Functional Fiber Increases Insulin Sensitivity By Modulating Intestinal Microbiota

With the improvement in living standards,metabolic syndrome characterized by obesity,hypertension,hyperlipidemia and hyperglycemia has become a major challenge affecting human health.Insulin resistance is an important pathophysiological basis that causes metabolic disorders and then metabolic syndrome.In recent years,studies have revealed that compared with normal people,the intestinal microbiota of pregnant and obese individuals has changed significantly,which promotes the occurrence of chronic low-degree inflammation,oxidative stress and insulin resistance,but the specific mechanism has not been fully clarified.Dietary fiber is an important regulator of intestinal microbes,which is beneficial to the stability of intestinal microecology.A new type of functional fiber?FF?is created previously by our research group and plays an important role in improving the intestinal microbiota composition and reproductive performance of sows.However,it has not been directly measured the effect of FF on insulin sensitivity of perinatal sows by glucose tolerance test?GTT?and meal test,as well as the link between insulin sensitivity and reproductive performance of sows.In addition,studies have confirmed that intermittent fasting can effectively improve obesity and related metabolic disorders,increase insulin sensitivity,but whether intermittent fasting combined with dietary fiber can synergistically improve the insulin sensitivity of obese individuals by regulating intestinal microbiota,is also worthy of further study.In this study,pregnant sows were used to study whether FF could affect host metabolism and improve insulin sensitivity so as to improve reproductive performance by regulating intestinal microbiota;obese mice were used as animal model to study the effect of functional fiber on intestinal microbiota,insulin sensitivity and weight;to explore whether FF combined with intermittent fasting can synergistically regulate the metabolism of obese mice.The main research contents and results are as follows:Part ? The effect of functional fiber on the reproductive performance and perinatal insulin sensitivity of sowsIn this study,the effects of FF on reproductive performance,process oxidative stress,perinatal insulin sensitivity and intestinal flora of sows were studied.99 sows were randomly divided into three groups and fed one of gestation diets supplemented with 0%?control group,CON?,1%?test group 1,T1?and 2%?test group 2,T2?FF.The feeding amount of sows in each group was the same during pregnancy.The litter performance of sows and growth performance of piglets were measured.Meal test was carried out on the109^th day of gestation and the 3^rd day of lactation,GTT was carried out on the 110^th and4^th day of gestation.Blood plasmas were collected on the 30^th,60^th,90^th,109^th day of gestation and the 3^rd,7^th and 21^th day of lactation to detect the oxidative stress indexes;fecal samples were collected on the 30^th,60^th,109^th day of gestation and the 3^rd day of lactation to detect fecal specific bacterial amounts.The main results are as follows:1. Due to the same feed intake of sows in each group during gestation,there were no significant differences in body weight and back-fat thickness among the three groups during the entire experiment?P>0.05?.There were no significant differences in total litter size,live litter size and litter weight at birth among the three groups?P>0.05?,but the piglet birth weight and rate of weak piglets?<800 g,%?in T1 and T2 significantly increased and decreased,respectively,compared to CON?P<0.01?.With the same litter size after cross-foster,T1,not T2,significantly increased the average daily gain of piglets during the whole lactation,as well as weights of 7-and 21-day old piglet compared to CON?P<0.05?.These results indicate that dietary supplementation of FF during gestation improves the performance of piglets at birth and weaning.2.Compared to CON,T2 significantly reduced the blood glucose levels before meal and 15-240 min after meal on G109d?P<0.05?,and reduced the glucose AUC in meal test on G109 d and L3 d?P<0.01?;T2 significantly decreased blood glucose levels of20-50 min after glucose infusion and glucose AUC in GTT on G110 d?P<0.05?.This indicates that 2%FF improves insulin sensitivity of perinatal sows.3.Compared to CON,T1 and T2 significantly decreased the levels of malondialdehyde on the day of delivery and 8-hydroxy-deoxyguanosine at day 109 of gestation?P<0.05?,and increased