High density lipoprotein as a source of cholesterol for adrenal steroidogenesis: a study in individuals with low plasma HDL-C.

Few studies have addressed the delivery of lipoprotein-derived cholesterol to the adrenals for steroid production in humans. While there is evidence against a role for low-density lipoprotein (LDL), it is unresolved whether high density lipoprotein (HDL) contributes to adrenal steroidogenesis. To study this, steroid hormone profiles in urine were assessed in male subjects suffering from functional mutations in ATP binding cassette transporter A1 (ABCA1) (n = 24), lecithin:cholesterol acyltransferase (LCAT) (n = 40), as well as in 11 subjects with low HDL cholesterol (HDL-C) without ABCA1/LCAT mutations. HDL-C levels were 39% lower in the ABCA1, LCAT, and low HDL-C groups compared with controls (all P < 0.001). In all groups with low HDL-C levels, urinary excretion of 17-ketogenic steroids was reduced by 33%, 27%, and 32% compared with controls (all P < 0.04). In seven carriers of either type of mutation, adrenocorticotropic hormone (ACTH) stimulation did not reveal differences from normolipidemic controls. In conclusion, this study shows that basal but not stimulated corticosteroid metabolism is attenuated in subjects with low HDL-C, irrespective of its molecular origin. These findings lend support to a role for HDL as a cholesterol donor for basal adrenal steroidogenesis in humans.

Plasma was obtained after an overnight fast and stored at Ϫ 80°C. Total cholesterol, LDL-C, HDL-C, and triglyceride levels were analyzed using commercially available enzymatic methods (Randox, Antrim, UK and Wako, Neuss, Germany) on a Cobas Mira autoanalyzer (Roche, Basel, Switzerland). Free cholesterol and total cholesterol were measured before and after precipitation of apoB-containing lipoproteins using phosphotungstic acid (Sigma) and a commercially available enzymatic assay (Diasys) on a Selectra autoanalyzer (Sopachem). ACTH was determined by an immunoluminometric assay (Nichols Institute, Los Angeles, CA). Aldosterone was measured using a radioimmunoassay (Siemens, Los Angeles, CA).

Stimulated adrenal steroidogenesis
Random subgroups of seven ABCA1 and seven LCAT mutation carriers consented to an ACTH stimulation study (cosyntropin or tetracosactin, 0.25 mg/ml; Novartis Pharma B.V., Arnhem, The Netherlands). After an overnight fast, participants underwent cosyntropin testing at 0900 h. Two baseline blood samples were obtained, 15 min and 1 min before administration of the 1 g cosyntropin bolus. Subsequent blood samples were drawn 30 min and 60 min after cosyntropin administration. Plasma cortisol levels were measured by enzyme immunoassay (Siemens Medical Solutions, Los Angeles, CA), and cortisol binding globulin (CBG) levels were measured with a commercial radioimmunoassay (Siemens Medical Solutions). Free cortisol levels were calculated using the method described by Coolens, van Baelen, and Heyns ( 24 ).

Statistical analysis
Unpaired Student's t -test was performed for analysis of continuous data with a normal distribution. In case of a skewed distribution, data were log-transformed prior to t -testing. Categorical data were assessed by 2 testing. A P value of <0.05 was considered statistically signifi cant. A linear regression model was used to correct for differences in LDL-C and statin use.

Population characteristics
We enrolled 24 and 40 carriers of loss-of-function mutations in either ABCA1 or LCAT . Two of the ABCA1 mutation study it was shown that low HDL-C in such patients was associated with attenuated adrenal responses to synthetic adrenocorticotropic hormone (ACTH) ( 7 ). In support of this, others reported a high incidence of adrenal failure in critically ill individuals with liver disease, with HDL-C being the only variable predictive of adrenal insuffi ciency ( 8 ).
Using adrenal cells, it has been suggested that HDL is the preferred lipoprotein for cholesterol delivery to the adrenal gland ( 9 ) and in accordance, scavenger receptor type B1 (SRB1)-mediated cholesterol uptake from HDL has been shown to be the predominant source of cholesterol in mice (10)(11)(12)(13). In line , it has been shown that mice lacking SRB1 display an impaired adrenal glucocorticoid stress response ( 14,15 ), lending support to a major role for HDL as a cholesterol donor in mice. In humans, we also showed that adrenal function was compromised in individuals with a functional mutation in SCARB1, the gene encoding SRB1 ( 16 ). While this study showed that cholesterol delivery to the adrenals via the HDL-SRB1 pathway is important for adrenal steroidogenesis in humans, it is unclear whether plasma HDL-C levels are associated with adrenal steroidogenesis in humans. To investigate this, we assessed basal and ACTH-stimulated adrenal cortical function in males with low HDL-C due to mutations in either ATP binding cassette transporter 1 (ABCA1) ( 17 ) or lecithin:cholesterol acyltransferase (LCAT) and in subjects with low HDL-C without mutations in ABCA1/LCAT, as well as in normolipidemic controls ( 18 ). We hypothesized that in subjects with low HDL-C levels, adrenal function would be compromised irrespective of the molecular origin of the low HDL-C.

