Colonic Phospholipid Research Articles

Abstract 5256: Intracellular lipid droplet quantity as a biomarker for obesity associated resistance to omega-3 fatty acid colon cancer prevention

Abstract Rationale: Obesity induces resistance to Omega-3 (ω-3) FA anticarcinogenesis effect. Obesity associated excessive fatty acid intracellular concentration results in excess triglycerides stored as intracellular lipid droplets, saturated acyl-co-A transport of FA to triglycerides resulting in excess substrate for cyclooxygenase metabolism with increased formation of PGE2 and other inflammatory eicosanoids. Methods: Five weeks old male F344 rats were randomized equally into two diet groups. One cohort fed a western diet of ω-6 fatty acids (EPA:ω6 ratio of 0) and other fed an ω-3 fatty acid supplemented diet (EPA:ω6 ratio of 0.4). One half from each diet group were further treated with azoxymethane/dextran sulfate sodium (AOM/DSS) and then euthanized at 21 weeks after the carcinogen induction. Snap frozen colon tissue were assayed for Prostaglandins and fatty acids. OCT fixed colonic tissue stained with oil red O (ORO) to detect lipid droplets which were quantified using Aperio image analysis. From the second set, rat colons were opened longitudinally and fixed in 10% Formalin. Tumors were identified and enumerated using stereomicroscope to get tumor count, incidence and volume. These tissue were subjected to hematoxylin & eosin staining for histological evaluation of colonic tumors. Results: The ω-3 FA diet significantly increased the percent of eicosapentaenoic acid (EPA) in total fatty acids in both untreated and AOM/DSS-treated rats. EPA in the colonic tissue was increased with ω-3 diet as compared to ω-6 from 1.4±0.8 to 2.6±1.0* in untreated rats and from 1.3±1.3 to 3.3±1.7* in AOM/DSS-treated rats (*p<0.005). PGE2 increased with AOM/DSS treatment. The ω-3 diet decreased PGE2 in colonic mucosa of untreated rats from 132±64 in ω-6 to 78±17 in ω-3 and 150±74 in ω-6 to 92±43 in ω-3 with AOM/DSS treatment. Most tumors were found in the distal colon (DC) with ω-3 diet significantly reducing the number of tumors (p value=0.0011). The ω-3 diet significantly reduced colonic adenoma volume and multiplicity. When tumors were stratified by size and location, there was a significant effect of diet in the DC, with ω-3 diet reducing the number of tumors in the 1-3mm range (p value=0.0018), 3-5mm (p value=0.0041) and >5mm (p value=0.0055). ω-3 also reduced the number of 3-5mm size tumors in the cecum (p value=0.0377). On preliminary quantification of lipid droplets, there was a reduction in the positive pixels divided by the total pixels of ORO stained colonic tissue of ω-3 rats (0.004 ±0.01 as compared to 0.013±0.07 in ω-6 rats; p value=0.0121) Conclusions: Dietary ω-3 supplementation that increases the EPA:AA ratio in colonic tissue phospholipids reduces excess intracellular FA concentrations reflected by a reduction in size and number of lipid droplets. Lipid droplet number and size may be useful as a biomarker for effective reduction of obesity associated inflammation and carcinogenesis risk. Citation Format: Muhammad N. Aslam, Nithya Sridhar, Maheen Nadeem, Becky R. Simon, Jianwei Ren, Ananda Sen, James Varani, William L. Smith, Zora Djuric, Dean E. Brenner. Intracellular lipid droplet quantity as a biomarker for obesity associated resistance to omega-3 fatty acid colon cancer prevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5256. doi:10.1158/1538-7445.AM2017-5256

Dietary supplementation of arachidonic acid increases arachidonic acid and lipoxin A4 contents in colon, but does not affect severity or prostaglandin E2 content in murine colitis model

