Modified AIN-93G Diet Research Articles

Dietary Milk Phospholipids Increase Body Fat and Modulate Gut Permeability, Systemic Inflammation, and Lipid Metabolism in Mice

The study aimed at how dietary milk polar lipids affect gut permeability, systemic inflammation, and lipid metabolism during diet-induced obesity (DIO). C57BL/6J mice (n = 6x3) were fed diets with 34% fat as energy for 15 weeks: (1) modified AIN-93G diet (CO); (2) CO with milk gangliosides (GG); (3) CO with milk phospholipids (MPL). Gut permeability was assessed by FITC-dextran and sugar absorption tests. Intestinal tight junction proteins were evaluated by Western blot. Plasma cytokines were measured by immunoassay. Body composition was assessed by magnetic resonance imaging. Tissue lipid profiles were obtained by thin layer chromatography. Hepatic expression of genes associated with lipid metabolism was assessed by RT-qPCR. MPL increased the efficiency of converting food into body fat and facilitated body fat accumulation compared with CO. MPL and GG did not affect fasting glucose or HOMA-IR during DIO. MPL increased while GG decreased plasma TG compared with CO. MPL decreased phospholipids subclasses in the muscle while increased those in the liver compared with CO. GG and MPL had little effect on hepatic expression of genes associated with lipid metabolism. Compared with CO, MPL decreased polar lipids content in colon mucosa. Small intestinal permeability decreased while colon permeability increased and then recovered during the feeding period. High-fat feeding increased plasma endotoxin after DIO but did not affect plasma cytokines. MPL and GG did not affect plasma endotoxin, adipokines and inflammatory cytokines. After the establishment of obesity, MPL increased gut permeability to large molecules but decreased intestinal absorption of small molecules while GG tended to have the opposite effects. MPL and GG decreased mannitol and sucralose excretions, which peaked at d 45 in the CO group. MPL decreased occludin in jejunum mucosa compared with CO. GG and MPL did not affect zonula occludens-1 in gut mucosa. In conclusion, during DIO, milk GG decreased gut permeability, and had little effect on systemic inflammation and lipid metabolism; MPL facilitated body fat accumulation, decreased gut permeability, did not affect systemic inflammation.

Dietary milk polar lipids modulate gut barrier integrity and lipid metabolism in C57BL/6J mice during systemic inflammation induced by Escherichia coli lipopolysaccharide

The focus of this work is the role milk polar lipids play in affecting gut permeability, systemic inflammation, and lipid metabolism during acute and chronic inflammation induced by a single subcutaneous injection of lipopolysaccharide. Three groups of C57BL/6J mice were fed: modified AIN-93G diet with moderate level of fat (CO); CO with milk gangliosides (GG); CO with milk phospholipids (MPL). The MPL did not prevent a gut permeability increase upon LPS stress but increased the expression of tight junction proteins zonula occludens-1 and occludin in colon mucosa. The GG prevented the gut permeability increase upon LPS stress. The MPL decreased absolute and relative liver mass and decreased hepatic gene expression of acetyl-CoA carboxylase 2 and 3-hydroxy-3-methylglutaryl-CoA reductase. The GG increased hepatic gene expression of acetyl-CoA acyltransferase 2. In conclusion, milk GG protected the intestinal barrier integrity but had little effect on systemic inflammation and lipid metabolism; milk MPL, conversely, had complex effects on gut permeability, did not affect systemic inflammation, and had beneficial effect on hepatic lipid metabolism.

