AIN-93G Control Diet Research Articles

Supplementation of Vitamin D3 and Fructooligosaccharides Downregulates Intestinal Defensins and Reduces the Species Abundance of Romboutsia ilealis in C57BL/6J Mice.

The activation of the vitamin D receptor (VDR) in the ileum has been shown to regulate Paneth cell-specific defensins, a large family of antimicrobial peptides; hence, this may serve as a potential mechanism to maintain intestinal homeostasis. Previously, we have demonstrated that a combination of vitamin D3 (VD) and fructooligosaccharides (FOSs) upregulates colonic Vdr in mice. Here, we aim to examine the effect of VD, alone or in combination with FOSs, on intestinal barrier integrity and the secretion of antimicrobial peptides, as well as the gut microbial community. Male and female C57BL/6J mice at 6 weeks old were randomized into three groups to receive the following dietary regimens (n = 10/sex/group) for 8 weeks: (1) standard AIN-93G control diet (CTR), (2) CTR + 5000 IU vitamin D3 (VD), and (3) VD + 5% fructooligosaccharides (VF). VD and VF differentially regulated the mRNA expressions of tight junction proteins in the colon and ileum. VF suppressed the upregulation of colonic ZO-1 and occludin, which was induced by VD supplementation alone. In the ileum, occludin but not ZO-1 was upregulated 20-fold in the VF-treated mice. While VD did not alter the mRNA expressions of Vdr and defensins in the ileum, these targets were downregulated by VF. Microbial analysis further reveals a shift of microbial beta diversity and a reduction in Romboutsia ilealis, a pathobiont, in VF-treated mice. Though the implications of these phenotypical and microbial changes remain to be determined, the administration of FOSs in the presence of VD may serve as an effective dietary intervention for maintaining intestinal homeostasis.

Abstract 762: The combination of EPA and naproxen suppresses colon tumorigenesis via altering the TME, and promoting epithelial maturation in cancerous crypts

Abstract Introduction: Colorectal cancer (CRC) is a leading cause of cancer-associated mortality. Our recent study in Pirc rats revealed that the combination of eicosapentaenoic acid and sodium naproxen exhibited additive tumor protection. While the results from this preclinical study were impressive, showing tumor suppression in excess of 95%, potential mechanisms underlying this tumor protection remain unclear. The purpose of this study was to determine the transcriptomic changes resulting in the tumor suppression afforded by the combination of EPA and naproxen supplementation in the diet. Methods: Six week-old Pirc rats were fed either a control AIN-93G diet, or AIN-93G diet supplemented with 2% EPA combined with 200 ppm naproxen, for a total of 20 weeks. Global transcriptomic analysis was performed using RNA sequencing (RNA-Seq) of colon tumors and tumor-adjacent normal tissue from 26-week-old Pirc rats. 100-bp paired-end sequencing was performed on an Illumina NovaSeq to a depth of 25 million reads per sample. Reads were trimmed with Trimmomatic-v0.39, and reads were mapped to the mRatBN7.2.105 rat reference genome using HISAT2. Differentially expressed genes were calculated using DESeq2, and gene set enrichment analysis (GSEA) were performed using Rstudio. Results: DESeq2 identified 809 differentially expressed genes (DEGs, 2-fold change > 1, FDR < 0.05) between large tumors from AIN-93G fed rats versus suppressed tumors from rats fed the combination of EPA and naproxen (490 up-regulated, 319 down-regulated). GSEA analysis revealed activation of inflammatory pathways including IL-17 signaling, JAK-STAT signaling, complement activation, cytokine signaling, ribosome biogenesis, and cell cycle were enriched in untreated control. In contrast, suppressed tumors from animals supplemented with EPA and naproxen were enriched in pathways including negative regulation of Wnt signaling, cell fate specification, ion channel complexes, and nutrient absorption. Furthermore, suppressed tumors shared significant GSEA pathway overlap with normal tissue, leading us to believe the combination of EPA and naproxen may be preventing tumorigenesis in part by maintaining cells in a differentiated state. Conclusion and Future directions: Inflammation plays a key role in tumorigenesis and promoting cancer stem cell niches in tumors. According to our analysis, the combination of EPA and naproxen attenuated cancer-associated inflammation and reduced proliferation in part by augmenting tumor differentiation. Future studies will utilize single-nuclei RNA-Seq (snRNA-Seq) to determine at a single cell resolution the transcriptomic changes of epithelial and stromal populations within the TME. This work was supported by National Cancer Institute task order 75N91019F00132. Citation Format: Ryan Beach, Michael Martinez, Vignesh Gunasekharan, Daniel W. Rosenberg. The combination of EPA and naproxen suppresses colon tumorigenesis via altering the TME, and promoting epithelial maturation in cancerous crypts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 762.

