| Objective:In this paper,A-type crystallized resistant starch(ARS)and B-type crystallized resistant starch(BRS)prepared by enzymatic debranching method in combination with recrystallized regulation technique were used as research objects.Type 2 diabetes mellitus(T2DM)mouse model was established by high-fat and high-sugar diet combined with streptozotocin(STZ).To study the effect of recrystallized RS3 in regulating abnormal blood glucose and its effect on intestinal flora structure.In addition,the regulation mechanism of glycolipid metabolism in T2 DM mice was further studied.Methods:118 male C57BL/6J mice(5-6 weeks old,20±2 g body weight,SPF)were adaptively fed for one week.A randomized group was selected as a blank control(n = 13)based on body weight and fasting glucose,while the remaining mice were treated with a high-fat,high-sugar diet combined with intraperitoneal injection of STZ to establish a T2 DM model.The criteria for determining the success of T2 DM modeling were: mice with fasting glucose >11.1 mmol/L or random glucose >16.7 mmol/L with polyuria were identified as T2 DM mice.By the end of modeling and excluding very low weight and dead mice,the remaining mice were randomly divided into 7 groups: model group(MOD),positive control group(MET),low-dose A-type crystallized resistant starch group(LARS),medium-dose A-type crystallized resistant starch group(MARS),high-dose A-type crystallized resistant starch group(HARS),high-dose B-type crystallized resistant starch group(BRS),and combined A-type and B-type crystallized resistant starch intervention group(ABRS).D12450 H standard diet was given to the CON group.The remaining groups were given D12451 high-fat diet.The mice were gavaged orally for 10 weeks.The blood was centrifuged at 4 °C for 15 min at 4000 ×g and then frozen,while the rest of the tissue was stored after rapid liquid nitrogen snap-freezing at-80 °C.Recrystallized structure and morphological characteristics of ARS and BRS were characterized by X-ray diffractometer and scanning electron microscope;the fasting blood glucose(FBG)and random blood glucose(RBG)of mice was measured by glucometer;the lipid levels were measured by triglyceride(TG),total cholesterol(TC),low-density lipoprotein(LDL-C)and high-density lipoprotein(HDL-C)kits.Elisa kits such as tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)were used to detect the levels of inflammatory factors.Serum hormone levels were measured using Elisa kits for glucagon-like peptide-1(GLP-1),glucagon(GC),and Peptide YY(PYY).Serum insulin levels were measured using fasting insulin(FINS)kit.16 S r RNA was used to detect the composition of the intestinal microbiota in the cecal contents;the composition of shortchain fatty acids(SCFA)in the cecum contents was measured by gas chromatography.Liquid chromatography-mass spectrometry(LC/MS)was used to detect non-targeted metabolomic analysis of the liver.RNA-Seq sequencing was used to analyze the expression of colon-related differential genes.Results:Structural characterization results show that ARS has high crystallinity and relatively dense particles;BRS has lower crystallinity than ARS and microporosity exists between particles.The results of blood glucose and FINS showed that compared with the MOD group,the FBG and FINS of mice in group LARS,MARS and HARS were significantly lower,with FBG reduced by 30.7%,43.1% and 37.0%,respectively(P<0.01),and FINS reduced by 19.15%,20.85% and 18.73%,respectively(P<0.05).Compared with the group MOD,homeostasis model assessment-insulin resistance index(HOMA-IR),homeostasis model assessment-insulin sensitivity(HOMA-IS),and homeostasis model assessment-β-cell function index(HOMA-β)were significantly changed in mice with RS3 intervention.In particular,in the MARS group,HOMA-β and HOMA-IS levels were elevated by 80.48%(P<0.05)and 42.86%(P<0.05),TC and LDL-C decreased by 18.55%(P<0.01)and 35.11%(P<0.05),GLP-1 and PYY increased by 16.33% and 40.26%,respectively,and GC decreased