BackgroundArachidonic acid (ARA) is an essential fatty acid and a major constituent of biomembranes. It is converted into various lipid mediators, such as prostaglandin E2 (PGE2) and lipoxin A4 (LXA4). The effects of dietary ARA on colon maintenance are unclear because PGE2 has both mucosal protective and proinflammatory effects, and LXA4 has an anti-inflammatory role. Our objective is to clarify the effects of dietary ARA on an experimental murine colitis model.MethodsC57BL/6 mice were fed three types of ARA diet (0.075%, 0.15% or 0.305% ARA in diet), DHA diet (0.315% DHA) or control diet for 6 weeks, and were then administered dextran sodium sulphate (DSS) for 7 days to induce colitis. We evaluated colitis severity, fatty acid and lipid mediator contents in colonic tissue, and the expression of genes related to lipid mediator formation.ResultsARA composition of colon phospholipids was significantly elevated in an ARA dose-dependent manner. ARA, as well as DHA, did not affect colitis severity (body weight loss, colon shortening, diarrhea and hemoccult phenomena) and histological features. PGE2 contents in the colon were unchanged by dietary ARA, while LXA4 contents increased in an ARA dose-dependent manner. Gene expression of cyclooxygenase (COX)-1 and COX-2 was unchanged, while that of 12/15-lipoxgenase (LOX) was significantly increased by dietary ARA. ARA composition did not correlate with neither colon length nor PGE2 contents, but significantly correlated with LXA4 content.ConclusionThese results suggest that dietary ARA increases ARA and LXA4 contents in colon, but that it has no effect on severity and PGE2 content in a DSS-induced murine colitis model.

Perinatal lipid nutrition alters early intestinal development and programs the response to experimental colitis in young adult rats

The long-chain polyunsaturated n-6 and n-3 fatty acids are essential nutrients in membrane biogenesis and regulate gene expression via their eicosanoid metabolites. We investigated whether the n-6 and n-3 fatty acid supply as determined by maternal diet alters colonic phospholipid fatty acids, intestinal morphology, and epithelial barrier permeability during milk feeding with lasting effect on mucosal responsiveness to dinitrobenzene sulfonic acid (DNBS)-induced colitis in young adulthood. Female rats were fed diets with 20% energy from safflower oil (SO) or canola oil (CO), or 8% fish oil (FO) plus 2% SO (10% FO) or 18% FO plus 2% SO (20% FO) throughout gestation and lactation and offspring weaned to a standard diet at 21 days of age. At 15 days of age, pups in the 20% and 10% FO groups had lower 20:4n-6 and higher 20:5n-3 and 22:6n-3 in colon phospholipids (P < 0.01), shorter crypts (P < 0.05), and higher paracellular permeability than SO or CO groups. At 3 mo of age, male offspring in the FO groups showed lasting reduction of crypt depth and a heightened inflammatory response to DNBS. We demonstrate that early decreased colon 20:4n-6 with increased n-3 fatty acids impairs intestinal barrier development and sensitizes the colon response to inflammatory insults later in life.

Dietary Lipids in Early Development and Intestinal Inflammatory Disease

Inflammatory bowel diseases are life-long reoccurring inflammatory disorders of the gastrointestinal tract and have been increasing in incidence in recent decades, notably in the pediatric population. Although genetic predisposition remains an important factor, this increased incidence most likely reflects an environmental change. One potential contributor to this is the change in dietary fat intake, with dietary intake of n-6 polyunsaturated fatty acids (PUFAs) following a similar temporal pattern to the change in inflammatory bowel disease incidence. Dietary n-6 PUFAs comprise a major, modifiable, environmental factor known to promote a heightened inflammatory response through a number of pathways, including their role as precursors for synthesis of eicosanoids and their inhibitory effect on the synthesis of the n-3 PUFAs eicosapentanoic acid and docosahexanoic acid. The increase in n-6 PUFA intake affects individuals of all ages, with fetal PUFA accretion and infant dietary PUFA intake from breast milk reflecting maternal dietary intake. A high level of n-6 PUFA in milk results in increased n-6 PUFA in colonic phospholipids and an exaggerated inflammatory response to chemically induced colitis. Conversely, during development, a diet low in n-6 PUFAs and high in n-3 PUFAs increases colonic n-3 fatty acids, attenuates the inflammatory response, and lowers colonic damage. High dietary n-6 PUFA intake may be an important environmental modifier that contributes to inflammatory bowel diseases.

Intestinal responsiveness to experimental colitis in young rats is altered by maternal diet