the levels of total superoxide dismutase on the day 7 of lactation?P<0.01?;in addition,T1 significantly decreased the level of reactive oxygen species in umbilical cord blood on the day of delivery?P<0.01?.These results indicate that dietary supplementation of FF during gestation relieves oxidative stress of perinatal sows.4.Compared to CON,the fecal amount of Clostridium perfringens in sows on day109 of gestation significantly decreased in T1 and T2?P<0.01?,the amount of Escherichia coli on days of 60 and 109 of gestation was significantly decreased in T1?P<0.05?.Compared to CON and T1,the amount of Lactobacillus on day 109 of gestation was the highest in T2?P<0.05?.These results indicate that dietary supplementation of FF during gestation regulates the composition of intestinal microbes in the middle and later gestation,increases the amount of beneficial microbes and reduces the amount of harmful microbes.Part ? The effects of functional fiber on the weight and insulin sensitivity of high-fat diet-induced obese miceThis experiment investigated the effects of dietary supplementation with functional fiber on body weight,glucose and lipid metabolism,systemic inflammation,intestinal barrier,and intestinal microbial composition of obese mice.110 C57BL/6 mice?4-5 week old,male?were fed with a high-fat diet?HFD?for 10 weeks,and 42 obese mice were obtained.According to the principle of similar average body weight and standard deviation,21 out of 42 obese mice were selected and randomly divided into 3 groups:Negative control group?NC?,test group 1?T1?and test group 1?T2?fed with high-fat diet supplemented with 0%,4%and 6%.7 normal mice fed with low-fat diet?LFD?were considered as positive control group?PC?.All obese mice received HFD for 12 cycles?6days/cycle??P1 phase?and then switched to regular diets for 4 cycles?P2 phase?;mice from PC group were treated with LFD and normal chow diet,respectively,in two stages.The weight of each mouse was recorded every cycle,and feed intake was recorded daily.In the 2^ndcycle of P2 phase,serum was collected 2 hours after a meal to analyze the levels of satiety hormones peptide YY?PYY?and glucagon-like peptide 1?GLP-1?.In the 12^thcycle of P1 phase and the 4^thcycle of P2 phase,fasting serum was collected to analyze lipid profile?triglyceride?TG?,nonesterified fatty acid?NEFA?,low-density lipoprotein-cholesterol?LDL-C?,high-density lipoprotein-cholesterol?HDL-C??,and to analyze the levels of inflammatory factors at P2 phase.Two days after blood collection,GTT and insulin tolerance test?ITT?were performed.Fecal samples were collected at the end of P1 and P2 phases for analyzing microbiota composition by 16S rRNA sequencing technology,and determining levels of short-chain fatty acids?SCFAs?.At the end of the study,mice were sacrificed.H&E staining was used to observe the morphological changes of colon and epididymal white adipose tissue.Brd U staining was used to observe the proliferation of colonic epithelial cells.Oil red O staining was used to measured liver fat content.RT-PCR was used to measured the expression of colon tight junction protein m RNA.The main results are as follows:1.At P1 phase,the energy intake and body weight of the mice in the T1 and T2 were not significantly different from that of NC?P>0.05?,which was significantly higher than that of PC?P<0.01?.At P2 phase,the energy intake and body weight of T1 and T2 were significantly lower than that of NC,and significantly higher than that of PC?P<0.01?;The levels of PYY and GLP-1 in the medium were significantly higher than those in NC?P<0.01?.At the end of the test,the lee's index,white subcutaneous and visceral white fat pad weight,epididymal white fat cell area,liver fat content and other obesity-related indicators of the mice in groups 1 and 2 were significantly lower than those in NC?P<0.05?,while No significant difference from PC?P>0.05?.The above results show that the addition of functional fiber to the ordinary diet increases the satiety of obese mice and reduces energy intake,thereby effectively achieving weight loss;while the addition of functional fiber to the high-fat diet cannot achieve the above effect.2. At P1 phase,there were no significant differences in TG,NEFA,LDL-C,HDL-C among all groups?P>0.05?.At P2 phase,the LDL-C concentration in t was significantly lower than that in NC?P<0.05?,and there was no significant difference from PC?P>0.05?;while the HDL-C concentration was significantly higher than PC Group and negative control group?P<0.01?.At P1 phase,the ITT glucose AUC of