CLA reduces antigen-induced histamine and PGE(2) release from sensitized guinea pig tracheae

Conjugated linoleic acid (CLA) has been shown to enhance immune reactions such as lymphocyte blastogenesis and delayed-type hypersensitivity. We investigated the role of CLA in type I (immediate) hypersensitivity, using a guinea pig tracheal superfusion model for measuring antigen-induced airway smooth muscle contraction and inflammatory mediator release. Female Hartley guinea pigs were fed a diet supplemented with 0.25 g corn oil or linoleic acid/100 g of diet (control) or 0.25 g CLA/100 g of diet for at least 1 wk before and during active sensitization to ovalbumin antigen. Tracheae from sensitized guinea pigs were suspended in air-filled water-jacketed (37 degrees C) tissue chambers in a superfusion apparatus. Tracheae were superfused with buffer containing antigen, and tissue contraction was recorded. Superfusate was collected at 90-s intervals for evaluation of histamine and PGE(2) release. CLA did not affect antigen-induced tracheal contractions when expressed as gram contraction per gram tissue. CLA significantly reduced antigen-induced histamine and PGE(2) release. CLA appears to decrease release of some inflammatory mediators during type I hypersensitivity reactions.     

10t,12c-conjugated linoleic acid inhibits lipopolysaccharide-induced cyclooxygenase expression in vitro and in vivo

Previous data demonstrated that conjugated linoleic acid (CLA) reduced eicosanoid release from select organs. We hypothesized that one active CLA isomer was responsible for the reduced prostaglandin release and that the mechanism was through the inhibition of inducible cyclooxygenase-2 (COX-2). Here, we examined the effects of 10t,12c-CLA and 9c,11t-CLA on COX-2 protein/mRNA expression, prostaglandin E(2) (PGE(2)) production, and the mechanism by which CLA affects COX-2 expression and prostaglandin release. The COX-2 protein expression level was inhibited 80% by 10t, 12c-CLA and 26% by 9c,11t-CLA at 100 microM in vitro. PGE(2) production was decreased from 5.39 to 1.12 ng/2 x 10(6) cells by 10t,12c-CLA and from 5.7 to 4.5 ng/2 x 10(6) cells by 9c,11t-CLA at 100 microM. Mice fed 10t,12c-CLA but not 9c,11t-CLA were found to have a 34% decrease in COX-2 protein and a 43% reduction of PGE(2) release in the lung. 10t,12c-CLA reduced COX-2 mRNA expression level by 30% at 100 microM in vitro and by 30% in mouse lung in vivo. Reduced COX-2 mRNA was attributable to an inhibition of the nuclear factor kappaB (NF-kappaB) pathway by 10t,12c-CLA. These data suggested that the inhibition of NF-kappaB was one of the mechanisms for the reduced COX-2 expression and PGE(2) release by 10t,12c-CLA.     

Release of prostaglandins from healthy and sensitized guinea-pig lung and trachea by histamine.

The effects of histamine and its antagonists on the release of prostaglandin E and F2alpha (PGE and PGF2alpha) and the 15-keto-13,14-dihydro PGF2alpha/E (metabolites) were examined in minced and whole perfused guinea pig lung. Lung fragments released considerable amounts of prostaglandins into the incubation media with time alone: parenchyma more PGF2alpha than PGE, trachea more PGE than PGF2alpha. The levels of PGF2alpha found in the filtrates of both tissues on per gram basis were about the same, whereas the concentrations of PGE were several fold higher in the media of incubated trachea. In contrast to lung, trachea released only trace amounts of metabolites. These differences in synthesis and turnover are probably of importance for maintenance of the adequate ventilation-perfusion ratios. The process of sensitization caused a significant increase in the outflows of PGF2alpha and metabolites from the lung fragments. The PGE to PGF2alpha ratio was decreased in both parenchymal and tracheal tissues. Increased spontaneous release of prostaglandins was also found in whole perfused sensitized lung. This was consistent with the hypothesis that sensitization with antigen alters the biochemical properties of the organism. Incubation of lung fragments with histamine had only a small additional effect on the liberation of prostaglandins, since the baseline release was high due to the trauma of mincing. However, histamine perfusion of whole lung caused severalfold increase in the outflows of prostaglandins. Pretreatment with pyrilamine (histamine receptor 1 antagonist) decreased the subsequent release of PGF2alpha by histamine. On the other hand, pretreatment with metiamide (histamine receptor 2 antagonist) diminished the subsequent release of PGE. It is suggested that stimulation of histamine receptor 1 is predominantly (but not solely) related to the synthesis of PGF2alpha, and stimulation of the receptor 2 is related to the synthesis of PGE.     

