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Service de Génétique Médicale (H.L., S.P.W., G.M.), Département d’Obstétrique (M.B.), Hôpital Ste-Justine, Montréal, Canada H3T 1C5; and Service d’endocrinologie (L.R., M.C., N.G.-P.), Département de Médecine, Université de Sherbrooke, Sherbrooke, Canada J1H 5N4
Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Faculty of Medicine, Université de Sherbrooke, 3001 12th Avenue North, Sherbrooke, Québec, Canada J1H 5N4. E-mail: n.gallo{at}courrier.usherb.ca.
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Abstract |
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Introduction |
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In the adrenal, free cholesterol is derived from two sources, de novo synthesis from acetyl-coenzyme A (6) or, to a greater extent, hydrolysis of cholesteryl esters stored in lipid droplets (7). The conversion of cholesteryl esters into free cholesterol may play an important role in controlling cholesterol availability for steroidogenesis. Free cholesterol is obtained by receptor-mediated endocytosis of low-density lipoprotein (LDL) (8) and cleavage of cholesteryl esters by lysosomal acid lipase and transport to the cytoplasm by a mechanism involving the Nieman Pick-C1 and Pick-C2 proteins (NPC 1 and NPC 2) (9), followed by reesterification in the cytoplasm by acetyl-coenzyme A/cholesterol acyl transferase (ACAT) (10, 11, 12, 13, 14). Cholesteryl esters derived from high-density lipoproteins (HDLs) can also be transferred to the cell interior via scavenger receptor B1. There is accumulating evidence indicating that lipid droplets are an important morphological characteristic reflecting the rate of steroid hormone production (15).
Hormone-sensitive lipase (HSL, E.C.3.1.1.3) (16, 17, 18, 19) is a fatty acyl hydrolase that cleaves fatty acyl esters of cholesterol, steroid hormones, and glycerol (20). Until recently, research on HSL focused primarily on its role in adipose tissue, in which HSL mediates the release of fatty acids from triglycerides in response to various stimuli, including ß-adrenergic stimulation. Following protein kinase A-dependent phosphorylation, HSL is translocated from the cytoplasm to the surface of lipid droplets from which it exerts its catalytic function (18).
HSL is also expressed in nonadipose tissues including skeletal muscle, myocardium, pancreatic ß-cells, testicle, and adrenal gland (21, 22). In bovine adrenal, evidence suggests that HSL is the major cholesteryl esterase, consistent with a role for HSL in adrenal steroidogenesis (10, 18). Furthermore, steroid hormones can also be formed from fatty acyl esters, the hydrolysis of which has been hypothesized to yield a presynthesized, immediately available pool of steroid hormones (20, 23).
The adrenal gland consists of two endocrine tissues with distinct functions. The cortex synthesizes and secretes steroids, and the medulla produces catecholamines and several neuropeptides (4). The adult cortex is composed of three well-identified layers. The external zona glomerulosa is composed of four to five layers of cells arranged in clusters surrounding a central capillary. Adjacent is the zona fasciculata, which consists of parallel and radial columns of large cells separated by sinusoids; finally, adjacent to the medulla, the zona reticularis, forming an irregular network of small cells. Although all adrenocortical cells have the capacity to produce corticosterone in rodents, only the zona glomerulosa produces aldosterone (24).
It has previously been difficult to directly study the importance of cholesteryl and steroid ester hydrolysis in the normal structure and function of the adrenal gland. We recently reported the creation of a line of gene-targeted mice with complete deficiency of HSL (25). The aim of the present study was to investigate whether HSL deficiency affects adrenal gland morphology and function under basal conditions and following ACTH stimulation.
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Materials and Methods |
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Previous studies have shown that the homozygous HSL-/- mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue. HSL-/- mice have normal body weight but reduced abdominal fat mass, compared with normal littermates (25). In addition, the HSL-/- mice were normoglycemic and normoinsulinemic under basal conditions but showed a 30% reduction of circulating free fatty acids with respect to control and heterozygous animals after an overnight fast. HSL-null mice were also glucose intolerant and displayed a lack of a rise in plasma insulin after a glucose challenge, suggesting that they are insulin resistant (26).
