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Articles

Targeted Disruption of Stearoyl-CoA Desaturase1 Gene in Mice Causes Atrophy of Sebaceous and Meibomian Glands and Depletion of Wax Esters in the Eyelid¹

Makoto Miyazaki^*, Weng Chi Man^* and James M. Ntambi

Departments of ^* Biochemistry and Nutritional Sciences, University of Wisconsin, Madison, WI 53706

²To whom correspondence should be addressed. E-mail: ntambi{at}biochem.wisc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Stearoyl-CoA desaturase (SCD) is a microsomal rate-limiting enzyme in the cellular synthesis of monounsaturated fatty acids (MUFA), mainly oleate (18:1) and palmitoleate (16:1), which are the major MUFA of membrane phospholipids, cholesterol esters and triglycerides. Three well-characterized isoforms of SCD, SCD1, SCD2 and SCD3, exist in mice. To investigate the physiologic functions of SCD1, we generated SCD1 null (SCD1-/-) mice. The skin and eyelid of SCD1-/- mice are deficient in triglycerides and cholesterol esters, and the eyelid also is deficient in wax esters. Furthermore, the eyelid and skin of SCD1-/- mice have higher levels of free cholesterol. SCD1-/- mice develop cutaneous abnormalities and narrow eye fissure with atrophic sebaceous and meibomian glands. Consumption of diets containing high levels of oleate, failed to restore the levels of triglycerides, cholesterol esters and wax esters in SCD1-/- mice to the levels found in the eyelid of wild-type mice. These results reveal a physiologic role of SCD in cholesterol homeostasis as well as in the de novo biosynthesis of cholesterol esters, triglycerides and wax esters required for normal skin and eyelid function.

KEY WORDS: • stearoyl-CoA desaturase • meibomian gland • sebaceous gland • wax ester • eyelid • mice

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Stearoyl-CoA desaturase (SCD)³ is a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids (MUFA). It catalyzes the 9-cis desaturation of acyl-CoA substrates; the preferred substrates are palmitoyl-CoA and stearoyl-CoA, which are converted to palmitoleyl-CoA and oleoyl-CoA, respectively (1) . The resulting MUFA are substrates for incorporation into membrane phospholipids, triglycerides and cholesterol esters. Studies in mice and rats have shown that SCD expression is highly regulated by diet, hormonal factors, developmental processes, temperature, metals, alcohol, peroxisomal proliferators and phenolic compounds, resulting in the alteration of the fatty acid composition of membrane phospholipids, triglycerides and cholesterol esters (2) . Effects on the composition of membrane phospholipids ultimately determine membrane fluidity, whereas the effects on the composition of cholesterol esters and triglycerides can affect lipoprotein metabolism and adiposity. Thus, the regulation of SCD is of considerable physiologic importance, and alteration in SCD activity has been implicated in a wide range of disorders including diabetes, atherosclerosis, cancer and obesity (2 3 4) .

A number of mammalian SCD genes have been cloned. Two SCD genes have been cloned in rats and four have been cloned from mice, three of which (SCD1, SCD2 and SCD3) are well characterized (3 ,5 6 7 8) . A single human SCD gene that is highly homologous to the rat and mouse SCD genes was cloned and characterized (9) . Despite the fact that the mouse, rat and human SCD genes are structurally similar, sharing 87% nucleotide sequence identity in the coding regions, some portions of their 5' flanking regions differ, resulting in divergent tissue-specific gene expression. In some mouse tissues such as the skin, the three SCD gene isoforms are expressed (8) . The physiologic importance of having two or more SCD isoforms expressed in the same tissue is not currently known but could be related to the substrate specificity of each SCD isoform or the means by which cells compartmentalize lipid biosynthesis for specific functions (10) .

