Analysis of sex differences in dietary copper-fructose interaction-induced alterations of gut microbial activity in relation to hepatic steatosis - PubMed (original) (raw)

Analysis of sex differences in dietary copper-fructose interaction-induced alterations of gut microbial activity in relation to hepatic steatosis

Ming Song et al. Biol Sex Differ. 2021.

Abstract

Background: Inadequate copper intake and increased fructose consumption represent two important nutritional problems in the USA. Dietary copper-fructose interactions alter gut microbial activity and contribute to the development of nonalcoholic fatty liver disease (NAFLD). The aim of this study is to determine whether dietary copper-fructose interactions alter gut microbial activity in a sex-differential manner and whether sex differences in gut microbial activity are associated with sex differences in hepatic steatosis.

Methods: Male and female weanling Sprague-Dawley (SD) rats were fed ad libitum with an AIN-93G purified rodent diet with defined copper content for 8 weeks. The copper content is 6 mg/kg and 1.5 mg/kg in adequate copper diet (CuA) and marginal copper diet (CuM), respectively. Animals had free access to either deionized water or deionized water containing 10% fructose (F) (w/v) as the only drink during the experiment. Body weight, calorie intake, plasma alanine aminotransferase, aspartate aminotransferase, and liver histology as well as liver triglyceride were evaluated. Fecal microbial contents were analyzed by 16S ribosomal RNA (16S rRNA) sequencing. Fecal and cecal short-chain fatty acids (SCFAs) were determined by gas chromatography-mass spectrometry (GC-MS).

Results: Male and female rats exhibit similar trends of changes in the body weight gain and calorie intake in response to dietary copper and fructose, with a generally higher level in male rats. Several female rats in the CuAF group developed mild steatosis, while no obvious steatosis was observed in male rats fed with CuAF or CuMF diets. Fecal 16S rRNA sequencing analysis revealed distinct alterations of the gut microbiome in male and female rats. Linear discriminant analysis (LDA) effect size (LEfSe) identified sex-specific abundant taxa in different groups. Further, total SCFAs, as well as, butyrate were decreased in a more pronounced manner in female CuMF rats than in male rats. Of note, the decreased SCFAs are concomitant with the reduced SCFA producers, but not correlated to hepatic steatosis.

Conclusions: Our data demonstrated sex differences in the alterations of gut microbial abundance, activities, and hepatic steatosis in response to dietary copper-fructose interaction in rats. The correlation between sex differences in metabolic phenotypes and alterations of gut microbial activities remains elusive.

Keywords: Copper; Fructose; Gut microbiota; Nonalcoholic fatty liver disease; Sex.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

Body weight and calorie intake throughout the 8 weeks of the experiment. Male and female weanling Sprague-Dawley rats were fed with adequate or marginal copper diet and had free access to deionized water or deionized water containing 10% fructose (w/v) for 8 weeks as described in the “Materials and Methods” section. Data represent means ± SD (n = 7–8). Cu, copper; A, adequate copper diet; AF, adequate copper diet +10% fructose (w/v) in the drinking water; M, marginal copper diet; MF, marginal copper diet +10% fructose (w/v) in the drinking water

Fig. 2

Effects of dietary copper-fructose interaction on plasma ALT, AST, liver histology, and fat accumulation. a Plasma ALT and AST. b Representative photos of liver histology using H&E staining. c Hepatic triglyceride. CuAF female rats had macrosteatosis (arrows) around the portal area. Microsteatosis (arrowheads) was observed in female CuMF rats as well as in some male rats as indicated. Data represent means ± SD (n = 7–8). Statistical significance was set at p ≤ 0.05. P values displayed are for the factors copper (Cu), fructose (F), and interaction (Cu × F) using two-way ANOVA followed with Tukey’s multiple comparisons test. A, adequate copper diet; AF, adequate copper diet +10% fructose (w/v) in the drinking water; M, marginal copper diet; MF, marginal copper diet +10% fructose (w/v) in the drinking water

Fig. 3

Effects of dietary copper and fructose on gut bacterial diversity and abundance. a Alpha-diversity: alpha rarefaction curves with each treatment using observed OTU measure and Shannon index. b Beta-diversity: weighted and unweighted UniFrac. c Taxonomic composition (percentage) of the gut microbiota at the phylum level. Cu, copper; A, adequate copper diet; AF, adequate copper diet +10% fructose (w/v) in the drinking water; M, marginal copper diet; MF, marginal copper diet +10% fructose (w/v) in the drinking water. M (first letter in the group name), male; F (first letter in the group name), female

Fig. 4

Relative abundance of gut microbiota at the genus level. Heatmap showing the abundance of 73 fecal gut microbes in a Male rats and b Female rats. Data represent means ± SD (n = 7–8). Statistical significance was set at p ≤ 0.05. P values displayed are for the factors copper (Cu), fructose (F), and interaction (Cu × F) by two-way ANOVA with Tukey’s multiple comparisons test. * versus CuA; # versus CuAF; $ versus CuM. A, adequate copper diet; AF, adequate copper diet +10% fructose (w/v) in the drinking water; M, marginal copper diet; MF, marginal copper diet +10% fructose (w/v) in the drinking water

Fig. 5

Linear discriminant analysis (LDA) effect size (LEfSe) analysis identifies differentially abundant taxa induced by dietary copper and fructose. Cladogram and histogram with LDA score ≥ 2 showing the features with differential abundance of taxa between groups in a male rats and b female rats (Wilcoxon rank-sum test). c Venn diagram. Each circle’s diameter in the cladogram is proportional to the taxon’s abundance. From the outer circle to the inner circle, the circles represent phyla, class, order, family, and genus. Differentially abundant taxa in specific groups were represented in different colors with the exception that yellow represents non-significant in the cladogram. M, male; F, female; Cu, copper; A, adequate copper diet; AF, adequate copper diet +10% fructose (w/v) in the drinking water; M, marginal copper diet; MF, marginal copper diet +10% fructose (w/v) in the drinking water

Fig. 6

Alterations of cecal and fecal SCFA levels induced by dietary copper and fructose. a Total SCFA levels. b SCFA levels (C2–C4). c Percentage of total SCFAs. Data represent means ± SD (n = 7–8). Statistical significance was set at p ≤ 0.05. P values displayed are for the factors copper (Cu), fructose (F), and interaction (Cu × F) by two-way ANOVA with Tukey’s multiple comparisons test. * versus CuA; # versus CuAF; $ versus CuM. Cu, copper; A, adequate copper diet; AF, adequate copper diet +10% fructose (w/v) in the drinking water; M, marginal copper diet; MF, marginal copper diet +10% fructose (w/v) in the drinking water. C2, acetic acid; C3, propionic acid; C4, butyric acid

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Analysis of sex differences in dietary copper-fructose interaction-induced alterations of gut microbial activity in relation to hepatic steatosis - PubMed (original) (raw)