fat-1 transgene expression prevents cell culture-induced loss of membrane n-3 fatty acids in activated CD4+ T-cells

In order to evaluate the effects of fatty acids on immune cell membrane structure and function, it is often necessary to maintain cells in culture. However, cell culture conditions typically reverse alterations in polyunsaturated fatty acid (PUFA) composition achieved by dietary lipid manipulation. Therefore, we hypothesized that T-cells from transgenic mice expressing the Caenorhabditis elegans n-3 desaturase (fat-1) gene would be resistant to the culture-induced loss of n-3 PUFA and, therefore, obviate the need to incorporate fatty acids or homologous serum into the medium. CD4⁺ T-cells were isolated from (i) control wild type (WT) mice fed a safflower oil-n-6 PUFA enriched diet (SAF) devoid of n-3 PUFA, (ii) fat-1 transgenic mice (enriched with endogenous n-3 PUFA) fed a SAF diet, or (iii) WT mice fed a fish oil (FO) based diet enriched in n-3 PUFA. T-cell phospholipids isolated from WT mice fed FO diet (enriched in n-3 PUFA) and fat-1 transgenic mice fed a SAF diet (enriched in n-6 PUFA) were both enriched in n-3 PUFA. As expected, the mol% levels of both n-3 and n-6 PUFA were decreased in cultures of CD4⁺ T-cells from FO-fed WT mice after 3 d in culture. In contrast, the expression of n-3 desaturase prevented the culture-induced decrease of n-3 PUFA in CD4⁺ T-cells from the transgenic mice. Carboxyfluorescein succinidyl ester (CFSE) -labeled CD4⁺ T-cells from fat-1/SAF vs. WT/SAF mice stimulated with anti-CD3 and anti-CD28 for 3 d, exhibited a reduced (P<0.05) number of cell divisions. We conclude that fat-1-containing CD4⁺ T-cells express a physiologically relevant, n-3 PUFA enriched, membrane fatty acid composition which is resistant to conventional cell culture-induced depletion.     

Mammary tumor development is directly inhibited by lifelong n-3 polyunsaturated fatty acids

IntroductionDespite the advocacy that diet may be a significant contributor to cancer prevention, there is a lack of direct evidence from epidemiological and experimental studies to substantiate such claims. Experimental studies suggest that n-3 polyunsaturated fatty acids (n-3 PUFA) from marine oils may reduce breast cancer risk, however, findings are equivocal. Thus, in this study, novel transgenic mouse models were employed to provide, for the first time, direct evidence for an anti-cancer role of n-3 PUFA in mammary tumorigenesis.Methodsfat-1 Mice, which are capable of endogenous n-3 PUFA synthesis, were bred with mouse mammary tumor virus (MMTV)-neu(ndl)-YD5 mice, an aggressive breast cancer model. The resultant offspring, including novel hybrid progeny, were assessed for tumor onset, size and multiplicity as well as n-3 PUFA composition in mammary gland and tumor tissue. A complementary group of MMTV-neu(ndl)-YD5 mice were fed n-3 PUFA in the diet.ResultsMice expressing MMTV-neu(ndl)-YD5 and fat-1 displayed significant (P<.05) reductions in tumor volume (~30%) and multiplicity (~33%), as well as reduced n-6 PUFA and enriched n-3 PUFA in tumor phospholipids relative to MMTV-neu(ndl)-YD5 control mice. The effect observed in hybrid progeny was similarly observed in n-3 PUFA diet fed mice.ConclusionUsing complementary genetic and conventional dietary approaches we provide, for the first time, unequivocal experimental evidence that n-3 PUFA is causally linked to tumor prevention.     

