HDL is redundant for adrenal steroidogenesis in LDLR knockout mice with a human-like lipoprotein profile
2016; Elsevier BV; Volume: 57; Issue: 4 Linguagem: Inglês
10.1194/jlr.m066019
ISSN1539-7262
AutoresMenno Hoekstra, Miranda Van Eck,
Tópico(s)Estrogen and related hormone effects
ResumoThe contribution of HDL to adrenal steroidogenesis appears to be different between mice and humans. In the current study, we tested the hypothesis that a difference in lipoprotein profile may be the underlying cause. Hereto, we determined the impact of HDL deficiency on the adrenal glucocorticoid output in genetically modified mice with a human-like lipoprotein profile. Genetic deletion of APOA1 in LDL receptor (LDLR) knockout mice was associated with HDL deficiency and a parallel increase in the level of cholesterol associated with nonHDL fractions. Despite a compensatory increase in the adrenal relative mRNA expression levels of the cholesterol synthesis gene, HMG-CoA reductase, adrenals from APOA1/LDLR double knockout mice were severely depleted of neutral lipids, as compared with those of control LDLR knockout mice. However, basal corticosterone levels and the adrenal glucocorticoid response to stress were not different between the two types of mice. In conclusion, we have shown that HDL is not critical for proper adrenal glucocorticoid function when mice are provided with a human-like lipoprotein profile. Our findings provide the first experimental evidence that APOB-containing lipoproteins may facilitate adrenal steroidogenesis, in an LDLR-independent manner, in vivo in mice. The contribution of HDL to adrenal steroidogenesis appears to be different between mice and humans. In the current study, we tested the hypothesis that a difference in lipoprotein profile may be the underlying cause. Hereto, we determined the impact of HDL deficiency on the adrenal glucocorticoid output in genetically modified mice with a human-like lipoprotein profile. Genetic deletion of APOA1 in LDL receptor (LDLR) knockout mice was associated with HDL deficiency and a parallel increase in the level of cholesterol associated with nonHDL fractions. Despite a compensatory increase in the adrenal relative mRNA expression levels of the cholesterol synthesis gene, HMG-CoA reductase, adrenals from APOA1/LDLR double knockout mice were severely depleted of neutral lipids, as compared with those of control LDLR knockout mice. However, basal corticosterone levels and the adrenal glucocorticoid response to stress were not different between the two types of mice. In conclusion, we have shown that HDL is not critical for proper adrenal glucocorticoid function when mice are provided with a human-like lipoprotein profile. Our findings provide the first experimental evidence that APOB-containing lipoproteins may facilitate adrenal steroidogenesis, in an LDLR-independent manner, in vivo in mice. Glucocorticoids are produced from the common steroidogenic precursor, cholesterol. It is generally accepted that under basal conditions sufficient amounts of cholesterol are acquired from endogenous synthesis by the adrenals. However, it has become evident from studies in genetically modified mice that lipoproteins, in particular HDLs, are important for delivering cholesterol substrate to adrenocortical cells under high steroidogenic pressure conditions, like stress. More specifically, probucol-induced lowering of both HDL-cholesterol and LDL-cholesterol levels in mice is associated with a >55% decrease in the glucocorticoid response to endotoxemia (1Hoekstra M. Korporaal S.J. Li Z. Zhao Y. Van Eck M. Van Berkel T.J. Plasma lipoproteins are required for both basal and stress-induced adrenal glucocorticoid synthesis and protection against endotoxemia in mice.Am. J. Physiol. Endocrinol. Metab. 2010; 299: E1038-E1043Crossref PubMed Scopus (25) Google Scholar). Studies in LCAT and APOA1 knockout mice, respectively, have indicated that a specific decrease in plasma HDL-cholesterol levels is associated with a 25–50% decrease in the maximal adrenal glucocorticoid output (2Hoekstra M. Korporaal S.J. van der Sluis R.J. Hirsch-Reinshagen V. Bochem A.E. Wellington C.L. Van Berkel T.J. Kuivenhoven J.A. Van Eck M. LCAT deficiency in mice is associated with a diminished adrenal glucocorticoid function.J. Lipid Res. 2013; 54: 358-364Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 3Plump A.S. Erickson S.K. Weng W. Partin J.S. Breslow J.L. Williams D.L. Apolipoprotein A-I is required for cholesteryl ester accumulation in steroidogenic cells and for normal adrenal steroid production.J. Clin. Invest. 