Original Article

Journal of Investigative Dermatology (1998) 111, 139–148; doi:10.1046/j.1523-1747.1998.00246.x

The Activity of HMG-CoA Reductase and Acetyl-CoA Carboxylase in Human Apocrine Sweat Glands, Sebaceous Glands, and Hair Follicles Is Regulated by Phosphorylation and by Exogenous Cholesterol

Cheryl D W Smythe, Michael Greenall* and Terence Kealey

  1. Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, U.K.;
  2. *Department of Surgery, The John Radcliffe Hospital, Oxford, U.K.

Correspondence: Dr Cheryl Smythe, 1Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR, U.K.

Received 18 October 1997; Revised 3 February 1998; Accepted 26 February 1998.

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Abstract

Human apocrine and sebaceous glands function to secrete lipids, predominantly triglycerides, fatty acids, cholesterol and its esters, and, in the sebaceous gland, squalene. The enzymes that catalyze the important regulatory steps in cholesterol and fatty acid biosyntheses, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase, respectively, were therefore studied in isolated human skin appendages, and their relevant kinetic parameters determined. The enzyme activities that were observed can account for previously described rates of incorporation of radiolabeled substrates into the appropriate lipids by glands in vitro. Reduced enzyme activities following homogenization in the presence of fluoride indicated that both of these enzymes in skin appendages are inactivated by phosphorylation. The activity of the enzyme known to catalyze this phosphorylation, the AMP-activated protein kinase, was also measured. Compactin was shown to inhibit HMG-CoA reductase in homogenates of these appendages. Conversely, incubation of whole sebaceous glands with compactin resulted in the stimulation of enzyme activity, which suggests that these appendages can respond to diminishing cholesterol levels. The effect of exogenous low density lipoprotein and 25-hydroxycholesterol on HMG-CoA reductase activity from skin appendages was investigated. HMG-CoA reductase activity in both apocrine and sebaceous glands was reduced following incubation with either low density lipoprotein or 25-hydroxycholesterol. Low density lipoprotein receptor and lipoprotein lipase mRNA expression was also detected in skin appendages. These results indicate that apocrine and sebaceous glands have the capacity to sequester dietary cholesterol and fatty acids that may have important implications for the understanding of both acne and axillary odor.

Keywords:

AMP-activated protein kinase, LDL, lipogenesis

Abbreviations:

AMPK, AMP-activated protein kinase; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; 25-HC, 25-hydroxycholesterol; WEM, William's E medium

The major, if not sole function of human apocrine sweat glands and sebaceous glands is lipid secretion. Apocrine lipid is associated with the generation of axillary odour (Shelley et al. 1953), whereas sebaceous lipid appears to be important in the pathogenesis of acne vulgaris (Pochi & Strauss 1964;Cunliffe & Shuster 1969). Given the clinical significance of apocrine and sebaceous lipid secretion, it is therefore important to understand the factors that regulate the production of these lipids.

The human hair follicle also contains lipids. Although present in small quantities, both cholesterol and cholesterol sulfate are integral to the cell membrane complex, where they are important for the structural stability of the hair as shown in wool (Brosche et al. 1994). Low concentrations of these lipids is associated with poor hair stability as observed in Werner's syndrome (Brosche et al. 1994). A knowledge of cholesterol metabolism within the hair follicle would therefore be useful in understanding its biology.

Analysis of skin surface lipids from different body sites has shown that sebum is a complex mixture of lipids, the major components of which are triglycerides, free fatty acids (57.5%), wax esters (26%), squalene (12%), and cholesterol and its esters (4.5%) (Greene et al. 1970). Freshly secreted apocrine sweat contains cholesterol (76%), fatty acids (19.2%), and wax esters (3.6%) (Leyden et al. 1981).

[14C]Acetate incorporation studies have shown that isolated apocrine and sebaceous glands can synthesize these lipids de novo (Guy et al. 1996;Barth et al. 1989); however, the central lipogenic enzymes have not been thoroughly investigated. Furthermore, it is not clear whether, in situ, skin appendages either synthesize these lipids de novo, sequester circulating lipids, or exhibit some combination of both. We have therefore characterized the important regulatory enzymes of cholesterol and fatty acid biosyntheses, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase, respectively (Rodwell et al. 1976;Wakil et al. 1983), in isolated human apocrine sweat glands, sebaceous glands, and hair follicles.

HMG-CoA reductase and acetyl-CoA carboxylase are both regulated by complex mechanisms, some of which are co-ordinated. In rat liver the AMP-activated protein kinase (AMPK), for example, catalyzes the phosphorylation of a specific serine residue within each of the enzymes, which results in their inactivation. Reactivation is catalyzed by protein phosphatase 2A (reviewed in Hardie 1992). Furthermore, both enzymes are regulated by exogenous cholesterol (Goldstein & Brown 1990;Lopez et al. 1996). In most cells, cholesterol may be biosynthesized de novo or exogenous cholesterol in the form of low density lipoprotein (LDL) may be taken up via the LDL receptor. When cholesterol is abundant, HMG-CoA reductase is downregulated at the levels of gene expression, translation, and degradation (Brown & Goldstein 1986). The cholesterol downregulation of acetyl-CoA carboxylase also occurs at the level of gene expression (Lopez et al. 1996). Acetyl-CoA carboxylase activity is further modulated allosterically by citrate, and by fatty acyl esters that are derived from triglycerides through the action of lipoprotein lipase (Wakil et al. 1983).

In this study, we have measured relevant kinetic parameters and major regulatory mechanisms of HMG-CoA reductase and acetyl-CoA carboxylase in skin appendages. Together these data suggest that HMG-CoA reductase and acetyl-CoA carboxylase from human skin appendages are very similar, if not identical, to those isolated from other species and human sources. We have also exploited the sterol regulation of HMG-CoA reductase to determine whether skin appendages have the capacity to sequester exogenous cholesterol. These experiments suggest that apocrine and sebaceous glands, but not hair follicles, can sequester cholesterol from their environment. The identification of lipoprotein lipase mRNA in both apocrine and sebaceous glands indicates that both glands can also sequester exogenous fatty acids.