Comparison of maternal versus postweaning ingestion of a high fat, high sucrose diet on depression-related behavior, novelty reactivity, and corticosterone levels in young, adult rat offspring

Consumption of a Western-type diet, high in fat and sugar, by mothers as well as maternal weight gain and obesity during gestation and lactation may impact offspring risk for mood and cognitive disorders. The objective of this study was to determine if ingestion of a high fat, high sucrose (HFS) diet by rat dams during gestation and lactation or by their pups after weaning impacted these behaviors and stress responsivity in young, adult offspring. To accomplish this, dams consumed either a 45% fat/high sucrose (HFS) diet or the AIN93G control diet during gestation and lactation. At weaning, pups from dams that consumed the HFS diet were weaned to the control diet. Pups from dams assigned to the control diet were weaned to either the control or HFS diet. Pup behavioral testing began at 10 weeks of age. Pups whose dams consumed the HFS diet during gestation and lactation exhibited increased depression-related behavior and baseline serum corticosterone levels, but no difference in peak levels in response to stress. Male pups of these dams displayed increased working memory during acquisition of the holeboard task and tended to exhibit more anxiety-related behavior in the elevated O-maze test. Regardless of when consumed, the HFS diet increased novelty reactivity in the open field test. These data indicate that diet but not maternal weight gain during gestation impacts offspring behavior and elevates stress hormone levels. Also, regardless of when consumed, the HFS diet increases novelty reactivity, a risk factor for depression and addiction.

Broccoli sprouts delay prostate cancer formation and decrease prostate cancer severity with a concurrent decrease in HDAC3 protein expression in TRAMP mice

Background: Cruciferous vegetables have been associated with the chemoprevention of cancer. Epigenetic regulators have been identified as important targets for prostate cancer chemoprevention. Treatment of human prostate cancer cells with sulforaphane (SFN), a chemical from broccoli and broccoli sprouts, inhibits epigenetic regulators such as histone deacetylase (HDAC) enzymes, but it is not known whether consumption of a diet high in broccoli sprouts impacts epigenetic mechanisms in an in vivo model of prostate cancer. Objective: In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, we tested the hypothesis that a broccoli sprout diet suppresses prostate cancer, inhibits HDAC expression, alters histone modifications, and changes the expression of genes regulated by HDACs. Methods: TRAMP mice were fed a 15% broccoli sprout, or control AIN93G diet; tissue samples were collected at 12 and 28 weeks of age. Results: Mice fed broccoli sprouts had detectable levels of SFN metabolites in liver, kidney, colon, and prostate tissues. Broccoli sprouts reduced prostate cancer incidence, and progression to invasive cancer by 11- and 2.4-fold at 12 and 28 weeks of age, respectively. There was a significant decline in HDAC3 protein expression in the epithelial cells of prostate ventral and anterior lobes at 12 weeks. Broccoli sprout consumption also decreased histone H3 lysine 9 tri-methylation in the ventral lobe (12 week), and decreased histone H3 lysine 18 acetylation in all prostate lobes (28 weeks). A decline in p16 mRNA levels, a gene regulated by HDAC3, was associated with broccoli sprouts consumption, but no significant changes were noted at the protein level. Conclusions: Broccoli sprout intake caused a decline in prostate cancer occurrence and HDAC3 protein expression in the prostate, extending prior work that implicated loss of HDAC3/corepressor interactions as a key preventive mechanism by SFN in vivo.

Comparative effects of human-equivalent low, moderate, and high dose oral prednisone intake on autoimmunity and glucocorticoid-related toxicity in a murine model of environmental-triggered lupus.

Autoimmune diseases can be triggered by environmental toxicants such as crystalline silica dust (cSiO2). Here, we characterized the dose-dependent immunomodulation and toxicity of the glucocorticoid (GC) prednisone in a preclinical model that emulates onset and progression of cSiO2-triggered lupus. Two cohorts of 6-wk-old female NZBWF1 mice were fed either control AIN-93G diet or one of three AIN-93G diets containing prednisone at 5, 15, or 50 mg/kg diet which span human equivalent oral doses (HED) currently considered to be low (PL; 5 mg/d HED), moderate (PM; 14 mg/d HED), or high (PH; 46 mg/d HED), respectively. At 8 wk of age, mice were intranasally instilled with either saline vehicle or 1 mg cSiO2 once weekly for 4 wk. The experimental plan was to 1) terminate one cohort of mice (n=8/group) 14 wk after the last cSiO2 instillation for pathology and autoimmunity assessment and 2) to maintain a second cohort (n=9/group) to monitor glomerulonephritis development and survival. Mean blood concentrations of prednisone's principal active metabolite, prednisolone, in mice fed PL, PM, and PH diets were 27, 105, 151 ng/ml, respectively, which are consistent with levels observed in human blood ≤ 12 h after single bolus treatments with equivalent prednisone doses. Results from the first cohort revealed that consumption of PM, but not PL diet, significantly reduced cSiO2-induced pulmonary ectopic lymphoid structure formation, nuclear-specific AAb production, inflammation/autoimmune gene expression in the lung and kidney, splenomegaly, and glomerulonephritis in the kidney. Relative to GC-associated toxicity, PM diet, but not PL diet, elicited muscle wasting, but these diets did not affect bone density or cause glucosuria. Importantly, neither PM nor PL diet improved latency of cSiO2-accelerated death. PH-fed mice in both cohorts displayed robust GC-associated toxicity including body weight loss, reduced muscle mass, and extensive glucosuria 7 wk after the final cSiO2 instillation requiring their early removal from the study. Taken together, our results demonstrate that while moderate doses of prednisone can reduce important pathological endpoints of cSiO2-induced autoimmunity in lupus-prone mice, such as upstream ectopic lymphoid structure formation, these ameliorative effects come with unwanted GC toxicity, and, crucially, none of these three doses extended survival time.