Differential regulation of the release of tumor necrosis factor-alpha and of eicosanoids by mast cells in rat airways after antigen challenge

BACKGROUND: Rat trachea display a differential topographical distribution of connective tissue mast cells (CTMC) and mucosal mast cells (MMC) that may imply regional differences in the release of allergic mediators such as tumor necrosis factor-alpha (TNF-alpha) and eicosanoids. AIM: To evaluate the role of CTMC and MMC for release of TNF-alpha and eicosanoids after allergenic challenge in distinct segments of rat trachea. MATERIALS AND METHODS: Proximal trachea (PT) and distal trachea (DT) from ovalbumin (OVA)-sensitized rats, treated or not with compound 48/80 (48/80) or dexamethasone, were incubated in culture medium. After OVA challenge, aliquots were collected to study release of TNF-alpha and eicosanoids. RESULTS: Release of TNF-alpha by PT upon OVA challenge peaked at 90 min and decayed at 6 and 24 h. Release from DT peaked at 30-90 min and decayed 6 and 24 h later. When CTMC were depleted with 48/80, OVA challenge exacerbated the TNF-alpha release by PT at all time intervals, while DT exacerbated TNF-alpha levels 6 and 24 h later only. Dexamethasone reduced TNF-alpha production after 90 min of OVA challenge in PT and at 3 and 6h in DT. OVA challenge increased prostaglandin D2) in DT and leukotriene B4 in both segments but did not modify prostaglandin E2 and leukotriene C4 release. CONCLUSION: OVA challenge induces TNF-alpha release from MMC, which is negatively regulated by CTMC. The profile of TNF-alpha and eicosanoids depends on the time after OVA challenge and of the tracheal segment considered.     

Impaired lipid accumulation by trans10, cis12 CLA during adipocyte differentiation is dependent on timing and length of treatment

Conjugated linoleic acids (CLAs) are a group of polyunsaturated fatty acids found in ruminant products, where the predominant isomers are cis9, trans11 (c9,t11) and trans10, cis12 (t10,c12) CLA. We have previously shown that t10,c12 CLA prevents lipid accumulation in mature adipocytes in part by acting as a peroxisome proliferator-activated receptor gamma (PPAR gamma) modulator. The objective of this study was to further establish the molecular mechanisms underlying the attenuating effect on lipid accumulation by t10,c12 CLA, with focus on time point and duration of treatment during adipogenesis. We have shown that t10,c12 CLA treatment has its most attenuating effect early (day (D) 0-6) during differentiation. Treatment during this period is sufficient to prevent lipid accumulation in mature adipocytes. The adipogenic marker genes PPAR gamma and CCAAT/enhancer binding protein alpha (C/EBP alpha) are both down-regulated after treatment within the period from D0-6, while additional treatment also down-regulates the expression of sterol regulatory element binding protein-1c (SREBP-1c), liver X receptor alpha (LXR alpha), fatty acid binding protein (aP2), fatty acid translocase (CD36) and insulin-sensitive glucose transporter 4 (GLUT4). These effects of t10,c12 CLA reflect the subsequent attenuation of lipid accumulation observed in mature adipocytes. Interestingly, the early B-cell factor (O/E-1), which is known to promote adipogenesis and to be involved in control of genes important for terminal adipocyte differentiation, is unaffected by treatment of t10,c12 CLA. Taken together, our data indicate that inhibition of lipid accumulation induced by t10,c12 CLA treatment during adipocyte differentiation is associated with a tight regulatory cross-talk between early (PPAR gamma and C/EBP alpha) and late (LXR alpha, aP2 and CD36) adipogenic marker genes.     

