Jaya T. Venkatraman

Effects of n−3 and n−6 fatty acids on the activities and expression of hepatic antioxidant enzymes in autoimmune-prone NZB×NZW F1 mice

Menhaden fish oil (FO) containing n−3 fatty acids dramatically extends the life span and delays the onset and progression of autoimmune disease in (NZB×NZW)F1 (B/W) female mice as compared to those fed corn oil (CO) rich in n−6 lipids. As an inefficient antioxidant defense system has been linked to autoimmune diseases, the present study was undertaken to determine whether the protective action of n−3 lipids is mediated through their antioxidant defense system. Weanling B/W mice were fed a nutritionally adequate, semipurified diet containing CO or krill oil (KO) or FO at 10% level (w/w)ad libitum until the mice were 6.5 months old. All diets contained the same level of vitamin E (21.5 mg/100 g diet). We compared the effects of feeding n−6 and n−3 lipids on survival, kidney disease, hepatic microsomal lipid composition, peroxidation, and on the activity and mRNA expression of the antioxidant enzymes catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in 6.5-month-old B/W mice. The results showed that when compared to livers from CO-fed mice, livers from KO- and FO-fed mice showed: (i) significantly higher (P<0.001) activities and expression of CAT, GSH-Px and SOD; (ii) significantly lower (P<0.001) arachidonic acid (20∶4n−6) and linoleic acid (18∶2n−6) and higher (P<0.001) eicosapentaenoic acid (20∶5n−3) and docosahexaenoic acid (22∶6n−3) levels in hepatic microsomes; and (iii) significantly lower (P<0.001) estimated peroxidation indices and thiobarbituric acid reactive substances generation. The data indicate that one of the mechanisms through which the n−3 lipids delay the onset of autoimmune diseases in B/W mice may be through maintenance of higher activities and expression of hepatic antioxidant enzymes.

ROLE OF OMEGA-3 FATTY ACIDS IN HEALTH AND DISEASE

Abstract Dietary lipid interventions have an important role in modulating the onset of autoimmunity, cardiovascular diseases and cancer. Many studies caused out in the past have established the adverse effects of saturated fats in humans and in animal models. Based on these adverse effects, the consumption of vegetable oils containing both monounsaturated omega (ω)-9 and polyunsaturated fany acids (rich in 18:2 ω-6) is rising significantly in the United States. The increased consumption of many vegetable oils particularly of ω-6 series is however to be viewed as pro-inflammatory and is suspected as one of the possible causes for the gradual rise in certain malignant tumors, rheumatoid arthritis and autoimmune diseases primarily due to the increased production of pro-inflammatory cytokines although its increased usage has reduced cardiovascular disease nearly 30% in the United States. Diets based on ω-6 enriched oils can increase the level of linoleic acid in tissue phosphoglycerides and are able to reduce cholesterol levels, yet these lipids usually tend to elevate excessive arachidonic acid (20:4 ω-6) levels. In contrast, ω-3 fatty acid-enriched fish oil (FO) and/or ω-3 precursors from certain vegetable oils (linolenic acid, 18:3 ω-3) are found to provide protection against cardiovascular disease, rheumatoid arthritis, cancer and possibly against the severity of viral infections. Nutritional modification of cellular functions by dietary lipids with a balanced ratio of ω-6 and ω-3 fatty acids offers an attractive avenue to correct, modify and/or prevent many patho-physiological processes in health and disease state and to reduce toxicity of drugs in many patients. The mediation of such effects is thought to be primarily achieved through alterations of cellular membranes composition and other endogenous lipid stores which may modify the functional activity of various receptors on plasma membranes. In summary, the protective effects of ω-3 lipids have been explained based on changes in eicosanoid synthesis and the reduced risk of sudden death from cardiac arrhythmia, increased protection from ischemic myocardium, improved myocardial function and reduction of other cardiovascular and autoimmune disease risks. However, well-designed studies are still required to further define the key role of both combination of ω-6 and ω-3 fatty acids, from marine and vegetable sources, both as a supplement to infant nutrition specifically for optimizing the development of cognitive function, and also as preventive measure for reducing the incidence of diseases of aging in rapidly growing elderly populations.

