The incidence of STZ-HFD induced HCC is significantly higher in male mice than in female mice

STZ-primed neonatal mice fed with HFD resulted in HCC at week 20. In 100% of the male mice (n = 8), HCC liver tumors were observed (Fig. 1, arrowhead). However, we observed that 1 out of 8 female mice developed liver tumors and the number of tumors in the single female mouse was significantly lower than those found in male mice (Fig. 1A and B). Regardless of sex, liver to body weight ratio, fasting serum glucose, serum triglyceride (TG), serum lipopolysaccharide (LPS), ALT, alpha-fetoprotein (AFP), and mRNA expression of Collagen type I (Col I) and Glypican-3 (Gpc-3) were significantly higher in STZ-HFD-exposed mice than the controls (Fig. 1C). When grouped by sex, no significant differences were observed in controls whereas male mice that underwent STZ-HFD intervention had statistically significant higher liver to body weight ratio, fasting serum glucose, serum TG, serum LPS, ALT, AFP, mRNA levels of Gpc-3 and Col I relative to females. Based on our results, the incidence of HCC in male STZ-HFD mice was 100% vs. a 12.5% HCC incidence observed in female STZ-HFD mice, thus revealing a clear sex disparity for development of HCC.

Figure 1: Pathophysiological features of NASH–HCC model mice. (A) Representative macroscopic photographs of mouse livers. Arrowheads indicate HCCs; (B) H&E stained liver sections from control and STZ-HFD mice from males and females. Bar = 72 μm; (C) Bar plots of liver to body weight ratio, serum levels of fasting glucose, TG, LPS, ALT and AFP, and mRNA levels of Col I and Gpc-3 in liver. a, p < 0.05, compared to controls; b, p < 0.05, females compared to males. Full size image

STZ-HFD intervention induced significant alteration in gut microbiota

To monitor shifts in the composition of fecal microbiota in the development of HCC, Illumina MiSeq sequencing was performed. In total, 969585 valid sequences were generated and a total of 639057 reads (average of 31953 ± 3692 S.D. reads per sample) were obtained for 20 samples (n = 5 in each group) after quality control. A total of 1159 operational taxonomic units (OTUs) were then identified by grouping reads at the 97% similarity level. The Shannon and Chao1 indices all reached stable values as indicated by the observed plateaus seen in for each group (Supplementary Fig. S1A,B). This indicated that most of the bacterial richness, ie., the number of taxa (species) present in a sample at a particular phylogenetic level (Chao1 index) and diversity, ie., a metric that combines both richness and the evenness of abundance of different taxa (Shannon index) in these communities were covered (Supplementary Fig. S1A,B). The Rarefaction curves revealed that although new rare phylotypes would be expected with additional sequencing, most of the diversity had already been captured as each curve has started to plateau (Supplementary Fig. S1C). Compared with the controls, the STZ-HFD group exhibited lower alpha-diversity as indicated by Chao1 (t test, P = 0.005), ACE (t test, P = 0.006) and Shannon (t test, P = 0.14) for both males and females (Supplementary Table S1). The Simpson (t test, P = 0.04) index is also a measure of diversity and was also significantly different between STZ-HFD mice and controls but the interpretation of this index with respect to our data is that control mice had a slightly higher value indicating more dominance from one taxa relative to the STZ-HFD groups. This was confirmed at the level of phylum in Fig. 2A. ACE or Chao1 were significantly different between control and STZ-HFD group in female mice but with no significant difference in male mice, highlighting sex differences in community richness with STZ-HFD female mice showing lower community richness relative to males. This result is also evident from the rarefaction curve (Supplementary Fig. S1C). In contrast, a significant difference in the Simpson index was only observed between control and STZ-HFD male mice (Supplementary Table S1). All of the indices describing microbiota α-diversity were found be significant when comparing STZ-HFD male vs. female mice. Female STZ-HFD mice scored lower in both diversity and richness relative to the male STZ-HFD mice. These results highlight the sex specific shifts in gut microbiota that occurred upon STZ-HFD treatment.

Figure 2 (A) Bar charts summarizing overall microbial composition of control (n = 5) and STZ-HFD mice (n = 5) at phylum level. (B) Fold change of gut microbiota at the phylum level in males relative to females in both control group and STZ-HFD group. (C) Taxonomic representation of statistical differences in relative abundances between STZ-HFD-exposed female and male mice. Linear discriminant analysis Effect Size (LEfSe) was conducted on relative taxonomic abundances from phylum until genus level. Differences are represented in the colour of the most abundant class (red: female, green: male, yellow: non-significant (p < 0.05)). Each circle’s diameter is proportional to the taxon’s abundance. (D) Bar charts of representative gut microbiota involved in BA metabolism with significant change due to STZ-HFD intervention. a,bp < 0.05, model vs. normal or normal male vs. normal female or model male vs model female (Mean ± SE). (E) OPLS-DA scores plot (R2X = 0.766, R2Y = 0.957, Q2 = 0.721) of mouse gut mcirobiota profiles involved in BA metabolism for classification by sex and STZ-HFD treatment. Full size image

