Glycerol Regulates Stratum Corneum Hydration in Sebaceous Gland Deficient (Asebia) Mice
2003; Elsevier BV; Volume: 120; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2003.12134.x
ISSN1523-1747
AutoresJoachim W. Fluhr, Mao‐Qiang Man, Barbara E. Brown, Philip W. Wertz, Debra Crumrine, John P. Sundberg, Kenneth R. Feingold, Peter M. Elias,
Tópico(s)Dermatology and Skin Diseases
ResumoThe only known function of human sebaceous glands is the provocation of acne. We assessed here whether sebum influences stratum corneum hydration or permeability barrier function in asebia J1 and 2 J mice, with profound sebaceous gland hypoplasia. Asebia J1 mice showed normal permeability barrier homeostasis and extracellular lamellar membrane structures, but they displayed epidermal hyperplasia, inflammation, and decreased (>50%) stratum corneum hydration, associated with a reduction in sebaceous gland lipids (wax diesters/monoesters, sterol esters). The triglyceride content of both asebia and control stratum corneum was low, consistent with high rates of triglyceride hydrolysis within the normal pilosebaceous apparatus, despite high rates of triglyceride synthesis. Although a mixture of synthetic, sebum-like lipids (sterol/wax esters, triglycerides) did not restore normal stratum corneum hydration to asebia skin, topical glycerol, the putative product of triglyceride hydrolysis in sebaceous glands, normalized stratum corneum hydration, and the glycerol content of asebia stratum corneum was 85% lower than in normal stratum corneum. In contrast, another potent endogenous humectant (urea) did not correct the abnormality. The importance of glycerol generation from triglyceride in sebaceous glands for stratum corneum hydration was demonstrated further by (i) the absence of sebaceous-gland-associated lipase activity in asebia mice, whereas abundant enzyme activity was present in the glands of control mice; and (ii) the inability of high concentrations of topical triglyceride to correct the hydration abnormality, despite the presence of abundant lipase activity in asebia stratum corneum. These results show that sebaceous-gland-derived glycerol is a major contributor to stratum corneum hydration. The only known function of human sebaceous glands is the provocation of acne. We assessed here whether sebum influences stratum corneum hydration or permeability barrier function in asebia J1 and 2 J mice, with profound sebaceous gland hypoplasia. Asebia J1 mice showed normal permeability barrier homeostasis and extracellular lamellar membrane structures, but they displayed epidermal hyperplasia, inflammation, and decreased (>50%) stratum corneum hydration, associated with a reduction in sebaceous gland lipids (wax diesters/monoesters, sterol esters). The triglyceride content of both asebia and control stratum corneum was low, consistent with high rates of triglyceride hydrolysis within the normal pilosebaceous apparatus, despite high rates of triglyceride synthesis. Although a mixture of synthetic, sebum-like lipids (sterol/wax esters, triglycerides) did not restore normal stratum corneum hydration to asebia skin, topical glycerol, the putative product of triglyceride hydrolysis in sebaceous glands, normalized stratum corneum hydration, and the glycerol content of asebia stratum corneum was 85% lower than in normal stratum corneum. In contrast, another potent endogenous humectant (urea) did not correct the abnormality. The importance of glycerol generation from triglyceride in sebaceous glands for stratum corneum hydration was demonstrated further by (i) the absence of sebaceous-gland-associated lipase activity in asebia mice, whereas abundant enzyme activity was present in the glands of control mice; and (ii) the inability of high concentrations of topical triglyceride to correct the hydration abnormality, despite the presence of abundant lipase activity in asebia stratum corneum. These results show that sebaceous-gland-derived glycerol is a major contributor to stratum corneum hydration. asebia J asebia 2 J aquaporin 3 free fatty acid peroxisomal proliferator activator α asebia stearyl CoA desaturase 1 (protein) stearyl CoA desaturase 1 (gene) transepidermal water loss triglyceride Cutaneous sebaceous glands deliver