Fat Of Mice Research Articles

Reduced Colitis-Associated Colon Cancer inFat-1(n-3 Fatty Acid Desaturase) Transgenic Mice

Bioactive food components containing n-3 polyunsaturated fatty acids (PUFA) modulate multiple determinants that link inflammation to cancer initiation and progression. Therefore, in this study, fat-1 transgenic mice, which convert endogenous n-6 PUFA to n-3 PUFA in multiple tissues, were injected with azoxymethane followed by three cycles of dextran sodium sulfate (DSS) to induce colitis-associated cancer. Fat-1 mice exhibited a reduced number of colonic adenocarcinomas per mouse (1.05 +/- 0.29 versus 2.12 +/- 0.51, P = 0.033), elevated apoptosis (P = 0.03), and a decrease in n-6 PUFA-derived eicosanoids, compared with wild-type (wt) mice. To determine whether the chemoprotective effects of n-3 PUFA could be attributed to its pleiotropic anti-inflammatory properties, colonic inflammation and injury scores were evaluated 5 days after DSS exposure followed by either a 3-day or 2-week recovery period. There was no effect of n-3 PUFA at 3 days. However, following a 2-week recovery period, colonic inflammation and ulceration scores returned to pretreatment levels compared with 3-day recovery only in fat-1 mice. For the purpose of examining the specific reactivity of lymphoid elements in the intestine, CD3(+) T cells, CD4(+) T helper cells, and macrophages from colonic lamina propria were quantified. Comparison of 3-day versus 2-week recovery time points revealed that fat-1 mice exhibited decreased (P < 0.05) CD3(+), CD4(+) T helper, and macrophage cell numbers per colon as compared with wt mice. These results suggest that the antitumorigenic effect of n-3 PUFA may be mediated, in part, via its anti-inflammatory properties.

Cyclooxygenase‐2 expression is reduced in the cortex of the fat‐1 mouse

Prostaglandins produced from arachidonic acid (AA, 20:4 n‐6) contribute to neuroinflammation which is believed to play an important role in neurological diseases such as Alzheimer's Disease. Growing evidence suggests that n‐3 polyunsaturated fatty acids (PUFA) have protective effects by inhibiting inflammatory processes. In this study, the effect of brain n‐3 PUFA on the protein expression of calcium‐dependant cytosolic phospholipase A2 (cPLA2) and cyclooxygenase‐2 (COX‐2), two enzymes of the AA cascade, was assessed in the transgenic fat‐1 mouse. This mouse is capable of endogenously converting n‐6 to n‐3 PUFA. Wildtype (WT) and fat‐1 mice (n=5/genotype) were terminated at 12 weeks of age. The brain was dissected onto cortex, hippocampus and cerebellum. Phospholipid fatty acid composition was determined by thin layer and gas chromatography, while cortical cPLA2 and COX‐2 were determined by western blotting. Phospholipid fractions in all brain regions were enriched in 22:6 n‐3 and reduced in n‐6 PUFA in fat‐1 as compared to WT mice. PLA2 expression was lower in fat‐1 mice, but not significantly different. However, COX‐2 protein levels were 25% lower in fat‐1 as compared to WT mice (p=0.02). Our results suggest that this model could be used to investigate mechanisms by which n‐3 PUFA regulate neuroinflammation.

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.

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.

Omega‐3 fatty acids alleviate chemically induced acute hepatitis by suppression of cytokines†‡

Cytokines such as tumor necrosis factor alpha (TNF-alpha) are key factors in liver inflammation. Supplementation with essential omega-3 polyunsaturated fatty acids (n-3 PUFA) has been demonstrated to lower TNF-alpha and IL-1 production in mononuclear cells. An inflammation-dampening effect has been observed with increased omega-3 fatty acid supplementation in several inflammatory diseases. In this study, we used the transgenic fat-1 mouse, expressing a Caenorhabditis elegans desaturase endogenously forming n-3 PUFA from n-6 PUFA, to analyze the effect of an increased n-3 PUFA tissue status in the macrophage-dependent acute D-galactosamine/lipopolysaccaride (D-GalN/LPS) hepatitis model. We show less severe inflammatory liver injury in fat-1 mice with a balanced n-6/n-3 PUFA ratio as evidenced by reduced serum alanine aminotransferase levels and less severe histological liver damage. This decreased inflammatory response was associated with decreased plasma TNF-alpha levels and with reduced hepatic gene expression of TNF-alpha, IL-1beta, IFN-gamma and IL-6 in fat-1 mice, leading to a decreased rate of apoptosis in livers from fat-1 animals, as measured by DAPI-staining. The results of this study offer evidence for an inflammation dampening effect of omega-3 polyunsaturated fatty acids in the context of liver inflammation.