the experimental group 2 was significantly lower than that of NC?P<0.05?,and there was no significant difference from PC?P>0.05?;the experimental group 1 was not significantly different from PC and NC?P>0.05?.At P2 phase,the fasting 6-hour blood glucose levels and ITT glucose AUC in the experimental groups 1 and 2 were significantly lower than those in the positive and negative control groups?P<0.05?.The above results indicate that 6%functional fibers can increase insulin sensitivity in obese mice at P1 and P2 phases,and promote cholesterol metabolism at P2 phase.3. The thickness of colonic mucosa in T1 and T2 was significantly higher than that in the NC?P<0.01?,but was similar to PC?P>0.05?;the depth of the crypts in T2 was significantly higher than that in PC and NC?P<0.05?.The m RNA expression of Claudin-2 gene?pore formation protein?in colon in test group 1 and 2 was significantly lower than that in NC?P<0.05?,but no significant difference from PC?P>0.05?;The m RNA expression level of ZO-1 gene?tight junction protein??P<0.05?and the number of Brd U positive staining cells of colon?P<0.05?in T2 were significantly higher than that of the NC,but no significant difference from PC?P>0.05?.The detection of systemic inflammation index found that the serum adiponectin concentrations in T1 and T2 were significantly higher than PC and NC?P<0.01?,and the leptin concentration was significantly lower than NC?P<0.01?,but no significant difference from PC?P>0.05?;the concentrations of tumor necrosis factor-?,interleukin-6 and lipopolysaccharide in the serum of T2 were significantly lower than that of NC?P<0.01?,extremely significant higher than PC?P<0.01?.The above results indicate that 6%FF improve the intestinal barrier function of obese mice and then relieve systemic inflammation.4.The results of fecal SCFAs showed that at P1 phase,the content of butyrate,isobutyrate,and isovalerate in T1?P<0.05?and propionate,butyrate,valerate,isobutyrate,The content of isovalerate was significantly lower than that of NC?P<0.05?,and no significant difference from PC?P>0.05?.At P2 phase,there was no significant difference in fecal SCFAs between T1,PC and NC?P>0.05?.The contents of acetate,propionate and total SCFAs in T2 was significantly higher than that in PC and NC?P<0.05?.The above results indicate that 6%FF regulates the metabolic activity of fecal microbiota in obese mice,which affects the production of SCFAs and increases the production of acetate and propionate during the normal diet feeding phase.5.16S rRNA sequencing showed that in P1 and P2 phases,the total number and?diversity of fecal microbitoa in NC were not significantly different from those in PC?P>0.05?,but Firmicutes/Bacteroidetes?F/B?ratio in NC is significantly higher than PC?P<0.05?,SCFA-producing bacteria such as Ruminococcus,Dorea and Oscillospira were significantly higher than PC?P<0.05?,the symbiotic beneficial bacteria Allobaculum and S24-7 are significantly lower than PC?P<0.05?.At P1 phase,the total number and?diversity of fecal microbiota in T2 were not significantly different from NC?P>0.05?,and the F/B ratio was significantly lower than NC?P<0.05?;at P2 phase,the total number of microbiota in T2 was significantly higher than that in NC?P<0.05?.SCTAs-producing bacteria such as Allobaculum and S24-7 were significantly higher than NC?P<0.05?.potentially harmful bacteria such as Dehalobacterium,Strptococcus,Desulfovibrio were significantly lower than NC?P<0.05?.The above results indicate that 6%FF change the facal microbiota composition,reduce F/B ratio,increase the relative abundance of beneficial bacteria and reduce the relative abundance of harmful bacteria.6.Spearman correlation analysis showed that decreased abundance genera Roseburia,Ruminococcus,Coprococcus,Oscillospira,Dehalobacterium,Streptococcus in T2 were positively correlated with weight,body weight gain and insulin resistance(fasting 6h blood glucose and AUC of glucose in ITT?P<0.05?.The increased abundance genera Allobaculum and S24-7 were negatively correlated with body weight and insulin resistance?P<0.05?.Part ? The effect of functional fiber on microbiota composition of different intestinal segments about high fat diet induced obese miceAt the end experiments,we collected the contents of duodenum,jejunum and ileum and fecal samples of the mice in part II experiment for 16S rRNA sequence to analysis microbiota composition.Results were as follows:1 The microbiota composition of lower gut?cecum and colon?and fecal microbiota of all mice showed decreased phylum and genus