sebum to the skin surface through a process of continuous, holocrine secretion. Sebum comprises a complex lipid mixture that is enriched in wax monoesters and diesters, with substantial species-specific differences in triglycerides (TGs), sterol esters, free fatty acids (FFAs), and squalene (Thody and Shuster, 1989Thody A.J. Shuster S. Control and function of sebaceous glands.Physiol Rev. 1989; 69: 383-416Crossref PubMed Scopus (306) Google Scholar;Stewart and Downing, 1991Stewart M.E. Downing D.T. Chemistry and function of mammalian sebaceous lipids.Adv Lipid Res. 1991; 24: 263-301Crossref PubMed Google Scholar). Although a variety of functions have been proposed for cutaneous sebaceous-gland-derived lipids (Nicolaides, 1974Nicolaides N. Skin lipids: Their biochemical uniqueness.Science. 1974; 186: 19-26Crossref PubMed Scopus (457) Google Scholar;Thody and Shuster, 1989Thody A.J. Shuster S. Control and function of sebaceous glands.Physiol Rev. 1989; 69: 383-416Crossref PubMed Scopus (306) Google Scholar), the predominant view holds that the principal role of sebum in humans is negative, i.e., its well-accepted pathophysiologic role in the provocation of acne (Kligman, 1963Kligman A.M. The use of sebum.Advances in Biology of the Skin: the Sebacous Glands. 1963Crossref Google Scholar;Stewart and Downing, 1991Stewart M.E. Downing D.T. Chemistry and function of mammalian sebaceous lipids.Adv Lipid Res. 1991; 24: 263-301Crossref PubMed Google Scholar). Because sebaceous-gland-impoverished skin, such as that in prepubertal children, exhibits normal basal barrier function, it has been assumed that sebum does not influence epidermal permeability barrier function (Kligman, 1963Kligman A.M. The use of sebum.Advances in Biology of the Skin: the Sebacous Glands. 1963Crossref Google Scholar). But the barrier recovery kinetics of sebaceous-gland-poor versus-enriched skin after acute insults has not been examined, and this form of stress test is a more sensitive indicator of permeability barrier status than are basal assessments (Feingold and Elias, 1999Feingold K.R. Elias P.M. The environmental interface: Regulation of permeability barrier homeostasis.in: Loden M. Maibach H.I. Dry Skin and Moisturizers: Chemistry and Function. CRC Press, Boca Raton, FL1999: 45-58Google Scholar). Moreover, products of Meibomian glands (modified sebaceous glands) influence barrier function in conjunctival epithelia (Bron and Tiffany, 1998Bron A.J. Tiffany J.M. The Meibomian glands and tear film lipids. Structure, function, and control.Adv Exp Med Biol. 1998; 438: 281-295Crossref PubMed Scopus (109) Google Scholar). Furthermore, after sebum is deposited on the surface of the SC, it inspissates into the interstices (Kligman and Shelley, 1958Kligman A.M. Shelley W.B. An investigation of the biology of the human sebaceous gland.J Invest Dermatol. 1958; 30: 99-125Abstract Full Text PDF PubMed Scopus (99) Google Scholar) and interacts with the extra-cellular lamellar bilayer system (Sheu et al., 1999Sheu H.M. Chao S.C. Wong T.W. Yu-Yun Lee J. Tsai J.C. Human skin surface lipid film: An ultrastructural study and interaction with corneocytes and intercellular lipid lamellae of the stratum corneum.Br J Dermatol. 1999; 140: 385-391Crossref PubMed Scopus (77) Google Scholar). There, its constituent fatty acids, which lack essential fatty acids, could dilute or replace the linoleic acid in SC acylceramides, as proposed for follicular epithelia (Stewart et al., 1986Stewart M.E. Grahek M.O. Cambier L.S. Wertz P.W. Downing D.T. Dilutional effect of increased sebaceous gland activity on the proportion of linoleic acid in sebaceous wax esters and in epidermal acylceramides.J Invest Dermatol. 1986; 87: 733-736Abstract Full Text PDF PubMed Google Scholar). As a net result of one or more of these processes, sebum could influence permeability barrier homeostasis. To assess two potential functions of cutaneous sebaceous glands, we compared permeability barrier homeostasis and SC hydration in two closely related models where sebaceous glands are largely absent, the asebia mouse. Asebia mice display not only profound sebaceous gland hypoplasia, but also other cutaneous abnormalities, including mild scaling, patchy scarring alopecia, and epidermal hyperplasia (Gates and Karasek, 1965Gates A.H. Karasek M. Hereditary absence of sebaceous glands in the mouse.Science. 