Long-time high-sucrose intake induced metabolic dysfunction via disrupting the balance of intestinal microenvironment

Long-time high-sucrose intake induced metabolic dysfunction via disrupting the balance of intestinal microenvironment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Long-time high-sucrose intake induced metabolic dysfunction via disrupting the balance of intestinal microenvironment Xiaojuan Wang, Zhipeng Li, Lili Zhang, Haiyu Guan, Dongyu Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3849756/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Sucrose, a common sweeter, is frequently added to drink and food, and excess intake of sucrose is really harmful to our health. This study aimed to reveal the potential mechanisms of high-sucrose induced metabolic dysfunction. Sixty mice were divided into two groups, respectively, the normal group (Nor group) and high-sucrose group (HS group). 16S rDNA and untargeted metabolomics technologies were used to analyzed the dynamic changes of gut microbiota and metabolites in colon contents of young mice, middle age mice and old mice. Long-time high-sucrose intake induced fat deposition and metabolic disorder, along with a completely different gut microbiota map. The energy metabolism related gene expression of the gut microbiome was downregulated, but potential pathogen gene expression was upregulated by long-time high-sucrose intake. Furthermore, mice in the HS were utilized less amino acids and long chain fatty acids, with effecting tryptophan metabolism by producing less indole, more 5-HT and kynurenine. Also less secondary bile acids, and more TMA/TMAO were produced by gut microbes in HS group. These results demonstrated that long-time high-sucrose intake leads to fat deposition and metabolic disorder via disrupting the balance of intestinal microenvironment. Biological sciences/Microbiology Health sciences/Endocrinology Health sciences/Endocrinology/Endocrine system and metabolic diseases high-sucrose metabolic dysfunction intestinal microenvironment gut microbiota intestinal metabolites Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction As an important component of the human diet, sugars were strongly loved by human beings, but have been shown to be harmfully associated with a variety of risk factors for a long time, mainly including obesity 1,2 , diabetes 3 , cardiovascular disease 4 , hyperuricaemia 5 , gout 6 , dental caries 7 , and some cancers 8 . A recent study reported that reducing the consumption of sugars to below 25 g/day is recommended to reduce the adverse effect 9 . However, the intake of sugar is immensely over the health standards for many people, with a cup of milk tea contained about 100g sugar and a bottle of coke contained about 54g sugar. Furthermore, the consumption of sugar sweetened beverages is still increase in the word. The Coca-Cola company revealed that the consumption of sugar-sweetened beverages increased by 17% in 2021 and 11.2% in 2022 10 . The annual sales of milk tea in China were only 800 million yuan in 2015, but increased to 66 billion yuan in 2020 9 . Sugar-sweetened beverages refer to the beverages with added sugar or sweeteners that have been prevalent worldwide, especially among younger people 4 . A cross sectional survey conducted among Chinese school students showed that sugar sweetened beverages provide 10–15% of the total calorie consumption of school students, with less than 10% of total daily energy intake provided by added sugar were recommended by World Health Organization (WHO) 11,12 . Overtake of sugars has a tightly relationship with obese, and excess sugars were converted to fat and accumulated in our body 9 . With the increase of consumption of sugars, the prevalence of obesity is rising, and more than one billion individuals worldwide who are currently obese 13 . In China, half of adults are overweight, comparing with a high prevalence of type 2 diabetic mellitus (T2DM), non-alcoholic fatty liver disease and cardiovascular disease 14 . Globally, although many governments have initiated actions to reduce the consumption of sugar in the last few years, however, no significant effects were produced 15 . The concepts of diets impacted the composition of gut microbes and gut microbes are critical component of digestion, breaking down complex carbohydrates, proteins, and to a lesser extent fats that reach the lower gastrointestinal tract, were widely accepted by researchers 16,17 . A lot of studies have found that high-fat diets via disordering the intestinal microecological balance influenced the human health 17 . Gut metabolites, including trimethylamine N-oxide (TMAO) 18 , short fatty acids (SCFAs) 19 , indoles 20 , secondary bile acids (SBAs) 21 , and so on, are produced by gut microbes and immediately impacted the physiological and pathological processes of our body 22,23 . Sucrose, a familiar disaccharide, is the most used sweeter in our daily life 2,13,24 . However, the studies about the influences of high-sucrose diets to intestinal microecological balance are rare. In this study, we will feed mice lengthen out to 18 months, and observe the changes of gut microbes and gut metabolites from young mice (YM) to middle age mice (MM), and finally to old age mice (OM) with 16S rRNA gene sequencing approach and UHPLC-QTOF/MS-based untargeted metabolomics analysis. Furthermore, the effects of long-time high-sucrose intake to the intestinal microecology were also been revealed in this study. Results Long- time high-sucrose intake decrease amnio acids and lipids intake but increased energy intake Two groups, named Nor group and HS group were designed in this study, and 3 subgroups, respectively, YM subgroup, MM subgroup and OM subgroup, were in every group (Fig. 1 A). Mice in the HS group drunk more sucrose water, then took less fodder. Correspondingly, mice in the normal group took more protein and lipids (Fig. 1 B-E). Sucrose was belonged to carbohydrate, then, mice in the HS group took more carbohydrate (Fig. 1 F). Due to the high-sucrose water contained large energy, the energy intake of the mice in the HS group largely surpassed the normal mice (Fig. 1 G). The middle age mice take more fodder and water, so with more protein and lipids, and carbohydrate than young mice and old age mice in both Nor and HS groups, but no significant difference between each subgroup (Fig. 1 B-G). Our study proved that mice in the HS groups took more energy with more intake of carbohydrate but less intake of protein and lipid. Long-time high-sucrose intake induced metabolic dysfunction Some studies have demonstrated that high-sucrose diet could increase the fat deposition and induce the metabolic disorder 25 . Our study agreed with above results. Compared to the normal mice, the weight of mice in the HS group was increased by 14.2% (YM group), 20.0% (MM group) and 30.0% (OM group) (Fig. 2 A). Correspondingly, the fat index and liver index, which reflected the fat deposition, were greatly increased after long-time drinking sucrose water, especially in the elder mice (Fig. 2 B-C). Our results also observed that elder mice have bad metabolic capability, then, with less increase of energy intake and more fat accumulation (Fig. 2 A-C). The metabolism of glucose and lipid was damaged after long-time consumption of high-sucrose (Fig. 2 D-F). With the increase of age, the FBG was tardily increase and the mice in the HS degenerated more ( Fig. 2 D ) . Compared to the normal group, the FBG of mice in the HS group was respectively increased by 15.1% (YM group), 21.9% (MM group) and 22.6% (OM group). The HOMA-IR index which reflects the insulin resistance, was significantly increased by sucrose intervention from the young mice to old mice (Fig. 2 E). OGTT results confirmed that the mice in the HS group had a bad glucose intolerance (Fig. 2 F), and the AUC of OGTT was increased after treating with sucrose. Our study also testified that long-time high-sucrose intake could induce dyslipidemia. After treating with sucrose, TG, TC and LDL-C were increased significantly not only in the OM group but also in the YM and MM group, but HDL-C was not changed significantly ( Fig. 2 G ) . Compare to the Nor group, the transaminases of the middle and old age mice, including ALT and AST, were increased in the HS group, but no significant changes in the young mice (Fig. 2 H). Similar from the above results, CRE and UA were increased in the middle and old age mice after drinking sucrose water (Fig. 2 H). Our results revealed that long-time excess sucrose intake was really harmful the health of body, with fat accumulation, blood glucose increase, dyslipidemia, and damage of hepatic and renal function. Long-time high-sucrose intervened mice have a different gut microbiota map The gut microbiota analysis discovered that high-sucrose greatly changed the gut microbiota and a completely different gut microbiota PCA map was found between the Nor and HS groups on the OTU level (Fig. 3 A). The PCA analysis and cluster analysis found that young mice, middle age mice and old age mice had a distinguishable gap, especially for mice in the HS group (Fig. 3 A-B). shannon index, ace index and chao index, which reflected community richness and diversity, were calculated. To our surprised, mice in the HS group were observed a higher community richness and diversity, and middle age mice had the highest shannon, ace and chao indexes (Fig. 3 C). Some studies found that high sugar diets decreased the community richness and diversity, which was differ from our study 26 . More deeply researches should be finished to explore why the results in our study are inconsistent with others. Bacteroidetes and Firmicutes were the main phyla in every subgroup. The gut microbes changed with age and mice in the middle age had the highest Bacteroidetes proportion and the lowest Firmicutes proportion not only in the Nor group but also in the HS group (Fig. 3 D, Fig. S1 A ). Correspondingly, the ratio o f Firmicutes / Bacteroidetes was significantly decreased in the middle age (Fig. 3 D). The mice in the HS group had a lower relative abundance of Firmicutes and a higher relative abundance of Bacteroidetes than mice in the Nor group at the middle and old age, with lower ratio of Firmicutes / Bacteroidetes. The decrease of Firmicutes / Bacteroidetes ratio was commonly found in the T2DM, obesity and coronary heart disease 27 . Besides, phylum Epsilonbacteraeota , Proteobacteria and Verrucomicrobia occupied an important proportion in all groups ( Fig. S1 ). Epsilonbacteraeota , mainly consist by Helicobacter (genus level), was greatly increased by high-sucrose, while Verrucomicrobia , mainly consist