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The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP−/− mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP−/− mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP+/+ and ACBP−/− mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP−/− mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP−/− mice display a ~50% increased transepidermal water loss compared with ACBP+/+ mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function.
The acyl-CoA binding protein (ACBP)/diazepam binding inhibitor (DBI) (Entrez Gene ID: 13167) is a 10 kDa intracellular protein that specifically binds medium and long chain acyl-CoA esters (C14–C22) with very high affinity (Kd~1–10 nM) (1, 2). The protein is expressed in all eukaryotic species and in all mammalian tissues investigated (3–5); however, expression levels differs markedly between different tissues and cell types examined, with particularly high levels in epithelial cell types and in cells with a high turn-over of fatty acids (FA) (6). Consistently with this, we have shown that the ACBP gene is activated by lipogenic transcription factors, such as the peroxisome proliferator-activated receptor γ (7) and members of the sterol-regulatory element binding protein family (7–10). In vitro studies indicate that ACBP plays a role in transport of acyl-CoA esters and may deliver acyl-CoA esters to phospholipid (11–13), glycerolipid (14), and cholesteryl ester synthesis (15). In addition, by sequestration of acyl-CoA esters, a number of acyl-CoA feedback-inhibited proteins, including FA synthetase (3), acetyl CoA carboxylase, and long chain acyl-CoA synthetase (16), are relieved of acyl-CoA inhibition in vitro.
Studies with targeted disruption of the yeast ACBP (Acb1p) gene have shown that ACBP is important for synthesis of very long chain fatty acids (VLCFA) and sphingolipids (17). Furthermore, Acb1p-depleted cells show accumulation of autophagocytotic vesicles and multilobed vesicles and display a strongly perturbed plasma membrane structure, indicating that lack of ACBP severely affects vesicular trafficking in yeast (17–19).
In mammalian cells, knockdown of ACBP has been reported to lead to a diminished potential of preadipocytes to undergo differentiation (20) and of hepatoma cells to express key enzymes in cholesterol and FA catabolic metabolism (21). Furthermore, reports have indicated that ACBP affects the transcriptional activity of nuclear hormone receptors by both interacting directly with the receptors (22) and by binding ligands (23).
Recently, we reported specific targeting of the ACBP gene in C57BL/6JBomTac mice (24). ACBP−/− mice are viable and fertile and are born in a normal Mendelian ratio. However, the ACBP−/− mice suffer from a poor adaptation to weaning and go through a crisis with decreased growth rate around that time. We have shown that the mice suffer from a delayed adaptation of the liver to weaning due to a delayed induction of lipogenic pathways. In contrast to our data, Kier and colleagues reported that targeted disruption of ACBP leads to embryonic preimplantation lethality (25). The reason for this is unclear, but it may be caused by differences in targeting strategies. Kier and coworkers deleted a large part of the Acbp proximal promoter region, which might have interfered with the regulation of other genes in the region, whereas our targeting approach involved only deletion of parts of introns 1 and 2 and exon 2, as described in Ref. 24.
During our studies, we also observed a pronounced skin and fur phenotype that is macroscopically similar to the phenotype of mice carrying a ~400 kb pleiotropic deletion on chromosome 1, a region containing six genes, including the ACBP gene (26, 27). The deletion was reported to increase lethality on the B6 background but not on a mixed 129/B6 background (28). The mutant mice on a mixed 129/B6 genetic background show sebocyte hyperplasia and sparse, matted, reddish hair with a greasy appearance. Examination of the fur lipid content revealed a decreased content of triacylglycerols and a concomitant increase of an unidentified lipid species. Furthermore, hyperplasia of sebocytes in nose skin and skin from lower thorax was reported. Interestingly, ectopic expression of ACBP could at least partially rescue the skin phenotype of these mice, indicating that it is the deletion of the ACBP locus that causes the macroscopic changes in the skin and fur phenotype. Our observations in ACBP−/− mice confirm this notion. To better understand how ACBP depletion affects skin and fur properties, we set out to investigate the biochemical changes responsible for the macroscopic phenotype.
Here we report for the first time that specific targeting of the ACBP gene in mice leads to a significant decrease in the very long chain free fatty acids (VLC-FFA) content and changes in the biophysical properties of the stratum corneum. Importantly, this situation leads to a significantly compromised epidermal barrier function. Our results indicate that ACBP is involved in processes that lead to production of VLC-FFAs via complex phospholipids in the lamellar body lipids.