The existence of multiple SCD genes in mouse and rat tissues makes it difficult to determine the role of each gene in lipid metabolism. Most previous studies have assessed SCD gene function by measuring mRNA expression but have not differentiated which SCD isoform is responsible for the altered total SCD activity. A clue to the physiologic role of the SCD1 gene and its endogenous products (MUFA) has come from recent studies of the asebia (ab^j) mutant mouse strain (11) , which has an extensive natural deletion in the SCD1 gene. Another mutant strain (ab^2j), which shows a splice junction deletion of exon 2 in the SCD1 gene and is allelic with the ab^j, has recently been described (12) . The homozygous ab^2j SCD1 mutant mouse is similar to the ab^j mouse except for the extent of epidermal thickness, scaling and epidermal permeability barrier as measured by transepidermal water loss methods (12) . The basis for the difference in phenotype between these two mutant mouse strains is not presently understood but has been attributed to modification of gene effects by strain background (12) . To further explore the physiologic roles of the SCD gene isoforms, we generated mice with a targeted disruption of the SCD1 gene. Our results indicate that SCD1 plays a major role in the de novo synthesis of triglycerides, cholesterol esters and wax esters required for normal skin and eyelid function.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Generation of the SCD1 knockout mice.

Mouse genomic DNA for the targeting vector was cloned from the 129/SV genomic library. The targeting vector construct was generated by insertion of a 1.8-kb Xba I/Sac I fragment with 3' homology as a short arm and 4.4-kb Cla I/Hind III fragment with 5' homology cloned adjacent to the neo expression cassette. The construct also contains a herpes simplex virus thymidine kinase cassette 3' to the 1.8-kb homology arm, allowing positive/negative selection. The targeting vector was linearized by Not I and electroporated into embryonic stem cells. Selection with geneticin and gancyclovior was performed. The clones resistant to both geneticin and gancyclovior were analyzed by Southern blot after EcoRI restriction enzyme digestion and hybridized with a 0.4-kb probe located downstream of the vector sequences. For polymerase chain reaction (PCR) genotyping, genomic DNA was amplified with primer A (5'-GGGTGAGCATGGTGCTCAGTCCCT-3'), which is located in exon 6, primer B (5'-ATAGCAGGCATGCTGGGGAT-3'), which is located in the neo gene (425-bp product, targeted allele) and primer C (5'-CACACCATATCTGTCCCCGACAAATGTC-3'), which is located downstream of the targeting gene (600-bp product, wild-type allele). PCR conditions were 35 cycles, each of 45 s at 94°C, 30 s at 62°C and 1 min at 72°C. The targeted cells were microinjected into C57Bl/6 blastocysts to generate the chimeric mice. The chimeric mice were then crossed with 129/SvEv Taconic females to generate SCD1-/- mice having a genetic background of SV129. Mice were maintained on a 12-h dark/light cycle and were fed a nonpurified diet (5008 test diet; PMI Nutrition Internatinal, Richmond, IN; hyyp://www.labdiet.com/5008.htm) or semipurified diets containing 5 g/100 g soybean oil (control diet) or a high oleate oil (high 18:1 diet) (4) . The semipurified diet was purchased from Harlan Teklad (Madison, WI) and contained (per 100 g diet): 20 g vitamin-free casein, 5.0 g soybean oil or high oleate oil, 0.35 g L-cystine, 13.2 g Maltodextrin, 51.7 g sucrose, 5.0 g cellulose, 3.5g mineral mix (AIN-93) (13) , 1.0 g vitamin mix (AIN-93) (13) and 0.3 g choline bitartrate. The fatty acid composition of the experimental oils was determined by gas-liquid chromatography (GLC). The soybean oil contained (per 100 g oil): 11 g palmitic acid (16:0), 23 g oleic acid [18:1(n-9)], 53 g linoleic acid [18:2(n-6)] and 8 g linolenic acid [18:3(n-3)]. The high oleate oil contained (per 100 g oil): 7 g 16:0, 50 g 18:1(n-9), 35 g 18:2(n-6) and 5 g 18:3(n-3). Homozygous (SCD1-/-), heterozygous (SCD1+/-) and wild-type (SCD1+/+) mice were housed and bred in a pathogen-free barrier facility of the department of Biochemistry operating on a 12-h light:dark cycle. The breeding of these mice is in accordance with the protocols approved by the animal care research committee (ACRC) of the University of Wisconsin-Madison.

Materials.