Effects of lipid-esterified conjugated linoleic acid isomers on platelet function: evidence for stimulation of platelet phospholipase activity

The effects of four conjugated linoleic acid (CLA) isomers on in vitro collagen-induced human platelet aggregation and thromboxane (TXB(2), the inactive metabolite of the proaggregatory TXA(2)) production were examined. As the free fatty acid (FFA), 9t, 11t-CLA was the most effective inhibitor of these two processes (I(50)s of 2.2 and 4 microM, respectively) and the 9c, 11c-CLA was the least effective (I(50)s of 8.3 and 37 microM) of the isomers tested. When platelets were preesterified with either 25 microM 9t, 11t-CLA or 9c, 11c-CLA, CLA incorporation in total platelet lipids increased from 0.24% to 0.31% and 0.38%, and most of this increase was found to be in the phosphatidyl choline and phosphatidyl ethanolamine subclasses. The decrease in arachidonic acid (AA) content in total fatty acids or phospholipids was an order of magnitude greater. Furthermore, no significant differences between platelets prelabeled with either 9t, 11t- or 9c, 11c-CLA in the inhibition of collagen-induced aggregation and TXB(2) formation were observed. However, platelets prelabeled with 9c, 11c-CLA stimulated basal TXB(2) production (4-fold) which was not observed with platelets pretreated with either 9t, 11t-CLA, linoleic acid or stearic acid. This enhancement was associated with a 2.4-5-fold increase in the release of endogenous AA. Our results suggest that the presence of a conjugated cis, cis double bond appears to change the lipid environment sufficiently to stimulate the basal platelet phospholipase activity, which in turn increases the formation of TXB(2).     

Biological activities of conjugated fatty acids: conjugated eicosadienoic (conj. 20:2delta(c11,t13/t12,c14)), eicosatrienoic (conj. 20:3delta(c8,t12,c14)), and heneicosadienoic (conj. 21:2delta(c12,t14/c13,t15)) acids and other metabolites of conjugated linoleic acid

The elongated form of conjugated linoleic acid (CLA), conjugated eicosadienoic acid (CEA, conj. 20:2delta(c11,t13/t12,c14)), was generated from CLA by liver microsomal fractions. Subsequent testing showed that dietary CEA significantly reduced body fat, and increased lean mass similar to CLA when compared to controls. CEA also decreased lipoprotein lipase activity and triacylglyceride, and increased glycerol release in 3T3-L1 adipocytes, correlated with the trans-12,cis-14 isomer, but CEA required a longer incubation period than cells treated with CLA. Based on the fact that CEA fed animals had CLA in tissue, we suggest that the effect of CEA is due to the CLA converted from CEA in the system. The delta-6 desaturated and elongated form of trans-10,cis-12 CLA (conjugated eicosatrienoic acid, CETA, conj. 20:3delta(c8,t12,c14)) inhibited LPL activity and increased glycerol release but was less active than trans-10,cis-12 CLA or CEA. The 21-carbon conjugated fatty acid, conjugated heneicosadienoic acid (CHDA, conj. 21:2delta(c12,t14/c13,t15)), was not active on LPL inhibition, triacylglyceride, or glycerol release in 3T3-L1 adipocytes. We also provide evidence that CLA was metabolized to conjugated dodecadienoic acid (conj. 12:2delta(c3,t5/t4,c6)). In addition, there were indications of the presence of conjugated tetradecadienoic acid (conj. 14:2delta(c5,t7/t6,c8)), suggesting that CLA can be metabolized through fatty acid beta-oxidation. This is the first work to report the presence of conjugated 12 and 14 carbon fatty acids, originated from CLA, and the biological activities of CEA, CETA and CHDA.     

t10c12-CLA maintains higher bone mineral density during aging by modulating osteoclastogenesis and bone marrow adiposity

Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Earlier, we showed that CLA (equal mixture of c9t11-CLA and t10c12-CLA) could protect age-associated bone loss by modulating inflammatory markers and osteoclastogenesis. Since, c9t11-CLA and t10c12-CLA isomers differentially regulate functional parameters and gene expression in different cell types, we examined the efficacy of individual CLA isomers against age-associated bone loss using 12 months old C57BL/6 female mice fed for 6 months with 10% corn oil (CO), 9.5% CO + 0.5% c9t11-CLA, 9.5% CO + 0.5% t10c12-CLA or 9.5% CO + 0.25% c9t11-CLA + 0.25% t10c12-CLA. Mice fed a t10c12-CLA diet maintained a significantly higher bone mineral density (BMD) in femoral, tibial and lumbar regions than those fed CO and c9t11-CLA diets as measured by dual-energy-X-ray absorptiometry (DXA). The increased BMD was accompanied by a decreased production of osteoclastogenic factors, that is, RANKL, TRAP5b, TNF-alpha and IL-6 in serum. Moreover, a significant reduction of high fat diet-induced bone marrow adiposity was observed in t10c12-CLA fed mice as compared to that of CO and c9t11-CLA fed mice, as measured by Oil-Red-O staining of bone marrow sections. In addition, a significant reduction of osteoclast differentiation and bone resorbing pit formation was observed in t10c12-CLA treated RAW 264.7 cell culture stimulated with RANKL as compared to that of c9t11-CLA and linoleic acid treated cultures. In conclusion, these findings suggest that t10c12-CLA is the most potent CLA isomer and it exerts its anti-osteoporotic effect by modulating osteoclastogenesis and bone marrow adiposity.     