Modulation of age-related alterations in membrane composition and receptor-associated immune functions by food restriction in fischer 344 rats

Food restriction is known to modulate aging and age-associated immune functions in rodents. In an attempt to understand the mechanism(s) through which food restriction delays age-associated loss of certain immune functions, lipid composition of spleen cells as well as binding of spleen cells to interleukin-2 (IL-2) and insulin were analyzed in four month-old and 19-month-old ad libitum fed (AL) and food-restricted (FR) Fischer-344 male rats. The results revealed that although AL-fed rats did not show a difference in age-related changes for IL-2 and insulin binding, the number of binding sites were significantly increased in the spleen cells of 19-month-old FR animals when compared with those of the 19-month-old AL group. When spleen cell phospholipid fractions were analyzed for fatty acid composition, the spleen cells from FR animals consistently revealed higher linoleic acid (18:2) levels and significantly lower arachidonic acid (20:4) and long chain fatty acid, docosatetraenoic acid (22:4) levels in the phosphatidylcholine and phosphatidylethanolamine fractions than the spleen cells of the AL rats. Further, spleen cell plasma membranes of FR rats also exhibited similar changes showing higher 18:2 and lower 20:4 and 22:4 levels than the AL animals. Finally, spleen cells obtained from 19-month-old FR animals produced higher levels of IL-2 and lesser prostaglandin E2 when compared to 19-month-old AL animals. The above observations suggest that one of the mechanisms through which food restriction may delay the loss of age-associated immune functions is through modulation of the fatty acyl composition of phospholipid fractions of spleen cell membranes. This modification may facilitate binding of IL-2 and insulin to their receptors and thus may improve T cell proliferation and prevent or delay age-related loss in immune functions.

Genotype effects on the antioxidant enzymes activity and mRNA expression in liver and kidney tissues of autoimmune-prone MRL/McJ-lpr/lpr mice

Congeneic pairs of MRL/lpr and MRL/++ (+/+) mice differ in incidence of autoantibodies, lymphoproliferative disease and survival, characteristics that are linked to immunological abnormalities. MRL/lpr mice have a significantly shorter life span compared to +/+ mice. Because a weak antioxidant defense and an increased generation of free radicals are generally implicated in the severity of many autoimmune disease, the present study was undertaken to compare the influence of genotype on lipid composition, lipid peroxidation and expression of mRNA, and activity of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in the livers and kidneys of these mice. The expression of SOD, GSH-Px and CAT mRNAs was significantly higher (P < 0.05) in the livers of +/+ mice, while in the kidneys only SOD expression was found significantly higher in +/+ mice when compared to MRL/lpr mice. Further, the activity of cytosolic SOD and GSH-Px was also found significantly higher (P < 0.001) in the livers of +/+ mice. Both livers and kidneys of MRL/lpr mice exhibited significantly higher levels of arachidonic acid (20:4(n-6)), significantly higher generation of thiobarbituric acid reactive substances (TBARS) and higher estimated peroxidation index than the +/+ mice. In addition, the MRL/lpr mice had higher levels of serum anti-cardiolipin antibodies. In summary, the results from the present study indicate that besides several immune-related abnormalities, the MRL/lpr mice may exhibit their inability to cope with oxidative stress due to a poor antioxidant defense system.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue specific regulation of transforming growth factor beta by omega-3 lipid-rich krill oil in autoimmune murine lupus

We have previously reported that hybrid New Zealand female mice [(NZBxNZW) F1 or BW) fed a diet enriched in ω-3 lipid-rich fish oil vs. ω-6 lipid-rich corn oil show delayed development of autoimmune lupus nephritis and longer life span. The present study was carried out to explore the possible beneficial effects of oil from Antarctic krill (Euphausia superba) as an alternative source of ω-3 lipids. Weanling BW mice were fed a nutritionally adequate semipurified diet supplemented with either 10% (wt/wt) krill oil (KO) or corn oil (CO). Cross-sectional studies were carried out on kidneys and spleens at 3.5 and 6.5 months of age. Our results indicate that KO prolonged life span (CO, 266.7 days ± 12.5; KO, 330.2 days ± 19.2; P<0.001) and delayed the onset of proteinuria. Splenocytes from KO mice displayed greater proliferative responses to mitogen (concavalin A), and significantly lower Pgp-1+ cells in both CD4+ and CD8+ T cell subsets. Lipid extracts of splenocytes from KO fed mice revealed higher levels of eicosapentaenoic (20:5ω-3; EPA) and docosahexaenoic (22:6ω-3; DHA) acids; EPA suppresses prostaglandin synthesis. Further, Northern blot analysis showed decreased expression of the oncogene c-ras (1.5-fold, P<0.05) in the spleens of KO fed mice. The expression of transforming growth factor β1 (TGFβ1) was higher in spleen cell extracts (3.5-fold; P<0.025), but lower in kidney extracts (5.97-fold; P<0.025) of KO fed mice. The data indicate that dietary supplementation with KO modulates expression of TGFβ in an organ specific manner. In the spleen, TGFβ could be immunosuppressive, whereas its expression in the kidney may be pathological and proinflammatory. In summary, dietary KO, like fish oil, can suppress the development of autoimmune murine lupus, and its effects on inflammatory mediators are organ specific.

