Martha A. Belury

CONJUGATED LINOLEIC ACID MODULATES HEPATIC LIPID COMPOSITION IN MICE

Conjugated linoleic acid (CLA) is a chemoprotective fatty acid that inhibits mammary, colon, forestomach, and skin carcinogenesis in experimental animals. We hypothesize that the ubiquitous chemoprotective actions of dietary CLA in extrahepatic tissues are dependent upon its role in modulating fatty acid composition and metabolism in liver, the major organ for lipid metabolism. This study begins to evaluate the role of CLA in lipid metabolism by determining the modulation of fatty acid composition by CLA. Female SENCAR mice were fed semipurified diets containing 0.0% (Diet A), 0.5% (Diet B), 1.0% (Diet C), or 1.5% (Diet D) CLA (by weight) for six weeks. Mice fed Diets B, C, and D exhibited lower body weights and elevated amounts of extractable total lipid in livers compared with mice fed diets without CLA (Diet A). Analyses of the fatty acid composition of liver by gas chromatography revealed that dietary CLA was incorporated into neutral and phospholipids at the expense of linoleate in Diets B, C, and D; oleate increased and arachidonate decreased in neutral lipids of CLA diet groups. In addition, increasing dietary CLA was associated with reduced linoleate in hepatic phospholipids. In an in vitro assay, CLA was desaturated to an unidentified 18:3 product to a similar extent as linoleate conversion to γ-linolenate (9.88, and 13.63%, respectively). These data suggest that CLA may affect metabolic interconversion of fatty acids in liver that may ultimately result in modified fatty acid composition and arachidonate-derived eicosanoid production in extrahepatic tissues. In addition to determining how dietary CLA modulates eicosanoid synthesis, further work is needed to identify enzymatic products that may result from desaturation of CLA.

Dietary conjugated linoleic acid modulation of phorbol ester skin tumor promotion

The fatty acid derivative conjugated dienoic linoleate (CLA) has been shown to inhibit initiation and postinitiation stages of carcinogenesis in several experimental animal models. The goal of the present study was to determine the role of increasing levels of dietary CLA in mouse skin tumor promotion elicited by 12-O-tetradecanoylphorbol-13-acetate (TPA). Mice were fed control (no CLA) diet during initiation, then switched to diets containing 0.0%, 0.5%, 1.0%, or 1.5% (wt/wt) CLA during skin tumor promotion by TPA. Body weights of mice fed 0.5%, 1.0%, or 1.5% CLA were similar to each other but were significantly lower (p < 0.05) than weights of mice fed no CLA (0.0%) throughout promotion. A reduction in papilloma incidence was observed in mice fed 1.5% CLA from Weeks 8 to 24 compared with mice fed diets containing 0.0-1.0% CLA (p < 0.05). Twenty-four weeks after tumor promotion was begun, diets containing 1.0% and 1.5% CLA inhibited tumor yield (4.94 and 4.35 tumors/mouse, respectively) compared with diets without CLA (0.0% CLA, 6.65 tumors/mouse, p < 0.05) or 0.5% CLA (5.92 tumors/mouse, p < 0.05). These data indicate that CLA inhibits tumor promotion in a manner that is independent of its anti-initiator activity. Further studies are warranted in identifying cellular mechanisms that are likely to be involved with the antipromoter effects of CLA.

Conjugated linoleic acid activates peroxisome proliferator-activated receptor α and β subtypes but does not induce hepatic peroxisome proliferation in Sprague–Dawley rats

Since conjugated linoleic acid (CLA) has structural and physiological characteristics similar to peroxisome proliferators, we hypothesized that CLA would activate peroxisome proliferator-activated receptor (PPAR). We compared the effects of dietary CLA (0.0, 0.5, 1.0 and 1.5% by weight) with a peroxisome proliferator (0.01% Wy-14,643) in female and male Sprague-Dawley (SD) rats. Dietary CLA had little effect on body weight, liver weight, and hepatic peroxisome proliferation, compared to male rats fed Wy-14,643 diet. Lipid content in livers from rats fed 1.5% CLA and Wy-14,643 diets was increased (P < 0.01) when compared to rats fed control diets regardless of gender. Hepatic acyl-CoA oxidase (ACO) mRNA levels were increased 3-fold in male rats fed 1.5% CLA diet compared to rats fed control diets while Wy-14,643 supported approximately 30-fold ACO mRNA accumulation. A similar response was observed for liver fatty acid-binding protein (L-FABP) mRNA. The effect of dietary treatments on hepatic PPAR-responsive genes in female rats was weaker than in male rats. The (9Z,11E)-CLA isomer activated PPAR alpha in transfected cells to a similar extent as Wy-14,643, whereas the furan-CLA metabolite was comparable to bezafibrate on activating PPAR beta. These data suggest that while CLA was able to activate PPARs it is not a peroxisome proliferator in SD rats.