Dietary Oyster (Crassostrea gigas) Extract Ameliorates Dextran Sulfate Sodium-Induced Chronic Experimental Colitis by Improving the Composition of Gut Microbiota in Mice.

Previously, we have reported that the intake of oyster extract (OE), prepared from Pacific oysters (Crassostrea gigas), can attenuate symptoms of dextran sulfate sodium (DSS)-induced acute experimental colitis in mice. Herein, we aimed to evaluate whether OE intake ameliorates chronic experimental colitis induced by repeated DSS administration in mice. Male C57BL/6J (4-week-old) mice were fed either the standard diet AIN93G (control diet) or the control diet containing 5.0% (w/w) OE (OE diet). After 21 days of diet feeding, chronic experimental colitis was induced by three cycles of 2.0% (w/w) DSS solution administration (5 days), followed by distilled water (5 days). Mice fed OE alleviated the shortened colonic length, increased the relative weight of the spleen, colonic histopathological score (regeneration), and blood in the stool score compared with mice fed control diet. A tendency to improve the α-diversity of fecal microbiota, which was exacerbated by colitis, was observed in mice fed OE. Correlation analysis suggested that the anti-colitis effect of OE intake could be related to the valeric acid content and relative abundances of Ruminococcus and Enterococcus in the feces. In conclusion, OE could ameliorate DSS-induced chronic experimental colitis by improving the gut environment, including the microbiota community and SCFA composition.

Abstract LB164: Combination of naproxen and a novel longer acting eicosapentaenoic acid analogue provide synergistic tumor protection in polyposis in rat colon (PIRC) model

Abstract The purpose of this study was to determine whether or not Naproxen and novel TP-252 combine for synergistic tumor protection in the PIRC model. Colorectal Cancer (CRC) is the second leading cause of cancer-related deaths in the United States. Patients with the genetic disorder Familial Adenomatous Polyposis (FAP) develop hundreds of polyps that unless removed by prophylactic colectomy, will progress to CRC at an early age. In recent decades, non-steroidal anti-inflammatory drugs (NSAIDs) and ω-3 marine fish oils such as eicosapentaenoic acid (EPA), have been evaluated for their chemopreventative potential in delaying the onset of CRC in high-risk patients. In this study, we examined the tumor protective effect of Naproxen alone, or in combination with EPA in the intestinal tumorigenesis Apcam1137 PIRC rat model. Since the free fatty acid form of EPA (FFA-EPA) is readily oxidized, we included a novel longer acting chemically stabilized form of EPA (magnesium L-lysinate bis-eicosapentaenoate), TP-252. Rats were fed a modified AIN-93G diet containing 200 ppm Naproxen, alone or in combination with two dose levels of TP-252 (1.5%, or 3%) or a single concentration of FFA-EPA (2%) by weight for 20 weeks. Rats that received 200 ppm Naproxen alone showed reduced tumor number by 66% (p < 0.0001) and size by 83% (p < 0.0001), respectively, compared to rats fed AIN-93G (control diet). However, when Naproxen was combined with high-dose TP-252 (3%), tumor number and size were reduced by 95% (p < 0.0001) and 98% (p < 0.0001) respectively, when compared to control AIN-93G diet. To elucidate potential mechanisms of tumor protection, a comprehensive lipidomic analysis was performed on colon tissue and plasma to determine potential metabolic changes associated with drug treatment. Naproxen or EPA alone resulted in significantly reduced arachidonic acid (AA)-derived ω-6 eicosanoids (PGE2, LTB4, and 5-HETE), but the combination of Naproxen and EPA yielded the most significant reduction that was also correlated with tumor protection. In addition, groups receiving EPA, either as FFA or TP-252, had a dose-dependent increase in anti-inflammatory EPA-derived ω-3 eicosanoids, as well as a panel of bioactive resolvins. Our lipidomic analysis also uncovered several lipid metabolites, including PGE3, RvE2, and 5-HEPE, that were correlated with tumor protection. Further studies should be focused on dose optimization, and the role of these lipid mediators in CRC initiation and progression. (Supported by the NCI Prevent Program 75N91019D00019, Task Order 75N91019F00132). Citation Format: Ryan Beach, Daniel W. Rosenberg, Altaf Mohammed. Combination of naproxen and a novel longer acting eicosapentaenoic acid analogue provide synergistic tumor protection in polyposis in rat colon (PIRC) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB164.