1996; 97: 2660-2671Crossref PubMed Scopus (164) Google Scholar). Moreover, disruption of (adrenal-specific) HDL receptor function in mice is associated with a 40–50% decrease in the adrenocortical steroidogenic capacity (4Hoekstra M. Ye D. Hildebrand R.B. Zhao Y. Lammers B. Stitzinger M. Kuiper J. Van Berkel T.J. Van Eck M. Scavenger receptor class B type I-mediated uptake of serum cholesterol is essential for optimal adrenal glucocorticoid production.J. Lipid Res. 2009; 50: 1039-1046Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 5Hoekstra M. van der Sluis R.J. Van Eck M. Van Berkel T.J. Adrenal-specific scavenger receptor BI deficiency induces glucocorticoid insufficiency and lowers plasma very-low-density and low-density lipoprotein levels in mice.Arterioscler. Thromb. Vasc. Biol. 2013; 33: e39-e46Crossref PubMed Scopus (33) Google Scholar). Male carriers of functional mutations in the HDL biogenesis genes, ABCA1 and LCAT, display a decrease in the 24 h urinary excretion rate of adrenal-derived steroids (6Bochem A.E. Holleboom A.G. Romijn J.A. Hoekstra M. Dallinga-Thie G.M. Motazacker M.M. Hovingh G.K. Kuivenhoven J.A. Stroes E.S. High density lipoprotein as a source of cholesterol for adrenal steroidogenesis: a study in individuals with low plasma HDL-C.J. Lipid Res. 2013; 54: 1698-1704Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). However, basal and stimulated plasma cortisol levels are similar in HDL-deficient male ABCA1 and LCAT mutation carriers and their normolipidemic controls (6Bochem A.E. Holleboom A.G. Romijn J.A. Hoekstra M. Dallinga-Thie G.M. Motazacker M.M. Hovingh G.K. Kuivenhoven J.A. Stroes E.S. High density lipoprotein as a source of cholesterol for adrenal steroidogenesis: a study in individuals with low plasma HDL-C.J. Lipid Res. 2013; 54: 1698-1704Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Furthermore, both the urinary glucocorticoid excretion rate and cortisol response to corticotropin are unaltered in females with genetically low HDL (7Bochem A.E. Holleboom A.G. Romijn J.A. Hoekstra M. Dallinga G.M. Motazacker M.M. Hovingh G.K. Kuivenhoven J.A. Stroes E.S. Adrenal function in females with low plasma HDL-C due to mutations in ABCA1 and LCAT.PLoS One. 2014; 9: e90967Crossref PubMed Scopus (10) Google Scholar). The presence of relatively low HDL-cholesterol levels is, thus, in striking contrast to what is observed in mice: not consistently associated with glucocorticoid insufficiency in humans. In vitro studies have suggested that both HDL and APOB-containing lipoproteins, i.e., VLDL and LDL, can theoretically supply cholesterol to adrenocortical cells (8Xing Y. Rainey W.E. Apolzan J.W. Francone O.L. Harris R.B. Bollag W.B. Adrenal cell aldosterone production is stimulated by very-low-density lipoprotein (VLDL).Endocrinology. 2012; 153: 721-731Crossref PubMed Scopus (27) Google Scholar, 9Higashijima M. Nawata H. Kato K. Ibayashi H. Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells.Endocrinol. Jpn. 1987; 34: 635-645Crossref PubMed Scopus (35) Google Scholar, 10Higashijima M. Kato K. Nawata H. Ibayashi H. Studies on lipoprotein and adrenal steroidogenesis: II. Utilization of low density lipoprotein- and high density lipoprotein-cholesterol for steroid production in functioning human adrenocortical adenoma cells in culture.Endocrinol. Jpn. 1987; 34: 647-657Crossref PubMed Scopus (20) Google Scholar, 11Rainey W.E. Rodgers R.J. Mason J.I. The role of bovine lipoproteins in the regulation of steroidogenesis and HMG-CoA reductase in bovine adrenocortical cells.Steroids. 1992; 57: 167-173Crossref PubMed Scopus (24) Google Scholar). Importantly, human subjects exhibit a markedly different lipoprotein profile as compared with mice. The majority of cholesterol in humans is carried by LDL, while the murine lipoprotein profile is characterized by relatively low to absent levels of cholesterol associated with VLDL/LDL in the context of normal HDL-cholesterol levels. The relative importance of HDL-associated cholesterol as steroidogenic substrate can, thus, hypothetically be different between these two specific species due to the fact that human plasma, as compared with murine plasma, contains additional potential cholesterol sources, i.e., LDL and VLDL. To provide