Depressive symptoms, omega-6:omega-3 fatty acids, and inflammation in older adults.

Objective: To address how interactions between polyunsaturated fatty acid (PUFA) levels and depressive symptoms were related to proinflammatory cytokine synthesis. Depression and stress promote proinflammatory cytokine production. Dietary intakes of omega-3 (n-3) and omega-6 (n-6) PUFAs also influence inflammation; high n-6:n-3 ratios enhance proinflammatory cytokine production, although n-3 has anti-inflammatory properties. Methods: Blood samples from 43 older adults (mean age = 66.67 years, SD = 10.09) provided data on PUFAs and tumor necrosis factor (TNF)-&agr;, interleukin (IL)-6, and IL-6 soluble receptor (sIL-6r). Depressive symptoms were assessed by the Center for Epidemiological Studies Depression Scale. Results: Depressive symptoms and n-6:n-3 ratios worked together to enhance proinflammatory cytokines beyond the contribution provided by either variable alone, with substantial variance explained by their interaction: 13% for IL-6 and 31% for TNF-&agr;, whereas full models accounted for 18% and 40%, respectively. Although predicted cytokine levels were consistent across n-6:n-3 ratios with low depressive symptoms, higher n-6:n-3 ratios were associated with progressively elevated TNF-&agr; and IL-6 levels as depressive symptoms increased. Higher levels of sIL-6r were associated with higher n-6:n-3 ratios. Six individuals who met the criteria for major depressive disorder had higher n-6:n-3 ratios and TNF-&agr;, IL-6, and sIL-6r levels than those who did not meet the criteria; excluding these six individuals reduced the variance explained by the depressive symptoms and n-6:n-3 ratio interaction. Conclusions: Diets with high n-6:n-3 PUFA ratios may enhance the risk for both depression and inflammatory diseases. AA = arachidonic acid; BMI = body mass index; CES-D = Center for Epidemiological Studies Depression Scale; DHA = docosahexanoic acid; EPA = eicosapentaenoic acid; IL-6 = interleukin-6; sIL-6r = IL-6 soluble receptor; n-3 = omega-3; n-6 = omega-6; NF-&kgr;B = nuclear factor kappa B; PSQI = Pittsburgh Sleep Quality Index; PUFA = polyunsaturated fatty acid; TNF-&agr; = tumor necrosis factor-&agr;.

Dietary conjugated linoleic acid induces peroxisome-specific enzyme accumulation and ornithine decarboxylase activity in mouse liver☆

Abstract Previous studies have shown that the dietary fatty acids, conjugated linoleic acids (CLA), inhibit carcinogenesis in the colon, mammary gland, forestomach, and skin. Several properties of this chemoprotective polyunsaturated fatty acid suggest it will act as an hepatic peroxisome proliferator. This study evaluated the effect of dietary CLA on the accumulation of enzymes associated with peroxisome proliferation in rodent liver. Female SENCAR mice were fed one of four semipurified diets containing 5% corn oil without CLA (“control diet”) or supplemented with incremental levels of CLA (0.5%, 1.0% or 1.5% by weight of diets) for 6 weeks. Hepatic mRNA levels of several enzymes known to be induced during peroxisome proliferation [i.e., acyl-CoA oxidase (ACO), cytochrome P4504A1 (CYP4A1), and liver fatty acid binding protein (FABP)] were measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Diets containing 0.5%, 1.0% or 1.5% CLA were associated with approximately 6-, 9-, and 9-fold increases in ACO mRNA, respectively, compared with mRNA levels in mice fed the control diet. The steady state levels of FABP and CYP4A1 mRNA accumulation were maximal in animals fed 1.0% CLA diets and less magnified in mice fed 1.5% CLA diets. Western blot analysis revealed that the relative abundance of ACO protein in livers of mice fed CLA-containing diet groups (0.5%, 1.0%, and 1.5% CLA) were 2.5, 3.0, and 3.0 times the level ACO protein of mice fed the control diet (0.0% CLA). Because most peroxisome proliferators are considered nongenotoxic hepatocarcinogens in rodents, the effect of dietary CLA on ornithine decarboxylase (ODC) activity, a measure of cell proliferation and tumor promotion, was quantified. Activity on hepatic ODC was increased by approximately 10-fold for mice fed 1.0% and 1.5% diets, respectively, compared with those fed the control or 0.5% CLA diets. These data suggest that CLA displays the typical peroxisome proliferation response, i.e., induction of ACO, CYP4A1 and FABP accumulation and cell proliferation in rodent liver.