Recruitment of study participants
Male subjects with HDL-C levels below the fi fth percentile were screened for mutations in ABCA1 and LCAT ( 17,19 ), of which the functionality was assessed in previously published studies ( 17,20,21 ). For the current study, we enrolled 24 carriers of mutations in the ABCA1 gene. We furthermore enrolled 40 male carriers of mutations in the LCAT gene. In addition, subjects with similarly reduced HDL-C levels without mutations in ABCA1 and LCAT were included (n = 11). As a control group, normolipidemic age-matched male individuals were recruited by advertisement. None of the included individuals used medication interfering with steroid metabolism. The study was approved by the institutional review board of the Academic Medical Center, Amsterdam, The Netherlands and all participants provided written informed consent.

Questionnaire and biochemical measurements
Medical history, cardiovascular risk factors, use of medication, and family history of cardiovascular disease were assessed using a questionnaire. Brachial artery blood pressures were measured using an oscillometric blood pressure device (Omron 705IT, Hoofddorp, The Netherlands). Hypertension was defi ned as 1 ) use of antihypertensive medication, or 2 ) a systolic blood pressure at visit above 140 mm Hg and/or diastolic blood pressure above 90 mm Hg.
Detailed analysis of the concentrations of cholesteryl esters and free cholesterol in the total cholesterol fraction and the isolated HDL-C fraction, in a subset of patients, is provided in Table 2 .
Compared with controls, hypertension was more prevalent among LCAT mutation carriers ( P = 0.01). Statin use was more prevalent in the ABCA1 and LCAT mutation carriers compared with the control group. The low HDL-C group had similar baseline characteristics compared with the ABCA1 and LCAT groups.

Basal adrenal steroidogenesis
Compared with the control group, we identifi ed lower 24 h urinary excretion of 17-KS in carriers of mutations in ABCA1 (33%, P = 0.003; Fig. 1A ), in LCAT (27%, P = 0.01; carriers were compound heterozygotes while three of the LCAT mutation carriers were homozygotes. None of the participants were referred to our clinic for symptoms of adrenal dysfunction. Individuals were matched to male controls for age. In parallel, we also included subjects with equally low HDL-C levels without mutations in ABCA1 or LCAT . Demographic, clinical, and biochemical characteristics of all low HDL-C groups as well as controls are listed in Table 1 . As expected, HDL-C levels were 39% lower in carriers of ABCA1 or LCAT mutations and in the low HDL-C group, compared with normolipidemic controls ( P < 0.001). LDL-C levels were 25% lower in carriers of ABCA1 mutations ( P = 0.003), 11% lower in carriers of LCAT mutations (n.s.), and 12% lower in the low HDL-C group ( P = 0.34 and P = 0.39) compared with controls .  ( 16 ) did not reveal symptoms of clinically relevant adrenal dysfunction. These fi ndings are in line with those in mice, and tissue culture showing that the supply of plasma lipoprotein-derived cholesterol is used for basal adrenal steroidogenesis, but that this pathway is not able to respond to acute stress (27)(28)(29). For a proper quick response, endogenous adrenal cortisol reserves are secreted upon stimulation. Because steroidogenic tissues are rapidly depleted of cortisol following stimulation, replenishing is thought to occur via uptake of cholesterol from lipoproteins. We previously reported that individuals with reduced SRB1 function displayed mild adrenal insuffi ciency in addition to a reduced urinary excretion of steroid hormones ( 16 ). Thus reduced SRB1 function on adrenal cells has a larger impact on adrenal steroidogenesis when compared with low levels of plasma HDL-C caused by reduced ABCA1 or LCAT function.
There were no differences in urinary excretion of free cortisol. However, particularly in the lower ranges the quantifi cation of free cortisol has been shown to be less reliable as compared with the quantifi cation of full urinary steroid metabolites in diagnosing the presence of hypocortisolism ( 30,31 ).
In accordance with LCAT's mechanism of action, cholesteryl ester concentration is lower in LCAT mutation carriers, with the most pronounced effect in homozygous carriers. Because adrenal steroidogenesis was not further decreased in homozygous carriers compared with heterozygous carriers, there is no indication that the adrenal gland has a preference for cholesteryl esters as a substrate for steroidogenesis. This is supported by the fact that a similar reduction in steroid metabolites was observed in subjects with low HDL-C not related to defects in LCAT.
Plasma levels of ACTH did not differ between the low HDL-C group and controls. The latter most likely refl ects Fig. 1B ), and in the low HDL-C group (30%, P = 0.04 and 34%, P = 0.02; Fig. 1 ). These differences remained statistically signifi cant after correcting for differences in plasma LDL-C and statin use using a linear regression model ( P = 0.01 and P = 0.02 for carriers of ABCA1 mutations or LCAT mutations vs. controls, respectively). The mean 17-KS steroid excretion of 14.1 mol/24 h in the ABCA1 mutation carriers was below the reference values for 17-KS in the appropriate age group ( 23 ).
The lower levels of urinary 17-OHCS in both ABCA1 and LCAT mutation carriers, as well as the low HDL-C group, did not reach statistical signifi cance ( P = 0.11 and P = 0.15, respectively; Fig. 1A, B ). The full panel of steroid metabolites is presented in Fig. 2A

