Characterization of an arachidonic acid-deficient (Fads1 knockout) mouse model - PubMed (original) (raw)
Characterization of an arachidonic acid-deficient (Fads1 knockout) mouse model
Yang-Yi Fan et al. J Lipid Res. 2012 Jul.
Abstract
Arachidonic acid (20:4(Δ5,8,11,14), AA)-derived eicosanoids regulate inflammation and promote cancer development. Previous studies have targeted prostaglandin enzymes in an attempt to modulate AA metabolism. However, due to safety concerns surrounding the use of pharmaceutical agents designed to target Ptgs2 (cyclooxygenase 2) and its downstream targets, it is important to identify new targets upstream of Ptgs2. Therefore, we determined the utility of antagonizing tissue AA levels as a novel approach to suppressing AA-derived eicosanoids. Systemic disruption of the Fads1 (Δ5 desaturase) gene reciprocally altered the levels of dihomo-γ-linolenic acid (20:3(Δ8,11,14), DGLA) and AA in mouse tissues, resulting in a profound increase in 1-series-derived and a concurrent decrease in 2-series-derived prostaglandins. The lack of AA-derived eicosanoids, e.g., PGE₂ was associated with perturbed intestinal crypt proliferation, immune cell homeostasis, and a heightened sensitivity to acute inflammatory challenge. In addition, null mice failed to thrive, dying off by 12 weeks of age. Dietary supplementation with AA extended the longevity of null mice to levels comparable to wild-type mice. We propose that this new mouse model will expand our understanding of how AA and its metabolites mediate inflammation and promote malignant transformation, with the eventual goal of identifying new drug targets upstream of Ptgs2.
Figures
Fig. 1.
Genotyping of Fads1 knockout mice. (A) Mice were cloned from an embryonic stem cell line (IST11525H2) carrying the Fads1 allele disrupted by the insertion of a gene trap vector (Omnibank Vector 76) in the first intron. The insertion disrupts transcription of the gene. (B) DNA isolated from mouse tail was genotyped by PCR. Fads1 gene product (310 bp) was found only in Wt and Het mice; V76 (Omnibank Gene Trap Vector 76) gene product (270 bp) was found only in Het and Null mice. Refer to Materials and Methods for primer sets and details describing the gene trapping technique.
Fig. 2.
Fads1 and Fads2 gene expression in knockout mice. (A) Colonic mucosa and (B) liver RNA were extracted, and RT-qPCR was performed to measure gene expression levels. Data were normalized by 18S expression, mean ± SEM, n = 3. Values not sharing the same letter indicate significance within respective groups (P < 0.05).
Fig. 3.
Kaplan-Meier survival curves of Fads1 mice. (A) Fads1 null mice exhibited low viability when fed a standard AA-free diet; n = 37 for Wt, n = 44 for Het, n = 11 for Null. (B) Dietary supplementation with AA (0.1 and 0.4%, w/w) partially reversed the Fads1 null mouse phenotype; n = 5 for Null + 0.1% AA, n = 3 for Null + 0.4% AA. Supplementation with 2.0% AA completely rescued the Null phenotype; n = 4 for Null ± 2% AA.
Fig. 4.
Tissue levels of AA and DGLA. Total phospholipids were isolated from mouse (A) colon mucosa, (B) liver, (C) splenocytes, and (D) serum, and fatty acid profiles were measured by GC-MS; mean ± SEM, n = 3. Values not sharing the same letter indicate a significant difference within respective fatty acids (P < 0.05).
Fig. 5.
Levels of DGLA and AA-derived prostaglandins. Prostaglandins (PGE1 and PGE2) extracted from (A) colon mucosa, (B) small intestine mucosa, and (C) lung were measured by LC-MS; mean ± SEM, n = 3. Values not sharing the same letter indicate a significant difference within respective prostaglandins (P < 0.05).
Fig. 6.
Effect of Fads1 deletion on colonic cell proliferation. Levels of cell proliferation in mouse colonic crypts were measured by the EdU Click-It assay. Data are expressed as (A) total number of cells per crypt and (B) percentage of EdU-labeled cells relative to the total number of cells per crypt; mean ± SEM, n = 3. Values not sharing the same letter indicate significant differences (P < 0.05).
Fig. 7.
Levels of PI(4,5)P2 in Fads1 knockout mouse T cells. Splenic CD4+ T cells were isolated, and the levels of PI(4,5)P2 were quantified by indirect anti-PIP2 ELISA. Data were normalized to wild-type (Wt) at time 0; mean ± SEM, n = 3. Values not sharing the same letter indicate significant differences (P < 0.05).
Fig. 8.
Immune cell populations in Fads1 knockout mice. Mononuclear cells were isolated from pooled spleens and mesenteric and inguinal lymph nodes. CD3+ T cell and MHCII cell populations were further analyzed by flow cytometry. Data are reported as (A) total number of mononuclear cells/mouse, (B) percentage of CD3+ T cells in the mononuclear cells, and (C) percentage of MHCII cells in the mononuclear cell fraction; mean ± SEM, n = 3. Values not sharing the same letter indicate significant differences (P < 0.05).
Fig. 9.
Levels of T-cell-derived inflammatory cytokines. T cells were isolated from pooled spleens and mesenteric and inguinal lymph nodes, and then stimulated with 5 μg/ml of plate-bound anti-CD3 plus 20 μg/ml of soluble anti-CD28 for 24 h. Culture supernatants were collected, and cytokine levels were measured using a Bio-Plex 200 System (Bio-Rad). Only selected cytokines are shown; mean ± SEM, n = 3. Values not sharing the same letter indicate significant differences (P < 0.05).
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