and increased observed OTUs and Chao1index than upper gut?P<0.01?,while?diversity was significant different between upper and lower gut.Firmicutes and Proteobacteria were the most abundant in ileum and cecum,respectively,and Bacteroides showed lowest abundance in cecum?P<0.01?.2 Principal coordinate analysis of microbiota composition between different intestinal segments in PC showed remarkable distinction among ileum,cecum,colon and fecal?P<0.01?,and the difference of microbiota composition between individual was greatest in ileum and was most similar in colon.Analysis of different bacteria in adjacent intestinal segments indicated that Ruminococcus and Oscillospira which are carbohydrate metabolic bacteria in cecum were more abundant than that in ileum,while compared to ileum the abundance of Peptoniphilus and Leuconostoc which are protein which are carbohydrate metabolic bacteria were lower in cecum.On the other hand,Bacteroides and Parabacteroides which are carbohydrate metabolic bacteria in colon were more abundant than that in cecum.Compared to colon,the abundance of facultative aerobes such as Salinicoccus?Bacillus?Yaniella were higher in fecal and obligate anaerobes such as Dehalobacterium was obviously lower?P<0.05?.3 Principal coordinate analysis of microbiota composition between different intestinal segments in NC showed significant difference between upper and lower intestinal?P<0.05?.Analysis of different bacteria in adjacent intestinal segments indicated that the abundance of some beneficial microbiota,Parabacteroides,actobacillus and Faecalibacterium,in ileum were lower than that in duodenum,and Staphylococcus,Streptococcus,Ochrobactrum and Serratia,which are harmful microbe,were less abundant in cecum than in ileum?P<0.05?.?diversity showed no difference in both control groups?P>0.05?,and?diversity was obviously different in duodenum,ileum,colon and fecal?P<0.05?,meanwhile Proteobacteria was more abundant in fecal compared to PC?P<0.05?.These results indicated that high fat diet changed the microbiota composition of whole intestinal segments beginning with duodenum.4 Principal coordinate analysis of microbiota composition between different intestinal segments in T2 showed remarkable difference between upper and lower intestinal segments?P<0.05?Analysis of different bacteria in adjacent intestinal segments indicated that Mucispirillum,Prevotella,Bacteroides and Allobaculum related to carbohydrate metabolism were abundant in cecum than in ileum?P<0.05?,and Coprococcus,Oscillospira and Ruminococcus belong to Phylum Firmicutes related to carbohydrate metabolism and harmful microbe Desulfovibrio were less abundant in colon than that in cecum?P<0.05?.?diversity showed no difference?P>0.05?,and?diversity was obviously distinguished in cecum,colon and fecal microbiota between Exp2 T2 and NC?P<0.05?;meanwhile compared to NC,Firmicutes was less abundant in colon and fecal?P<0.05?,and Actinobacteria was more abundant in fecal?P<0.05?.These results indicated that 6%FF changed the microbiota composition of lower intestinal beginning with cecum selectively enriching fiber degrading bacteria.Part ?Effect of functional fiber combined with intermittent fasting on insulin sensitivity of obese mice induced by high-fat diet21 out of 42 mice obtained in part?were randomly assigned in 3 groups:every other day fasting?EODF?group?control group 2,CON2?,EODF plus 4%FF supplementation group?test group 1,T1?and EODF plus 6%FF supplementation group?test group 2,T2?.Taken the negative control group in the test of part II as the ad libitum group?control group 1,CON1?.All mice received high-fat diets for 12 cycles?6days/cycle??P1 phase?and then switched to regular diets for 4 cycles?P2 phase?for exploring the synergistic effects of EODF and FF supplementation on body weight,body fat,glycolipid metabolism,intestinal barrier,facal SCFAs and microbiota composition in obesity mice.The main results are as follows:1.Energy intake and body weight of mice at P1 and P2 stages,as well as obesity-related indicators at the end of test including the lee's index,weights of subcutaneous and visceral white fat pads,area of epididymal white adiposcyte and liver fat content,were significantly lower than CON1?P<0.05?.At P1 stage,the energy intake and body weight of T1 and T2 were not significantly different from those in CON2?P>0.05?;at P2 stage,the energy intake and body weight of T2 was significantly lower than CON2?P<0.01?.In P2 phase,serum concentration of PYY in T2 was significantly higher than that in CON2?P<0.05?,and