第一部分

基于“肠道菌群-短链脂肪酸-GPR43”途径探究

生命早期运动干预编程成年期代谢的机制

【研究目的】

生命早期运动干预可影响成年期的代谢状态，但具体作用机制不明。新近研究显示母体肠道菌群代谢产物短链脂肪酸（Short-chain fatty acids，SCFAs）可透过胎盘作用于胚胎重要代谢器官的特异性G蛋白偶联受体（G protein-coupled receptors，GPRs），影响靶器官的代谢调控功能，进而编程子代成年期代谢健康。本研究旨在通过生命早期运动干预的无菌小鼠模型明确肠道菌群在运动跨代代谢编程中的关键作用，再通过靶向代谢组学阐明短链脂肪酸是否是肠道菌群介导跨代调控的重要媒介，进一步利用胚胎期小鼠肝脏转录组学探寻“肠道菌群-SCFAs-GPR43”途径下游的关键靶标分子，最后在体外细胞实验中进行验证。

【研究方法】

1. 建立生命早期运动干预的无菌小鼠模型：

选用雌性C57BL/6小鼠，分别在无菌（Germ free，GF）与无特定病原体（Specific pathogen free，SPF）环境中饲养，并给予正常饮食。随后，将小鼠随机分为4组，即SPF静止组、SPF运动干预组、GF静止组和GF运动干预组。其中，运动干预采用自主跑轮的方式，干预时间包括孕前3周和整个孕期。GF和SPF雌鼠分别与相应环境中正常饮食并处于正常活动状态的C57BL/6雄鼠进行交配。雄性子鼠出生后均转为SPF环境下的哺乳期ICR雌鼠代乳，断乳后予正常饮食，12周龄后予45%高脂饮食挑战至24周龄。

2. 探索生命早期运动的代际代谢保护作用是否受肠道菌群影响：

每周监测母鼠运动量和体重，于孕中晚期（胚胎期14.5天，embryonic day 14.5，E14.5）行口服葡萄糖耐量试验（Oral glucose tolerance test，OGTT），在即将分娩时（E18.5）处死，记录产仔数和仔鼠出生体重，称量母鼠肝脏、胰腺和脂肪组织重量，取外周血分离获取血清，测定空腹血糖（Fasting blood glucose，FBG）、胰岛素、瘦素、脂联素和血脂谱的水平。雄性子鼠出生后每周监测体重，每月行OGTT，24周龄时行胰岛素耐量试验，处死时称量肝脏和脂肪组织重量，取外周血分离获取血清，测定FBG、胰岛素和血脂谱的水平。

3. 探究肠道菌群在介导生命早期运动干预编程成年期代谢中的分子机制：

获取上述小鼠模型中母鼠的盲肠内容物，利用16S rRNA测序技术对肠道微生物群落结构进行分析，明确孕前和孕期运动干预是否可富集产SCFAs菌。同时获取母鼠的血清和胎盘，通过靶向代谢组学检测母鼠盲肠内容物、血清和胎盘中SCFAs的变化，重点关注在三者中一致改变的SCFA。获取上述小鼠模型中E18.5的子鼠肝脏，采用qRT-PCR方法检测胎肝SCFAs特异性受体GPR43及分化发育和代谢调控相关基因的表达，进一步通过RNA-seq考察胎肝中转录组的变化，明确发挥关键作用的下游调控基因。

4. 体外实验探明SCFA通过GPR43对肝细胞代谢的调节作用：

乙酸钠干预小鼠AML12肝细胞，通过qRT-PCR方法检测细胞模型GPR43和关键下游调控基因Angptl8的表达水平。

【研究结果】

1. 孕前及孕期运动不仅带来自身代谢获益，同时对子代具有代谢保护效益：

孕前及孕期运动显著减轻E18.5 SPF孕鼠腹股沟皮下脂肪重量，增加肩胛间棕色脂肪重量；降低FBG及空腹胰岛素水平，增加胰岛素敏感性；增加高密度脂蛋白胆固醇水平；降低瘦素水平，增加脂联素水平；未对其体重及孕期增重、产仔数、子代出生体重和E14.5糖耐量造成显著影响。

