Six-week-old C57BL/6 mice were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, Guangdong, China). All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University. The female mice were randomly divided into three groups: (1) the control group, which received a normal diet (10 kcal% fat, 3.85 kcal/g; Guangdong Medical Laboratory Animal Center; batch No. D12450B) and untreated drinking water; (2) the model group, which received a high-fat diet (45 kcal% fat, 4.73 kcal/g; Guangdong Medical Laboratory Animal Center; batch No. D12450B) and drinking water supplemented with 10% (w/v) fructose (Fru, 4 kcal/g; Yongjin Biotechnology Co., Guangzhou, Guangdong, China; batch No. BWJ4341-2016). This group was also named the HFD group; and (3) the treatment group (HFD + YP), which received a daily oral gavage of YP powder (Shanghai Yuanye Bio-Technology Co., Shanghai, China; batch No. S24912) dissolved in sterile water and administered while maintained on an HFD, starting after mating. The dosage of YP was 200 mg/kg body weight per day. The control group and HFD groups were given an equal amount of sterile water by gavage after mating. After a six-week dietary intervention, female mice were mated with lean male mice. Mating was confirmed by the presence of a vaginal mucus plug the following morning, which was designated as gestational day (GD) 0. Based on pilot experiments and power analysis, each group required n = 6 mice to achieve 80% power to detect a 30% difference at significance levels of $\alpha$ = 0.05.

Mice underwent an oral glucose tolerance test (OGTT) at GD15 after a 12-hour fast. Each mouse was orally gavaged with glucose at a dose of 2 g/kg body weight, prepared in normal saline. Blood samples were collected from the vein at 0, 30, 60, 90, and 120 minutes after glucose administration to measure the glucose concentration.

Blood glucose levels were quantified using a glucose assay kit (Solarbio, Beijing, China; batch No. BC2495) in accordance with the manufacturer’s protocol, which follows the glucose oxidase method. Serum insulin levels were measured with a mouse ultrasensitive insulin ELISA kit (Wuhan Colorful Gene Biotech Co., Ltd., Wuhan, China; batch No. JYM0351Mo). The HOMA-IR was calculated using the following formula: HOMA-IR = (fasting glucose $\times$ fasting insulin)/22.5.

3T3-L1 murine preadipocytes were purchased from Cas9X Biotechnology Company (Suzhou, Jiangsu, China; batch No. TCM-C702). This cell line has been authenticated by short tandem repeat analysis, morphological observation, and adipogenic induction, and it tested negative for mycoplasma contamination. 3T3-L1 cells in the logarithmic growth phase were seeded into culture vessels at a density of 2.0 $\times$ 10⁴ cells/cm² at 37 °C and 5% CO₂ until reaching 90–100% confluency. Adipogenic differentiation kit (Cas9X biotechnology; Suzhou, Jiangsu, China; batch No. EFMX-D102) used for adipogenic induction by alternative replacement of the induction and maintenance medium. The timing for terminating cell induction was determined based on the number and size of lipid droplets formed during the process, followed by staining for identification. Cells were first fixed with 4% neutral formaldehyde solution at room temperature for 30 to 60 min, stained with red oil O solution for 30 min, and evaluated under a microscope. The glucose tolerance of 3T3-L1 adipocytes was assessed by performing glucose uptake tests under an insulin stimulation at a dosage of 100 nM for 30 min, using the Glucose Uptake-Glo^TM assay kit (Promega, Madison, WI, USA; batch No. J1341).