1965; 148: 1471-1473Crossref PubMed Scopus (78) Google Scholar;Josefowicz and Hardy, 1978Josefowicz W.J. Hardy M.H. The expression of the gene asebia in the laboratory mouse. I. Epidermis and dermis.Genet Res. 1978; 31: 53-65Crossref PubMed Scopus (22) Google Scholar;Sundberg and King, 1996Sundberg J.P. King Jr, L.E. Mouse models for the study of human hair loss.Dermatol Clin. 1996; 14: 619-632Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Although recent studies have shown that mutations in the gene that encodes stearyl CoA desaturase 1 (SCD1) are responsible for both the asebia J1 and asebia 2 J phenotypes (Zheng et al., 1999Zheng Y. Eilertsen K.J. Ge L. et al.Scd1 is expressed in sebaceous glands and is disrupted in the asebia mouse.Nat Genet. 1999; 23: 268-270Crossref PubMed Scopus (197) Google Scholar;Sundberg et al., 2000Sundberg J.P. Boggess D. Sundberg B.A. Eilertsen K. Parimoo S. Filippi M. Stenn K. Asebia-2J (Scd1 (ab2J)): A new allele and a model for scarring alopecia.Am J Pathol. 2000; 156: 2067-2075Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), the basis for sebaceous gland hypoplasia in these models is not known. We report here that, despite the presence of epidermal hyperplasia, asebia J1 mice display normal permeability barrier homeostasis but a profound abnormality in SC hydration. The hydration abnormality in asebia skin could be attributed further to decreased glycerol generation due to a lack of endogenous, sebaceous-gland-derived TG and associated lipase activity. The hydration abnormality in asebia mice could explain the increased propensity of sebum-depleted skin sites of humans to develop eczematous skin disorders, particularly when subjected to xeric stress. Male asebia J1 mice (ABJ/Le-ScdabJ/ScdabJ) (The Jackson Laboratory, Bar Harbor, ME) and their normal heterozygote (+/abJ) and wild-type (+/+ or +/?) littermates, between 6 and 10 wk of age, were utilized for most studies. Asebia 2 J (ab2J/ab2J, (The Jackson Laboratory) mice were utilized in some experiments, where indicated. All mice were maintained in the Animal Care Facility of the Veterans Affairs Medical Center, San Francisco, in a temperature- and humidity-controlled room, and fed standard laboratory chow (Teklad 22/5 Rodent Diet) and acidified tap water ad libitum. All functional studies were performed in the Animal Care Facility, and mice were anesthetized with chloral hydrate and/or isoflorane. SC hydration was quantitated as changes in electrical capacitance in arbitrary units (Corneometer CM 820, Courage & Khazaka, Cologne, Germany) in asebia J1 and 2 J and wild-type mice, over skin sites (mean of three measurements in each animal) that had been shaved 24 h previously. Corneometry (capacitance measurements) provides a reliable and reproducible measure of SC hydration (e.g.,Fluhr et al., 1999Fluhr J.W. Gloor M. Lehmann L. Lazzerini S. Distante F. Berardesca E. Glycerol accelerates recovery of barrier function in vivo.Acta Derm Venereol. 1999; 79: 418-421Crossref PubMed Scopus (157) Google Scholar;Gloor et al., 2002Gloor M. Fluhr J. Lehmann L. Gehring W. Thieroff-Ekerdt R. Do urea/ammonium lactate combinations achieve better skin protection and hydration than either component alone?.Skin Parmacol Appl Skin Physiol. 2002; 15: 35-43Crossref PubMed Scopus (16) Google Scholar). The slight flakiness of untreated asebia SC did not alter the reproducibility of measurements taken from or adjacent to previously shaved sites. In some animals, immediately after capacitance measurements, basal transepidermal water loss (TEWL) was measured with an electrolytic water analyzer (MEECO, Warrington, PA). The barrier was then abrogated over three separate sites in each animal by sequential cellophane (Scotch-type®, 3M, Minneapolis, MN) tape strippings until TEWL levels exceeded 2 mg per cm2 per h. Permeability barrier recovery rates were compared in asebia J1 (n=17) versus wild-type (+/?) (n=10) mice 3 and 6 h after acute disruption. To assess the potential role of sebum and its products in SC hydration, we performed the following replenishment studies: (i) a synthetic sebaceous lipid