Compromised epidermal barrier stimulates Harderian gland activity and hypertrophy in ACBP−/− mice
2015; Elsevier BV; Volume: 56; Issue: 9 Linguagem: Inglês
10.1194/jlr.m060780
ISSN1539-7262
AutoresSigne Bek, Ditte Neess, Karen Dixen, Maria Bloksgaard, Ann-Britt Marcher, J. Chemnitz, Nils J. Færgeman, Susanne Mandrup,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoAcyl-CoA binding protein (ACBP) is a small, ubiquitously expressed intracellular protein that binds C14-C22 acyl-CoA esters with very high affinity and specificity. We have recently shown that targeted disruption of the Acbp gene leads to a compromised epidermal barrier and that this causes delayed adaptation to weaning, including the induction of the hepatic lipogenic and cholesterogenic gene programs. Here we show that ACBP is highly expressed in the Harderian gland, a gland that is located behind the eyeball of rodents and involved in the production of fur lipids and lipids used for lubrication of the eye lid. We show that disruption of the Acbp gene leads to a significant enlargement of this gland with hypertrophy of the acinar cells and increased de novo synthesis of monoalkyl diacylglycerol, the main lipid species produced by the gland. Mice with conditional targeting of the Acbp gene in the epidermis recapitulate this phenotype, whereas generation of an artificial epidermal barrier during gland development reverses the phenotype. Our findings indicate that the Harderian gland is activated by the compromised epidermal barrier as an adaptive and protective mechanism to overcome the barrier defect. Acyl-CoA binding protein (ACBP) is a small, ubiquitously expressed intracellular protein that binds C14-C22 acyl-CoA esters with very high affinity and specificity. We have recently shown that targeted disruption of the Acbp gene leads to a compromised epidermal barrier and that this causes delayed adaptation to weaning, including the induction of the hepatic lipogenic and cholesterogenic gene programs. Here we show that ACBP is highly expressed in the Harderian gland, a gland that is located behind the eyeball of rodents and involved in the production of fur lipids and lipids used for lubrication of the eye lid. We show that disruption of the Acbp gene leads to a significant enlargement of this gland with hypertrophy of the acinar cells and increased de novo synthesis of monoalkyl diacylglycerol, the main lipid species produced by the gland. Mice with conditional targeting of the Acbp gene in the epidermis recapitulate this phenotype, whereas generation of an artificial epidermal barrier during gland development reverses the phenotype. Our findings indicate that the Harderian gland is activated by the compromised epidermal barrier as an adaptive and protective mechanism to overcome the barrier defect. The acyl-CoA binding protein (ACBP)/diazepam binding inhibitor, (entrez gene ID: 13167) is a 10kDa intracellular protein binding medium- and long-chain acyl-CoA ester (C14–C22) with high affinity and specificity (1Rosendal J. Ertbjerg P. Knudsen J. Characterization of ligand binding to acyl-CoA-binding protein.Biochem. J. 1993; 290: 321-326Crossref PubMed Scopus (135) Google Scholar). The protein is evolutionarily conserved and is expressed in all eukaryotic cell types investigated (2Burton M. Rose T.M. Faergeman N.J. Knudsen J. Evolution of the acyl-CoA binding protein (ACBP).Biochem. J. 2005; 392: 299-307Crossref PubMed Scopus (123) Google Scholar). However, expression levels differ markedly between different cell types. In general, lipogenic cell types as well as epithelial cells involved in transport functions display high levels of ACBP expression, whereas heart, lung, muscle, and spleen have low expression levels (3Bovolin P. Schlichting J. Miyata M. Ferrarese C. Guidotti A. Alho H. Distribution and characterization of diazepam binding inhibitor (DBI) in peripheral tissues of rat.Regul. Pept. 1990; 29: 267-281Crossref PubMed Scopus (132) Google Scholar, 4Neess D. Kiilerich P. Sandberg M.B. Helledie T. Nielsen R. Mandrup S. ACBP:a PPAR and SREBP modulated housekeeping gene.Mol. Cell. 