the feed intake on the day of eating 1-2 h and 0-24 h in T2 was significantly lower than that in CON2?P<0.05?.The above results show that EODF significantly reduces the energy intake and obesity of obese mice;6%FF supplementation to the ordinary diet,but not HFD,can enhance the satiety of EODF mice,and further reduce energy intake,as well as body weight.2.At P1 phase,the serum TG and LDL-C concentrations in T1 and T2 were significantly lower than those in the CON1?P<0.01?,and there was no significant difference from CON2?P>0.05?;at P2 phase,the serum LDL-C concentration in T1 and T2 was significantly lower than that in the CON1?P<0.01?,and there was no significant difference from the control group 2?P>0.05?.At P1 phase,the ITT glucose AUC of T1was significantly lower than that of CON 1?P<0.01?,and there was no significant difference from CON2?P>0.05?,T2 was extremely significantly lower than CON1 and CON2?P<0.01?;At P2 phase,the ITT glucose AUC of T1 and T2 was significantly lower than that of CON1 and CON2?P<0.01?.The above results indicate that 6%FF addition enhances insulin sensitivity in EODF mice.3.The results of colon tissue morphology showed that there were no significant differences in mucosal layer thickness,crypt depth,and m RNA expression of tight junction protein gene of colon tissue?P>0.05?.The number of Brd U positive staining cells in CON2,T1 and T2 was significantly higher than that in CON1?P<0.01?,and T2was significantly higher than that in CON2?P<0.01?,indicating that EODF promoted the renewal of colonic epithelial cells,6%FF supplementation reinforces this effect.4. The measurement results of fecal SCFAs showed that in P1 phase,the concentrations of acetate,propionate,butyrate,valerate and isovalerate and total SCFAs in CON2 were significantly lower than those in CON1?P<0.01?.There was no significant difference in the SCFA concentrations between T1 and T2 compared with CON2?P>0.05?;in P2 phase,there was no significant difference in the SCFA concentration between the CON2 and CON1?P>0.05?.The concentration of acetate in T1 and the concentrations of acetate,butyrate and total SCFAs in T2 were significantly higher than those in CON2?P<0.05?.The above results indicate that EODF significantly reduces the production of SCFAs in the intestine of obese mice at P1 phase;FF supplementation significantly increases the intestinal production of SCFAs at P2 phase.5. In P1 and P2 phases,compared with the control group 1,there was no significant difference in total amount of fecal microbes and?diversity in CON2?P>0.05?,but there was a significant difference in?diversity in CON2?P<0.05?.In P1 phase,the relative abundances of beneficial bacteria Lactobacillus and S24-7 in CON2 were significantly higher than those in CON1?P<0.05?;in P2 phase,the relative abundances of probiotic bacteria such as Lactobacillus and Paraprevotella in CON2 were significantly higher than CON1 in P2 phase?P<0.05?,and potentially harmful bacteria such as Staphylococcus,Helicobacter,and Desulfovibrio in CON2 were significantly lower than CON1?P<0.05?.In P1 and P2 phases,the total number of fecal microbes in T2 was not significantly different from that in CON2?P>0.05?,but the microbial alpha diversity in P2 phase was significantly higher than that in CON2?P<0.01?,and the F/B ratio in P2 phase was significantly lower than CON2?P<0.01?.The relative abundances of S24-7,Lactobacillus,and Bifidobacterium in T2 were significantly higher than those in the CON1?P<0.05?,and no significant difference from CON2?P>0.05?.Correlation analysis showed that S24-7,Lactobacillus and Bifidobacterium were significantly negatively correlated with obesity and insulin resistance of mice?P<0.05?.The above results indicates that FF combined with EODF may improve insulin sensitivity by synergistically promoting the enrichment of beneficial bacteria in intestine.In summary,the main conclusions of this study are as follows:?1?FF improves reproductive performance by improving insulin sensitivity of perinatal sows.FF can increase insulin sensitivity of obese mice under high-fat diet and ordinary diet,but has no weight loss under high-fat diet?2?FF selectively enrichs the fiber degrading bacteria in the intestinal tract beginning from caecum segment,improve intestinal barrier function,and then relieve LPS-driven systemic inflammation.?3?EODF combined with FF further reduces energy intake,reduces obesity,promotes the accumulation of intestinal beneficial bacteria Lactobacillus,Bifidobacterium,S24-7,and improves the insulin sensitivity of mice.