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MATERIALS AND METHODS

Materials

William's E medium, L-glutamine, trace element mix, penicillin, and streptomycin were supplied by Gibco (Romford, Essex, U.K.). Dulbecco's phosphate buffered saline and Earle's medium were from Sigma (Poole, Dorset, U.K.). Thin layer chromatography plates were obtained from Whatman (Maidstone, Kent, U.K.) and all radiochemicals were from Amersham (Slough, Berks, U.K.). Kyro EOB was a very kind gift from Dr. B. Middleton (Department of Biochemistry, Nottingham University, U.K.). All solvents were supplied by BDH (Lutterworth, Leics, U.K.). LDL was a very generous gift from Ms. C. Marchant and Ms. N. Law (Dr M. Mitchinson's laboratory, Department of Pathology, Cambridge University, U.K.). Compactin was obtained from Fluka Chemicals (Gillingham, Dorset, U.K.). Super RT enzyme was supplied by HT Biotechnology (Cambridge, U.K.), dNTP were from Pharmacia (St. Albans, Herts, U.K.), Taq from Bioline (London, U.K.), and oligo dTs from Promega (Southampton, U.K.). All other reagents were from Sigma. All reagents used were of the highest grade available.

Isolation of apocrine glands

Thin pieces of apparently normal axillary skin were taken during staging for breast cancer from female patients aged from 35 to 60, at the John Radcliffe Hospital, Oxford. Ethical committee permission was obtained. After removing excess subcutaneous fat, the skin was placed in Dulbecco's phosphate buffered saline (PBS) and examined using an Olympus binocular dissecting microscope. Large apocrine glands within the fat layer were microdissected out with a scalpel blade and a pair of stainless steel Ideal-tek no. 5 microforceps, and placed in PBS Figure 1a). The remainder of the skin was cut into pieces 0.5 cm2, PBS was added, and the skin was sheared with sharp scissors (Kealey et al. 1986). Apocrine glands were picked out, snap frozen, and stored in liquid nitrogen unless stated otherwise.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Isolated human skin appendages. Apocrine gland (a), sebaceous gland (b), and hair follicles (c) were isolated from human skin as described in Materials and Methods. Scale bars: 0.2 mm.

Full figure and legend (128K)

Where appropriate, apocrine glands were maintained at 37°C in a humidified 5% CO2:95% air atmosphere, on polycarbonate filters in William's E medium (WEM) supplemented with 100 U penicillin per ml, 100 mug streptomycin per ml, 2 mM glutamine, 2% fetal calf serum, 10 mug insulin per ml, and 10 ng hydrocortisone per ml, unless stated otherwise.

Isolation of sebaceous glands

Pieces of apparently normal skin were obtained from female patients aged between 18 and 50 undergoing breast reduction surgery at Addenbrooke's Hospital, Cambridge. Pieces of 2 cm2 were placed in a Petri dish and covered with PBS. Sebaceous glands Figure 1b) were microdissected out and placed in Earle's medium prior to snap freezing as described for apocrine glands.

Sebaceous glands were also isolated from thin pieces of apparently normal midline chest skin obtained from male and female patients aged between 20 and 75 undergoing cardiac surgery at the Papworth Hospital, Papworth. Ethical committee permission was obtained. These glands were isolated by shearing (Kealey et al. 1986) and snap frozen as described above.

Where appropriate, sebaceous glands were maintained at 37°C in a humidified 5% CO2:95% air atmosphere, on polycarbonate filters in WEM without phenol red, containing 11.1 mM glucose, 2 mM glutamine, 100 U penicillin per ml, 100 mug streptomycin per ml, 2.5 mug fungizone per ml, 10 mug insulin per ml, 10 mug transferrin per ml, 10 ng hydrocortisone per ml, 10 ng sodium selenite per ml, 3 nM triiodothyronine, 1% trace element mix, and 10 mug bovine pituitary extract per ml (Guy et al. 1996).

Isolation of hair follicles

Human anagen hair follicles were isolated from scalp skin from females undergoing facelift surgery (Philpott et al. 1990). The subdermal adipose layer was removed from the dermis with a scalpel blade. Hair follicles were pulled out of the adipose tissue using microforceps and placed in PBS Figure 1c). Follicles were individually snap frozen immediately after isolation unless stated otherwise.

Where appropriate, hair follicles were maintained at 37°C in a humidified 5% CO2:95% air atmosphere in WEM supplemented with 100 U penicillin per ml, 100 mug streptomycin per ml, 2 mM glutamine, 10 mug insulin per ml, and 10 ng hydrocortisone per ml.

Individual experiments were carried out on glands or follicles isolated from single subjects. Glands or follicles from several subjects were never pooled.

RNA isolation and reverse transcription

Total RNA was isolated from skin appendages using the method ofChomczynski & Sacchi (1987). Single stranded cDNA was prepared from skin appendage total RNA using oligo dT primers (Chomczynski & Sacchi 1987). For the cDNA preparation, each tube contained 2.6 mug RNA, 0.1 mg oligo dT primer per ml, 1 mM dNTP, 20 U RNasin, 20 U Super RT in a volume of 30 mul. The RNA and oligo dT primers were preincubated at 68°C for 3 min and then placed on ice for a further 2 min before adding the other components. The complete mixture was incubated at 42°C for 1 h, and the reaction stopped by heating the mixture to 80°C for 10 min.

PCR of LDL receptor and lipoprotein lipase cDNA

LDL receptor and lipoprotein lipase cDNA were amplified as previously described byWang et al. (1989). Fifteen picomoles each of 5'-TCTGTCTCGAGGGGTAGCTG-3' and 5'-CAATGTCTCACCAAGCTCTG-3' LDL receptor primers, or 15 pmol each of 5'-GAGATTTCTCTGTATGGCACC-3' and 5'-CTGCAAATGAGACACTTTCTC-3' lipoprotein lipase primers, were added to the PCR mixture. The mixture was overlaid with 20 mul of mineral oil and amplified with a thermal sequencer (PTC-100, MJ Research, Watertown, MA) for 35 cycles. The amplification profile involved denaturation at 95°C for 30 s, primer annealing at 55°C for 30 s, and extension at 72°C for 1 min.