SPF运动干预组雄性子代在幼年期（4周龄）即展现出比SPF静止组更好的糖耐量，这种优势在成年期（12周龄）仍可观察到。生命早期运动在一定程度上保护成年期雄性子鼠免受高脂饮食诱导的体重增长、糖代谢紊乱和肝功能受损，与SPF静止组相比，SPF运动组在高脂饮食挑战下的OGTT曲线下面积更小，实验终点时腹股沟皮下脂肪和肝脏的相对重量更轻，FBG、空腹胰岛素水平更低，胰岛素敏感性更高，谷草转氨酶和谷丙转氨酶水平降低。

2. 孕前及孕期运动的代际代谢改善作用在GF环境中消失：

孕前及孕期运动显著增加E18.5 GF孕鼠总胆固醇和甘油三酯水平，有增加其产仔数的趋势，未对其体重及孕期增重、FBG、空腹胰岛素、胰岛素敏感性及E14.5糖耐量造成显著影响。

GF运动组的雄性子代出生体重有增加的趋势，但后续发育阶段至12周龄期间，两组体重变化无统计学差异。于12周龄予高脂饮食挑战后，与GF静止组相比，GF运动组的体重显著增加，但两组间重要代谢器官的相对重量、糖耐量及胰岛素耐量则无明显差异。

3. 肠菌源SCFAs可能是介导生命早期运动改善成年期代谢的关键媒介：

孕前和孕期运动可调节SPF E18.5孕鼠的肠道菌群，增加其物种丰富度及群落多样性，并显著富集产SCFAs菌属，包括Desulfovibrio和Roseburia等。相应地，与SPF静止组相比，SPF运动组孕鼠盲肠内容物中乙酸、丁酸及戊酸水平显著升高，其中乙酸在血清和胎盘中也呈现出与此一致的组间差异；而缺失肠道菌群的GF两组间则未见盲肠内容物中SCFAs水平的统计学差异。此处，SPF运动组胎肝中SCFAs的特异性受体GPR43表达显著高于SPF静止组；与之相反，运动干预在GF组中未引起胎肝GPR43表达水平的改变。

4. “乙酸-GPR43/Angptl8”可能是关联“肠-肝轴”的重要途径：

SPF组E18.5胎肝RNA-seq发现，生命早期运动显著下调胎肝中Angptl8的表达；相反，GF组胎肝Angptl8的表达没有显著差异。

进一步选用运动干预后在母体盲肠内容物、外周循环及胎盘组织均呈现一致变化趋势的乙酸干预AML12小鼠肝细胞，发现无论在正常培养条件或棕榈酸加油酸诱导的脂毒性条件下，乙酸钠均可增加GPR43 mRNA的表达水平并抑制Angptl8的表达。

【研究结论】

肠道菌群是生命早期运动干预产生成年期代谢保护效应的必要条件；

母代孕前及孕期运动运动使其肠道内富集产SCFAs菌进而增加循环中SCFAs浓度，透过胎盘，作用于胚胎肝脏的特异性受体GPR43，可能通过Angptl8影响其代谢调节功能，最终编程子代成年期代谢健康。

 

第二部分

肠道菌群在介导生命早期饮食干预调控成年期白色脂肪棕色化中的作用

【研究目的】

在包括宫内发育和新生儿期的生命早期阶段，营养过剩会显著提升成年后发生代谢异常的风险，染料木素（Genistein，GEN）饮食干预可有效逆转这一不良“代谢记忆”。新近研究发现，肠道菌群可能通过诱导白色脂肪棕色化来发挥代谢保护效应，但其具体机制不明。本研究拟通过动物模型探究GEN饮食干预对白色脂肪棕色化的影响以及肠道菌群是否在其中发挥关键作用，进而探明“肠道菌群-白色脂肪组织棕色化轴”在介导生命早期GEN干预调控成年期代谢中的潜在机制。

