Glycosidic Bond Rearrangement In Starch Molecules And Effects Of Its Product On Glucose And Lipid Metabolism Of Mice

Starch is an important component in human diet and the dominant source of calories for body activity.Reducing starch digestibility is beneficial to glucose homeostasis and human health,and therefore it has become a research focus of carbohydrate nutrition and related fields in recent years.The 1,4-α-glucan branching enzyme(GBE,EC 2.4.1.18)can catalyze the hydrolysis of α-1,4 glycosidic bonds in starch molecules to produce linear short chains,which can be connected to the receptor chains via α-1,6 glycosidic bonds.This process rebuilds starch molecules and hardly introduces new chemical groups or other types of glycosidic bonds.This approach to modifying starch yields more products and fewer byproducts and has received extensive attention.Unfortunately,no studies have directly demonstrated that these products deriving from GBE-catalyzed glycosidic bond rearrangement show health benefits.The mechanisms for their effects on glucose and lipid metabolism of body are still unclear.In the present study,the GBE from Geobacillus thermoglucosidans STB02(Gt-GBE)was employed to rebuild normal corn starch(NCS)molecules.The digestion property of the modified product was investigated using both in vitro and in vivo methods.Additionally,the effects of enzymatically rebuilding starch molecules in the daily diet on glucose and lipid metabolism of body and related mechanisms were explored using type 2 diabetic mice.The results may reveal a novel direction and effective strategy for type 2 diabetes management.The main results of the present study are as follows.Firstly,Gt-GBE was employed to rebuild NCS molecules.The fine structure of the modified product was comprehensively characterized.Proton nuclear magnetic resonance spectroscopic analysis showed that the ratio of α-1,6 glycosidic bonds in the modified product increased to 7.51%,which was 135.4% higher than that of native NCS.Additionally,the product did not contain other types of glycosidic bonds,except for α-1,4 and α-1,6 glycosidic bonds.High-performance size-exclusion chromatography further revealed that NCS molecules were slightly hydrolyzed during the GBE-catalyzed glycosidic bond rearrangement.The modified product exhibited a detectable reducing level(dextrose equivalent value = 2.08),which is the typical characteristic of maltodextrin.Moreover,the branching pattern analysis demonstrated that the modified product contained more abundant short branches polymerized by 2～8 glucose units and smaller proportion of external chains,as compared with NCS.A thorough β-amylolysis proved that the modified product displayed a dense internal framework,which is rich in short internal chains polymerized by 3～6 glucose units.The average external and internal chain length were 14.8% and 16.6% lower,respectively,than those of NCS.As such,the Gt-GBE-modified product showed a short-clustered molecular structure and was named as short-clustered maltodextrin(SCMD).Secondly,the differences in digestive property between SCMD and DE 2 maltodextrin(MD)were compared using both in vitro and in vivo methods.These differences were expected to reflect the roles of GBE-catalyzed glycosidic bond rearrangement,rather than hydrolysis,in reducing starch digestibility and improve postprandial glucose homeostasis,as both SCMD and MD showed similar molecular weight.The Englyst assay revealed that slight hydrolysis hardly affected in vitro digestibility of NCS.By comparison,the rapidly digestible fraction of SCMD was 20.7% lower than that of NCS,while the slowly digestible fraction was 1.58-fold greater.Moreover,as compared with MD,SCMD could block the binding and continuous attack of pancreatic α-amylase on inner chain,due to its dense and highly-branched internal framework.This led to the survival of multi-branched α-limit dextrin with high degree of polymerization,which was difficult to bind with mucosal α-glucosidase.As a result,SCMD was slowly digested and absorbed in small intestinal brush-border as simulated by Caco-2 cell monolayer.Significant decreases in postprandial glycemic and insulinemic responses were therefore detected in ICR mice following SCMD intake,compared with those in the control(MD).This indicated that SCMD required less insulin to regulate postprandial glucose homeostasis.Additionally,SCMD stimulated the sustained release of glucagon-like peptide-1(GLP-1)and peptide tyrosine-tyrosine(PYY)in ICR mice.Four hours after administration,the plasma GLP-1 and PYY levels of SCMD-fed mice were 27.4% and 