At the phylum level, the majority of the bacterial phyla identified in the fecal samples were encompassed by Bacteriodetes (73.1% in control male mice and 67.4% in control female mice, 59.9% in STZ-HFD male mice and 57.3% in STZ-HFD female mice, on average) and Firmicutes (18.9% in control male mice and 26.5% in control female mice, 24.3% in STZ-HFD male mice and 13.5% in STZ-HFD female mice, on average) as depicted in Fig. 2A. This is also reflected by the relatively high Simpson index (Supplementary Table S1).

The relative amounts measured for other bacteria were; (1) Proteobacteria (6.7% in control male mice and 5.7% in control female mice, 13.9% in STZ-HFD male mice and 28.5% in STZ-HFD female mice, on average), (2) Deferribacteres (1.0% in control male mice and 0.2% in control female mice, 1.0% in STZ-HFD male mice and 0.3% in STZ-HFD female mice, on average), and (3) Actinobacteria (0.1% in control male mice and 0.1% in control female mice, 0.7% in STZ-HFD male mice and 0.1% in STZ-HFD female mice, on average). Twenty weeks of HFD feeding induced widespread changes in gut microbial community structure at the phylum level, with abundances of Proteobacteria increased and abundances of Bacteroidetes decreased in all mice. Interestingly, Firmicutes were decreased significantly after 20 weeks of HFD feeding in female mice, in contrast to a significantly increased Firmicutes population in male mice. The ratio of Firmicutes to Bacteroidetes was markedly increased upon HFD in male mice (0.26 to 0.41) and decreased significantly in female mice (0.39 to 0.24). Verrucomicrobia was significantly decreased in male mice but was increased in female mice. As shown in Fig. 2B, differences in gut microbiota at the phylum level were observed between males and females in the controls and the difference remained after STZ-HFD intervention.

Identification of bacterial taxa abundances associated with STZ-HFD intervention and sex

Microbial compositions of STZ-HFD in male and female mice were compared by applying the linear discriminant analysis (LDA) effect size (LEfSe) algorithm on relative taxonomic abundances at different phylogenetic levels (from phylum until genus level). When compared to controls (Supplementary Fig. S2A,B), STZ-HFD mice showed decreased abundance of Coriobacteriaceae, Bacteroidaceae, Paraprevotellaceae, Prevotella, Lactobacillus, Lactobacillaceae, Anaerostipes, Coprobacillus, and Erysipelotrichaceae. On the other hand, Corynebacterium, Corynebacteriaceae, Rhodococcus, Nocardiaceae, Streptophyta, Bacillus, Bacillaceae, Staphyiococcus, Aerococcus, Enterococcus, Allobaculum, Erysipelotrichales, Klebsiella, Acinetobacter, Pseudomonadales, Enterobacteriales and Turicibacteraies were significantly increased in STZ-HFD-exposed mice, compared to control mice, based on the alpha-values for the factorial Kruskal-Wallis test between groups (p < 0.05) and the logarithmic LDA score (>2.0). Next, sex-dependent differences in taxa were identified by directly comparing STZ-HFD exposed males with STZ-HFD-exposed females (Fig. 2C and Supplementary Fig. S2C). This revealed a higher abundance of Corynebacterium, Corynebacteriaceae, Rhodococcus, Nocardiaceae, Adlercreutzia which belong to the phylum Actinobacteria, Bacillus, Bacillaceae, Staphylococcus, and Staphylococcaceae within the class of Bacilli, Desulfovibrio and Desulvibrionales within the phylum of Proteobacteria, and Clostrodium within the phylum of Firmicutes in male mice when compared to female mice. In particular, we observed that the bacteria involved in BA metabolism were different between males and females and became significantly different after STZ-HFD intervention (Fig. 2D).

As revealed by the OPLS-DA scores plot established using gut microbiota involved in BA metabolism (R2X = 766, R2Y = 0.957, Q2(cum) = 0.721), the control male, control female and STZ-HFD female mice were located in the first and second quadrant while STZ-HFD male mice were located at the fourth quadrant away from the controls (Fig. 2E).

We also performed the MANOVA on the first three weighted microbial PCoA axes and found that the influence of STZ-HFD intervention (p < 0.0001), sex (p = 0.001) and the interaction of STZ-HFD intervention and sex were significant (p < 0.0001) per the Wilks’ test. Thus far we have established; (1) that male mice are more susceptible to HCC, (2) that there are significant sex disparities in gut microbiota in STZ-HFD treated mice, (3) significant differences at the phylum level exist between male and females both in control and STZ-HFD mice, (4) significant differences in BA metabolizing microbiota were present in male vs. female mice for both control and STZ-HFD groups.