Natural Vitamin E Supplementation during Pregnancy in Rats Increases RRR-α-Tocopherol Stereoisomer Proportion and Enhances Fetal Antioxidant Capacity, Compared to Synthetic Vitamin E Administration

Introduction: Low dietary intake of vitamin E is a global public health issue. RRR-α-tocopherol (RRR-αT) is the only naturally occurring vitamin E stereoisomer, but the equimolecular mixture of all eight stereoisomers, synthetic vitamin E (S-αT), is commonly consumed. The objective of this study was to evaluate bioavailability and antioxidant activity of RRR-αT versus S-αT, in both mother and fetus, after maternal supplementation during pregnancy. Methods: Female rats (7 weeks of age) received a modified AIN-93G diet supplemented with 75 IU/kg of RRR-αT (NVE, n = 20) or S-αT (SVE, n = 17). At delivery, the levels of αT, stereoisomer distribution, and antioxidant capacity were analyzed in maternal and fetal plasma. Results: NVE administration significantly increased the proportion of RRR-αT stereoisomer in maternal and fetal plasma. The percentage of RRR-αT increased from 32.76% to 88.33% in maternal plasma, and 35.25% to 97.94% in fetal plasma, in the NVE group compared to SVE. Fetal plasma from the NVE group was found to have higher total antioxidant capacity compared to SVE. Lastly, fetal plasma RRR-αT stereoisomer percentage was positively associated with expression levels of scavenger receptor class B type 1 (SR-B1) in the placenta. Conclusions: Both natural and synthetic sources of vitamin E showed similar bioavailability. Still, NVE supplementation increased the proportion of RRR-αT and promoted higher antioxidant activity in fetal plasma at birth. Placental SR-B1 might be involved in the stereoselective transfer of RRR-αT stereoisomer across the placenta and may improve αT bioactivity in the fetus.

Abstract A005: Identification of lipid pharmacodynamic biomarkers in FAP model

Abstract Familial Adenomatous Polyposis (FAP) is a genetic disorder characterized by the development of numerous polyps in the colon, that unless removed by colectomy, inevitably progress to colorectal cancer (CRC). Although there is currently no FDA-approved treatment to delay colectomy, a common target for chemoprevention is cyclooxygenase-2 (COX-2). Non-steroidal anti-inflammatory drugs (NSAIDs) and omega-3 fatty acids are known to alter COX metabolism and affect downstream eicosanoids involved in CRC progression. In this study, we examined the chemopreventive efficacy of the NSAID naproxen alone, or in combination with TP-252, a novel, chemically-stable analogue of the omega-3 fatty acid, eicosapentaenoic acid (EPA), in the intestinal tumorigenesis Apcam1137 PIRC rat model. Over the course of this 20-week study, rats were fed a modified AIN-93G diet supplemented with 200 ppm naproxen, alone or in combination with TP-252 (1.5%, or 3% by weight). Rats treated with 200 ppm naproxen had 66% (p < 0.0001) fewer colon tumors than controls (AIN-93G) and an 81% (p < 0.0001) reduction in tumor size. 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 controls. Since the primary mechanisms of action for naproxen and EPA target COX metabolic activity, we performed a comprehensive lipidomic analysis on colon tissue and plasma to determine lipidomic changes associated with drug treatment. Increased formation of several EPA-derived eicosanoids, including 9-HEPE, 12-HEPE, 18-HEPE, and resolvin E2, whose mucosal levels correlated with both reduced tumor number and volume, were observed. These metabolites were also concomitantly elevated in the plasma of treated animals, representing potential pharmacodynamic (PD) biomarkers of CRC prevention. Future studies will decipher potential mechanisms that underlie these EPA-derived PD biomarkers and their role in tumor suppression. (Supported by the NCI Prevent Program 75N91019D00019, Task Order 75N91019F00132). Citation Format: Ryan Beach, Altaf Mohammed, Daniel W. Rosenberg. Identification of lipid pharmacodynamic biomarkers in FAP model [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A005.