Abstract 2571: Paternal epigenetic regulation contributes to the prevention of estrogen receptor-negative mammary cancer with combined broccoli sprouts and green tea polyphenols consumption in transgenic mice

Abstract Breast cancer is one of the leading causes of cancer-related death among women in the United States. It is widely appreciated that abnormal epigenetic alterations act as one of the primary contributors to breast cancer initiation and progression. Parental, including maternal and paternal, nutritional interventions have shown considerable impact on fetal development leading to different susceptibility of offspring to various chronic diseases. Moreover, combinatorial dietary administration to parents could be more efficacious in ameliorating epigenetic aberrations and changing cancer risk in an individual's later life. The bioactive dietary broccoli sprouts (BSp) and green tea polyphenol (GTP) are important epigenetic modulators that can prevent various cancers. Our study focusses on investigating the effect of paternal dietary BSp and GTP administration on mammary cancer prevention of their offspring. Two spontaneous breast cancer transgenic mouse models, C3(1)/SV40 and Her2/neu, were employed in this study. Male C3(1)/SV40 or Her2/neu transgenic mice were randomly assigned into four groups and treated with: control AIN-93G diet, 26% BSp (w/w) in food pellets, 0.5% GTPs (w/v) in drinking water or combined BSp and GTPs from 3 wks of age until 10 wks of age. Treated male mice of different groups were then mated with non-treated female mice. Female pups were selected after their weaning and tumor growth was monitored weekly until the termination of the experiment. Tumor- and epigenetic-related protein expression and histone modifications (methylation and acetylation) were measured. Our study indicated that paternal BSp or/and GTPs administration suppressed tumor growth, decreased tumor incidence, and delayed tumor latency compared with the control in both mouse models. Overall, paternal dietary intervention displayed more efficacy for mammary tumor prevention in C3(1)/SV40 mice and the combination group in this model also showed synergistically effects. Subsequent analysis with the C3(1)/SV40 mouse tumors demonstrated that paternal BSp or/and GTPs treatments upregulated the expression of tumor suppressor proteins, such as P16 and P53, and down-regulated tumor promoting proteins, such as MYC and BMI1. Moreover, altered DNA methylation, histone methylation and acetylation levels were also observed. These results suggested that paternal exposure to BSp and GTPs may contribute to ER-negative mammary tumor prevention in their offspring through epigenetic regulations and the combined addition of bioactive botanicals could be a more promising approach for coping with breast cancer initiation and progression in humans. Citation Format: Shizhao Li, Huixin Wu, Trygve Tollefsbol. Paternal epigenetic regulation contributes to the prevention of estrogen receptor-negative mammary cancer with combined broccoli sprouts and green tea polyphenols consumption in transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2571.

Dietary Arginine Supplementation during Gestation and Lactation Increases Milk Yield and Mammary Lipogenesis in Rats.

Arginine, an essential amino acid during the reproductive period, has been shown to enhance lactation performances in livestock. Whether it could help mothers with breastfeeding difficulties is not known. This study aimed to determine whether dietary arginine supplementation would enhance milk production in rat dams nursing large 12-pup litters and, if so, what mechanisms are involved. In 3 series of experiments, differing in dam killing timing, 59 primiparous, pregnant Sprague-Dawley rats (mean±SD weight: 254±24.7g) were randomly assigned to receive either 1) an AIN-93G diet supplemented with l-arginine at 2.0% (ARG diet), through lactation and gestation (AGL group); 2) a control AIN-93G diet including at 3.5% an isonitrogenous mix of amino acids that are not essential for lactation (MA diet), during gestation and lactation (MA group); or 3) the MA diet during gestation and the ARG diet during lactation (AL group). Milk flow was measured using deuterated water enrichment between days 11 and 18. Plasma hormones and mammary expression of genes involved in lactation were measured using ELISA and qRT-PCR, respectively, at lactation days 12, 18, or 21 in the 3 experiments. Data were analyzed by ANOVA. Dam food intake, pup weight gain, milk flow normalized to dam weight, and milk fat concentration were 17%, 9%, 20%, and 20% greater in the AGL group than in the MA group, respectively (P<0.05). Genes involved in lipogenesis and lipid regulation were overexpressed ≤2.76-fold in the mammary gland of AGL dams compared with MA dams (P<0.05) and plasma leptin concentration was 39% higher (P=0.008). Milk flow and composition and mammary gene expression of the AL group did not differ from those of the MA group, whereas milk fat concentration and flow were 26% and 37% lower than in the AGL group, respectively. Arginine supplementation during gestation and lactation enhances milk flow and mammary lipogenesis in rats nursing large litters.

Impact of Carotenoid Cleaving Enzymes on Lycopene Accumulation in Transgenic Mice