【研究方法】

1. GEN干预抗生素清扫小鼠模型：

雌性C57BL/6J小鼠随机分为6组：正常饮食对照组、正常饮食+GEN干预组和正常饮食+GEN+抗生素干预组；以及高脂饮食对照组、高脂饮食+GEN干预组和高脂饮食+GEN+抗生素干预组。共干预20周，前10周单纯60%高脂饮食干预，后10周加给抗生素和（或）GEN干预（灌胃，30mg/kg/d）。通过在小鼠饮水中添加四联抗生素（0.5g/L万古霉素、1g/L甲硝唑、1g/L硫酸新霉素和1g/L氨苄西林）来清除肠道菌群。每周记录小鼠体重、摄食量和饮水量。在GEN干预前、干预第5周和第10周行腹腔注射葡萄糖耐量试验。实验结束后取血清检测血糖血脂生化指标，收集盲肠内容物进行16S rRNA测序，在腹股沟皮下脂肪组织和肩胛间棕色脂肪组织中采用qRT-PCR方法检测棕色化/产热相关基因的表达，通过Spearman相关性分析评估介导GEN改善代谢的关键肠道菌属相对丰度与棕色化/产热相关基因表达水平以及糖耐量曲线下面积之间的关系。

2. 生命早期GEN饮食干预跨代小鼠模型：

C57BL/6J雌性小鼠被随机分配至3组：正常饮食组、高脂饮食组和高脂饮食添加GEN组（GEN添加剂量为0.6g/kg体重）。饮食干预贯穿孕前3周、妊娠期及哺乳期。孕前期干预结束后，将上述各组雌鼠分别与同龄、正常饮食且自由活动的C57BL/6J雄性小鼠合笼。子代小鼠在断乳后统一给予正常饮食，并持续喂养至24周龄。雌性子鼠处死后取腹股沟皮下脂肪组织用qRT-PCR和MassArray方法检测棕色化相关基因的表达和甲基化水平，收集盲肠内容物进行非靶向代谢组学并基于液相色谱-串联质谱平台对参与甲硫氨酸循环的代谢物进行定量，使用Spearman相关性分析评估差异盲肠代谢物是否参与棕色化相关基因的甲基化并影响其表达。

【研究结果】

无论何种饮食，补充GEN均可明显改善糖耐量并显著增加产热相关基因Ucp1和PGC1α在脂肪组织中的表达，而抗生素清除肠道菌群则抵消了上述代谢获益。此外，在补充GEN的正常饮食瘦小鼠中，显著富集的产SCFAs菌Blautia、Ruminiclostridium_5和Ruminiclostridium_9与棕色化标志物和糖耐量显著相关。而在高脂饮食诱导的肥胖小鼠中，补充GEN后显著增加的产SCFAs菌Ruminiclostridium、Rikenella和Clostridium_sensu_stricto_1同样也与宿主代谢改善有关。

生命早期GEN饮食干预显著逆转上调了成年期雌性小鼠因母代高脂饮食所导致的棕色化标志基因下调，且这一现象与这些基因转录起始位点附近区域甲基化水平的降低相一致。同时，生命早期的GEN摄入改变了成年期的盲肠代谢物谱，促进了胆汁酸的产生，并减少了参与甲硫氨酸循环的代谢物含量。此外，上述差异代谢物的相对丰度与棕色化标志基因的甲基化和表达水平显著相关。

【研究结论】

补充GEN可改善雌性小鼠糖代谢并促进脂肪组织棕色化/产热标志基因表达，肠道菌群在其中扮演关键角色。

生命早期GEN饮食干预可能通过改变成年期雌性小鼠肠道菌群的组成，导致盲肠代谢物的变化，降低白色脂肪组织中棕色化标志基因的甲基化水平，从而促进白色脂肪棕色化，最终影响其代谢健康。

 

Part 1

Exploring the Mechanisms of Early-Life Exercise Intervention in Programming Adult Metabolism via the “Gut Microbiota-Short Chain Fatty Acids-GPR43” Pathway