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Depletion of the ACBP ortholog Acb1 in Saccharomyces cerevisiae results in altered membrane structures, accumulation of vesicles, and compromised synthesis of very-long-chain fatty acids and sphingolipids (11Faergeman N.J. Feddersen S. Christiansen J.K. Larsen M.K. Schneiter R. Ungermann C. Mutenda K. Roepstorff P. Knudsen J. Acyl-CoA-binding protein, Acb1p, is required for normal vacuole function and ceramide synthesis in Saccharomyces cerevisiae.Biochem. J. 2004; 380: 907-918Crossref PubMed Scopus (70) Google Scholar, 12Gaigg B. Neergaard T.B. Schneiter R. Hansen J.K. Faergeman N.J. Jensen N.A. Andersen J.R. Friis J. Sandhoff R. Schroder H.D. et al.Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae.Mol. Biol. Cell. 2001; 12: 1147-1160Crossref PubMed Scopus (119) Google Scholar). We recently generated mice with targeted disruption of the Acbp gene (ACBP−/−) (13Neess D. Bloksgaard M. Bek S. Marcher A.B. Elle I.C. Helledie T. Due M. Pagmantidis V. Finsen B. Wilbertz J. et al.Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.J. Biol. Chem. 2011; 286: 3460-3472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These mice are born in a Mendelian ratio and are visually indistinguishable from their heterozygous and ACBP+/+ littermates until around 16 days of age, where the ACBP−/− mice develop a matted and greasy fur. This fur phenotype persists throughout life and is at older age accompanied by scaling of the skin and occasional hair loss. Further investigations demonstrated that the epidermal barrier function is compromised in both young and adult ACBP−/− mice (14Bloksgaard M. Bek S. Marcher A.B. Neess D. Brewer J. Hannibal-Bach H.K. Helledie T. Fenger C. Due M. Berzina Z. et al.The acyl-CoA binding protein is required for normal epidermal barrier function in mice.J. Lipid Res. 2012; 53: 2162-2174Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), which, by analogy with other models (15Li W. Sandhoff R. Kono M. Zerfas P. Hoffmann V. Ding B.C. Proia R.L. Deng C.X. Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice.Int. J. Biol. Sci. 2007; 3: 120-128Crossref PubMed Scopus (127) Google Scholar, 16Miyazaki M. Dobrzyn A. Elias P.M. Ntambi J.M. Stearoyl-CoA desaturase-2 gene expression is required for lipid synthesis during early skin and liver development.Proc. Natl. Acad. Sci. USA. 2005; 102: 12501-12506Crossref PubMed Scopus (108) Google Scholar, 17Stone S.J. Myers H.M. Watkins S.M. Brown B.E. Feingold K.R. Elias P.M. Farese Jr, R.V. Lipopenia and skin barrier abnormalities in DGAT2-deficient mice.J. Biol. Chem. 2004; 279: 11767-11776Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar), may be caused by the observed significant reduction of very-long-chain free fatty acids in the stratum corneum (14Bloksgaard M. Bek S. Marcher A.B. Neess D. Brewer J. Hannibal-Bach H.K. Helledie T. Fenger C. Due M. Berzina Z. et al.The acyl-CoA binding protein is required for normal epidermal barrier function in mice.J. Lipid Res. 2012; 53: 2162-2174Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Recently, we demonstrated that conditional targeting of the Acbp gene in keratinocytes of mice (K14-ACBP−/−) recapitulates the defect epidermal barrier function, arguing that this defect is caused by lack of ACBP in the epidermis per se (18Neess D. Bek S. Bloksgaard M. Marcher A.B. Faergeman N.J. Mandrup S. Delayed hepatic adaptation to weaning in ACBP-/- mice is caused by disruption of the epidermal barrier.Cell Reports. 2013; 5: 1403-1412Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In addition to the skin and hair phenotype, the ACBP−/− mice suffer from a delayed adaptation to weaning, which includes a delayed hepatic upregulation of the lipogenic gene program at weaning (13Neess D. Bloksgaard M. Bek S. Marcher A.B. Elle I.C. Helledie T. Due M. Pagmantidis V. Finsen B. Wilbertz J. et al.Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.J. Biol. Chem. 2011; 286: 3460-3472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). This is caused by suppression of the activity of the sterol-regulatory element binding proteins possibly as a result of increased hepatic accumulation of triacylglycerol and cholesteryl esters (13Neess D. Bloksgaard M. Bek S. Marcher A.B. Elle I.C. Helledie T. Due M. Pagmantidis V. Finsen B. Wilbertz J. et al.Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.J. Biol. Chem. 2011; 286: 3460-3472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Intriguingly, we recently showed that K14-ACBP−/− mice recapitulate the delayed hepatic adaptation to weaning, demonstrating that lack of ACBP in the epidermis leads to suppression of the lipogenic and cholesterogenic gene programs in the liver. Importantly, the hepatic phenotype could be rescued by establishing an artificial barrier using either Vaseline or latex, thereby for the first time establishing a physiological link between the epidermal barrier function and hepatic gene expression and metabolism (18Neess D. Bek S. Bloksgaard M. Marcher A.B. Faergeman N.J. Mandrup S. Delayed hepatic adaptation to weaning in ACBP-/- mice is caused by disruption of the epidermal barrier.Cell Reports. 2013; 5: 1403-1412Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The Harderian gland, originally discovered by Harder in 1694, is located behind the eyeball of most terrestrial vertebrates. The gland contains more than 20% lipids per wet weight, where neutral lipids constitute 55–57% of total lipids (19Tvrzicka E. Rezanka T. Krijt J. Janousek V. Identification of very-long-chain fatty acids in rat and mouse harderian gland lipids by capillary gas chromatography-mass spectrometry.J. Chromatogr. A. 1988; 431: 231-238Crossref PubMed Scopus (16) Google Scholar). The main lipid species in the murine Harderian gland is monoalkyl diacylglycerol (MADAG) (19Tvrzicka E. Rezanka T. Krijt J. Janousek V. Identification of very-long-chain fatty acids in rat and mouse harderian gland lipids by capillary gas chromatography-mass spectrometry.J. Chromatogr. A. 1988; 431: 231-238Crossref PubMed Scopus (16) Google Scholar, 20Kasama K. Blank M.L. Snyder F. Identification of 1-alkyl-2-acyl-3-(2′,3′- diacylglycerol)glycerols, a new type of lipid class, in harderian gland tumors of mice.J. Biol. Chem. 1989; 264: 9453-9461Abstract Full Text PDF PubMed Google Scholar), which is secreted in an exocrine fashion onto the external surface of the cornea (21Satoh Y. Gesase A.P. Habara Y. Ono K. Kanno T. Lipid secretory mechanisms in the mammalian harderian gland.Microsc. Res. Tech. 1996; 34: 104-110Crossref PubMed Scopus (15) Google Scholar). Although the function(s) of the Harderian gland is still not completely understood, it is well established that lipids produced in the Harderian gland lubricate and protect the cornea and are used for grooming of the fur (22Cohn S.A. Histochemical observations on the Harderian gland of the albino mouse.J. Histochem. Cytochem. 1955; 3: 342-353Crossref PubMed Scopus (69) Google Scholar). In some species, lipids from the Harderian gland have been shown to provide thermoregulation (23Thiessen D.D. Kittrell E.M. The Harderian gland and thermoregulation in the gerbil (Meriones unguiculatus).Physiol. Behav. 1980; 24: 417-424Crossref PubMed Scopus (81) Google Scholar, 24Harolw H.J. The Influence of Hardarian gland removal and fur lipid removal on heat loss, and water flux to and from the skin of muskrats, Ondotra zibethicus.Physiol. Zool. 1984; 57: 349-356Crossref Google Scholar, 25Shanas U. Terkel J. Grooming secretions and seasonal adaptations in the blind mole rat (Spalax ehrenbergi).Physiol. Behav. 