Identification of oncogenic signatures in the inflammatory colon of C57BL/6 mice fed a high-fat diet

Adoption of an obesogenic diet such as a high-fat diet (HFD) results in obesity, bacterial dysbiosis, chronic inflammation, and cancer. Gut bacteria and their metabolites are recognized by interleukin-1 (IL-1R)/toll-like receptors (TLRs) which are essential to maintain intestinal homeostasis. Moreover, host extracellular microRNAs (miRNAs) can alter bacterial growth in the colon. Characterization of the underlying mechanisms may lead to identifying fecal oncogenic signatures reflecting colonic health. We hypothesize that an HFD accelerates the inflammatory process and modulates IL-1R/TLR pathways, gut microbiome, and disease-related miRNA in the colon. In this study, 4-week-old C57BL/6 mice were fed a modified AIN93G diet (AIN, 16% energy fat) or an HFD (45% energy fat) for 15 weeks. In addition to increased body weight and body fat composition, the concentrations of plasma interleukin 6 (IL-6), inflammatory cell infiltration, β-catenin, and cell proliferation marker (Ki67) in the colon were elevated > 68% in the HFD group compared to the AIN group. Using a PCR array analysis, we identified 14 out of 84 genes with a ≥ 24% decrease in mRNA content related to IL-1R and TLR pathways in colonic epithelial cells in mice fed an HFD compared to the AIN. Furthermore, the content of Alistipes bacteria, the Firmicutes/Bacteroidetes ratio, microRNA-29a, and deoxycholic and lithocholic acids (secondary bile acids with oncogenic potential) were 55% greater in the feces of the HFD group compared to the AIN group. Collectively, this composite, a multimodal profile may represent a unique HFD-induced fecal signature for colonic inflammation and cancer in C57BL/6 mice.

LCHP Diet Enriched with Cholesterol Promotes Non-Alcoholic Fatty Liver Disease in Wistar Rats

The aim of this study was to analyze the effect of a low-carbohydrate–high-protein diet, enriched with cholesterol (LCHPch), on body weight, lipid metabolism, as well as kidney and liver function in rats. Wistar rats (N = 18, male) were randomly allocated into experimental groups and fed a modified AIN-93G diet with the addition of 1% cholesterol (AINch, WDch and LCHPch diets) for 8 weeks. Despite the lack of significant differences in the final body weight and liver weight of animals, the kidneys of rats in the LCHPch group were considerably heavier compared to the control group. Serum total cholesterol and the sum of low- and very-low-density fractions of cholesterol as well as ALT activity were significantly increased in the LCHPch rats in comparison to the AINch group. Simultaneously, the highest content of liver fat was obtained for animals from LCHPch group. It was also shown that both WDch and LCHPch diets significantly changed fatty acid profile in the adipose tissue of rats compared to control, with a significant increase in SFA and MUFA and a decrease in PUFA. Non-alcoholic fatty liver disease was observed in rats fed not only WDch but also LCHPch diets. The detailed mechanism still needs to be investigated to prevent the organism from the harmful effects of macronutrient dietary imbalance.

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.

Slc39a4 in the small intestine predicts zinc absorption and utilization: a comprehensive analysis of zinc transporter expression in response to diets of varied zinc content in young mice

Zinc homeostasis is primarily maintained by zinc transporters that regulate zinc uptake and efflux in the small intestine; however, the relative contribution of the many zinc transporters identified (Slc39a1-14, Slc30a1-10) to dietary zinc absorption and utilization remains unknown. The objective of this study was to determine the expression of Slc39a1-14 and Slc30a1-10 in the small intestine and their relative contribution to dietary zinc absorption in mice. Five-week-old male C57BL/6J mice were fed modified AIN-93G diets containing <1, 30, or 100ppm zinc (n=15 mice/diet). Following 1 week of feeding, mice were given an oral gavage containing 67Zn and liver and plasma isotope appearance was determined 6-h later by ICP-MS. Expression of Slc39a1-14 and Slc30a1-10 was determined in mucosa from duodenum, jejunum, and ileum. Plasma and liver total zinc concentrations were not different after one week of feeding (P>.05). Liver and plasma appearance of 67Zn was greater in mice fed <1ppm compared to the 30ppm (P<.0001) and 100ppm (P<.0001) zinc diets. With the exception of Slc39a2, Slc39a12, Slc30a3, and Slc30a8, the remaining zinc transporters were expressed across all diets and intestinal segments. Expression of Slc39a4, Slc39a11, and Slc30a6 changed with diet (Pdiet<.05 for all); expression of Slc39a5, Slc39a7, Slc39a11, Slc39a14, Slc30a1, Slc30a2, Slc30a4, Slc30a5, Slc30a7, and Slc30a10 changed by intestinal segment (Psegment<.05 for all). Slc39a4 was the only transporter positively associated with liver (r2=0.316, P<.001) and plasma (r2=0.189, P<.01) 67Zn appearance. Although most zinc transporters are expressed in the small intestine, intestinal Slc39a4 predicts fractional zinc absorption and utilization in young mice.