Possible mechanisms through which dietary lipids, calorie restriction, and exercise modulate breast cancer

Though the exact cause of breast cancer still remains a mystery, years of investigations have suggested that several dietary and endocrine-related factors may induce and/or could modulate the growth of breast cancer. Epidemiologic and experimental evidence have indicated a close association between high-fat diets and increased incidence of breast cancer.1–3 Furthermore, several immunologic functions, including the levels of growth factors, cytokines, and sex steroid hormones, may be altered or regulated by dietary lipids.4,5 Excessive fat in the diet has been reported to enhance the growth of both spontaneously occurring and chemically induced colon and mammary tumors, as well as accelerated growth of transplantable carcinomas.6–8 Diets containing high levels of ω-6 fatty acids derived from vegetable fats appear to enhance tumorigenesis, while ω-3-containing lipids, either from vegetable or marine origin, or low levels of fat in the diet can diminish tumorigenesis. Recently, a number of mechanisms have been proposed to explain the modulation of mammary tumorigenesis in experimental animals by increasing the levels of dietary fats.9,10 Initiation and promotion have been linked to immune suppression,11 prostaglandin production,12,13 free radical formation,14 membrane fluidity changes,15 intracellular transport system modulation,16 increased caloric utilization,17 increased mammotrophic hormone secretion,18 and cytokine changes.19,20 Over-expression of oncogenes and certain growth factors are other mechanisms that have been linked to dietary changes that may influence mammary tumori-genesis.

Micronutrient and lipid interactions in cancer

Several hypotheses about the possible relationship between diet, micronutrients, and cancer prevention have emerged in recent years primarily from the results of epidemiologic studies.I Variations in diet and nutrition practices among different populations have been associated with increased or decreased cancer risk depending upon the availability of foods and the level of individual nutrient and calorie intake or their close interactions. 9 In the case of dietary fat intake, the American public consumes nearly 40% of their total calories from the dietary fats and oils. However, the recent declining death rate from heart disease in the U.S.A. is mainly due to reduced saturated fat and cholesterol intake and reduction in smoking habits. In contrast to the reduced saturated fat intake, there is, however, an 8-IO-fold increase in polyunsaturated fats (n-6) since 1900, which is currently suspected to influence the incidence of certain endocrine-dependent cancers, primarily based on studies carried out in animal models. In 1986, the American Cancer Society estimated 930,000 new cancer cases and 472,000 deaths from all forms of cancer in the U.S.A. 1O The roles of specific dietary components and their interacting mechanisms on immune deficiency leading to initiation or promotion of neoplastic and/or autoimmune disease are still not well understood. 11.12 However, we propose that there are two potential possibilities: 1) absence of adequate antioxidant supplementation to the dietary fats to prevent lipid peroxidation and/or decreased amount of micronutrients intake due to the increased consumption of refined foods, and 2) the type of fatty acids that incorporate into the cell membranes and their interaction with membrane proteins could modulate the signal transduction mechanism leading to abnormal gene activation. The intake of various essential micronutrients has been suggested to decrease cancer risk and its severity by modifying specific phases of carcinogenesis.P Several animal studies indicate that of the micronutrients implicated in cancer prevention, vitamins A, C, D, and E, and minerals such as calcium and selenium show the most promise in modifying the growth of tumor cells. Interaction of appropriate lipids and micronutrients may act in several ways. They can inhibit cancer initiation or promotion by acting at the molecular level. Certain micronutrients may act as antioxidants and inhibit activation of protooncogenes, detoxify carcinogenic agents, and prevent formation of carcinogenic substances. Micronutrients may also enhance the functional activities of the immune system and its interacting mechanisms composed of T cells, B cells, macrophages and natural killer (NK) cells of the host by enhancing various kinds of cytokine production. II In this paper we shall describe briefly the role of vitamins A, C, D, and E, selenium, zinc, and

Insulin binding to liver nuclei from lean and obese mice is altered by dietary fat

