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

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.


Introduction
Metabolic syndrome is characterized by a cluster of pathologies consisting of hypertension, insulin resistance, abdominal obesity, and dyslipidemia [1]. Obesity further contributes to the development of a multitude of hepatic and gastrointestinal (GI) disorders including nonalcoholic fatty liver disease (NAFLD) [2]. Characterized by steatosis (excessive buildup of fat), NAFLD is the most prevalent chronic liver disorder in the world [3], affecting over 65 million Americans with an estimated annual financial burden of $103 billion [4]. Concurrent with the rise of obesity and type 2 diabetes [5], the increase in NAFLD is strongly associated with consumption of a Westernized diet [6]. Hallmarked by high-fat and low-dietary fiber intake, this dietary pattern also modulates the gut microbiome, increasing intestinal dysbiosis, gut wall permeability, and translocation of microbial products (e.g., endotoxins from Gram-negative bacteria) into the circulation [7][8][9][10].
ese events can ultimately initiate and sustain proinflammatory and oxidative cascades that can lead to liver damage and disease [11,12]. In turn, liver products (e.g., bile acids) can influence the gut microbiota composition and intestinal barrier integrity [13,14]. erefore, the so-called "gut-liver axis" is increasingly recognized as an important factor in the etiology of metabolic syndrome and NAFLD [11,15]. Due to the complexity of this interaction, many open research questions have emerged, particularly in relation to overall diet and dietary components.
Previously, we reported that short-term consumption of a high-fat diet (HFD; 60% kcal fat) by male Sprague-Dawley rats for 6 weeks promoted simple steatosis through an increase of hepatic lipid accumulation [16]. Furthermore, hepatic injury, lipopolysaccharide (LPS) concentrations, and cecal nuclear factor kappa B (NF-κB) and interleukin-1β (IL-1β) expression were significantly increased in HFD rats [16]. Additionally, we found that 6-week HFD increased the abundance of Gram-positive bacteria, which has been associated with metabolic disease [16]. A similar study by Jensen et al. indicated that 16 weeks of HFD (60% fat, 20% carbohydrate) consumption resulted in extensive steatosis and inflammation in male Sprague-Dawley rats [5]. Moreover, hepatic dysfunction was confirmed in these HFDfed rats along with increased levels of liver triacylglycerol and inflammatory markers [5]. Together these findings suggest that an HFD can have profound deleterious effects on liver and GI health.
A first-line treatment recommended for NAFLD and the traditional treatment for obesity and associated comorbidities are dietary intervention [17]. Unfortunately, the success of these interventions is transient, as weight regain and regression to previous health status and poor dietary habits are well-characterized [18]. erefore, it is important to identify more effective strategies for prevention and treatment of metabolic syndrome and NAFLD. Natural food products and their active components may offer achievable alternatives for the treatment of NAFLD with few adverse effects. e use of natural products and herbal medicines for the management of metabolic diseases continues to rise in popularity in Western societies [19]. Prior research found that a soil-derived mineral extract decreased weight gain, blood glucose, and glycated hemoglobin in a diabetic animal model [20]. Additionally, a recent study from our laboratory demonstrated that an organic mineral complex (OMC) derived from plant and soil fractions prevented symptoms of metabolic syndrome and liver injury (evidenced by lower ALT activity) and mitigated the rise in circulating endotoxin levels induced by consumption of a 10-week HFD in male Sprague-Dawley rats [21].
OMC is a complex of minerals, trace elements, organic acids (particularly fulvic acid), nitrates, and various microbial degradation products from plant and animal origins. Fulvic acid is notable as it has been shown to reduce the release of proinflammatory mediators after exposure to LPS in human monocytes [22] and alter the microbiota of soil ultimately promoting nutrition uptake by plants [22]. However, very little is known about whether these soilderived products affect the composition and function of the gut microbiota. erefore, the objective of the present study was to further investigate the effects of OMC on the gut-liver axis of these animals by determining its effects on HFD-induced pathophysiological changes and gut microbiome composition in six-week-old male Sprague-Dawley rats. We hypothesized that OMC supplementation would mitigate risk factors associated with hepatic and GI inflammation induced by a 10-week HFD.