A 10% Tomato Diet Selectively Reduces Radiation-Induced Damage in TRAMP Mice

BackgroundTomatoes contain carotenoids that have the potential to alter the effects of external beam radiation therapy (EBRT). ObjectivesWe hypothesized that dietary lyophilized tomato paste (TP) would reduce apoptosis within carotenoid-containing nonneoplastic tissues in EBRT-treated TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice. MethodsMale TRAMP mice (n = 73) were provided an AIN-93G diet or a modified AIN-93G diet containing 10% TP (wt:wt) at 4 wk of age. Prostate tumor growth was monitored by ultrasound. The caudal half of the mouse was irradiated with 7.5 Gy (Rad) or 0 Gy (sham) at 24 wk of age or after the tumor volume exceeded 1000 mm3 with a Cobalt-60 source. Mice were euthanized 24 h postradiation. Carotenoids and α-tocopherol were measured by HPLC and compared by a t test. Tissues were assessed for radiation-induced changes (hematoxylin and eosin) and apoptosis [cleaved caspase-3 (CC3)] and compared by Kruskal–Wallis test or Freedman–Lane's permutation test. ResultsSerum concentrations of lycopene (52% lower), phytoene (26% lower), and α-tocopherol (22% lower) were decreased in TP-fed irradiated mice (TP-Rad) compared with TP-fed sham mice (P < 0.05). CC3 scores increased within the prostate tumor with radiation treatments (P < 0.05), but were not affected by tomato consumption. In nonneoplastic tissues, TP-Rad had a lower percentage of CC3-positive cells within the cranial (67% lower) and caudal (75% lower) duodenum than irradiated mice on the control diet (Rad) (P < 0.005). Likewise, CC3 scores within the dorsolateral prostate of TP-Rad trended toward lower scores than for Rad (P = 0.07). ConclusionsTP selectively reduces radiation-induced apoptosis in extratumoral tissues without decreasing radiation-induced apoptosis within the prostate tumor in TRAMP mice. Additional studies are needed to confirm and expand upon these findings.

Baked cod consumption delayed the development of kidney and liver dysfunction and affected plasma amino acid concentrations, but did not affect blood pressure, blood glucose or liver triacylglycerol concentrations in obese fa/fa Zucker rats.

Obesity is associated with changes in amino acid metabolism, and studies show that ingestion of fish proteins influence amino acid composition in plasma and urine, in addition to affecting risk factors for metabolic syndrome. Since the majority of fish proteins consumed by humans are as fish fillet, it is of interest to investigate if cod fillet intake affects amino acid composition and metabolic disorders. We hypothesized that a modified AIN-93G diet containing cod fillet would affect amino acid compositions in plasma and urine in obese rats, and also affect risk factors for metabolic syndrome when compared to rats fed a regular AIN-93G diet with casein as the protein source. Obese Zucker fa/fa rats, a rat model of metabolic syndrome, received diets containing 25% protein from lyophilized baked cod fillet and 75% protein from casein (Baked cod diet), or a Control diet with casein for four weeks. The Baked cod diet affected the amino acid composition in plasma, with e.g., lower glycine, histidine, homoarginine, homocysteine, methionine, proline and tyrosine concentrations, but did not affect amino acid concentrations in urine. The concentrations of markers for kidney and liver dysfunction were lower in the Baked cod group, however blood pressure development, fasting and postprandial glucose, and hepatic triacylglycerol concentrations were similar to the Control group. To conclude, substituting 25% of dietary protein with baked cod fillet affected concentrations of some amino acids in plasma and delayed development of kidney and liver dysfunction, but did not affect blood pressure, glucose concentration or fatty liver.