experimental proof for this hypothesis in the current study, we determined the impact of HDL deficiency on the adrenal glucocorticoid output in genetically modified mice that contain a human-like lipoprotein profile. APOA1 knockout mice lacking a functional APOA1 protein (12Vaessen S.F. Veldman R.J. Comijn E.M. Snapper J. Sierts J.A. van den Oever K. Beattie S.G. Twisk J. Kuivenhoven J.A. AAV gene therapy as a means to increase apolipoprotein (Apo) A-I and high-density lipoprotein-cholesterol levels: correction of murine ApoA-I deficiency.J. Gene Med. 2009; 11: 697-707Crossref PubMed Scopus (16) Google Scholar) were provided on a hyperlipidemic LDL receptor (LDLR) knockout background (13Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1271) Google Scholar) by Dr. J. A. Kuivenhoven from the Amsterdam Medical Center (Amsterdam, The Netherlands). These APOA1/LDLR double knockout (DKO) mice were subsequently inbred to maintain an in-house colony. Male APOA1/LDLR DKO (N = 14) and control LDLR single knockout (SKO) mice (N = 14) were maintained on a regular chow diet. Throughout the experiment, both types of mice were housed in the same climate-controlled stable with a 12 h/12 h dark-light cycle and handled identically. At 4 months of age, ad libitum-fed age-matched mice (N = 6 per genotype) were bled at 9:00 AM from the tail to obtain a basal plasma corticosterone measurement. Subsequently, these six mice per genotype were injected intraperitoneally with a sublethal dose (50 μg/kg) of lipopolysaccharide from Salmonella minnesota R595 and euthanized 2 h later to measure the maximum endotoxemia-related plasma corticosterone response (1Hoekstra M. Korporaal S.J. Li Z. Zhao Y. Van Eck M. Van Berkel T.J. Plasma lipoproteins are required for both basal and stress-induced adrenal glucocorticoid synthesis and protection against endotoxemia in mice.Am. J. Physiol. Endocrinol. Metab. 2010; 299: E1038-E1043Crossref PubMed Scopus (25) Google Scholar, 14Cai L. Ji A. de Beer F.C. Tannock L.R. van der Westhuyzen D.R. SR-BI protects against endotoxemia in mice through its roles in glucocorticoid production and hepatic clearance.J. Clin. Invest. 2008; 118: 364-375Crossref PubMed Scopus (124) Google Scholar). The remaining mice (N = 8 per genotype) were subjected to overnight fasting by food deprivation from 5:00 PM onwards. At 9:00 AM the next morning, mice were bled via the tail for fasting plasma corticosterone and blood glucose measurements. After anesthesia by subcutaneous injection with a mix of 70 mg/kg bodyweight xylazine, 1.8 mg/kg bodyweight atropine, and 350 mg/kg bodyweight ketamine, mice were bled via retro-orbital bleeding, euthanized, and subjected to whole body perfusion with ice-cold PBS. Adrenals were collected free of surrounding fat, weighed, and stored at −20°C or fixed overnight in 3.7% neutral-buffered formalin solution (Formalfixx; Shandon Scientific Ltd, UK). All animal work was approved by the Leiden University Animal Ethics Committee and performed in compliance with the Dutch government guidelines and Directive 2010/63/EU of the European Parliament. Corticosterone levels in tail blood plasma were determined using the corticosterone 3H RIA kit from ICN Biomedicals according to the protocol from the supplier. Plasma concentrations of free cholesterol and cholesterol esters were determined using enzymatic colorimetric assays. The cholesterol distribution over the different lipoproteins in plasma was analyzed by fractionation of 30 μl pooled plasma of each mouse genotype using a Superose 6 column (3.2 × 30 mm, Smart-system; Pharmacia). Total cholesterol content of the effluent was determined using enzymatic colorimetric assays. Blood glucose levels were routinely measured using a calibrated Accu-Check glucometer (Roche Diagnostics, Almere, The Netherlands) Lipids from adrenals were extracted using the method of Bligh and Dyer (15Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar). After dissolving the lipids in 1% Triton X-100, the contents of free cholesterol and cholesterol esters were determined using enzymatic colorimetric assays and expressed as micrograms per milligram of protein. Seven micrometer cryosections were prepared on a Leica CM3050-S cryostat. Cryosections were routinely stained with hematoxylin (Sigma) and Oil red O (Sigma) for lipid visualization. Gene expression analysis was performed essentially as described (16Hoekstra M. Kruijt