OMC Isolate.
e current study used a proprietary OMC obtained from the study sponsor Isagenix International, LLC (Gilbert, AZ). A compositional analysis of OMC has been described previously [21]. Briefly, OMC is a dietary supplement extracted from several mineral mines in North America and is isolated and manufactured by Mineral Biosciences, LLC (Goodyear, AZ). Similar to the natural substance, shilajit, OMC is composed of over 50 minerals (predominantly calcium, sulfur, potassium, sodium, and magnesium), nitrates, and fulvic acid [21].

Animal Models.
is study is a continuation of published work from our laboratory using six-week-old male Sprague-Dawley rats (140-160 g, n � 36) randomly divided into two groups that received either standard Chow diet (Teklad Global, 24% protein, 58% carbohydrate, and 18% fat (in % kcal), Indianapolis, IN) or HFD containing 20% protein, 20% carbohydrate, and 60% kcal from fat (Cat. No. D12492, Research Diets Inc., New Brunswick, NJ) for 10 weeks [21]. Rats in each group were administered 0 (control), 0.6, or 3.0 mg/mL OMC in their drinking water throughout the study. Food and OMC-treated water were replaced every 2-3 days to prevent spoiling. Animals were allowed free access to water and food ad libitum. At the end of the 10-week study, animals were euthanized (sodium pentobarbital, 200 mg/kg, i.p.) [21]. All procedures were approved by the Arizona State University Institutional Animal Care and Use Committee.

Oil Red O Staining.
Samples of liver tissue were embedded in OCT compound, frozen in isopentane cooled by dry ice, and stored at −80°C until analysis. Lipid droplets and neutral fat were stained using a commercially available Oil Red O staining kit according to the manufacturer's protocol (ScyTek Laboratories, Inc., Logan, UT, USA). Briefly, tissue sections (12 μm) were collected onto color frost microscope slides (VWR, Radnor, PA, USA) using a cryostat (Leica Biosystems CM1950, Buffalo Grove, IL, USA). Sections were incubated with propylene glycol for 5 mins at room temperature followed by heated Oil Red O solution for 10 min. Tissue sections were then treated with 85% propylene glycol for 1 min and rinsed with distilled water. Mayer's hematoxylin (Lillie's modification) was used to counterstain sections, and slides were cover slipped using aqueous mounting medium (Cat No. AML060, ScyTek Laboratories, Inc.), following washes in tap water and distilled water. Images of slides were collected at 40x magnification using an EVOS FL Auto Imaging System from Life Technologies (AMAFD1000, Carlsbad, CA, USA).

Liver Triacylglycerol
Assay. Liver triacylglycerol concentrations were measured according to the methods of Jouihan [24]. Briefly, 100-300 mg tissue (n � 5 rats per group) was digested in 350 μL ethanolic KOH (1 : 2 ratio of 30% potassium hydroxide and ethanol) overnight at 55°C. e total volume was adjusted to 1000 μL with 50% ethanol, and the mixture was vortexed, then centrifuged at 13,000 rpm for 5 mins. e supernatant was transferred to a clean microcentrifuge tube, and the volume adjusted to 1200 μL with 50% ethanol, then vortexed. A 200 μL aliquot was transferred to a new tube to which 215 μL 1 M magnesium chloride was added, and the mixture was vortexed and then placed on ice for 10 minutes. After centrifuging for 5 minutes at 13,000 rpm, the supernatant was transferred to a new tube to be used for the assay. Liver free glycerol concentrations were determined using a commercially available kit per the manufacturer's protocol (Sigma Aldrich, St. Louis, MO, USA) and triacylglycerol content was determined from the free glycerol concentrations using the following equation: triacylglycerol (mg/g tissue) was calculated as follows: [glycerol] (mg/dl) * (10/30) * (415/200) * 0.012 (dL)/tissue mass (g).

Illumina Sequencing and Microbial Sequence Analyses.
e Microbiome Analysis Laboratory at Arizona State University implemented QIIME analyses and Illumina sequencing to analyze microbial taxonomy from week 0 and week 10 fecal samples as previously described [21,25]. Subsequently, microbial differences were examined using linear discriminant analysis of effect size (LEfSe) analyses implemented within the online Galaxy module (http:// huttenhower.sph.harvard.edu/galaxy/) explained by Segata et al. [25]. Relative abundance percentages were then calculated from taxonomic data generated from QIIME.

