Body weight changes of male and female rats

Body weight changes of male and female rats are shown in Table 1. When the weight gains of male and female rat groups were compared at the end of the experiment period, no statistically significant difference was found between the male rat groups. In female rats, the highest weight gain was observed in the F group, and the lowest weight gain was observed in the M group. When compared between sexes, it was observed that weight gain was higher in male rats compared to female rats (Table 1).

Table 1 Comparison of body weight changes of male and female rats (C, n = 6; CMC, n = 4; M, n = 6; F, n = 8; FM, n = 8). Intergroup analysis: #Different from M group, one-way ANOVA, post-hoc Bonferroni test (p < 0.05). Cross-sex analysis: adifferent from the same group of females, Student t-test (p < 0.05)Serum biochemistry findings of male and female ratsSerum glucose, insulin, HOMA-IR, and TG values

In male and female rats, serum glucose, insulin, HOMA-IR, and TG levels increased in the F group and decreased in the FM group (Table 2; Fig. S1a and b, Fig. S2a and b, Fig. S3a and b, Fig. S4a and b). Although a decreasing trend in glucose levels was observed in the FM group of male rats compared to the F group, it was not statistically significant (Table 2; Fig. S1a). When compared between sexes, serum glucose levels were found to be lower in male F and FM groups compared to the same female groups, and serum insulin level was lower in the male M group than in the female M group (Table 2). There was no statistically significant difference between the HOMA-IR values of male and female rat groups, and the TG level was lower in the male FM group than in the same female group (Table 2).

Table 2 Serum Glucose (mg/dL), Insulin (pmol/L), HOMA-IR, and TG (mg/dL) values of male and female rats (C, n = 6; CMC, n = 4; M, n = 6; F, n = 8; FM, n = 8). Intergroup analysis: *Different from C group, +different from CMC group, #different from M group, &different from FM group, one-way ANOVA, post-hoc Bonferroni test (p < 0.05). Cross-sex analysis: aDifferent from the same group of females, Student t-test (p < 0.05)Inflammation markers measured in ileal tissue of male and female rats

It was found that IL-1β level was higher in the F group compared to the C group in both male and female rats (Table 3; Fig. S5a and b). It was observed that the IL-1β level decreased in the male FM group compared to the F group (Table 3; Fig. S5a). When male and female rats were compared, the IL-1β level of the male FM group was lower than that of the female FM group (Table 3). In male rats, the TNF-α level, which increased in the fructose group compared to the control group, decreased in the FM group (Table 3; Fig. S6a). In female rats, there was no statistically significant difference between the groups (Table 3; Fig. S6b). When compared between sexes, TNF-α levels were lower in males than females in the CMC group and higher in the F group (Table 3). When NF-κB levels were analyzed in male and female rats, no statistically significant difference was found between groups and sexes (Table 3; Fig. S7a and b).

Table 3 IL-1β (pg/mg), TNF-α (pg/mg), and NF-κB (pg/mg) values measured in the ileum tissue of male and female rats (C, n = 6; CMC, n = 4; M, n = 6; F, n = 8; FM, n = 8). Intergroup analysis: *Different from C group, +different from CMC group, #different from M group, &different from FM group, one-way ANOVA, post-hoc Bonferroni test (p < 0.05). Cross-sex analysis: aDifferent from the same group of females, Student t-test (p < 0.05)Histopathological analysis findings in ileum tissue of male and female ratsHistomorphometric measurements of ileum tissue of male and female rats

In male and female rats, the villus length of the F group was found to be lower than the Cgroup. On the other hand, villus length was higher in the FM group compared to the F group (Table 4; Fig. S8a and b). When male and female rats were compared, villus length was lower in males than females in the FM group but higher in the F group (Table 4).