Fecal samples were collected at GD16 in individual sterilized cages and immediately frozen in liquid nitrogen. DNA was extracted according to the instructions of the DNA extraction kits (TaKaRa Bio, Beijing, China; batch No. 9765). The integrity and purity of the DNA were detected by 1% agarose gel electrophoresis, and the concentration and purity of the DNA were detected by the NanoDrop One microvolume spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). PCR amplification and product electrophoresis were performed using genomic DNA as a template, and primers with barcodes and Premix Tag (TaKaRa Bio; Beijing, China; batch No. RR004A) were used for PCR amplification based on selected sequencing regions. Gene Tools Analysis Software (Cambridge, UK; Version 4.03.05.0, SynGene) was used to quantify PCR product concentrations. The volume of each sample was adjusted to ensure equal mass, and the PCR products were pooled. Mixed PCR products were purified using the E.Z.N.A. Gel Extraction and Gel Recovery Kit (Omega Bio-Tek, Guangzhou, Guangdong, China; batch No. D2500). Target DNA fragments were eluted with TE buffer. Library construction was then performed using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA; batch No. E7645) standard process. The high-throughput sequencing platform HiSeq was utilized for online sequencing, and the raw image data file was converted into a raw sequencing dataset (Raw Reads) through base recognition (Base Calling) analysis. The results were stored in the FASTQ file format, which contains the sequencing sequence (Reads) and its corresponding quality information. Alpha diversity, which reflects the richness and evenness of microbial communities within a sample, was evaluated using the Shannon and Simpson indices. Beta diversity, which measures compositional differences between microbial communities across samples, was evaluated using Principal Coordinates Analysis (PCoA) based on the Bray-Curtis distance matrix. Additionally, Analysis of Similarities (ANOSIM) was employed to determine the significance of differences in microbial communities between groups. Analysis of species differences across multiple groups was based on Kruskal-Wallis rank sum test. The false discovery rate (FDR) was used for multiple-testing correction.

The data are presented as mean $\pm$ SD. We validated the assumption of normality using both the Shapiro-Wilk test and visual inspection of the quantile-quantile (Q-Q) plot. The homogeneity of variances was assessed using the Brown-Forsythe test. A p-value greater than 0.05 indicated that the assumptions were met, and a one-way analysis of variance (ANOVA) was used to detect statistical significance, followed by Tukey’s multiple comparisons test. A Kruskal-Wallis rank sum method was used for non-parametric test, followed by Dunn’s multiple comparisons test. A p-value less than 0.05 was considered statistically significant. Statistical analysis and graph plotting were conducted with GraphPad Prism (version 9, GraphPad software Inc., San Diego, CA, USA).

The experimental protocol for the animal study is shown in Fig. 1A. The HFD intervention was initiated six weeks before pregnancy and was maintained throughout the pregnancy. Fasting blood glucose was measured, and an OGTT was performed before mating to exclude individuals with pre-existing diabetes. All variables of interest satisfied the assumption of normality according to the Shapiro-Wilk test (all p $>$ 0.3). The Brown-Forsythe test confirmed that the assumption of homogeneity of variance was not violated (all p $>$ 0.4). Consequently, ANOVA was applied for group comparisons. Significant weight gains in the pre-pregnancy stage were observed in groups that underwent HFD treatment, including the HFD group and HFD + YP group, compared to the control group (Fig. 1B). Moreover, HFD-treated mice exhibited a significant weight gain during early and mid-pregnancy (Fig. 1C). An OGTT at GD15 was conducted to determine the changes in glucose tolerance during pregnancy. The area under the curve (AUC) for the OGTT of the HFD group was significantly increased compared to that in the control group (Fig. 1D,E). To further assess the effects of HFD on glucose metabolism, the level of fasting serum glucose and insulin was measured at GD16 and used for calculating HOMA-IR. Compared to the control group, mice in the HFD group had a higher HOMA-IR (1.54 $\pm$ 0.76 vs. 1.40 $\pm$ 0.94). These findings supported that mice subjected to HFD were more susceptible to insulin resistance, exhibited glucose intolerance, and developed GDM.

Fig. 1.

YP inhibits the progression of GDM. (A) Schematic design of the experiment. (B) Weight gain during intervention before pregnancy. (C) Weight gain during pregnancy. (D) Oral glucose tolerance tests (OGTT). (E) AUC of OGTT. * p 0.05; ** p 0.01; *** p 0.001; **** p 0.0001; ns, not significant. GDM, gestational diabetes mellitus; AUC, area under the curve; YP, Yam Polysaccharide; HFD, high-fat and fructose diet.

