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Am J Physiol Endocrinol Metab 295: E820-E831, 2008. First published July 29, 2008; doi:10.1152/ajpendo.90206.2008
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Disturbed cholesterol homeostasis in hormone-sensitive lipase-null mice

Céline Fernandez,1 Marie Lindholm,2 Morten Krogh,3 Stéphanie Lucas,1 Sara Larsson,1 Peter Osmark,1 Karin Berger,1 Jan Borén,4 Barbara Fielding,5 Keith Frayn,5 and Cecilia Holm1

1Department of Experimental Medical Science, 2Department of Clinical Sciences, and 3Department of Theoretical Physics, Lund University, Lund; 4Sahlgrenska Center for Cardiovascular and Metabolic Research/Wallenberg Laboratory, Department of Molecular and Clinical Medicine, The Sahlgrenska Academy at Göteborg University, Gothenburg, Sweden; and 5Oxford Centre for Diabetes, Endocrinology, and Metabolism, University of Oxford, Oxford, United Kingdom

Submitted 1 February 2008 ; accepted in final form 22 July 2008


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transcriptomics analysis revealed that genes involved in hepatic de novo cholesterol synthesis were downregulated in fed HSL-null mice that had been on a high-fat diet (HFD) for 6 mo. This finding prompted a further analysis of cholesterol metabolism in HSL-null mice, which was performed in fed and 16-h-fasted mice on a normal chow diet (ND) or HFD regimen. Plasma cholesterol was elevated in HSL-null mice, in all tested conditions, as a result of cholesterol enrichment of HDL and VLDL. Hepatic esterified cholesterol content and ATP-binding cassette transporter A1 (ABCA1) mRNA and protein levels were increased in HSL-null mice regardless of the dietary regimen. Unsaturated fatty acid composition of hepatic triglycerides was modified in fasted HSL-null mice on ND and HFD. The increased ABCA1 expression had no major effect on cholesterol efflux from HSL-null mouse hepatocytes. Taken together, the results of this study suggest that HSL plays a critical role in the hydrolysis of cytosolic cholesteryl esters and that increased levels of hepatic cholesteryl esters, due to lack of action of HSL in the liver, are the main mechanism underlying the imbalance in cholesterol metabolism in HSL-null mice.

lipoproteins; unsaturated fatty acids; high-fat diet; microarray

HORMONE-SENSITIVE LIPASE (HSL) is best known as the enzyme hydrolyzing acylglycerols stored in adipose tissue (49). In addition to acylglycerols, it hydrolyzes several other lipids, including cholesteryl esters, retinyl esters, lipoidal esters, and water-soluble esters (25, 47). HSL is widely expressed, and besides adipose tissue it is present in steroidogenic tissues, liver, skeletal muscle, intestinal mucosa, macrophages, and pancreatic β-cells (49). Its role in nonadipose tissues is not completely understood, but the phenotype revealed by the characterization of several independently generated HSL-null mouse lines in recent years suggests that HSL has several functions in addition to its role in adipocyte lipolysis (14, 22, 26, 42). Thus, the picture of HSL being a multifunctional enzyme involved in lipid metabolism and lipid signaling is emerging.

One feature shared among all characterized HSL-null models is lack of obesity (14, 22, 26, 42). HSL-null mice have in fact been shown to be resistant to diet-induced obesity (17). Despite the lack of obesity, an inflammatory response characterized by macrophage infiltration and increased necrotic-like cell death has been observed in white adipose tissue of HSL-null mice (7, 16). Adipose tissue inflammation is a condition frequently observed in the obese state and a possible trigger of obesity-associated insulin resistance and its complications, such as nonalcoholic fatty liver disease. The HSL-null mouse line established in our laboratory is indeed characterized by insulin resistance observed at the level of the liver as well as skeletal muscle and adipose tissue (22). However, conflicting data have been reported for other HSL-null mouse strains showing increased hepatic insulin sensitivity (27, 48).

Besides the lack of obesity, other features consistently observed in the different HSL-null mouse lines are decreased levels of plasma nonesterified fatty acids (NEFA) and increased levels of plasma cholesterol (15, 42, 48). Decreased levels of plasma NEFA result from impairment of adipocyte lipolysis and are, as expected, most pronounced in the fasted state. In the model established by Haemmerle et al. (15), the decreased levels of plasma NEFA have been shown to result in decreased hepatic synthesis of VLDL, which in turn is presumably the main cause of the low plasma triglyceride levels observed in HSL-null mice (11, 15, 17, 48). The elevation of plasma cholesterol, shown to be accompanied by increased HDL cholesterol (15), is intriguing. Noteworthy, neutral cholesterol ester hydrolase activity is abolished, or at least dramatically reduced, in all HSL-expressing tissues, including the liver, with macrophages being an exception showing only slightly reduced cholesterol ester hyhdrolase activity (9, 13, 22, 26). Lipase activity, on the other hand, is intact or much less reduced in nonadipose tissues of HSL-null mice (9, 13, 22, 26). This suggests that HSL may act as a cholesterol ester hydrolase in many tissues, whereas its function as a lipase may be confined to adipose tissue.