Radioactive [-³²P]dCTP (111 TBq/mmol) was obtained from Dupont (Wilmington, DE). TLC plates (TLC Silica Gel G60) were from Merck (Darmstadt, Germany). [1-¹⁴C]-stearoyl-CoA was purchased from American Radiolabeled Chemicals (St Louis, MO). Immobilon-P transfer membranes were from Millipore (Danvers, MA). Enhanced chemiluminescence (ECL) Western blot detection kit was from Amersham-Pharmacia (Piscataway, NJ). All other chemicals were purchased from Sigma (St Louis, MO).

Lipid analysis.

Total lipids were extracted from tissues according to the method of Bligh and Dyer (14) , and phospholipids, wax esters, free cholesterol, triglycerides and cholesterol esters were separated by silica gel high performance TLC (HPTLC). Petroleum hexane/diethyl ether/acetic acid (80:30:1, v/v/v) or benzene/hexane (65:35, v/v) was used as a developing solvent (15) . Spots were visualized by 5.0 mmol/L 2',7'-dichlorofluorescein in 95% ethanol or by 0.63 mol/L cupric sulfate containing 0.89 mol/L phosphoric acid. The wax ester, cholesterol ester and triglyceride spots were scraped, 1 mL of 5% HCl-methanol added and heated at 100°C for 1 h (4) . The methyl esters were analyzed by GLC using cholesterol heptadecanoate, triheptadecanoate and heptadecanoic acid as internal standards. Free cholesterol (Free Choleterol C, Wako Chemicals, Japan), total cholesterol (Cholesterol CII, Wako) and triglyceride [Triglycerides (INT) 20, Sigma] contents of eyelid and skin were determined by enzymatic assays.

Isolation and analysis of RNA.

Total RNA was isolated from livers using the acid guanidinium-phenol-chloroform extraction method (16) . Total RNA (20 µg) was separated by 1.0% agarose/2.2 mol/L formaldehyde gel electrophoresis and transferred onto nylon membrane. The membrane was hybridized with ³²P-labeled SCD1 and SCD2 probes. A pAL15 probe was used as control for equal loading (4) .

SCD activity assay.

Stearoyl-CoA desaturase activity was measured in liver microsomes essentially as described (17) . Tissues were homogenized in 10 volumes of 100 mmol/L potassium buffer, pH 7.4. The microsomal membrane fractions (100,000 x g pellet) were isolated by sequential centrifugation. Reactions were performed at 37°C for 5 min with 100 µg of protein homogenate and 60 µmol/L of [1-¹⁴C]-stearoyl-CoA (60,000 dpm), 2 mmol/L of NADH and 100 mmol/L of Tris/HCl buffer (pH 7.2). After the reaction, fatty acids were extracted and then methylated with 5% HCl/methanol. Saturated fatty acid and MUFA methyl esters were separated by 100 g/L AgNO₃-impregnated TLC using hexane/diethyl ether (9:1) as developing solution. The plates were sprayed with 5.0 mmol/L 2',7'-dichlorofluorescein in 95% ethanol and the lipids were identified under UV light. The fractions were scraped off the plate, and the radioactivity was measured using a liquid scintillation counter. The enzyme activity was expressed as nmol/(min · mg protein).

Immunoblotting.

Pooled liver membranes from three mice of each group were prepared as described (18) . The same amount of protein (25 µg) from each fraction was subjected to 100 g/L SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P transfer membranes at 4°C. After blocking with 10 g/100 mL nonfat milk in Tris buffered saline buffer (pH 8.0) plus Tween at 4°C overnight, the membrane was washed and incubated with rabbit anti-rat SCD as primary antibody and goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate as the secondary antibody. Visualization of the SCD protein was performed with an ECL Western blot detection kit (Amersham-Pharmacia).

Histology.

Tissues were fixed with neutral-buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin as described (19) .

Statistical analysis.

Stastistical analysis of the data was carried out using StaView (Abacus Concepts, Berkeley, CA). Data were analyzed using Student’s t test or one-way ANOVA followed by Fisher’s least significant difference test. A difference of P < 0.05 was considered significant. Values are presented as means ± SD.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Targeted disruption of the SCD1 gene.

