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Faculty of Veterinary Medicine, Department of Infectious diseases and Immunology, Utrecht University, NetherlandsDepartment of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, South Africa
Faculty of Veterinary Medicine, Department of Clinical Sciences of Companion Animals, Utrecht University, NetherlandsFaculty of Veterinary Medicine, Department of Infectious diseases and Immunology, Utrecht University, Netherlands
Canine atopic dermatitis (AD) is a chronic inflammatory (skin) disease which shares
several characteristics with its human counterpart e.g. the genetic predisposition
to develop the disease, the early age of onset, the predilection sites of the affected
skin and similarities in immunopathogenic mechanisms [
] reviewed the immune dysregulation of canine AD and stated that, similar to human
AD, it is probably the resultant of a systemic component, the atopic constitution,
and a tissue-specific component, i.c. altered reactivity of the skin [
Epicutaneous sensitization with Dermatophagoides farinae induces generalized allergic
dermatitis and elevated mite-specific immunoglobulin E levels in a canine model of
atopic dermatitis.
]. With respect to altered skin reactivity both allergen-specific cellular mechanisms
and an impaired epidermal barrier in atopic subjects likely contribute to the onset
and perpetuation of AD in man [
]. In the beginning of the last century it was proposed that AD is related to abnormal
FA metabolism since linoleic acid (LA) deficiency in human and rodents leads to marked
abnormalities of the skin of AD patients [
]. It has been well established that in AD patients LA concentrations tend to be elevated
in blood and adipose tissue, however, several studies reported that the levels of
downstream metabolites of LA and also of α-linolenic acid (ALA) were found to be reduced
[
]. Both delta-5-desaturase (FADS1) and delta-6-desaturase (FADS2) are responsible for
the synthesis of highly unsaturated n-3 and n-6 FA from LA and ALA (Fig. 1.). Thus deficit amounts of LA and ALA metabolites in AD have been attributed to reduced
Δ-6- and Δ-5 desaturase activity [
]. Human, rat and guinea pig epidermis have been shown to lack enzymatic activity of
both desaturases which implies that several important members of epidermal fatty acids,
e.g. arachidonic acid (AA), are derived from extra-epidermal sites [
]. To date very few studies focused on the characteristics and metabolism of skin lipids
in dogs with respect to a possible epidermal lipid barrier defect in canine AD. We
hypothesize that an abnormal lipid metabolism contributes to the pathogenesis of canine
AD, potentially as a result of a defect in the epidermal lipid barrier. The aim of
the present study was to find evidence for this association in dogs by the analysis
of the mRNA expression of these enzymes and the PUFA composition in non-lesional skin
(NLS) and lesional skin (LS) of atopic dogs in comparison to healthy controls. Gene
expression levels of Δ-5 desaturase (FADS1) and Δ-6 desaturase (FADS2) were measured
by quantitative PCR in biopsies from non-lesional and lesional skin of canine AD patients
(n = 28) and from control skin of healthy dogs (n = 7). The mRNA expression level of FADS1 was significantly lower in lesional skin compared
to healthy control skin (5.5-fold) and non-lesional atopic skin (4-fold) (Fig. 2 (A) ). With respect to FADS2 mRNA expression a significant decrease (1.5-fold) was found
in lesional AD skin when compared to non-lesional AD skin (Fig. 2B). Both FADS1 and FADS2 show high correlation in expression in non-lesional (n = 0.57; p < 0.01) as well as lesional skin (n = 0.47; p < 0.05). We assume that the low mRNA levels found for FADS1 and FADS2 in lesional skin
coincide with a lower enzymatic activity as is shown in literature [
]. Our study implies that suppression already takes place at the mRNA level. It is
plausible that the decreased mRNA expression of Δ-5 desaturase in atopic skin results
from changed regulation by SREBP-1c and PPAR-α. These transcription factors play important
roles in the regulation of both Δ-5 and Δ-6 desaturases [
]. Based on the pathway of PUFA biosynthesis it was expected that a decrease in Δ-5-desaturase
activity would lead to reduced production of AA from DGLA and of EPA from eicosatetraenoic
acid (ETA) (Fig. 1). In contrast our FA analysis showed significant increased levels not only of AA,
but also of EPA in lesional skin when compared to non-lesional skin (Fig. 2C). Besides the transcription factors mentioned, (dietary) PUFA are major regulators
of the expression levels of both Δ-5 and Δ-6 desaturase indicating that these enzymes
are involved in feedback regulation with respect to the production of e.g. AA, EPA
and DHA [
]. This PUFA inhibition of desaturases is mediated by SREBP-1c whereby PUFA suppress
the target gene transcription (e.g. of desaturases) by reducing the active form of
SREBP-1c [
]. The decreased expression of Δ-5 desaturase mRNA observed in our study might be explained
by the high amounts of AA and EPA found in lesional skin. In this respect also the
decreased mRNA expression level for Δ-6 desaturase in lesional skin if compared to
non-lesional skin (NLS; p ≤ 0.001) could have been expected. However, this expression is not comparable to that
of for Δ-5 desaturase mRNA (p ≤ 0.0001). An explanation might be that the final enzymatic step in the synthesis of
AA and EPA is the direct result of Δ-5 desaturase activity and thus these products
might be more important in negative feedback regulation of Δ-5 than of Δ-6 desaturase.