mixture, containing a TG (tripalmitate, 250 μg), a wax monoester (stearyl stearate, 125 μg), and a cholesterol ester (cholesterol palmitate, 25 μg), or (ii) tripalmitate alone (1%, 5%, and 10%) in propylene glycol:ethanol (7:3 vol) versus vehicle alone was applied to 2.5 or 4 cm2 areas on contralateral flanks of asebia J1 or 2 J and wild-type control mice (n=5 or 6 animals each) twice daily for 3 d; (iii) 0.05%, 0.1%, 1.0%, 2.5%, or 10% aqueous glycerol or water alone was applied twice daily to 4 cm2 areas on the flanks of asebia J1 or 2 J mice (n=4 animals in each glycerol group) for 3 d. Finally, SC hydration was compared after 10% glycerol versus 10% urea applications to contralateral flanks of asebia 2 J animals (n=4) each twice daily for 4 d, as above. In all studies, SC hydration was measured daily by corneometry immediately before the next topical application (≈12 h after the last prior topical application). At the end of all studies, tape strippings or biopsies were obtained for light microscopy, lipid analysis, and/or lipase cytochemistry. Biopsies were obtained from asebia J1 versus control mice under basal conditions from skin sites that had been shaved 24 h earlier. Skin pieces were minced into pieces of 0.5 mm3, and processed for light and electron microscopy. Electron microscopy samples were prefixed in half-strength Karnovsky's fixative, postfixed in either osmium tetroxide (OsO4) or ruthenium tetroxide (RuO4), and embedded in an epoxy resin (Hou et al., 1991Hou S.Y. Mitra A.K. White S.H. Menon G.K. Ghadially R. Elias P.M. Membrane structures in normal and essential fatty acid-deficient stratum corneum: Characterization by ruthenium tetroxide staining and X-ray diffraction.J Invest Dermatol. 1991; 96: 215-223Abstract Full Text PDF PubMed Google Scholar). To assess potential sites where TG to glycerol hydrolysis could occur, we performed ultrastructural cytochemistry for acid lipase, as described previously (Rassner et al., 1997Rassner U.A. Crumrine D.A. Nau P. Elias P.M. Microwave incubation improves lipolytic enzyme preservation for ultrastructural cytochemistry.Histochem J. 1997; 29: 387-392Crossref PubMed Scopus (32) Google Scholar). Ultrathin sections were viewed in a Zeiss 10 A electron microscope, operated at 60 kV, after further contrasting with lead citrate and uranyl acetate. One-half micron sections of epoxy-embedded samples were stained with 1% aqueous toluidine blue for light microscopy and to visualize mast cell metachromasia. Full-thickness mouse skin was excised from shaved asebia J1 and control animals, and incubated with 10 mM ethylenediamine tetraacetic acid (EDTA), pH 7.4, at 37°C for 30 min to separate epidermis from dermis (Grubauer et al., 1989Grubauer G. Feingold K.R. Harris R.M. Elias P.M. Lipid content and lipid type as determinants of the epidermal permeability barrier.J Lipid Res. 1989; 30: 89-96Abstract Full Text PDF PubMed Google Scholar). Nucleated epidermal cells were separated from SC by further incubation with 0.5% trypsin in phosphate-buffered saline for 2 h, followed by vortexing. SC sheets were freeze-dried, weighed, and total lipids were extracted (Bligh and Dyer, 1959Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can J Biochem Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar), dried, weighed, and stored at –70°C until analyzed. Neutral lipids were separated from polar lipids by high performance thin layer chromatography, using a CAMAG linomat IV Autospotter (CAMAG Scientific, Washington, NC), extracted, and solubilized in chloroform:methanol (2:1 vol). Nonpolar lipids were then fractionated and quantitated (Law et al., 1995Law S. Wertz P.W. Swartzendruber D.C. Squier C.A. Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy.Arch Oral Biol. 1995; 40: 1085-1091Abstract Full Text PDF PubMed Scopus (148) Google Scholar). Briefly, 10×10 cm glass-backed TLC plates, coated with 0.25 mm thick silica gel G (Adsorbosil-plus-1; Alltech Associates, Deerfield, IL), were washed with chloroform:methanol (2:1) and activated in a 110°C oven; the adsorbent was scored into 6 mm wide lanes. Calibrated glass capillary tubes were used to apply 5 μl samples and chromatograms were developed in n-hexane:benzene (1:1 vol) to 95 cm. Finally, chromatograms were developed to 5 cm with hexane:ethyl ether:acetic acid (70:30:1), charred, and quantitated by photodensitometry. Lipid standards were obtained from Sigma (St. Louis, MO). Asebia J1 and 2 J mice as well as wild-type littermates were carefully shaved 24 h before collecting total SC by pooling sequential tape strips down to the glistening layer in all groups (D-Squame disks, CuDerm Corporation, Dallas, TX). Because of differences in SC thickness and cohesion (=protein per stripping) between the two groups, data reflect total glycerol/total SC protein from equivalent surface areas. The disks from each site were pooled (four to six disks each) and soaked in 500 μl 1% Triton-X100 (Sigma) in water, sonicated 5 min, and then stored overnight at 4°C. Blank D-Squame disks were treated similarly as a background control in all assays. Samples were vortexed thoroughly and 50 μl aliquots of each solution were taken to assay glycerol content, using a previously described radiometric method (Newsholme, 1974Newsholme E.A. Glycerol.in: Bergmeyer H.U. Methods of Enzymatic Analysis. 2nd edn. Academic Press, San Francisco, CA1974: 1409-1414Google Scholar). Glycerol standard curves ranging from 0.0 to 8.8 nmol per ml were generated. Each assay volume contained 0.05 μCi of glycerol [14C(U)], 160 mCi per mmol (American Radiolabeled Chemicals, St. Louis, MO), 0.002 U of glycerol kinase from Bacillisstearothermophilus (85 units per mg at 25°C with adenosine-5′-triphosphate and glycerol as substrate), and 0.3 mg adenosine-5′-triphosphate (Roche Molecular Biochemicals, Indianapolis, IN). A final concentration of 0.5% Triton X-100 was maintained in standard curve as well as samples. Incubation times were 4 min at 27°C, and protein assays were performed on the remaining disks and soak solutions, as described previously (Fluhr et al., 2001Fluhr J.W. Kao J. Jain M. Ahn S.K. Feingold K.R. Elias P.M. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.J Invest Dermatol. 2001; 117: 44-51Crossref PubMed Google Scholar). Statistical analyses were performed with Prism 3 software (GraphPad, San Diego, CA), using paired and unpaired Student's t tests. Where a non-Gaussian distribution occurred, a nonparametric Wilcoxon test was used. As described previously (Gates and Karasek, 1965Gates A.H. Karasek M. Hereditary absence of sebaceous glands in the mouse.Science. 1965; 148: 1471-1473Crossref PubMed Scopus (78) Google Scholar;Josefowicz and Hardy, 1978Josefowicz W.J. Hardy M.H. The expression of the gene asebia in the laboratory mouse. I. Epidermis and dermis.Genet Res. 1978; 31: 53-65Crossref PubMed Scopus (22) Google Scholar), asebia J1 mice displayed marked sebaceous gland hypoplasia, hyperkeratosis, epidermal hyperplasia with a normal-appearing granular layer by light microscopy, and increased numbers of mast cells in the dermis (Figure 1a vs b). Basal TEWL levels were comparable in mutant versus control animals (data not shown). Following acute barrier abrogation by cellophane tape stripping, the kinetics of barrier recovery were similar in asebia J1 versus control mice at 3 and 6 h (Figure 3a). Extracellular lamellar membrane structures, a further parameter of permeability barrier status, also appeared completely normal in RuO4 postfixed asebia J1 SC (Figure 2). Likewise, there were no differences in the lamellar body secretory system in asebia J1 versus control mice (not shown). In contrast, asebia J1 mice displayed ≈50% reduction in SC hydration assessed by electrical capacitance (p<0.0001) (Figure 3b). Together, these studies demonstrate normal epidermal barrier function and lamellar membranes in asebia J mice, despite the presence of sebaceous gland hypoplasia, evidence of epidermal hyperplasia, and mast cell proliferation and profound abnormalities in SC hydration.Figure 3Barrier recovery is normal, but asebia J1 mice display abnormal SC hydration.Panel A: Permeability barrier was disrupted by sequential tape stripping in asebia J1 (n=6) versus wild-type (n=4) mice (three to four sites were assessed on each animal, yielding a total n of 10–12). TEWL rates were raised to ≥4 mg per cm2 per h, and then percent barrier recovery was compared 3 and 6 h after acute disruption. The differences were not statistically significant