Table 4 Villus length (µm), villus width (µm), and crypt depth (µm) measurements of ileum tissue of male and female rats (C, n = 6; CMC, n = 4; M, n = 6; F, n = 8; FM, n = 8). Intergroup analysis: *Different from C group, +different from CMC group, #different from M group, βdifferent from F group,&different from FM group, one-way ANOVA, post-hoc Bonferroni test (p < 0.05). Cross-sex analysis: aDifferent from the same group of females, Student t-test (p < 0.05)

There was no statistically significant difference between the groups when the width of the villus was evaluated in male and female rats (Table 4; Fig. S9a and b). When compared between sexes, villus width was higher in males than females in all groups (Table 4).

The crypt depth was lower in the F group of female rats compared to the C and FM groups (Table 4; Fig. S10b). When compared between sexes, crypt depth was higher in males than females in the F and FM groups (Table 4).

Histopathological evaluation of inflammation of the ileum tissue of male and female rats

In both male and female rats, the inflammation score was higher in the F group compared to the C group. In addition, the inflammation score was lower in the FM group compared to the F group in both sexes (Fig. 1a and b).

Fig. 1

Inflammation scores of the ileum tissue of male (a) and female (b) rats (C, n = 6; CMC, n = 4; M, n = 6; F, n = 8; FM, n = 8). Intergroup analysis: *Different from C group, +different from CMC group, #different from M group, &different from FM group, one-way ANOVA, post-hoc Bonferroni test (p < 0.05)

When male and female rats were compared, it was found that the inflammation score of the female FM group was lower than the male FM group. There is no statistically significant difference between other groups.

As a result of histological scoring of ileum tissues, inflammation and epithelial/crypt damage scores of male and female rats in the F group showed a statistically significant increase compared to the C and FM groups, while no tissue regeneration was observed. It was also noted that in addition to inflammation, degeneration of epithelial tissue and crypt structures decreased (with scoring) in the FM group compared to the F group (Fig. 2a and b).

Fig. 2

Histopathological observation of the ileum tissue of male (a) and female (b) rat groups. Epithelial tissue (⬅) and crypt ( ) were observed in all experimental groups. The F and FM groups showed inflammatory cell infiltration (►)and damage to epithelial tissue and crypt structures (➨). Inflammation was absent in the C, CMC, and M groups. Compared with the F group, the ileum of the FM group showed mild inflammatory cells and degeneration of epithelial tissue and crypt structures (Hematoxylin&Eosin, × 200)

Figures 2a and 2b show, respectively, that the male and female groups in the ileum tissues. The ileum histological structure was normal, with integrity of the epithelium, villi, and crypts in the C, CMC, and M groups. No pathological changes, as well as the absence of inflammatory infiltrate are in these groups. Fructose-induced epithelial tissue and crypt structures damage and inflammatory cell infiltrate compared to the control groups.

As a result of histological scoring of ileum tissues, inflammation and epithelial/crypt damage scores of male and female rats in the F group showed a statistically significant increase compared to the C and FM groups, while no tissue regeneration was observed. It was also noted that in addition to inflammation, degeneration of epithelial tissue and crypt structures decreased (with scoring) in the FM group compared to the F group (Fig. 2a and b).

Immunohistochemical analysis findings in ileum tissue of male and female rats

Zonulin, Occludin, Claudin-1, Claudin-2, ZO-1, E-Cadherin, and F-Actin immunoreactivity in ileum tissues of the experimental groups were evaluated by measuring the percentage of immunopositive areas with immunohistochemical analysis.

There was no statistically significant difference between male and female rat groups in the percentage of Zonulin immunopositive area (Table 5; Fig. 3a and b). The percentage of Zonulin immunopositive area was higher in the F and FM groups in male rats than in female rats (Table 5).

Table 5 Zonulin, Occludin, Claudin-1, Claudin-2, ZO-1, E-Cadherin, and F-Actin immunoreactivities in ileum samples of male and female rats (C, n = 6; CMC, n = 4; M, n = 6; F, n = 8; FM, n = 8). Intergroup analysis: *Different from C group, +different from CMC group, #different from M group, &different from FM group, one-way ANOVA, post-hoc Bonferroni test (p < 0.05). Cross-sex analysis: adifferent from the same group of females, Student t-test (p < 0.05)Fig. 3

