Retinol Esterification by DGAT1 Is Essential for Retinoid Homeostasis in Murine Skin

Abstract

Retinoic acid (RA) is a potent signaling molecule that is essential for many biological processes, and its levels are tightly regulated by mechanisms that are only partially understood. The synthesis of RA from its precursor retinol (vitamin A) is an important regulatory mechanism. Therefore, the esterification of retinol with fatty acyl moieties to generate retinyl esters, the main storage form of retinol, may also regulate RA levels. Here we show that the neutral lipid synthesis enzyme acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) functions as the major acyl-CoA:retinol acyltransferase (ARAT) in murine skin. When dietary retinol is abundant, DGAT1 deficiency results in elevated levels of RA in skin and cyclical hair loss; both are prevented by dietary retinol deprivation. Further, DGAT1-deficient skin exhibits enhanced sensitivity to topically administered retinol. Deletion of the enzyme specifically in the epidermis causes alopecia, indicating that the regulation of RA homeostasis by DGAT1 is autonomous in the epidermis. These findings show that DGAT1 functions as an ARAT in the skin, where it acts to maintain retinoid homeostasis and prevent retinoid toxicity. Our findings may have implications for human skin or hair disorders treated with agents that modulate RA signaling.

FIGURE 1.

Altered retinoid homeostasis in Dgat1^-/- mice. a, reduced in vitro ARAT activity in whole skin of Dgat1^-/- mice (age 7 weeks, n = 7/genotype). ^*, p < 0.001 versus wild type. Retinyl esters (RE), triacylglycerols (TG), and cholesterol esters (CE) are the respective products of the ARAT, DGAT, and acyl-CoA:cholesterol acyltransferase reactions. b, retinol (ROL) and all-trans-retinoic acid (atRA) concentrations are increased in whole skin in Dgat1^-/- mice fed the retinoid-sufficient (RS) diet but not in those fed the retinoid-deficient (RD) diet (age 7.5–14 weeks, n = 4–6/genotype). ^*, p < 0.05 versus wild type; ^**, p < 0.001; #, p < 0.05 versus retinoid-sufficient diet. c, serum retinol concentrations are similar in wild-type and Dgat1^-/- mice (age 7.5–14 weeks, n = 4–6/genotype). ^*, p < 0.001 versus retinoid-sufficient diet. d, hepatic retinyl ester and retinol concentrations are similar in wild-type and Dgat1^-/- mice (age 7.5–14 weeks, n = 4–6/genotype). ^*, p < 0.05; ^**, p < 0.01; #, p = 0.01; †, p < 0.001 versus retinoid-sufficient diet. e, RA target gene expression is increased in the whole skin of Dgat1^-/- mice fed a retinoid-abundant chow diet. mRNA levels were quantified by real time PCR (age 7 weeks, n = 5–6/genotype). ^*, p < 0.01; ^**, p < 0.05 versus wild type.

FIGURE 2.

Enhanced epidermal response to topical retinol treatment in Dgat1^-/- mice. a, enhanced epidermal hyperplasia in Dgat1^-/- skin after topical retinol treatment. Retinol (50, 100, or 200 nmol dissolved in 100 μl of ethanol or ethanol alone was applied once topically to dorsal skin. Skin was harvested 4 days after treatment, and sections were stained with hematoxylin-eosin (age 6 months, n = 3/genotype/dose). Scale bars, 50 μm. b, increased epidermal thickness in Dgat1^-/- skin treated with retinol. Mice were age 6 months, n = 3/genotype. ^*, p = 0.0143; ^**, p = 0.01; ^***, p < 0.001 versus vehicle-treated Dgat1^-/- skin. c, increased susceptibility of Dgat1^-/- skin to retinol-induced irritation. Scaling, cracking, and crusty lesions were prominent in Dgat1^-/- skin. Retinol (100 μl of 1 nmol/μl in ethanol) was applied topically once daily for 3 consecutive days to the dorsal cephalad skin. Ethanol was applied to the dorsal caudal skin (age 11 weeks, n = 5/genotype).

FIGURE 3.