J.K. Van Eck M. Van Berkel T.J. Specific gene expression of ATP-binding cassette transporters and nuclear hormone receptors in rat liver parenchymal, endothelial, and Kupffer cells.J. Biol. Chem. 2003; 278: 25448-25453Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Equal amounts of RNA were reverse transcribed and real-time quantitative PCR analysis was subsequently executed on the cDNA using an ABI Prism 7500 apparatus (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Acidic ribosomal phosphoprotein P0 (36B4), GAPDH, succinate dehydrogenase complex subunit A flavoprotein (SDHA), peptidylprolyl isomerase A (PPIA), β-2-microglobulin (B2M), transferrin receptor (TFRC), and β-actin (ACTB) were used as housekeeping genes for normalization. Statistical analysis was performed using GraphPad InStat software (San Diego, CA, http://www.graphpad.com). Normality of the experimental groups was confirmed using the method of Kolmogorov and Smirnov. The significance of differences was calculated using a two-tailed unpaired t-test or two-way ANOVA where appropriate. Probability values less than 0.05 were considered significant. Regular chow diet-fed LDLR knockout mice exhibit a highly similar lipoprotein profile to that observed in normolipidemic humans (13Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1271) Google Scholar). To investigate whether the contribution of HDL to adrenal glucocorticoid output is different in mice with a human-like lipoprotein profile, we determined the impact of genetic HDL deficiency in mice on a LDLR knockout genetic background. Hereto, HDL-deficient APOA1 knockout mice were crossbred with LDLR SKO mice to generate the respective APOA1/LDLR DKO mice. As can be appreciated from Fig. 1A, plasma free and total cholesterol levels did not significantly differ between regular chow diet-fed male DKO and SKO mice. However, lipoprotein distribution analysis on pooled plasma (Fig. 1B) revealed that DKO mice exhibited a highly similar reduction in plasma HDL-cholesterol levels (−65%), as previously noted in APOA1 knockout mice on a wild-type background (3Plump A.S. Erickson S.K. Weng W. Partin J.S. Breslow J.L. Williams D.L. Apolipoprotein A-I is required for cholesteryl ester accumulation in steroidogenic cells and for normal adrenal steroid production.J. Clin. Invest. 1996; 97: 2660-2671Crossref PubMed Scopus (164) Google Scholar). DKO mice showed a parallel 89% increase in levels of cholesterol associated with VLDL particles, as compared with their respective APOA1-containing SKO controls (Fig. 1B). As a result, the plasma nonHDL-cholesterol over HDL-cholesterol ratio was, thus, markedly higher in DKO mice as compared with SKO mice (Fig. 1C). Previous studies by the group of Dr. Mary Sorci-Thomas have suggested that, after a short-term (4 h) fasting period, the adrenals of DKO mice are severely depleted of cholesterol esters, despite the fact that DKO mice still carry ∼30% of the normal amount of HDL-associated cholesterol in APOE-enriched HDL particles (17Zabalawi M. Bhat S. Loughlin T. Thomas M.J. Alexander E. Cline M. Bullock B. Willingham M. Sorci-Thomas M.G. Induction of fatal inflammation in LDL receptor and ApoA-I double-knockout mice fed dietary fat and cholesterol.Am. J. Pathol. 2003; 163: 1201-1213Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Quantification of the adrenal lipid stores revealed that both free cholesterol (−21%; P = 0.002) and cholesterol ester (−51%; P = 0.005) levels were also markedly lower in the adrenals of our DKO mice, as compared their SKO controls, after an overnight fast (Fig. 2A), suggesting that the adrenal lipid depletion effect associated with APOA1 deficiency is independent of the metabolic/stress state. Oil red O staining of adrenal cryostat sections further verified adrenal lipid depletion. Adrenals from SKO mice had abundant neutral lipids in their adrenal cortex. In contrast, an equally low extent of lipid accumulation was microscopically detected within cortical cells of DKO adrenals (Fig. 2B), as previously noted in glucocorticoid insufficient LCAT knockout mice and probucol-treated C57BL/6 mice (1Hoekstra M. Korporaal S.J. Li Z. Zhao Y. Van Eck M. Van Berkel T.J. Plasma lipoproteins are required for both basal and stress-induced adrenal glucocorticoid synthesis and protection against endotoxemia in mice.Am. J. Physiol. Endocrinol. Metab. 