Figure 1A shows the strategy that was used to disrupt the SCD1 gene. The mutant mice were viable and fertile, and bred with predicted Mendelian distributions. PCR of genomic DNA using the designated primers showed a band of 425 bp (Fig. 1 B) that would be expected in the SCD1-/- mice. The SCD1+/- DNA showed both the 425- and 600-bp fragments, whereas the wild-type DNA showed the expected 600-bp fragment. To determine whether the expression of the SCD1 gene was ablated, we performed Northern blot analysis of mRNA isolated from several tissues (Fig. 1 C). SCD1 mRNA was expressed in liver, eyelid, white adipose tissue (WAT) and skin of wild-type mice, reduced by 50% in SCD1+/- mice and undetectable in the SCD1-/- mice. SCD2 mRNA was expressed at similar levels in the eyelid, WAT, skin, brain and eyeball of both SCD1-/- mice and wild-type mice but undetectable in the liver, consistent with published results (2) . SCD enzyme activity in liver, as measured by the rate of conversion of [1-¹⁴C]stearoyl-CoA to [1-¹⁴C]oleate (Fig. 1 D), was high in the wild-type mice, intermediate in the SCD1+/- and undetectable in the total extracts of livers of the SCD1-/- mice. Consistent with the Northern blot results, Western blot analysis showed high immunoreactive SCD protein in liver from the wild-type, intermediate in the SCD1+/- and none in the SCD1-/- mice (Fig. 1 D). Table 1 shows the fatty acid composition of liver, eyelid, WAT, skin, brain and eyeball. The relative amount of palmitoleate [16:1(n-7)] in liver from SCD1-/- mice was 55% lower (P < 0.05) than in wild-type mice, whereas that of oleate [18:1(n-9)] was 35% lower (P < 0.05). The relative levels of palmitoleate in WAT and skin of the SCD1-/- mice were >70% lower than in wild-type mice (WAT, P < 0.01; skin, P < 0.01), whereas the decrease in the level of oleate in these tissues was 20% (WAT, P < 0.001; skin, P < 0.05). These changes in the levels of MUFA resulted in greater levels of the corresponding saturated fatty acids (18:0 and 16:0) and lower desaturation indices, indicating a reduction in desaturase activity. The relative levels of 16:1(n-7) and 18:1(n-9) in liver, eyelid, WAT and skin of SCD+/- mice were between those of wild-type and SCD1-/- mice. In contrast to the liver, WAT and skin, the brain, which expresses predominantly the SCD2 isoform, had a similar fatty acid composition and unaltered desaturation index both in the wild-type and SCD1-/- mice. We conclude that our disruption resulted in mice null for the SCD1 gene and that the expression of SCD2 isoform does not compensate for SCD1 deficiency.

View larger version (21K): [in this window] [in a new window]   Figure 1. Generation of the SCD1 null mice. (A) Targeting strategy for disruption of the SCD1 gene. A neomycin-resistant cassette replaced the 6 exons of the gene by homologous recombination, resulting in the replacement of the complete coding region of the SCD1 gene. Gene-targeting events were verified by Southern blot analysis using EcoRI and probe 1 or 2 or by polymerase chain reaction (PCR) analysis. (B) PCR analysis of DNA isolated from different genotypes of F1 offspring using appropriate primers. The 425-bp fragment detected represents the mutated allele. In breeding heterozygotes, wild type, heterozygotes and homozygotes were born in Mendelian fashion (+/+:+/-:-/- = 21:43:20). (C) Northern blot analysis for the expression of SCD1 and 2 mRNA. Total RNA (20 µg) isolated from the liver, eyelid, white adipose tissue (WAT), brain, eyeball and skin of wild-type, SCD1+/- and SCD1-/- mice pooled from 5 mice of each group was subjected to Northern blot analysis followed by hybridization with labeled probes specific for SCD1 and SCD2 cDNA. A cDNA probe for pAL15 (16) was used to confirm equal loading. (D) SCD enzyme activity and immunoblot analysis of SCD from livers of wild-type, SCD1+/- and SCD1-/- mice. For enzyme activity, aliquots of microsome fraction (100 µg) from livers of each group were incubated with a reaction mixture containing [1-14C]stearoyl-CoA for 5 min. The products were saponified and acidified and the fatty acids were extracted and separated by TLC. Enzyme activity is represented as nanomoles of substrate desaturated per milligram of protein per minute. Data are denoted as the mean ± SD (n = 3). For immunoblot analysis, aliquots of membrane fraction (80 µg) from pooled livers of each group were subjected to 10% SDS polyacrylamide gel electrophoresis followed by detection with SCD antibody.