STZ-HFD resulted in significantly higher levels of hepatic BAs in male mice than in female mice

Given the significant sex-associated differences in BA metabolizing microbiota, we next investigated the hepatic BA profiles in the mice. STZ-HFD treatment led to significantly altered liver BA concentrations in both sexes (Fig. 3A) as revealed by the OPLS-DA scores plot established using hepatic BA data (R2X = 0.801, R2Y = 0.739, Q2(cum) = 0.607). The hepatic BAs, 3-ketodeoxycholic acid (3-ketoDCA), taurocholic acid (TCA), taurolithocholic acid (TLCA), taurochenodeoxycholic acid (TCDCA), and 7-ketodeoxycholic acid (7-ketoDCA), were significantly increased in STZ-HFD-exposed mice compared to controls (Fig. 3 and Supplementary Fig. S3). More substantial increases in hepatic BA levels were observed in male STZ-HFD mice. Moreover, the increase in TLCA was only observed in males exposed to STZ-HFD and decreased levels of TLCA was observed, but with no statistical significance, in females (Fig. 3B). Among the significantly altered liver BAs, TCA, TCDCA, TLCA, 7-ketoDCA and 3-ketoDCA were significantly higher in males than in females after STZ-HFD intervention (Fig. 3C).

Figure 3: Hepatic BA profiles are significantly different between males and females upon STZ-HFD exposure. (A) OPLS-DA scores plot (R2X = 0.801, R2Y = 0.739, Q2 = 0.607) of mouse hepatic BA profiles for classification by sex and STZ-HFD treatment. (B) Heatmap showing the fold change values of mean concentration of BAs for STZ-HFD model group compared to control group in males and females and for males compared to females in control group and model group. (C) Bar plots of representative BAs with significant change due to STZ-HFD intervention. a,bp < 0.05, model vs. normal or normal male vs. normal female or model male vs model female (Mean ± SE). (D) The mRNA expression of genes in normal group and STZ-HFD intervention group with quantitative real-time polymerase chain reaction (qRT-PCR) analysis in male and female mice. ap < 0.05, compared to controls; bp < 0.05, females compared to males. Full size image

STZ-HFD also led to significant increases in fecal and serum BA levels. Fecal BAs, TDCA, GLCA, GDCA, and GCA were increased in male STZ-HFD mice relative to control. The results were more variable for female mice with TDCA, GDCA and GCA showing increases with STZ-HFD and GLCA slightly but significantly decreased in the model vs. control (Supplementary Fig. S3). GDCA, TDCA, and GLCA, secondary, microbiota metabolized BAs were significantly higher in male relative to female model mice (Supplementary Fig. S3).

Serum concentrations of TCDCA, TCA, ACA, TLCA, 3-ketoDCA, and 7-ketoDCA were lower in control male vs. female mice (Supplementary Fig. S4). Notably, serum levels of TCDCA, ACA, 3-ketoDCA and 7-ketoDCA were found to be significantly higher in STZ-HFD treated male relative to female mice. This flip from low to high concentration of specific BAs in male relative to female model mice reminds us of the flip in abundance discussed earlier for the Firmicutes/Bacteriodetes ratio which showed the ratio to go from low in control to high in STZ-HFD males and vice versa in female mice. Both of these results indicate sex specific changes upon STZ-HFD treatment. In order to determine whether BA transport into and out of the liver was affected by STZ-HFD and was responsible for the greater increase in hepatic BAs for STZ-HFD male mice, we next examined mRNA expression for the BA transporter genes.

Sex disparity was found in the expression of hepatic BA transporter mRNA

A qRT-PCR analysis revealed that genes involved in hepatic BA transport and synthesis were significantly different between sexes. In STZ-HFD treated male mice, hepatic FXR expression was significantly decreased. In STZ-HFD female mice FXR showed a decreasing trend that was not statistically significant. FXR is known to regulate the SHP and thus, accompanying the decrease in FXR mRNA expression was a decrease in mRNA expression for SHP for both male and female STZ-HFD mice. A depressed expression of FXR mRNA could also explain decreased expression of mRNA for BA transporters. The expression of mRNA for the major BA uptake transporter, the sodium-taurocholate cotransporting polypeptide (NTCP), was suppressed by STZ-HFD treatment (Fig. 3D). In addition, the bile salt export pump (BSEP) mRNA was found to be significantly decreased in male model mice relative to control. The female model mice exhibited BSEP mRNA levels that were significantly decreased relative to control but significantly increased with respect to male model results. Thus, these alterations in BA transport may lead to increased BA accumulation in hepatocytes and BA-induced liver injury. The expression of mRNA for BA synthesis, CYP7A1 and CYP7B1, was sinificantly down-regulated after STZ-HFD intervention in male STZ-HFD mice relative to control but the smaller decrease observed for female STZ-HFD mice was not statistically significant. Notably, in female mice, no significant difference in the mRNA expression of hepatic SHP, CYP7A1, and CYP7B1 was found between model and normal mice (Fig. 3D).