Activation of Stat5 and induction of a pregnancy-like mammary gland differentiation by eicosapentaenoic and docosapentaenoic omega-3 fatty acids

The protective effect of early pregnancy against breast cancer can be attributed to the transition from undifferentiated cells in the nulliparous to the differentiated mature cells during pregnancy. Considerable evidence suggests strongly that the n-3 polyunsaturated fatty acid (PUFA) content of adipose breast tissue is inversely associated with an increased risk of breast cancer. Here, we report that there was a decrease in the n-6/n-3 PUFA ratio and a significant increase in concentration of n-3 PUFA docosapentaenoic acid and eicosapentaenoic acid in the pregnant gland. The functional role of n-3 PUFAs on differentiation was supported by the studies in the fat-1 transgenic mouse, which converts endogenous n-6 to n-3 PUFAs. Alternation of the n-6/n-3 ratio in favor of n-3 PUFA, and particularly docosapentaenoic acid, in the mammary gland of fat-1 mouse resulted in development of lobulo-alveolar-like structure and milk protein beta-casein expression, mimicking the differentiated state of the pregnant gland. Docosapentaenoic acid and eicosapentaenoic acid activated the Jak2/Stat5 signaling pathway and induced a functional differentiation with production of beta-casein. Expression of brain type fatty acid binding protein brain type fatty acid binding protein in virgin transgenic mice also resulted in a reduced ratio of n-6/n-3 PUFA, a robust increase in docosapentaenoic acid accumulation, and mammary differentiation. These data indicate a role of mammary derived growth inhibitor related gene for preferential accumulation of n-3 docosapentaenoic acid and eicosapentaenoic acid in the differentiated gland during pregnancy. Thus, alternation of n-6/n-3 fatty acid compositional ratio in favor of n-3 PUFA, and particularly docosapentaenoic acid and eicosapentaenoic acid, is one of the underlying mechanisms of pregnancy-induced mammary differentiation.     

The iFat1 transgene permits conditional endogenous n-3 PUFA enrichment both in vitro and in vivo

Fat-1 transgenic mice, which endogenously convert n-6 PUFA to n-3 PUFA, are a useful tool in health research; however with this model timing of n-3 PUFA enrichment cannot be directly controlled. To add such capability, the novel Cre-recombinase inducible fat-1 (iFat1) transgenic mouse has been developed. The aim of this study was to characterize the utility of the iFat1 transgene as a model of Cre-inducible endogenous n-3 PUFA enrichment. Functionality of the iFat1 transgene was screened both in vitro and in vivo. In the presence of Cre, the iFat1 transgene resulted in a balancing (p < 0.01) of the n-6/n-3 PUFA ratio within phospholipids in the human embryonic kidney 293T cell line. For in vivo analysis, iFat1 transgenic mice were crossed with the R26-Cre-ER^T2 (Tam-Cre) mouse line, a tamoxifen inducible Cre-expression model. Tam-Cre/iFat1 double hybrids were transiently treated with tamoxifen at 6–7 weeks, then terminated 3 weeks later. Tamoxifen treated mice had increased (p < 0.05) tissue n-3 PUFA and ≥two-fold reduction (p < 0.05) in the n-6/n-3 PUFA ratio of liver, kidney and muscle phospholipids relative to vehicle treated controls. Collectively these findings suggest that the iFat1 transgenic mouse may be a promising tool to help elucidate the temporal effects through which n-3 PUFA impacts health related outcomes.     

Seizure resistance in fat-1 transgenic mice endogenously synthesizing high levels of omega-3 polyunsaturated fatty acids

n-3 polyunsaturated fatty acids (PUFA), derived from marine oils, have been shown to protect against various neurological diseases. However, very little is known about their potential anticonvulsant properties. The objective of the present study was to determine whether enrichment of brain lipids with n-3 PUFA inhibits seizures induced by pentylenetetrazol. We demonstrate that increased brain levels of n-3 PUFA in transgenic fat-1 male mice, which are capable of de novo synthesis of n-3 PUFA from n-6 PUFA, increases latency to seizure onset by 45%, relative to wildtype controls ( p  = 0.08). Compared with wildtype littermates, transgenic fat-1 mice have significantly ( p  < 0.05) higher levels of docosahexaenoic acid and total n-3 PUFA in brain total lipid extracts and phospholipids. Levels of brain docosahexaenoic acid were positively correlated to seizure latency ( p  < 0.05). These findings demonstrate that n-3 PUFA have anticonvulsant properties and suggest the possibility of a novel, non-drug dietary approach for the treatment of epilepsy.     