Increasing evidence suggests that fetal and neonatal nutrition impacts later health. Aims of the present study were to determine the effect of maternal dietary fat composition on intestinal phospholipid fatty acids and responsiveness to experimental colitis in suckling rat pups. Female rats were fed isocaloric diets varying only in fat composition throughout gestation and lactation. The oils used were high (8%) in n-3 [canola oil (18:3n-3)], n-6 (72%) [safflower oil (18:2n-6)], or n-9 (78%) [high oleic acid safflower oil (18:1n-9)] fatty acids, n = 6/group. Colitis was induced on postnatal day 15 by intrarectal 2,4-dinitrobenzene sulfonic acid (DNBS) administration with vehicle (50% ethanol) and procedure (0.9% saline) controls. Jejunal and colonic phospholipids and milk fatty acids were determined. The distal colon was assessed for macroscopic damage, histology, and MPO activity. The 18:2n-6 maternal diet increased n-6 fatty acids, whereas the 18:3n-3 diet increased n-3 fatty acids in milk and pup jejunal and colonic phospholipids. Maternal diet, milk, and pup intestinal n-6-to-n-3 fatty acid ratios increased significantly in order: high 18:3n-3 < high 18:1n-9 < high 18:2n-6. DNBS administration in pups in the high 18:2n-6 group led to severe colitis with higher colonic damage scores and MPO activity than in the 18:1n-9 and 18:3n-3 groups. High maternal dietary 18:3n-3 intake was associated with colonic damage scores and MPO activity, which were not significantly different from ethanol controls. We demonstrate that maternal dietary fat influences the composition of intestinal lipids and responsiveness to experimental colitis in nursing offspring.

Aldosterone alters the phospholipid composition of rat colonocytes

Previous studies have shown that aldosterone treatment of amphibian epithelial cells results not only in stimulation of Na + absorption but also in changes in phospholipid composition which are necessary for the mineralocorticoid action of aldosterone. The present study was designed to investigate the effect of aldosterone on phospholipids of mammalian epithelia. Phospholipid and fatty acid composition was examined in colonic epithelium (mineralocorticoid target tissue) and thymus (non-mineralocorticoid but glucocorticoid target tissue) of rats which had received aldosterone or vehicle by a miniosmotic pump for 7 days. Aldosterone increased the mass of colonic phospholipids relative to cellular proteins with concomitant changes in the percentage distribution of fatty acids, whereas the relative distribution of membrane phospholipds was not changed. Phosphatidylcholine increased the content of polyunsaturated and decreased that of monounsaturated fatty acids, which predominantly reflected the accretion of arachidonic and a decrease in oleic and palmitoleic acids. Within the phosphatidylethanolamine subclass, pretreatment of rats with aldosterone decreased the content of monounsaturated fatty acids (predominantly oleic and palmitoleic acid) and of n-3 fatty acids, and increased the content of saturated fatty acids (palmitic acid). The saturated-to-nonsaturated fatty acid ratio also significantly increased after aldosterone treatment. No changes in thymic phospholipids were seen. The results are consistent with the contention that aldosterone specifically modulates phospholipid concentration and metabolism in mineralocorticoid target tissue. The changes in phospholipid content and its fatty acid composition during the fully developed effect of aldosterone may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.

Lack of promotion of colon carcinogenesis by high-oleic safflower oil.

The nonpromoting effect of olive oil on colon carcinogenesis has been attributed to its high oleic acid content, whereas a positive association of monounsaturated fat in beef tallow with colon tumors has been reported. The effect of constituents other than fatty acids could not be neglected in these experiments. In order to minimize the effects of minor constituents in the oils, the authors compared conventional safflower oil with oil from a mutant strain of safflower that is rich in oleic acid. ICR mice were treated with 1,2-dimethylhydrazine (DMH, 20 mg/kg body weight every week for 12 weeks) and then were fed either a high-fat diet (23.5% by weight), containing safflower oil (HF-LA) or high-oleic safflower oil (HF-OA), or a low-fat diet (5% by weight), containing safflower oil (LF-LA) or high-oleic safflower oil (LF-OA). The test diets were continued until termination of the experiment at 30 weeks after the first administration of DMH. Fatty acid composition of colon phospholipids was determined by gas-liquid chromatography-mass spectrometry. Tumor multiplicity in animals fed the HF-OA diet was indistinguishable from that in animals fed LF-LA or LF-OA. In contrast, animals fed the HF-LA diet had a significantly higher incidence of colon tumors (mostly adenocarcinomas) than the other groups. Fatty acid profiles of colon phospholipids reflected those of the diet. Animals fed a HF-LA diet showed a marked decrease of nervonic acid (C24:1, n-9) in the colon sphingomyelin. These data indicate that oleic acid does not enhance DMH-induced colon carcinogenesis in mice, even when they are fed a high-fat diet.

Comparative Effects of Dietary Fat Manipulation on Fatty Acid Composition of Rat Stomach, Jejunum, and Colon Phospholipids.