by Akkermansia (genus level), was greatly decreased by high-sucrose diet (Fig. 3 D). Furthermore, the relative abundance of Verrucomicrobia gradually decreased with age in all groups. The mice in old age HS group have the highest Helicobacter count and lowest Akkermansia count (Fig. 2 D, File S1 ). Above results may indicate that excess sucrose intake and gradually increased age have the ability to increase the harmful bacteria and decrease the beneficial bacteria. On the family level, we found that the differential flora between Nor and HS group were changed by age. The mainly differential families were Ruminococcaceae , Bacteroidaceae , Akkermansiaceae , Burkholderiaceae and Clostridiales_vadinBB60_group at the young age subgroups. However, Muribaculaceae , Lachnospiraceae and Helicobacteraceae were become the mainly differential families at the middle age and old age subgroups ( Fig. S1 B) . Muribaculaceae and Helicobacteraceae were increased, and Lachnospiraceae and Akkermansiaceae were decreased by sucrose at every period (Fig. 3 E). Long-time high-sucrose intake increase potential pathogen gene expression and decrease energy metabolism related gene expression BugBase phenotypic prediction was executed in our study. In the HS group, we found that the ratio of anaerobe/aerobe was greatly downregulated, but the ratio of gram negative/gram positive was upregulated in the middle age and old age mice (Fig. 4 A-B). The relative abundance of potential pathogen gene expression was upregulated by the high-sucrose of every period, but significant difference was observed at the old age groups (Fig. 4 C). We also found that the ratio of anaerobe/aerobe was arisen with the age in the Nor group, but was declined with age in the HS group. The change of gram negative/ gram positive had a contrary tendency. However, the relative abundance of potential pathogen gene in gut microbes was persistently arisen with age, and the oldest mice in the HS group expressed the most potential pathogen genes. COG function analysis found that the energy metabolism related genes of the gut microbiome in the HS groups were downregulated (Fig. 4 D). high-sucrose intake could reduce the gut microbe’s gene expression of transport and metabolism in carbohydrate, amino acid and lipid (Fig. 4 E). The gene expression of energy metabolism in gut microbes declined with age in both groups. The results told us long-time high-sucrose intake might reduce the ability of gut microbes to metabolize energy. Long-time high-sucrose intake effects gut macronutrients utilization A different gut metabolites map was observed from PCA analysis in the Nor and HS groups at every age group (Fig. 5 A). Above results indicated that long-time high-sucrose intake could influence the metabolism in gut, which was agreed with the results of high-sucrose intake and age effected the gene expression of gut microbes (Fig. 5 A). Three macronutrients including carbohydrates, fatty acids and amino acids were digested in the intestinal, and influenced by gut microbiota 16,28 . In this study, mice in HS group were taken large sucrose water, so less fodder were taken. Correspondingly, less fatty acids and amino acids but more carbohydrates (sucrose belonged to carbohydrate) were taken in the HS group (Fig. 1 ). Compare to the Nor group, carbohydrates reminded less in the HS group with a bluer heatmap (Fig. 5 B), and amino acids and fatty acids reminded more with a redder heatmap (Fig. 5 C-D). Considering more carbohydrates and less amnio acids and fatty acids were taken by mice in the HS group, above results indicated that more carbohydrates but less fatty acids and amino acids were utilized in the HS mice. Twenty amino acids, which are used to synthesize human proteins, were picked out in this study ( Fig S2 ). All the 20 amnio acids showed higher concentration in the HS group ( Fig S2 ). Aromatic amino acids (AAAs), including phenylalanine, tyrosine and tryptophan, and branched chain amino acids (BCAAs), including valine, leucine and isoleucine, were remained more in the colonic contents of mice in the HS group, which meant that high-sucrose intake reduced amino acids utilization, especially for utilization of AAAs and BCAAs, by host or gut microbes (Fig. 5 F). Long-time high-sucrose intake effected gut tryptophan metabolism Thousands of gut metabolites were produced or transformed by gut microbiota 28 . Tryptophan and its metabolites have key roles in diverse physiological processes 29 . Three mainly metabolic pathways, including 5-HT pathway, kynurenine pathway and indole pathway, were observed in the progress of tryptophan metabolism 29 . Similarly, less tryptophan was utilized in the mice of HS group, to our surprise, not all the metabolites of tryptophan were decrease by the high-sucrose water (Fig. 6 A-D). 5-HT and kynurenic acid were increased in the HS group (Fig. 6 B-C). 5-HT, also called serotonin, which has the ability to excite nerve and promote gastrointestinal motility 30 , was greatly increased by high-sucrose intake (Fig. 6 C). This result could use to explain why sweet make people happy. This alter vanished in the old mice, which means that the feeling of happiness would passivate after exposing under the high-sucrose condition for a long time. Kynurenic acid, which have the ability to protective nerve and resist depression (Fig. 6 E), has the similar tendency 31 . Indole and indole derivatives, belonged to ligands of aryl hydrocarbon receptor (AhR) and synthesized by gut microbes with tryptophan, are benefit for our health via the abilities to improve intestinal barrier disorder and glucose and lipid dysmetabolism, reduce inflammation and atherosclerosis 29 (Fig. 6 E). However, high-sucrose intake greatly reduced the production of indole and indole derivatives, which might be induced metabolic diseases (Fig. 6 A). Long-time high-sucrose intake effected gut bile acids metabolism Bile acids included primary bile acds (PBAs) and SBAs. PBAs were produced in liver with cholesterol, and are transformed into SBAs through the intestinal flora 32 . Bile acids not only are vital to glucose and lipid metabolism but also are important signal molecules to inflammation and energy expenditure (Fig. 7 E) 21,32 . PBAs, mainly included CA, β-MCA, CDCA and GCA, in the HS group, CA was decreased, but β-MCA was increased (Fig. 7 A, Fig. 7 C). SBAs, mainly including UDCA, DCA and LCA, were decreased in the HS group (Fig. 7 B, Fig. 7 D). Totally, the changes of bile acid pool might reduce the energy expenditure, and promote the inflammatory response, glucose and lipid dysmetabolism (Fig. 7 E). Long-time high-sucrose intake effected gut TMA-TMAO metabolism In our study, we found that TMA, a gut metabolite of L-carnitine or choline, is gradually increase with age, and mice in the HS group have a higher concentration of TMA in colonic contents (Fig. 8 A). TMA translates into trimethylamine oxide (TMAO) mainly in liver by FMO and little in the gut by microbes 18 . TMAO, which is famous for accelerating the process of atherosclerosis and chronic kidney diseases (CKD), and disordering glucose metabolism and cholesterol absorption 33 , also increased by high-sucrose diet (Fig. 8 B- 8 C). We also observed that old mice utilized more L-Carnitine or choline, which produced more TMA and TMAO. Discussion In this study, it was demonstrated that long-time high-sucrose intake induced fat deposition and metabolic disorder, which had been proved to be harmful to our health. We also found that mice in different age had a different composition of gut microbiota and gut metabolites. Furthermore, long time high-sucrose intake greatly changed the utilization of nutrient, which led to the changes of gut microbes and gut metabolites, and finally induced the imbalance of gut microenvironment (Fig. 9 ). Diets affect the composition of gut microbiota, which has been widely accepted by people 34 . Excessive intake of sugars can cause many health problems, such as T2DM, obesity, NAFLD, via altering microbial ecology 35 . Sucrose is the most commonly used sweetener, but its effects to gut microbial metabolism is short of research. Firmicutes and Bacteroidetes are the predominant bacterial phyla colonizing the healthy human gut 36 . high-sucrose intake induced decrease of Firmicutes , but increase of Bacteroidetes in colonic contents of mice. Correspondingly, the Firmicutes to Bacteroidetes ration was reduced by long time high-sucrose intake. The decrease of Firmicutes / Bacteroidetes ratio in obesity was observed in many clinic studies, and had become as a specific microbial signature of obesity, T2DM and NAFLD 27 . Excessive intake of sucrose might be one of the reasons for metabolic dysfunction patients with a lower Firmicutes / Bacteroidetes ratio. At the family level, Muribaculaceae and Helicobacteraceae (belonged to Bacteroidetes ) were increased, and Lachnospiraceae and Akkermansiaceae (belonged to Firmicutes ) were decreased by high-sucrose at every period. Akkermansiaceae , a famous gut microbial family, is benefit for our health, and the lack or decreased abundance of this commensal bacterium was linked with multiple diseases 37,38 . Akkermansia muciniphila (a species of Akkermansiaceae ), as a potential probiotic in treatment of metabolic disease, has been explored in many studies 38,39 . Our study firstly reported that long-time intake of high-sucrose can reduce the abundance of Akkermansiaceae . Lachnospiraceae are comprised 58 genera and several unclassified strains, and Blautia , Coprococcus , Dorea , Lachnospira , Oribacterium , Roseburia , and L-Ruminococcus are the main genera that have been detected in the human intestine 40 . The intestinal microorganisms, belonged to Lachnospiraceae , have the ability to produce SCFAs, SPBs and indole derivatives with amnio acids, saccharides and long fatty acids 40 . The abundance of Lachnospiraceae was decreased by high-sucrose intake, which might lead to the reduce of production in beneficial metabolites for the host 40 . Helicobacter pylori , Helicobacter heilmannii , Helicobacter labacensis and Helicobacter burdigaliensis associated with human digestive diseases, are main species of Helicobacteraceae 41 . Muribaculaceae , also named family S24-7, are dominant in the mouse gut microbiota, but has not been cultured until recently. Hence, the function of Muribaculaceae to the host is unclear 42 . Our study showed that high-sucrose diet upregulated the relative abundance of Helicobacteraceae and Muribaculaceae , which was indicated that Muribaculaceae might relate to the progress of diseases. high-sucrose drink changed the condition of gut microenvironment, with more sucrose and lower value of PH. Hence, the gut microbes, which are good at utilizing sucrose as energy source and adapt lower PH environment, proliferate more. The gene function analysis found that potential pathogen was upregulated by high-sucrose intervention. After a long-time high-sucrose intervention, the energy metabolism related gene expression was not upregulated, but was downregulated. Because of the changes of gut microbiota, the production of indoles and indole derivatives which are generated by gut microbes with tryptophan, is decreased, but the production of 5-HT and kynurenine are increased. 