N-3 PUFAs Protect against Aortic Inflammation and Oxidative Stress in Angiotensin II-Infused Apolipoprotein E-/- Mice

Abdominal aortic aneurysm is associated with infiltration of inflammatory cells into the aortic wall. The inflammatory response is also evident in animal models, such as apolipoprotein E-deficient (ApoE^-/-) mice that have been infused with angiotensin II, prior to development of aortic aneurysm. Since omega-3 polyunsaturated fatty acids (n-3 PUFAs) and their metabolites have anti-inflammatory and pro-resolving activity, we hypothesised that dietary supplementation with n-3 PUFAs would protect against inflammatory processes in this mouse model. Twenty C57 and 20 ApoE^-/- 3-4 week old male mice were supplemented with a low (0.14%, n = 10/group) or high (0.70%, n = 10/group) n-3 PUFA diet for 8 weeks before 2-day infusion with 0.9% saline or angiotensin II (1000 ng/kg/min). Four ApoE^-/- mice on the low n-3 PUFA diet and none of the ApoE^-/- mice on the high n-3 PUFA diet showed morphological evidence of abdominal aortic dissection. The plasma concentration of the n-3 PUFA metabolite, resolvin D1 was higher in angiotensin II-infused ApoE^-/- mice fed the high, compared to the low n-3 PUFA diet. The number of neutrophils and macrophages infiltrating the abdominal aorta was elevated in ApoE^-/- mice on the low n-3 PUFA diet, and this was significantly attenuated in mice that were fed the high n-3 PUFA diet. Most neutrophils and macrophages were associated with dissected aortas. Immunoreactivity of the catalytic subunit of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, Nox2, and superoxide were elevated in ApoE^-/- mice that were fed the low n-3 PUFA diet, and this was also significantly attenuated in mice that were fed the high n-3 PUFA diet. Together, the findings indicate that supplementation of ApoE^-/- mice with a diet high in n-3 PUFA content protected the mice against pro-inflammatory and oxidative stress responses following short-term infusion with angiotensin II.     

Improvement of polyunsaturated fatty acids synthesis by the coexpression of CYB5 with desaturase genes in Saccharomyces cerevisiae

Since Saccharomyces cerevisiae contains Δ9 fatty acid desaturase (OLE1) as a sole fatty acid desaturase, it produces saturated and monounsaturated fatty acids of 16- and 18-carbon compounds. We showed earlier that Kluyveromyces lactis Δ12 (KlFAD2) and ω3 (KlFAD3) fatty acid desaturase genes enabled S. cerevisiae to make also polyunsaturated fatty acids (PUFAs), linoleic (18:2n-6), and α-linolenic (18:3n-3) acids. Unlike Δ9 fatty acid desaturase Ole1p, the two added fatty acid desaturases (KlFAD2and KlFAD3) do not contain a cytochrome b5 domain, and we now report on effects of the overexpression of K. lactis and S. cerevisiae cytochrome b5 (CYB5) genes as well as temperature effects on PUFA synthesis. Without extra cytochrome b5, while PUFA synthesis is significant at low temperature (20 °C), it was marginal at 30 °C. Overexpression of cytochrome b5 at 20 °C did not affect the fatty acid synthesis so much, but it significantly enhanced the synthesis of PUFA at 30 °C.     