Zonulin, Occludin, Claudin-1, Claudin-2, ZO-1, E-Cadherin and F-Actin immunoreactivity of male (a) and female (b) rat groups (DAB-Hematoxylin, X400)

There was no statistically significant difference in the percentage of Occludin and Claudin-1 immunopositive areas between male rat groups (Table 5; Fig. 3a). In female rats, the percentage of Occludin and Claudin-1 immunopositive areas was found to be lower in the F group than the C group and they were higher in the FM group compared to the F group (Table 5; Fig. 3b). When compared between sexes, the percentage of Occludin immunopositive area was found to be higher in males than females in all groups, although it was not significant in the FM group. It was observed that the percentage of Claudin-1 immunopositive area was lower in male C and FM groups than in the same female groups (Table 5).

The percentage of Claudin-2 immunopositive area was lower in the F group of male rats compared to the C and FM groups (Table 5; Fig. 3a), and there was no statistically significant difference between the female rat groups (Table 5; Fig. 3b). It was found that the percentage of Claudin-2 immunopositive area of the male F group was lower than the female F group (Table 5).

There was no statistically significant difference between the groups in the percentage of ZO-1 immunopositive areas in male rats (Table 5; Fig. 3a). In female rats, the percentage of ZO-1 immunopositive areas was lower in the F group compared to the C group (Table 5; Fig. 3b). It was observed that the percentage of ZO-1 immunopositive area was higher in the male F group compared to the female F group (Table 5).

There was no statistically significant difference between the groups in the percentage of E-Cadherin immunopositive area in male and female rats (Table 5; Fig. 3a and b). It was observed that the percentage of E-Cadherin immunopositive area in the male F group was lower than in the same female group (Table 5).

In male rats, the percentage of F-Actin immunopositive area was lower in the F group than in the C group (Table 5; Fig. 3a). In female rats, there was no statistically significant difference between the groups (Table 5; Fig. 3b). The percentage of F-Actin immunopositive area was higher in male rats compared to female rats in the C and FM groups (Table 5).

Gut microbiota analysis findings

After examining 64 samples (32 in the male group and 32 in the female group), a total of 8 481 079 readings were carried out in these samples. Among male rat groups, while 684 325 readings were obtained in the C group (n = 6), 608 752 readings in the CMC group (n = 4), 486 629 readings in the M group (n = 6), 1 251 101 readings in the F group (n = 8) and 807 519 readings in the FM group (n = 8), among the female rat groups; 777 755 readings were obtained in the C group (n = 6), 535 473 readings in the CMC group (n = 4), 1 692 113 readings in the M group (n = 6), 821 912 readings in the F group (n = 8) and 815 500 readings in the FM group (n = 8).

Alpha and beta diversity analysis

In this study, the alpha diversity of gut microbiota obtained from male and female rat groups was interpreted using Chao1 and Shannon indices. These indices are useful tools for understanding species diversity in ecosystems. The Chao1 index provides an estimated species richness by consideringthe impact of rare species, while the Shannon index measures diversity by consideringthe evenness between species.

According to the Chao1 and Shannon indices, alpha diversity, which refers to ASV richness and microbial abundance, increased in the M group compared to the FM and C groups in males. Although there were changes in other groups in terms of microbial findings, no significant difference was observed (Fig. 4a and Fig. S12a; Tables S2 and S5). In female rat groups, no statistically significant difference was observed (Fig. S11b and S12b; Tables S3 and S6). When the male and female rat groups were compared, it was observed that the diversity scale increased in males and narrowed in females, but it was not statistically significant (Fig. 4b and Fig. S12c; Tables S4 and S7).