by 14.97%(P<0.01).Compared with the MOD group,the serum glucose levels in the RS3 intervention group decreased significantly,with reductions of 42.08% and 30.34%in the MET and HARS groups(P<0.01),respectively.In addition,with the extension of the intervention time,the body weight of mice in the LARS,MARS and HARS groups gradually increased and the liver lipid droplet area was significantly smaller than that of the MOD group.Analysis of the cecum contents flora showed that MARS and HARS more significantly increased the composition of beneficial intestinal bacteria,increased the relative abundance of Desulfobacterota,Bacteroidetes,Lactobacillus,Lachnoclostridium and Desulfovibrio,suppressed the relative abundance of the thick-walled phylum and decreased the ratio of thick-walled to Bacteroidetes abundance(F/B)compared to the LARS group.SCFA results showed a significant increase in acetic acid content in all intervention groups compared to the MOD group(P<0.05),propionic acid in the LARS,HARS and ABRS groups(P<0.05)and butyric acid in the MARS and BRS groups(P<0.05).The Analysis of the cecum contents flora showed that MARS and HARS more significantly increased the composition of beneficial intestinal bacteria,increased the relative abundance of Desulfobacterota,Bacteroidetes,Lactobacillus spp.,Lachnoclostridium spp.and Desulfovibrio spp.,suppressed the relative abundance of the thick-walled phylum and decreased the ratio of thick-walled to Bacteroidetes abundance(F/B)compared to the LARS group.SCFA results showed a significant increase in acetic acid content in all intervention groups compared to the MOD group(P<0.05),propionic acid in the LARS,HARS and ABRS groups(P<0.05)and butyric acid in the MARS and BRS groups(P<0.05).The hepatic metabolomic results showed that MARS and HARS significantly increased the content of metabolites such as methionine,L-cystine and glutathione and regulated metabolites related to branched-chain amino acids,bile acid metabolism and vitamin digestion and absorption in mice,mainly involving the diabetic-induced amino acid metabolic pathways(lysine metabolism,glutamate metabolism,arginine and proline metabolism),and ether lipid metabolic pathways.Transcriptomic analysis results showed that the relative expression levels of FPKM of Tnf-α(Tumor necrosis factor-α)and Socs1(Suppressor of cytokine signalling-1)were significantly decreased(P<0.05)in the MARS group compared with the MOD group,and the relative expression levels of FPKM of Irs3(Insulin receptor substrate-3)and Lipe(Lipase hormone-sensitive)had significantly higher relative FPKM expression levels(P<0.05).PI3K-Akt,NF-κB,AMPK,type 2 diabetes and insulin resistance pathway inhibition status was improved after ARS intervention,presumably related to amino acid metabolism(taurine and hypotaurine metabolism),lipid metabolism(regulation of adipocyte lipolysis,fat digestion and absorption).Finally showed that ARS mainly improved blood glucose and insulin resistance in T2 DM mice through multiple pathways.Conclusions:After RS3 intervention,T2 DM mice had lower blood glucose and lipid levels,ARS significantly improved insulin resistance,and increased acetic acid,propionic acid and butyric acid levels.The relative abundance of beneficial bacteria in the MARS and HARS groups(such as Bacteroidetes,Lactobacillus spp.,Desulfovibrio spp.and Lachnoclostridium spp.,etc.).The type 2 diabetes and insulin resistance pathways were inhibited in the MOD group of mice,and RS3 improved pathway inhibition and reduced fat deposition,with MARS and HARS significantly modulated cysteine and methionine metabolism,glycerophospholipid metabolism,and phosphate and hypophosphate metabolic pathways.In addition,RS3 may improve insulin resistance and alleviate the disease state in T2 DM mice by inhibiting PI3K-Akt,NF-κB,AMPK,type 2 diabetes and insulin resistance pathways through the expression of Tnf-α,Socs1,Irs3 and Lipe genes. |
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