The decreased hepatic insulin sensitivity observed in our strain of HSL-null mice, together with the intriguing elevation of plasma cholesterol, prompted us to perform a transcriptomics analysis of the liver of HSL-null mice to gain insight into the mechanisms underlying these features of the HSL-null mouse model and to learn more about the role of HSL in the liver. The most striking result upon this analysis was the downregulation of eight genes encoding enzymes catalyzing successive steps in de novo synthesis of cholesterol. On the basis of these findings, the major aim of the present study was to perform a detailed analysis of cholesterol homeostasis in HSL-null mice. The results obtained support a role for HSL in the cytosolic hydrolysis of hepatic cholesteryl esters and furthermore suggest that HSL, via this role, is important for the maintenance of cholesterol homeostasis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. HSL-null mice were generated by targeted homologous recombination of the HSL gene in 129SV-derived embryonic stem cells, as described elsewhere (13). HSL-null and wild-type control mice were littermates and had a mixed genetic background from the inbred strains C57BL/6J and SV129. The animals were maintained in a temperature-controlled room (22°C) on a 12:12-h light-dark cycle. Female mice were employed throughout this study. Mice, 8–10 wk of age, were fed ad libitum a normal chow diet (ND) containing 4.8% fat and 0.005% cholesterol or a high-fat diet (HFD) containing 35.9% fat and 0.03% cholesterol (product nos. D12310 [GenBank] and D12309 [GenBank] , respectively; Research Diets, New Brunswick, NJ), as indicated in the figure legends, before the in vitro analyses were performed. Mice were killed by cervical dislocation, and tissues were rapidly dissected, snap-frozen, and stored in liquid nitrogen or at –80°C until analysis. The studies were approved by the local Animal Ethics Committee at Lund University.

Microarray analysis and gene ontology analysis. Equal amounts of liver samples from four to six HSL-null or wild-type fed mice were pooled, and RNA was extracted according to Chomczynski and Sacchi (6). Total RNA was purified using RNeasy kit (Qiagen). Biotinylated cRNA was prepared, following the protocol from Affymetrix and hybridized to mouse Affymetrix MG-U74A-v2 chips. The chips were washed, scanned, and subsequently analyzed with the Affymetrix Genechip MAS version 5.0 software. The chips were standardized by global scaling to a target intensity of 100. To identify genes differently expressed, three parameters that are calculated independently were combined: detection call, change call, and signal log ratio of base 2. To be considered down- or upregulated, a gene had to be detected in at least one of two groups, the expression level between two groups had to be identified as increased or decreased, and finally, the signal log ratio had to be ≥1 or no more than –1 respectively, which corresponds to a fold change of ≥2 or no more than –2. The total set of data is accessible through the GEO repository as series IDGSE10067.

The 12,488 genes and expressed sequence tags present on the chip were mapped to 4,589 gene ontology (GO) categories (18) with the use of the web server ACID (31). The genes were ranked in decreasing order of absolute value of logarithmic fold change between HSL-null mice on HFD and wild-type mice on HFD; i.e., the genes with the largest fold changes, up or down, had the lowest ranks. A Wilcoxon rank-sum test was employed for each GO category with the use of the publicly available program Catmap (2). Catmap outputs, among other things, 1) P values calculated as the probability that a random ordering of the genes produces a lower, or equally low, Wilcoxon rank sum as the ordering investigated, 2) false discovery rates for lists of GO categories, and 3) a receiver operating characteristic (ROC) area for each category, which is a normalized form of the Wilcoxon rank sum. The ROC area ranges from zero to one; the ROC area is one when the Wilcoxon rank sum attains its minimum and zero when the Wilcoxon rank sum attains its maximum. For large GO categories, the P value can be very small even for ROC areas far below one (but >0.5).