Standard laboratory chemicals as well as 6-lauroyl-2-(N,N-dimethylamino)naphthalene (LAURDAN), fluorescein, and 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) were from Invitrogen (Denmark); DMSO was from Fluka. Primary rabbit-anti rat ACBP antiserum was kindly provided by Dr. Karsten Kristiansen, Copenhagen University. Secondary antibody for immunostaining of paraffin-embedded tissue sections was EnVision+ System-HRP labeled anti rabbit polymer (Dako, Copenhagen, Denmark). Veet® hair removal cream was from Reckitt Benckiser, and IsoFlo®Vet was from Orion Pharma Animal Health, Denmark. Lipid standards: N-18:0 Phytosphingosine/N-stearoyl 4-hydroxysphinganine was from Avanti Polar Lipids (860610P). Docosanoic-7,7,8,8-d4 acid, tetracosanoic-9,9,10,10-d4 acid, and hexacosanoic-12,12,13,13-d4 Acid were from CDN Isotopes (D-5709, D-6167, and D-6145, respectively). Nu-Chek 18-4A was from Nu-Chek Prep, Inc. Triheptadecanoin (3xC17:0 triacylglyceride, 33-1700-9) and heptadecanoic acid (C17:0, 10-700-13) were from Larodan. Cholesterol (C8667-5G) and 1-O-palmityl-2,3-dipalmitoyl-rac-glycerol (H3260) were from Sigma-Aldrich.
Mice with targeted disruption of the ACBP gene were generated as described (24, 29). Mice carrying the targeted ACBP gene were identified using primer pairs i) 5′-agg atc tcc tgt cat ctc acc ttg ctc ctg and 5′-aag aac tcg tca aga agg cga tag aag gcg (~500 bp fragment within the neoR cassette) and ii) 5′-agg atc tcc tgt cat ctc acc ttg ctc ctg and 5′-gta tct gct cat cta ttc ggc ttg g (~1,200 bp fragment spanning the 3′ region of the neoR cassette to the 5′ region of intron 2). The ACBP+/+ allele was identified using primers 5′-ggg tcc ggg aag ggt tgg agc and 5′-ggc gct tca cct cct cag cgg (spanning ~1,140 bp within the region 5′ of the initiator codon to the 5′ region within exon 2). Mice used for experiments within this work were 3- to 4-month-old mice from backcross generations 12–14 on the C57BL/6JBomTac genetic background. Mice were housed at the Biomedical Laboratory, University of Southern Denmark, under standard laboratory conditions including 12 h light/dark cycle, free access to feed (altromin 1324 for maintenance, altromin 1314 for breeding and lactation) and water in a room with ~55% relative humidity at 22°C ± 3°C. Breeding of transgenic mice and animal experiments were approved by the Danish Animal Experiment Inspectorate, and all animal experiments were conducted in conformity with the PHS policy on humane care and use of laboratory animals (30).
Paraffin sections were mounted on SuperFrostPlus® microscopic slides, dried over night at room temperature, and stored at 4°C. ACBP immunohistochemical staining with rabbit-anti-ACBP antiserum was performed as described (31) using heat-induced epitope retrieval in Tris-EGTA buffer.
Mice were euthanized, and 3 µl 0.1 mM LAURDAN in DMSO were placed on the surface of the inside of the excised ear. Tissue was left incubating for ~30 min at room temperature in a slightly humidified chamber. Cover slips were wetted with water vapor to enhance contact with the tissue. LAURDAN generalized polarization (GP) measurements (32) were used to evaluate the organization of the extracellular lipids within stratum corneum, as described by (33). LAURDAN is an amphiphilic probe that preferentially partitions into lipid membranes. The fluorescence emission properties are sensitive to the water dipolar relaxation process that occurs in the probe's environment. Briefly, the energy of the LAURDAN excited singlet state progressively decreases when the extent of dipolar relaxation process is augmented, red shifting the probe's emission spectrum, i.e., decreasing the GP function to lower values (see Equation 1). The extent of water dipolar relaxation is normally related to the lipid packing of the studied membrane being low when the membrane packing is tight (e.g., membranes in the gel phase) (34). LAURDAN GP measurements were performed using the microscopy instrumental setup described in Ref. 35. Likewise, LAURDAN GPs were calculated using the formula:
The correction factor G was calculated by acquiring GP images of a known LAURDAN reference solution in the microscope at the same instrumental conditions used in the tissue sections (for further detail, see Ref. 35). LAURDAN (2 µM) in DMSO was used, with the predetermined GP = 0.011. The reference's GP was measured as described Ref. 35.
Mouse ear was incubated with 3 µl 0.1 mM BCECF in ethanol for 1 h at room temperature in vivo. Labeling was repeated every 15 min during the 1 h incubation time. Mice were then euthanized, and the ear was immediately covered with a water vapor-wetted cover slip to enhance contact with the tissue. Lifetime of the probe in the stratum corneum was determined to evaluate the local proton activity in the tissue, as described by Ref. 36. Measurements were performed using the same multiphoton excitation microscope described above. Fluorescence lifetimes were measured in the frequency domain using a field programmable gate array (FPGA) card for detection. The FPGA and the microscope are controlled by software developed by the Laboratory for Fluorescence Dynamics (37). As a lifetime reference, we used 5 mM fluorescein in ammonium acetate solution pH 9.2 (lifetime 4.05 ns). Excitation wavelength was 820nm and emission light was collected through a 525 ± 25 nm filter. Solutions experiments were carried out to validate measurement and calculation methods (10 µM BCECF in 0.2M Na2HPO4/0.1M citrate with 3 mM KCl/140 mM NaCl). BCECF lifetime and species fractions were determined using the phasor plot in the SimFCS software (38). Fractional intensities were determined from solutions containing fully protonated or deprotonated BCECF (pH below 4 and above 8, respectively). An indication of the local proton activity in the skin was then estimated from the lifetime images as described by Ref. 36, using Equation 2, where pKa ~7, Ii is the intensity obtained for the ith species and fi is the species fraction (i = BCECF or BCECFH):
Although our measurements are referenced to solutions displaying different pHs, we decided to correlate the quantity pH in Equation 2 with the activity of protons. This assumption take into account that the concentration of water in skin stratum corneum is far from being in excess, a requirement to use the term pH. Therefore, a decrease in the proton activity indicates that the measured apparent pH from Equation 2 increases (and vice versa).