Objective

Early-life exercise intervention can influence the metabolic state in adulthood; however, the underlying mechanisms remain unclear. Recent studies have shown that short-chain fatty acids (SCFAs), metabolic products derived from the maternal gut microbiota, can cross the placenta and act on specific G protein-coupled receptors (GPRs) in key embryonic metabolic organs. This interaction modulates the metabolic regulatory functions of target organs, ultimately programming the metabolic health of the offspring in later life. The present study aims to (1) clarify the critical role of the gut microbiota in mediating exercise-induced transgenerational metabolic programming using a germ-free (GF) mouse model, (2) determine via targeted metabolomics whether SCFAs serve as key mediators in this process, (3) explore downstream target molecules of the “gut microbiota-SCFAs-GPR43” pathway in embryonic liver transcriptomes, and (4) validate these findings with in vitro cell experiments.

Methods

Establishment of a GF Mouse Model for Early-Life Exercise Intervention

The mouse model of early-life exercise intervention was established using C57BL/6 female mice maintained under either GF or specific pathogen-free (SPF) conditions. All mice received a standard diet and were randomly assigned to one of four groups: SPF sedentary group, SPF exercise intervention group, GF sedentary group, and GF exercise intervention group. Exercise intervention was conducted using voluntary running wheels for three weeks prior to conception and throughout pregnancy. GF and SPF female mice were mated with normal-diet, free-moving C57BL/6 males the corresponding environmental conditions. Upon birth, all male offspring were transferred to lactating ICR females for nursing under SPF condition. After weaning, they were maintained on a standard diet until 12 weeks of age, at which point they were challenged with a 45% high-fat diet (HFD) until 24 weeks.

Exploring whether the Metabolic Benefits of Early-Life Exercise Intervention Depend on Gut Microbiota

Maternal exercise levels and body weight were monitored weekly. An oral glucose tolerance test (OGTT) was conducted during mid-gestation (embryonic day 14.5, E14.5). At E18.5 (just before parturition), dams were sacrificed to record litter size, pup birth weights, and to measure the weights of liver, pancreas, and adipose tissues. Peripheral blood was collected for serum separation to determine serum metabolic parameters, including fasting blood glucose (FBG), insulin, leptin, adiponectin, and lipid profiles.

The body weight of male offspring was monitored weekly. OGTTs were performed monthly, and an insulin tolerance test was conducted at 24 weeks of age. At the endpoint of experiment, liver and adipose tissues weights were measured, and serum was collected for FBG, insulin, and lipid profiles analyses.

Investigating the Molecular Mechanisms Underlying Gut Microbiota-Mediated Metabolic Programming Effects of Early-Life Exercise Intervention

Cecal contents from the dams were collected for 16S rRNA sequencing to characterize the gut microbiota, with particular focus on the enrichment of SCFAs-producing bacteria during prenatal exercise intervention. In parallel, maternal cecal contents, serum and placenta were analyzed using targeted metabolomics to assess changes in SCFAs levels, with an emphasis on those SCFAs that exhibited consistent alterations across these compartments.

Fetal livers collected at E18.5 were analyzed using quantitative reverse transcription PCR (qRT-PCR) to assess the expression of SCFAs-specific receptor GPR43, along with genes related to differentiation, development, and metabolic regulation. Moreover, transcriptomic changes in the fetal liver were investigated through RNA sequencing (RNA-seq) to identify the downstream regulatory genes that play a crucial role.

In Vitro Validation of SCFA-Mediated Hepatic Metabolic Regulation via GPR43

Mouse AML12 hepatocytes were treated with sodium acetate, and qRT-PCR was performed to assess the expression levels of GPR43 and Angptl8 under both normal culture condition and lipotoxic conditions induced by palmitic acid and oleic acid.

Results

Maternal exercise before and during pregnancy provided metabolic benefits to both mothers and offspring.

In SPF dams at E18.5, prenatal exercise significantly reduced the weight of inguinal subcutaneous adipose tissue (iWAT) and increased that of interscapular brown adipose tissue (BAT); it also lowered FBG and fasting insulin levels, enhanced insulin sensitivity, decreased leptin levels, increased adiponectin and high-density lipoprotein cholesterol levels—without significantly affecting body weight, total gestational weight gain, litter size, pup birth weights, or glucose tolerance at E14.5.