Abstract Objectives To evaluate the role of β-carotene oxygenase 1 (BCO1) and BCO2 on lycopene tissue distribution. Methods Three-week old C57BL/6 male and female mice (wild type [WT], Bco1−/−, Bco2−/−, Bco1−/− × Bco2−/− double knock out [DKO]) were divided into groups based on genotype (n = 16 per group split evenly by sex) and fed a powdered AIN 93G control diet for 2 weeks. After this period, mice were gavaged daily for 2 weeks with 1 mg of lycopene dissolved in cottonseed oil. 12 h-fasted mice were then sacrificed, and liver, serum, heart, kidney, intestine, gonadal adipose, prostate, spleen, and testes were harvested. Tissues were preserved in liquid nitrogen and stored at −80 until analyses. We measured lycopene levels in all samples by using high-performance liquid chromatography. Data analyses were performed using two-way ANOVA, followed by the Sidaks test with a statistical significance threshold of P &amp;lt; 0.05. Results Female mice showed higher lycopene levels in the intestine (P &amp;lt; 0.045) and liver (P &amp;lt; 0.007) irrespective of genotype, while male mice had higher lycopene levels in serum (P &amp;lt; 0.004). Intestine, serum, and kidneys exhibited higher lycopene levels in DKO mice compared to all other genotypes (P &amp;lt; .0001), while having higher lycopene levels in testes (P &amp;lt; 0.0001) compared to Bco2−/− and WT mice and adipose (P &amp;lt; 0.005) only in comparison to Bco2−/− mice. DKO exhibited higher lycopene levels in the spleen compared to Bco1−/− mice (P &amp;lt; 0.02). Lycopene levels in the liver (P &amp;lt; 0.0001) were higher in Bco2−/− mice compared to Bco1 −/− and DKO mice, while Bco1−/− mice had lower hepatic lycopene levels compared to all other genotypes. Conclusions Female mice accumulated higher lycopene levels in most tissues compared to males. These results were consistent when data were corrected by total tissue weight. The data suggest the absence of BCO2 favors carotenoid accumulation in many extrahepatic tissues, an effect that is enhanced in the absence of both carotenoid cleaving enzymes. Funding Sources Internal funding, University of Illinois Urbana Champaign.

Metabolomics Profiling Revealed Double-Edged Metabolic Effects of Functionalized Wheat Bran in Mouse

Abstract Objectives Wheat bran is rich in bioactive components, mainly dietary fiber, micronutrients, and phytochemical contents. However, the health-promoting effects of wheat bran is restricted by the high-degree crosslinking of dietary fiber and low bioavailability of phytochemicals. The present study aims to improve the functions of wheat bran by increasing the levels of soluble fiber and free ferulic acid from optimized processing to examine the metabolic effects of consuming functionalized wheat bran (FWB) in mice. Methods Control wheat bran (CWB) was processed by an optimized combination of milling, high pressure pulverization, and alkali treatment, leading to dramatic increases of soluble fiber and free ferulic acid. Three groups of male mice were fed the control AIN93G diet, and two modified AIN93G diets containing 10% of CWB and 10% FWB, respectively, for 7 days. The effects of CWB and FWB on the mouse metabolome were determined through the LC-MS based metabolomics analysis of feces, liver, serum, and urine samples. Results The processing dramatically improved the function-associated physicochemical properties of wheat bran, including the increases of soluble fiber content and viscosity by milling and high-pressure pulverization and the elevation of free ferulic acid by alkali treatment. FWB feeding elevated microbial SCFAs production, promoted the excretion of bile acids and cholesterol in feces, modified the lipidome in the liver, decreased triacylglycerols and cholesterol in serum, and increased the levels of ferulic acid and microbial metabolites in urine. On the other hand, FWB feeding resulted in the increases of free amino acids in feces and the decreases of essential amino acids, choline and its metabolites in the liver. Conclusions The optimized processing dramatically increased the soluble fiber and free ferulic acid contents of wheat bran, resulting in improved hypocholesterolemic and antioxidant functions of FWB. However, the bioavailability of nutrients, including essential amino acids and choline, and the homeostasis of lipidome could be negatively affected by FWB diet. These double-edged metabolic effects warrant further investigations on how to achieve the balance between the functionalization of bioactive components and the disruption of nutrient bioavailability in wheat bran processing. Funding Sources NIFA project MIN-18–125.

P054 Modulation of the Gut Microbiota-Farnesoid X Receptor Axis Improves Deoxycholic Acid-induced Intestinal Inflammation in Mice

Abstract Background Inflammatory bowel disease (IBD) is associated with gut dysbiosis and dysregulation of bile acid metabolism. A high luminal content of deoxycholic acid (DCA) with consumption of a Westernized diet is implicated in the pathogenesis of IBD. The aim of the study is to explore the role of intestinal microbiota and bile acid metabolism in mice with DCA-induced intestinal inflammation. Methods 4-week-old wild-type C57BL mice were fed with AIN-93G (control diet), AIN-93G+0.2% DCA, AIN-93G+0.2% DCA+6 weeks of fexaramine (FXR agonist), or AIN-93G+0.2% DCA+antibiotic cocktail for 24 weeks. Histopathology, Western blotting, and qPCR were performed on the intestinal tissue. Fecal microbiota was analyzed by 16S rDNA sequencing. Fecal bile acid and short-chain fatty acid (SCFA) levels were analyzed by chromatography. Results Gut dysbiosis and enlarged bile acid pool were observed in DCA-treated mice, accompanied by a lower farnesoid X receptor (FXR) activity in the intestine. Administration of fexaramine mitigated DCA-induced intestinal injury, restored intestinal FXR activity, activated fibroblast growth factor 15, and normalized bile acid metabolism. Furthermore, fexaramine administration increased the abundance of SCFA-producing bacteria. Depletion of the commensal microbiota with antibiotics decreased the diversity of the intestinal microbiota, attenuated bile acid synthesis, and reduced intestinal inflammation induced by DCA. Conclusion DCA induced-intestinal inflammation is associated with alterations of gut microbiota and bile acid profiles. Interventions targeting the gut microbiota-FXR signaling pathway may reduce DCA-induced intestinal disease.