Restriction analysis

Amplified LDL receptor cDNA was digested with AflII at 37°C for 2 h in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9 at 25°C, and 100 mug bovine serum albumin per ml. Amplified lipoprotein lipase cDNA was digested with MnlI at 37°C for 2 h in 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9 at 25°C, and 100 mug bovine serum albumin per ml. Digested PCR products were visualized using ethidium bromide staining on an sodium dodecyl sulfate polyacrylamide gel electrophoresis gel.

HMG-CoA reductase assay

As the amount of human skin available was limited, sufficient material could not be obtained from any single skin case to purify the protein. Therefore, HMG-CoA reductase activity was determined in unpurified homogenates that contained the whole appendage. Glands or follicles from individual subjects were suspended in 20 mM Tris-HCl, 50 mM KCl, 2 mM ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 2 mM ethylenediamine tetraacetic acid (EDTA), pH 7.4 at 20°C, 5 mM dithiothreitol, 50 muM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 0.25% (vol/vol) Kyro EOB (Goldstein et al. 1983), and were immediately homogenised on ice, using 20 strokes of a Jencons Mini glass homogeniser.

The homogenate was assayed in duplicate for HMG-CoA reductase activity using a radiochemical method (Shapiro et al. 1974;Easom & Zammit 1984b). For each assay, 25 mul of a mix containing 100 mM KH2PO4, 195 mM KCl, 25 mM EDTA, pH 7.25, 110 muM [3–14C]-HMG-CoA (80000 dpm per nmol), 60 mM glucose-6-phosphate, 12 mM dithiothreitol, 1 unit glucose-6-phosphate dehydrogenase, and 4 mM NADP was used. Control incubations lacked NADP. The assay mixture was preincubated for 5 min at 37°C, and the reaction started by the addition of 50 mul of homogenate. Following various incubation times, 25 mul of 5 M HCl was added to stop the reaction. The assay tubes were then incubated for 30 min to complete the spontaneous conversion of [14C]mevalonate to [14C]mevalonolactone. [5–3H]Mevalonolactone (10000 dpm) was added to each sample, which was then centrifuged to remove precipitated protein. Mevalonolactone (1 mg per ml) was added to the supernatant, an aliquot was applied to a silica gel LK5D thin layer chromatography plate, and chromatographed in toluene/acetone (1:1). The product was visualized upon contact with iodine, and the stained area scraped from the plate into a scintillation vial. Four milliliters of Optisafe scintillant was added and the vial counted in a Packard 1500 Tri-Carb liquid scintillation analyzer with a dual label programme for3H and14C. Recovery of the [3H]mevalonolactone marker was used to correct the value of [14C]mevalonolactone product. Results are expressed as pmoles14C incorporated into mevalonolactone per min per mg of total protein. Under these conditions, apocrine, sebaceous, and hair follicle HMG-CoA reductase activities were linear with respect to both time (for at least 10 min) and protein (0–130 mug).

Acetyl-CoA carboxylase assay

Glands from individual subjects were homogenised on ice in 250 mM mannitol, 100 mM Tris-HCl, 1 mM EDTA, pH 7.2 at 4°C, 1 mM dithiothreitol, 1 mM benzamidine, 4 mug soybean trypsin inhibitor per ml using 20 strokes of a Jencons Mini glass homogeniser. Glands were assayed for acetyl-CoA carboxylase activity using a method previously described byHalestrap & Denton (1973). For each assay, which was carried out in duplicate, 10 mul of homogenate was added to 90 mul of 100 mM Tris-HCl, pH 7.4 at 37°C, 0.3 mM acetyl-CoA, 4 mM ATP, 2 mM MgCl2, 1% (wt/vol) bovine serum albumin, and 20 mM [14C]NaHCO3 (0.3 Ci per mol), which had been preincubated for 5 min at 37°C. Control incubations did not contain acetyl-CoA. The reaction was stopped at various time points using 50 mul of 6.5% perchloric acid. Each sample was left on ice for at least 5 min and then centrifuged to precipitate denatured protein. A volume of 125 mul of the supernatant was transferred to a scintillation vial and dried at 80°C for at least 1 h. The residue was dissolved in 200 mul of water and 1 ml of Optisafe scintillation fluid added. After thorough mixing, each vial was counted in a Packard 1500 Tri-Carb liquid scintillation analyzer for14C. Results are expressed as nmoles14C incorporated into acid-stable product per min per mg of total protein. Under these conditions, apocrine and sebaceous acetyl-CoA carboxylase activities were linear with respect to both time (for at least 90 s) and protein (0–40 mug).

AMP-activated protein kinase assay

The kinase was measured using the method described byCarling et al. (1991). Glands from individual subjects were homogenised in 50 mM HEPES, pH 7.0, 1 mM EDTA, 1 mM dithiothreitol, 50 mM NaF, 10% (wt/vol) glycerol. Homogenates were assayed in duplicate using the synthetic His-Met-Arg-Ser-Ala-Met-Ser-Gly Leu-His-Leu-Val-Lys-Arg-Arg peptide, which has been shown to be specifically phosphorylated by the AMP-activated protein kinase (Davies et al. 1989), in a total volume of 25 mul. The reaction was started by the addition of [32P]ATP (200 muM) (200–500 cpm per pmol) to 20 mul of assay mix that contained 40 mM HEPES buffer (pH 7.0), 400 muM peptide, 200 muM AMP, 80 mM NaCl, 8% glycerol, 0.8 mM EDTA, 0.8 mM dithiothreitol, and 5 mM MgCl2. Control incubations did not contain the peptide substrate. Following incubation for 10 min at 30°C, 15 mul aliquots were removed and spotted on 1 cm times 1 cm squares of phosphocellulose paper (P81, Whatman) that were dropped into 500 ml 1% (vol/vol) H3PO4. The squares were washed for 20 min in three changes of 1% H3PO4, then washed in acetone, air-dried, and following immersion in 10 ml of Optisafe scintillant, counted in a Packard 1500 Tri-Carb liquid scintillation analyzer. Results are expressed as nmoles32P incorporated into the His-Met-Arg-Ser-Ala-Met-Ser-Gly Leu-His-Leu-Val-Lys-Arg-Arg peptide per min per mg of total protein. Under these conditions, apocrine and sebaceous AMP-activated protein kinase activities were linear with respect to both time (for at least 10 min) and protein (0–20 mug).