Endogenous synthesis of n-3 polyunsaturated fatty acids in Fat-1 mice is associated with increased mammary gland and liver syndecan-1

Long chain n-3 PUFA have been shown to have chemopreventive properties against breast cancer through various mechanisms. One pathway, studied in human breast cancer cell lines, involves upregulation of the proteoglycan, syndecan-1 (SDC-1) by n-3 PUFA-enriched LDL. Using Fat-1 mice that are able to convert n-6 to n-3 PUFA, we tested whether SDC-1 level in vivo is elevated in mammary glands due to endogenously synthesized rather than LDL-derived n-3 PUFA. Female Fat-1 and wild type (wt) mice were fed an n-6 PUFA- enriched diet for 7 weeks. Fatty acid analysis of plasma lipoproteins showed that total n-6 PUFA reflected dietary intake similarly in both genotypes (VLDL, 36.2±2.2 and 40.9±3.9; LDL, 49.0±3.3 and 48.1±2.0; HDL, 54.6±1.2 and 58.2±1.3, mean ± SEM percent of total fatty acids for Fat-1 and wt animals respectively). Lipoprotein percent n-3 PUFA was also similar between groups. However, phospholipids and triglycerides extracted from mammary and liver tissues demonstrated significantly higher n-3 PUFA and a corresponding decrease in the ratio n-6/n-3 PUFA in Fat-1 compared to wt mice. This was accompanied by higher SDC-1 in mammary glands and livers of Fat-1 mice, thus demonstrating that endogenously synthesized n-3 PUFA may upregulate SDC-1 in the presence of high dietary n-6 PUFA.     

The Fat-1 Mouse has Brain Docosahexaenoic Acid Levels Achievable Through Fish Oil Feeding

Fat-1 transgenic mice endogenously convert n-6 to n-3 polyunsaturated fatty acids (PUFA). The aims of this study were to test whether a) fish oil feeding can attain similar brain n-3 PUFA levels as the fat-1 mouse, and b) fat-1 mouse brain docosahexaenoic acid (22:6n-3; DHA) levels can be potentiated by fish oil feeding. Fat-1 mice and their wildtype littermates consumed either a 10% safflower oil (SO) or a 2% fish oil and 8% safflower oil chow (FO). Brain total lipid and phospholipid fraction fatty acids were analyzed using GC-FID. Wildtype mice fed FO chow had similar brain levels of DHA as fat-1 mice fed SO chow. Fat-1 mice fed FO chow had similar brain n-3 PUFA levels as fat-1 mice fed SO chow. In conclusion, brain levels of DHA in the fat-1 mouse can be obtained by and were not further augmented with fish oil feeding.     

Associations of maternal prenatal dietary intake of n-3 and n-6 fatty acids with maternal and umbilical cord blood levels

Maternal n-3 and n-6 polyunsaturated fatty acid (PUFA) status may influence birth outcomes and child health. We assessed second trimester maternal diet with food frequency questionnaires (FFQs) (n=1666), mid-pregnancy maternal erythrocyte PUFA concentrations (n=1550), and umbilical cord plasma PUFA concentrations (n=449). Mean (SD) maternal intake of total n-3 PUFA was 1.17 g/d (0.43), docosahexaenoic and eicosapentaenoic acids (DHA+EPA) 0.16 g/d (0.17), and total n-6 PUFA 12.25 g/d (3.25). Mean maternal erythrocyte and cord plasma PUFA concentrations were 7.0% and 5.2% (total n-3), 5.0% and 4.6% (DHA+EPA), and 27.9% and 31.4% (total n-6). Mid-pregnancy diet–blood and blood–blood correlations were strongest for DHA+EPA (r=0.38 for diet with maternal blood, r=0.34 for diet with cord blood, r=0.36 for maternal blood with cord blood), and less strong for n-6 PUFA. The FFQ is a reliable measure of elongated PUFA intake, although inter-individual variation is present     

Effects of different dietary fatty acids profiles on the growth performance and body composition of juvenile tench (<Emphasis Type="Italic">Tinca tinca</Emphasis> (L.))