Naked (Veet® treated) skin from the belly was used for lipid analyses and the FA elongation assay. Tissue was left 4–5 days after hair removal prior to isolation for lipid analyses. For isolation of epidermis, tissue was left floating on 2.5 mg/ml Dispase II (Roche) in Hanks BSS (w/o phenol red, Gibco 14025) at 4°C over night. Thereafter, epidermis was gently lifted away from dermis (under a micro-dissection microscope), washed once in distilled water, and freeze-dried to determine dry tissue weight. Stratum corneum was isolated after overnight floating on 1% trypsin solution (cell culture grade). Cells from the viable epidermis were removed from stratum corneum sheets by repeated rigorous shaking. Each sample preparation was checked under the microscope to ensure lack of epidermal cells. Fur for lipid analyses was obtained from the body of mice by shaving with an ethanol-cleaned electrical shaver.
Ceramides were extracted as previously described (39) with slight modifications. Stratum corneum was extracted three times with n-hexan/ethanol 95:5 (v/v) under ultrasonication for 20 min. Pooled extracts from the same animal were filtered using a 0.45 μm PTFE syringe filter (Carl Roth GmbH and Co. KG, pore size) and evaporated under N2. The residue was resolved in 1 ml chloroform/methanol 2:1 (v/v) and stored at −30°C until analysis. Samples were analyzed using TLC. The preparation of the plate and the application of samples were carried out as previously described (40). Samples were run in duplicate with a ceramide mixture consisting of ceramide AP (L- and D-conformation), CerNS, CerNP, and CerEOS, applied in different amounts (20–800 ng) for calibration.
Epidermal and stratum corneum lipid extracts were diluted according to dry tissue weight, and lipid extracts corresponding to 100 µg starting material from each mouse were pooled. Aliquots of the pooled extracts were subjected to TLC. Lipid extracts were run parallel to 5 µg Nu-Chek 18-4A and either 5 µg cholesterol standard or 5 µg heptadecanoic acid standard. Plates were developed twice in petroleumether/diethylether/acetic acid 70/30/1 (v/v/v). For visualization of cholesterol, plates were stained with 10% FeCl3 in 5% CH3COOH and 5% H2SO4 (pink/purple for cholesterol) and baked at 100°C for ~5 min. For FA visualization, plates were stained with 10% CuSO4 in 8% H2PO3 and were baked at 180°C for ~8 min.
Following addition of internal standard (1 nmol/mg epidermis and stratum corneum dry weight N-18:0 Phytosphingosine or 2nmol/mg fur triheptadecanoin) lipids were extracted by a modified Bligh and Dyer (41) method. Dry tissue was homogenized in a total volume of 1.9 ml chloroform:methanol:0.88% KClaq 2:1:0.8 (v/v/v). Following homogenization, the ratio of solvents was changed to chloroform:methanol:0.88% KClaq 1:1:0.9 (v/v/v), and samples were mixed rigorously. Following centrifugation, the lower phase was transferred to a new tube, and the upper phase reextracted twice with synthetic lower phase. The combined lower phases were washed once with synthetic upper phase and dried under a stream of nitrogen. Lipids were resuspended in synthetic lower phase and filtered through a solvent resistant 0.45 µm filter prior to analysis.
Lipid were loaded on to a normal phase PVA SIL HPLC column (YMC Europe GmbH, D-46514 Scherbeck, Germany, 1 × 150 mm) in 3 µl synthetic lower phase and eluted at 50 µl/min. Solvents used for elution were A [hexane-isopropanol (98:2)]; B [methyl tert-butyl ether (MTBE)-isopropanol (45:55)] and C (methanol). All three contained 0,1% triethanolamine (TEA) and equimolar formic acid. The elution gradient was changed linearly over time to give ratios A:B:C as follows: 0 min, 12:88:0; 3 min, 9,8:74,2:16; 4 min, 7,6:61,4:31; 5 min, 0:0:100; 11 min, 0:0:100; 13 min, 0:100:0; 17 min, 12:88:0; and 24 min, 12:88:0. Ions in the effluent were ionized by electrospray ionization with an electrode potential of 3,500 V and the masses of negative ions were detected by a Bruker Esquire-LC quadrupole ion trap mass spectrometer. This method was used for quantification against the internal ceramide standard of epidermal and stratum corneum ceramides as well as stratum corneum free FAs.