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.

Dietary Supplementation of Foxtail Millet Ameliorates Colitis-Associated Colorectal Cancer in Mice via Activation of Gut Receptors and Suppression of the STAT3 Pathway.

Coarse cereal intake has been reported to be associated with reduced risk of colorectal cancer. However, evidence from intervention studies is absent and the molecular basis of this phenomenon remains largely unexplored. This study sought to investigate the effects of foxtail millet and rice, two common staple grains in Asia, on the progression of colitis-associated colorectal cancer (CAC) and define the mechanism involved. In total, 40 BALB/c mice were randomized into four groups. The Normal and azoxymethane/dextran sodium sulfate (AOM/DSS) groups were supplied with an AIN-93G diet, while the millet- and rice-treated groups were supplied with a modified AIN-93G diet. Compared to the AOM/DSS-induced CAC mice supplemented with rice, an increased survival rate, suppressed tumor burden, and reduced disease activity index were observed in the millet-treated group. The levels of IL-6 and IL-17 were decreased in the millet-treated group compared to both the AOM/DSS and AOM/DSS + rice groups. Millet treatment inhibited the phosphorylation of STAT3 and the related signaling proteins involved in cell proliferation, survival and angiogenesis. These beneficial effects were mediated by the activation of gut receptors AHR and GPCRs via the microbial metabolites (indole derivates and short-chain fatty acids) of foxtail millet. Moreover, millet-treatment increased the abundance of Bifidobacterium and Bacteroidales_S24-7 compared to the rice-treated mice. This study could help researchers to develop better dietary patterns that work against inflammatory bowel disease (IBD) and for CAC patients.

Milk polar lipids modulate lipid metabolism, gut permeability, and systemic inflammation in high-fat-fed C57BL/6J ob/ob mice, a model of severe obesity

Dynamic interactions between lipid metabolism, gut permeability, and systemic inflammation remain unclear in the context of obesity. Milk polar lipids, lipids derived from the milk fat globule membrane, could positively affect the aforementioned obesity-related endpoints. This study aimed to test the hypotheses that milk polar lipids will reduce gut permeability, systemic inflammation, and liver lipid levels, and differentially affect the hepatic expression of genes associated with fatty acid synthesis and cholesterol regulation in preexisting obesity. We fed 3 groups of C57BL/6J ob/ob mice (n = 6 per group) for 2 wk: (1) a modified AIN-93G diet (CO) with 34% fat by energy; (2) CO with milk gangliosides (GG) at 0.2 g/kg of diet; and (3) CO with milk phospholipids (PL) at 10 g/kg of diet. The GG and PL were provided as semi-purified concentrates and replaced 2.0% and 7.2% of dietary fat by energy. The GG and PL did not affect total food intake, weight gain, fasting glucose, or gut permeability. The PL decreased liver mass and the mesenteric fat depot compared with the CO. The GG increased tight junction protein occludin in colon mucosa compared with the CO. The GG and PL decreased tight junction protein zonula occludens-1 in jejunum mucosa compared with the CO. Plasma endotoxin increased during the study but was unaffected by the treatments. Compared with the CO and GG, the PL increased plasma sphingomyelin and plasma IL-6. The GG and PL differentially regulated genes associated with lipid metabolism in the liver compared with the CO. Regarding general effects on lipid metabolism, the GG and PL decreased lipid levels in the liver and the mesenteric depot, and increased lipid levels in the plasma. Diet consumption decreased significantly when the ob/ob mice were kept in metabolic cages, which were not big enough and resulted in unwanted animal deaths. Future studies may keep this in mind and use better metabolic equipment for ob/ob mice. In conclusion, dietary milk polar lipids may have limited beneficial effects on gut barrier integrity, systemic inflammation, and lipid metabolism in the context of severe obesity.