Protein determination

The concentration of protein in apocrine, sebaceous, and hair follicle homogenates was determined using the method ofBradford (1976) with lyophilized bovine serum albumin as the standard.

Statistical analysis of data

Data were analyzed using a t test unless stated otherwise. Where data were compared with control values of 100%, a one sample t test was used.

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RESULTS

Isolated skin appendages Figure 1 shows a freshly isolated apocrine gland (a), a sebaceous gland (b), and hair follicles (c).

HMG-CoA reductase is active and regulated by phosphorylation in skin appendages

The in situ phosphorylation status of HMG-CoA reductase may be estimated if the tissue is rapidly cooled, then homogenised in the presence of fluoride and kinase inhibitors (Easom & Zammit 1984a). Therefore, apocrine glands, sebaceous glands, and hair follicles were snap frozen in liquid nitrogen immediately after isolation before assaying for HMG-CoA reductase activity. The appendages were rapidly homogenised in the absence or presence of 50 mM fluoride, incubated for 1 h at 37°C, and the homogenates were assayed under conditions of maximum velocity (Vmax) for "total" and "expressed" HMG-CoA reductase activities, respectively. Figure 2 shows that HMG-CoA reductase is present and active in apocrine glands, sebaceous glands, and hair follicles, fractions of which are phosphorylated, giving rise to activation ratios (expressed/total activities) of 30%, 63%, and 34%, respectively.

Figure 2.
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"Expressed" and "total" HMG-CoA reductase activities from apocrine glands, sebaceous glands, and hair follicles. Apocrine glands, sebaceous glands, and hair follicles were homogenised in the presence or absence of fluoride, incubated for 1 h at 37°C, and then assayed for "expressed" and "total" HMG-CoA reductase activities, respectively, as described in Materials and Methods. Results are the mean of n = 5 for apocrine and sebaceous data, and n = 3 for hair follicle data, each carried out in duplicate. Error bars: SEM. Twenty apocrine glands, 10 sebaceous glands, and 30 follicles were used per treatment, per experiment.

Full figure and legend (19K)

The addition of exogenous protein phosphatase 2A (40 mU per ml) to homogenates, followed by incubation for 1 h at 37°C in the absence of fluoride, did not give rise to any further activation of HMG-CoA reductase, relative to that seen in "total" controls. This indicates that the incubation of homogenates at 37°C in the absence of fluoride is sufficient to completely dephosphorylate, and thus fully activate the enzyme. This has been previously demonstrated in murine epidermis (Proksch et al. 1990).

Freezing did not have a detrimental effect on HMG-CoA reductase activity, as the activity of sebaceous glands homogenised following snap freezing did not differ significantly from the activity measured in glands that had not been frozen (fresh 909 plusminus 151 pmol per min per mg, frozen 1020 plusminus 119 pmol per min per mg, mean plusminus SEM, n = 5).

HMG-CoA reductase kinetic parameters

To determine the kinetic parameters for sebaceous gland, apocrine gland, and hair follicle HMG-CoA reductase, initial rates (V0) were measured at increasing S-HMG-CoA concentration in the absence of fluoride. Values for the Michaelis–Menten constant (Km) and the Vmax were calculated by fitting the Michaelis–Menten equation to the initial rate data, and are shown in Table 1, along with values obtained from other animals and tissues.


Appendage HMG-CoA reductase is inhibited by compactin

Compactin has been shown to be a potent competitive inhibitor of HMG-CoA reductase activity in various cell-free systems (Brown et al. 1978;Kaneko et al. 1978). Figure 3(a) shows a concentration-dependent inhibition of HMG-CoA reductase activity from apocrine glands, sebaceous glands, and hair follicles in response to compactin. The results are expressed as percentage of controls, which were incubated in the absence of compactin, as the values of HMG-CoA reductase activity from individuals varied widely (coefficient of variation 30–46%), but nevertheless showed a consistent inhibition. The effects of the inhibitor can be observed at 1 ng per ml, a concentration at which apocrine and hair follicle enzymes are significantly inhibited relative to controls. Compactin appears to have an equally potent effect on each of the appendages, significantly lowering HMG-CoA reductase activity to 30% of controls at 1000 ng per ml. In contrast to cell-free systems, it has been previously shown that administration of HMG-CoA reductase inhibitors to rats results in the stimulation of HMG-CoA reductase expression in the liver (Liscum et al 1983), which is reflected in an increase in enzyme activity, as observed in human fibroblast cultures (Brown et al. 1978), although cholesterol biosynthesis is inhibited. It has been suggested that this increase in activity is a feed-forward response to sterol deprivation resulting from the inhibition of HMG-CoA reductase activity (Brown et al. 1978). Incubation of whole sebaceous glands with 10 ng per ml compactin, which were then washed before homogenization, resulted in a significant increase in HMG-CoA reductase activity relative to controls Figure 3b). Only at the higher concentration of 1000 ng per ml was there a significant inhibition of activity relative to controls.

Figure 3.
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Compactin inhibits HMG-CoA reductase activity in skin appendages. (a) HMG-CoA reductase activity was measured in cell-free preparations of apocrine glands, sebaceous glands, and hair follicles that contained the appropriate concentration of compactin as described in Materials and Methods. Fifty apocrine glands, 30 sebaceous glands, and 100 hair follicles were used per experiment. Controls were incubated with 0.1% dimethylsulfoxide. *Significant difference from controls (p < 0.05); **significant difference from controls (p < 0.01). Results are expressed as the percentage of control activity, n = 3, each carried out in duplicate. Error bars: SEM. (b) Sebaceous glands from individual subjects were maintained overnight with increasing concentrations of compactin. Following washing in PBS to remove the compactin, glands were snap frozen, homogenised, and assayed for HMG-CoA reductase activity, as described in Materials and Methods. Five sebaceous glands were used for each concentration. stSignificant difference from controls (p < 0.01). Results are expressed as the percentage of control activity, n = 6, each carried out in duplicate. Error bars: SEM.