Fig. 4

Demonstration of microbiome differences between male and female groups with boxplots and dot plots. Alpha and beta diversity plots to show the differences in microbiota structure between groups (C, n = 6; CMC, n = 4; M, n = 6; F, n = 8; FM, n = 8); a Chao1 diversity indices of the gut microbiota of male rat groups. b Shannon diversity indices of the gut microbiota of male/female rat groups (F = Female (Green), M = Male (Blue)) (Boxes indicate first and third quartiles, dashed lines indicate upper and lower whiskers and horizontal thick lines indicate median). c-d-e 2D PCoA plots between groups of male, female, and male/female rats (each dot represents a sample)

Beta diversity analysis used to assess individual taxonomic differences is usually performed using beta diversity indices such as Jaccard, Bray–Curtis, or Sørensen. These indices measure species composition similarities or differences. This study, beta diversity measurement was performed using Principal Coordinates Analysis (PCoA) based on Bray–Curtis distances. According to PCoA, the F group showed different clustering compared to the C group in both male and female rats (Fig. 4c and d). It was observed that the FM group clustered differently compared to the F group in male rats. Metformin altered the gut microbiota of the FM group, causing it to move away from the C group (Fig. 4c). In female rats, there was a close clustering of the F and FM groups and metformin slightly changed the gut microbiota of the FM group, but this was insufficient to approximate it to the C group (Fig. 4d). There was a difference between male and female rats in terms of beta diversity measured by PCoA based on Bray–Curtis distance (Fig. 4e).

Adonis (Table S8), Multiple Response Permutation Procedure (MRPP) (Table S9), Non-Metric Multidimensional Scaling (NMDS) (Table S10), and Anosim (Fig. S13) analyses were used for the statistical analysis of beta diversity sample groupings. According to the analysis results, the groupings differ and are correct to some extent.

Adonis analysis gives information about whether grouping is sufficient or not. Likewise, MRPP analysis is also used for this purpose. When the analyses in Table 8 is examined statistically, it is seen that the p values are significant and the groupings made in our study are sufficient. In MRPP analysis, A values provide information about whether groups have different characteristics; positive values of A indicate that the groups have different properties. According to MRPP analysis, A values are positive, and the groups created in our study have different characteristics (Table S9).

The Anosym analysis confirms the Adonis and MRPP analysis results while also providing information about whether the grouping is valuable. According to Anosym analysis, to say that there is a difference between groups, "R", which can take values between -1 and 1, must have a value above zero. When the R-value takes a value between -1 and 0, the groups are considered to be similar to each other, and when it takes a value between 0 and 1, they are considered to be different from each other; the closer the R-value is to 0, the more similar the groups are to each other. The R values of male rat groups (Fig. S13a), female rat groups (Fig. S13b), and sex-based groups (Fig. S13c), were calculated as 0.24, 0.382, and 0.046, respectively, and it is seen that the groupings are different from each other in our study.

According to NMDS, the grouping is considered correct when the R2 value is above 0.8. According to the NMDS stress analysis based on Bray–Curtis distances, R2 values are above 0.8, and the statistical groupings made in our study were made correctly (Table S10).

When the analysis results are taken together, it is possible to say that the groups created for the study have different characteristics from each other and that the groupings are made correctly and are sufficient.

Taxonomic evaluation

Taxonomic bar graphs were used to analyze the composition of the gut microbiota at the phylum level. The taxonomic distribution (abundance) at the phylum level is given in Fig. 5a in male rat groups and in Fig. 5b in female rat groups, respectively.

Fig. 5

Taxonomic relative abundance distribution at phylum level between a) male and b) female rat groups (C, n = 6; CMC, n = 4; M, n = 6; F, n = 8; FM, n = 8). TM7: Candidatus Saccharibacteria (TM7) is a division under a large bacterial phylum. Saccharibacteria, formerly known as TM7, is a major bacterial lineage. “Other” is formed by summing the relative abundances of phyla with relative abundances below 1%

It was observed that the relative abundance of Firmicutes phylum was higher in male rats in the F group than in the M group, and in female rats in the F and FM groups compared to the C and CMC groups (Fig. 6a). The relative abundance of the Bacteroidetes phylum in the F and FM groups was lower in male rats than in the M group, and in female rats compared to the C, CMC and M groups (Fig. 6b). The F/B ratio of male rats was higher in the F group than in the M group. In female rats, it was found to be higher in the F and FM groups than in the C group and also in the F group compared to the CMC group (Fig. 6c). When compared between sexes, the relative abundance of the Firmicutes phylum was lower in male M and FM groups than in the same female groups. There was no significant difference between male and female rat groups in terms of Bacteroidetes and F/B ratios (Table S1).