2010; 299: E1038-E1043Crossref PubMed Scopus (25) Google Scholar, 2Hoekstra M. Korporaal S.J. van der Sluis R.J. Hirsch-Reinshagen V. Bochem A.E. Wellington C.L. Van Berkel T.J. Kuivenhoven J.A. Van Eck M. LCAT deficiency in mice is associated with a diminished adrenal glucocorticoid function.J. Lipid Res. 2013; 54: 358-364Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). An efficient feedback system exists that modulates the expression of genes involved in cholesterol synthesis and uptake in response to changes in intracellular cholesterol levels [reviewed by Sato and Takano (18Sato R. Takano T. Regulation of intracellular cholesterol metabolism.Cell Struct. Funct. 1995; 20: 421-427Crossref PubMed Scopus (37) Google Scholar)]. Quantitative real-time PCR was employed to uncover possible compensatory gene regulation. No change was noted, as compared with SKO adrenals, in the relative mRNA expression level of the HDL receptor, SR-BI, in DKO adrenals (Fig. 2C). In addition, genetic APOA1 deficiency was not associated with a difference in relative mRNA expression levels of hormone-sensitive lipase (HSL), ACAT-1, and steroidogenic acute regulatory protein (STAR) that are respectively involved in the de- and re-esterification of cholesterol and intracellular mobilization of cholesterol to the steroidogenic pathway (Fig. 2C). However, we did observe a marked increase (425%; P < 0.001; Fig. 2C) in the mRNA expression of the enzyme, HMG-CoA reductase, in DKO adrenals. It thus appears that, in a human-like lipoprotein context, HDL deficiency in mice is associated with depletion of adrenal cholesterol stores despite a compensatory increase in intra-adrenal cholesterol synthesis. Levels of the primary glucocorticoid, corticosterone, were measured in plasma under basal and stressed conditions to verify whether the depletion of adrenal cholesterol esters also executed a negative impact on the overall steroid output. In line with the general notion that lipoprotein-derived cholesterol is not required for the synthesis of glucocorticoids under low steroidogenic conditions, plasma corticosterone levels were similar in nonstressed ad libitum-fed SKO and DKO mice (Fig. 3A). Food deprivation is a powerful inducer of an adrenal glucocorticoid response in mice (19Bates H.E. Campbell J.E. Ussher J.R. Baggio L.L. Maida A. Seino Y. Drucker D.J. Gipr is essential for adrenocortical steroidogenesis; however, corticosterone deficiency does not mediate the favorable metabolic phenotype of Gipr(-/-) mice.Diabetes. 2012; 61: 40-48Crossref PubMed Scopus (31) Google Scholar, 20Petersen H.H. Andreassen T.K. Breiderhoff T. Bräsen J.H. Schulz H. Gross V. Gröne H.J. Nykjaer A. Willnow T.E. Hyporesponsiveness to glucocorticoids in mice genetically deficient for the corticosteroid binding globulin.Mol. Cell. Biol. 2006; 26: 7236-7245Crossref PubMed Scopus (102) Google Scholar). Overnight fasting resulted in a significant 6.6-fold increase (P < 0.001 vs. basal) in circulating corticosterone levels in SKO mice, as anticipated. Strikingly, corticosterone levels were virtually identical in both groups of fasted mice (256 ± 21 ng/ml for DKO vs. 269 ± 15 ng/ml for SKO; P > 0.05). HDL deficiency thus does not seem to be associated with glucocorticoid insufficiency in mice with a human-like lipoprotein. In agreement with a normal metabolic glucocorticoid action in HDL-deficient mice, DKO mice did not display hypoglycemia, as compared with SKO mice under fasting conditions (Fig. 3B). The induction of endotoxemia is associated with a concomitant rise in the plasma level of glucocorticoids (21Suzuki S. Nakano K. LPS-caused secretion of corticosterone is mediated by histamine through histidine decarboxylase.Am. J. Physiol. 1986; 250: E243-E247PubMed Google Scholar, 22Hoekstra M. Frodermann V. van den Aardweg T. van der Sluis R.J. Kuiper J. Leukocytosis and enhanced susceptibility to endotoxemia but not atherosclerosis in adrenalectomized APOE knockout mice.PLoS One. 2013; 8: e80441Crossref PubMed Scopus (11) Google Scholar). In further support of a similar maximal steroidogenic capacity of the adrenals in the two types of mice, equally high levels of corticosterone (∼250 ng/ml; Fig. 3A) were detected in the plasma of SKO and DKO mice after induction of endotoxemia through injection of a sublethal dose of lipopolysaccharide. In the current study, we tested the hypothesis that a difference in lipoprotein profile between mice and humans can explain the relative importance of HDL-cholesterol as substrate for adrenal steroidogenesis. A 70% reduction in plasma HDL-cholesterol levels in APOA1 SKO mice is associated with a severe depletion of adrenal cholesterol ester stores and a concomitant impairment of the adrenal glucocorticoid response to stress (3Plump A.S. Erickson S.K. Weng W. Partin J.S. Breslow J.L. Williams D.L. Apolipoprotein A-I is required for cholesteryl ester accumulation in steroidogenic cells and for normal adrenal steroid production.J. Clin. Invest. 