Juvenile tench (initial weight of about 57 g) were fed feed supplemented with fish oil (group FO), linseed oil (group LO), peanut oil (group PO), or rapeseed oil (group RO) containing 47% protein and 12% fat for 55 days. The inclusion of the tested oils was 50 g kg⁻¹ (42% total crude lipids in diets). No significant differences were noted in the fish growth performance. The proximate composition of the whole fish bodies and the viscera (water, protein, fat, ash) was similar in all the dietary treatments (P > 0.05). Differences were noted only with regard to the ash content of the fillets (P < 0.05). The analysis of the fatty acids profiles of tench (whole fish) indicated there were significant differences in the total content of monoenoic and polyenoic (PUFA) acids. Significant differences were also noted with regard to n-3 PUFA and n-6 PUFA. Consequently, the ratio of n-3/n-6 acids ranged from 1.6 (group PO) to 2.08 (group LO; P < 0.05). The feed applied was not confirmed to have had an impact on the fatty acids profile of the tench fillets. There was a statistically significant intergroup difference in the content of saturated fatty acids (SFA) in tench viscera. In the fish fed vegetable oils supplemented diets, the level of SFA was lower (P < 0.05).     

Marine fish oil is more potent than plant-based n-3 polyunsaturated fatty acids in the prevention of mammary tumors

Marine-derived n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to inhibit mammary carcinogenesis. However, evidence regarding plant-based α-linolenic acid (ALA), the major n-3 PUFA in the Western diet, remains equivocal. The objective of this study was to examine the effect of lifelong exposure to plant- or marine-derived n-3 PUFAs on pubertal mammary gland and tumor development in MMTV-neu(ndl)-YD5 mice. It is hypothesized that lifelong exposure to n-3 PUFA reduces terminal end buds during puberty leading to delayed tumor onset, volume and multiplicity. It is further hypothesized that plant-derived n-3 PUFAs will exert dose-dependent effects. Harems of MMTV-FVB males were bred with wild-type females and fed either a (1) 10% safflower (10% SF, n-6 PUFA, control), (2) 10% flaxseed (10% FS), (3) 7% safflower plus 3% flaxseed (3% FS) or (4) 7% safflower plus 3% menhaden (3% FO) diet. Female offspring were maintained on parental diets. Compared to SF, 10% FS and 3% FO reduced (P<.05) terminal end buds at 6 weeks and tumor volume and multiplicity at 20 weeks. A dose-dependent reduction of tumor volume and multiplicity was observed in mice fed 3% and 10% FS. Antitumorigenic effects were associated with altered HER2, pHER-2, pAkt and Ki-67 protein expression. Compared to 10% SF, 3% FO significantly down-regulated expression of genes involved in eicosanoid synthesis and inflammation. From this, it can be estimated that ALA was 1/8 as potent as EPA+DHA. Thus, marine-derived n-3 PUFAs have greater potency versus plant-based n-3 PUFAs.     

A Metabolomic Analysis of Omega-3 Fatty Acid-Mediated Attenuation of Western Diet-Induced Nonalcoholic Steatohepatitis in LDLR -/- Mice

Background

Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease and a risk factor for cirrhosis, hepatocellular carcinoma and liver failure. Previously, we reported that dietary docosahexaenoic acid (DHA, 22:6,n-3) was more effective than eicosapentaenoic acid (EPA, 20:5,n-3) at reversing western diet (WD) induced NASH in LDLR^-/- mice.

Methods

Using livers from our previous study, we carried out a global non-targeted metabolomic approach to quantify diet-induced changes in hepatic metabolism.