Fig. 6

The graphs of a) Firmicutes b) Bacteroidetes c) F/B ratio d) Proteobacteria e) Verrucomicrobia, and f) Actinobacteria relative abundance in the intestinal microbiota of male and female rat groups (C, n = 6; CMC, n = 4; M, n = 6; F, n = 8; FM, n = 8). Intergroup analysis: *Different from C group, +different from CMC group, #different from M group, βdifferent from F group, &different from FM group, one-way ANOVA, post-hoc Bonferroni test (p < 0.05)

There was no significant difference in the relative abundance of the Proteobacteria phylum between male rat groups. The relative abundance of the Proteobacteria phylum was found to be higher in the C and M groups of female rats than in the F and FM groups (Fig. 6d). While the relative abundance of the Proteobacteria phylum was lower in males compared to females in the M group, it was higher in the FM group (Table S1).

There was no significant difference in the relative abundance of the Verrucomicrobia phylum between the groups in male and female rats (Fig. 6e). In females, Verrucomicrobia phylum could not be detected in group F as a result of the applied method. Therefore, when compared between sexes, the Verrucomicrobia phylum's relative abundance was higher in the male F group than in the female F group (Table S1).

The relative abundance of the Actinobacteria phylum was higher in male rats in the F and FM groups than in the C, CMC, and M groups. In female rats, it was lower in the M group compared to the C, CMC, and F groups. In both male and female rats, it was found to be lower in the FM group than in the F group (Fig. 6f). The relative abundance of the Actinobacteria phylum was lower in the CMC group and higher in the F and FM groups in males compared to females (Table S1).

The LEfSe analysis method was applied for the graphical representation of the taxa that were differentially abundant between the groups and their effect sizes and phylogenetic relationships. According to the LEfSe analysis, the graphs created show microbial communities whose relative abundances differ notably at all taxonomic levels in the gut microbiota of male, female, and sex-based rat groups (Fig. 7a, b, c). Among the microbial communities in question, those whose LDA score is higher than the logarithmic threshold value of 2 are defined as biomarkers. In our study, according to LEfSe analysis, at the genus and species level.

Fig. 7

LDA scoring graph created to show significant microorganism differences between male and female rat groups (C, n = 6; CMC, n = 4; M, n = 6; F, n = 8; FM, n = 8) (LDA score > 2, P < 0.05); a Male rat groups b) Female rat groups c) Male and female together

In male rats, Ruminococcus, Butyricococcus, Lactobacillus hamsteri, and Butyricococcus pulliaceorium differed in the CMC group; Ruminococcus flavefaciens differed in the M group; Bifidobacterium pseudolongum differed in the F group; Bifidobacterium, Corynebacterium, Mucispirillum, Lactobacillus helveticus, Lactobacillus reuteri, Corynebacterium lubricantis, Corynebacterium variable, Bacteroides acidifaciens, and Mucispirillum schaedleri differed in the FM group (Fig. 7a).

In female rats, Ruminococcus differed in the CMC group; Parabacteroides, Alistipes, Aggregatibacter, Bacteroides acidifaciens, Bacteroides ovatus, Alistipes finegoldi, and Aggregatibacter pneumotropica differed in the M group; Akkermansia, Collinsella, Akkermansia muciniphilla, Lactobacillus helveticus, and Lactobacillus reuteri differed in the F group (Fig. 7b).

According to the grouping of male and female rats among themselves, Faecalibacterium, Collinsella, Prevotellave, Alicyclobacillus, Lactobacillus reuteri, Faecalibacterium prausnitzii, Collinsella aerofaciens, Prevotella copri, and Alicyclobacillus ferripilum differed in females; Akkermansia, Corynebacterium, Alcaligenes, Akkermansia muciniphila, Corynebacterium lubricantis, Corynebacterium stationis, Bacteroides uniformis, Corynebacterium variabile, Bifidobacterium animalis, and Alcaligenes faecalis differed in males (Fig. 7c).