Statistical Analyses
Data are expressed as means ± SEM. Data were analyzed using SigmaPlot (Systat Software Version 10.0, San Jose, CA, USA). Western blot, liver triacylglycerol/glycerol, and CML data were analyzed by two-way ANOVA with diet (Chow and HFD) and treatment (dose of OMC) as factors. Average relative abundance of phyla was calculated by taking the individual phylum absolute abundance per sample, dividing by the sample sum of absolute abundances, then transformed to a percentage. Relative abundance data were analyzed by one-way ANOVA followed by Bonferroni post hoc tests. Week 0 and Week 10 Firmicutes-to-Bacteroidetes ratios were calculated by dividing the relative abundance of Firmicutes by that of Bacteroidetes for all groups and analyzed by two-way ANOVA followed by Tukey post hoc tests. Statistical significance was considered for p < 0.05 for all analyses. LEfSe analysis was conducted using Kruskal-Wallis sum-rank tests, Wilcoxon rank-sum tests, and linear discriminant analysis as previously described [25].

Tight Junction Protein Expression in the Cecum.
10-week HFD consumption did not affect the expression of the tight junction protein occludin in the cecum (the cecum: Figure 2(a); df � 1, F � 0.166, and p � 0.686; the small intestine: Figure 2(b) df � 1, F � 1.330, and p � 0.259). Additionally, OMC supplementation did not influence the expression of occludin in the cecum (df � 2, F � 0.512, and p � 0.605). In contrast, low-dose OMC supplementation increased protein expression of occludin in the small intestine of both Chow-and HFD-fed animals (df � 2, F � 3.421, and p � 0.047).

Firmicutes-to-Bacteroidetes Ratio.
e Firmicutes : Bacteroidetes ratio was calculated as a marker of gut dysbiosis. e F/B ratio was lower in fecal samples from HFD-fed rats treated with 0.6 mg/mL OMC as compared to untreated HFD rats (Figure 10; two-way ANOVA, Tukey post hoc test, p � 0.0416). Additionally, "time" was a significant source of variation (two-way ANOVA, df � 1, F � 9.932, and p � 0.0038).