The SCD1-/- mice exhibited cutaneous abnormalities and a narrow eye fissure, which started around the weaning age (3–4 wk). The abnormalities became more severe with aging. The SCD1-/- mice had a thinner hair coat than the wild-type control mice. The hair loss in the SCD1-/- mice was apparent throughout the whole skin area. Histological examination of the skin of the SCD1-/- mice revealed atrophy of the sebaceous glands, whereas the wild-type mice had prominent and well-differentiated sebaceous glands (compare Fig. 2A for wild-type with Fig. 2 B for SCD1-/-). Figure 2 C is a higher magnification of the sebaceous gland of a wild-type mouse, showing a foamy appearance of the cytoplasm due to the presence of lipid droplets, the sebum. In contrast, at a higher magnification, the cytoplasm of the atrophic sebaceous gland in the SCD1-/- mouse had lost the foamy appearance (Fig. 2 D), suggesting depletion of sebum lipids. The SCD1-/- mice had narrower eye fissures than wild-type mice. Histological examination of the eyelid of the SCD1-/- mice revealed atrophy of the meibomian gland, a specialized sebaceous gland (compare Fig. 3B for the SCD1-/- mice with 3A for wild type) that secretes meibum, the oily material that prevents the corneal surface of the eye from drying out. At a higher magnification (Fig. 3 C), the cytoplasm of the meibomian gland of the wild-type control mouse appears foamy due to the presence of meibum, whereas the meibomian gland of the SCD1-/- mouse lacks the foamy appearance (Fig. 3 D) due to depletion of the meibum lipids. No abnormalities were found in cornea and retina of the SCD1-/- mice (data not shown).

View larger version (131K): [in this window] [in a new window]   Figure 2. Histology of skin of wild-type and SCD1-/- mice. The mice were 2-mo-old males. (A) Hematoxylin and eosin (H&E) staining of a wild-type mouse shows normal epidermal structure with a well-differentiated sebaceous gland (arrowhead). (B) H&E staining of skin of a SCD1-/- mouse shows atrophic sebaceous gland (arrowhead). (C) Higher magnification (x400) of the sebaceous gland of the wild-type mouse. Note the foamy appearance of the cytoplasm. (D) Higher magnification (x400) of the sebaceous gland of the SCD1-/- mouse showing atrophic sebaceous gland. The foamy appearance of the cytoplasm seen in the wild type has disappeared.

View larger version (157K): [in this window] [in a new window]   Figure 3. Histology of the eyelid of wild-type and SCD1-/- mice. The mice were 2-mo-old males. (A) Hematoxylin and eosin (H&E) staining of a wild-type mouse shows a normal well-differentiated meibomian gland (arrowhead). Intralobular duct (d). (B) H&E staining of the eyelid of a SCD1-/- mouse shows atrophic meibomian glands (arrowhead). (C) Higher magnification (x400) of the meibomian gland of the wild-type mouse. Note the foamy appearance of the cytoplasm. (D) Higher magnification (x400) of the atrophic meibomian gland of the SCD1-/- mouse. The foamy appearance observed in the wild-type meibomian gland has disappeared.