5-HT pathway, kynurenine pathway and indole pathway were the main metabolic pathway of tryptophan 43 . 5-HT pathway and kynurenine pathway exist in body, leading to the generation of biologically active compounds, such as serotonin, melatonin and niacin 30,43 . While, indole pathway exists in the gut, leading to improvement of intestinal barrier disorder, glucose and lipid dysmetabolism, inflammation and atherosclerosis 43 . After long-time sucrose intake, 5-HT and kynurenine pathways were increased, but indole pathway was decreased. This phenomenon can be explained that the ability of gut microbes to utilize tryptophan is decreased under the high-sucrose condition, thus, the generation of indole and indole derivatives is reduced. However, the reasons of 5-HT and kynurenine pathways increased by sucrose treatment are complicated, which deserve more attention to explore. Gut metabolites, including indoles, SBAs, SCFAs, TMA/TMAO and so on, are produced by gut microbes with food residue in gut. Changes of gut microbes and nutrients intake induced changed of gut metabolites. Because of more intake of sucrose, other nutrients, including amnio acids, long chain fatty acids and saccharides, were utilized less, but carnitine and choline, which can be used for generating TMA by gut microbes, were utilized more in the high-sucrose treated mice. Correspondingly, more TMA and TMAO (TMAO is formed from TMA in liver) were detected in the colonic contents of high-sucrose diet mice groups. Clinical effects of TMAO includes alteration of cholesterol and sterol metabolism, progression of chronic kidney diseases, atherosclerosis, heart attack, metabolic syndrome and T2DM [ 31 ]. Above results indicated that long-time high-sucrose intake might increase the risk of TMAO-related diseases. Other side, SPBs, such as UDCA, DCA and especially LCA, were decreased under high-sucrose condition. UDCA, usually is used for treating cholestatic liver disease with its ability to promote the secretion of bile aids 44 . Recently, a famous study found that UDCA could protect from SARS-CoV-2 infection by reducing ACE2 45 . DCA and LCA are effective ligands for farnesoid X receptor and G protein-coupled receptor 5, and have favor regulations of diabetes, effective ligands for farnesoid X receptor and G protein-coupled receptor 5, and have favor regulations of diabetes 32 . In this study, we found that the concentration of UDCA, DCA and LCA was the lowest in the old mice colonic contents, and long-time high-sucrose drink could significantly decrease the SPBs, especially the concentration of LCA. Our study reveals that long-time high-sucrose intake is really harmful to health, with excess energy intake led to metabolic dysfunction and changes of nutrients intake led to disorder of gut microbes and metabolites (Fig. 9 ). Finally, mice after a long-time high-sucrose intervention showed an imbalance intestinal microenvironment (Fig. 9 ). Hence, it is really important for us to reduce the intake of sucrose-sweetened beverages. Materials and methods Chemicals and reagents Sucrose, methanol, anhydrous ethanol, sodium hydroxide and trifluoroacetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Isoflurane was purchased from Shandong Keyuan Pharmaceutical Co., Ltd. (Jinan, China). The normal diet (nutritional ingredients was tabled in Table S1 ), is purchased from from Beijing Ke-ao-xie-li Feed Co., Ltd. Insulin ELISA Kit (EZRMI-13 K, Merck Millipore Co., Ltd, Darmstadt, Germany), Stool DNA Kit (DP328, TIAGEN biotech Co., Ltd., Beijing, China).Total cholesterol (TC, E-BC-K109-M), triglyceride (TG, E-BC-K261-M), high-density lipoprotein cholesterol (HDL-C, E-BC-K221-M), low-density lipoprotein cholesterol (LDL-C, E-BC-K205-M) colorimetric assay kit were purchased from Elabscience Biotechnology Co.,Ltd (Wuhan, China). Animal experiments A total of 60 male SPF C57BL/6J mice (8week, 22-25g) were purchased from Hunan SJA Laboratory animal Co., Ltd. (Changsha, China, License number: SCXK2020-0002) and raised in Department of Laboratory Animals, Central South University (Changsha, China) with under a relative humidity of 50 ± 15%, a temperature of 25 ± 2°C, a 12 hours dark-light cycle with sterile water and diet, and 5 mice living in one cage. All animal experiments were approved by the animal ethics committee of institutional animal care and use committee of central south university (Changsha, China, permit number: 2021sydw0112). All methods were performed in accordance with the relevant guidelines and regulations. Also, the study is reported in accordance with ARRIVE guidelines. Sixty mice were divided into two groups (30 mice per group), named normal group (Nor group) and high-sucrose group (HS group). All the mice were fed with normal diets. Mice in the normal group were drunk a normal pure water and mice in the HS group were drunk a sucrose water and a pure water (7:00 to 19:00 pure water, 19:00 to next day 7:00 sucrose water, 20%, w:v). All the mice were intervened at the same surroundings, and tested the weight, food intake and water drink every week. Ten mice of each group were sacrificed from inhaling isoflurane anaesthetized rats by opening the chest at 4 months (Yong mice YM), 12 months (Middle-age mice), 24 months (Old Mice, OM). The purposes of this experiment are observed the variation of gut microbiota and intestinal metabolites from the young to old mice, and looking for changes in gut microbes and metabolites after feeding with normal and sucrose drink ( Figure. 1A ). Biochemical assays Fast blood glucose (FBG, fasting for 8 hours) was measured with a glucometer (Accu-Chek (R) Active, Roche Diagnostics GmbH, Mannheim, Germany) by angular vein sampling. An oral glucose tolerance test (OGTT) was performed before the mice being sacrificed, and the method was followed before studies 21,46 . TC, TG, HDL-C, LDL-C, and serum glucose were detected followed the manufacturer’s instructions. Fasting serum insulin was quantified by ELISA kits. Homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated as before study 21 . 16S rRNA gene sequencing analysis Colonic contents samples of mice were collected for 16S rRNA gene analysis. Briefly, the steps of the analysis were DNA extraction, DNA quality detection, 16S rRNA gene amplification, purified the PCR products, PE library construction, Illumina sequencing and bioinformatics analysis 21,46 . This part was analyzed at the Shanghai Majorbio BioPharm Technology Co., Ltd.. The detail was saved in File S1 . Untargeted metabolomics analysis of intestinal metabolites Colonic contents samples were used for untargeted metabolomics analysis with UHPLC-QTOF-MS technology. The method was conducted as previously described 19 . Briefly, the methods concluded sample preprocessing, QC preparation, LC-MS/MS mass spectrometry analysis and data processing. This part was detected at the Shanghai Applied Protein Technology Co., Ltd.. The detail was saved in File S1 . Statistical analysis SPSS 23.0 and R software were used statistical analysis. GraphPad Prism 9.0 software was used for graphical presentation. The heatmaps were drawn using R software (pheatmap package). One-way ANOVA was performed to investigate alterations among three or more groups, Student’s t-tests was performed for comparing the data between only two groups. The date of heatmap was normalized with the z-score method (z=(x-µ)/σ). Abbreviations AUC Area under curve β-MCA Beta-Muricholic acid CA Cholic acid CDCA Chenodeoxycholic acid CRE Creatinine DCA Deoxycholic acid FBG Fast blood glucose HDL-C High-density lipoprotein cholesterol HOMA-IR Homeostasis model assessment of insulin resistance LCA Lithocholic acid LDL-C Low-density lipoprotein cholesterol MDCA Murideoxycholic acid NEG Negative OGTT Oral glucose tolerance test OTU Operational taxonomic unit PBAs Primary bile acids PCA Principal component analysis POS Positive SBAs Secondary bile acids SCFAs Short fatty acids TC Total cholesterol T2DM Type 2 diabetic mellitus TG Triglyceride TMA trimethylamine TMAO Trimethylamine N-oxide UA Uric acid UCA Ursodeoxycholic acid UDCA Ursodeoxycholic acid. Declarations Ethical approval All animal experiments were approved by the animal ethics committee of institutional animal care and use committee of central south university (Changsha, China, permit number: 2021sydw0112). Conflicts of interest The authors have declared that no competing interests exist. Author Contribution X.W, Z.L, and Y.Y wrote the main manuscript. X.W, L.Z and Y.Y completed the animal experiment and collected the data. X.W, H.G , Y. M and D.L prepared the figures. D.W, X. N and Y.Y revised the manuscript. X.N and Y.Y designed the experiment and obtained funding. All authors reviewed the manuscript and contributed to the article and approved the submitted version. Acknowledgements This work was financially supported by Natural Science Foundation of Guangdong Province ( 2022A1515110232, 2023A1515012246) Data availability The 16S rRNA gene sequences data and the metagenomic sequences data that supported the fndings of this study are publicly available at the NIH National Center for Biotechnology Information Sequence Read Archive (SRA) with BioProject ID PRJNA1063852. References Qin, P. et al. 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Biomedicine & pharmacotherapy 131, 110669, doi: 10.1016/j.biopha.2020.110669 (2020). Additional Declarations No competing interests reported. Supplementary Files FigS1.pdf FigS2.pdf FileS1.docx FileS2.xlsx FileS3.xlsx TableS1.