ACTH stimulation test
ACTH (
-1-24 corticotropin) (Cortrosyn, Organon Canada Ltd., Scarborough, Ontario, Canada), 0.8 µg/g body weight (25 µg/30 g per 0.2 ml) was injected ip to conscious fed mice. Then 100 µl blood were collected from the tail vein in heparinized capillary tubes before and 1 h after ACTH injection, centrifuged at 5100 x g for 20 min at 4 C, and plasma was stored at -20 C until steroid measurements by RIA.
Metabolite and hormone measurements
Corticosterone and aldosterone were measured directly in plasma with commercial RIA kit (corticosterone kit, Medicorp, Montréal, Québec, Canada) and aldosterone (Intermedico, Montréal, Québec, Canada). Glycemia was measured with a portable blood glucose meter (Medisense, Inc., Bedford, MA).
Histological procedures
For phase-contrast microscopy, adrenal glands were immersed immediately after removal in 4% paraformaldehyde for 24 h, embedded in paraffin, cut into 3-µm sections, and dehydrated through xylene and graded alcohol series for standard hematoxylin-eosin staining. Slides were mounted in nonaqueous mounting media (VectaMount, Vector Laboratories, Burlingame, CA). Images were observed using an Eclipse 300 microscope (Nikon, Montréal, Québec, Canada) equipped with a CoolSnap color digital camera. Acquired images were processed and analyzed using Adobe Photoshop 4.0 (Delray Beach, FL).
For electron microscopy, mice were anesthetized with sodium pentobarbital (Somnitol, MTC Pharmaceuticals, Hamilton, Canada). Mice were perfused-fixed through the heart with 5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.4). After the perfusion, adrenal glands were removed, cut into 1-mm3 pieces, processed, embedded in Epon, and prepared for electron microscopy as described previously (3).
The number and volume of lipid droplets were calculated as described by Nussdorfer et al. (27). The estimation of the average volume of lipid droplets was based on the assumption of a spherical shape because phase-contrast or electron microscopy shows circular structures. The volume was calculated by the formula 4/3 
(DLD/2) (3). The diameter distribution (DLD) was determined on the electron micrographs as indicated in Table 1
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Results |
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, A and B). In contrast, fasciculata cells from HSL-/- mice appeared hypertrophied because of an accumulation of intracellular lipid droplets (Fig. 1B). Larger aggregates of lipid droplets, which we termed syncytial lipoid structures (SLS), occurred at the junction between the zona reticularis and medulla (Fig. 1C), sometimes invading the medulla. Of note, chromaffin cells themselves were not modified (Fig. 1C, black arrow). When compared with the outer portion of adrenals from control mice (Fig. 2, A and B), cells adjacent to zona glomerulosa of HSL-/- mice display an abundance of small lipid droplets, which engorge the cell, displacing and compressing the nucleus against the cell membrane, a phenotype found in all cells (Fig. 2C, arrow). Deeper in the cortex, lipid droplets were larger (Fig. 2D, black arrows) and small SLS appeared (Fig. 2D, white arrows).
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), but their number was decreased (3.5-fold decrease, Table 1). Moreover, lipid droplets were often irregular, with a tendency to fuse (Fig. 3, A and B). The zona fasciculata was characterized by a progressive enlargement of the lipid droplets from the outer to the inner portions (Fig. 4). Near the zona glomerulosa, several cells, but not all (one of four in Fig. 4B), had fewer (Table 1) and larger (3.1-fold increase, Table 1) lipid droplets than cells from similar regions of control HSL+/+ mice (Fig. 4A). Deeper in the gland, lipid droplet size was increasingly heterogeneous, with some cells being occupied by fused lipid droplets (Fig. 4D) and a small number of large vacuoles near zona reticularis (Fig. 4E). Moreover, cell membranes appeared irregular; the pericapillary spaces were enlarged, with the presence of microvilli whose number and length increased from the periphery to the central portion of the cortex (arrows, Fig. 4, C–E). Finally, at the junction between zona reticularis and medulla, the syncytial lipoid structures were revealed to be aggregates of damaged cells in which lipids accumulated as rectangular crystalloid bodies and cytoplasmic structures were profoundly altered (Fig. 4, F and G).