We measured free cholesterol, cholesterol ester, triglycerides and wax ester content in the skin and eyelid. HPTLC of lipids extracted from the skin of wild-type mice showed that the skin contained very high levels of triglycerides (Fig. 4 A), whereas the eyelid was very rich in wax esters (Fig. 4 C). The triglycerides, cholesterol ester, and wax ester levels were markedly reduced in both the skin and eyelid of the SCD1-/- mice compared with the wild-type control mice. Intermediate levels were observed in the lipids of the heterozygote mice (SCD1+/-). A separate TLC analysis using a solvent system consisting of hexane/benzene (45:65, v/v) was performed to separate (based on R_f values) cholesterol esters from wax esters and also to resolve the wax diesters and triesters (15) . As shown, the diester was the major wax ester in the eyelid (Fig. 4 D). The skin also contained wax diesters (Fig. 4 B) but at much lower levels than the eyelid diesters. The cholesterol ester concentrations in eyelid and skin of SCD1-/- mice were 73% (P < 0.001) and 43% (P < 0.05) lower, respectively, than in wild-type mice, whereas free cholesterol levels in skin and eyelid were 57% (P < 0.001) and 93% (P < 0.001) greater, respectively (Table 2 ). The total wax ester concentration in the eyelid of the SCD1-/- mice compared with the wild-type mice was 72% lower (P < 0.001). The total wax ester concentration of the skin was too low for accurate measurement. The triglyceride concentrations in the skin and eyelid were 53% (P < 0.001) and 60% (P < 0.001) lower, respectively, than in wild-type mice.

View larger version (53K): [in this window] [in a new window]   Figure 4. High performance TLC (HPTLC) of lipids extracts from skin (A and B) and eyelids (C and D) of wild-type and SCD1-/- mice. Total lipid extracts were pooled and analyzed by HPTLC. Equivalent amounts of lipid extract (from 0.5 mg of eyelid or skin) were loaded in each lane. Each lane represents lipids from eyelid and skin samples of two mice. In A and C, the solvent system was hexane/ether/acetic acid (90:25:1). In B and D, the solvent system used to resolve the wax esters was benzene/hexane (65:35). Std, standards.

View larger version (18K): [in this window] [in a new window]   Figure 5. The concentrations of monounsaturated fatty acids in the total lipid fraction, the cholesterol ester, wax ester, and triglyceride fractions (A) and in the phospholipid fraction (B) of eyelid of wild-type and SCD1-/- mice. Lipid extracts in (A) were pooled and analyzed by TLC, methyl esterified and quantitated by gas-liquid chromatography (GLC). Values are means ± SD, n = 3. *Significantly different (P < 0.05) from wild-type mice.

High levels of dietary 18:1(n-9) did not alter triglyceride, cholesterol ester and wax ester levels in the eyelid of SCD1-/- mice.

The cellular oleate or palmitioleate used for cholesterol ester, triglyceride and wax ester synthesis in mouse eyelid can be synthesized either de novo or by desaturation of exogenous stearate or palmitate derived from the diet. To determine whether dietary oleate could substitute for the endogenously synthesized oleate and restore the levels of triglycerides, esters and wax esters to the levels found in the eyelid of the wild-type mouse control, we supplemented the semipurified mouse diet with high levels of oleate as triolein. This diet was then fed to the SCD1-/- mice for 2 wk, long enough to ensure equilibration of lipid pools. Total eyelid extracts were prepared; the lipid fractions were analyzed by TLC and the fatty acid composition was analyzed by GLC. Feeding diets supplemented with triolein to the SCD1-/- mice did not increase the levels of triglycerides, cholesterol esters or wax esters (Fig. 6A ). GLC analysis also showed that the levels of 18:1(n-9) were not increased in the total lipid fraction of the eyelid (Fig. 6 B). The mice maintained the eye fissures and the thin hair coat phenotype of the SCD1-/- mice that consumed the semipurified diet. No changes in phenotype or in levels of neutral lipids were observed when the SCD1-/- mice were fed diets rich in 16:1 or a combination of 18:1 and 16:1 (data not shown). These results suggest that the endogenously synthesized 18:1 or 16:1 by SCD constitute a pool of MUFA required for the synthesis of the eyelid or skin cholesterol esters and triglycerides and further confirm the requirement for these fatty acids in the synthesis of eyelid wax diesters.