In SPF male offspring of maternal exercise (OSE) group, improved glucose tolerance was observed as early as 4 weeks of age compared to those in the maternal sedentary (OSS) group, and this advantage persisted into adulthood (12 weeks). Early-life exercise partially protected adult male offspring from HFD-induced obesity, glucose intolerance, and liver dysfunction. Specifically, under HFD challenge, OSE group exhibited a smaller area under the curve (AUC) of OGTT, reduced iWAT and liver weights, lower FBG and fasting insulin levels, improved insulin sensitivity, and decreased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels compared to the OSS group.

The Intergenerational Metabolic Benefits of Maternal Exercise were Abolished in GF Condition.

In contrast, the intergenerational metabolic benefits of exercise intervention were not observed under GF conditions. In GF dams, exercise intervention before and during pregnancy significantly increased total cholesterol and triglyceride levels at E18.5 and showed an increased trend in litter size. There were no significant differences in body weight, total gestational weight gain, FBG, fasting insulin, insulin sensitivity, or glucose tolerance at E14.5.

Although GF male offspring in the exercise (OGE) group tended to have higher birth weights, weight gain from postnatal to 12 weeks of age did not differ statistically between groups. After an HFD challenge at 12 weeks, OGE group exhibited significantly higher body weights compared to the GF maternal sedentary (OGS) group; however, there were no significant differences in the relative weights of major metabolic organs, glucose tolerance, or insulin tolerance between the two groups.

SCFAs derived from Gut Microbiota may be the Critical Mediators of the Metabolic Improvements induced by Early-Life Exercise Intervention.

Prenatal exercise intervention modulated the gut microbiota composition of dams by increasing species richness and community diversity, and significantly enriched SCFAs-producing genera, such as Desulfovibrio and Roseburia. Correspondingly, levels of acetic acid, butyric acid, and valeric acid in the cecal contents were significantly elevated in the SPF exercise group, with acetic acid also exhibiting a consistent and significant intergroup difference in serum and placenta. In the absence of gut microbiota (GF groups), no statistically significant differences in cecal SCFAs levels were observed between the exercise and sedentary groups.

Moreover, fetal liver from the OSE group exhibited significantly higher GPR43 expression than the OSS group, whereas no such change was detected in the GF groups.

The “Acetate-GPR43/Angptl8” Appears to be a Crucial Pathway Linking the “Gut-liver” Axis.

RNA-seq analysis of E18.5 fetal liver from SPF groups revealed that early-life exercise intervention significantly downregulated Angptl8 expression in fetal liver, whereas no significant changes were observed in the GF groups. In vitro, sodium acetate treatment of AML12 hepatocytes significantly upregulated GPR43 mRNA expression and downregulated Angptl8 expression under both normal and lipotoxic conditions.

Conclusions

Gut microbiota is essential for the metabolic protective effects of early-life exercise intervention in adulthood.

Maternal exercise before and during pregnancy enriches enteral SCFA-producing bacteria, increasing circulating SCFAs concentrations, which cross the placenta and activate the specific receptor GPR43 in fetal liver. This may regulate metabolic function via Angptl8, ultimately programming improved metabolic health in the offspring during adulthood.

 

Part 2

The Role of Gut Microbiota in Mediating Early-Life Dietary Intervention-Induced White Fat Browning in Adulthood

Objective

Over-nutrition during early life (intrauterine development and neonatal period) significantly increases the risk of metabolic disorders in adulthood. Genistein (GEN), a dietary isoflavone, has been shown to reverse this detrimental "metabolic memory". Growing evidence indicated that gut microbiota may exert metabolic protective effects by promoting white adipose tissue browning (WFB), yet the underlying mechanisms remain unclear. This study aimed to investigate the impact of GEN intervention on WFB and the pivotal role of gut microbiota in mediating these effects, further elucidating the "gut microbiota-WAT browning axis" as a potential mechanism through which early-life GEN intervention regulates adult metabolic health.