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, from zona glomerulosa of control mice, illustrated normal mitochondria with elongated cristae and lipid droplets surrounded by dense material. In the HSL-/- mice (Fig. 5B), the number of mitochondria cristae increased, with a mean value of 8.22 ± 0.81 (n = 56) for the HSL-/- adrenals, compared with 5.54 ± 0.4 (n = 56) per mitochondria in the control adrenal glands (P < 0.001), and they appeared dilated. In addition, the surface of the lipid droplets in HSL-/- cells was devoid of dense material.
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Plasma corticosterone and aldosterone levels under basal conditions and following ACTH stimulation
Steroid hormone levels were measured before and 1 h after ACTH administration in male and female animals to evaluate the capacity of the HSL-/- adrenal glands to respond to acute ACTH stimulation. In 6-month-old male animals, the mean basal levels of corticosterone were slightly lower, but not statistically significant, in HSL-/- mice, compared with HSL+/+ mice (0.34 ± 0.05 vs. 0.47 ± 0.08 nmol/ml, respectively, n = 9, P = 0.10). After ACTH stimulation, these levels were significantly lower in HSL-/- mice, compared with controls (0.70 ± 0.04 vs. 1.02 ± 0.04 nmol/ml, respectively, n = 9, P < 0.001) (Fig. 6A). Similar observations were found in HSL-/- females, in which basal levels of corticosterone were not significantly different but following ACTH stimulation were significantly lower than levels in normal controls. This was observed both in mice aged 6 months (Fig. 6B) and 2 months (post-ACTH corticosterone levels, HSL+/+, 1.43 ± 0.06 nmol/ml; HSL-/-, 1.03 ± 0.07 nmol/ml; P = 0.001). Aldosterone levels were assessed in females. Levels did not differ under basal conditions between the two strains of animals (1.02 ± 0.14 vs. 1.00 ± 0.18 pmol/ml). Lower mean aldosterone levels were observed in HSL-/- mice with respect to HSL+/+ animals, but this was not significant (data not shown).
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Discussion |
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Lipid accumulation in the zona fasciculata and formation of cholesterol-laden lipoid structures in zona reticularis also occurs in other states of decreased cholesterol utilization. Examples include chronic treatment with aminoglutethimide, an inhibitor of the cholesterol enzyme P450scc (29) and congenital lipoid adrenal hyperplasia in humans and mice, a disorder caused by hereditary deficiency of the StAR protein (30). However, in contrast to these conditions, HSL-deficient mice do not accumulate cholesterol or other lipids in lysosomes (15, 29). The observations in HSL deficiency are consistent with predictions of the metabolic role of HSL, suggesting that in HSL deficiency, cholesterol esters accumulate in lipid droplets and are not delivered into the cytoplasm, as is the case when the deficiency is due to a metabolic block. The observation of increased lipid accumulation and necrotic lipoid cells in the perimedullar region and medulla supports the migration theory of adrenocortical cell renewal and differentiation (31), which posits that young zona fasciculata cells arise at the zona glomerulosa junction and then migrate through zona fasciculata and zona reticularis. The finding of degenerative cellular aggregates suggests that intracellular lipid accumulation became toxic for steroidogenic cells. However, syncytial lipoid structures invade the medulla without affecting the morphology of adjacent chromaffin cells, suggesting that the syncytial lipoid structures and their contents are not themselves toxic for neighboring cells.
Our observations permit several predictions to be made of the metabolic consequences of HSL deficiency, many of which can now be tested directly. The finding of dilated pericapillary spaces and an increased number and length of microvilli suggest a compensatory change to increase HDL and/or LDL uptake, as observed under ACTH stimulation (15, 29). However, the modest decrease in steroidogenesis in HSL-/- mice contrasts with the severe histological changes. The persistent steroidogenesis can be explained by an increased steroidogenic activity of the intact cells, as suspected by the ultrastructural modifications shown in mitochondria of the HSL-/- zona glomerulosa cells in which cristae appeared dilated and increased in number. It differs strikingly from StAR-deficient mice, which die during the first days of life because of markedly deficient adrenal and gonadal steroidogenesis (32, 33). In mice, HDL normally provide most of the free cholesterol for steroid production, with lesser contributions from LDL and de novo synthesis (28, 34, 35, 36). It is plausible that the lack of cholesteryl ester hydrolysis may stimulate a compensatory decrease in ACAT activity or increases in LDL uptake or in endogenous cholesterol synthesis (Fig. 7). Of note, the high level of circulating cholesterol in HSL-deficient mice (25, 37) could further enhance lipid accumulation in adrenocortical cells. In HSL deficiency, ACTH stimulation, which produces a coordinated increase of lipoprotein uptake and processing (38) and cholesterol synthesis (39, 40, 41), may increase lipid accumulation and hence be deleterious in HSL-deficient mice.