View larger version (17K): [in this window] [in a new window]   Figure 6. Lipids and monounsaturated fatty acid concentrations in eyelids of wild-type and SCD1-/- mice fed high oleate diets. At 6 wk of age, the mice were fed a control (5 g soybean oil/100 g) diet or high oleate (5 g 18:1/100 g) diet for 2 wk. (Panel A) Total lipids were extracted from eyelids of wild-type and SCD1-/- mice. Lipid extracts were analyzed by high performance TLC (HPTLC). Equivalent amounts of lipid extract (from 0.5 mg eyelid) were loaded in each lane. Each lane represents lipids from eyelid of one mouse. Std; standards. (Panel B) Total lipids were extracted from 2 mg of harderian gland. The monounsaturated fatty acid contents were quantified by gas-liquid chromatography. Values are means ± SD, n = 3. Means with a different superscript letter are significantly different (P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To further explore the physiologic roles of the SCD gene isoforms and to reveal other phenotypes not previously characterized in the ab^j and ab^j2 mice, we generated mice with a targeted disruption of the SCD1 gene. Like the ab^j and ab^2j, the SCD1-/- mice exhibited cutaneous abnormalities with atrophic sebaceous gland and narrow eye fissure with atrophic meibomian glands. However, unlike the asebia (ab^j) and ab^2j, which have reduced levels of cholesterol in their skin, the skin and eyelid of our SCD1-/- mice had significantly greater levels of free cholesterol than the wild-type mice. The SCD-/- mice, like the ab^j mice, had decreased concentrations of liver cholesterol and triglyceride, as well as plasma VLDL-triglycerides (data not shown). However, differences were noted in the levels of lipoproteins compared with the previously reported plasma lipoprotein profile of ab^j mice (4) and these will be reported elsewhere. These differences could also be due to mouse strain backgrounds modifying gene effects. In this study, a more extensive analysis of the changes in lipid and fatty acid composition of several tissues was carried out. Figures 4 5 6 and Table 2 show that the SCD1-/- mice were deficient in eyelid triglycerides, cholesterol esters and wax esters. These deficiencies in the lipid pools can be attributed to decreases in the endogenous levels of 18:1 and 16:1. The increase in the levels of 18:2(n-6) observed in the phospholipid fraction (Fig. 5 B) indicates that SCD1 deficiency alters the acyl chain composition of membrane phospholipids.

The cutaneous abnormalities in the SCD1-/- mice as well as those observed in the ab^j and ab^2j mice are similar to those reported in transgenic mice overexpressing human apolipoprotein (apo)CI (21) and in mice deficient in acyl-CoA:cholesterol acyl transferase enzyme (ACAT-1) (19) . ApoC1 activates lipoprotein lipase to facilitate VLDL clearance from plasma, but an excess of apoC1 has been shown to impair hepatic uptake of VLDL (21) . Like the SCD-/- mice (Figs. 2 , 3) , apoCI mice exhibit atrophic sebaceous and meibomian glands as well as a decrease in wax diester and triglycerides of the epidermis. The skin of mice overexpressing apoC1 also has increased levels of free cholesterol but unlike our SCD1-/- mice, the cholesterol ester levels in these tissues are not altered. Like the SCD-/- mice, the ACAT-1–deficient mice are deficient in cholesterol ester and have more free cholesterol in the eyelid with atrophy of the meibomian glands. The deficiency in cholesterol ester of the SCD1-/- mice is most likely due to lack of MUFA produced by SCD as substrates of ACAT, but the relationship between endogenously synthesized MUFA and the phenotype of mice with high expression of apoC1 is less clear. However, the phenotypic feature that the SCD1-/-, ACAT-1-/- and mice overexpressing human apoC1 have in common is the increased levels of free cholesterol either in the eyelid or skin or both. Because excess free cholesterol can lead to cell death (22) , it is tempting to speculate that atrophy of the sebaceous and meibomian glands may be due to an increase in the amount of cellular free cholesterol in these glands rather than the reduced levels of sebum and meibum.

