The role of dietary arachidonic acid and docosahexaenoic acid in preventing the phenotype observed with highly unsaturated fatty acid deficiency

Abstract

The physiological roles of highly unsaturated fatty acids (HUFA), mainly arachidonic acid (AA, 20:4ω6) and docosahexaenoic acid (DHA, 22:6ω3), are not completely understood. In order to study specific functions for AA and DHA, a delta-6 desaturase knockout (D6D-/-) mouse was created. D6D is a key enzyme in synthesizing HUFA from the precursor dietary essential fatty acids, linoleic acid (LA, 18:2ω6) or α-linolenic acid (ALA, 18:3ω3). By disrupting D6D expression, LA and ALA provided in the diet will not be metabolized to HUFA. Phenotype of the D6D-/- mouse is therefore specific to lack of AA and/or DHA and consists of ulcerative dermatitis, male infertility, gastrointestinal ulcers, and hepatic lipidosis. New insight on specific AA and DHA roles was established through dietary prevention of HUFA deficiency phenotype. The absence of a D6D isozyme had to be assessed before further characterizing HUFA roles with the D6D-/- mouse model. The presence of a D6D isozyme would interfere with the creation of HUFA deficiency. The primary D6D isozyme candidate was Fads3 gene due to its increased gene expression in D6D-/- liver and homology to the Fads2 gene that encodes for D6D. Cloning and transfection of Fads3 into cultured HEK293 cells confirmed lack of D6D activity (Chapter 3). The order of appearance of D6D-/- phenotype due to HUFA deficiency had yet to be determined. A D6D-/- time course study (Chapter 4) characterized the mouse at different ages in order to follow sequence of HUFA deficiency pathology. The amount of HUFA in D6D-/- at weaning was comparable to control mouse indicating the presence of HUFA stores that most likely result from HUFA passed on from the mother. Subsequent HUFA depletion with age correlated with severity of D6D-/- phenotype. Male infertility, gastrointestinal erosions, and hepatic lipidosis are the first observed HUFA deficiency phenotype to appear at 6 weeks of age, followed by impaired antibody response at 9 weeks, and ulcerative dermatitis by 21 weeks of age. HUFA supplementation studies helped determine specific roles for AA and DHA in preventing HUFA deficiency phenotype. Hepatic lipidosis was prevented by either AA or DHA (Chapter 5). AA essentiality was specific to skin and gastrointestinal function since DHA supplementation was unsuccessful in preventing ulcerative dermatitis or gastrointestinal ulcers (Chapter 6). DHA essentiality was specific to male reproduction as indicated by full restoration of spermatogenesis, sperm counts, and sperm motility (Chapter 7). The role of DHA in spermatogenesis is related to acrosome biogenesis, a process which relies on vesicle fusion (Chapter 8). The immune system (Chapter 9) was further characterized following up on splenomegaly and thymic atrophy observations of the first characterization of the D6D-/-. HUFA deficiency results in decreased antibody response indicating essentiality for HUFA in immune function. In summary, these studies showed for the first time a specific requirement for AA in skin, and of DHA in male reproduction. The mechanism behind DHA requirement in male fertility has been linked to acrosome biogenesis. Future research done with the D6D-/- mouse model will help develop hypothesis on other potential mechanisms behind the essentiality of AA and DHA. Understanding how HUFA maintain tissue homeostasis will help in the development of treatments for diseases that result from an altered essential fatty acid metabolism