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3849756","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":266985230,"identity":"5bd379cb-89e0-4e60-a57a-e04c6415b0d6","order_by":0,"name":"Xiaojuan Wang","email":"","orcid":"","institution":"IntegratedHospital of Traditional Chinese Medicine, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojuan","middleName":"","lastName":"Wang","suffix":""},{"id":266985231,"identity":"49f03014-6f67-4ad1-97c4-963cc43ebd32","order_by":1,"name":"Zhipeng Li","email":"","orcid":"","institution":"IntegratedHospital of Traditional Chinese Medicine, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhipeng","middleName":"","lastName":"Li","suffix":""},{"id":266985232,"identity":"ea7404c5-669b-4c7c-aa52-04796b224e79","order_by":2,"name":"Lili Zhang","email":"","orcid":"","institution":"IntegratedHospital of Traditional Chinese Medicine, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Zhang","suffix":""},{"id":266985233,"identity":"2b77cb19-481a-4858-8593-c0771a5d2101","order_by":3,"name":"Haiyu Guan","email":"","orcid":"","institution":"IntegratedHospital of Traditional Chinese Medicine, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Haiyu","middleName":"","lastName":"Guan","suffix":""},{"id":266985234,"identity":"810b35ba-86df-4d9d-a404-85f9dfd8352d","order_by":4,"name":"Dongyu Li","email":"","orcid":"","institution":"IntegratedHospital of Traditional Chinese Medicine, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dongyu","middleName":"","lastName":"Li","suffix":""},{"id":266985235,"identity":"6c50825a-6b64-4e32-a912-15a791e6ce31","order_by":5,"name":"Yenan Mo","email":"","orcid":"","institution":"IntegratedHospital of Traditional Chinese Medicine, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yenan","middleName":"","lastName":"Mo","suffix":""},{"id":266985236,"identity":"7e5c0b8a-35a9-4e3a-be63-6aedf50e3ed3","order_by":6,"name":"Dongsheng Wang","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"prefix":"","firstName":"Dongsheng","middleName":"","lastName":"Wang","suffix":""},{"id":266985237,"identity":"deea9aad-24f2-4d3d-b89c-696e1fb7aa96","order_by":7,"name":"Xiaoli Nie","email":"","orcid":"","institution":"IntegratedHospital of Traditional Chinese Medicine, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Nie","suffix":""},{"id":266985238,"identity":"639dfa98-081d-4d4e-be6e-19d5c3110ca5","order_by":8,"name":"Ye Yao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYNACg/88/OyNjQ8+EK+lgllGsudws+EM4rWcYbYxuJHeJs1BlJOOHz4m8bGNjcfg5sMGaQYGOzndBkJazqSlSc5s4+GRvJ3YYFzAkGxsdoCQlgM5ZtK8bRI8fEAtyTMYDiRuI6jl/Bsz6b9tBjwMNw82HOYhSssNoC0MZxJ4BG4wNjYTpUXyxrNky56KAzySPYnNjDMMiPAL3/nkgzd+GByw52c//vzHhwo7OYJaFA4wsEgguZOAchCQb2BgJiGZjIJRMApGwYgEAJKTR3pdxgh7AAAAAElFTkSuQmCC","orcid":"","institution":"IntegratedHospital of Traditional Chinese Medicine, Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ye","middleName":"","lastName":"Yao","suffix":""}],"badges":[],"createdAt":"2024-01-10 08:50:06","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-3849756/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3849756/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49672630,"identity":"ff7b10d3-3c9f-4312-bd31-958f9570f6ee","added_by":"auto","created_at":"2024-01-16 09:11:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116172,"visible":true,"origin":"","legend":"\u003cp\u003eLong-time high-sucrose intake influence protein, lipid, carbohydrate and energy intake. A: experiment design. B: food intake. C: water intake. D: protein intake. E: lipid intake. F: carbohydrate intake. G: energy intake. N=10 mice per/subgroup. Data are mean ±SEM. Statistical analysis was performed using Student-t test, * p \u0026lt; 0.05, ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/86c6b3040ca5f3fb1b2c3e2a.jpg"},{"id":49672637,"identity":"94eb2b38-e96a-4339-87d4-ac9b22b64fe0","added_by":"auto","created_at":"2024-01-16 09:11:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":351336,"visible":true,"origin":"","legend":"\u003cp\u003eLong-time high-sucrose intake induced fat accumulation, hyperglycemia, dyslipidemia and damage of hepatic and renal function. A: weight. B: liver index. C: fat index. D: FGB. E: HOMA-IR. F: OGTT. G: serum lipid levels. H:\u003cstrong\u003e \u003c/strong\u003ehepatic and renal function. N=10 mice per/subgroup. Data are mean ± SEM. Statistical analysis was performed using Student-t test, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.01, ns: not significant. ALT, alaninetransaminase; AST, alutamic oxalacetic transaminase; CRE, creatinine; FGB, fast blood glucose; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance index; LDL-C, low-density lipoprotein cholesterol; OGTT, blood glucose levels of oral glucose tolerance test; TC, total cholesterol; TG, triglyceride; UA, uric acid.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/74b0e35198704582938b0a7c.jpg"},{"id":49673106,"identity":"e85f6a9c-b812-458d-bfd7-fd8daf742075","added_by":"auto","created_at":"2024-01-16 09:19:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":262338,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of high-sucrose intake on gut microbiota modulation. A: PCA of the microbial composition at on the OTU level, B: cluster analysis of the microbial composition at on the OTU level, C: alpha diversity indexes, D: relative abundance of main phyla, E: Relative abundance of main families. N=10 mice per/subgroup. Data are normalized with log2, Data are mean ± SEM. Statistical analysis was performed using Student-t test, *\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.05\u003cem\u003e,\u003c/em\u003e ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01. PCA: principal component analysis; OUT: operational taxonomic unit.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/0c46cb75a1c5a8d369916615.jpg"},{"id":49672631,"identity":"864e0662-5024-454a-93f3-4a0bf86d98d4","added_by":"auto","created_at":"2024-01-16 09:11:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":228079,"visible":true,"origin":"","legend":"\u003cp\u003ethe effects of high-sucrose intake on gut microbiota gene function prediction. A: ratio of anaerobe/aerobe, B: ratio of gram negative/gram positive, C: relative abundance of potential pathogen, D: barplot of COG function classfication, E: relative abundance of gene expression on energy metabolism.N=10 mice per/subgroup. Data are normalized with log10, Data are mean ± SEM. Statistical analysis was performed using Student-t test, * p \u0026lt; 0.05, ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/73e87a532dd9ba9763cae2f7.png"},{"id":49672643,"identity":"1dad57c0-83ff-4d7a-83eb-f884801c7e92","added_by":"auto","created_at":"2024-01-16 09:11:28","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1990663,"visible":true,"origin":"","legend":"\u003cp\u003ehigh-sucrose intake changed the utilization of three macronutrients. A: PCA of gut metabolites, B: heatmap of carbohydrates, C: heatmap of fatty acids, D heatmap of amnio acids, E: relative concentration of aromatic amino acids, F: relative concentration of branched chain amino acids. N=10 mice per/subgroup. Data in heat map are normalized with z-score method (z=(x-μ)/σ), and data in Fig 5A, Fig 5E and Fig 5F are normalized with log2. Data are mean ± SEM. Statistical analysis was performed using Student-t test, * p \u0026lt; 0.05, ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/a77926489bcbe77adbfc959b.jpg"},{"id":49672636,"identity":"56d68ff3-7759-430e-b569-a42e50ce0e05","added_by":"auto","created_at":"2024-01-16 09:11:28","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":287841,"visible":true,"origin":"","legend":"\u003cp\u003ehigh-sucrose intake effected gut tryptophan metabolism. A: indole pathway, B: kynurenine pathway, C:5-HT pathway, D: heatmap of tryptophan metabolites, E: the effects of high-sucrose intake on tryptophan metabolism. N=10 mice per/subgroup. Data in Figure 6A-C are normalized with log2. Data in heatmap are calculated with area under the curve, and normalized with z-score method (z=(x-μ)/σ). Data are mean ± SEM. Statistical analysis was performed using Student-t test, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/e235cdadab67515700f11183.jpg"},{"id":49673110,"identity":"75b10d15-db30-41c0-a49f-b9a764041e8d","added_by":"auto","created_at":"2024-01-16 09:19:28","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":145158,"visible":true,"origin":"","legend":"\u003cp\u003ehigh-sucrose intake effected gut bile acid metabolism. A: relative concentration of PBAs, B: relative concentration of SBAs, C: AUC of PBAs, D: AUC of SBAs. E: the effects of high-sucrose intake on bile acids metabolism. N=10 mice per/subgroup. Data are normalized with log2. Data are mean ± SEM. Statistical analysis was performed using Student-t test, * p \u0026lt; 0.05, ** p \u0026lt; 0.01. CA: cholic acid, CDCA: chenodeoxycholate, DCA: deoxycholic acid, GCA: chenodeoxycholate, LCA: lithocholic acid, PBAs: primary bile acids, UDCA: ursodeoxycholic acid, SBAs: secondary bile acids, β-MCA: β-muricholic acid.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/38576204d07373d3aeeeac83.jpg"},{"id":49672633,"identity":"49448907-29b9-46e8-88de-3c5f6852f34d","added_by":"auto","created_at":"2024-01-16 09:11:28","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":99349,"visible":true,"origin":"","legend":"\u003cp\u003ehigh-sucrose intake effected gut TMA-TMAO metabolism. A: relative concentration of TMA related metabolites, B: AUC of TMA related metabolites, C: the effects of high-sucrose on TMA-TMA metabolism. N=10 mice per/subgroup. Data are normalized with log2. Data are mean ±SEM. Statistical analysis was performed using Student-t test, * p \u0026lt; 0.05, ** p \u0026lt; 0.01. TMA: trimethylamine, TMAO: trimethylamine N-oxide.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/4fea9a0e32d09b1f4ac72c33.jpg"},{"id":49672641,"identity":"e2b34aeb-97ac-4360-8305-ce0fa4ac7127","added_by":"auto","created_at":"2024-01-16 09:11:28","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":261328,"visible":true,"origin":"","legend":"\u003cp\u003eLong-time high-sucrose intake induced metabolic dysfunction via disrupting the balance of intestinal microenvironment.