We hypothesized that feeding high levels of dietary oleate and palmitoleate could correct the deficiency in triglyceride, cholesterol ester and wax ester in the eyelids of SCD1-/- mice. However, upon supplementing the semipurified diets with high levels of oleate as triolein and feeding this diet to the SCD1-/- mice for 2 wk, we found no increase in the levels of triglycerides, cholesterol esters or wax esters (Fig. 6 A). GLC analysis also showed that the levels of 18:1(n-9) were not increased in the total lipid fraction of the eyelid (Fig. 6 B). Dietary oleate could not increase the levels of wax diester, cholesterol ester and triglyceride in the skin of the SCD1-/- mice (data not shown). No changes in phenotype were observed when the SCD1-/- mice were fed diets rich in 16:1 or a combination of 18:1 and 16:1. These feeding experiments argue against the idea that dietary intake is ultimately the major lipid source of the MUFA that are incorporated into cellular neutral lipids. The present data are consistent with our previous concept (4) that endogenously synthesized oleate and palmitoleate, which arise from SCD activity in the endoplasmic reticulum, provide the bulk of the monounsaturated fatty acid pool and are preferred substrates for the enzymes of triglyceride, cholesterol and wax ester biosynthesis. It is possible that dietary MUFA and the SCD-synthesized MUFA are functionally compartmentalized into distinct lipid pools, thereby suggesting a mechanism of neutral lipid biosynthesis that is unknown at this time. The deficiencies in the neutral lipids exist despite the expression of the SCD2 gene in the eyelid of SCD1-/- mice (Fig. 1 C), suggesting that SCD2 cannot compensate for the SCD1 deficiency in this tissue.

It was reported recently that chronic blepharitis and dry eye syndrome, which constitute one of the most common and frustrating eye disease conditions in humans, are due to lipid abnormalities in meibum (23 24 25 26) , but the nature of these lipid abnormalities has not been well characterized. It has been found, however, that meibum from patients with meibomian keratoconjunctivitis has decreased levels of oleic acid, a major product of SCD, whereas that from patients with meibomian seborrhea has increased levels of oleic acid (26) . These observations, together with our present study, suggest that the alteration of SCD activity in the eyelid can be implicated in human eye diseases. Meibum is a mixture of triglycerides, cholesterol esters and wax esters. It is possible that these neutral lipids synergistically endow this semiliquid mixture with the physical properties, such as correct viscosity and melting point, necessary to prevent the corneal surface of the eye from drying out by retarding evaporation of tear moisture. Thus, the deficiencies in meibum, together with the dysfunction of meibomian glands, are most likely the cause of the narrow eye shape or dry eye syndrome observed in SCD1-/- mice and humans, respectively. The eyelids of SCD1-/- mice deficient in meibum have more frequent bacterial infections than those of wild-type control mice, suggesting that eyelid meibum may be toxic to some pathogenic microorganisms, although this property remains to be established.

In conclusion, the characterization of the targeted SCD knockout mouse we have generated allows another experimental model, in addition to the naturally occurring models of SCD1 disruption, with which to study lipid metabolism in both normal and disease states. The studies reported here provide substantial evidence that SCD1 gene expression plays a major role in triglyceride, cholesterol ester and wax ester biosynthesis in the skin and eyelid. The enzymes that synthesize triglycerides, cholesterol esters and wax esters require 18:1 and 16:1, the endogenous products of SCD, as substrates. Thus, the SCD1 gene may be a major checkpoint in the processes of cholesterol homeostasis, lipoprotein and neutral lipid metabolism. The ocular complications exhibited by the SCD1 knockout mouse and the failure of high levels of dietary MUFA to repair these defects strongly suggest differences in the metabolic functions of de novo synthesized and dietary MUFA. The studies described here may have broad implications for potential use of the SCD1 gene as a target in the treatment of human eye and skin diseases.

left rule

	ACKNOWLEDGMENTS

 
We thank Juris Ozols (University of Connecticut Health Center, Farmington, CT) for the antibody to the rat liver microsomal stearoyl-CoA desaturase. We thank Alan Attie for useful discussions.

	FOOTNOTES

 
¹ Supported by American Heart Association and by NIEHS-National Institutes of Health training grant ES09090-04 to the University of Wisconsin-Madison EHS Center for Developmental & Molecular Toxicology.

³ Abbreviations used: ACAT, acyl-CoA:cholesterol acyl transferase enzyme; apo, apolipoprotein; ECL, enhanced chemiluminescence; GLC, gas-liquid chromatography; HPTLC, high performance TLC; MUFA, monounsaturated fatty acids; PCR, polymerase chain reaction; SCD, stearoyl-CoA desaturase; TBS, Tris buffered saline; WAT, white adipose tissue; +/+, wild-type; +/-, heterozygous; -/- homozygous.

Manuscript received April 5, 2001. Initial review completed May 15, 2001. Revision accepted June 23, 2001.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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