For quantification of epidermal free FAs, one fourth of the extracted epidermal lipids were derivatized to acyl-choline esters essentially as described in Ref. 42. Before processing, 4 nmol of each of the following deuterated FA standards, palmitic-7,7,8,8-d 4 acid, docosanoic-7,7,8,8-d 4 acid, tetracosanoic-9,9,10,10-d 4 acid, and 2 nmol hexacosanoic-12,12,13,13-d 4 acid, was added to the one-fourth extract. The residue was dissolved in acetonitrile/water/formic acid (200 µl, 50:50:0.025, v/v/v) and analyzed in a QSTAR Hybrid LC/MS/MS Quadrupole TOF system and quantified against deuterated internal FA standards. Epidermis total FA (TFA) content was quantified similarly after acidic hydrolysis (43) of another one-fourth extract. Before processing, 12 nmol of each of the following deuterated FA standards, palmitic-7,7,8,8-d 4 acid, docosanoic-7,7,8,8-d 4 acid, tetracosanoic-9,9,10,10-d 4 acid, and 5 nmol hexacosanoic-12,12,13,13-d 4 acid, was added to the one-fourth extract.
Full thickness hairless skin explants from the abdominal region were isolated and incubated in 10 mM EDTA in Dulbecco's PBS without calcium and magnesium. Following 1 h preincubation, 7 μCi/ml [1-14C]oleic acid (PerkinElmer) complexed to BSA (1:4, BSA:FA) was added to each explant and incubation was continued for an additional 4 h. Explants were then transferred to ice cold 10 mM EDTA in PBS to stop the reaction (44). Epidermis was isolated and lipids extracted as described above. Extracted lipids were resuspended in synthetic lower phase containing 50 mg/l butylated hydroxyl toluene, according to wet weight of epidermis. Lipid extracts were hydrolyzed and FAs were converted to FA methyl esters (FAME) by incubation with 1ml 2.5% H2SO4 in water-free methanol (SUPELCO) for 5 h at 80°C. FAMEs were extracted by addition of hexane and water followed by centrifugation. The upper phase was isolated and dried under a steam of N2. FAMEs were resuspended in chloroform and separated by reverse-phase TLC (KC18 TLC PLATES (Whatman, Ref. 45) as previously described (46). The radiolabeled FAMEs were detected using a Molecular Dynamics Storage phosphor screen and Typhoon Trio Scanner (Amersham Biosciences) and quantified by ImageQuant 5.0. The detected FAMEs were normalized to [1-14C]oleic acid content.
Fur lipid extracts were dried under a stream of nitrogen and dissolved in 7.5 mM ammonium acetate in chloroform/methanol/2-propanol (1:2:4, v/v/v). Fur lipid mass spectrometric analysis was performed on a hybrid LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a robotic nanoflow ion source TriVersa (Advion BioSciences Ltd, Ithaca NY) using chips with 4.1 mm nozzle diameter. The ion source was controlled by Chipsoft 6.4 software (Advion BioSciences) and operated at the ionization voltage of 0.96 kV and gas pressure 1.25 psi. Plates with lipid extracts were chilled to 4°C. MS survey scans were acquired in positive ion mode using the Orbitrap analyzer operated under the target mass resolution of 100,000 (full width at half maximum, FWHM), defined at m/z 400 under automatic gain control set to 1e5 as the target value. Epidermal and stratum corneum lipid extracts were dried under a stream of nitrogen, dissolved in chlorofom:methanol 1:2 with 5% isopropanol and 5 mM acetic acid and analyzed on a QSTAR Hybrid LC/MS/MS Quadrupole TOF mass spectrometer. Injection was through NanoES capillaries, Proxeon, Medium (cat. no. ES380), samples were ionized by 900V, and data were acquired during 2 min with scan range m/z 150–1,400. The monoalkyl-diacylglycerol and triacylglycerol content was quantified against the triheptadecanoin internal standard.
Hair was removed from the abdominal skin of the mice by electrical shaving followed by 150 s treatment with Veet® hair removal cream. Throughout the experiment, standard wooden bedding was replaced with Kleenex® tissues. Transepidermal water loss (TEWL) was determined using a DermaLab® TEWL probe (Cortex Technology, Denmark) in accordance with the guidelines of the Standardization Group of the European Dermatitis Society (47). Skin conductance was measured using a DermaLab® MOIST probe. Three- to four-month-old male mice were anesthetized with IsoFlo®Vet (2.75% in 350ml/O2/min, Orion Pharma Animal Health, Denmark), and measurements were conducted on naked skin on the abdomen of the mice. TEWL data were collected when mean TEWL and MOIST values were equal within groups (Student t-test) for two consecutive days after (≥5 days) Veet® treatment (data not shown). Furthermore, mice were only included in the experiment when no damage was visible on the skin (redness, edema, irritation, scaling). To avoid any effect of the anesthetics on the TEWL values, TEWL measurements were initiated ≥ 6 min after induction of anesthesia. During measurements, mice were placed on a temperature-controlled electrical blanket to avoid hypothermia.