Adrenal response to cosyntropin
In a cosyntropin stimulation test, the cortisol response to physiological levels of ACTH was measured as an approach to assess adrenal cortical function ( 25,26 ). The peak serum cortisol response to ACTH was comparable in ABCA1 and LCAT mutation carriers and did not differ from controls ( P = 0.10 and P = 0.87 respectively, Fig. 3A, B ). Also peak plasma levels of free cortisol, taking into account possible differences in CBG levels ( 16 ), were not different. Plasma lipid levels did not differ signifi cantly before and after cosyntropin testing (data not shown).

DISCUSSION
The present study demonstrates that under normal conditions male individuals with low levels of HDL-C, irrespective of molecular origin, have signifi cantly lower urinary Fig. 1. Twenty-four hour urinary steroid excretion in male ABCA1 (A) and LCAT (B) mutation carriers compared with age-matched male controls. Data are presented as mean ± SD. p 1 , the uncorrected P value for Student's t -test; p 2 , the P value corrected for LDL-C; p We would like to discuss three limitations of our study. First, adrenal cells express ABCA1 ( 32 ). Thus reduced adrenal ABCA1 expression could conceptually lead to accumulation of cholesterol in adrenal cells, compromising the fact that the decrease in urinary steroid metabolites is only mild, thereby precluding a compensatory ACTH increase. In line , none of the study participants reported signs of hypocortisolism.  intra-adrenal signaling and steroidogenesis due to cholesterol toxicity as proposed for pancreatic ␤ -cell dysfunction in individuals carrying ABCA1 mutations ( 33,34 ). Although we cannot exclude this possibility, mutations in LCAT as well as low HDL-C in noncarrier individuals are associated with similar reductions in HDL-C and urinary steroid metabolite excretion, making an ABCA1-specifi c effect implausible.
A second limitation is that carriers of ABCA1 mutations showed signifi cantly lower levels of plasma LDL-C levels in addition to low levels of HDL-C. This could mean that combined low levels of HDL-C and LDL-C account for the effects observed, but as already discussed, a role for LDL-C in human adrenal corticoid production is unlikely (4)(5)(6). In line, statistical corrections for the observed reductions in LDL-C did not affect outcome.
Third, in the urinary steroid metabolites, the 17-KS were particularly decreased in carriers compared with controls. Because 17-KS are metabolites derived largely from the androgenic pathway, this fi nding hints toward the preferential use of HDL-derived cholesterol in adrenal steroidogenesis. Further studies are needed to dissect whether, and to what extent, HDL-derived cholesterol contributes to steroidogenesis in either adrenal or testicular steroid production.
In conclusion, we demonstrate that basal adrenal steroidogenesis is compromised in males with low levels of plasma HDL-C, establishing a role for HDL-derived cholesterol in adrenal steroidogenesis in humans.