Full figure and legend (47K)

HMG-CoA reductase activity decreases in maintained appendages

We, and others, have previously shown that sebaceous glands exhibit improved rates of lipogenesis following overnight maintenance, compared with rates seen immediately after isolation (Cassidy et al. 1986;Middleton et al. 1988). This has been attributed to the glands needing time to "recover" following the "trauma" of isolation. To determine whether this pattern of lipogenesis following isolation is reflected in enzyme activities, apocrine glands were either snap frozen immediately after isolation or maintained overnight before freezing. Maintained glands had only 30% of total enzyme activity compared with those frozen immediately after isolation (Table 2). The expressed activity of maintained glands also fell significantly to 46% of controls (p < 0.01, n = 3).


A more detailed time course of HMG-CoA reductase activity in maintained apocrine glands showed that the total enzyme activity initially increased with maintenance. A comparison with glands frozen immediately after isolation, demonstrated that enzyme activity peaked at 135% after 4 h maintenance. Enzyme activity then fell sharply over the next 8 h to 30%.

Although a similar time course was not performed for sebaceous glands, due to a shortage of material, sebaceous gland total activity following overnight maintenance fell to 57% of that seen in glands frozen immediately after isolation (Table 2). The reduced activity observed here would not appear to be due to poor maintenance, as acetyl-CoA carboxylase activity does not fall under the same conditions (data not shown). Under these conditions, acetyl-CoA carboxylase may have a longer half-life than that of HMG-CoA reductase.

Comparison of HMG-CoA reductase activity in maintained hair follicles with those frozen immediately after isolation shows a different trend from that observed in glands (Table 2). In hair follicles, enzyme activity increased to 160% of controls upon maintenance.

We proposed that the reduced activity observed in overnight maintained glands might be a result of cholesterol accumulation. Such inhibition might not occur in hair follicles as they have a lower enzyme activity, which is less likely to lead to cholesterol accumulation. To test this hypothesis, sebaceous glands were maintained in the absence of glucose. Under such conditions, sebaceous glands have a lower lipogenic capacity1 and therefore synthesize less cholesterol; however, glands maintained under these conditions exhibited a further reduction in HMG-CoA reductase activity compared with controls (Table 2). Thus, cholesterol accumulation by the glands does not appear to explain the reduced HMG-CoA reductase activity following overnight maintenance.

Effect of patient age and sex on HMG-CoA reductase activity

During the course of this study sebaceous glands were isolated from patients of both sexes and a wide range of ages. Figure 4 summarizes the HMG-CoA reductase activity measured. The intra-individual variation was small (coefficient of variation = 3%); however, there was considerable variability across patients, with the activity from glands taken from identically aged patients varying up to 5-fold. None the less, a multiple linear regression of HMG-CoA reductase activity by age and sex gave an overall fit of R2 = 0.10, with a negative contribution of age, although this did not reach significance (regression coefficient = –4.67, p = 0.17). This indicates that glands isolated from older patients have a lower HMG-CoA reductase activity. This decline in enzyme activity may reflect the decline in sebum production that is known to occur with age (Cunliffe 1989a). There was virtually no difference in HMG-CoA reductase activity between the sexes (p = 0.99).

Figure 4.
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Sebaceous gland HMG-CoA reductase activity decreases with age. Sebaceous glands isolated from males and females were homogenized, and HMG-CoA reductase activity measured, as described in Materials and Methods. At least five glands were used per data point.

Full figure and legend (10K)

Acetyl-CoA carboxylase is active and regulated by phosphorylation in apocrine and sebaceous glands

The degree to which acetyl-CoA carboxylase is phosphorylated in vivo in isolated apocrine and sebaceous glands was estimated as described above for HMG-CoA reductase. Figure 5 demonstrates that apocrine glands expressed 59% of the total activity, whereas sebaceous glands expressed 26%, when assayed under Vmax conditions. Figure 5 also demonstrates that acetyl-CoA carboxylase activity from both apocrine and sebaceous glands is sensitive to levels of citrate, an allosteric activator of the enzyme in other systems (Moss & Lane 1971).

Figure 5.
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"Expressed" and "total" acetyl-CoA carboxylase activities from apocrine glands and sebaceous glands. Apocrine glands (a) and sebaceous glands (b) were homogenised in the presence or absence of fluoride, incubated for 1 h at 37°C, and then assayed for "expressed" and "total" acetyl-CoA carboxylase activities, respectively, in the presence of either 0.5 mM citrate or 10 mM citrate as described in Materials and Methods. n = 3, each carried out in duplicate. Error bars: SEM. Fifteen apocrine glands and 15 sebaceous glands were used per treatment, per experiment.

Full figure and legend (56K)

Acetyl-CoA carboxylase activities measured in homogenates incubated with protein phosphatase 2A (40 mU per ml) for 1 h at 37°C, were not significantly different from those incubated in its absence. This would indicate that, when incubated at 37°C in the absence of fluoride, the endogenous phosphatases are sufficiently active to completely dephosphorylate the enzyme.

Acetyl-CoA carboxylase kinetic parameters

To determine the kinetic parameters for apocrine and sebaceous acetyl-CoA carboxylase, initial rates were measured at increasing acetyl-CoA concentration in the absence of fluoride. Values for Km and Vmax were calculated by fitting the Michaelis–Menten equation to the initial rate data, and are shown in Table 3, together with values obtained from other animals and tissues.


The kinetic parameters for apocrine and sebaceous gland acetyl-CoA carboxylase with respect to citrate were also determined by fitting the Hill equation to the initial rate data measured at increasing citrate concentration. Values obtained for Ka, Vmax, and the Hill coefficient (h) are shown in Table 3, together with values obtained from other animals and tissues. The Hill coefficient is often used as an index of cooperativity, with positive cooperativity occurring when h > 1. The value of 1.2 calculated for each of the glandular enzymes, therefore suggests that citrate promotes acetyl-CoA carboxylase activity in a cooperative fashion in these appendages.