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During preparation of this manuscript, Kraemer et al. (42) reported that neutral cholesteryl esterase activity was severely deficient in an independently derived line of HSL-deficient mice, directly showing HSL to be the major adrenocortical neutral cholesteryl esterase. In contrast with their earlier report (37), they also found a statistically significant decrease in post-ACTH corticosterone levels in HSL-deficient mice, compared with controls, although this was present only in females. This article also provided further evidence for an increase in cholesteryl ester content in female adrenals of normal rats, with respect to normal male controls. Although the morphological changes in HSL-deficient adrenals were less severe in males than in females, we found highly significant differences between post-ACTH levels of corticosterone levels in both sexes. This discrepancy between the results of Osuga et al. (37) and Kraemer et al. (42) and our results is difficult to explain. It may involve the age of mice (we studied 6-month-old males), whereas age was not specified in the most recent publication of Kraemer et al. (42) and was less than 3 months in the previous report (37). However, in 2-month-old HSL-deficient female mice, we observed a similar low level of post-ACTH corticosterone increase as in older mice. The differences may also be due to parental background effects retained in each strain during crosses to the C57BL background. In any case, the recent report of Kraemer et al. (42) provides independent confirmation of the importance of the morphological and functional observations presented here.
To date, HSL deficiency has not been described in humans. The adrenal manifestations of human HSL deficiency are expected to be qualitatively similar to those of mice but possibly less severe. Endogenous synthesis, by which cholesterol might bypass the lipid droplet and be used directly for steroidogenesis, accounts for only about 4% of adrenal steroidogenesis in mice, a species in which uptake of cholesterol esters from HDL predominates (34, 35, 36, 43). In mice, recent studies confirm the importance of HDL as the main plasma lipoprotein because HDL-deficient mice with mutant apolipoprotein A1 have a reduced steroidogenic response to stress (28). By comparison, experiments conducted with cultured human adrenal cells indicated that LDL uptake and endogenous cholesterol synthesis account for a larger fraction of steroidogenesis (44).
In addition to progressive lipoid degeneration and possible insufficiency of the adrenal cortex, HSL-deficient humans may have similar abnormalities in others tissues as HSL-/- mice, including abnormal adipose tissue histology and function (25), reduced glucose-stimulated insulin secretion (26), and male infertility caused by azoospermia (45). Of note, infertility in HSL-deficient male mice does not result from abnormal steroid hormone synthesis (37, 45) but rather from a direct role of HSL in spermatogenesis (45). HSL deficiency could reasonably be considered in patients suspected of congenital lipoid adrenal hyperplasia in whom StAR deficiency cannot be documented, particularly if this is associated with abnormalities in other tissues similar to those of murine HSL deficiency.
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Acknowledgments |
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Footnotes |
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Abbreviations: ACAT, Acetyl-coenzyme A/cholesterol acyl transferase; ACTH, adrenocorticotropin; HDL, high-density lipoprotein; HSL, hormone-sensitive lipase; LDL, low-density lipoprotein; NPC, Nie-man Pick-C; SLS, syncytial lipoid structures; StAR, steroidogenic acute regulatory protein.
Received March 25, 2002.
Accepted for publication May 21, 2002.
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References |
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-hydroxylase and 3 ß-hydroxysteroid dehydrogenase in the integration of gonadal and adrenal steroidogenesis via the 
5 and 4 pathways of steroidogenesis in mammals. Biol Reprod 56:789–799[CrossRef][Medline]
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) and after 1-h ACTH administration (0.8 µg/g) (
) in 6-month-old male (A) and female (B) animals. Results are a mean ± SE of nine animals. **, P < 0.001, the statistical significance between control and ACTH-stimulated mice or between ACTH-stimulated HSL+/+ and HSL-/- mice.