Data are presented as mean ± SEM unless otherwise indicated. Data were analyzed by Student's t-test or one way ANOVA unless otherwise stated. Software for statistical analyses was SAS® Statistical Analysis Systems (SAS) Analyst application (SAS release version 9.1, 2002–2004) by SAS Institute Incorporated, Cary, NC, and the GraphPad Prism software. Level of significance was preset to 0.05.
We previously reported the generation of mice with targeted disruption of ACBP (24). These mice are born in a normal Mendelian ratio and show similar fertility and lifespan as ACBP+/+ mice; however, they display a poor adaptation to weaning and need special care during that time (24). Furthermore, the mice develop a clear macroscopic skin and fur phenotype from ~16 days of age (Fig. 1A). The fur becomes greasy and matted, and at the time of weaning, the ACBP−/− mice are easily distinguished from ACBP+/+ and ACBP+/− littermates. The different appearance of the fur is sustained throughout life; however, the fur becomes less greasy and develops in some areas a reddish brown color (Fig. 1B). Furthermore, most mice develop alopecia and scaling of the skin in the naked areas with age (Fig. 1C). This phenotype is macroscopically similar to that of mice with the nm1054 mutation that deletes ACBP and at least five other genes (26–28).
Evaluation of the relative expression of ACBP in the different layers of the skin showed that ACBP expression is highest in the suprabasal layers, especially stratum spinosum (Fig. 2A); however, expression is observed in all live cells in the epidermis (Fig. 2A). In sebocytes, we observed a low-to-intermediate staining, whereas keratinocytes along the hair shaft display more prominent staining (Fig. 2B). Sebocytes lining the sebaceous glands are more stained compared with the differentiated cells in the center of the gland (Fig. 2B). This pattern of expression in mouse skin is similar to that reported for human skin (48). Sections from ACBP−/− mice (Fig. 2C) as well as negative (IgG) controls (data not shown) showed no staining.
Histological examination of the skin from ACBP−/− mice and ACBP+/+ controls revealed that there are no morphological differences between genotypes in either the thick epidermis of the foot (Fig. 2D, E) or the skin from the back (Fig. 2F, G). We have examined the abundance and appearance of sebocytes and pilosebaceous units during skin and hair development and did not observe developmental defects or other differences in sebocytes or pilosebaceous units between adult ACBP+/+ and ACBP−/− mice (data not shown). We furthermore examined the expression of the classical differentiation markers keratin 5, keratin 10, and loricrin in thin (back skin) and thick (foot sole) epidermis and found no differential expression or morphological differences in the epidermis when comparing ACBP−/− mice and ACBP+/+ controls (Fig. 3). Keratin 5 expression was confined to the basal undifferentiated keratinocytes (Fig. 3A, B), whereas keratin 10 expression was seen throughout the differentiated live cell layers of epidermis (Fig. 3C, D) as well as stratum corneum. Loricrin expression was, as expected, highest in the stratum granulosum (Fig. 3E, F). Finally, ultrastructural studies using transmission electron microscopy did not reveal obvious defects in the ACBP−/− mouse skin (data not shown).
To investigate whether disruption of the ACBP gene affected the biophysical properties of the skin, we performed a characterization of the skin surface by two-photon excitation microscopy. It has previously been reported that the lateral organization of stratum corneum lipid membranes would have pronounced consequences for the barrier capacity of the skin (49). Thus, we first evaluated the lateral organization of lipid membranes existing in the skin stratum corneum by labeling excised mouse ear tissue with LAURDAN and evaluating it under a two-photon excitation microscope. The LAURDAN GP function is sensitive to the extent of water dipolar relaxation processes (and water content) occurring at the membrane interface where the probe is located (50). The LAURDAN GP values measured in ACBP+/+ and ACBP−/− mice indicate that in both cases the contributing membranes within the stratum corneum region display tight lateral packing, i.e., corresponding to gel-like membranes in model systems (GP above 0.5, see Ref. 32 and references therein). However, a significant increase in LAURDAN GP values in the stratum corneum membranes of ACBP−/− mice compared with ACBP+/+ controls is observed (Fig. 4A). This indicates that the water content of the stratum corneum extracellular lipid matrix in the ACBP−/− mice is lower than in ACBP+/+ controls. Consistent with previous observations (50, 51) showing that membranes with a low extent of water dipolar relaxation processes display more narrow GP histograms, the LAURDAN GP histogram across each image is narrower in ACBP−/− compared with ACBP+/+ controls (Fig. 4B, further illustrated by Fig. 4C, D).