AMPK is active in apocrine and sebaceous glands

The enzyme responsible for the phosphorylation of HMG-CoA reductase and acetyl-CoA carboxylase, AMPK, has been measured in various rat tissues, and high levels of activity appear to reflect a highly lipogenic tissue (Davies et al. 1989). AMPK activity was therefore measured in apocrine and sebaceous glands, using a peptide substrate specific for the kinase. Activities observed for apocrine and sebaceous glands were 0.04 nmol per min per mg and 0.44 nmol per min per mg, respectively.

HMG-CoA reductase activity is downregulated by exogenous cholesterol in apocrine and sebaceous glands

Apocrine glands and sebaceous glands were incubated for 4 h with either 5 mug LDL per ml or 5 mug 25-hydroxycholesterol (25-HC) per ml, values approximating those previously used byMarsden & Middleton (1989) with sebaceous glands and byGoldstein & Brown (1973) with fibroblasts. The glands were then snap frozen. Hair follicles were incubated overnight with either 5 mug LDL per ml or 5 mug 25-HC per ml. For these experiments, fetal calf serum in apocrine maintenance medium was replaced with lipoprotein deficient serum. Figure 6(a) shows that incubation with LDL and 25-HC significantly reduced apocrine HMG-CoA reductase activity to 80% and 72% of control values, respectively. Similarly, sebaceous gland HMG-CoA reductase activity was downregulated following incubation with either 5 mug LDL per ml or 5 mug 25-HC per ml to 81% and 77% of controls, respectively Figure 6b).

Figure 6.
Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

LDL downregulates HMG-CoA reductase activity in apocrine glands, sebaceous glands, but not hair follicles. Apocrine glands (a) and sebaceous glands (b) were isolated and maintained for 4 h with either 5 mug LDL per ml or 5 mug 25-HC per ml. Hair follicles (c) were maintained overnight with either 5 mug LDL per ml or 5 mug 25-HC per ml. Controls were incubated with the appropriate carrier. Following freezing, appendages were assayed for HMG-CoA reductase activity as described in Materials and Methods. Results are expressed as a percentage of controls, and n = 7 for apocrine glands, n = 7 for sebaceous glands and follicles with LDL, n = 11 for sebaceous glands with 25-HC, and n = 4 for follicles 25-HC, each carried out in duplicate. *Significant difference from control values (p < 0.05); **significant difference from control values (p < 0.02). Error bars: SEM.

Full figure and legend (29K)

Figure 6 also shows that incubation of hair follicles (c) with 5 mug LDL per ml resulted, unexpectedly, in a stimulation of HMG-CoA reductase activity relative to controls; however, incubation with 5 mug 25-HC per ml significantly reduced HMG-CoA reductase activity to 88% of controls.

LDL receptor mRNA is expressed in appendages

To determine whether the lack of response of hair follicle HMG-CoA reductase to exogenous LDL was due to an absence of LDL receptors, receptor expression was investigated using reverse transcriptase polymerase chain reaction. Figure 7 shows a typical gel of the PCR amplification of a segment of LDL receptor from cDNA that has been reverse transcribed from total RNA isolated from follicles, and also apocrine and sebaceous glands. Negative control lanes, where cDNA was absent from the amplification reaction, were empty (not shown). A single band corresponding to a 258 bp product was obtained using LDL receptor primers. The identity of this product was confirmed by restriction enzyme analysis. Digestion with the enzyme AflII yielded product lengths of identical sizes to those predicted from the published human LDL receptor sequence (Yamamoto et al. 1984). Therefore, apocrine glands, sebaceous glands, and hair follicles express mRNA for the LDL receptor.

Figure 7.
Figure 7 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

LDL receptor mRNA is expressed in apocrine glands, sebaceous glands, and hair follicles. PCR amplification using human LDL receptor primers of cDNA reverse transcribed from mRNA isolated from apocrine glands (lanes 1 and 2), sebaceous glands (lanes 3 and 4), and hair follicles (lanes 5 and 6), was carried out as described in Materials and Methods. Phi X174 Hae III markers ranging in size from 72 to 310 bp were run in parallel (lane M). Lanes 2, 4, and 6 contain PCR amplified cDNA that has been digested with the restriction enzyme AflII. This gel is representative of five experiments, with 10 appendages per experiment.

Full figure and legend (29K)

Lipoprotein lipase mRNA is expressed in appendages

To investigate whether apocrine and sebaceous glands have the capacity to sequester fatty acids from the circulation, a segment of lipoprotein lipase cDNA was amplified from cDNA that had been reverse transcribed from glandular total RNA. A single band corresponding to a 276 bp product was obtained using lipoprotein lipase primers Figure 8). A restriction digest of the lipoprotein lipase PCR product with the enzyme MnlI yielded product lengths of identical sizes to those predicted from the published human lipoprotein lipase sequence (Wion et al. 1987). Therefore, both apocrine glands and sebaceous glands express mRNA for lipoprotein lipase.

Figure 8.
Figure 8 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Lipoprotein lipase mRNA is expressed in apocrine glands and sebaceous glands. PCR amplification using human lipoprotein lipase primers of cDNA reverse transcribed from mRNA isolated from apocrine (lanes 1 and 2) and sebaceous glands (lanes 3 and 4), was carried out as described in Materials and Methods. Phi X174 Hae III markers ranging in size from 118 to 310 bp were run in parallel (lane M). Lanes 2 and 4 contain PCR amplified cDNA that has been digested with the restriction enzyme MnlI. This gel is representative of five experiments, with 10 appendages per experiment.

Full figure and legend (39K)

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DISCUSSION

The skin and its appendages have been shown to be important sites of de novo lipid synthesis, as determined by the in vivo incorporation of radiolabeled substrates into cholesterol and nonsaponifiable lipids in both rats and primates (Feingold et al. 1982,1983). The skin also makes an important contribution to the total body synthesis of fatty acids, with murine epidermis exhibiting half the rate of liver fatty acid synthesis (Grubauer et al. 1987). Few studies, however, have looked at the individual steps important in the control of these synthetic pathways. Goldstein, Brown, and colleagues assayed plucked human scalp hairs and found HMG-CoA reductase highly active (Brannan et al. 1974).Proksch et al. (1990) detailed the activity of HMG-CoA reductase in murine skin, and on examining its activity in intact, stripped, and protected skin, they showed that the transepidermal water barrier regulates the concentration and activation state of the epidermal enzyme. Murine epidermal acetyl-CoA carboxylase activity is similarly regulated (Ottey et al. 1995).