at either time point. Panel B: Hydration of SC was measured by electrical capacitance with a corneometer, and expressed in arbitrary units ±SEM. The differences between asebia J1 (n=12) and control (n=8) mice are highly significant (p<0.0001).View Large Image Figure ViewerDownload (PPT)Figure 2Asebia J1 SC reveals normal extracellular membrane structures. Comparable SC extracellular lamellar membranes and desmosomes are seen in the SC of asebia J1 (A) and control (N) mice (arrows). RuO4-postfixed. Bar: 0.25 μm.View Large Image Figure ViewerDownload (PPT) We next assessed the lipid biochemical basis for the abnormal SC hydration in asebia J mice. Whereas the total lipid content was not altered in asebia J versus control SC, the content of the major nonpolar lipid species, namely, wax diesters, wax esters, sterol esters, was decreased significantly (Figure 4a, b), consistent with the absence of sebaceous glands (Nikkari, 1974Nikkari T. Comparative chemistry of sebum.J Invest Dermatol. 1974; 62: 257-267Crossref PubMed Scopus (110) Google Scholar). FFA levels also appeared to be reduced in asebia SC, but the difference did not achieve statistical significance (FFA would continue to be generated in asebia epidermis from catabolism of epidermal phospholipids). Yet, substantial sterol/wax esters and some TGs were present not only in control but also in the SC of asebia J1 mice (Figure 4b). Finally, there was also a slight but statistically significant increase in free sterols (Figure 4b), as noted in previous studies (Wilkinson and Karasek, 1966Wilkinson D.I. Karasek M.A. Skin lipids of a normal and mutant (asebic) mouse strain.J Invest Dermatol. 1966; 47: 449-455Abstract Full Text PDF PubMed Scopus (49) Google Scholar;Arndt, 1996Arndt K.A. Cutaneous Medicine and Surgery: An Integrated Program In Dermatology. W.B. Saunders, Philadelphia, PA1996Google Scholar;Sundberg et al., 2000Sundberg J.P. Boggess D. Sundberg B.A. Eilertsen K. Parimoo S. Filippi M. Stenn K. Asebia-2J (Scd1 (ab2J)): A new allele and a model for scarring alopecia.Am J Pathol. 2000; 156: 2067-2075Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). These studies demonstrate a significant depletion, but not an elimination, of nonpolar lipids of presumed sebaceous gland origin in the SC of asebia J1 mice. The abnormality in hydration in asebia J1 SC suggests that sebaceous-gland-derived products could influence this process. Hence, we next applied a mixture of synthetic, sebum-like lipids, i.e., a wax monoester (stearyl stearate), a sterol ester (cholesterol palmitate), and a TG (tripalmitate), to the skin of asebia J1 and age/sex-matched wild-type control mice. Despite repeated applications, this mixture did not correct the hydration abnormality in asebia J1 mice (Table I).Table IEffects of a sebaceous lipid-like mixture on SC hydration (arbitrary units±SEM)AnimalPretreatment1 d3 dControl (n=6)49.8±2.352.6±4.757.8±4.5Asebia J1 (n=6)29.8±2.824.5±1.626.5±1.6To assess the potential role of sebaceous gland lipids in SC hydration, a lipid mixture (40 μl) containing TG (250 μg), stearyl stearate (125 μg), and cholesterol palmitate (25 μg) in a 40 μl propylene glycol:ethanol (7:3 vol) vehicle versus vehicle alone was applied twice daily to 4 cm2 areas on opposite flanks of asebia J and control mice (n=6 each) for 3 d. During these studies, SC hydration was measured daily by corneometry immediately before and 12 h after the last prior application. There were no significant changes in hydration with lipid applications to either asebia or control mice. The differences between asebia and control mice remained significant at all time points (p<0.001). Open table in a new tab To assess the potential role of sebaceous gland lipids in SC hydration, a lipid mixture (40 μl) containing TG (250 μg), stearyl stearate (125 μg), and cholesterol palmitate (25 μg) in a 40 μl propylene glycol:ethanol (7:3 vol) vehicle versus vehicle alone was applied twice daily to 4 cm2 areas on opposite flanks of asebia J and control mice (n=6 each) for 3 d. During these studies, SC hydration was measured daily by corneometry immediately before and 12 h after the last prior application. There were no significant changes in hydration with lipid applications to either asebia or control mice. The differences between asebia and control mice remained significant at all time points (p<0.001). The inability of topical sebaceous lipids to correct the hydration abnormality suggested that glycerol, a well-known humectant, generated as a result of the high rates of TG synthesis and hydrolysis that occur within the pilosebaceous follicle (Nicolaides and Wells, 1957Nicolaides N. Wells G.C. On the biogenesis of the free fatty acids in human skin surface lipids.J Invest Dermatol. 