Copper-fructose interaction-induced metabolic effects exhibit sex dimorphism (23, 25). Sex specific alterations of gut microbiota in response to a specific diets have been demonstrated in a variety of studies (57–59). Given that the gut microbiota play a causal role in driving the development of metabolic diseases, we aimed to determine whether sex-specific alterations of the gut microbiota are linked to hepatic steatosis. Our data showed that sex differences do exist in the gut microbiota, gut microbiota metabolites such as SCFAs, and hepatic steatosis following dietary copper and fructose exposure. Female rats exhibited more pronounced alterations in the abundance of various taxa than that did male rats at multiple taxa levels, including phylum, family and genus. The number of distinct abundant taxa identified by LEfSe was also higher in female rats than in male rats. In addition, SCFAs were decreased to a greater extent in female rats compared to male rats, particularly in CuMF group. Moreover, female rats with an adequate copper diet developed mild, but apparent steatosis after 8 weeks of added fructose feeding (CuAF), but female CuMF rats, which showed the most significantly altered gut microbial activity, did not. Therefore, the altered gut microbial activity does not correlate with the hepatic fat accumulation.
SCFAs are the end products of microbial fermentation of indigestible fiber, and they play a critical role in energy homeostasis and metabolism (60). In our study, we found significantly decreased SCFAs, particularly butyrate, concomitant with the reduced butyrate producers, Lachnospiraceae and Ruminococcaceae (61), in CuMF female rats, implying the most significantly altered gut microbial activities in this group. We found mild hepatic steatosis in CuAF female rats; thus, it is unlikely that this hepatic steatosis is attributable to the metabolic effects of gut microbiota. Accelerated de novo lipogenesis (DNL) is known to contribute to fructose-induced hepatic steatosis (62, 63). However, the underlying mechanisms are unclear. A recent study demonstrated a two-point mechanism leading to fructose-induced hepatic steatosis. One part is gut bacteria derived acetate which serves as a substrate for acetyl-CoA synthesis via acyl-CoA synthetase short chain family member 2 (ACSS2) in the liver. Second, fructose metabolism in hepatocytes activates a signal leading to lipogenic gene expression (64). Interestingly, the most significantly changed SCFAs occurred in CuMF rats, in which exacerbated liver injury and steatosis were seen in our previous study when rats were exposed to a high fructose diet via 30% fructose (w/v) in the drinking water and sucrose-enriched diet (AIN-76) (21). This finding suggests that hepatic steatosis may be related to the amount of fructose intake. In support of this, a recent study demonstrated that dietary fructose is primarily metabolized in the small intestine and only excess fructose intake spills over to the colon microbiota and liver (65). Previous studies showed that either inhibition of fructose metabolism in the liver (66) or elimination of gut microbiota by antibiotics (67) protected against fructose induced hepatic steatosis, indicating that fructose metabolism in both liver and gut microbiota is required to facilitate the development of steatosis. When a large amount of fructose intake saturates the capacity of the small intestine metabolism, presumably excess fructose will proceed to the colon, the gut microbiota and liver. However, the priority of excess fructose to be distributed and metabolized in colon microbiota or liver or other tissues is unclear when a modest amount of fructose was ingested. It has been shown that dietary copper-fructose interaction exacerbates copper deficiency-induced metabolic syndrome, likely due to impaired intestinal copper absorption because of excess fructose ingestion (21, 68). Whether the extent of interaction relates to the relative amounts of copper and/or fructose, and subsequent metabolic effects remain largely unknown and warrant further study.
Despite significantly changed gut microbiota and SCFAs in CuMF rats, only a few of the female rats developed modest steatosis in CuAF group, suggesting the altered gut microbial activities were not sufficient to lead to a significant phenotype change in the current study. At this point, it seems that hepatic steatosis and the shifts in gut microbial activity are not correlated. Of note, Porphyromonadaceae and Parabacteroides are two of the microbiota signatures associated with CuAF in female rats, although with relative low abundance (1.52%), which is different from male rats identified by LEfSe. Whether increased abundance of Porphyromonadaceae and Parabacteroides plays a causal role in fructose-induced hepatic steatosis needs to be examined.
Sex difference in fructose-induced metabolic effects are mixed (24, 69, 70). In contrast to previous studies on copper-fructose interactions (23, 25, 26), our results showed that female rats are relative sensitive to fructose-induced hepatic steatosis. The discrepancy may be attributed to several factors. First is the dose of copper and fructose. A lower dose of copper (0.6 ppm) and a higher dose of fructose (30–62%) were used in Field’s as well as in Morrell’s studies (23, 26). It appeared that males are more sensitive to the deleterious effects of copper deficiency. In our study, marginal copper diet (1.5 ppm) and 10% fructose (w/v) in the drinking water were used, presumably leading to less pronounced copper-fructose interactions and metabolic effects than previous studies (23, 26). Second, the activities of fructose-metabolizing enzymes and intermediate metabolites differed by sex and copper level (71). In fact, the activities of liver enzymes involved in lipogenesis was affected not only by the type of carbohydrate, but also by the quantity (72). Lastly, differences in facilities, diet components and species as well as experimental durations may all contribute to discrepancy (25, 73, 74).
In support of our results, a previous study demonstrated that weanling female rats exhibit a higher rate of acetate incorporation into lipids in liver compared to male rats (75), suggesting a higher lipogenic capacity in female rats. However, there is species difference in driving the lipogenic enzyme activity in response to carbohydrate (72). In human studies, fructose-induced increase in hepatic DNL and decrease in fatty acid oxidation was more pronounced in men and premenopausal women than in postmenopausal women (28, 63, 76, 77). Sex hormones are the known factors regulating sex dimorphism of fructose-related metabolic effects (7). However, the molecular underpinnings remain elusive. Recent studies showed that GLUT8 mediates distinct metabolic effects between males and females in response to dietary fructose (29, 30, 78). GLUT8 is a dual-specificity glucose and fructose transporter, which was found to be abundantly expressed in both murine and human liver and intestine (30, 78, 79). Interestingly, while GLUT8 mutation does not alter intestinal fructose absorption in male mice (29), it enhances intestinal fructose absorption in female mice, which was associated exacerbated hypertension, hyperinsulinemia, and hyperlipidemia when fed with high-fructose diet (30). Conversely, GLUT8-deficient male mice are protection from high-fructose diet-induced dyslipidemia, glucose intolerance and hypertension (29). These studies revealed an important molecular mechanism underlying the tissue-specific and sex-specific divergence in response to fructose.
A potential limitation of the current study is the one time analysis of gut microbiota and hepatic steatosis. Although female rats displayed earlier development of steatosis, it is difficult to predict the ultimate severity of steatosis and disease progression. Since male rats exhibit decreased diversity of gut microbiome, and given that the microbial gene richness is associated with inflammation, insulin resistance and dyslipidaemia (80, 81), it is plausible that male rats develop steatosis with a prolonged duration on experimental regime. Thus, long-term and multiple time points evaluation will provide more accurate profiles of disease progression in the context of sex difference. However, sex differences observed in animal studies are under strictly defined experimental conditions. Therefore, a caveat must be noted when extrapolating animal data to human, as humans have much more complex genetic and environmental factors than experimental animals.
Perspectives and Significance
In summary, our current study provides evidence of sex-specific alterations in gut microbial activities and hepatic steatosis in response to dietary copper-fructose interaction in a rat model. However, sex differences in the liver and gut do not seem to be related. Future studies deciphering the molecular mechanisms would help us better understand sex-specific responses to dietary copper-fructose interactions.