\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/1f2705e507caf2d91f0079d5.jpg"},{"id":50075262,"identity":"72849e1a-3216-4e39-bc6b-af9d883d2b0d","added_by":"auto","created_at":"2024-01-24 05:39:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1768030,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/8c27a787-2e47-495e-93e7-9ca1f85a33a0.pdf"},{"id":49673108,"identity":"b2160756-eca4-48a7-9583-e1b126189457","added_by":"auto","created_at":"2024-01-16 09:19:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":238675,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/7868429b3a8ec7e5512aa5cd.pdf"},{"id":49673429,"identity":"ea6bf0fd-93d7-44c3-9ed3-b14012569220","added_by":"auto","created_at":"2024-01-16 09:27:28","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":554506,"visible":true,"origin":"","legend":"","description":"","filename":"FigS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/558b1faa0a0587af3031fc31.pdf"},{"id":49673104,"identity":"fac1cdc6-8180-4b42-88c7-902599afa6c4","added_by":"auto","created_at":"2024-01-16 09:19:28","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16055,"visible":true,"origin":"","legend":"","description":"","filename":"FileS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/689623d425c64bb355fec39d.docx"},{"id":49673105,"identity":"e9c866f7-0ca4-4ea9-b33d-57d57b7089e6","added_by":"auto","created_at":"2024-01-16 09:19:28","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":390033,"visible":true,"origin":"","legend":"","description":"","filename":"FileS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/675a102e489a1d87a7950bfc.xlsx"},{"id":49672645,"identity":"220fe756-e452-4b2e-bd80-a12964aa398e","added_by":"auto","created_at":"2024-01-16 09:11:29","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16993769,"visible":true,"origin":"","legend":"","description":"","filename":"FileS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/4b57f3435624a8ca2ae4d6b6.xlsx"},{"id":49673912,"identity":"35d4988d-a363-4ee2-ae06-e6277375a786","added_by":"auto","created_at":"2024-01-16 09:35:28","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":69097,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3849756/v1/78d0902e7042a69b6e884c4c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Long-time high-sucrose intake induced metabolic dysfunction via disrupting the balance of intestinal microenvironment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs an important component of the human diet, sugars were strongly loved by human beings, but have been shown to be harmfully associated with a variety of risk factors for a long time, mainly including obesity\u003csup\u003e1,2\u003c/sup\u003e, diabetes\u003csup\u003e3\u003c/sup\u003e, cardiovascular disease\u003csup\u003e4\u003c/sup\u003e, hyperuricaemia\u003csup\u003e5\u003c/sup\u003e, gout\u003csup\u003e6\u003c/sup\u003e, dental caries\u003csup\u003e7\u003c/sup\u003e, and some cancers\u003csup\u003e8\u003c/sup\u003e. A recent study reported that reducing the consumption of sugars to below 25 g/day is recommended to reduce the adverse effect\u003csup\u003e9\u003c/sup\u003e. However, the intake of sugar is immensely over the health standards for many people, with a cup of milk tea contained about 100g sugar and a bottle of coke contained about 54g sugar. Furthermore, the consumption of sugar sweetened beverages is still increase in the word. The Coca-Cola company revealed that the consumption of sugar-sweetened beverages increased by 17% in 2021 and 11.2% in 2022\u003csup\u003e10\u003c/sup\u003e. The annual sales of milk tea in China were only 800\u0026nbsp;million yuan in 2015, but increased to 66\u0026nbsp;billion yuan in 2020\u003csup\u003e9\u003c/sup\u003e. Sugar-sweetened beverages refer to the beverages with added sugar or sweeteners that have been prevalent worldwide, especially among younger people\u003csup\u003e4\u003c/sup\u003e. A cross sectional survey conducted among Chinese school students showed that sugar sweetened beverages provide 10\u0026ndash;15% of the total calorie consumption of school students, with less than 10% of total daily energy intake provided by added sugar were recommended by World Health Organization (WHO)\u003csup\u003e11,12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOvertake of sugars has a tightly relationship with obese, and excess sugars were converted to fat and accumulated in our body\u003csup\u003e9\u003c/sup\u003e. With the increase of consumption of sugars, the prevalence of obesity is rising, and more than one billion individuals worldwide who are currently obese\u003csup\u003e13\u003c/sup\u003e. In China, half of adults are overweight, comparing with a high prevalence of type 2 diabetic mellitus (T2DM), non-alcoholic fatty liver disease and cardiovascular disease\u003csup\u003e14\u003c/sup\u003e. Globally, although many governments have initiated actions to reduce the consumption of sugar in the last few years, however, no significant effects were produced\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe concepts of diets impacted the composition of gut microbes and gut microbes are critical component of digestion, breaking down complex carbohydrates, proteins, and to a lesser extent fats that reach the lower gastrointestinal tract, were widely accepted by researchers\u003csup\u003e16,17\u003c/sup\u003e. A lot of studies have found that high-fat diets via disordering the intestinal microecological balance influenced the human health\u003csup\u003e17\u003c/sup\u003e. Gut metabolites, including trimethylamine N-oxide (TMAO)\u003csup\u003e18\u003c/sup\u003e, short fatty acids (SCFAs)\u003csup\u003e19\u003c/sup\u003e, indoles\u003csup\u003e20\u003c/sup\u003e, secondary bile acids (SBAs)\u003csup\u003e21\u003c/sup\u003e, and so on, are produced by gut microbes and immediately impacted the physiological and pathological processes of our body\u003csup\u003e22,23\u003c/sup\u003e. Sucrose, a familiar disaccharide, is the most used sweeter in our daily life\u003csup\u003e2,13,24\u003c/sup\u003e. However, the studies about the influences of high-sucrose diets to intestinal microecological balance are rare.\u003c/p\u003e \u003cp\u003eIn this study, we will feed mice lengthen out to 18 months, and observe the changes of gut microbes and gut metabolites from young mice (YM) to middle age mice (MM), and finally to old age mice (OM) with 16S rRNA gene sequencing approach and UHPLC-QTOF/MS-based untargeted metabolomics analysis. Furthermore, the effects of long-time high-sucrose intake to the intestinal microecology were also been revealed in this study.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLong- time high-sucrose intake decrease amnio acids and lipids intake but increased energy intake\u003c/h2\u003e \u003cp\u003eTwo groups, named Nor group and HS group were designed in this study, and 3 subgroups, respectively, YM subgroup, MM subgroup and OM subgroup, were in every group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Mice in the HS group drunk more sucrose water, then took less fodder. Correspondingly, mice in the normal group took more protein and lipids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E). Sucrose was belonged to carbohydrate, then, mice in the HS group took more carbohydrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Due to the high-sucrose water contained large energy, the energy intake of the mice in the HS group largely surpassed the normal mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The middle age mice take more fodder and water, so with more protein and lipids, and carbohydrate than young mice and old age mice in both Nor and HS groups, but no significant difference between each subgroup (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-G). Our study proved that mice in the HS groups took more energy with more intake of carbohydrate but less intake of protein and lipid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLong-time high-sucrose intake induced metabolic dysfunction\u003c/h2\u003e \u003cp\u003eSome studies have demonstrated that high-sucrose diet could increase the fat deposition and induce the metabolic disorder \u003csup\u003e25\u003c/sup\u003e. Our study agreed with above results. Compared to the normal mice, the weight of mice in the HS group was increased by 14.2% (YM group), 20.0% (MM group) and 30.0% (OM group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Correspondingly, the fat index and liver index, which reflected the fat deposition, were greatly increased after long-time drinking sucrose water, especially in the elder mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Our results also observed that elder mice have bad metabolic capability, then, with less increase of energy intake and more fat accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003eThe metabolism of glucose and lipid was damaged after long-time consumption of high-sucrose (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). With the increase of age, the FBG was tardily increase and the mice in the HS degenerated more \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Compared to the normal group, the FBG of mice in the HS group was respectively increased by 15.1% (YM group), 21.9% (MM group) and 22.6% (OM group). The HOMA-IR index which reflects the insulin resistance, was significantly increased by sucrose intervention from the young mice to old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). OGTT results confirmed that the mice in the HS group had a bad glucose intolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), and the AUC of OGTT was increased after treating with sucrose.\u003c/p\u003e \u003cp\u003eOur study also testified that long-time high-sucrose intake could induce dyslipidemia. After treating with sucrose, TG, TC and LDL-C were increased significantly not only in the OM group but also in the YM and MM group, but HDL-C was not changed significantly \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. Compare to the Nor group, the transaminases of the middle and old age mice, including ALT and AST, were increased in the HS group, but no significant changes in the young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Similar from the above results, CRE and UA were increased in the middle and old age mice after drinking sucrose water (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eOur results revealed that long-time excess sucrose intake was really harmful the health of body, with fat accumulation, blood glucose increase, dyslipidemia, and damage of hepatic and renal function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLong-time high-sucrose intervened mice have a different gut microbiota map\u003c/h2\u003e \u003cp\u003eThe gut microbiota analysis discovered that high-sucrose greatly changed the gut microbiota and a completely different gut microbiota PCA map was found between the Nor and HS groups on the OTU level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The PCA analysis and cluster analysis found that young mice, middle age mice and old age mice had a distinguishable gap, especially for mice in the HS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). shannon index, ace index and chao index, which reflected community richness and diversity, were calculated. To our surprised, mice in the HS group were observed a higher community richness and diversity, and middle age mice had the highest shannon, ace and chao indexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Some studies found that high sugar diets decreased the community richness and diversity, which was differ from our study\u003csup\u003e26\u003c/sup\u003e. More deeply researches should be finished to explore why the results in our study are inconsistent with others.