The mRNA expression of FXR, CYP7B1, BSEP and SHP, was lower and expression of NTCP and CYP7A1 were higher in normal female mice when compared to normal male mice. The expression of the above-mentioned genes was less altered in female mice than in male mice when exposed to STZ-HFD (Fig. 3D).

Hepatic expression of miRNAs was significantly different between STZ-HFD treated male and female mice

Since the expression of miRNAs are different between men and women with HCC and can be regulated by BAs20,21,22, we further analyzed miRNAs in liver tissues of male and female mice from the STZ-HFD model group and control group. As shown in Fig. 4, the tumor suppressive miRNAs, miR-26a, miR-26a-1, miR-192, miR-122, miR-22, and miR-125b were lower, whereas the tumor-promoting miRNAs, miR-10b and miR-99b were higher in males than in females in both the STZ-HFD group and the control group. As expected, the expression of tumor-suppressive miRNAs were decreased whereas the tumor-promoting miRNAs were increased much more in male mice than in female mice after STZ-HFD treatment, which presumably facilitated the development of liver tumors in male model mice.

Figure 4: Hepatic expression of tumor suppressive miRNAs, miR-26a, miR-26a-1, miR-192, miR-122, miR-22 and miR-125b, and tumor promoting miRNAs, miR-10b and miR-99b in NASH-HCC model male and female mice. Data are presented as the mean ± S.E. *p < 0.05, model compared to control. Full size image

BA-binding resin treatment can prevent HCC in male mice with recovered levels of differentially expressed BAs, gut microbiota and miRNAs

The levels of BAs including TCA, TCDCA, TLCA, 3-keto DCA, and 7-keto DCA, and the gut microbiota including Corynebacterium, Corynebacteriaceae, Rhodococcus, Nocardiaceae, Adlercreutzia, Bacillus, Bacillaceae, Staphylococcus, Staphylococcaceae, Lactobacillales, Desulfovibrio, Desulvibrionales, Clostrodium, and Clostridiales, were much higher in male STZ-HFD mice than in female STZ-HFD mice. The miRNAs were also significantly different between males and females. In a separate study using the STZ-HFD mice model we used a BA sequestrant, cholestyramine, to remove the intestinal BAs in male mice. We observed that depletion of secondary BAs in the intestine by cholestyramine prevented the STZ-HFD male mice from developing tumors, none in the cholestyramine treatment group (n = 8) developed tumor while all of the mice in the model group (n = 8) developed liver tumors (Fig. 5A and B). After cholestyramine administration, the levels of BAs, TCA, TCDCA, TLCA, 3-keto DCA and 7-keto DCA, were significantly decreased in the liver (Fig. 5C). The abnormal gut microbial profile and miRNAs were also normalized with cholestyramine intervention (Fig. 5D and E).

Figure 5 (A) Representative macroscopic photographs of livers. Arrowheads indicate HCC. (B) H&E stained liver sections from normal, NASH-HCC and NASH-HCC-cholestyramine mice at week 20. Bar = 90 μm. (C) Levels of BAs significantly increased in NASH-HCC mice were attenuated after cholestyramine treatment. (D) Relative abundances of altered Clostridium, Desulfovibrio, Staphylococcus, Adlercreutzia, Corynebacterium and Lactobacillius were normalized after cholestyramine treatment. (E) Expression of altered miRNAs, miR-26a, miR-26a-1, miR-192, miR-122, miR-22, miR-125b, miR-10b and miR-99b were normalized after cholestyramine treatment. Full size image

The BA metabolic profiles were significantly different between men and women

Results from our recently published data18 showed that the serum BA levels including TCA, TCDCA, TLCA, 7-keto DCA, 3-keto DCA, DCA and GCA were significantly different between healthy men and women, similar to the mice data (Supplementary Fig. S4). To verify the findings from the animal studies that differentially expressed BAs impact liver carcinogenesis in a sex dependent manner, we profiled the serum BAs in age and BMI matched liver disease patients and healthy participants of men and women. Serum BA measurement in liver fibrosis (n = 30, 15 males and 15 females aged 50–75 years), cirrhosis (n = 40, 20 males and 20 females aged 50–75 years), and HCC (n = 40, 30 males and 10 females aged 50–75 years) patients and healthy participants (n = 40, 20 males and 20 females aged 50–75 years) showed that the levels of BAs differentially expressed between healthy men and women were significantly increased in patients (both sexes) but with higher fold changes in men than in women in the development of liver disease (Fig. 6 and Supplementary Fig. S4).