1996; 97: 2660-2671Crossref PubMed Scopus (164) Google Scholar). The APOE-rich HDL particles remaining in these mice (23Plump A.S. Azrolan N. Odaka H. Wu L. Jiang X. Tall A. Eisenberg S. Breslow J.L. ApoA-I knockout mice: characterization of HDL metabolism in homozygotes and identification of a post-RNA mechanism of apoA-I up-regulation in heterozygotes.J. Lipid Res. 1997; 38: 1033-1047Abstract Full Text PDF PubMed Google Scholar) are apparently not able to compensate for the lack of cholesterol supplied by APOA1-containing particles for steroidogenesis. Although it cannot be excluded that the APOE-rich HDL is a poor substrate for adrenal cholesterol delivery, we anticipate that the adrenal cholesterol insufficiency observed in APOA1 SKO mice is primarily the result of an overall too low amount of HDL particles being present in the circulation. Genetic variations in the APOA1 gene have also been associated with HDL deficiency in humans (24Wada M. Iso T. Asztalos B.F. Takama N. Nakajima T. Seta Y. Kaneko K. Taniguchi Y. Kobayashi H. Nakajima K. et al.Marked high density lipoprotein deficiency due to apolipoprotein A-I Tomioka (codon 138 deletion).Atherosclerosis. 2009; 207: 157-161Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 25Tilly-Kiesi M. Zhang Q. Ehnholm S. Kahri J. Lahdenperä S. Ehnholm C. Taskinen M.R. ApoA-IHelsinki (Lys107→0) associated with reduced HDL cholesterol and LpA-I:A-II deficiency.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1294-1306Crossref PubMed Scopus (43) Google Scholar, 26Matsunaga T. Hiasa Y. Yanagi H. Maeda T. Hattori N. Yamakawa K. Yamanouchi Y. Tanaka I. Obara T. Hamaguchi H. Apolipoprotein A-I deficiency due to a codon 84 nonsense mutation of the apolipoprotein A-I gene.Proc. Natl. Acad. Sci. USA. 1991; 88: 2793-2797Crossref PubMed Scopus (97) Google Scholar). However, due to the limited number of subjects with genetic APOA1 deficiency, the specific contribution of APOA1-containing HDL particles to adrenal steroidogenesis remains to be determined in the human setting. In the current study, we observed that APOA1 deficiency in mice with a human-like lipoprotein, i.e., on a LDLR knockout (hyperlipidemic) background, is associated with a similar 65% reduction in plasma HDL-cholesterol levels and adrenal cholesterol depletion, as observed in APOA1 knockout mice on a wild-type (normolipidemic) background. In contrast, APOA1/LDLR DKO mice do not suffer from glucocorticoid insufficiency, as their maximal glucocorticoid output is similar to that of HDL-containing single LDLR knockout controls. It thus appears that the presence of a human-like lipoprotein profile alleviates the glucocorticoid insufficiency associated with APOA1 deficiency in mice. Studies by Plump et al. (3Plump A.S. Erickson S.K. Weng W. Partin J.S. Breslow J.L. Williams D.L. Apolipoprotein A-I is required for cholesteryl ester accumulation in steroidogenic cells and for normal adrenal steroid production.J. Clin. Invest. 1996; 97: 2660-2671Crossref PubMed Scopus (164) Google Scholar) have suggested that adrenals from APOA1 knockout mice are still able to respond to stress, although to a minor extent as compared with those of wild-type mice, due to compensatory upregulation of pathways that are normally of minor importance, such as cholesterol uptake by the LDLR and de novo cholesterol synthesis. A 5-fold increase in the gene expression of HMG-CoA reductase in the adrenals of DKO mice was detected under fasting stress conditions, which suggests that de novo cholesterol synthesis is stimulated to compensate for the loss of HDL-cholesterol. In contrast to our DKO mice, HDL-deficient LCAT knockout mice do display a diminished adrenal glucocorticoid function despite a marked 6-fold increase in adrenal HMG-CoA reductase expression (2Hoekstra M. Korporaal S.J. van der Sluis R.J. Hirsch-Reinshagen V. Bochem A.E. Wellington C.L. Van Berkel T.J. Kuivenhoven J.A. Van Eck