Long-term dietary intake of excessive vitamin A impairs spermatogenesis in mice.

Vitamin A and its derivatives contribute to many physiological processes, including vision, neural differentiation, and reproduction. Vitamin A deficiency causes early cessation of spermatogenesis, characterized by a marked depletion of germ cells. However, there has been no clear understanding about the role of chronic intake of vitamin A excess (VAE) in spermatogenesis. The objective of this study was to investigate whether chronic intake of VAE diet causes arrest of spermatogenesis. To examine the effects of VAE on spermatogenesis, we used ICR male mice fed with control (AIN-93G purified diet: 4 IU/g) diet or VAE (modified AIN-93G diet with VAE: 1,000 IU/g) diet for 7 weeks (from 3 to 10 weeks of age). At 10 weeks of age, the retinol concentration in the testes of VAE mice was significantly higher than that of control mice. Testicular cross sections from control mice contained a normal array of germ cells, while the seminiferous tubules from VAE mice exhibited varying degrees of testicular degeneration. Daily sperm production in VAE testes was dramatically decreased compared to that in control testes. Sperm viability, motility, and morphology were also impaired in VAE mice. Furthermore, we examined the effects of VAE on the expression of genes involved in retinoid signaling and spermatogenesis to determine the underlying molecular mechanisms. Therefore, we are the first to present results describing the long-term dietary intake of VAE impairs spermatogenesis using a mouse model.

Bone structure is largely unchanged in growing male CD-1 mice fed lower levels of vitamin D and calcium than in the AIN-93G diet

BackgroundCalcium (Ca) and vitamin D (vit D) in the AIN-93G diet may be higher than required for healthy bone development, and mask the potential benefit of a dietary intervention. ObjectiveThe objective was to determine if lower levels of Ca and vit D than is present in the AIN-93G diet supports bone development in growing male CD-1 mice. MethodsWeanling male CD-1 mice were randomized to modified AIN-93G diets containing either 100 (Trial 1) or 400 (Trial 2) IU vit D/kg diet within one of two or three Ca levels (0.35, 0.30, or 0.25% Ca diet in Trial 1 or 0.35% or 0.25% in Trial 2) or the AIN-93G diet (1000 IU/kg vit D and 0.5% Ca) from weaning to 4 months of age (n = 13–15/group). At 2 and 4 months of age, BMD and structural properties of the tibia were analyzed in vivo. Structure of lumbar vertebra 4 (L4) and mandible, and femur strength were assessed ex vivo at age 4 months. ResultsThere were no differences in tibia, L4, and mandible structure between the AIN-93G diet and the 0.35% Ca groups at either vit D level. A few structure outcomes were compromised with the 0.25 and/or 0.3% Ca diets but there were no differences in femur biomechanical strength compared to AIN-93G group in either Trial. ConclusionAt 400 or 100 IU vit D/kg diet, Ca can be lowered to 0.35% without detriment to BMD or bone structure while bone strength is not altered at lower Ca (0.25%) compared to CD-1 mice fed AIN-93G diet. Because of genetic variation in CD-1 mice among different breeding facilities, results in CD-1 mice from other facilities may differ from the present study.

A novel bioactive derivative of eicosapentaenoic acid (EPA) suppresses intestinal tumor development in ApcΔ14/+ mice.