Modulation of the Gut Microbiota-farnesoid X Receptor Axis Improves Deoxycholic Acid-induced Intestinal Inflammation in Mice.

Inflammatory bowel disease (IBD) is associated with gut dysbiosis and dysregulation of bile acid metabolism. A high luminal content of deoxycholic acid (DCA) with consumption of a Westernised diet is implicated in the pathogenesis of IBD. The aim of the study is to explore the role of intestinal microbiota and bile acid metabolism in mice with DCA-induced intestinal inflammation. Wild-type C57BL mice, 4 weeks old, were fed with AIN-93G (control diet), AIN-93G+0.2% DCA, AIN-93G+0.2% DCA+6 weeks of fexaramine (FXR agonist), or AIN-93G+0.2% DCA+antibiotic cocktail, for 24 weeks. Histopathology, western blotting, and qPCR were performed on the intestinal tissue. Faecal microbiota was analysed by 16S rDNA sequencing. Faecal bile acid and short chain fatty acid (SCFA) levels were analysed by chromatography. Gut dysbiosis and enlarged bile acid pool were observed in DCA-treated mice, accompanied by a lower farnesoid X receptor (FXR) activity in the intestine. Administration of fexaramine mitigated DCA-induced intestinal injury, restored intestinal FXR activity, activated fibroblast growth factor 15, and normalised bile acid metabolism. Furthermore, fexaramine administration increased the abundance of SCFA-producing bacteria. Depletion of the commensal microbiota with antibiotics decreased the diversity of the intestinal microbiota, attenuated bile acid synthesis, and reduced intestinal inflammation induced by DCA. DCA induced-intestinal inflammation is associated with alterations of gut microbiota and bile acid profiles. Interventions targeting the gut microbiota-FXR signalling pathway may reduce DCA-induced intestinal disease.

Abstract 3461: Psyllium protects against high fructose diet induced exacerbation of colitis and colitis associated colorectal carcinogenesis

Abstract Patients with longstanding Inflammatory Bowel Disease, including ulcerative colitis and Crohn's disease, are at an increased risk of developing colorectal cancer (CRC). Over the past three decades, there has been a significant increase in the consumption of fructose, a monosaccharide that has been linked to increased growth of experimental intestinal tumors. Additionally, recent evidence shows that feeding a diet high in fructose worsens acute experimental colitis. The aims of the current study were to 1) determine if a high fructose diet (HFrD) exacerbated chronic colitis in mice and led to enhanced colorectal tumorigenesis and 2) evaluate the potential chemopreventive properties of dietary fiber in this context. To carry this out, the azoxymethane (AOM)/DSS model of colitis-associated colorectal neoplasia was utilized in male C57BL/6J mice. In comparison to control AIN93G diet, an isocaloric diet supplemented with fructose led to worse chronic colitis including weight suppression, worse diarrhea, colon shortening and a higher histologic score. Tumor incidence, number and size were all increased (Ps&amp;lt;0.001) in the HFrD compared to control diet group. Because eliminating fructose from the diet is impractical, it is important to identify other potential dietary constituents that may minimize its harmful effects. Dietary fiber has been suggested to be beneficial for reducing the severity of colitis. Therefore, we next determined whether the severity of HFrD-associated acute colitis was altered by dietary psyllium, pectin, inulin or cellulose fibers. In comparison to the other three fibers, psyllium protected against acute DSS- induced colitis. It also led to a significant shift in the fecal microbiota. Finally, we tested whether psyllium would attenuate the worsening of chronic colitis and colitis-associated colorectal neoplasia mediated by HFrD. Using the AOM/DSS model, we found that psyllium fiber protected against the exacerbation of chronic colitis mediated by HFrD with improved diarrhea, bleeding and histologic score. Consistent with its ability to suppress HFrD-mediated exacerbation of chronic colitis, psyllium markedly suppressed the increase in colorectal tumor incidence, number and size in mice that received the HFrD. Taken together, these results indicate that feeding a HFrD exacerbated chronic colitis leading to increased colitis-associated colorectal tumorigenesis. Supplemental psyllium fiber attenuated these procarcinogenic effects of a HFrD. Citation Format: Srijani Basu, Ryohei Nishiguchi, David C. Montrose, Hannah Staab, Xi Kathy Zhou, Hanhan Wang, Melanie Johncilla, Rhonda K. Yantiss, Andrew Dannenberg. Psyllium protects against high fructose diet induced exacerbation of colitis and colitis associated colorectal carcinogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3461.