Discussion
With the increasing incidence of gastrointestinal and metabolic complications in Westernized countries, natural health products are increasingly being considered as alternative treatments for these diseases. Indeed, gut microbiota-targeted therapies have been suggested for NAFLD, including the use of probiotics, prebiotics, antibiotics, and some active components found in herbal medicines [26]. A prior study from our laboratory demonstrated that OMC prevents HFD-induced increases in circulating LPS, a marker of gut integrity [21]. Based on this observation, the goal of the present study was to examine the effects of OMC on HFD-induced gastrointestinal and liver pathophysiological outcomes in these rats. A potential limitation of the present study is the relatively small sample size, which reflects the research design of the original work [21] and is consistent with sample sizes included in a similar study examining the effects of a 6-week HFD on the gut microbiome [16].
e results from the current study show that (1) a 10week HFD induced gut dysbiosis, as well as hepatic steatosis and accumulation of advanced glycation end products (CML) in young male Sprague-Dawley rats; (2) although OMC did not prevent hepatic steatosis, it was effective at preventing HFD-induced changes to the gut microbiome and hepatic CML levels; and (3) OMC may help prevent gut leakiness as it promoted the expression of occludin in the small intestine. Together these findings are suggestive of a potential pathological link between the gut microbiota and liver induced by HFD, which may be prevented by supplementation with OMC. e gut-liver axis is increasingly recognized as an important factor in the development of metabolic syndrome and NAFLD. Key features of this connection include gut wall permeability, translocation of microbial products [7][8][9][10], and propagation of proinflammatory and oxidative cascades that can lead to liver damage and disease [11,12]. Additionally, products from the gut microbiota can impact the liver and, in turn, liver products can influence the gut microbiota composition and gut barrier integrity [13,14]. e major drivers of increased gut permeability include inflammation and dysbiosis [27,28], which have been linked to consumption of a high-fat Western diet [9,29,30]. Prior studies indicate that high-fat feeding can reduce the expression of tight junction proteins occludin and zonaoccludens (ZO)-1 and also increase plasma endotoxemia in C57bl6/J mice [31], whereas protein expression of occludin was not significantly different between groups and low-dose OMC supplementation was associated with higher expression of occludin in the proximal small intestine of both the Chow and HFD-fed rats. Although not analyzed in the current study, this effect may be due to the presence of nitrate in OMC. Nitrate is reduced to nitrite in the oral cavity then nitric oxide (NO) in the stomach [32]. Previous studies have shown that NO may act as a nutrient for the lower microbiome [33] and prevent intestinal barrier dysfunction by protecting tight junction proteins from reactive oxygen species through redox reactions [32,34]. However, an increase in intestinal permeability facilitates passive translocation of luminal LPS or LPS-containing bacteria into the lamina propria, promoting greater translocation of LPS into blood (termed endotoxemia) [35]. For this reason, circulating LPS concentration is a surrogate marker for assessing in vivo intestinal permeability [36]. As our prior study showed OMC prevented HFD-induced increases in circulating LPS [21], it is possible that OMC may similarly strengthen the intestinal barrier through increased expression of occludin.
Prior studies have shown that high-fat feeding is linked to increased expression of proinflammatory cytokines in the gastrointestinal tract [16,37,38]. However, in the current study, consumption of an HFD for 10 weeks did not increase the expression of inflammatory markers (NF-κB (p65) and IL-1β) in the cecum or proximal small intestine. In studies of C57BL/6 mice, consumption of an HFD for 2-16 weeks only increased TNF-α in the ileum [39]. Tumor necrosis factor-α is an important inducer of NF-κB (p65) [40], and an earlier study indicated that LPS can activate intestinal NF-κB with the support of neutrophils and endogenous TNF-α [41]. However, in the current study, TNF-α was undetectable in plasma samples. us, it is possible that HFD intake for 10 weeks likewise did not increase intestinal TNF-α or activation of NF-κB, although it is possible that HFD-intake   altered the expression of other proinflammatory cytokines in the gut or elsewhere in the body. In fact, others have shown that diet-induced obesity and metabolic syndrome are marked by an increase in proinflammatory markers secreted from adipose tissue [42]. In addition, studies have shown that HFD consumption can increase LPS which is translocated from the gut and subsequently participates in the inflammatory responses in adipose tissue [43]. Our prior  study likewise showed increased plasma LPS following 10week HFD in these same study animals [21]. erefore, it is possible that inflammatory cytokines were altered in adipose tissue, although that was not examined in the present study. Elevated circulating LPS can accelerate lipid accumulation [44] and promote liver injury [45]. Moreover, hepatic lipid accumulation leads to increased endogenous formation of CML, likely due to increased lipid peroxidation [46] and inflammation [47]. CML levels are reportedly correlated with the severity of liver disease [48], and advanced glycation end products have been suggested to play a critical role in the pathogenesis of NAFLD [49]. One mechanism through which CML affects cellular function is binding to the receptor for AGEs and activating intracellular signaling pathways such as NF-κB [50,51], which could contribute to the pathogenesis of NAFLD. Findings from the present study showed lower CML levels in HFD rats receiving OMC supplementation without significantly affecting lipid accumulation in the liver.