Omega-3 supplementation lowers inflammation and anxiety in medical students: A randomized controlled trial

Observational studies have linked lower omega-3 (n-3) polyunsaturated fatty acids (PUFAs) and higher omega-6 (n-6) PUFAs with inflammation and depression, but randomized controlled trial (RCT) data have been mixed. To determine whether n-3 decreases proinflammatory cytokine production and depressive and anxiety symptoms in healthy young adults, this parallel group, placebo-controlled, double-blind 12-week RCT compared n-3 supplementation with placebo. The participants, 68 medical students, provided serial blood samples during lower-stress periods as well as on days before an exam. The students received either n-3 (2.5 g/d, 2085 mg eicosapentaenoic acid and 348 mg docosahexanoic acid) or placebo capsules that mirrored the proportions of fatty acids in the typical American diet. Compared to controls, those students who received n-3 showed a 14% decrease in lipopolysaccharide (LPS) stimulated interleukin 6 (IL-6) production and a 20% reduction in anxiety symptoms, without significant change in depressive symptoms. Individuals differ in absorption and metabolism of n-3 PUFA supplements, as well as in adherence; accordingly, planned secondary analyses that used the plasma n-6:n-3 ratio in place of treatment group showed that decreasing n-6:n-3 ratios led to lower anxiety and reductions in stimulated IL-6 and tumor necrosis factor alpha (TNF-α) production, as well as marginal differences in serum TNF-α. These data suggest that n-3 supplementation can reduce inflammation and anxiety even among healthy young adults. The reduction in anxiety symptoms associated with n-3 supplementation provides the first evidence that n-3 may have potential anxiolytic benefits for individuals without an anxiety disorder diagnosis. ClinicalTrials.gov identifier: NCT00519779.

Conjugated linoleic acid reduces arachidonic acid content and PGE2 synthesis in murine keratinocytes

Dietary conjugated linoleic acid (CLA) is associated with decreased 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced tumor promotion in mouse skin. In addition, CLA decreases TPA-induced prostaglandin E synthesis and ornithine decarboxylase activity in cultured keratinocytes compared with linoleic acid (LA) and arachidonic acid (AA). When LA or CLA was added to keratinocyte cell cultures, the amounts of each of these cellular fatty acids increased significantly in a dose-dependent manner. Furthermore, LA treatment was associated with increased cellular AA while the AA content of keratinocytes was reduced when cultures were treated with CLA. Moreover, CLA (16 microg/ml) was more potent than LA at decreasing the level of 14C-AA incorporated into cellular phosphatidylcholine. In order to determine the effect of CLA on arachidonate-derived PGE2, the release of 14C-AA and 14C-PGE2 synthesis was measured in cultures pre-treated with LA/14C-AA or CLA/14C-AA for 12 h. The amount of 14C-AA release induced by TPA in CLA/14C-AA pre-treated cultures was significantly lower than cultures pre-treated with LA/14C-AA. Furthermore, TPA-induced 14C-PGE2 was significantly lower in cultures pre-treated with CLA/14C-AA compared with cultures pre-treated with LA/14C-AA. The effects of LA and CLA on AA composition of phospholipids and subsequent arachidonate-derived PGE2 synthesis will provide insight into the anti-promoter mechanisms of CLA.