Abnormal immune responses in fa/fa Zucker rats and effects of feeding conjugated linoleic acid

Objective: The objective of this study was to characterize immune function in the fa/fa Zucker rat, and to determine the effects of feeding conjugated linoleic acid (CLA) isomers on immune function. Methods and Procedures: Lean and fa/fa Zucker rats were fed for 8 weeks nutritionally complete diets with different CLA isomers (%wt/wt): control (0%), c9t11 (0.4%), t10c12 (0.4%), or MIX (0.4% c9t11 + 0.4% t10c12). Isolated splenocytes were used to determine phospholipid (PL) fatty acid composition and cell phenotypes, or stimulated with mitogen to determine their ability to produce cytokines, immunoglobulins (Ig), and nitric oxide (NO). Results: Splenocyte PL of fa/fa rats had a higher proportion of total monounsaturated fatty acids and n ?3 polyunsaturated fatty acids (PUFA), and lower n ?6 PUFA and n ?6‐to‐n ?3 PUFA ratio (P < 0.05). Feeding CLA increased the content of CLA isomers into PL, but there were lower proportions of each CLA isomer in fa/fa rats. Splenocytes of fa/fa rats produced more amounts of IgA, IgG, and IgM, NO, and interleukin‐1β (IL‐1β), IL‐6, and tumor necrosis factor‐α (TNF‐α) (P < 0.05). Obese rats fed the t10c12 diet produced less TNF‐α and IL‐1β (lippopolysaccharide (LPS), P < 0.05). Splenocytes of fa/fa rats produced less concanavalin A (ConA)‐stimulated IL‐2 (P < 0.0001) than lean rats, except fa/fa rats fed the c9t11 diet (P < 0.05). Discussion: The c9t11 and t10c12 CLA isomers were incorporated into the membrane PL of the fa/fa Zucker rat, but to a lesser extent than lean rats. Splenocytes of obese rats responded in a proinflammatory manner and had reduced T‐cell function and feeding the t10c12 and c9t11 CLA isomers may improve some of these abnormalities by distinct methods.     

Inter-organ proteomic analysis reveals insights into the molecular mechanisms underlying the anti-diabetic effects of cis-9, trans-11-conjugated linoleic acid in ob/ob mice

cis‐9, trans‐11‐Conjugated linoleic acid (c9 t11 CLA) exerts anti‐diabetic effects by improving systemic insulin sensitivity and inflammation. Levels of CLA in beef can be increased by feeding cattle on pasture. This study aimed to explore the efficacy of a CLA‐rich diet (0.6% w/w c9 t11 CLA), presented as beef enriched with CLA or beef supplemented with synthetic CLA (c9 t11 CLA), for 28 days on molecular biomarkers of the metabolic syndrome, and adipose, hepatic, and skeletal muscle proteome in male ob/ob mice. Despite equal weight gain, CLA‐fed mice had lower plasma glucose, insulin, non‐esterified fatty acid, triacylglycerol and interleukin‐6, and higher adiponectin concentrations than controls. c9 t11 CLA induced differential regulation of redox status across all tissues, and decreased hepatic and muscle endoplasmic reticulum stress. CLA also modulated mechanistic links between the actin cytoskeleton, insulin signalling, glucose transport and inflammation in the adipose tissue. In the liver and muscle, c9 t11 CLA improved metabolic flexibility through co‐ordination between carbohydrate and energy metabolism. c9 t11 CLA may ameliorate systemic insulin sensitivity in obesity‐induced diabetes by altering cellular stress and redox status, and modulating nutrient handling in key insulin‐sensitive tissues through complex biochemical interplay among representative proteomic signatures.     