To further investigate the biophysical properties of the skin tissue, we examined the local proton activity of the skin outermost layer; i.e., the stratum corneum. Mouse ear skin was labeled with the BCECF fluorescent probe, the fluorescence lifetime of which is sensitive to the protonation of the probe (52). The apparent stratum corneum pH is increased from ~5.5 ± 0.1, as observed in tissue from ACBP+/+ mice, to ~5.9 ± 0.1 in tissue from ACBP−/− mice (Fig. 5A), with a slightly lower pH locally in the intercellular space (~5.3 ± 0.1 in ACBP+/+ and ~5.8 ± 0.1 in ACBP−/− mice, Fig. 5B). These values are in agreement with previous observations in mouse skin (53). Representative images are presented in Fig. 5C–F and show that the local proton activity is decreased in the skin in general as well as in the intercellular space. The alterations of the natural proton gradient observed for ACBP−/− mice (with respect to the ACBP+/+ controls) correlate with a lower water content of the stratum corneum intercellular space in ACBP−/− mice (measured with LAURDAN GP function), suggesting potential alterations in the skin tissue's water gradient.
As described previously, lateral organization of membranes composed of stratum corneum lipids is highly dependent on both pH (54) and lipid composition (55). We therefore determined the overall lipid composition of the stratum corneum and epidermis. By means of TLC, we detected no significant differences between ACBP+/+ and ACBP−/− mice in overall FFA or cholesterol content in isolated stratum corneum and epidermis (Fig. 6A, B). In addition, we quantified the content of ceramides by MS as well as TLC without detecting significant quantitative or qualitative differences in stratum corneum or epidermis (Fig. 6C and supplementary Figs. I, A–E, and II, A–F).
To further investigate the composition of lipid subspecies, we determined the content of FFAs. The major FFA species in isolated stratum corneum are longer than 20 carbons in length, primarily C24 and C26 FAs (56, 57), and derive primarily from hydrolysis of complex phospholipids in the lamellar bodies (58–60). Using MS, we were able to quantify the C20–C28 FFA content (Fig. 7); however, for FAs shorter than C20 we had too high background contamination from plastics and solvents to quantify correctly; and for FAs longer than C28, the signal was too low and variable for quantification. Interestingly, despite the lack of differences in the overall FFA content of the stratum corneum (Fig. 6A), we found a remarkable >50% decrease in the content of VLC-FFAs in stratum corneum of ACBP−/− mice compared with ACBP+/+ mice (Fig. 7A). Notably, the VLC-FFA profile as well as the total VLCFA content in whole thickness epidermis was similar between genotypes (Fig. 7B, C), indicating that lack of ACBP specifically affects the FFA composition in the stratum corneum where the VLCFAs are more abundant (Fig. 7A). The only significant difference in epidermal FFA content between ACBP+/+ and ACBP−/− mice is the increase in C20:1 FA (Fig. 7B), which may derive from MADAGs produced by the sebaceous glands (61, 62).
Because the disruption of ACBP specifically affected the abundance of VLCFA in the stratum corneum, we speculated that the abundant presence of ACBP in stratum spinosum may be a requirement for elongation of FAs to be used for establishment of the extracellular lipid lamellae in stratum corneum. To investigate whether disruption of ACBP affects FA elongation per se, we performed an elongation activity assay in skin explants by incubating these with radiolabeled FAs. However, we did not detect significant differences in the elongation activity between ACBP+/+ and ACBP−/− skin explants (Fig. 7D).These results indicate that lack of ACBP perturbs the formation VLC-FFA by mechanism other than FA chain elongation.
To evaluate whether the sebum lipid composition was changed, thereby being a possible source of the increased content of C20:1FA in epidermis, we quantified the content of neutral lipids within isolated stratum corneum, whole thickness epidermis, and fur. It became evident from TLC analyses that a lipid specie less polar than TAGs is highly abundant in the lipid extracts from stratum corneum, epidermis, and fur (data not shown). On the basis of its molecular weight as determined with four digits of precision of the m/z, we identified this lipid as monoalkyl-diacylglycerol (MADAG) using lipid MS (data not shown). As this species is particularly enriched in sebum that is secreted along the hairs (61), we included fur lipid extracts in the MS analyses of the MADAG and TAG content in the skin. Interestingly, in the stratum corneum and epidermis of ACBP−/− mice, the MADAG content is significantly increased, while the content of TAGs is unchanged (Fig. 8A, B). In fur lipid extracts from ACBP−/− mice, we found significantly increased amounts of MADAG lipids compared with extracts from ACBP+/+ mice, and concomitant with this, we observed a decreased content of TAGs (Fig. 8C). These results indicate that MADAG synthesis is significantly increased in the sebocytes, leading to dramatically increased levels of MADAGs in the skin and on the hair of ACBP−/− mice.