Familial adenomatous polyposis (FAP) is a genetic disorder characterized by the development of hundreds of polyps throughout the colon. Without prophylactic colectomy, most individuals with FAP develop colorectal cancer at an early age. Treatment with EPA in the free fatty acid form (EPA-FFA) has been shown to reduce polyp burden in FAP patients. Since high-purity EPA-FFA is subject to rapid oxidation, a stable form of EPA compound has been developed in the form of magnesium l-lysinate bis-eicosapentaenoate (TP-252). We assessed the chemopreventive efficacy of TP-252 on intestinal tumor formation using ApcΔ14/+ mice and compared it with EPA-FFA. TP-252 was supplemented in a modified AIN-93G diet at 1, 2 or 4% and EPA-FFA at 2.5% by weight and administered to mice for 11 weeks. We found that administration of TP-252 significantly reduced tumor number and size in the small intestine and colon in a dose-related manner and as effectively as EPA-FFA. To gain further insight into the cancer protection afforded to the colon, we performed a comprehensive lipidomic analysis of total fatty acid composition and eicosanoid metabolites. Treatment with TP-252 significantly decreased the levels of arachidonic acid (AA) and increased EPA concentrations within the colonic mucosa. Furthermore, a classification and regression tree (CART) analysis revealed that a subset of fatty acids, including EPA and docosahexaenoic acid (DHA), and their downstream metabolites, including PGE3 and 14-hydroxy-docosahexaenoic acid (HDoHE), were strongly associated with antineoplastic activity. These results indicate that TP-252 warrants further clinical development as a potential strategy for delaying colectomy in adolescent FAP patients.

Colonic aberrant crypt formation accompanies an increase of opportunistic pathogenic bacteria in C57BL/6 mice fed a high-fat diet

The increasing worldwide incidence of colon cancer has been linked to obesity and consumption of a high-fat Western diet. To test the hypothesis that a high-fat diet (HFD) promotes colonic aberrant crypt (AC) formation in a manner associated with gut bacterial dysbiosis, we examined the susceptibility to azoxymethane (AOM)-induced colonic AC and microbiome composition in C57/BL6 mice fed a modified AIN93G diet (AIN, 16% fat, energy) or an HFD (45% fat, energy) for 14 weeks. Mice receiving the HFD exhibited increased plasma leptin, body weight, body fat composition and inflammatory cell infiltration in the ileum compared with those in the AIN group. Consistent with the gut inflammatory phenotype, we observed an increase in colonic AC, plasma interleukin-6, tumor necrosis factor-α, monocyte chemoattractant protein-1 and inducible nitric oxide synthase in the ileum of the HFD-AOM group compared with the AIN-AOM group. Although the HFD and AIN groups did not differ in bacterial species number, the HFD and AIN diets resulted in different bacterial community structures in the colon. The abundance of certain short-chain fatty acid (SCFA) producing bacteria (e.g., Barnesiella) and fecal SCFA (e.g., acetic acid) content were lower in the HFD-AOM group compared with the AIN and AIN-AOM groups. Furthermore, we identified a high abundance of Anaeroplasma bacteria, an opportunistic pathogen in the HFD-AOM group. Collectively, we demonstrate that an HFD promotes AC formation concurrent with an increase of opportunistic pathogenic bacteria in the colon of C57BL/6 mice.

Saturation of SERCA's lipid annulus may protect against its thermal inactivation

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps are integral membrane proteins that catalyze the active transport of Ca2+ into the sarcoplasmic reticulum, thereby eliciting muscle relaxation. SERCA pumps are highly susceptible to oxidative damage, and cytoprotection of SERCA dampens thermal inactivation and is a viable therapeutic strategy in combating diseases where SERCA activity is impaired, such as muscular dystrophy. Here, we sought to determine whether increasing the percent of saturated fatty acids (SFA) within SERCA's lipid annulus through diet could protect SERCA pumps from thermal inactivation. Female Wistar rats were fed either a semi-purified control diet (AIN93G, 7% soybean oil by weight) or a modified AIN93G diet containing high SFA (20% lard by weight) for 17 weeks. Soleus muscles were extracted and SERCA lipid annulus and activity under thermal stress were analyzed. Our results show that SERCA's lipid annulus is abundant with short-chain (12–14 carbon) fatty acids, which corresponds well with SERCA's predicted bilayer thickness of 21 Å. Under control-fed conditions, SERCA's lipid annulus was already highly saturated (79%), and high-fat feeding did not increase this any further. High-fat feeding did not mitigate the reductions in SERCA activity seen with thermal stress; however, correlational analyses revealed significant and strong associations between % SFA and thermal stability of SERCA activity with greater %SFA being associated with lower thermal inactivation and greater % polyunsaturation and unsaturation index being associated with increased thermal inactivation. Altogether, these findings show that SERCA's lipid annulus may influence its susceptibility to oxidative damage, which could have implications in muscular dystrophy and age-related muscle wasting.