Dietary Melibiose Regulates Th Cell Response and Enhances the Induction of Oral Tolerance

We examined how dietary melibiose affected the T-helper (Th) cell responses induced by an orally fed antigen in ovalbumin (OVA)-specific T cell receptor transgenic mice (OVA 23-3). Dietary melibiose markedly decreased the Th2 type responses as shown by a significant decrease in the interleukin (IL)-4 production and T cell proliferative response induced by sensitization from the 7-d oral administration of OVA. It was additionally observed that the Th1 type responses tended to decrease. We therefore examined the effect of melibiose feeding on the induction of immunological tolerance induced by the oral administration of an antigen (oral tolerance). The Th cell responses induced in BALB/c mice by subcutaneous immunization with OVA were suppressed by the prior oral administration of OVA. Such responses in the OVA-fed and immunized mice were further diminished by dietary melibiose. These results suggest that dietary melibiose strongly affected the Th cell responses to an ingested antigen, and further demonstrate the potential of melibiose to enhance the induction of oral tolerance.     

Dietary melibiose regulates th cell response and enhances the induction of oral tolerance

We examined how dietary melibiose affected the T-helper (Th) cell responses induced by an orally fed antigen in ovalbumin (OVA)-specific T cell receptor transgenic mice (OVA 23-3). Dietary melibiose markedly decreased the Th2 type responses as shown by a significant decrease in the interleukin (IL)-4 production and T cell proliferative response induced by sensitization from the 7-d oral administration of OVA. It was additionally observed that the Th1 type responses tended to decrease. We therefore examined the effect of melibiose feeding on the induction of immunological tolerance induced by the oral administration of an antigen (oral tolerance). The Th cell responses induced in BALB/c mice by subcutaneous immunization with OVA were suppressed by the prior oral administration of OVA. Such responses in the OVA-fed and immunized mice were further diminished by dietary melibiose. These results suggest that dietary melibiose strongly affected the Th cell responses to an ingested antigen, and further demonstrate the potential of melibiose to enhance the induction of oral tolerance.     

t10,c12 Conjugated linoleic acid induces compensatory growth after immune challenge

Previous work demonstrated that feeding commercial preparations of conjugated linoleic acid (CLA) [a 50:50 mixture of c9,t11 and t10,c12 CLA (cCLA)] partially overcame lipopolysaccharide (LPS)-induced growth depression. The objective of this study was to determine which CLA isomer was responsible for the reduction of LPS-induced growth depression. Dietary cCLA supplementation for 3 weeks protected mice from LPS-induced weight loss 24 h after injection compared to mice fed isocaloric and isonitrogenous control diets supplemented with either corn oil (CO) or a mixture of CO and olive oil. Dietary c9,t11 or t10,c12 CLA led to body weight loss intermediate to controls and cCLA. After LPS-induced weight loss, the t10,c12 CLA fed mice regained weight faster than the control or c9,t11 CLA fed mice. Dietary t10,c12 CLA and cCLA reduced plasma tumor necrosis factor 2 h after LPS stimulation. While neither c9,t11 nor t10,c12 CLA isomers alone protected from immune-induced weight loss, the t10,c12 CLA isomer induced compensatory gain.     

Reduction in basal adenylate cyclase activity during the immunologic release of histamine from guinea pig lung.

Cyclic AMP has been implicated in the regulation of the immunologic release of histamine from lung and other tissues and cell types. The mechanism whereby intracellular levels of cAMP are altered during mediator release was investigated. Measurements of histamine, adenylate cyclase, and cAMP phosphodiesterase activities were made in actively and passively sensitized guinea pig lung after challenge with antigen. A transient decrease in basal adenylate cyclase activity occurred which returned to control levels after histamine release. There was no change in cAMP phosphodiesterase activity determined at substrate concentrations of 1 mM and 0.01 mM. The adenylate cyclase response did not occur under the following conditions: 1) incubation of nonsensitized lung with antigen, 2) incubation of sensitized lung with antigen in the absence of extracellular calcium, and 3) incubation of nonsensitized lung with compound 48/80. These observations indicate 1) the adenylate cyclase response and the immunologic release of histamine are intimately related, and 2) the reduction in intracellular levels of cAMP which have been reported to occur during immunologic histamine release are mediated via adenylate cyclase.     

