Table grape consumption reduces adiposity and markers of hepatic lipogenesis and alters gut microbiota in butter fat-fed mice

Abstract

Our objective was to determine if consuming table grapes reduces adiposity and its metabolic consequences and alters gut microbiota in mice fed a high-fat (HF), butter-rich diet. C57BL/6J mice were fed a low-fat (LF) diet or HF diet with 3% or 5% grapes for 11weeks. Total body and inguinal fat were moderately but significantly reduced in mice fed both levels of grapes compared to their controls. Mice fed 5% grapes had lower liver weights and triglyceride levels and decreased expression of glycerol-3-phosphate acyltransferase (Gpat1) compared to the 5% controls. Mice fed 3% grapes had lower hepatic mRNA levels of peroxisome proliferator-activated receptor gamma 2, sterol-CoA desaturase 1, fatty-acid binding protein 4 and Gpat1 compared to the 3% controls. Although grape feeding had only a minor impact on markers of inflammation or lipogenesis in adipose tissue or intestine, 3% of grapes decreased the intestinal abundance of sulfidogenic Desulfobacter spp. and the Bilophila wadsworthia-specific dissimilatory sulfite reductase gene and tended to increase the abundance of the beneficial bacterium Akkermansia muciniphila compared to controls. In addition, Bifidobacterium, Lactobacillus, Allobaculum and several other genera correlated negatively with adiposity. Allobaculum in particular was increased in the LF and 3% grapes groups compared to the HF-fed controls. Notably, grape feeding attenuated the HF-induced impairment in epithelial localization of the intestinal tight junction protein zonula occludens. Collectively, these data indicate that some of the adverse health consequences of consuming an HF diet rich in saturated fat can be attenuated by table grape consumption.

Keywords: Grapes; Intestines; Microbiota; Obesity; Steatosis.

Figure 1

Adiposity indices of C57BL/6J mice fed a low fat (LF) diet or high fat (HF), butter-rich diets with or without 3% or 5% powdered grapes for 10 weeks. (A) Body fat percentages were measured at week 5 and week 10 using dual energy x-ray absorptiometry (DEXA). (B) At week 11, epididymal, inguinal, retroperitoneal, and mesenteric white adipose tissue (WAT) depots were excised and weighed. The weights of the epididymal, inguinal, retroperitoneal, and mesenteric depots were measured, and their sum labelled total WAT. Means ± SEM without a common lowercase letter differ (p<0.05) using one-way ANOVA and Student’s t test. Means ± SEM (n=9–10) sharing the symbol “*” differ using the Bonferroni’s adjustment (p<0.01). 3S, HF diet containing 3% sugar; 3G, HF diet containing 3% grapes; 5S, HF diet containing 5% sugar; 5G, HF diet containing 5% grapes.

Figure 2

Glucose tolerance tests (GTT)s of C57BL/6J mice fed a low fat (LF) diet or high fat (HF), butter-rich diets with or without 3% or 5% powdered grapes for 10 weeks. At weeks 3, 6, and 9, GTTs were conducted on mice fasted for 8 h and injected i.p. with a 20% glucose solution. Data are expressed as total area under the curve (AUC) for the GTTs. Means ± SEM (n=9–10) without a common lowercase letter differ (p<0.05) using one-way ANOVA and Student’s t test. 3S, HF diet containing 3% sugar; 3G, HF diet containing 3% grapes; 5S, HF diet containing 5% sugar; 5G, HF diet containing 5% grapes.

Figure 3

Liver weights (A), liver triglyceride levels (B), ratio of liver weight to body weight (BW) (C), Oil red-O staining of liver, and the expression of several lipogenic genes in liver (E) of C57BL/6J mice fed a low fat (LF) diet or high fat (HF), butter-rich diets with or without 3% or 5% powdered grapes for 10 weeks. qPCR was conducted to measure mRNA abundance of genes associated with hepatic lipogenesis. Means ± SEM (n=9–10) without a common lowercase letter differ (p<0.05) using one-way ANOVA and Student’s t test. 3S, HF diet containing 3% sugar; 3G, HF diet containing 3% grapes; 5S, HF diet containing 5% sugar; 5G, HF diet containing 5% grapes.

Figure 4

The expression of markers of inflammation and lipid metabolism in epididymal and inguinal WAT of C57BL/6J mice fed a low fat (LF) diet or high fat (HF), butter-rich diets with or without 3% or 5% powdered grapes for 10 weeks. qPCR was conducted to measure mRNA abundance of genes associated with inflammation and lipid metabolism in epididymal (EPI; visceral) (A, C) and inguinal (ING; subcutaneous) (B, D) WAT depots. Means ± SEM (n=9–10) without a common lowercase letter differ (p<0.05) using one-way ANOVA and Student’s t test. 3S, HF diet containing 3% sugar; 3G, HF diet containing 3% grapes; 5S, HF diet containing 5% sugar; 5G, HF diet containing 5% grapes.