Basal Diet Determined Long-Term Composition of the Gut Microbiome and Mouse Phenotype to a Greater Extent than Fecal Microbiome Transfer from Lean or Obese Human Donors

The Western dietary pattern can alter the gut microbiome and cause obesity and metabolic disorders. To examine the interactions between diet, the microbiome, and obesity, we transplanted gut microbiota from lean or obese human donors into mice fed one of three diets for 22 weeks: (1) a control AIN93G diet; (2) the total Western diet (TWD), which mimics the American diet; or (3) a 45% high-fat diet-induced obesity (DIO) diet. We hypothesized that a fecal microbiome transfer (FMT) from obese donors would lead to an obese phenotype and aberrant glucose metabolism in recipient mice that would be exacerbated by consumption of the TWD or DIO diets. Prior to the FMT, the native microbiome was depleted using an established broad-spectrum antibiotic protocol. Interestingly, the human donor body type microbiome did not significantly affect final body weight or body composition in mice fed any of the experimental diets. Beta diversity analysis and linear discriminant analysis with effect size (LEfSe) showed that mice that received an FMT from obese donors had a significantly different microbiome compared to mice that received an FMT from lean donors. However, after 22 weeks, diet influenced the microbiome composition irrespective of donor body type, suggesting that diet is a key variable in the shaping of the gut microbiome after FMT.

Epigenomic Regulation of SATB2 Links Maternal Obesity to Impaired Osteoblast Differentiation (P11-034-19)

ObjectivesNutritional status during intrauterine and/or early postnatal life has substantial influences on adult offspring health. However, evidence on the impact of high fat diet (HFD)-induced maternal obesity on regulation of fetal bone development is sparse. Thus, we investigated the effects of maternal obesity in rodent and human cells on epigenetic regulation of osteoblast differentiation. MethodsFirst, female Sprague-Dawley rats were fed either a low-fat AIN-93G control diet or a high fat diet (HFD) (45% fat calories) for 10 wk starting at 6 wk of age. Lean (from control diet) and obese (from HFD) female rats were then time-impregnated (n = 6 per group) by control diet fed male rats. At gestational day 18.5 (E18.5), all fetuses were taken and embryonic osteogenic calvarial cells (EOCCs) were isolated. Second, human osteo-progenitors of mesenchymal stem cells were isolated from umbilical cord following delivery from pregnant mothers. ResultsWe found epigenetic regulation of polycomb-regulated gene Ezh2 (Enhancer of zeste homolog 2) in embryonic rats from HFD obese rat dams. Increased enrichment of repressive histone mark H3K27me3 on the gene body of SATB2 (ChIP Seq analysis) was associated with aberrant differentiation of EOCCs to mature osteoblasts. Knocking down Ezh2 in EOCCs and ST2 cells increased SATB2 expression; on the other hand, Ezh2 overexpression in EOCCs and ST2 cells decreased SATB2 expression. These data were consistent with ChIP experimental results showing strong association between H3K27me3, Ezh2 and SATB2. Second, human mesenchymal stem cells (MSCs) from umbilical cord (UC) were isolated following delivery from obese/overweight (pre-pregnancy BMI ≥ 25 kg/m2) and control (pre-pregnancy BMI between 19–25 kg/m2) pregnant mothers. We found: 1) UC-MSCs from pregnant obese/overweight mothers showed increased Ezh2 expression and decreased SATB2 mRNA expression, which was concurrent lower osteoblastogenesis potential in EOCCs; 2) ChIP experiments using H3K27me3 IP (immune-precipitation) showed significant association between H3K27me3, Ezh2 and SATB2. ConclusionsThese findings indicate maternal HFD-induced obesity-associated decrease of fetal pre-osteoblastic cell differentiation is under epigenetic control through SATB2 expression. Funding SourcesSupported by USDA-ARS Project.

Broccoli Sprouts Delay Prostate Cancer Formation and Decrease Prostate Cancer Severity with a Concurrent Decrease in HDAC3 Protein Expression in Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) Mice

BackgroundCruciferous vegetables have been associated with the chemoprevention of cancer. Epigenetic regulators have been identified as important targets for prostate cancer chemoprevention. Treatment of human prostate cancer cells with sulforaphane (SFN), a chemical from broccoli and broccoli sprouts, inhibits epigenetic regulators such as histone deacetylase (HDAC) enzymes, but it is not known whether consumption of a diet high in broccoli sprouts impacts epigenetic mechanisms in an in vivo model of prostate cancer. ObjectiveIn the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, we tested the hypothesis that a broccoli sprout diet suppresses prostate cancer, inhibits HDAC expression, alters histone modifications, and changes the expression of genes regulated by HDACs. MethodsTRAMP mice were fed a 15% broccoli sprout or control AIN93G diet; tissue samples were collected at 12 and 28 wk of age. ResultsMice fed broccoli sprouts had detectable amounts of SFN metabolites in liver, kidney, colon, and prostate tissues. Broccoli sprouts reduced prostate cancer incidence and progression to invasive cancer by 11- and 2.4-fold at 12 and 28 wk of age, respectively. There was a significant decline in HDAC3 protein expression in the epithelial cells of prostate ventral and anterior lobes at age 12 wk. Broccoli sprout consumption also decreased histone H3 lysine 9 trimethylation in the ventral lobe (age 12 wk), and decreased histone H3 lysine 18 acetylation in all prostate lobes (age 28 wk). A decline in p16 mRNA levels, a gene regulated by HDAC3, was associated with broccoli sprout consumption, but no significant changes were noted at the protein level. ConclusionsBroccoli sprout intake was associated with a decline in prostate cancer occurrence and HDAC3 protein expression in the prostate, extending prior work that implicated loss of HDAC3/ corepressor interactions as a key preventive mechanism by SFN in vivo.