1996; 60: 653-656Crossref PubMed Google Scholar). Thus, harderianectomy leads to a decrease in the ability of the muskrat and Mongolian gerbil to defend their core body temperature in cold water (23Thiessen D.D. Kittrell E.M. The Harderian gland and thermoregulation in the gerbil (Meriones unguiculatus).Physiol. Behav. 1980; 24: 417-424Crossref PubMed Scopus (81) Google Scholar, 24Harolw H.J. The Influence of Hardarian gland removal and fur lipid removal on heat loss, and water flux to and from the skin of muskrats, Ondotra zibethicus.Physiol. Zool. 1984; 57: 349-356Crossref Google Scholar). It was shown that the majority of the fur lipids originate from the Harderian gland in Mongolian gerbils; however, similar findings could not be observed in rats, hamsters, or mice (23Thiessen D.D. Kittrell E.M. The Harderian gland and thermoregulation in the gerbil (Meriones unguiculatus).Physiol. Behav. 1980; 24: 417-424Crossref PubMed Scopus (81) Google Scholar). Thus, the function of the Harderian gland in thermo-protection in other rodents is unclear. In addition, the gland has been implicated in pheromone production and sexual behavior in some rodents (26Thiessen D.D. Harriman A.E. Harderian gland exudates in the male Meriones unguiculatus regulate female proceptive behavior, aggression, and investigation.J. Comp. Psychol. 1986; 100: 85-87Crossref PubMed Scopus (53) Google Scholar, 27Payne A.P. The attractiveness of Harderian gland smears to sexually naive and experienced male golden hamsters.Anim. Behav. 1979; 27: 897-904Crossref PubMed Scopus (45) Google Scholar, 28Payne A.P. Pheromonal effects of Harderian gland homogenates on aggressive behaviour in the hamster.J. Endocrinol. 1977; 73: 191-192Crossref PubMed Scopus (69) Google Scholar). Here we show that lack of ACBP in mice stimulates the activity and hypertrophy of the Harderian gland during weaning, and we present evidence indicating that this occurs as a defense mechanism in response to the impaired epidermal barrier of these mice. Generation of mice with constitutive (13Neess D. Bloksgaard M. Bek S. Marcher A.B. Elle I.C. Helledie T. Due M. Pagmantidis V. Finsen B. Wilbertz J. et al.Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.J. Biol. Chem. 2011; 286: 3460-3472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and conditional (18Neess D. Bek S. Bloksgaard M. Marcher A.B. Faergeman N.J. Mandrup S. Delayed hepatic adaptation to weaning in ACBP-/- mice is caused by disruption of the epidermal barrier.Cell Reports. 2013; 5: 1403-1412Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) targeting of the Acbp gene have previously been described. The mice were housed under standard conditions at ∼55% relative humidity at 22 ± 3°C as described elsewhere (13Neess D. Bloksgaard M. Bek S. Marcher A.B. Elle I.C. Helledie T. Due M. Pagmantidis V. Finsen B. Wilbertz J. et al.Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.J. Biol. Chem. 2011; 286: 3460-3472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Cold exposure was carried out in a 4°C refrigerated room with normal light cycles and ad libitum access to food. Vaseline was applied as described previously (18Neess D. Bek S. Bloksgaard M. Marcher A.B. Faergeman N.J. Mandrup S. Delayed hepatic adaptation to weaning in ACBP-/- mice is caused by disruption of the epidermal barrier.Cell Reports. 2013; 5: 1403-1412Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In brief, Vaseline was applied on the core body of the mice twice a day from 7 days of age to 28 days of age. The mice were weaned at 21 days of age as described (13Neess D. Bloksgaard M. Bek S. Marcher A.B. Elle I.C. Helledie T. Due M. Pagmantidis V. Finsen B. Wilbertz J. et al.Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.J. Biol. Chem. 2011; 286: 3460-3472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Breeding of transgenic mice and all animal experiments were approved by the Danish Animal Experiment Inspectorate. For determination of mRNA levels, tissues were processed, and real-time PCR was carried out as previously described (13Neess D. Bloksgaard M. Bek S. Marcher A.B. Elle I.C. Helledie T. Due M. Pagmantidis V. Finsen B. Wilbertz J. et al.Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.J. Biol. Chem. 2011; 286: 3460-3472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Primers used are: Acbp fwd: 5′ TTTCGGCATCCGTATCACCT; Acbp rew: 5′ TTTGTCAAATTCAGCCTGAGACA; TfIIb fwd: 5′ GTTCTGCTCCAACCTTTGCCT; TfIIb rev: 5′ TGTGTAGCTGCCATCTGCACTT; Adhaps fwd: 5′ TCCTATGGCTTGATGTGTCC; Adhaps rev: 5′ GTCCTGTTATCCCAGCCTCT; Dhapat fwd: 5′ GGTGTGTGTGAATGAAGAAGG; Dhapat rev: 5′ TGAAGGACAGCATGAGGAAG; Acc fwd: 5′ AACTTGCCAGAGCAGAAGGCA; Acc rev: 5′ GGATCTACCCAGGCCACATTG; Fasn fwd: 5′ TGCCAGCGTGCAATGATG; Fasn rev: 5′ CCTTTGAAGTCGAAGAAGAAGAGA. Preparation of whole cell extracts, Western blotting, and ECL were carried out as described previously (13Neess D. Bloksgaard M. Bek S. Marcher A.B. Elle I.C. Helledie T. Due M. Pagmantidis V. Finsen B. Wilbertz J. et al.Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.J. Biol. Chem. 2011; 286: 3460-3472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Primary antibodies used were rabbit antiserum against ACBP (1:2,000) and rabbit anti-human TFIIB C-18 (1:1,000) (sc-225; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Secondary antibody used was HRP-conjugated swine anti-rabbit IgG (1:1,000) (Dako, Denmark). Paraffin sections (5 µm) were mounted on Super Frost Plus® microscopic slides. Before staining, sections were dried for 60 min at 60°C, paraffin was removed with xylene, and sections were rehydrated with graded ethanol. Slides were then incubated in Mayer's hematoxylin (1 mg/ml in water) for 10 min and quickly in 1% HCl in 70% ethanol and rinsed for 10 min in flowing tap water before staining with eosin (2 min in 2% eosin in water). After staining, the slides were rinsed in water, dehydrated (2 × 1 min in 96% ethanol, 3 × 1 min in 99% ethanol, 2 × 5 min in xylene), and subsequently mounted. Adult mice were submitted to perfusion fixation with 2.5% glutaraldehyde in PBS (pH 7.4). Biopsies were postfixed in 1% osmium tetroxide (pH 7.2) for 2 h at 4°C, dehydrated in ethanol, and embedded in Araldite. Semithin sections (0.5 µm) were stained with toluidin blue for light microscopy. The area of acinar cells, the number of vesicles per cell and per µm2, and the diameter of the vesicles were measured in 0.5 µm sections stained with osmium tetroxide and toluidin blue using the Leica IM500 measurement application (100× magnification). Sections of glands from 3 month old ACBP−/− (n = 6) and ACBP+/+ (n = 8) mice were evaluated. For each animal, the size and number of vesicles in a minimum of 10 acinar cells were determined. For determination of de novo lipid synthesis in the Harderian gland, adult mice (n = 8 for each genotype and condition) were injected i.p. with 0.04 µCi/g mouse 14C-acetic acid ([1-14C] acetic acid, sodium salt; Amersham Biosciences) in 0.9% saline and euthanized 1 h later by cervical dislocation. The Harderian glands were removed, snap-frozen in liquid nitrogen, and stored at −80°C. Total lipids were extracted from one Harderian gland using standard Bligh and Dyer extraction (29Bligh 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). Lipids from equal amounts of tissue were loaded onto silica plates (Merck High Performance Thin-Layer Chromatography [HPTLC]) and left to air dry for 15 min. The plates were developed in a horizontal development chamber in a solvent mixture of hexane-diethyl ether-acetic acid (v/v/v) (80:30:1) for the separation of neutral lipids. Band intensities were determined by densitometry. The visualization of 14C-labeled lipids and the total lipids was carried out as previously described (13Neess D. Bloksgaard M. Bek S. Marcher A.B. Elle I.C. Helledie T. Due M. Pagmantidis V. Finsen B. Wilbertz J. et al.Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.J. Biol. Chem. 2011; 286: 3460-3472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Harderian glands were isolated from adult ACBP−/− (n = 7) and ACBP+/+ (n = 7) mice and washed in Krebs ringer buffer (Sigma) containing 0.5% fatty acid-free BSA (Sigma). Immediately thereafter, similar-sized explants (approximately half a gland) were incubated for 2 h in Krebs ringer buffer with 1% fatty acid-free BSA containing 0.5 µCi/ml 14C-acetic acid ([1-14C] acetic acid, sodium salt; Amersham Biosciences) at 37°C. Explants were removed and washed, and the weight was determined. Lipids were extracted from the media as described above. The extracted lipids were diluted relative to the explant weight (equal volume per tissue weight) and analyzed by HPTLC as described above. The free level of choline in the Harderian gland was measured using a Choline/Acetylcholine Quantification Kit (MAK056-1KT; Sigma). Harderian glands from adult mice were homogenized in supplied assay buffer, and the level of choline was determined according to the manufacturer by fluorometry using a FLOUstar Omega fluorescence plate reader (BMG Labtech) and normalized to protein content as determined by a PierceTM BCA Protein Assay kit (23227; Thermo Scientific). Plasma from fed adult mice were collected and separated as described elsewhere (13Neess D. Bloksgaard M. Bek S. Marcher A.B. Elle I.C. Helledie T. Due M. Pagmantidis V. Finsen B. Wilbertz J. et al.Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.J. Biol. Chem. 2011; 286: 3460-3472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The triiodothyronine (T3) level was analyzed using a FLOUstar Omega fluorescence plate reader (BMG Labtech) and a mouse/rat T3 ELISA kit (T3043T-100, Calbiotech). ACBP−/− mice consistently display swollen eyelids and increased secretion from the eye cavity (Fig. 1A). This phenotype is visible from around weaning and persists throughout adulthood. Interestingly, when we examined the eye cavity of adult mice, we found that the lipid-producing Harderian gland is approximately twice as large in ACBP−/− mice compared with ACBP+/+ mice (Fig. 1B). Examination of the expression of ACBP in this gland showed that, similar to other lipogenic tissues (Fig. 1E) (4Neess D. Kiilerich P. Sandberg M.B. Helledie T. Nielsen R. Mandrup S. ACBP:a PPAR and SREBP modulated housekeeping gene.Mol. Cell. Biochem. 2006; 284: 149-157Crossref PubMed Scopus (33) Google Scholar, 30Alho H. Fremeau Jr, R.T. Tiedge H. Wilcox J. Bovolin P. Brosius J. Roberts J.L. Costa E. Diazepam binding inhibitor gene expression: location in brain and peripheral tissues of rat.Proc. Natl. Acad. Sci. USA. 1988; 85: 7018-7022Crossref PubMed Scopus (97) Google Scholar, 31Alho H. Harjuntausta T. Schultz R. Pelto-Huikko M. Bovolin P. Immunohistochemistry of diazepam binding inhibitor (DBI) in the central nervous system and peripheral organs: its possible role as an endogenous regulator of different types of benzodiazepine receptors.Neuropharmacology. 1991; 30: 1381-1386Crossref PubMed Scopus (63) Google Scholar), ACBP is expressed at very high levels in Harderian gland of ACBP+/+ mice. In contrast, ACBP is completely absent in the Harderian gland of ACBP−/− mice (Fig. 1C, D). In mice, the Harderian gland is not fully developed at birth. It becomes functional from around 14 days of age when the pups open their eyes; however, the gland continues to grow and mature until adulthood (32Shirama K. Hokano M. Electron-microscopic studies on the maturation of secretory cells in the mouse Harderian gland.Acta Anat. (Basel). 