Insulin binding to the plasma membrane is known to be altered by modifying the membrane composition through dietary treatment. As insulin binding receptors are also present on nuclear membrane, this study was undertaken to investigate if specific binding of insulin to the liver nuclei is altered by diet. 8-wk-old female C57 B 6J lean and ob/ob mice were fed semipurified diets containing 20% (w/w) fat of either high or low polyunsaturated-to-saturated (P/S) fatty acid ratio for 4 wk. Liver nuclei were prepared, insulin binding was measured and nuclear phospholipids were isolated for lipid analysis. Insulin binding was highest in nuclei prepared from lean mice fed a high P/S diet. Specific binding of insulin to nuclei prepared from obese mice was also increased by the high P/S diet, but to a lesser extent compared to lean mice. Feeding a high P/S diet increased polyunsaturated fatty acid content of membrane phospholipids from both lean and ob/ob mice. Obese mice were characterized by higher levels of arachidonic acid and lower levels of linoleic acid in phosphatidylcholine. The present study establishes that insulin binding to liver nuclei is increased by feeding a high P/S diet, and that insulin binding to liver nuclei from obese mice is lower than from lean mice.

Influence of food restriction and aging on the binding of insulin to liver nuclei in Fischer-344 rats

Aging is generally associated with the development of insulin resistance. Food restriction has been accepted as a powerful modulator of the aging process, though the underlying mechanisms are not yet elucidated. The present study was carried out to determine the effect of food restriction on the binding of insulin to liver nuclei of Fischer-344 rats ranging in age from 3–24 months. After isolating nuclei from livers of 3m, 6m, 10m, 12m and 24m ad libitum-fed and food-restricted rats, 100 μg of nuclear protein was incubated with 0.2 ng of 125I-insulin and 0.2 to 2,000 ng/200 μl unlabeled insulin at 23°C for 2 hr. Scatchard analysis of the data revealed that insulin binding to nuclei was highest at 6m of age and an age-associated decline occurred in the binding of insulin to liver nuclei. The maximum number of binding sites for insulin in the liver nuclei of 3m, 6m, 10m and 24m old animals were 24±2, 37.5±2, 26±2.5, 14±3 for the ad libitum-fed group and 8.3±3, 38.5±2.5, 28.5±3, and 24.5±3 ng/mg protein for the food-restricted groups, respectively. The above data suggest that food restriction can delay the loss of insulin binding to liver nuclei. These observations suggest that food restriction may be an important modulator of insulin binding to liver nuclei which may have a functional role in delaying age-associated insulin resistance.

Differential effects of ω-6 and ω-3 fatty acids on interleukin-2 production and mRNA expression by EL-4.IL-2 cells*

Our studies with lupus-prone (NZBxNZW)F 1 (B/W) female mice have indicated that dietary ω --3 lipids (menhaden oil) significantly extend the life span and delay the onset of autoimmune disease, while ω --6 lipids (corn oil) shorten the life span by accelerating the onset and progression of autoimmune disease, probably by altering the cell and subcellular membrane fatty acid composition. To understand the mechanisms through which ω --6 (linoleic acid, 18:2, and arachidonic acid, 20:4) and ω --3 (eicosapentanoic acid, 20:5, and docosahexanoic acid, 22:6) lipids exert their differential effects, we have studied the effects of these fatty acids in vitro on cell proliferation, peroxidation, interleukin-2 (IL-2) production, IL-2 mRNA levels, and surface IL-2 receptor (IL-2R) expression in an IL-2 producing mouse lymphoma cell line (EL-4.IL-2: EL-4). When EL-4 cells were cultured in the presence of individual ω --6 and ω --3 fatty acids (at a final concentration of 10 μg/mL), the respective fatty acid was found to incorporate into the cells at a significant level, and no adverse effects were noted either on the viability of the cells or on the de novo DNA synthesis. In addition, lipid peroxidation, as measured by the generation of thiobarbituric acid-reactive substances, was significantly higher ( P ω --6 as compared with control cells (to which no fatty acid was added). Also, 20:4 ω --6 significantly inhibited ( P ω --6 was at the gene level, as seen by an inhibition in phorbol-12-myristate-13-acetate induced IL-2 mRNA levels by 20:4 ω --6. Compared with saturated fatty acids, both ω --6 and ω --3 lipids induced higher IL-2R surface expression, as seen by flow cytometry. These studies suggest that dietary ω --3 lipids lower membrane lipid peroxidation. and thereby may preserve normal immunological functions that may delay the course of autoimmune disease in B/W mice.