There is substantial evidence that VLCFAs are important for the epidermal permeability barrier. First, investigations in reconstituted skin lipid mixtures have demonstrated that the FA chain length distribution dramatically influences lipid membrane lateral organization (55). Second, knockout of FA elongases expressed in the epidermis has been shown to cause impairment of the epidermal barrier (63–66). Thus, the significant decrease in VLC-FFA in the stratum corneum combined with the changes in the biophysical properties of the tissue prompted us to determine the rate of water loss across the skin as a measure of the permeability barrier function. To allow measurements on naked skin, hair was removed from the abdomen of 3- to 4-month-old mice by shaving followed by Veet® treatment, after which the skin was allowed to recover for > 5 days until mean TEWL and MOIST values were equal within groups for two consecutive days (data not shown). Interestingly, ACBP−/− mice had a ~50% increased water permeability compared with ACBP+/+ controls (Fig. 9). Similar results, but with higher experimental variation, were obtained on shaved, non-Veet®-treated skin (data not shown). A similar trend, although not as pronounced, was observed in 3-week-old mice, indicating that impairment of the epidermal barrier is initiated at early age but worsens with age (mean TEWL ± SEM 4.77 ± 0.11 [ACBP+/+, n = 10) and 5.18 ± 0.12 (ACBP−/−, n = 11) at age 3 weeks]. Thus, disruption of the ACBP gene significantly impairs the epidermal permeability barrier in both young and adult mice, but the epidermal dysfunction is exacerbated in the adult mice.
In this article, we show that targeted disruption of ACBP in mice leads to a clearly distinguishable skin and fur phenotype with greasy and matted fur when the mice are approximately 16 days old. At older ages, the skin becomes dry and scaly, and some of the fur is lost. Careful histological examinations of skin from 3-week-old and 3-month-old mice show no gross differences in skin morphology, structure of sebaceous glands, or number of hair follicles between ACBP−/− and ACBP+/+ mice. Importantly, our data show for the first time that the stratum corneum of ACBP−/− mice display altered biophysical properties and a significant decrease in VLC-FFA concomitant with a compromised epidermal barrier function.
The phenotype of the ACBP−/− mice show several similarities to that of the previously reported nm1054 deletion mutants (26). These mice lack a ~400 kb fragment of chromosome 1, including the Acbp locus and at least five other genes (26–28), and were reported to have sparse, reddish, matted hair with a greasy appearance. Similar to our data from ACBP−/− mice, the lipids associated with the fur of nm1054 mutant mice were also different from the fur lipids of wild-type mice. Fleming and colleagues (26) showed a decreased amount of TAG and an increased amount of a nearly comigrating lipid species that they were unable to identify. This lipid was most likely MADAG, which we identified in this study. In addition, dependent on the anatomical site of the biopsy, the nm1054 mutant mice were reported to have an increased number of sebocytes associated with most pilosebaceous units (26). We have carefully investigated the development, abundance, and appearance of pilosebaceous units in the skin from the back, but we did not detect any differences between ACBP+/+ and ACBP−/− adult mice. Additional phenotypic characteristics, such as hydrocephaly and anemia, were observed in the nm1054 mutant mice, and on a C57BL/6J background, the nm1054 mutation led to significant prenatal lethality as well as increased lethality of newborns (28). On the basis of our results from careful characterization of ACBP−/− mice during both early backcross generations and in our present congenic strain (C57BL/6JBomTac), we conclude that these phenotypic characteristics are most likely related to the deletion of genes other than the ACBP gene.
Consistent with the increased transepidermal water loss, the LAURDAN GP data indicate that there is a significant decrease in the water content of ACBP−/− mouse stratum corneum membranes, as inferred from the higher LAURDAN GP values. Subtle changes in the permeability properties of the membranes participating in the barrier can be caused by lipid compositional changes. For example, it has been reported that the characteristic FFA composition of the stratum corneum membranes is a very important factor in promoting hydrocarbon chain mixing, defining lipid mixing properties and membrane stability (55, 67). Similarly, the increased apparent pH detected in the stratum corneum of ACBP−/− mice may also modulate the stability of the membranes by enhancing ionization of the FAs. This will increase repulsion between polar head groups at the membrane interface, causing local instabilities that may lead to increased permeability across the membranes. This is consistent with the increased TEWL observed in ACBP−/− mice.
In terms of lipid composition, we did not detect major differences between ACBP+/+ and ACBP−/− mice in the total content of the classical skin lipids. Thus, cholesterol, overall FFA, and ceramide contents are not significantly different between genotypes in either the stratum corneum or the epidermis. Interestingly however, the stratum corneum of ACBP−/− mice contains significantly lower levels of several VLC-FFA species compared with ACBP+/+ mice. Our analyses allowed only for quantification of FAs up to 28 carbons in length; however, it is likely that the content of FFAs with longer chain length is decreased as well. This decrease in VLC-FFA may be responsible for the changes in the physical properties (apparent pH, water content) within the stratum corneum lipid membranes.
Despite the decrease in VLC-FFA in stratum corneum, we did not detect differences in the elongation activity of epidermal explants, indicating that lack of ACBP does not affect FA elongation per se. ACBP may instead be required for incorporation of VLCFAs into complex lipids, such as phospholipids, that are excreted in the lipid lamellar bodies and serve as the main source of extracellular VLC-FFA in stratum corneum (58–60, 68–70).