A Herbal Formula HT051, a Combination of Pueraria lobata and Rehmannia glutinosa, Prevents Postmenopausal Obesity in Ovariectomized Rats.

Menopause is strongly associated with an increased risk of metabolic dysfunctions due to the decline in estrogen. Here, we hypothesized that dietary HT051, containing the roots of Pueraria lobata and Rehmannia glutinosa, has beneficial effects on ovariectomized (OVX) rats by regulating lipid metabolism. Forty-eight female Sprague-Dawley rats were randomly divided into 4 groups: sham-operated (Sham), OVX, OVX with low-dose HT051 supplementation, and OVX with high-dose HT051 supplementation. The rats were fed with a modified AIN-93G diet or an HT051-containing modified AIN-93G diet for 8 weeks. Body weight, fat mass, and serum levels of total cholesterol, triglyceride, glucose, alanine transaminase, and aspartate transaminase decreased in HT051-fed OVX rats. Dietary HT051 supplementation significantly decreased the mRNA expression of lipogenesis-related genes, including sterol regulatory element-binding protein 1c and fatty acid synthase, and increased the mRNA expression of β-oxidation-related genes, including peroxisome proliferator-activated receptor and carnitine palmitoyl transferase 1 in the liver of OVX rats. Moreover, the expression of genes involved in adipogenesis and inflammation was significantly lower in the adipose tissue of OVX rats fed with HT051 than in the OVX group. These findings suggest that HT051 may be a potential natural alternative for the management of postmenopausal metabolic dysfunctions.

Protection of the brain through supplementation with larch arabinogalactan in a rat model of vascular dementia.

BACKGROUND/OBJECTIVESVascular dementia (VaD) caused by reduced blood supply to the brain manifests as white matter lesions accompanying demyelination and glial activation. We previously showed that arabinoxylan consisting of arabinose and xylose, and arabinose itself attenuated white matter injury in a rat model of VaD. Here, we investigated whether larch arabinogalactan (LAG) consisting of arabinose and galactose could also reduce white matter injury.MATERIALS/METHODSWe used a rat model of bilateral common carotid artery occlusion (BCCAO), in which the bilateral common carotid arteries were exposed and ligated permanently with silk sutures. The rats were fed a modified AIN-93G diet supplemented with LAG (100 mg/kg/day) for 5 days before and 4 weeks after being subjected to BCCAO. Four weeks after BCCAO, the pupillary light reflex (PLR) was measured to assess functional consequences of injury in the corpus callosum (cc). Additionally, Luxol fast blue staining and immunohistochemical staining were conducted to assess white matter injury, and astrocytic and microglial activation, respectively.RESULTSWe showed that white matter injury in the the cc and optic tract (opt) was attenuated in rats fed diet supplemented with LAG. Functional consequences of injury reduction in the opt manifested as improved PLR. Overall, these findings indicate that LAG intake protects against white matter injury through inhibition of glial activation.CONCLUSIONSThe results of this study support our hypothesis that cell wall polysaccharides consisting of arabinose are effective at protecting white matter injury, regardless of their origin. Moreover, LAG has the potential for development as a functional food to prevent vascular dementia.