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacteroidetes\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e were the main phyla in every subgroup. The gut microbes changed with age and mice in the middle age had the highest \u003cem\u003eBacteroidetes\u003c/em\u003e proportion and the lowest \u003cem\u003eFirmicutes\u003c/em\u003e proportion not only in the Nor group but also in the HS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). Correspondingly, the ratio o\u003cem\u003ef Firmicutes\u003c/em\u003e/\u003cem\u003eBacteroidetes\u003c/em\u003e was significantly decreased in the middle age (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The mice in the HS group had a lower relative abundance of \u003cem\u003eFirmicutes\u003c/em\u003e and a higher relative abundance of \u003cem\u003eBacteroidetes\u003c/em\u003e than mice in the Nor group at the middle and old age, with lower ratio of \u003cem\u003eFirmicutes\u003c/em\u003e/\u003cem\u003eBacteroidetes.\u003c/em\u003e The decrease of \u003cem\u003eFirmicutes\u003c/em\u003e/\u003cem\u003eBacteroidetes\u003c/em\u003e ratio was commonly found in the T2DM, obesity and coronary heart disease\u003csup\u003e27\u003c/sup\u003e. Besides, phylum \u003cem\u003eEpsilonbacteraeota\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eVerrucomicrobia\u003c/em\u003e occupied an important proportion in all groups (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). \u003cem\u003eEpsilonbacteraeota\u003c/em\u003e, mainly consist by \u003cem\u003eHelicobacter\u003c/em\u003e (genus level), was greatly increased by high-sucrose, while \u003cem\u003eVerrucomicrobia\u003c/em\u003e, mainly consist by \u003cem\u003eAkkermansia\u003c/em\u003e (genus level), was greatly decreased by high-sucrose diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, the relative abundance of \u003cem\u003eVerrucomicrobia\u003c/em\u003e gradually decreased with age in all groups. The mice in old age HS group have the highest \u003cem\u003eHelicobacter\u003c/em\u003e count and lowest \u003cem\u003eAkkermansia\u003c/em\u003e count (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cb\u003eFile S1\u003c/b\u003e). Above results may indicate that excess sucrose intake and gradually increased age have the ability to increase the harmful bacteria and decrease the beneficial bacteria.\u003c/p\u003e \u003cp\u003eOn the family level, we found that the differential flora between Nor and HS group were changed by age. The mainly differential families were \u003cem\u003eRuminococcaceae\u003c/em\u003e, \u003cem\u003eBacteroidaceae\u003c/em\u003e, \u003cem\u003eAkkermansiaceae\u003c/em\u003e, \u003cem\u003eBurkholderiaceae\u003c/em\u003e and \u003cem\u003eClostridiales_vadinBB60_group\u003c/em\u003e at the young age subgroups. However, \u003cem\u003eMuribaculaceae\u003c/em\u003e, \u003cem\u003eLachnospiraceae\u003c/em\u003e and \u003cem\u003eHelicobacteraceae\u003c/em\u003e were become the mainly differential families at the middle age and old age subgroups (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB)\u003c/b\u003e. \u003cem\u003eMuribaculaceae\u003c/em\u003e and \u003cem\u003eHelicobacteraceae\u003c/em\u003e were increased, and \u003cem\u003eLachnospiraceae\u003c/em\u003e and \u003cem\u003eAkkermansiaceae\u003c/em\u003e were decreased by sucrose at every period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLong-time high-sucrose intake increase potential pathogen gene expression and decrease energy metabolism related gene expression\u003c/h2\u003e \u003cp\u003eBugBase phenotypic prediction was executed in our study. In the HS group, we found that the ratio of anaerobe/aerobe was greatly downregulated, but the ratio of gram negative/gram positive was upregulated in the middle age and old age mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). The relative abundance of potential pathogen gene expression was upregulated by the high-sucrose of every period, but significant difference was observed at the old age groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We also found that the ratio of anaerobe/aerobe was arisen with the age in the Nor group, but was declined with age in the HS group. The change of gram negative/ gram positive had a contrary tendency. However, the relative abundance of potential pathogen gene in gut microbes was persistently arisen with age, and the oldest mice in the HS group expressed the most potential pathogen genes.\u003c/p\u003e \u003cp\u003eCOG function analysis found that the energy metabolism related genes of the gut microbiome in the HS groups were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). high-sucrose intake could reduce the gut microbe\u0026rsquo;s gene expression of transport and metabolism in carbohydrate, amino acid and lipid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The gene expression of energy metabolism in gut microbes declined with age in both groups. The results told us long-time high-sucrose intake might reduce the ability of gut microbes to metabolize energy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLong-time high-sucrose intake effects gut macronutrients utilization\u003c/h2\u003e \u003cp\u003eA different gut metabolites map was observed from PCA analysis in the Nor and HS groups at every age group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Above results indicated that long-time high-sucrose intake could influence the metabolism in gut, which was agreed with the results of high-sucrose intake and age effected the gene expression of gut microbes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThree macronutrients including carbohydrates, fatty acids and amino acids were digested in the intestinal, and influenced by gut microbiota\u003csup\u003e16,28\u003c/sup\u003e. In this study, mice in HS group were taken large sucrose water, so less fodder were taken. Correspondingly, less fatty acids and amino acids but more carbohydrates (sucrose belonged to carbohydrate) were taken in the HS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCompare to the Nor group, carbohydrates reminded less in the HS group with a bluer heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and amino acids and fatty acids reminded more with a redder heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). Considering more carbohydrates and less amnio acids and fatty acids were taken by mice in the HS group, above results indicated that more carbohydrates but less fatty acids and amino acids were utilized in the HS mice.\u003c/p\u003e \u003cp\u003eTwenty amino acids, which are used to synthesize human proteins, were picked out in this study (\u003cb\u003eFig \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). All the 20 amnio acids showed higher concentration in the HS group (\u003cb\u003eFig \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Aromatic amino acids (AAAs), including phenylalanine, tyrosine and tryptophan, and branched chain amino acids (BCAAs), including valine, leucine and isoleucine, were remained more in the colonic contents of mice in the HS group, which meant that high-sucrose intake reduced amino acids utilization, especially for utilization of AAAs and BCAAs, by host or gut microbes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLong-time high-sucrose intake effected gut tryptophan metabolism\u003c/h2\u003e \u003cp\u003eThousands of gut metabolites were produced or transformed by gut microbiota\u003csup\u003e28\u003c/sup\u003e. Tryptophan and its metabolites have key roles in diverse physiological processes \u003csup\u003e29\u003c/sup\u003e. Three mainly metabolic pathways, including 5-HT pathway, kynurenine pathway and indole pathway, were observed in the progress of tryptophan metabolism\u003csup\u003e29\u003c/sup\u003e. Similarly, less tryptophan was utilized in the mice of HS group, to our surprise, not all the metabolites of tryptophan were decrease by the high-sucrose water (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). 5-HT and kynurenic acid were increased in the HS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). 5-HT, also called serotonin, which has the ability to excite nerve and promote gastrointestinal motility\u003csup\u003e30\u003c/sup\u003e, was greatly increased by high-sucrose intake (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). This result could use to explain why sweet make people happy. This alter vanished in the old mice, which means that the feeling of happiness would passivate after exposing under the high-sucrose condition for a long time. Kynurenic acid, which have the ability to protective nerve and resist depression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), has the similar tendency\u003csup\u003e31\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIndole and indole derivatives, belonged to ligands of aryl hydrocarbon receptor (AhR) and synthesized by gut microbes with tryptophan, are benefit for our health via the abilities to improve intestinal barrier disorder and glucose and lipid dysmetabolism, reduce inflammation and atherosclerosis \u003csup\u003e29\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). However, high-sucrose intake greatly reduced the production of indole and indole derivatives, which might be induced metabolic diseases (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLong-time high-sucrose intake effected gut bile acids metabolism\u003c/h3\u003e\n\u003cp\u003eBile acids included primary bile acds (PBAs) and SBAs. PBAs were produced in liver with cholesterol, and are transformed into SBAs through the intestinal flora\u003csup\u003e32\u003c/sup\u003e. Bile acids not only are vital to glucose and lipid metabolism but also are important signal molecules to inflammation and energy expenditure (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE)\u003csup\u003e21,32\u003c/sup\u003e. PBAs, mainly included CA, β-MCA, CDCA and GCA, in the HS group, CA was decreased, but β-MCA was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). SBAs, mainly including UDCA, DCA and LCA, were decreased in the HS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Totally, the changes of bile acid pool might reduce the energy expenditure, and promote the inflammatory response, glucose and lipid dysmetabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eLong-time high-sucrose intake effected gut TMA-TMAO metabolism\u003c/h3\u003e\n\u003cp\u003eIn our study, we found that TMA, a gut metabolite of L-carnitine or choline, is gradually increase with age, and mice in the HS group have a