Figure 5

Ileal mucosa localization of the tight junction protein ZO-1 at the apical area of the ileum epithelium in C57BL/6J mice fed a low fat (LF) diet or high fat (HF), butter-rich diets with or without 3% or 5% powdered grapes for 10 weeks. Localization of ZO-1 was visualized by immunostaining of ileum samples, and the staining was quantified (n=3–4). Arrows indicate impaired localization of ZO-1 at the apical area of the ileum epithelium. Means ± SEM without a common lowercase letter differ (p<0.05) using one-way ANOVA and Student’s t test. 3S, HF diet containing 3% sugar; 3G, HF diet containing 3% grapes; 5S, HF diet containing 5% sugar; 5G, HF diet containing 5% grapes.

Figure 6

Abundance of sulfidogenic bacteria and A. muciniphilia in the intestinal mucosa or digesta of C57BL/6J mice fed a low fat (LF) diet or high fat (HF), butter-rich diets with or without 3% or 5% powdered grapes for 10 weeks. Abundance of B. wadsworthia-specific, functional gene target dissimilatory sulfate reductase (dsrA-Bw) and the targeted sulfidogenic bacterial genus Desulfobacter (DSB) species in the ileum mucosa (A). Abundance of A. muciniphilia in colonic mucosa and digesta and in cecal digesta (B). qPCR was conducted to measure abundance of 16S rRNA and functional genes. Means ± SEM (n=9–10) without a common lowercase letter differ (p<0.05) using one-way ANOVA and Student’s t test. 3S, HF diet containing 3% sugar; 3G, HF diet containing 3% grapes; 5S, HF diet containing 5% sugar; 5G, HF diet containing 5% grapes. Means ± SEM (n=9–10) without a common lowercase letter differ (p<0.05).

Figure 7

Observed bacterial taxa in cecal mucosa of C57BL/6J mice fed a low fat (LF) diet or high fat (HF), butter-rich diets with or without 3% or 5% powdered grapes for 10 weeks. (A) Rarefaction curves of observed species are shown. Samples were rarified to 15,000 sequencing reads per sample. (B) Above - Area Under the Curve (AUC) of rarefaction curves shown for each diet group. Below – Observed species at 15,000 sequencing reads for each diet group. 3S, HF diet containing 3% sugar; 3G, HF diet containing 3% grapes; 5S, HF diet containing 5% sugar; 5G, HF diet containing 5% grapes. Data are presented as means ± SEM (n=9–10) using one-way ANOVA and Dunn’s test for multiple comparisons.

Figure 8

Significantly altered relative abundances of microbial taxa (i.e., A- Firmicutes, B- Actinobacteria, and C- Tenericutes) found across C57BL/6J mice fed a low fat (LF) diet or high fat (HF), butter-rich diets with or without 3% or 5% powdered grapes for 10 weeks. Taxa shown were significantly altered based on Kruskal Wallis test run using QIIME software following filtering of OTUs that were not present in 50% of samples. FDR corrected p values based on this analysis are shown. To conduct multiple comparisons relative abundances were analyzed via ANOVA followed by Dunn’s test for multiple comparisons. 3S, HF diet containing 3% sugar; 3G, HF diet containing 3% grapes; 5S, HF diet containing 5% sugar; 5G, HF diet containing 5% grapes. Data are presented as means ± SEM (n=9–10). Unless otherwise indicated, asterisks show significant differences of HF diets with or without grapes compared to LF control.

Figure 9

The microbial relative abundances from C57BL/6J mice fed a low fat (LF) diet or high fat (HF), butter-rich diets with or without 3% or 5% powdered grapes for 10 weeks. (A) Top Left - PCA plot between low fat (LF) and HF + 3% grapes (HF3G) are shown. Top Right – Pareto plot showing percentage of variance explained by principal components. Below – Histogram showing individual genera that were negatively or positively correlated with LF and/or HF-3G diets. (B) Same as A, but showing PCA of HF-3G vs HF + 3% sugar (HF-3S) groups.

Figure 10

(A) Bifidobacterium spp. relative abundance is negatively correlated with body fat percentage (r = −0.53, p = 0.001) and inguinal fat pad weight (r = −0.48, p = 0.004) as determined by Spearman Correlation Analysis. (B) Heatmap showing relative abundances of taxa that were significantly correlated with body fat percentage and inguinal fat pad weight. Increasing intensity of red color signifies higher abundance and increasing intensity of green color signifies lower relative abundance of taxa shown. LF, low fat; HF, high fat; 3S, HF diet containing 3% sugar; 3G, HF diet containing 3% grapes; 5S, HF diet containing 5% sugar; 5G, HF diet containing 5% grapes.