Novel Organic Mineral Complex Prevents High-Fat Diet-Induced Changes in the Gut and Liver of Male Sprague-Dawley Rats

Abstract

Diet-induced obesity and metabolic syndrome are associated with the onset of gastrointestinal diseases, such as hepatic steatosis and gut inflammation. Prior research shows that a proprietary soil-derived organic mineral complex (OMC) prevents hyperglycemia, endotoxemia, and liver injury in rats fed a high-fat diet (HFD) for 10 weeks. The aim of this study was to further examine the effects of OMC on the liver and gastrointestinal health of these rats. Six-week-old male Sprague-Dawley rats (n = 36) were divided into two dietary groups: Chow or HFD fed for 10 weeks. Animals were further divided (n = 6/group) and administered 0, 0.6, or 3.0 mg/mL OMC in their drinking water. The 10-week HFD resulted in significant liver fat accumulation. Both OMC doses prevented hepatic increases in the glycation end product Nε-(carboxymethyl)lysine (CML) induced by HFD (p < 0.05). Low-dose OMC was associated with higher expression of occludin in the small intestine of rats fed either diet (two-way ANOVA, p < 0.042). Linear discriminant analysis (LDA) effect size (LEfSe) indicated significant differences in fecal microbial composition of untreated HFD-fed rats in comparison to untreated Chow rats at 10 weeks (LDA score > 2.0 : 18). After 10 weeks, untreated HFD-fed rats were also more abundant in bacteria associated with obesity and metabolic disease in comparison to corresponding week 0 samples (LDA score > 2.0 : 31), 10-week untreated Chow (LDA > 2.0 : 18), or 10-week OMC-treated HFD-fed rats (0.6 mg/mL; LDA > 2.0 : 80, 3.0 mg/mL; LDA > 2.0 : 8). Low-dose OMC prevented the HFD-induced increase in the Firmicutes-to-Bacteroidetes (F/B) ratio (p < 0.0416). Study animals treated with OMC exhibited no significant changes in the gut microbiota at week 10, although gut inflammatory biomarkers were not significantly altered by diet or OMC treatment. These results indicate that OMC supplementation ameliorates glycosylation reactions and modifies HFD-induced alterations in the intestinal microbiota.

Figure 1

Protein expression of inflammatory cytokines in the cecum and small intestine. (a) IL-1β protein expression in cecum homogenates from rats in all treatment groups (n = 6/group), p=0.702. Effects of OMC supplementation on IL-1β expression, p=0.602. HFD vs OMC interaction, p=0.783. (b) NF-κB (p65) protein expression in cecum homogenates from rats in all treatment groups (n = 6/group), p=0.362. Effects of OMC supplementation on NF-κB expression, p=0.803. HFD vs OMC interaction, p=0.855. (c) IL-1β protein expression in small intestine homogenates from rats in all treatment groups (n = 6/group), p=0.603. Effects of OMC supplementation on IL-1β expression, p=0.574. HFD vs OMC interaction, p=0.931. (d) NF-κB (p65) protein expression in small intestine homogenates from rats in all treatment groups (n = 6/group), p=0.746. Effects of OMC supplementation on NF-κB expression, p=0.304. HFD vs OMC interaction, p=0.586. Densitometry values were normalized to Coomassie for all samples, and data are expressed as mean ± SEM and analyzed by two-way ANOVA.

Figure 2

Occludin protein expression in the cecum and small intestine (SI). (a) Occludin protein expression in cecum homogenates from rats in all treatment groups (n = 5-6/group), p=0.686. Effects of OMC supplementation on occludin expression, p=0.605. Diet vs dose interaction, p=0.943. (b) Occludin protein expression in small intestine homogenates from rats in all treatment groups (n = 5-6/group), p=0.259. Effects of OMC supplementation on occludin expression, ^∗p < 0.05 Chow 0.6 mg/mL OMC vs Chow, and ^#p < 0.05 HFD 0.6 mg/mL OMC vs HFD. Diet vs dose interaction, p=0.891. Densitometry values were normalized to Coomassie for all samples. Data are expressed as mean ± SEM and analyzed by two-way ANOVA.

Figure 3

Liver Nε-(carboxymethyl)lysine concentrations. Data are expressed as mean ± SEM and analyzed by two-way ANOVA, n = 6/treatment group, ^#p < 0.05 vs. HFD untreated.