higher concentration of TMA in colonic contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). TMA translates into trimethylamine oxide (TMAO) mainly in liver by FMO and little in the gut by microbes\u003csup\u003e18\u003c/sup\u003e. TMAO, which is famous for accelerating the process of atherosclerosis and chronic kidney diseases (CKD), and disordering glucose metabolism and cholesterol absorption\u003csup\u003e33\u003c/sup\u003e, also increased by high-sucrose diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB- \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). We also observed that old mice utilized more L-Carnitine or choline, which produced more TMA and TMAO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, it was demonstrated that long-time high-sucrose intake induced fat deposition and metabolic disorder, which had been proved to be harmful to our health. We also found that mice in different age had a different composition of gut microbiota and gut metabolites. Furthermore, long time high-sucrose intake greatly changed the utilization of nutrient, which led to the changes of gut microbes and gut metabolites, and finally induced the imbalance of gut microenvironment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDiets affect the composition of gut microbiota, which has been widely accepted by people\u003csup\u003e34\u003c/sup\u003e. Excessive intake of sugars can cause many health problems, such as T2DM, obesity, NAFLD, via altering microbial ecology\u003csup\u003e35\u003c/sup\u003e. Sucrose is the most commonly used sweetener, but its effects to gut microbial metabolism is short of research. \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e are the predominant bacterial phyla colonizing the healthy human gut\u003csup\u003e36\u003c/sup\u003e. high-sucrose intake induced decrease of \u003cem\u003eFirmicutes\u003c/em\u003e, but increase of \u003cem\u003eBacteroidetes\u003c/em\u003e in colonic contents of mice. Correspondingly, the \u003cem\u003eFirmicutes\u003c/em\u003e to \u003cem\u003eBacteroidetes\u003c/em\u003e ration was reduced by long time high-sucrose intake. The decrease of \u003cem\u003eFirmicutes\u003c/em\u003e/\u003cem\u003eBacteroidetes\u003c/em\u003e ratio in obesity was observed in many clinic studies, and had become as a specific microbial signature of obesity, T2DM and NAFLD\u003csup\u003e27\u003c/sup\u003e. Excessive intake of sucrose might be one of the reasons for metabolic dysfunction patients with a lower \u003cem\u003eFirmicutes\u003c/em\u003e/\u003cem\u003eBacteroidetes\u003c/em\u003e ratio.\u003c/p\u003e \u003cp\u003eAt the family level, \u003cem\u003eMuribaculaceae\u003c/em\u003e and \u003cem\u003eHelicobacteraceae\u003c/em\u003e (belonged to \u003cem\u003eBacteroidetes\u003c/em\u003e) were increased, and \u003cem\u003eLachnospiraceae\u003c/em\u003e and \u003cem\u003eAkkermansiaceae\u003c/em\u003e (belonged to \u003cem\u003eFirmicutes\u003c/em\u003e) were decreased by high-sucrose at every period. \u003cem\u003eAkkermansiaceae\u003c/em\u003e, a famous gut microbial family, is benefit for our health, and the lack or decreased abundance of this commensal bacterium was linked with multiple diseases\u003csup\u003e37,38\u003c/sup\u003e. \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e (a species of \u003cem\u003eAkkermansiaceae\u003c/em\u003e), as a potential probiotic in treatment of metabolic disease, has been explored in many studies\u003csup\u003e38,39\u003c/sup\u003e. Our study firstly reported that long-time intake of high-sucrose can reduce the abundance of \u003cem\u003eAkkermansiaceae\u003c/em\u003e. \u003cem\u003eLachnospiraceae\u003c/em\u003e are comprised 58 genera and several unclassified strains, and \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eCoprococcus\u003c/em\u003e, \u003cem\u003eDorea\u003c/em\u003e, \u003cem\u003eLachnospira\u003c/em\u003e, \u003cem\u003eOribacterium\u003c/em\u003e, \u003cem\u003eRoseburia\u003c/em\u003e, and \u003cem\u003eL-Ruminococcus\u003c/em\u003e are the main genera that have been detected in the human intestine\u003csup\u003e40\u003c/sup\u003e. The intestinal microorganisms, belonged to \u003cem\u003eLachnospiraceae\u003c/em\u003e, have the ability to produce SCFAs, SPBs and indole derivatives with amnio acids, saccharides and long fatty acids\u003csup\u003e40\u003c/sup\u003e. The abundance of \u003cem\u003eLachnospiraceae\u003c/em\u003e was decreased by high-sucrose intake, which might lead to the reduce of production in beneficial metabolites for the host\u003csup\u003e40\u003c/sup\u003e. \u003cem\u003eHelicobacter pylori\u003c/em\u003e, \u003cem\u003eHelicobacter heilmannii\u003c/em\u003e, \u003cem\u003eHelicobacter labacensis\u003c/em\u003e and \u003cem\u003eHelicobacter burdigaliensis\u003c/em\u003e associated with human digestive diseases, are main species of \u003cem\u003eHelicobacteraceae\u003c/em\u003e\u003csup\u003e41\u003c/sup\u003e. \u003cem\u003eMuribaculaceae\u003c/em\u003e, also named family S24-7, are dominant in the mouse gut microbiota, but has not been cultured until recently. Hence, the function of \u003cem\u003eMuribaculaceae\u003c/em\u003e to the host is unclear\u003csup\u003e42\u003c/sup\u003e. Our study showed that high-sucrose diet upregulated the relative abundance of \u003cem\u003eHelicobacteraceae\u003c/em\u003e and \u003cem\u003eMuribaculaceae\u003c/em\u003e, which was indicated that \u003cem\u003eMuribaculaceae\u003c/em\u003e might relate to the progress of diseases.\u003c/p\u003e \u003cp\u003ehigh-sucrose drink changed the condition of gut microenvironment, with more sucrose and lower value of PH. Hence, the gut microbes, which are good at utilizing sucrose as energy source and adapt lower PH environment, proliferate more. The gene function analysis found that potential pathogen was upregulated by high-sucrose intervention. After a long-time high-sucrose intervention, the energy metabolism related gene expression was not upregulated, but was downregulated.\u003c/p\u003e \u003cp\u003eBecause of the changes of gut microbiota, the production of indoles and indole derivatives which are generated by gut microbes with tryptophan, is decreased, but the production of 5-HT and kynurenine are increased. 5-HT pathway, kynurenine pathway and indole pathway were the main metabolic pathway of tryptophan\u003csup\u003e43\u003c/sup\u003e. 5-HT pathway and kynurenine pathway exist in body, leading to the generation of biologically active compounds, such as serotonin, melatonin and niacin\u003csup\u003e30,43\u003c/sup\u003e. While, indole pathway exists in the gut, leading to improvement of intestinal barrier disorder, glucose and lipid dysmetabolism, inflammation and atherosclerosis \u003csup\u003e43\u003c/sup\u003e. After long-time sucrose intake, 5-HT and kynurenine pathways were increased, but indole pathway was decreased. This phenomenon can be explained that the ability of gut microbes to utilize tryptophan is decreased under the high-sucrose condition, thus, the generation of indole and indole derivatives is reduced. However, the reasons of 5-HT and kynurenine pathways increased by sucrose treatment are complicated, which deserve more attention to explore.\u003c/p\u003e \u003cp\u003eGut metabolites, including indoles, SBAs, SCFAs, TMA/TMAO and so on, are produced by gut microbes with food residue in gut. Changes of gut microbes and nutrients intake induced changed of gut metabolites. Because of more intake of sucrose, other nutrients, including amnio acids, long chain fatty acids and saccharides, were utilized less, but carnitine and choline, which can be used for generating TMA by gut microbes, were utilized more in the high-sucrose treated mice. Correspondingly, more TMA and TMAO (TMAO is formed from TMA in liver) were detected in the colonic contents of high-sucrose diet mice groups. Clinical effects of TMAO includes alteration of cholesterol and sterol metabolism, progression of chronic kidney diseases, atherosclerosis, heart attack, metabolic syndrome and T2DM [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Above results indicated that long-time high-sucrose intake might increase the risk of TMAO-related diseases.\u003c/p\u003e \u003cp\u003eOther side, SPBs, such as UDCA, DCA and especially LCA, were decreased under high-sucrose condition. UDCA, usually is used for treating cholestatic liver disease with its ability to promote the secretion of bile aids\u003csup\u003e44\u003c/sup\u003e. Recently, a famous study found that UDCA could protect from SARS-CoV-2 infection by reducing ACE2\u003csup\u003e45\u003c/sup\u003e. DCA and LCA are effective ligands for farnesoid X receptor and G protein-coupled receptor 5, and have favor regulations of diabetes, effective ligands for farnesoid X receptor and G protein-coupled receptor 5, and have favor regulations of diabetes\u003csup\u003e32\u003c/sup\u003e. In this study, we found that the concentration of UDCA, DCA and LCA was the lowest in the old mice colonic contents, and long-time high-sucrose drink could significantly decrease the SPBs, especially the concentration of LCA.\u003c/p\u003e \u003cp\u003eOur study reveals that long-time high-sucrose intake is really harmful to health, with excess energy intake led to metabolic dysfunction and changes of nutrients intake led to disorder of gut microbes and metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Finally, mice after a long-time high-sucrose intervention showed an imbalance intestinal microenvironment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Hence, it is really important for us to reduce the intake of sucrose-sweetened beverages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eSucrose, methanol, anhydrous ethanol, sodium hydroxide and trifluoroacetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Isoflurane was purchased from Shandong Keyuan Pharmaceutical Co., Ltd. (Jinan, China). The normal diet (nutritional ingredients was tabled in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), is purchased from from Beijing Ke-ao-xie-li Feed Co., Ltd. Insulin ELISA Kit (EZRMI-13 K, Merck Millipore Co., Ltd, Darmstadt, Germany), Stool DNA Kit (DP328, TIAGEN biotech Co., Ltd., Beijing, China).Total cholesterol (TC, E-BC-K109-M), triglyceride (TG, E-BC-K261-M), high-density lipoprotein cholesterol (HDL-C, E-BC-K221-M), low-density lipoprotein cholesterol (LDL-C, E-BC-K205-M) colorimetric assay kit were purchased from Elabscience Biotechnology Co.,Ltd (Wuhan, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003eA total of 60 male SPF C57BL/6J mice (8week, 22-25g) were purchased from Hunan SJA Laboratory animal Co., Ltd. (Changsha, China, License number: SCXK2020-0002) and raised in Department of Laboratory Animals, Central South University (Changsha, China) with under a relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;15%, a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, a 12 hours dark-light cycle with sterile water and diet, and 5 mice living in one cage. All animal experiments were approved by the animal ethics committee of institutional animal care and use committee of central south university (Changsha, China, permit number: 2021sydw0112). All methods were performed in accordance with the relevant guidelines and regulations. Also, the study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e \u003cp\u003eSixty mice were divided into two groups (30 mice per group), named normal group (Nor group) and high-sucrose group (HS group). All the mice were fed with normal diets. Mice in the normal group were drunk a normal pure water and mice in the HS group were drunk a sucrose water and a pure water (7:00 to 19:00 pure water, 19:00 to next day 7:00 sucrose water, 20%, w:v). All the mice were intervened at the same surroundings, and tested the weight, food intake and water drink every week. Ten mice of each group were sacrificed from inhaling isoflurane anaesthetized rats by opening the chest at 4 months (Yong mice YM), 12 months (Middle-age mice), 24 months (Old Mice, OM). The purposes of this experiment are observed the variation of gut microbiota and intestinal metabolites from the young to old mice, and looking for changes in gut microbes and metabolites after feeding with normal and sucrose drink (\u003cb\u003eFigure. 