Results

Livers from WD + olive oil (WD + O)-fed mice displayed histological and gene expression features consistent with NASH. The metabolomic analysis of 320 metabolites established that the WD and n-3 polyunsaturated fatty acid (PUFA) supplementation had broad effects on all major metabolic pathways. Livers from WD + O-fed mice were enriched in saturated (SFA) and monounsaturated fatty acids (MUFA), palmitoyl-sphingomyelin, cholesterol, n-6 PUFA, n-6 PUFA-containing phosphoglycerolipids, n-6 PUFA-derived oxidized lipids (12-HETE) and depleted of C_20-22 n-3 PUFA-containing phosphoglycerolipids, C_20-22 n-3 PUFA-derived oxidized lipids (18-HEPE, 17,18-DiHETE) and S-lactoylglutathione, a methylglyoxal detoxification product. WD + DHA was more effective than WD + EPA at attenuating WD + O-induced changes in NASH gene expression markers, n-6 PUFA and oxidized lipids, citrate and S-lactosyl glutathione. Diet-induced changes in hepatic MUFA and sphingolipid content were associated with changes in expression of enzymes involved in MUFA and sphingolipid synthesis. Changes in hepatic oxidized fatty acids and S-lactoylglutathione, however, correlated with hepatic n-3 and n-6 C_20-22 PUFA content. Hepatic C_20-22 n-3 PUFA content was inversely associated with hepatic α-tocopherol and ascorbate content and positively associated with urinary F2- and F3-isoprostanes, revealing diet effects on whole body oxidative stress.

Conclusion

DHA regulation of hepatic SFA, MUFA, PUFA, sphingomyelin, PUFA-derived oxidized lipids and S-lactoylglutathione may explain the protective effects of DHA against WD-induced NASH in LDLR^-/- mice.     

Preferential retention of algal carbon in benthic invertebrates: Stable isotope and fatty acid evidence from an outdoor flume experiment

1. According to the River Continuum Concept, headwater streams are richer in allochthonous (e.g. terrestrial leaves) than autochthonous (e.g. algae) sources of organic matter for consumers. However, compared to algae, leaf litter is of lower food quality, particularly ω-3 polyunsaturated fatty acids (n-3 PUFA), and would constrain the somatic growth, maintenance, and reproduction of stream invertebrates. It may be thus assumed that shredders, such as Gammarus, receive lower quality diets than grazers, e.g. Ecdyonurus, that typically feed on algae.
2. The objective of this study was to assess the provision of dietary PUFA from leaf litter and algae to the shredder Gammarus and the grazer Ecdyonurus. Three different diets (algae, terrestrial leaves, and an algae–leaf litter mix) were supplied to these macroinvertebrates in a flume experiment for 2 weeks. To differentiate how diet sources were retained in these consumers, algae were isotopically labelled with ¹³C.
3. Both consumers became enriched with ¹³C in all treatments, demonstrating that both assimilated algae. For Gammarus, n-3 PUFA increased, whereas n-6 PUFA stayed constant. By contrast, the n-3 PUFA content of Ecdyonurus decreased as a consequence of declining algal supply.
4. Results from compound-specific stable isotope analysis provided evidence that the long-chain n-3 PUFA eicosapentaenoic acid (EPA) in both consumers was more enriched in ¹³C than the short-chain n-3 PUFA α-linolenic acid, suggesting that EPA was taken up directly from algae and not from heterotrophic biofilms on leaf litter. Both consumers depended on algae as their carbon and EPA source and retained their EPA from high-quality algae.

     

Modulation of lipid peroxidation and antioxidant enzymes in murine salivary gland by dietary fatty acid ethyl esters