Methods

Mouse model of GEN intervention with antibiotic-induced microbiota depletion:

Female C57BL/6J mice were randomized into six groups: control diet (CD), CD+GEN, CD+GEN+antibiotics (ABX); high-fat diet (HFD), HFD+GEN, and HFD+GEN+ABX group. The experiment lasted 20 weeks, with the first 10 weeks involving a 60% HFD, followed by 10 weeks of HFD supplemented with GEN (30mg/kg/d by gavage) and/or antibiotic cocktail (0.5g/L vancomycin, 1g/L metronidazole, 1g/L neomycin sulfate, and 1g/L ampicillin) administered via drinking water. Body weight, food intake, and water consumption were recorded weekly. Intraperitoneal glucose tolerance tests (IPGTTs) were performed three times, first before the start of GEN and ABX intervention (baseline), second at 5 weeks after GEN and ABX intervention, and third at the end of the experiment. Upon completion of the intervention, serum samples were collected for glucolipid metabolic biochemical assays, cecal contents were collected for 16S rRNA sequencing, and expression levels of browning/thermogenesis-related genes in iWAT and interscapular BAT were assessed by qRT-PCR. Spearman correlation analyses were conducted to explore the relationship between the relative abundance of differential gut microbiota, the expression levels of browning/thermogenesis-related genes and the glucose tolerance.

2. Early-life GEN dietary intervention in a transgenerational mouse model:

Female C57BL/6J mice were randomly assigned to three groups: CD, HFD, and HFD+GEN (0.6g/kg diet) group. Dams were maintained on these diets for 3 weeks prior to mating and throughout gestation and lactation. After the initial 3-week dietary intervention, the female mice were mated with standard C57BL/6 males. After weaning, the female offspring were maintained on a normal diet until 24 weeks of age. At the end of the experiment, iWAT was collected for qRT-PCR and MassArray analysis to assess gene expression and DNA methylation levels of browning markers. Cecal contents were collected for untargeted metabolomic analysis and a target quantitative analysis of methionine cycle metabolites. Spearman correlation analyses were used to assess whether cecal metabolites are involved in the methylation of browning-related genes and influence their expression.

Results

The lean mice with a CD and GEN diet exhibited better glucose tolerance and higher expression of UCP1 and PGC1α in iWAT compared with those without GEN. Markedly enriched SCFAs-producing genera (Blautia, Ruminiclostridium_5 and Ruminiclostridium_9) in GEN-treated mice were significantly correlated with browning markers and glucose tolerance. In obese mice, GEN alleviated the detrimental effects of an HFD on glucose homeostasis and increased the expression levels of UCP1 and PGC1α in BAT. Obvious increases in SCFAs-producing genera (Ruminiclostridium, Rikenella, and Clostridium_sensu_stricto_1) by genistein were associated with metabolic improvement. However, depleting gut microbiota by ABX abolished these metabolic benefits.

Early-life GEN dietary intervention significantly reversed the downregulation of browning markers in female offspring caused by maternal HFD exposure. This effect was consistent with a reduction in DNA methylation levels near the transcription start sites of these genes. Furthermore, early-life GEN intake altered the cecal metabolites profile in adulthood, promoting the production of bile acids, potent regulators of glucolipid metabolism, and reducing metabolites involved in the methionine cycle, key methyl donors for the methylation process. The relative abundance of these differential metabolites was significantly correlated with the methylation and expression levels of browning markers.

Conclusions

Supplementation with GEN improved glucose metabolism in female mice and promoted the mRNA expression levels of browning/thermogenesis markers in adipose tissues, with the gut microbiota playing a crucial role in this process.

Early-life GEN dietary intervention led to changes in the composition of the gut microbiota in adult female mice, resulting in alterations in cecal metabolites (e.g., bile acids and methionine cycle intermediates) and a reduction in the methylation levels of browning markers in iWAT, thereby promoting the WFB and ultimately reprogramming metabolic health.