on sitagliptin was significantly higher than that of normal control group (P= 0.023), but slightly lower than the HC rats and the difference was not statistically significant (P= 0.062). After 4 weeks of drug intervention, blood glucose of rats with Gin treatment was lower than that of HC group and the difference was statistically significant (P= 0.002). Rats in sitagliptin group and HC group showed no significant difference in fasting glucose (P= 0.197). Fasting blood glucose in HC group was significantly higher than that in the normal control group (P= 0.011). After 8 weeks of intervention, Gin group had significantly lower fasting blood glucose than HC rats and the difference was statistically significant (P= 0.001).After 4 weeks of intervention, OGTT test was performed and we found AUC values of insulin and GLP-1 in Gin group tended to be higher than those of HC group (P= 0.06, P = 0.067, respectively). At that time the AUC value of GLP-1 in sitagliptin group was significantly higher than that in the HC group and the difference was statistically significant (P= 0.003). After 8 weeks of intervention, the AUC value of insulin in sitagliptin group was significantly higher than HC group (P= 0.013). At this point the AUC level of C-peptide in Gin group tended to be higher than HC group (P= 0.07), whereas AUC values of C-peptide and GLP-1 in sitagliptin group were significantly higher than those in the HC group (P= 0.009 and P= 0.034).Mechanism study:Both Gin and sitagliptin could ameliorate the expression of TNF-a, 3-NT, TGF-pl, CTGF and reduced the apoptosis of Pcells. Gin could more potently induce the expression of Nrf-2 than sitagliptin, and it could suppress PTEN and activate its downstream PI3K/Akt pathway, whereas sitagliptin had no significant effect on P13K/Akt pathway.[Conclusion] Our research shows that Gin could modulate plasma GLP-1 and GIP levels thereby regulate the glucose metabolism. Gln exerts hypoglycemic effect in not only IGT, diabetes with short duration, and also moderate to severe T2DM. The effect of the drug is similar to that of sitagliptin. Animal experiments further demonstrated hypoglycemic effect of Gln is not only due to its possible role in promoting the secretion of GLP-1 as well as pancreatic ? cell protection, but also related to its antioxidant, anti-inflammatory and anti-fibrotic activities. The mechanism regarding its protective effect on ? cells involves the possible suppressive effect on PTEN and activation of Nrf2. |
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