Decreased incorporation of either VLCFAs or linoleic acid into lamellar body lipids has been shown to compromise the epidermal permeability barrier in a number of mouse models. Interestingly, only a subset of these models suffer from a lethal phenotype, and for at least three of these, i.e. the acyl-CoA:diacylglycerol acyltransferase (DGAT) 2 (71), stearoyl CoA desaturase (SCD) 2 (72), and elongase of VLCFAs ELOVL 4 (63–65) knockout mouse models, the level of omega-hydroxylated ceramides is profoundly decreased in the skin. These mice die due to excessive dehydration within a few hours after birth. Contrary to these two mouse models, the ACBP−/− mice show normal levels of omega-hydroxylated ceramides in stratum corneum, which is consistent with the relatively mild barrier defect seen in the ACBP−/− mice.
In contrast to the lethal phenotype of DGAT2−/−, SCD2−/−, and ELOVL4−/− mice, other models such as ELOVL3−/− (66), SCD1−/− (73), or mice with essential FA deficiency (EFAD) (74–76), display a nonlethal phenotype with increased transepidermal water loss. Characteristic for these as well as for the ACBP−/− mice is the development of tousled fur, reduced fur content, change (browning) in coat color, and irritated, eczematous skin in adult mice (66). Despite the striking similarities between the skin and fur phenotypes of ACBP−/− and ELOVL3−/− mice, we did not detect changes in epidermal FA elongation in ACBP−/− mice, suggesting that ACBP affects different biochemical steps of the synthesis of VLC-FFA for the stratum corneum.
Consistent with an important role of ACBP in mouse skin, we show that ACBP is highly expressed in the suprabasal layers of epidermis and in the rim of the sebaceous glands. The expression profile of ACBP in mouse epidermis is in agreement with previous observations in human skin (48). In relation to the observed fur phenotype of the ACBP−/− mice, we observed a significant increase in MADAG levels in both epidermis and stratum corneum of ACBP−/− compared with ACBP+/+ mice. MADAGs found in the skin are primarily derived from sebocytes (61), and previous compositional characterization of skin surface lipids in mice have shown that FA C20:1 is the major FA in MADAGs produced by the sebaceous glands (61, 62). These lipids are only in rare diseased cases produced by keratinocytes (77, 78). We find increased MADAG levels in epidermis, stratum corneum, and fur of ACBP−/− mice. We propose that as MADAGs contain abundant amounts of FA C20:1, it is likely this FA is generated as a side effect to the increased MADAG synthesis. However, we cannot exclude other origins of this FA or the MADAG species in epidermis.
Consistent with the sebum being the main source of MADAG, examination of the fur lipids, which are derived almost exclusively from lipid biosynthesis in the sebaceous glands, showed a marked increase in MADAG content concomitant with a ~60% decrease in TAG content in ACBP−/− compared with ACBP+/+ mice. Thus, absence of ACBP appears to shift the balance to favor MADAG synthesis over TAG synthesis in the sebocytes. The reason for this shift in lipid classes is unknown; however, it is possible that the TAG synthesis is decreased because synthesis of MADAG is so dramatically increased in the absence of ACBP. In any case, the quantitative increase and compositional change in fur lipids are likely to be responsible for the greasy appearance of the fur. Furthermore, although sebum lipids are not generally considered key actors in the skin barrier (68, 70), it is possible that these lipids contribute to the altered biophysical properties of ACBP−/− mice stratum corneum.
In conclusion, the results reported here show that ACBP is required for normal epidermal barrier function. We demonstrate that lack of ACBP leads to significantly decreased levels of VLCFA in the stratum corneum, despite the presence of normal elongation activity in whole thickness epidermis, indicating that ACBP plays a role in FA trafficking in specific cell types in mammals, in this case the viable epidermis. The decrease in stratum corneum free FA chain length is likely to be directly related to the increased permeability of the epidermal barrier and hence the decreased polarity as well as the decreased local proton activity of the stratum corneum. Finally, our data support a role of ACBP in promoting TAG and limiting MADAG synthesis in the sebaceous glands. Future studies should address the exact biochemical role of ACBP for complex lipid synthesis in keratinocytes.
The authors thank Dr. Lars Norlen, Karolinska Institutet, Stockholm, Sweden, and Dr. Fernando Larcher, CIEMAT Epithelial Unit, Madrid, Spain, for discussions of data and critical reading of the manuscript. The authors thank Dr. Christer S. Ejsing, Department of Biochemistry and Molecular Biology, University of Southern Denmark, for recording the mass spectrometry data used for Fig. 8 and for critical reading of the manuscript. The authors are grateful to Dr. Karsten Kristiansen, Department of Biology, Copenhagen University, Denmark, for the ACBP antiserum.
This work was supported by the Danish National Research Foundation, which supported MEMPHYS Center for Biomembrane Physics (2001–2011), and by the Danish Molecular Biomedical Imaging Center (DaMBIC), the Danish Natural Science Research Council, the Danish Medical Science Research Council, the Lundbeck Foundation, the Novo Nordisk Foundation, the Novo Scholarship Program, and BioNET. The authors declare no conflicts of interest.
[S]The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of two figures.