Cyclical alopecia and excessive hair shedding in Dgat1^-/- mice. a, a Dgat1^-/- mouse photographed at 8, 9, and 11 weeks of age. Note the first appearance of alopecia (white box) in the dorsal caudal skin region at 9 weeks of age and complete regrowth of hair in the same region (white box) by 11 weeks of age. b, a Dgat1^-/- mouse photographed at 5 and 7 months of age. Note the different locations of alopecia (boxed areas) at two ages. c, adhesive tape test of dorsal hair loss in P28, P52, and P63 wild-type and Dgat1^-/- mice (n = 3/genotype/time point; each tape represents hair from a different mouse). d, quantification of hair removed by adhesive tape (n = 3/genotype/time point). ^*, p < 0.001 versus wild-type.

FIGURE 4.

Retinoid deprivation prevents alopecia in Dgat1^-/- mice. a, retinoid-depleted (retinoid-deficient diet) Dgat1^-/- mice (n = 6) did not develop alopecia by 13 weeks of age, whereas chow-fed Dgat1^-/- mice (n = 5) had prominent alopecia by 13 weeks of age. b, in retinoid-depleted Dgat1^-/- mice, resumption of the retinoid-sufficient diet at age 13 weeks induced alopecia within 8 days (n = 3/group). c, mRNA levels of CrbpI were lower, and CrabpII trended lower in whole skin in Dgat1^-/- mice fed the retinoid-deficient diet than in those fed the retinoid-sufficient diet (age 7.5–14 weeks, n = 2–6 mice/group). ^*, p < 0.001; ^**, p < 0.05 versus wild type; #, p < 0.05; †, p = 0.079 versus retinoid-sufficient diet. RD, retinoid-deficient; RS, retinoid-sufficient.

FIGURE 5.

Altered hair cycling in Dgat1^-/- mice. a, schematic of the first two postnatal hair cycles in Dgat1^-/- and wild-type mice (P44, n = 6/genotype; P63, n = 5/genotype; n = 3/genotype at all other time points). A, anagen (black); C, catagen (black gradient); T, telogen (white). b, precocious onset of second anagen in Dgat1^-/- mice. Dgat1^-/- and wild-type mice were shaved on P51 and photographed at various time points thereafter. Precocious anagen onset is evident in Dgat1^-/- mice by the appearance of new hair at P63 and complete regrowth of hair by P76 (n = 5/genotype). c, retinoid deprivation modulates the onset of the second anagen phase in Dgat1^-/- mice. Schematic time line depicting the onset of the second postnatal anagen phase in five retinoid-depleted Dgat1^-/- mice compared with the average age of onset of second anagen in chow-fed Dgat1^-/- and wild-type mice (32). T, telogen (white bar); A, anagen (black bar). RD, retinoid-deficient; RS, retinoid-sufficient.

FIGURE 6.

Effects of Dgat1 deficiency on retinoid homeostasis are epidermis-autonomous. a, specific deletion of Dgat1 in epidermis. Shown is PCR detection of the wild-type and floxed allele of Dgat1 and Cre transgene in genomic DNA. Interleukin-2 (IL-2) served as an internal PCR control. The absence of a PCR band indicates Cre-mediated recombination. b, absence of Dgat1 mRNA in the epidermis of K14-Cre⁺Dgat1^flox/flox (EP-D1KO) mice. mRNA levels of Dgat1 in the tail epidermis and WAT were quantified by real time PCR. (age 13 weeks, n = 3–6 mice/group). c, cyclical alopecia in EP-D1KO mice. Alopecia was detectable by 7.5 weeks of age. Note partial hair regrowth by 10.5 weeks of age. Alopecia was not observed in control mice. (n = 6/genotype). d, increased mRNA expression of RA target genes in whole skin of EP-D1KO mice. mRNA levels were quantified by real time PCR (age 13 weeks, n = 5/genotype). ^*, p < 0.0001; ^**, p = 0.0002 versus control.

FIGURE 7.

Model of how DGAT1 deficiency modulates RA signaling in skin. LRAT is the major pathway for maintaining adequate stores of retinol, as retinyl esters, in the skin. However, the ARAT activity of DGAT1 is important for generating retinyl esters when retinol levels are excessive and preventing retinol toxicity. In DGAT1 deficiency in murine skin, excess retinol is converted to retinaldehyde and subsequently to RAs, which modulate gene expression and hair cycling. If retinol is depleted from the diet, the effects of DGAT1 deficiency are minimized.