1957; 29: 423-433Crossref PubMed Scopus (59) Google Scholar;Thody and Shuster, 1989Thody A.J. Shuster S. Control and function of sebaceous glands.Physiol Rev. 1989; 69: 383-416Crossref PubMed Scopus (306) Google Scholar;Stewart and Downing, 1991Stewart M.E. Downing D.T. Chemistry and function of mammalian sebaceous lipids.Adv Lipid Res. 1991; 24: 263-301Crossref PubMed Google Scholar), could be the responsible hydrating component. Indeed, endogenous glycerol levels were profoundly reduced in the SC of asebia J1 mice (control, 0.8233±0.1991 nmol glycerol per μg SC protein, versus asebia, 0.1284±0.053 nmol glycerol per μg SC protein; p=0.0141). Based upon the demonstrated deficiency of this humectant in asebia SC, we next assessed whether glycerol applications alone could correct the asebia hydration abnormality. Four days of twice daily 10% glycerol applications completely reversed the hydration abnorm-alities in asebia J1 SC, and significantly, but incompletely, reversed the abnormality in asebia 2 J animals (Table II). Lower concentrations (i.e., from 0.1% to 5% glycerol) significantly increased SC hydration in asebia 2 J animals, but only concentrations above 1% exceeded the impact of the aqueous vehicle alone (Table II). In contrast, glycerol concentrations above 10% did not increase SC hydration further (not shown). The water vehicle alone slightly, but significantly, improved hydration in both asebia J1 and 2 J mice (Table II). In contrast, topical glycerol slightly but significantly reduced, rather than increased, SC hydration in normal wild-type mice (by ≈10%; p<0.01).Table IIEffects of glycerol on SC hydration in asebia J1 and 2 J versus control mice (arbitrary units±SEM)GroupsPretreatmentPost-treatmentSignificance versus baselineExperiment 1 (asebia J1 mice)Control+vehicle (water) (n=8)46.0±0.756.8±1.9p<0.0001Control+10% glycerol–50.9±1.5p<0.01Asebia+vehicle (water)24.1±1.431.4±2.3p<0.01Asebia+10% glycerol (n=8)–52.0±1.3p<0.0001Experiment 2 (asebia 2 J mice)Control+vehicle (water) (n=16)48.7±1.146.4±2.2NSControl+1% glycerol–45.6±1.5NSControl+5% glycerol–47.1±1.6NSControl+10% glycerol–48.4±1.8NSAsebia+vehicle (water) (n=16)15.2±1.119.3±1.3p<0.05Asebia+1% glycerol–21.3±2.5p<0.05Asebia+5% glycerol–25.8±1.9p<0.001Asebia+10% glycerol–31.1±1.6p<0.000110% aqueous glycerol or water alone was applied twice daily to 4 cm2 areas on the flanks of mice for 4 d, followed by measurement of SC hydration (see Methods) (n=16 sites each on four mice or as shown). Experiments 1 and 2 were performed in asebia J1 and 2 J mice respectively versus wild-type littermates. Open table in a new tab 10% aqueous glycerol or water alone was applied twice daily to 4 cm2 areas on the flanks of mice for 4 d, followed by measurement of SC hydration (see Methods) (n=16 sites each on four mice or as shown). Experiments 1 and 2 were performed in asebia J1 and 2 J mice respectively versus wild-type littermates. Finally, to ascertain whether the requirement for glycerol is specific or replaceable by another potent polar, endogenous, but chemically unrelated humectant, we compared SC hydration after applications of 10% glycerol versus 10% urea to contralateral flanks on asebia 2 J mice. As seen in Figure 5, only glycerol improved SC hydration in the asebia mice. These results show that the abnormal hydration in asebia J skin is largely reversed by topical glycerol, but not by sebaceous lipids or another potent endogenous humectant, urea. We next determined whether reduced generation of glycerol from TG within the pilosebaceous follicle accounts for the hydration abnormality. To evaluate potential sites of glycerol generation in the skin, we assessed lipase activity in the
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