Although the major function of apocrine and sebaceous glands is lipid secretion, no group, to our knowledge, has carried out a detailed investigation on the lipogenic enzymes involved, although a preliminary study of HMG-CoA reductase activity in sebaceous glands has been described by Langtry et al.2 Therefore, in this study we have looked at the important regulatory steps of cholesterol and fatty acid biosyntheses in human skin appendages. We have determined their activity and major regulatory mechanisms, and investigated whether exogenous lipids could contribute to the glands' secretion.

Our results indicate that a percentage of both HMG-CoA reductase and acetyl-CoA carboxylase from skin appendages is phosphorylated and therefore inactive in vivo. Nevertheless, the activity of the remaining dephosphorylated enzymes can account for the rate of incorporation of radiolabeled substrates into the appropriate lipids by glands in vitro (Barth et al. 1989;Guy et al. 1996). Comparable studies determining the rate of incorporation of [14C]acetate into the different lipid classes by isolated hair follicles have not been performed. The activation ratios observed here for both HMG-CoA reductase and acetyl-CoA carboxylase are comparable with those previously observed in murine epidermis (Proksch et al. 1990) and rat liver (Easom & Zammit 1984b;Davies et al. 1992).

It is hard to comment on any changes that may occur in enzyme concentration and activation state in the time between the removal of the skin from the patient, and isolation of the glands, but the results presented here are consistent with those measuring rates of incorporation of radiolabeled acetate into lipids by isolated glands, which in turn are consistent with rates of lipid production by the glands in vivo (Leyden et al. 1981;Kealey et al. 1986;Barth et al. 1989;Guy et al. 1996). The time of sampling is approximately midday, which would correspond to the rat diurnal high; however, when human hairs were plucked at various times and then assayed, no diurnal variation was observed (Brannan et al. 1974).

Maximum HMG-CoA reductase and acetyl-CoA carboxylase activities varied greatly between patients. This may reflect the known intrapatient variability in secretion rates and content in vivo (Cunliffe & Shuster 1969); however, Km and Ka values were comparable between patients. Comparison of the kinetic parameters determined for both HMG-CoA reductase and acetyl-CoA carboxylase from skin appendages with those previously observed in other tissues (Smith et al. 1986;Ness et al. 1986;Bianchi et al. 1990;Trumble et al. 1995), indicates that the skin appendage enzymes exhibit similar affinities for substrates and allosteric effectors as those previously described.

Previous studies have identified two isoforms of acetyl-CoA carboxylase with differential patterns of expression between tissues (Bianchi et al. 1990;Saddik et al. 1993;Trumble et al. 1995). It is not possible to determine from the data presented here which of the two human acetyl-CoA carboxylase isoforms are predominantly present in glands. Nevertheless, both isoforms have been cloned (Abu-Elheiga et al. 1995;Widmer et al. 1996), meaning that northern blotting using probes specific for each isoform would clarify the issue.

Specific inhibitors of HMG-CoA reductase, collectively known as statins, have proved very effective in the treatment of hypercholesterolaemia by lowering plasma total cholesterol and LDL levels, with a concomitant increase in the life expectancy of hypercholesterolaemic individuals (Plosker & McTavish 1995). We have shown that 10 ng per ml compactin, a statin, significantly inhibits skin appendage HMG-CoA reductase activity. Pharmacokinetic studies have shown that, following a typical 20 mg dose of a closely related HMG-CoA reductase inhibitor, pravastatin, the plasma concentration of the inhibitor was 26.5 ng per ml (McTavish & Sorkin 1991). Treatment with compactin may therefore affect sebaceous and apocrine gland function and secretion. Indeed, eczematous skin rashes have been reported in some patients following statin treatment (Krasovec et al. 1993), which the authors suggest is due to the inhibition of epidermal HMG-CoA reductase activity. The inhibition of sebaceous HMG-CoA reductase may be a contributing factor to this condition; however, it should be noted that more recently developed cholesterol-lowering drugs have been shown to have little effect on nonhepatic tissues such as skin (Dobs et al. 1991;Pedreno et al. 1994;Komai & Tsujita 1994).

The decline in HMG-CoA reductase activity in maintained apocrine and sebaceous glands compared with those frozen immediately after isolation is an interesting observation, particularly as whole sebaceous glands demonstrate increased rates of incorporation of radiolabeled acetate into cholesterol, cholesterol esters, and squalene following overnight maintenance (Cassidy et al. 1986;Middleton et al. 1988). Nevertheless, the residual enzyme activity following maintenance of both apocrine and sebaceous glands is still sufficient to account for total lipogenesis by glands in vitro (Barth et al. 1989;Leyden et al. 1981;Guy et al. 1996). Reduced activity does not appear to be due to poor maintenance as acetyl-CoA carboxylase activity does not fall under the same conditions.

A similar decline in HMG-CoA reductase activity with time has been observed in hepatocyte primary cultures (Hylemon et al. 1985). Activity measured in the microsomal fraction of these cultures decreased over 48 h after plating out, but then remained constant over the next 72 h. Why HMG-CoA reductase activity falls over time in both organ and cell cultures is not clear, and the level at which this occurs awaits investigation.

HMG-CoA reductase has been shown to be sensitive to the level of exogenous cholesterol in several different cell types. The mechanisms through which this occurs have been summarized byGoldstein & Brown (1990). Briefly, cholesterol enters the cell by receptor-mediated endocytosis through the LDL receptor and results in the stimulation of HMG-CoA reductase degradation, and inhibition of its transcription and translation.

We exploited this control mechanism to determine whether apocrine and sebaceous glands can utilize exogenous cholesterol. It appears that apocrine glands can indeed internalize exogenous cholesterol, as they responded to exogenous cholesterol by repressing the levels of HMG-CoA reductase activity to 80% of those maintained in the absence of sterols. These values are similar to those reported byGoldstein & Brown (1973) for cultured fibroblasts over the same time period. This downregulation suggests that apocrine secretion may contain dietary lipids, and may have important implications for the control of body odour.