1991; 140: 304-312Crossref PubMed Scopus (12) Google Scholar). We found that the size of the gland is similar in ACBP−/− and ACBP+/+ mice until just before weaning at 21 days of age (Fig. 1F). However, in the week after weaning, the size of the Harderian glands of ACBP−/− mice doubles, whereas gland size in ACBP+/+ mice remains relatively constant. This is true both when gland size is normalized to body weight (Fig. 1F) and when it is expressed only as gland weight (supplementary Fig. 1). This indicates that weaning initiates systemic and/or endogenous signals in the gland that leads to a major and rapid hypertrophy of the gland, which is maintained in adulthood. The Harderian gland consists of a simple tubuloalveolar system with acinar cells arranged in acini (33Shirama K. Hokano M. Harderian glands and their development in laboratory rats and Mice.in: S. Webb Harderian Glands. R. Hoffman M. Puig-Domingo R. Reiter In Harderian Glands. Springer Berlin Heidelberg, 1992: 25-51Crossref Google Scholar). This overall structure is maintained in the ACBP−/− mice (Fig. 2A); however, the acinar cells from ACBP−/− mice are larger (Fig. 2C) and contain fewer (Fig. 2A, E, F) but significantly larger vesicles (Fig. 2D) compared with glands from ACBP+/+ mice. Furthermore, lipid staining with osmium tetroxide demonstrated that the vesicles and the lumen contained more lipid in the ACBP−/− glands compared with ACBP+/+ glands (Fig. 2B). These data indicate that lack of ACBP causes hypertrophy and increased activity of the Harderian gland. The increased size and lipid filling of the vesicles in the Harderian gland of ACBP−/− mice prompted us to investigate the lipid content of the gland. We therefore carefully dissected the gland and extracted the lipids. Using HPTLC, we found that the major overall change in lipid classes was a decrease in the amount of MADAG, the main lipid product of the Harderian gland (19Tvrzicka E. Rezanka T. Krijt J. Janousek V. Identification of very-long-chain fatty acids in rat and mouse harderian gland lipids by capillary gas chromatography-mass spectrometry.J. Chromatogr. A. 1988; 431: 231-238Crossref PubMed Scopus (16) Google Scholar, 20Kasama K. Blank M.L. Snyder F. Identification of 1-alkyl-2-acyl-3-(2′,3′- diacylglycerol)glycerols, a new type of lipid class, in harderian gland tumors of mice.J. Biol. Chem. 1989; 264: 9453-9461Abstract Full Text PDF PubMed Google Scholar), in glands from ACBP−/− mice compared with the glands from ACBP+/+ mice (Fig. 3A and data not shown). The fatty acids used for synthesis of MADAG originate primarily from de novo fatty acid synthesis in the gland (34Miyazaki M. Kim H.J. Man W.C. Ntambi J.M. Oleoyl-CoA is the major de novo product of stearoyl-CoA desaturase 1 gene isoform and substrate for the biosynthesis of the Harderian gland 1-alkyl-2,3-diacylglycerol.J. Biol. Chem. 2001; 276: 39455-39461Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 35Seyama Y. Kasama T. Yasugi E. Park S-H. Kano K. S.Webb HarderianGlands Hoffman R. Puig-Domingo M. Reiter R. In Lipids in Harderian Glands and Their Significance. Springer, Berlin, Heidelberg1992: 195-217Google Scholar). In vivo pulse-labeling with 14C-acetic acid showed that de novo synthesis of MADAG in Harderian glands is significantly higher in ACBP−/− mice compared with ACBP+/+ mice (Fig. 3B). However, this increase occurred in the absence of changes in the mRNA expression of key enzymes involved in synthesis of MADAG (35Seyama Y. Kasama T. Yasugi E. Park S-H. Kano K. S.Webb HarderianGlands Hoffman R. Puig-Domingo M. Reiter R. In Lipids in Harderian Glands and Their Significance. Springer, Berlin, Heidelberg1992: 195-217Google Scholar), such as dihydroxyacetone phosphate acyltransferase 1-alkyl-dihydroxyacetone phosphate synthase (supplementary Fig. 2A, B). Similarly, there was no increase in the expression of genes encoding key enzymes in the synthesis of fatty acids, such as fatty acid synthase or acetyl-CoA carboxylase (supplementary Fig. 2C, D). This indicates that the increase in the synthesis of MADAG is not driven by increased expression of enzymes involved in synthesis of fatty acids but rathe
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