1A\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical assays\u003c/h2\u003e \u003cp\u003eFast blood glucose (FBG, fasting for 8 hours) was measured with a glucometer (Accu-Chek (R) Active, Roche Diagnostics GmbH, Mannheim, Germany) by angular vein sampling. An oral glucose tolerance test (OGTT) was performed before the mice being sacrificed, and the method was followed before studies\u003csup\u003e21,46\u003c/sup\u003e. TC, TG, HDL-C, LDL-C, and serum glucose were detected followed the manufacturer\u0026rsquo;s instructions. Fasting serum insulin was quantified by ELISA kits. Homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated as before study\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e16S rRNA gene sequencing analysis\u003c/h2\u003e \u003cp\u003eColonic contents samples of mice were collected for 16S rRNA gene analysis. Briefly, the steps of the analysis were DNA extraction, DNA quality detection, 16S rRNA gene amplification, purified the PCR products, PE library construction, Illumina sequencing and bioinformatics analysis\u003csup\u003e21,46\u003c/sup\u003e. This part was analyzed at the Shanghai Majorbio BioPharm Technology Co., Ltd.. The detail was saved in \u003cb\u003eFile S1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eUntargeted metabolomics analysis of intestinal metabolites\u003c/h2\u003e \u003cp\u003eColonic contents samples were used for untargeted metabolomics analysis with UHPLC-QTOF-MS technology. The method was conducted as previously described\u003csup\u003e19\u003c/sup\u003e. Briefly, the methods concluded sample preprocessing, QC preparation, LC-MS/MS mass spectrometry analysis and data processing. This part was detected at the Shanghai Applied Protein Technology Co., Ltd.. The detail was saved in \u003cb\u003eFile S1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSPSS 23.0 and R software were used statistical analysis. GraphPad Prism 9.0 software was used for graphical presentation. The heatmaps were drawn using R software (pheatmap package). One-way ANOVA was performed to investigate alterations among three or more groups, Student\u0026rsquo;s t-tests was performed for comparing the data between only two groups. The date of heatmap was normalized with the z-score method (z=(x-\u0026micro;)/σ).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArea under curve\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eβ-MCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBeta-Muricholic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCholic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCDCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChenodeoxycholic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCreatinine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeoxycholic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFast blood glucose\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHDL-C\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh-density lipoprotein cholesterol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHOMA-IR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHomeostasis model assessment of insulin resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLithocholic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDL-C\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLow-density lipoprotein cholesterol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMurideoxycholic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNEG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOGTT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOral glucose tolerance test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOTU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOperational taxonomic unit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrimary bile acids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrincipal component analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSBAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSecondary bile acids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSCFAs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eShort fatty acids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal cholesterol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eT2DM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eType 2 diabetic mellitus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTriglyceride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etrimethylamine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMAO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTrimethylamine N-oxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUric acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUrsodeoxycholic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUDCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUrsodeoxycholic acid.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthical approval\u003c/h2\u003e \u003cp\u003eAll animal experiments were approved by the animal ethics committee of institutional animal care and use committee of central south university (Changsha, China, permit number: 2021sydw0112).\u003c/p\u003e\u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eX.W, Z.L, and Y.Y wrote the main manuscript. X.W, L.Z and Y.Y completed the animal experiment and collected the data. X.W, H.G , Y. M and D.L prepared the figures. D.W, X. N and Y.Y revised the manuscript. X.N and Y.Y designed the experiment and obtained funding. All authors reviewed the manuscript and contributed to the article and approved the submitted version.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was financially supported by Natural Science Foundation of Guangdong Province ( 2022A1515110232, 2023A1515012246)\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe 16S rRNA gene sequences data and the metagenomic sequences data that supported the fndings of this study are publicly available at the NIH National Center for Biotechnology Information Sequence Read Archive (SRA) with BioProject ID PRJNA1063852.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQin, P. \u003cem\u003eet al.\u003c/em\u003e Sugar and artificially sweetened beverages and risk of obesity, type 2 diabetes mellitus, hypertension, and all-cause mortality: a dose-response meta-analysis of prospective cohort studies. 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Biomedicine \u0026amp; pharmacotherapy 131, 110669, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biopha.2020.110669\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2020.110669\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"high-sucrose, metabolic dysfunction, intestinal microenvironment, gut microbiota, intestinal metabolites","lastPublishedDoi":"10.21203/rs.3.rs-3849756/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3849756/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSucrose, a common sweeter, is frequently added to drink and food, and excess intake of sucrose is really harmful to our health. This study aimed to reveal the potential mechanisms of high-sucrose induced metabolic dysfunction. Sixty mice were divided into two groups, respectively, the normal group (Nor group) and high-sucrose group (HS group). 16S rDNA and untargeted metabolomics technologies were used to analyzed the dynamic changes of gut microbiota and metabolites in colon contents of young mice, middle age mice and old mice. Long-time high-sucrose intake induced fat deposition and metabolic disorder, along with a completely different gut microbiota map. The energy metabolism related gene expression of the gut microbiome was downregulated, but potential pathogen gene expression was upregulated by long-time high-sucrose intake. Furthermore, mice in the HS were utilized less amino acids and long chain fatty acids, with effecting tryptophan metabolism by producing less indole, more 5-HT and kynurenine. Also less secondary bile acids, and more TMA/TMAO were produced by gut microbes in HS group. These results demonstrated that long-time high-sucrose intake leads to fat deposition and metabolic disorder via disrupting the balance of intestinal microenvironment.\u003c/p\u003e","manuscriptTitle":"Long-time high-sucrose intake induced metabolic dysfunction via disrupting the balance of intestinal microenvironment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-16 09:11:23","doi":"10.21203/rs.3.rs-3849756/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d9c74de-8bc8-4881-b705-9cc21f19acf1","owner":[],"postedDate":"January 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28128020,"name":"Biological sciences/Microbiology"},{"id":28128021,"name":"Health sciences/Endocrinology"},{"id":28128022,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases"}],"tags":[],"updatedAt":"2024-01-24T05:31:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-16 09:11:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3849756","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3849756","identity":"rs-3849756","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}