ese results are corroborated with our previous findings showing decreased ALT, a marker for liver injury [21]. As the HFD rats displayed increased lipid accumulation in the liver, OMC supplementation may have a protective benefit against steatosis and NAFLD. e gut microbiome is implicated in the onset of many metabolic and inflammatory disorders. Consistent with prior studies of rodents [21,52], chronic high-fat feeding significantly increased liver triacylglycerol and altered the gut microbial communities in male Sprague-Dawley rats. HFD-induced pathogenic state results in an imbalance (dysbiosis) of Firmicutes and Bacteroidetes. Interestingly, the F/B ratio of untreated HFD-fed rats at 10 weeks was significantly higher than HFD-fed rats treated with the low dose of OMC. erefore, it is possible that fulvic acid, the main active component in OMC treatment [21], may prevent an increase in Firmicutes in the study animals. While very little information is known about the effects of fulvic acid, a 2017 study by Gao et al. found that 60-day fulvic acid feeding decreased the abundance of Firmicutes in the intestine of Paramisgurnus dabryanus [53]. Additionally, Gao et al. showed that fulvic acid can increase lysozyme levels in the intestine [53], which has been shown to significantly decrease Firmicutes levels in pregnant sows [54].
Results from LEfSe analyses showed differences in taxonomic structure between rats fed a Chow or HFD after 10 weeks. In comparison to Chow-fed rats, fecal samples from untreated HFD-fed rats were more enriched in Firmicutes (rc4_4, Dorea, Clostridium cocleatum, and schaedleri), Deferribacteres (Desulfovibrio, Deferribacteraceae, Deferribacterales, Deferribacteres, and Mucispirillum), Bacteroidetes (Rikenellaceae, Streptococcaceae, and acidifaciens), Proteobacteria (Desulfovibrio), and Tenericutes (Anaeroplasmatales, Anaeroplasma, and Anaeroplasmataceae) families and species at 10 weeks; all of which are associated with the development of inflammation, obesity, and type 2 diabetes [55][56][57][58][59][60]. Moreover, fecal samples from these HFDfed rats were more abundant in bacteria associated with high-fat diet intake from the phyla Bacteroidetes (Parabacteroides, a commensal microbe) [61], Firmicutes (Oscillospira, Ruminococcus, garvieae, gnvaus, Roseburia, and Peptococcaceae), Actinobacteria (Christensenellaceae, Coriobacteriia, and Coriobacteriales), and Proteobacteria (Enterobacteriales) in comparison to fecal samples collected at week 0. LEfSe analysis also revealed 10-week OMC supplementation (0.6 mg/mL and 3.0 mg/mL) prevented alterations in microbial composition induced by HFD-intake. ese findings were likely influenced by consumption of energy dense diets, including the ingestion of high  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. amounts of saturated fat [62]. In addition, these data are consistent with our prior work, which revealed that 6 weeks of HFD feeding increased the abundance of Firmicutes, Proteobacteria, Clostridiales, and Streptococcaceae [16]. Several taxa noted in other studies assessing the gut microbiome of humans with NAFLD or cirrhosis had increased abundance, including Streptococcaceae [63], Dorea [64,65], and Clostridium [66]. ese are notable findings as Streptococcaceae is known to induce persistent inflammation in the intestinal mucosa [67], and increased abundance of Streptococcaceae has been noted in other, similar research studies. For example, in a study that fed germ-free mice an HFD and inoculated them with feces from patients with NASH, the mice had markedly elevated serum levels of ALT, AST, endotoxin, IL-6, monocyte chemotactic protein 1, and hepatic triglycerides [68]. Similarly, increased abundance of Dorea has been reported in the gut microbiome of HFD-fed C57BL/6 male mice [68]. In a study assessing the gut microbial changes during liver disease progression using a streptozotocin-HFD induced NASH-HCC C57BL/6J mouse model that is highly relevant to human liver disease, Clostridium was found to be positively associated with increased LPS levels [69]. We also noted an increased abundance of the genus Ruminococcus induced by 10 weeks of high-fat intake when compared to baseline for these animals (i.e., week 0). In this genus, there are species that produce alcohol [70]. Endogenous ethyl alcohol from the gut can reach the liver by the portal vein, which could contribute to liver damage and aggravate the pathology of NAFLD [71]. Together, the increased abundance of these taxa in the gut microbiome of the HFD rats examined in the present study is suggestive of a state of gut dysbiosis as well as hepatic steatosis and glycation.
Consistent with prior research demonstrating that OMC supplementation may play a vital role in preventing several symptoms of metabolic syndrome [21], OMC was shown to prevent alterations in liver CML and the fecal microbial composition of rats fed HFD for 10 weeks. Due to the paucity of research on the effects of fulvic acid and other components of OMC on the gut microbiota, these results are difficult to explain mechanistically. OMC may be acting directly within the GI tract, as well as exerting indirect effects, such as through the liver. In the liver, fulvic acid can act as an antioxidant by uncoupling electron transport in liver mitochondria leading to a decrease in reactive oxygen species (ROS) production [72], which may explain the previous observation that OMC prevents HFD-induced increases in ALT activity [21].

Conclusion
In summary, the current study demonstrated that a 10-week HFD alters gut microbial composition and promotes hepatic steatosis; however, it did not elevate markers of gut inflammation. In addition, OMC supplementation prevented changes in CML levels and the gut microbiota, while increasing expression of occludin, a fundamental tight junction protein.
is study provides support for further investigation into therapeutic and preventive properties of this natural health product on the alleviation of metabolic disease risk factors in the gastrointestinal and hepatic systems.

Data Availability
e data used to support the findings of this study are available upon request.

Conflicts of Interest
AEM is employed by Isagenix International LLC. e authors have no conflicts of interest regarding the publication of this paper. AEM was not involved in the study design and was blinded as to the identity of samples during all analyses.