Inhibition of inflammatory response in transgenic fat-1 mice on a calorie-restricted diet

Both n − 3 fatty acids ( n − 3 FA) and calorie-restriction (CR) exert anti-inflammatory effects in animal models of autoimmunity and inflammation. In the present study we investigated the synergistic anti-inflammatory effects of n − 3 FA and CR on LPS-mediated inflammatory responses using fat-1 transgenic mice that generate n − 3 FA endogenously. Wild-type (WT) and fat-1 mice were maintained on ad libitum (AL) or CR (40% less than AL) diet for 5 mo; splenocytes were cultured in vitro with/without LPS. Our results show: (i) no difference in body weights between WT and fat-1 mice on AL or CR diets, (ii) lower n − 6/ n − 3 FA ratio in splenocytes from fat-1 mice on both AL and CR diets, (iii) significant reduction in NF-κB (p65/p50) and AP-1 (c-Fos/c-Jun) DNA-binding activities in splenocytes from fat-1/CR mice following LPS treatment, and (iv) significant reduction in κB- and AP-1-responsive IL-6 and TNF-α secretion following LPS treatment in splenocytes from fat-1/CR mice. The inhibition of LPS-mediated effects was more pronounced in fat-1/CR mice when compared to fat-1/AL or WT/CR mice. These data show that transgenic expression of fat-1 results in decreased pro-inflammatory n − 6 FA, and demonstrate for the first time that splenocytes from fat-1 mice on CR diet exhibit reduced pro-inflammatory response when challenged with LPS. These results suggest that n − 3 lipids with moderate CR may confer protection in autoimmune and inflammatory diseases.

Melanoma growth is reduced in fat-1 transgenic mice: impact of omega-6/omega-3 essential fatty acids.

An important nutritional question as to whether the ratio of omega-6 (n-6) to omega-3 (n-3) fatty acids plays a role in tumorigenesis remains to be clarified in well qualified experimental models. The recently engineered fat-1 mice, which can convert n-6 to n-3 fatty acids and have a balanced ratio of n-6 to n-3 fatty acids in their tissues and organs independent of diet, allow carefully controlled studies to be performed in the absence of potential confounding factors of diet and therefore are a useful model for elucidating the role of n-6/n-3 fatty acid ratio in tumorigenesis. We implanted mouse melanoma B16 cells into transgenic and WT littermates and examined the incidence of tumor formation and tumor growth rate. The results showed a dramatic reduction of melanoma formation and growth in fat-1 transgenic mice. The level of n-3 fatty acids and their metabolite prostaglandin E(3) (PGE(3)) were much higher (but the n-6/n-3 ratio is much lower) in the tumor and surrounding tissues of fat-1 mice than that of WT animals. The phosphatase and tensin homologue deleted on the chromosome 10 (PTEN) gene was significantly up-regulated in the fat-1 mice. In vitro experiments showed that addition of the n-3 fatty acid eicosapentaenoic acid or PGE(3) inhibited the growth of B16 cell line and increased the expression of PTEN, which could be partially attenuated by inhibition of PGE(3) production, suggesting that PGE(3) may act as an antitumor mediator. These data demonstrate an anticancer (antimelanoma) effect of n-3 fatty acids through, at least in part, activation of PTEN pathway mediated by PGE(3).

N‐3 polyunsaturated fatty acids endogenously synthesized in fat‐1 mice are enriched in the mammary gland

In this study, we determined the phospholipid FA composition in the mammary gland of the transgenic Fat-1 mouse. This is the first animal model developed that can endogenously synthesize n-3 PUFA. The synthesis of n-3 PUFA is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase from Caenorhabditis elegans, which utilizes n-6 PUFA as substrate. Wild-type and Fat-1 female mice were terminated at 7 wk of age and the fifth mammary gland was removed. Lipids were extracted and phospholipids were separated by TLC and converted to FAME for analysis by GC. There was no significant change in total saturated, monounsaturated, and PUFA composition. However, there was a significant increase in total n-3 PUFA and a corresponding decrease in n-6 PUFA. The major n-3 PUFA that were enriched included 20:5n-3 and 22:6n-3. The n-6 PUFA that were reduced included 20:4n-6, 22:4n-6, and 22:5n-6. Overall, these findings demonstrate that female Fat-i mice have elevated levels of n-3 PUFA in the mammary gland. Moreover, the n-3 desaturase products are the same n-3 PUFA found in fish oil, which have been shown to have chemoprotective properties against breast cancer. Therefore, this newly developed mouse model may be highly useful for investigating molecular and cellular mechanisms by which n-3 PUFA prevents and inhibits breast cancer growth.