Figure 4

Hepatic lipid accumulation. (a) Free glycerol and (b) triacylglycerol concentrations in liver samples isolated from n = 5 animals in each group. Data are expressed as mean ± SEM and analyzed by two-way ANOVA, ^∗p < 0.05 vs respective Chow control. (c) Representative images of Oil Red O stained hepatic tissues collected from male Sprague-Dawley rats in each treatment group. Frozen tissue sections were stained with Oil Red O to show neutral triglycerides and lipid content and counterstained with hematoxylin to show nuclei (blue). Livers from rats fed HFD for 10 weeks showed evidence of simple steatosis.

Figure 5

Differently abundant taxa after 10 weeks of either Chow or HFD. (a) Linear discriminate analysis of effect size (LEfSe) results depicting different microbial taxonomic signatures in fecal samples collected from male Sprague-Dawley rats fed an HFD (n = 5-6/group) at week 10 in comparison to Chow-fed samples at week 10. Fecal samples collected from HFD-fed rats were more abundant in the following phyla: Bacteroidetes, Firmicutes, Clostridium, Deferribacteres, Proteobacteria, and Tenericutes (LDA > 2.0 : 18). No other differences were detected. (b) Cladogram depicting differentially abundant microbial taxa in stool samples collected from male Sprague-Dawley rats fed a high-fat diet (n = 5-6) for 10 weeks. Microbial taxa enriched in HFD rats are shown in red.

Figure 6

Change in differently abundant taxa between fecal samples of HFD rats collected at week 0 and week 10. (a) Linear discriminate analysis of effect size (LEfSe) results depicting different microbial taxonomic signatures in fecal samples collected from male Sprague-Dawley rats fed HFD (n = 5-6/group) at week 10 in comparison to HFD week 0 samples. Fecal samples collected from HFD-fed rats at week 10 were more abundant in bacteria in the following phyla: Firmicutes, Proteobacteria, Bacteroidetes, Tenericutes, and Actinobacteria. No other differences were detected. (b) Cladogram depicting differentially abundant microbial taxa in stool samples collected from male Sprague-Dawley rats fed HFD (n = 5-6/group) for 10 weeks. Microbial taxa enriched in HFD rats are shown in red.

Figure 7

Effects of low-dose OMC on taxa abundance in rats fed HFD for 10 weeks. (a) Linear discriminate analysis of effect size (LEfSe) results depicting different microbial taxonomic signatures in fecal samples collected from male Sprague-Dawley rats fed a 10-week HFD (n = 6/group) in comparison to HFD-fed rats treated with 0.6 mg/mL OMC for 10 weeks. No other differences were detected. (b) Cladogram depicting differentially abundant microbial taxa in stool samples collected from male Sprague-Dawley rats fed HFD (n = 5-6/group) for 10 weeks. Microbial taxa enriched in HFD rats are shown in red.

Figure 8

Effects of high-dose OMC on taxa abundance in rats fed HFD for 10 weeks. (a) Linear discriminate analysis of effect size (LEfSe) results depicting different microbial taxonomic signatures in fecal samples collected from male Sprague-Dawley rats fed a 10-week HFD (n = 5-6/group) in comparison to HFD-fed rats treated with 3.0 mg/mL OMC for 10 weeks. No other differences were detected. (b) Cladogram depicting differentially abundant microbial taxa in stool samples collected from male Sprague-Dawley rats fed an HFD (n = 5-6/group) for 10 weeks. Microbial taxa enriched in HFD rats are shown in red.

Figure 9

Average percent relative abundance of phyla. (a) Relative abundance of microflora at phylum level in fecal samples of each group at week 0. Data were analyzed by one-way ANOVA followed by Bonferroni post hoc test found no significant differences between groups (n = 5-6/group). 14 phyla were identified. (b) Relative abundance of microflora at phylum level in fecal samples of each group at week 10. Data were analyzed by one-way ANOVA followed by Bonferroni post hoc test found no significant differences between groups (n = 5-6/group). 15 phyla were identified.

Figure 10

Firmicutes-to-Bacteroidetes ratio of all groups at week 0 and week 10. Data were analyzed by two-way ANOVA RM followed by Tukey post hoc test, ^∗denotes p < 0.0355 and p=0.0416 compared to Chow + 3.0 mg/mL and HFD + 0.6 mg/mL F/B ratio at week 10, respectively (n = 5-6/group).