Sebaceous gland HMG-CoA reductase activity was also downregulated by exogenous cholesterol. This suggests that dietary lipids may influence sebum lipid content, and supports the finding byNikkari et al. (1975). They determined the fraction of intravenously administered, labeled free, and esterified cholesterol that appeared on areas of skin rich in sebaceous glands, and found that between 29 and 46% of skin surface cholesterol is derived from plasma.

Our finding that sebaceous HMG-CoA reductase is downregulated by cholesterol is in contrast to that reported by Langtry et al.2 In their study no difference in HMG-CoA reductase activity was observed between glands cultured in either fetal calf serum, lipoprotein deficient serum, or high concentrations of LDL,2 despite evidence for LDL receptor activity. There are several reasons that might account for this discrepancy. Langtry et al. examined the effects of LDL over a 20 h incubation period; however, we have shown that HMG-CoA reductase activity falls dramatically after 4 h of maintenance, therefore any differences in activity upon the addition of cholesterol may not be apparent after this prolonged time. The effect of cholesterol over a longer period was not carried out due to a shortage of material. Alternatively, differences between these studies may be due to differences in the body site and donors from which the glands were isolated.

Together with evidence for LDL receptor expression, these data indicate that apocrine and sebaceous glands have the capacity to utilize exogenous lipid. To what extent exogenous cholesterol contributes to the pool of secreted lipids is an important question for the future.

In contrast to glands, HMG-CoA reductase activity from whole hair follicles was not downregulated by LDL, despite the expression of LDL receptors as shown by reverse transcriptase polymerase chain reaction. This supports an earlier observation by Goldstein, Brown, and colleagues. Using hair follicles plucked from individuals who differed greatly in their plasma cholesterol levels, they showed that HMG-CoA reductase activity in these follicles is not regulated by LDL in vivo (Brannan et al. 1974). In this study, LDL induced a significant stimulation of hair follicle HMG-CoA reductase activity. It is not clear why such an induction of enzyme activity occurs and further work is required to establish the mechanism.

The addition of 25-HC, an oxysterol that bypasses the LDL receptor and which is therefore capable of introducing cholesterol in the absence of LDL receptors (Brown et al. 1975), significantly reduced HMG-CoA reductase activity in isolated human hair follicles. These apparently discrepant results between different forms of cholesterol may be due to the presence of nonfunctional LDL receptors within follicles. Alternatively these results may be reconciled if the distribution of LDL receptors within the hair follicle is analogous to that within the skin (te Pas et al. 1991): absent from the outer components of the follicle that have differentiated from the basal lamina of the epidermis, but present on the relatively small number of dermally derived components. Any decrease in HMG-CoA reductase activity through the uptake of LDL by these dermally derived cells, would be masked by the lack of response in the majority of the cells assayed. Localization of the LDL receptor and its mRNA using immunocytochemistry and in situ hybridization, respectively, would confirm this hypothesis.

We have also demonstrated that apocrine and sebaceous glands have the enzymatic capacity to synthesize the secreted fatty acids and triglycerides. The presence of fatty acids in triglycerides that have unique positions of unsaturation and/or branched chains (Nicolaides et al. 1964), suggests that lipids are indeed synthesized by the gland in vivo; however, traces of the essential fatty acid linoleate have been found in human sebum, indicating that exogenous fatty acids also contribute to the sebaceous secretion (Stewart et al. 1989). Although lipoprotein lipase activity was not measured, we have identified lipoprotein lipase expression in apocrine and sebaceous glands, presumably the route through which glands sequester fatty acids from the circulation.

These studies have demonstrated that both apocrine glands and sebaceous glands have the capacity to take up exogenous lipids, implying that diet may influence axillary odor and acne. Any links between diet and apocrine activity have not been determined to our knowledge; however, studies investigating the influence of diet on acne have suggested that there is no link between diet and sebaceous gland activity (overviewed inCunliffe 1989b). Therefore any uptake of exogenous lipids by sebaceous glands may be negligible in comparison with their lipogenic activity, and not influence the pathology of acne. Further studies are now needed to determine the contribution exogenous lipids make to apocrine and sebaceous gland secretions, and whether exogenous lipids make a significant contribution to the glands' secretion in situ.

In summary, this study demonstrates that the enzymes important in regulating cholesterol and fatty acid metabolism, HMG-CoA reductase and acetyl-CoA carboxylase, respectively, are active in isolated human apocrine glands, sebaceous glands, and hair follicles, and enzyme activities are modulated by phosphorylation state. The enzyme responsible for this phosphorylation, the AMP-activated protein kinase, is also active within the lipogenic apocrine and sebaceous glands. Measurement of relevant kinetic parameters and means of regulation suggest that they are identical to those enzymes isolated from other species and human sources. Furthermore, it appears that both apocrine and sebaceous glands can sequester exogenous cholesterol and could potentially take up dietary fatty acids. These findings may have implications for the control of axillary odor and acne.

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Notes

1 Downie MMT, Kealey T: Intermediary metabolism of the human sebaceous gland. J Invest Dermatol 108:373, 1997 (abstr.)

2 Langtry JAA, Marsden JR, Middleton B, McLaughlan J: HMG-CoA reductase and LDL receptor function in human sebaceous glands. J Invest Dermatol 98:520, 1992 (abstr.)

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Acknowledgments

We thank Mr. B.A. Matty, FRCS, Mr. F.V. Nicolle, FRCS, and Mr. J. Bowen, FRCS of Harley Street, London and Sir Terence English, FRCS, Mr. J. Dunning, FRCS, Mr. S. Large, FRCS, Mr. S. Nashef, FRCS, Mr. J. Wallwork, FRCS, Mr. F. Wells, FRCS, and their colleagues at Papworth Hospital for supplying us with skin. We thank Dr. Carl Smythe of Dundee University for the kind gifts of protein phosphatase 2A and His-Met-Arg-Ser-Ala-Met-Ser-Gly Leu-His-Leu-Val-Lys-Arg-Arg peptide, and Dr. M. Green, Dr. G. Westgate, Dr. J. Beck, and Dr. F. van der Ouderaa of Unilever Research for useful discussions. This work was supported by Unilever Research.

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