Syzygium aromaticum-derived triterpenes modulate intestinal glucose handling in streptozotocin-induced diabetic rats.
Polyphagia in diabetes mellitus ascribed to elevated plasma ghrelin concentrations is associated with prolonged postprandial hyperglycaemia due to increased activities of intestinal carbohydrate hydrolyzing enzymes and glucose transporters. Postprandial hyperglycaemia is a major risk factor in the development of diabetic complications, and as such, should be managed to prevent chronic vascular complications. Previous studies in our laboratory have shown that Syzygium aromaticum-derived oleanolic acid (OA) and maslinic acid (MA) use various mechanisms to lower blood glucose concentrations in experimental diabetes. The effects of these triterpenes, however, on intestinal glucose handling remain unknown. Accordingly, this study was designed to investigate the effects of these triterpenes on intestinal glucose handling in STZ-induced diabetic rats. Materials and methods OA and MA were extracted from Syzygium aromaticum cloves using a previously validated protocol. Briefly, S. aromaticum-derived OA and MA were sequentially extracted with dichloromethane and ethyl acetate to obtain ethyl acetate solubles which contained mixtures of OA/ursolic acid (UA) and methyl maslinate/methyl corosolate. These solubles were purified by silica gel 60 column chromatography with hexane: ethyl acetate solvent systems to produce OA and MA. The structures of these triterpenes were confirmed by using 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy and were comparable with those previously reported in literature. The in vitro studies investigated the inhibitory effects of OA and MA against enzymes such as α-amylase, α-glucosidase and sucrase. Additionally, the effects of various concentrations of OA and MA (0.82 - 6.56 mmol/L) on intestinal glucose transport were investigated using the everted intestinal sacs protocol. The in vivo studies investigated the effects of OA and MA on intestinal carbohydrate handling in separate groups of non-diabetic and STZdiabetic male Sprague Dawley rats. These studies were subdivided into oral glucose tolerance (OGT) responses which were carried out over two hours following loading with various carbohydrates as well as sub-chronic studies that were carried out over 5-weeks where the rats were kept on standard rat chow. OGT responses were monitored in separate groups of nondiabetic and STZ-induced diabetic animals the rats treated with OA and MA (80 mg/kg, p.o.). The rats were loaded with monosaccharides, disaccharides and polysaccharides after an 18-hour fast. The sub-chronic studies investigated the effects of the triterpenes on blood glucose concentrations over 5-weeks in groups of non-diabetic and STZ-induced diabetic male Sprague- Dawley rats. In those animals in which the effects of OA/MA were investigated, the rats were administered with OA/MA (80 mg/kg, p.o.) twice daily. Blood glucose, body weights as well as food and water intake were assessed every third day for the duration of the experimental period. At the end of the experimental period, the rats were killed and blood was collected for plasma insulin and ghrelin measurements. Furthermore, mid portions of the small intestine were snap frozen in liquid nitrogen and stored in a BioUltra freezer at -70 °C for Western blot analysis of glucose transporters, carbohydrate hydrolyzing enzymes and ghrelin expression. Additionally, the effects of OA and MA on intestinal oxidative stress were evaluated through malondealhyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) measurements.Results The in vitro studies revealed that OA and MA possess inhibitory effects on the activity of α- amylase, α-glucosidase and sucrase comparable with those of the standard drug acarbose. In addition, OA and MA significantly (p<0.05) inhibited intestinal glucose transport in the everted intestinal sacs in a dose-independent manner. The OGT studies showed that OA and MA had no significant effects on blood glucose concentrations in non-diabetic rats loaded with the various carbohydrates by comparison with the non-diabetic control. However, the triterpene-treated STZdiabetic rats loaded with the various carbohydrate showed significant (p<0.05) reductions in blood glucose concentrations by comparison with untreated STZ-diabetic rats. OA and MA progressively reduced blood glucose concentration as well as food and water intake over the 5- week study in STZ-induced diabetic rats by comparison with untreated STZ-diabetic rats. Treatment with OA and MA had no effects on plasma insulin concentration in STZ-induced diabetic rats. However, these triterpenes significantly (p<0.05) reduced plasma ghrelin concentrations by comparison with untreated STZ-induced diabetic rats. Furthermore, rats treated with OA and MA showed significant (p<0.05) decreases in ghrelin, SGLT1, GLUT2, α- amylase and α-glucosidase expression in the gastrointestinal tract by comparison with untreated STZ-diabetic rats. This was accompanied by improvements in their intestinal antioxidant status as there were significant (p<0.05) reductions in MDA concentrations with significant (p<0.05) increases in SOD and GPx by comparison with the STZ-diabetic control. Additionally, OA and MA-treated rats showed significant (p<0.05) increases in intestinal glycogen concentrations with concomitant significant (p<0.05) increases in the intestinal expression of glycogen synthase by comparison with untreated STZ-diabetic animals. Discussion The results of the present study indicate that the blood glucose lowering effects of OA and MA in STZ -induced diabetic rats are mediated, in part, via modulating postprandial hyperglycaemia. These findings suggest that this is achieved through the ghrelin-mediated reduction in food intake leading to decreased expression of intestinal carbohydrate hydrolyzing enzymes as well as intestinal glucose transporters. This was followed by significant improvements in the antioxidant status in the rats suggesting that these triterpenes could, by preventing chronic postprandial hyperglycaemia, prevent the onset of the development of diabetic complications. The results of this study are of considerable importance as they suggest another mechanism for the anti-diabetic properties of the triterpenes and further explain the role of the gastrointestinal tract in the management of diabetes mellitus. Conclusion The results of the present study suggest that the S. aromaticum-derived triterpenes possess antidiabetic properties that arise, in part, through the modulation of intestinal glucose handling.