M. LCAT deficiency in mice is associated with a diminished adrenal glucocorticoid function.J. Lipid Res. 2013; 54: 358-364Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). From these combined findings, it can be concluded that such a 5- to -6-fold increase in adrenal HMG-CoA reductase expression is, by itself, not sufficient to overcome adrenal glucocorticoid insufficiency. All mice used in the current study did not express a functional LDLR, which excludes a compensatory role for LDLR-mediated cholesterol acquisition by adrenals in DKO mice. In vitro studies by Kraemer et al. (27Kraemer F.B. Shen W.J. Patel S. Osuga J. Ishibashi S. Azhar S. The LDL receptor is not necessary for acute adrenal steroidogenesis in mouse adrenocortical cells.Am. J. Physiol. Endocrinol. Metab. 2007; 292: E408-E412Crossref PubMed Scopus (26) Google Scholar) have suggested that the LDLR is of negligible importance for acute steroidogenesis by adrenocortical cells. Furthermore, the levels of corticosterone measured in the fasting state in male LDLR knockout mice in the current experiment are almost identical to those found in male wild-type mice in our previous studies (28Hoekstra M. Meurs I. Koenders M. Out R. Hildebrand R.B. Kruijt J.K. Van Eck M. Van Berkel T.J. Absence of HDL cholesteryl ester uptake in mice via SR-BI impairs an adequate adrenal glucocorticoid-mediated stress response to fasting.J. Lipid Res. 2008; 49: 738-745Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). We therefore consider it highly unlikely that the impact of APOA1/HDL deficiency on glucocorticoid output in vivo is dependent on the adrenal LDLR genotype. In our experimental setup, APOB-containing lipoproteins could not be cleared by the LDLR, which is normally suggested to be the primary route of cholesterol delivery by these circulating lipid/protein complexes. Considerable evidence is, however, present that SR-BI is also able to facilitate the uptake of cholesterol from APOB-containing lipoproteins. Initial in vitro studies by Swarnakar et al. (29Swarnakar S. Temel R.E. Connelly M.A. Azhar S. Williams D.L. Scavenger receptor class B, type I, mediates selective uptake of low density lipoprotein cholesteryl ester.J. Biol. Chem. 1999; 274: 29733-29739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and Stangl, Hyatt, and Hobbs (30Stangl H. Hyatt M. Hobbs H.H. Transport of lipids from high and low density lipoproteins via scavenger receptor-BI.J. Biol. Chem. 1999; 274: 32692-32698Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) showed that murine SR-BI is able to mediate the selective uptake of cholesterol esters from human LDL. Subsequent cell culture studies by Webb et al. (31Webb N.R. de Beer M.C. Yu J. Kindy M.S. Daugherty A. van der Westhuyzen D.R. de Beer F.C. Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I, but not apoB, in human apoB transgenic mice.J. Lipid Res. 2002; 43: 1421-1428Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) verified a similar interaction of SR-BI with autologous mouse LDL. In support of a parallel role for SR-BI in APOB-containing lipoprotein cholesterol delivery in vivo, the removal from the blood circulation and tissue uptake of β-migrating VLDL particles, LDL, and chylomicron-remnants has been shown to be significantly lower in mice lacking functional SR-BI expression (32Van Eck M. Hoekstra M. Out R. Bos I.S. Kruijt J.K. Hildebrand R.B. Van Berkel T.J. Scavenger receptor BI facilitates the metabolism of VLDL lipoproteins in vivo.J. Lipid Res. 2008; 49: 136-146Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 33Brodeur M.R. Luangrath V. Bourret G. Falstrault L. Brissette L. Physiological importance of SR-BI in the in vivo metabolism of human HDL and LDL in male and female mice.J. Lipid Res. 2005; 46: 687-696Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 34Out R. Kruijt J.K. Rensen P.C. Hildebrand R.B. de Vos P. Van Eck M. Van Berkel T.J. Scavenger receptor BI plays a role in facilitating chylomicron metabolism.J. Biol. Chem. 2004; 279: 18401-18406Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). As a result, SR-BI knockout mice not only display increased plasma levels of HDL-cholesterol, but also exhibit an increase in the amount of cholesterol carried by APOB-containing lipoproteins (35Van Eck M. Twisk J. Hoekstra M. Van Rij B.T. Van der Lans C.A. Bos I.S. Kruijt J.K. Kuipers F. Van Berkel T.J. Differential effects of scavenger receptor BI deficiency on lipid metabolism in cells of the arterial wall and in the liver.J. Biol. 