The phospholipid fatty acid composition of three parts of gastrointestinal tract (GIT) possessing different functions (stomach, jejunum, and colon) was examined to determine if these gut segments consistently respond to dietary fat. Three groups of eight weanling male Sprague-Dawley rats were fed one of three isocaloric, semi-synthetic, nutritionally adequate diets for six weeks. The fat type in the control diet was primarily beef tallow supplemented with sufficient linoleic acid (18: 2n-6) to prevent essential fatty acid deficiency, the n-6 diet contained high levels of 18:2n-6, and the n-3 diet provided high levels of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids with similar levels of 18:2n-6 as in the control diet. Feeding the n-3 diet resulted in the incorporation of 20:5n-3 and 22:6n-3 into all the GIT segments examined; however, the incorporation was significantly higher in the jejunum and the colon, compared with that in the stomach, phospholipids. The arachidonic acid (20:4n-6) content was lower in the stomach, jejunum, and colon phospholipids following the feeding of the n-3 diet. The 18: 2n-6 content was found to be higher following consumption of the n-3 diet compared with that found for the control diet. The 20:4n-6 content was significantly higher in the jejunum than in the colon or stomach phospholipids, whereas the 18:2n-6 content was consistently higher in all the GIT segments in animals fed the n-6 diet. This diet group also exhibited lower levels of monounsaturated fatty acids in the stomach and lower levels of saturated fatty acids in the jejunum and the colon phospholipids. Thus, dietary fat manipulations inconsistently influence the phospholipid fatty acid composition of various parts of the GIT, suggesting that the functioning of the stomach, jejunum, and colon may be affected differently by alterations in the type of dietary fat ingested.

An Enteral Formula Containing Fish Oil, Indigestible Oligosaccharides, Gum Arabic and Antioxidants Affects Plasma and Colonic Phospholipid Fatty Acid and Prostaglandin Profiles in Pigs ,

Evidence supports a pathogenic role of arachidonic acid–derived inflammatory mediators within the gastrointestinal tract of patients with inflammatory bowel disease. The purpose of this study was to assess the effects of an ulcerative colitis nutritional formula (UCNF) containing oligosaccharides, fish oil, gum arabic and antioxidants on plasma and colonic phospholipid fatty acid and prostaglandin profiles in pigs. Twenty-four growing barrows in two replications were equally randomized among four killing times (d 0, 7, 14 and 21), and one of two diets, a control and the UCNF. Diets contained comparable levels of protein, fat, and nonstructural carbohydrate and met 100% of the energy requirements of the pig. Intake and body weight were recorded daily while blood, urine and tissue samples were collected at time of kill. Within 1 wk of ingestion of the UCNF, the composition of plasma phospholipid fatty acids showed an increase in 20:5(n-3) and 22:6(n-3) (P < 0.0001) and a decrease in 20:4(n-6) and 18:2(n-6) (P < 0.0001). Similar effects were observed for the phospholipids in the colonic and cecal mucosa. Plasma prostaglandin E was unaffected by treatment, whereas thromboxane B2 and 6-keto-prostaglandin F1α levels were significantly decreased after 7 d of UCNF ingestion. Ingestion of the UCNF resulted in a suppression in the synthesis of proinflammatory prostaglandins by cecal and colonic mucosal cells. Levels of colonic and cecal prostaglandin E, 6-keto-prostaglandin F1α and thromboxane B2 were significantly decreased after 7 d of UCNF ingestion. These changes may have been mediated by rapid increases of (n-3) fatty acids into cellular phospholipids. Dietary supplementation with the UCNF may prove beneficial for patients with ulcerative colitis by modulating colonic prostaglandin synthesis.

Effect of Intestinal Resection on Phospholipid Class Distribution and Fatty Acid Composition of Mucosal Cells in the Rat Large Intestine

In this study we examined the effect of 50% distal small bowel resection (DSBR) on the lipid composition of rat colonic mucosa, including the fatty acid composition of major phospholipid classes, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), and sphingomyelin (SM). Phospholipid, free cholesterol, triacylglycerols, diacylglycerols, and cholesterol ester levels were not affected by the surgical operation. PE together with PC accounted for more than 60% of total phospholipid, in both groups of animals. The relative percentages of PE, PC, PI, SM, and cardiolipin (CL) remained unaffected by DSBR. However, a decrease in the content of LPC was observed in 50%-resected animals. Different fatty acid composition changes in the colonic phospholipid classes were observed after resection. PC fraction contained the highest amounts of saturated fatty acids, but monounsaturated fatty acids were present in high levels in PI fraction, in both groups of animals. DSBR produces different changes in the levels of linoleic and arachidonic acids. These results suggest that the lipid composition and the fatty acid profile of the different phospholipid fractions change in the adaptive response of colon to intestinal resection.