Omega-3 fatty acids, oxidative stress, and leukocyte telomere length: A randomized controlled trial

Shorter telomeres have been associated with poor health behaviors, age-related diseases, and early mortality. Telomere length is regulated by the enzyme telomerase, and is linked to exposure to proinflammatory cytokines and oxidative stress. In our recent randomized controlled trial, omega-3 (n-3) polyunsaturated fatty acid (PUFA) supplementation lowered the concentration of serum proinflammatory cytokines. This study assessed whether n-3 PUFA supplementation also affected leukocyte telomere length, telomerase, and oxidative stress. In addition to testing for group differences, changes in the continuous n-6:n-3 PUFA ratio were assessed to account for individual differences in adherence, absorption, and metabolism. The double-blind four-month trial included 106 healthy sedentary overweight middle-aged and older adults who received (1) 2.5g/day n-3 PUFAs, (2) l.25g/day n-3 PUFAs, or (3) placebo capsules that mirrored the proportions of fatty acids in the typical American diet. Supplementation significantly lowered oxidative stress as measured by F2-isoprostanes (p=0.02). The estimated geometric mean log-F2-isoprostanes values were 15% lower in the two supplemented groups compared to placebo. Although group differences for telomerase and telomere length were nonsignificant, changes in the n-6:n-3 PUFA plasma ratios helped clarify the interventions impact: telomere length increased with decreasing n-6:n-3 ratios, p=0.02. The data suggest that lower n-6:n-3 PUFA ratios can impact cell aging. The triad of inflammation, oxidative stress, and immune cell aging represents important pre-disease mechanisms that may be ameliorated through nutritional interventions. This translational research broadens our understanding of the potential impact of the n-6:n-3 PUFA balance. ClinicalTrials.gov identifier: NCT00385723.

Effect of dietary conjugated linoleic acid on phorbol ester-induced PGE2 production and hyperplasia in mouse epidermis.

Conjugated linoleic acid (CLA) is a chemoprotective fatty acid that inhibits phorbol ester-induced skin tumor promotion in mice. The goal of the present study was to determine potential chemoprotective mechanisms through which CLA may be acting. Mice were fed diets containing 0.0%, 0.5%, 1.0%, or 1.5% CLA (by wt) for six weeks. The epidermis was evaluated for fatty acid composition, vascular permeability, prostaglandin E2 (PGE2) production, hyperplasia, ornithine decarboxylase activity, and c-myc mRNA accumulation. Fatty acid analysis of mouse epidermis demonstrated a dose-dependent increase of CLA incorporation into phospholipids and neutral lipids. In mice topically treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), dietary CLA (1.5%) significantly (p < 0.05) reduced PGE2 synthesis (2-fold). Additionally, CLA lowered accumulation of c-myc mRNA, a gene commonly associated with regulating cell cycle components involved in cellular proliferation, although this trend was not significant. Vascular permeability was unaffected by dietary CLA. These data suggest that dietary CLA modulates TPA-induced tumor promotion through a mechanism involving PGE2 production; however, dietary CLA had a moderate effect on c-myc mRNA levels and little effect on TPA-induced hyperplasia and ornithine decarboxylase activity.

Anti-Aromatase Activity of Phytochemicals in White Button Mushrooms (Agaricus bisporus)

White button mushrooms (Agaricus bisporous) are a potential breast cancer chemopreventive agent, as they suppress aromatase activity and estrogen biosynthesis. Therefore, we evaluated the activity of mushroom extracts in the estrogen receptor-positive/aromatase-positive MCF-7aro cell line in vitro and in vivo. Mushroom extract decreased testosterone-induced cell proliferation in MCF-7aro cells but had no effect on MCF-10A, a nontumorigenic cell line. Most potent mushroom chemicals are soluble in ethyl acetate. The major active compounds found in the ethyl acetate fraction are unsaturated fatty acids such as linoleic acid, linolenic acid, and conjugated linoleic acid. The interaction of linoleic acid and conjugated linoleic acid with aromatase mutants expressed in Chinese hamster ovary cells showed that these fatty acids inhibit aromatase with similar potency and that mutations at the active site regions affect its interaction with these two fatty acids. Whereas these results suggest that these two compounds bind to the active site of aromatase, the inhibition kinetic analysis indicates that they are noncompetitive inhibitors with respect to androstenedione. Because only conjugated linoleic acid was found to inhibit the testosterone-dependent proliferation of MCF-7aro cells, the physiologically relevant aromatase inhibitors in mushrooms are most likely conjugated linoleic acid and its derivatives. The in vivo action of mushroom chemicals was shown using nude mice injected with MCF-7aro cells. The studies showed that mushroom extract decreased both tumor cell proliferation and tumor weight with no effect on rate of apoptosis. Therefore, our studies illustrate the anticancer activity in vitro and in vivo of mushroom extract and its major fatty acid constituents.