Antiatherogenic effect of Tiger nut tubers (Cyperus esculentus L.) supplemented diet in apolipoprotein E knockout mice

The present study was carried out to evaluate the physiological effect of tiger nut tubers on lipid metabolism and peroxidation stress in apolipoprotein E knockout (apoE-/-) mice. Six male and five female mice, 6-7 wk old, was fed AIN 93G control diet supplemented with 25 % of whole powder of tiger nut tubers for 11 wk. Measurements were done for plasma, liver, aorta and brain lipid fractions. Peroxidation stress in mice serum and brain, i.e. vitamin E, nitric oxide and TBARS were evaluated and aortic valve lesions area was also determined. The obtained results showed that, type of diet and mice gender did not affect mice growth parameters. Serum and liver lipids were lowered in male mice fed tiger nut diet than that fed the control diet. However, the effect of tiger nut diet on the peroxidation parameters was not prominent. The mice fed on chufa-diet resulted in significant reduction in the aortic arch atherosclerotic lesions in both gender compared to that fed the basal diet. Our results show that tiger nut tubers have antiatherogenic effect and contain a gender dependent factor which regulates lipid metabolism in apoE-/- mice.

Effect of conjugated linoleic acid and different type of dietary fat on serum lipid profile, liver enzymes activity and oxidative stress markers in Wistar rats

Nutritional recommendations emphasize the need to limit consumption of saturated fatty acids and to increase the intake of polyunsaturated fatty acids in the prevention of non-communicable chronic diseases, particularly cardiovascular diseases. Among the fatty acids with health-related effects on the body, conjugated fatty acids are mentioned (i.e. CLA). The current study was designed to determine the effects of conjugated linoleic acid (CLA) on serum lipid profile, glucose, liver enzymes activity (AST and ALT), malonic dialdehyde (MDA) as well as lipid hydroperoxide (LPO) concentrations in rats fed diet differing in type of dietary fat. Male Wistar rats were divided into six groups and fed the following diets: control AIN-93G diet contained soybean oil (O) and diets with modification of fat source: butter (B) and margarine (M). The experimental diets were supplemented with 1% of conjugated linoleic acid (O+CLA, B+CLA, M+CLA). After 21 days the blood was collected and lipid profile, glucose, liver enzymes, MDA as well as LPO were analyzed. The dietary treatments had no significant effect on the body weight and liver weight of the animals. The concentrations of total cholesterol (TC) and LDL+VLDL cholesterol were unchanged. Both experimental factors (fat source and CLA) had a significant influence on the TAG and HDL levels. Margarine (M) significantly increased the TAG concentration, whereas CLA had a significant impact on the TAG reduction (M+CLA). Glucose level was significantly decreased in all groups fed diets supplemented with CLA. Serum ALT significantly increased in all CLA groups. Fat source had statistically significant influence on the MDA concentration. The LPO level was significantly elevated in all CLA groups. There was statistically significant interaction of experimental factors (fat source and CLA supplementation) on LPO level. Margarine had an adverse effect on the rat’s lipid profile. However, in the group fed with margarine, the addition of CLA decreased the concentration of TAG. Regardless of the type of the dietary fat, CLA supplementation increased the level of LPO in the blood serum of animals.

The Anti-Diabetic Potential of Thermally Treated Garlic, Turmeric, and Ginger in Pre-Diabetic Male Wistar Rat Model

Spices (turmeric (T), ginger (GI), and garlic (GA) (TGG)) have been used for centuries for food preservation, flavors, and medicinal properties. Research suggests that TGG contain potent antioxidants that may prevent and/or delay chronic diseases such as cancer, diabetes, and heart diseases. Heat treatment of spices may potentially increase antioxidative activity by modifying the inherent chemical structure of potent antioxidative compounds in spices. The purpose of this study was to determine the anti-diabetic potential of thermally treated TGG on Wistar male rats. Two-week-old male Wistar rats were randomly assigned to 8 groups (N = 24, n = 3) including control AIN-93G Diet (C) and high fat (HFD) and high sugar (HS) (glucose 10%) diet and treatment HFD/HS diets containing T, GA, GI (1% and 2%) singly for 11 weeks. Weekly feed intake, body weight, and blood glucose levels were recorded. Rats were sacrificed at 13 wks. by CO2 asphyxiation. Liver, pancreas, adipose (thigh), cecum, femur, urine, and serum samples were collected for quantitative determination of detoxification and antioxidative enzyme analysis, bone mineralization, and cholesterol using standard protocol. Of spice-incorporated diets, rats fed turmeric (1%) exhibited the lowest reduction in blood glucose levels at 90 mg/dL compared to the control 58 mg/dL. Additionally rats fed TGG at both concentrations resulted in an induction of antioxidant (GSH) and antioxidant enzyme (GPx) activity with significantly (p ≤ 0.05) higher levels compared to the control. Serum total cholesterol levels were lower in spice-incorporated diets compared to control HFD/HS fed rats. Therefore, the use of thermal application on spices presents promise in potentiating the antioxidant effects and thereby their potential health promoting properties.