Decreased expression of both the alpha1- and alpha2-subunits of the Na-K-ATPase reduces maximal alveolar epithelial fluid clearance

Impaired epithelial sodium channel function predisposes to delayed resorption of pulmonary edema and more severe experimental lung injury, whereas even a small fraction of the normal Na-K-ATPase activity is thought to be sufficient to maintain normal ion transport. However, direct proof is lacking. Therefore, we studied baseline and cAMP stimulated alveolar fluid clearance (AFC) in mice with a 50% decrease in lung protein expression of the alpha(1)- and/or alpha(2)-subunit of the Na-K-ATPase. There was no difference in basal and stimulated AFC in alpha(1)(+/-) or alpha(2)(+/-) mice compared with wild-type littermates. Also, the compound heterozygous mice (alpha(1)(+/-)/alpha(2)(+/-)) had normal basal AFC. However, the combined alpha(1)(+/-)/alpha(2)(+/-) mice showed a significant decrease in cAMP-stimulated AFC compared with wild-type littermates (11.1 +/- 1.0 vs. 14.9 +/- 1.8%/30 min, P < 0.001). When exposed to 96 h of >95% hyperoxia, the decrease in stimulated AFC in the alpha(1)(+/-)/alpha(2)(+/-) mice was not associated with more lung edema compared with wild-type littermates (lung wet-to-dry weight ratio 6.6 +/- 0.9 vs. 5.9 +/- 1.1, respectively; P = not significant). Thus a 50% decrease in protein expression of the alpha(1)- or alpha(2)-subunits of the Na-K-ATPase does not impair basal or stimulated AFC. However, a 50% protein reduction in both the alpha(1)- and alpha(2)-subunits of the Na-K-ATPase produces a submaximal stimulated AFC, suggesting a synergistic role for alpha(1)- and alpha(2)-subunits in cAMP-dependent alveolar epithelial fluid clearance.     

Different effects of conjugated linoleic acid isomers on lipoprotein lipase activity in 3T3-L1 adipocytes

Conjugated linoleic acids (CLAs) are the positional and geometric isomers of linoleic acid. In the present study the effects of cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA ) on intracellular and heparin-releasable (HR-) lipoprotein lipase (LPL) activity in 3T3-L1 adipocytes were investigated. Cells were exposed to the two CLA isomers and linoleic acid, which were bound to bovine serum albumin (BSA). In the adipocytes insulin up-regulated and tumor necrosis factor alpha (TNFalpha) down-regulated HR-LPL activity, which corresponds with the findings in vivo. The experimental fatty acids at low concentrations (<30 μmol/L) moderately increased intracellular and HR-LPL activity. At a concentration of 100 μmol/L, c9,t11 CLA and t10,c12 CLA suppressed HR-LPL activity to 20 and 24% below the BSA control level, respectively, while linoleic acid had no effect unless its concentration was as high as 1000 μmol/L. Insulin abolished the inhibitory effect of c9,t11 CLA, but not of t10,c12 CLA. In the presence of insulin, t10,c12 CLA inhibited HR-LPL activity by 41% compared to BSA control. In contrast to TNFalpha, which suppressed both intracellular LPL and HR-LPL activity, CLAs suppressed HR-LPL activity without decreasing intracellular LPL activity. Additionally, t10,c12 CLA (100 μmol/L) partially prevented TNFalpha-induced decrease of intracellular LPL activity. These results indicate that CLAs differ from linoleic acid in regulating HR-LPL activity, and t10,c12 CLA appeared to be more effective than c9,t11 CLA.     

A dietary conjugated linoleic acid treatment that slows renal disease progression alters renal cyclooxygenase-2-derived prostanoids in the Han: SPRD-cy rat

A mixture of dietary conjugated linoleic acid (CLA) isomers reduces inflammation and mitigates disease progression in the Han:SPRD-cy rat model of chronic kidney disease. Since cyclooxygenase (COX) activities and prostanoid levels are higher in diseased kidneys in this rat, and dietary CLA can inhibit COX2 and prostanoid production in other tissues, the effects of dietary CLA were investigated. Kidney homogenates from normal and diseased Han:SPRD-cy rats were analyzed for prostanoid levels under various conditions: endogenous levels, steady-state levels (60-min incubations) and produced by COX isoforms. Thromboxane B(2) (TXB(2); TXA(2) metabolite), 6-keto-prostaglandin F(1α) (6-keto-PGF(1α); PGI(2) metabolite) and PGE(2) levels under these conditions were two- to ninefold higher in diseased kidneys. Dietary CLA resulted in ～32%-53% lower levels of prostanoids produced by total COX and COX2 activities in normal and diseased kidneys and partially mitigated alterations in COX2 protein levels associated with disease. The COX1 protein and activity were higher in renal disease, resulting in increased production of TXB(2) and 6-ketoPGF(1α), but not PGE(2). Dietary CLA had no effect on COX1, however. Disease resulted in up to twofold higher ratios of TXB(2)/6-ketoPGF(1α), TXB(2)/PGE(2) and 6-ketoPGF(1α) /PGE(2), and dietary CLA partially mitigated these increases under several conditions. Elevated levels of renal membrane associated cytosolic phospholipase A(2) in diseased kidneys also were reduced by 50% with CLA feeding. The effects of CLA feeding on COX2 protein levels and activity indicate that the beneficial effect of dietary CLA in this renal disorder is mediated in part via effects on COX2-derived prostanoids.     