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Adrenal glucocorticoid output is diminished in human subjects carrying a functional mutation in the SR-BI gene (38Vergeer M. Korporaal S.J. Franssen R. Meurs I. Out R. Hovingh G.K. Hoekstra M. Sierts J.A. Dallinga-Thie G.M. Motazacker M.M. et al.Genetic variant of the scavenger receptor BI in humans.N. Engl. J. Med. 2011; 364: 136-145Crossref PubMed Scopus (266) Google Scholar). Several heterozygote SR-BI P297S carriers actually show signs of adrenal dysfunction in spite of markedly increased plasma HDL-cholesterol levels (38Vergeer M. Korporaal S.J. Franssen R. Meurs I. Out R. Hovingh G.K. Hoekstra M. Sierts J.A. Dallinga-Thie G.M. Motazacker M.M. et al.Genetic variant of the scavenger receptor BI in humans.N. Engl. J. Med. 2011; 364: 136-145Crossref PubMed Scopus (266) Google Scholar). Given that, in the human situation, disruption of SR-BI function appears to be associated with a more extreme effect on the adrenal steroidogenic capacity than genetic lowering of HDL-cholesterol levels, it can be suggested that, in our current human-like lipoprotein setting, the impaired acquisition of cholesterol from APOA1-containing HDL particles can be fully compensated by enhanced cholesterol synthesis combined with SR-BI-mediated delivery of cholesterol from APOB-containing lipoproteins to the adrenals. Novel intervention strategies to reduce cardiovascular disease risk, such as proprotein convertase subtilisin/kexin type 9 (PCSK9) antibody treatment and statin/ezetimibe combination therapies, are aimed at reaching extremely low plasma LDL-cholesterol levels. No remarkable adrenal-associated events have been reported in meta-analyses of anti-PCSK9 antibody (39Zhang X.L. Zhu Q.Q. Zhu L. Chen J.Z. Chen Q.H. Li G.N. Xie J. Kang L.N. Xu B. Safety and efficacy of anti-PCSK9 antibodies: a meta-analysis of 25 randomized, controlled trials.BMC Med. 2015; 13: 123Crossref PubMed Scopus (199) Google Scholar) and statin/ezetimibe trials (40Luo L. Yuan X. Huang W. Ren F. Zhu H. Zheng Y. Tang L. Safety of coadministration of ezetimibe and statins in patients with hypercholesterolaemia: a meta-analysis.Intern. Med. J. 2015; 45: 546-557Crossref PubMed Scopus (20) Google Scholar). This may, at first sight, argue against our current working hypothesis that APOB-containing lipoproteins serve as primary cholesterol donors for steroidogenesis. However, one should take into account that: 1) adrenal dysfunction may only become evident under stress conditions; and 2) in-depth adrenal function testing is not common within these cardiovascular-oriented clinical trials. As such, inclusion of the adrenocorticotropic hormone (ACTH) stimulation test, the standard method to assess the maximal adrenal cortisol response, in trial protocols may aid in validating our hypothesis in the human setting. In light of our challenging concept, it is of interest to note that several case studies by Illingworth and colleagues (41Illingworth D.R. Kenny T.A. Connor W.E. Orwoll E.S. Corticosteroid production in abetalipoproteinemia: evidence for an impaired response ACTH.J. Lab. Clin. Med. 1982; 100: 115-126PubMed Google Scholar, 42Illingworth D.R. Kenny T.A. Orwoll E.S. Adrenal function in heterozygous and homozygous hypobetalipoproteinemia.J. Clin. Endocrinol. Metab. 1982; 54: 27-33Crossref PubMed Scopus (70) Google Scholar, 43Illingworth D.R. Orwoll E.S. Connor W.E. Impaired cortisol secretion in abetalipoproteinemia.J. Clin. Endocrinol. Metab. 1980; 50: 977-979Crossref PubMed Scopus (31) Google Scholar) have indicated that genetic LDL deficiency (abetalipoproteinemia) in humans is associated with subclinical adrenal insufficiency, as evident from an impaired ACTH-induced cortisol response and a lower urinary (free) cortisol excretion rate. In conclusion, we have shown that HDL is not critical for proper adrenal glucocorticoid function in mice with a human-like lipoprotein profile. Our findings contribute to a better understanding of the adrenal glucocorticoid function under human-like lipoprotein conditions and provide the first experimental evidence that APOB-containing lipoprotein fractions may facilitate adrenal steroidogenesis, in an LDLR-independent manner, in vivo. double knockout LDL receptor single knockout
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