Grape Polyphenols Attenuate Diet-Induced Obesity and Hepatic Steatosis in Mice in Association With Reduced Butyrate and Increased Markers of Intestinal Carbohydrate Oxidation

Abstract

A Western Diet (WD) low in fiber but high in fats and sugars contributes to obesity and non-alcoholic fatty liver disease (NAFLD). Supplementation with grape polyphenols (GPs) rich in B-type proanthocyanidins (PACs) can attenuate symptoms of cardiometabolic disease and alter the gut microbiota and its metabolites. We hypothesized that GP-mediated metabolic improvements would correlate with altered microbial metabolites such as short chain fatty acids (SCFAs). To more closely mimic a WD, C57BL/6J male mice were fed a low-fiber diet high in sucrose and butterfat along with 20% sucrose water to represent sugary beverages. This WD was supplemented with 1% GPs (WD-GP) to investigate the impact of GPs on energy balance, SCFA profile, and intestinal metabolism. Compared to WD-fed mice, the WD-GP group had higher lean mass along with lower fat mass, body weight, and hepatic steatosis despite consuming more calories from sucrose water. Indirect and direct calorimetry revealed that reduced adiposity in GP-supplemented mice was likely due to their greater energy expenditure, which resulted in lower energy efficiency compared to WD-fed mice. GP-supplemented mice had higher abundance of Akkermansia muciniphila, a gut microbe reported to increase energy expenditure. Short chain fatty acid measurements in colon content revealed that GP-supplemented mice had lower concentrations of butyrate, a major energy substrate of the distal intestine, and reduced valerate, a putrefactive SCFA. GP-supplementation also resulted in a lower acetate:propionate ratio suggesting reduced hepatic lipogenesis. Considering the higher sucrose consumption and reduced butyrate levels in GP-supplemented mice, we hypothesized that enterocytes would metabolize glucose and fructose as a replacement energy source. Ileal mRNA levels of glucose transporter-2 (GLUT2, SLC2A2) were increased indicating higher glucose and fructose uptake. Expression of ketohexokinase (KHK) was increased in ileum tissue suggesting increased fructolysis. A GP-induced increase in intestinal carbohydrate oxidation was supported by: (1) increased gene expression of duodenal pyruvate dehydrogenase (PDH), (2) a decreased ratio of lactate dehydrogenase a (LDHa): LDHb in jejunum and colon tissues, and (3) decreased duodenal and colonic lactate concentrations. These data indicate that GPs protect against WD-induced obesity and hepatic steatosis by diminishing portal delivery of lipogenic butyrate and sugars due to their increased intestinal utilization.

Keywords: Akkermansia muciniphila; Western diet; butyrate; energy expenditure; grape polyphenols; hepatic steatosis; intestinal metabolism; short chain fatty acids.

Figure 1

GPs improved body composition and decreased energy efficiency despite greater consumption of calories from carbohydrates. (A) Mean weekly body weight for each diet-group (n = 6–8). (B) Mean body composition, i.e., water, lean, and fat mass as % of body weight (BW), after 7 and 21 weeks of the diet-intervention (n = 6–8). (C) Mean area under the curve (AUC) of caloric intake from macronutrients from both food and sucrose water over the diet-intervention period (n = 6–8/group). Raw data from which AUCs of caloric intake were obtained are shown in Supplementary Figures 2B–E. (D) Energy efficiency (mg of body weight gained per kcal consumed) over the diet-intervention. (E) Fecal caloric density per gram of feces (wet wt.) and (F) total caloric output from feces per day, during week 17 of diet-intervention. (G) Calories absorbed per day (calories consumed—calories excreted) and (H) absorptive efficiency denotes the percentage of calories absorbed by intestine from calories consumed from food and sucrose water at week 17 of diet-intervention. (I) Cecal weight with contents on day of euthanasia (week 24 of diet-intervention). Data are presented as mean ± SD. For (B) significant difference between diet groups was determined by an unpaired, two-tailed, t-test with Welch's correction and for all other panels significant differences were determined by one-way ANOVA followed by Dunnett's multiple comparisons test with WD group as the control; black colored asterisks indicate statistical significance between the LFD vs. WD group and blue-colored asterisks indicate statistical significance between the WD vs. WD-GP group: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Figure 2

GP supplementation increased energy expenditure. (A) Energy expenditure (EE) as kcals expended per minute normalized to kilogram of body weight and (B) kilogram of lean mass during day and night phases at week 10 and 21 of the diet-intervention. Left two panels of A,B show between group comparisons at weeks 10 and 21, and the right two panels compare weeks 10 and 21 within group. (C) Respiratory exchange ratio (RER) at weeks 10 and 21 of diet intervention are shown over time in metabolic chamber. (D) Two-tailed Pearson Correlation Analysis between UCP1 mRNA levels in BAT and body weights at week 24. Data are presented as mean ± SD. Significant difference between diet-groups was determined by one-way ANOVA followed by Dunnett's multiple comparisons test using the WD group as control, except in A Day and Night graphs and in B where an unpaired, two-tailed, t-test with Welch's correction was used. Black colored asterisks indicate statistical significance between same diet groups or LFD vs. WD groups while blue-colored asterisks indicate statistical significance between the WD vs. WD-GP group ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. BW, Body weight.

Figure 3

GP supplementation diminished WD-induced hepatic steatosis. (A) Liver weights at endpoint, after 23 weeks of diet-intervention, (B) Liver weight as a percentage of body weight. (C) Mean percentage of liver coverage by lipid droplets per cross-section, as quantified from hematoxylin and eosin (H&E) staining (n = 3/group); (D) Representative images of murine livers from each diet group with corresponding H&E-stained liver sections (below) showing white lipid droplets; images were captured at 40× magnification. Organ weights as raw weight (left panel) and as percent of body weight (right panel) of (E) white adipose tissue (WAT), and (F) brown adipose tissue (BAT) on day of euthanasia (week 24 of diet-intervention). Data are presented as mean ± SD. Significant differences between groups were determined by one-way ANOVA followed by Dunnett's multiple comparisons test with the WD group as control. Black colored asterisks indicate statistical significance between the LFD vs. WD group and blue-colored asterisks indicate statistical significance between the WD vs. WD-GP group. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. BW, Body weight.

Figure 4

GP supplementation decreased intestinal concentrations of specific SCFAs and lactate. (A) Concentrations of individual SCFAs and BCFAs in colon content. (B) Total SCFA and BCFA concentration. (C) Ratio of acetate to propionate. (D) Lactate concentrations in duodenum and colon tissues and in cecal content. Data are presented as mean ± SD. Significant difference was determined by unpaired, two-tailed, t-test with Welch's correction; ^*p < 0.05, ^**p < 0.01.

Figure 5

Intestinal gene expression. qPCR analyses showing relative mRNA levels of indicated genes or gene ratios expressed in (A) duodenum (B) jejunum, (C) ileum and (D) colon tissues of mice fed WD or WD-GP (n = 6–8 mice per group). Data represent technical duplicates analyzed by 2^−ΔCT method. Data are presented as mean ± SD. Significant difference was determined by an unpaired, two-tailed, t-test with Welch's correction; ^*p < 0.05, ^**p < 0.01.

Figure 6

Illustration of genes and products involved in carbohydrate metabolism.