33.8% higher,respectively,than those of MD-fed mice.The released GLP-1 and PYY were thought to weaken the glycemic response to the next meal.In response to an exactly same second meal as control group(MD),mice that received an initial meal of SCMD had a 28.4% lower glycemic peak when compared with mice that received an initial meal of MD.Thirdly,with SCMD as principal dietary carbohydrate,the SCMD-containing diet was observed to cause no adverse effects on the feeding behavior and physiological status of normal C57BL/6J mice.Basing on this premise,effects of SCMD-containing diet on glucose and lipid metabolism,hepatic and renal function,and gut health of spontaneous type 2 diabetic db/db mice were investigated to explore the potential health benefits of SCMD.The results demonstrated that SCMD-containing diet,as compared to a diet with normal starch,greatly attenuated hyperglycemia,repaired insulin resistance and pancreatic islet function,and ameliorated glucose homeostasis of db/db mice.Moreover,SCMD-containing diet effectively relieved blood dyslipidemia,systemic inflammation and hepatic steatosis,and improved liver function of db/db mice.SCMD also served as a preferred dietary energy source,which enhanced energy expenditure and accelerated brown adipose activation of db/db mice.Further analysis revealed that SCMD-containing diet relieved occurrence and development of diabetic nephropathy.The renal injury,fibrosis and glomerular function were significantly improved.Additionally,SCMD-containing diet greatly ameliorated gut health of db/db mice.Especially,the butyrate level of SCMD-fed mice was over 26-fold greater than that of control group,and the relative abundance of Akkermansia(a genus regarded as beneficial to host health.)was approximately 232-fold greater.Fourthly,with a view to the molecular mechanism for SCMD’s effects on blood glucose homeostasis of db/db mice,MD and resistant dextrin(RD)were blended in a certain proportion to prepare a mixture(MD+RD),which contained a similar rapidly digestible fraction to that of SCMD.The effects of carbohydrate digestion and absorption in the distal small intestine on glucose homeostasis were analyzed using mice model.MD+RD-containing diet hardly improved the glucose homeostasis of db/db mice.After a 10-week intervention,the average fasting blood glucose level elevated steeply to 21.4 mmol/L.This might result from the limited contribution of MD+RD to inducing GLP-1 secretion from the ileum,as the fractions of MD+RD reaching the distal small intestine were difficult to be digested.By comparison,the fractions of SCMD reaching the distal small intestine,althouth they are similar to the those of MD+RD in quantity,could be further digested and absorbed.Thus,SCMD greatly triggered GLP-1 secretion from the ileum.The released GLP-1 effectively repaired pancreatic β-cell fuction and islets morphology.Further analysis revealed that the SCMD-induced GLP-1relieved insulin resistance,which promoted the insulin-mediated hepatic glucose uptake and glycogenesis of db/db mice.The glucose metabolism disorder of db/db mice was therefore repaired.The average fasting blood glucose level reduced to 9.3 mmol/L.Based on these results,it can be speculated that effects of SCMD on glucose homeostasis depended on GLP-1.Finally,in order to support the crucial role of dietary SCMD intake,the effects of SCMDcontaining diet and low-carbohydrate ketogenic diet on the lipid metabolic metabolism dysregulation and hepatic function disorder in type 2 diabetic db/db mice were investigated.The results demonstrated that SCMD-containing diet effectively relieved blood dyslipidemia and hepatic steatosis of db/db mice.Additionally,it greatly promoted hepatic glycogenesis via ameliorating hepatic insulin resistance.In contrast,the hepatic glycogenolysis and gluconeogenesis were decelerated.Further analysis revealed that the improving effects on hepatic metabolism depended on GLP-1 induced by SCMD and was independent of leptin signal.By comparison,ketogenic diet effectively promoted leptin release,while hardly impacted GLP-1 secretion.Unfortunately,such leptin cannot paly any biological roles in db/db mice with a knockout in the leptin receptor gene.On the contrary,high leptin levels accelerated hepatic glycogenolysis and gluconeogenesis and blocked the tricarboxylic acid cycle.Therefore,excess acetyl-Co A,which originated from dietary fat in ketogenic diet,was converted to ketone bodies.This process aggravated metabolic dysregulation in the liver and even induced severe hypercholesterolemia,hyperglycemia,and ketoacidosis of db/db mice.