Trans-10,cis-12-conjugated linoleic acid inhibits Caco-2 colon cancer cell growth

A commercially available mixture of conjugated linoleic acid (CLA) isomers decreases colon cancer cell growth. We compared the individual potencies of the two main isomers in this mixture [cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12)] and assessed whether decreased cell growth is related to changes in secretion of insulin-like growth factor II (IGF-II) and/or IGF-binding proteins (IGFBPs), which regulate Caco-2 cell proliferation. Cells were incubated in serum-free medium with different concentrations of the individual CLA isomers. t10c12 CLA dose dependently decreased viable cell number (55 +/- 3% reduction 96 h after adding 5 microM t10c12 CLA). t10c12 CLA induced apoptosis and decreased DNA synthesis, whereas c9t11 CLA had no effect. Immunoblot analysis of 24-h serum-free conditioned medium using a monoclonal anti-IGF-II antibody revealed that Caco-2 cells secreted both a mature 7,500 molecular weight (M(r)) IGF-II and higher M(r) forms of IGF-II. The levels of the higher M(r) and the mature form of IGF-II were decreased 50 +/- 3% and 22 +/- 2%, respectively, by 5 microM t10c12 CLA. c9t11 CLA had no effect. Ligand blot analysis of conditioned medium using 125I-labeled IGF-II revealed that t10c12 CLA slightly decreased IGFBP-2 production; c9t11 CLA had no effect. Exogenous IGF-II reversed t10c12 CLA-induced growth inhibition and apoptosis. These results indicate that CLA-inhibited Caco-2 cell growth is caused by t10c12 CLA and may be mediated by decreasing IGF-II secretion in Caco-2 cells.     

Acute exposure of L6 myotubes to cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid isomers stimulates glucose uptake by modulating Ca2+/calmodulin-dependent protein kinase II

Conjugated linoleic acid (CLA), a dietary fat, has been considered beneficial in metabolic syndrome. Despite several findings indicating that CLA improves glucose clearance, little information is available regarding the cellular dynamics of CLA on skeletal muscle. We sought to investigate the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in cis-9, trans-11(c9,t11) and trans-10, cis-12 (t10,c12) CLA isomer-mediated glucose transport by L6 myotubes. t10,c12-CLA stimulated both intracellular Ca(2+) release (Ca(i)(2+)) and CaMKII phosphorylation, whereas c9,t11-CLA showed only modest effects on both. Sequestering Ca(i)(2+) with BAPTA/AM abrogated the effect of both CLA isomers on Akt substrate-160 kDa (AS160) phosphorylation and glucose uptake by myotubes. Exposing myotubes to KN-93 or autocamtide 2-related inhibitory peptide to block CaMKII activity prevented both CLA isomers from inducing AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished CLA isomer-mediated glucose uptake. These results indicate that CLA isomers require Ca(i)(2+)-CaMKII to mediate glucose uptake. Evidence that CaMKII blockers inhibit t10,c12-CLA-mediated AMP-activated protein kinase (AMPK) activation indicated that CaMKII acts upstream of AMPK in response to t10,c12-CLA. Lastly, CLA isomers stimulated the formation of reactive oxygen species but had no effect on stress-activated protein kinase/c-jun NH(2)-terminal kinase. These data establish that t10,c12-CLA acts via Ca(i)(2+)-CaMKII-AMPK-AS160 to stimulate skeletal muscle glucose transport, whereas the mechanism of c9,t11-CLA remains unclear. Given that impairments in muscle glucose utilisation are apparent in metabolic syndrome, delineating the molecular mechanisms by which CLA isomers mediate muscle glucose uptake may identify new approaches to manage this condition.