GLA and stearidonic acid (SDA) are the direct metabolites of Δ-6 desaturase metabolism
and as FA analysis did not reveal a significantly higher amount of GLA in lesional
versus non-lesional skin this might explain why Δ-6 desaturase was not suppressed
by GLA to the same extent as Δ-5 desaturase by AA and EPA.
Fig. 1Pathway of biosynthesis and metabolism of polyunsaturated fatty acids. All mammals
can synthesize FA de novo from acetyl coenzyme A. The end product of this synthesis is palmitic acid (16: 0)
which can be elongated to stearic acid (18:0). Stearic acid can easily be converted
to oleic acid (18:1 n-9) by Δ-9 desaturase, universally present in cells of both plants and animals. Mammalian
cells lack Δ-12 desaturase and thus are not able to convert oleic acid to LA (LA;
18:2 n-6). They also lack Δ-15 desaturase, the enzyme responsible for the synthesis of α-linolenic
acid (ALA; 18:3 n-3) out of LA. To obtain these two essential FA (EFA) mammals are completely dependent
on their diet. Once consumed, these EFA can be metabolised to other FA. Thus, LA,
the parent precursor for the n-6 pathway, can be converted via γ-linolenic acid (GLA; 18:3 n-6) and dihomo-γ-linolenic acid (DGLA; 20:3 n-6) to arachidonic acid (AA; 20:4 n-6). For the n-3 pathway the same enzymes are used and the parent precursor of this pathway, α-linolenic
acid (ALA; 18:3 n-3) can be metabolised via stearidonic acid (SDA; 18:4 n-3) and eicosatetraenoic acid (20:4 n-3) to eicosapentaenoic acid (EPA; 20:5 n-3).
Fig. 2(A) Relative mRNA levels of Δ-5 desaturase (FADS1) in healthy skin, non-lesional atopic
skin (NLS) and lesional atopic skin (LS). The control biopsies were taken from healthy
dogs (n = 7) and the NLS and LS biopsies were taken from atopic dogs (n = 28). RNA was isolated and mRNA analysis was performed using Real-time qPCR. Data represent
mean ± SEM. A statistically significant difference in Δ-5 desaturase was noted between the
LS AD group versus the control group (ΔΔΔ p-value < 0.0001, Multivariate ANOVA) and between the LS AD group versus the NLS AD group (***p-value < 0.0001; repeated measures analysis of the GLM). (B) Relative mRNA levels of Δ-6 desaturase
(FADS2) in healthy skin, non-lesional atopic skin (NLS) and lesional atopic skin (LS).
A statistically significant difference in Δ-6 desaturase was noted between the LS
AD group versus NLS AD group (**p-value < 0.001, repeated measures analysis of the GLM). (C) Fatty acid composition (mean of
percentage ± SEM) of skin lipids in NLS and LS (*p-value ≤ 0.05 when NLS and LS are compared) as measured by gas chromatography of fatty acid
methyl esters. Fatty acid concentration is expressed as a percentage of total fatty
acids. Significant differences between NLS and LS were found for the amount of DGLA
(dihomo-gamma-linoleic acid), AA (arachidonic acid) and EPA (eicospentaenoic acid).
No significant differences were found for GLA (gamma linolenic acid) and ALA (α-linolenic
acid).
Epicutaneous sensitization with Dermatophagoides farinae induces generalized allergic dermatitis and elevated mite-specific immunoglobulin E levels in a canine model of atopic dermatitis.