Compared with the HFD group, the YP-treated group exhibited significantly lower levels of blood glucose at 30 min, 60 min, and 90 min during the OGTT, along with a smaller AUC (Fig. 1D,E). A notable decrease in HOMA-IR was also observed in the HFD + YP group compared to the HFD group (1.00 $\pm$ 0.50 vs. 1.54 $\pm$ 0.76). These findings suggested that YP may alleviate impaired glucose tolerance and insulin resistance in the GDM model. Additionally, the addition of YP did not significantly induce weight loss in the pregnancy compared to the HFD group (Fig. 1C).

Additionally, an ex vivo experiment was performed to evaluate the impact of YP on insulin resistance. Murine preadipocyte 3T3-L1 were induced to differentiate into adipocytes (Fig. 2A). Dexamethasone inhibited glucose uptake in adipogenically induced 3T3-L1 cells, indicating successful establishment of an insulin resistance model (Fig. 2B). YP was found to alleviate dexamethasone-induced impairment of glucose uptake in a dose-and time-dependent manner (Fig. 2C,D).

Fig. 2.

YP alleviated dexamethasone (DEX)-induced impaired glucose uptake in 3T3-L1 adipocytes. (A) Red oil O staining demonstrated the adipogenesis induction in 3T3-L1 cells, scale bar: 50 µm. (B) DEX inhibited glucose uptake in 3T3-L1 adipocytes compared to control group. (C,D) YP enhanced glucose uptake in 3T3-L1 adipocytes incubated with 2 µm DEX compared to control group. ns, not significant; DMEM, Dulbecco’s Modified Eagle Medium; PBS, phosphate buffered saline, * p 0.05, *** p 0.001.

Analysis of alpha diversity of GM among the three groups showed that the Shannon index was significantly higher in the HFD group than in the control group, whereas the Simpson index was lower. These findings indicated that the HFD-induced GDM model exhibited increased richness and decreased evenness of microbial communities compared with the control group. (Fig. 3A,B). In addition, YP prevented the HFD-induced increase in alpha diversity (Fig. 3A,B). Subsequently, the beta diversity of microbial communities among the three groups was investigated using PCoA, which revealed three distinct clusters corresponding to the intervention groups. The separation between the HFD and control groups was particularly pronounced, indicating HFD-induced alteration in GM composition. Importantly, YP administration induced additional alterations in the microbial community structure across samples (Fig. 3C).

Fig. 3.

YP changed the composition of gut microbiota (GM). (A) Shannon index in different groups. (B) Simpson index in different groups. (C) PCoA analysis. (D) Relative abundance of microbial species of the top 10 phyla with significant differences. (E) Relative abundance of microbial species of the top 10 genera with significant differences. (F) Relative abundance of microbial species of the top 20 genera with significant differences. ns, no significance. * p 0.05. PCoA, Principal Coordinates Analysis.

We analyzed the relative abundance of species across the three groups at the phylum level (Fig. 3D). The Firmicutes/Bacteroidetes (F/B) ratio was increased in both the HFD and HFD + YP groups compared with the control group (Fig. 3D). An increased F/B ratio is commonly observed in obese individuals [20]. The elevated F/B ratio in the HFD and HFD + YP groups indirectly reflects the effect of the metabolic alteration induced by HFD treatment and is consistent with the finding that YP did not attenuate the HFD-induced weight gain in pregnant mice. At the genus level, the relative abundance of Alloprevotella decreases in the HFD group compared with the control group but increases in the HFD + YP group. In contrast, the relative abundance of Lachnospiraceae NK4A136 group increased in the HFD group but declined with YP treatment (Fig. 3E,F). These findings suggest that Alloprevotella and Lachnospiraceae NK4A136 group may be potential microbial targets through which YP alleviates GM dysbiosis and abnormal glucose metabolism.