The present study was undertaken to investigate the effect of n-9, n-6, and n-3 dietary fatty acid ethyl esters on basal (uninduced) and Fe2+/ascorbate (induced) lipid peroxidation (LPO) in salivary gland (SG) of mice. Feeding n-3 ethyl ester polyunsaturated fatty acids (PUFA) increased the uninduced and induced LPO in SG homogenates. In contrast, feeding olive oil ethyl esters (n-9) significantly lowered the induced and uninduced LPO in SG tissue. Salivary gland susceptibility to LPO increased in the order of: olive oil < corn oil < safflower oil < n-3 ethyl esters. Olive oil esters in the diet increased primarily the 18:1 levels in SG tissue. Whereas feeding n-3 PUFA notably increased the superoxide dismutase (SOD) and catalase activities in SG homogenates, no significant changes were seen between n-9 and n-6 PUFA-fed mice. Lower levels of Vitamin E (Vit E) in the tissues of n-3 PUFA-fed mice indicate that the higher the dietary lipid unsaturation, the higher the requirement for Vit E in the diet. Our results indicate that, similar to other organs, salivary gland susceptibility to uninduced or induced oxidation depends on the source of dietary PUFA. In conclusion, feeding olive oil increases the resistance of SGs to induced and uninduced LPO.     

Fatty acid compositional changes during the embryonic development of the swimming crab,Portunus pelagicus (Portunidae: Decapoda)

Abstract

The fatty acid composition, moisture, and total lipid of the eggs from the swimming crab, Portunus pelagicus, at three different embryonic stages (within 24 h, during the eye placode stage and the final heart beat stage), were measured. Results showed that the moisture and lipid content significantly increased and decreased (p < 0.05), respectively, as the stages progressed. The most prevalent fatty acids that were initially deposited included C16:0, C18:1n-9, and C18:0, while the most consumed fatty acids were C22:5n-6, C22:5n-3, and C20:1n-7. Among the major fatty acid groups, polyunsaturated fatty acids (PUFA) and long-chain PUFA (LC-PUFA) were consumed more than saturated fatty acids and significantly more (p < 0.05) than monounsaturated fatty acids (p < 0.05). Meanwhile, n-3 PUFA was deposited in significantly higher amounts (p < 0.05) than n-6 PUFA, but both were consumed at similar amounts at 43.4% and 41.3%, respectively. The relatively low amount of C20:5n-3 and C22:6n-3 consumption may indicate these fatty acids were conserved, while the essential fatty acids C18:3n-3 and C18:3n-6 were consumed at high amounts. These findings may have implications for broodstock nutrition in order to formulate a well-balanced diet.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood–brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood–brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (−23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 μM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [³H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood–brain barrier, possibly via changes in GLUT1 protein expression and activity.     

Regulation of epithelial cell turnover and macrophage phenotype by epithelial cell-derived transforming growth factor beta1 in the mammary gland

Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates cell proliferation, apoptosis and immune system responses. In the breast, the mammary epithelium is the primary source of TGFB1 and increased expression is associated with increased breast cancer risk. This study was conducted to investigate the roles of epithelial cell-derived TGFB1 in regulation of epithelial cell activity and macrophage phenotype in the mammary gland. Tgfb1 null mutant and wildtype mammary epithelium was transplanted into contra-lateral sides of the cleared mammary gland of TGFB1 replete scid mice. Transplanted tissue was analysed for markers of proliferation and apoptosis to determine the effect of Tgfb1 null mutation on epithelial cell turnover, and was analysed by immunohistochemistry to investigate the location, abundance and phenotype of macrophages. The number of proliferating and dying ductal epithelial cells, determined by BrdU and TUNEL, was increased by 35% and 3.3-fold respectively in mammary gland transplanted with Tgfb1 null epithelium compared to wildtype epithelium (p < 0.05). Abundance of F4/80+ macrophages in between Tgfb1 null epithelial cells compared to wildtype epithelial cells was increased by 50%. The number of iNOS+ and CCR7+ cells in the stroma surrounding Tgfb1 null alveolar epithelium was increased by 78% and 2-fold respectively, and dendriform MHC class II+ cells within ductal epithelium were decreased by 30%. We conclude that epithelial cell-derived TGFB1 in the mammary gland has two functions: (1) regulation of cellular turnover of epithelial cells, and (2) regulation of local macrophage phenotype. These findings shed new light on the diversity of roles of TGFB1 in the mammary gland which are likely to impact on breast cancer risk.