Real-time quantitative PCR. Total liver RNA was isolated using RNeasy kit from fed and 16-h-overnight-fasted mice, and then 1 µg was treated with DNase I (DNase amplification grade; Invitrogen). The purified RNA was reverse transcribed using random hexamers (Amersham Biosciences) and SuperScript II reverse transcriptase (Invitrogen Life Technologies). PCR was performed with an ABI 7900 system (Applied Biosystems). The following mouse Taqman primers were used: Gapdh (Mm99999915_g1), ATP-binding cassette transporter A1 (ABCA1; Mm00442646_m1), ATP-binding cassette transporter G1 (Abcg1; Mm00437390_m1), low-density lipoprotein receptor-related protein 1 (Lrp1; Mm01160450_g1), and stearoyl-CoA desaturase 1 (Scd1; Mm00772290_m1). Additionally, SYBR Green primers for the following genes were used: hypoxanthine phosphoribosyltransferase 1 (Hprt1), ribosomal protein s29 (Rps29), 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, farnesyl diphosphate synthase (Fdps), LDL receptor, and scavenger receptor BI (SR-BI). Primers were designed using the software Primer Express 1.5 (Applied Biosystems), and the sequences are found in supplemental data. Relative abundance of mRNA was calculated after normalization by geometric averaging of three internal control genes (Gapdh, Hprt1, and Rps29) (40).

Plasma lipids and lipoprotein analysis. Blood samples were drawn by retroorbital puncture in isofluorane anasthetized mice, using EDTA as an anticoagulant. Mice were either refed (overnight fasting for 12 h followed by 2 h of free access to food) or fasted for 16 h overnight. The plasma concentrations of triglycerides, cholesterol, and NEFA were determined using commercially available kits (Thermo Trace; Wako Chemicals). Samples from pooled plasma (n = 12–14) were separated by fast performance liquid chromatography (FPLC) gel filtration (ÄKTA Explorer; GE Healthcare) with a Superose 6 HR 10/30 column. Aliquots of 200 µl were injected onto the column and separated with PBS buffer, pH 7.4, at a flow rate of 0.2 ml/min (28). Triglyceride and cholesterol in the collected fractions were assayed as stated above.

Western blot analysis. Liver samples were homogenized in lysis buffer, pH 7.0, containing 25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton, and 1X protease inhibitor cocktail (Complete Mini; Roche). Total protein concentration was measured by BCA assay (Pierce), and 50 µg of protein was subjected to SDS-polyacrylamide gel electrophoresis (8% polyacrylamide). After transfer to PVDF membranes (Invitrogen), blots were incubated with a primary antibody mouse anti-mouse/rat ABCA1 (Neuromics) according to the instructions of the manufacturer. As secondary antibody, a horseradish peroxidase-conjugated sheep anti-mouse IgG was used. Western blot analysis was performed using a chemiluminescence system (Luminol), and detection was made using a charge-coupled device camera (LAS 1000; Fuji). Band intensities were quantified using the ImageJ software (http://rsb.info.nih.gov/ij).

Liver cholesterol. Total hepatic lipid content was extracted from mice that were either fed or fasted overnight for 16 h (10). In brief, 50 mg of liver was homogenized in 1 ml of PBS, and an aliquot corresponding to 20 mg was transferred to a glass tube containing 2:1 chloroform-methanol (vol/vol) and incubated overnight at 4°C. After being washed twice with water, the organic phase was dried under nitrogen gas. The lipids were then redissolved in chloroform and separated by TLC in a phase consisting of ether-petroleum ether-acetic acid (15:84:1), and the lipid spots were detected using iodine vapour. Silica gel of samples corresponding to the spots of cholesterol and cholesterol oleate standards were scraped and washed three times in chloroform. The samples were then dried under nitrogen gas and suspended in isopropanol before free and esterified cholesterol were measured with the use of commercial kits (Free Cholesterol, Wako Chemicals and Infinity Cholesterol Liquid Stable Reagent kit; Thermo Electron).

Liver triglycerides. Acylglycerides were extracted from the liver of fed and 16-h overnight-fasted mice and quantified using a kit (Thermo Trace) (3).

Fatty acid profile of liver triglycerides. Hepatic lipids were extracted from fed and 16-h overnight-fasted mice. Briefly, samples of liver (~200 mg) were homogenized in 750 µl of saline and transferred to a 10-ml glass tube, and lipids were extracted in 2:1 chloroform-methanol (vol/vol) (10). Triglycerides were separated by solid-phase extraction, and fatty acid methyl esters prepared before the samples were analyzed by gas chromatography (4).

Cholesterol efflux from isolated hepatocytes. Hepatocytes were isolated from wild-type and HSL-null mice using a two-step collagenase perfusion method (5) and plated on collagen-coated six-well plates at a density of 2.0 x 106 cells/35-mm well. The cells were cultured in William's E medium with Glutamax supplemented with penicillin (50,000 IU/l), streptomycin (50 mg/l), 0.28 mM sodium ascorbate, 0.1 µM sodium selenite, 1 nM insulin, and 1 nM dexamethasone. The medium was changed 5 h after plating to remove nonattached cells. After overnight cell culture, cholesterol efflux study was performed according to Sahoo et al. (32). In brief, cells were washed twice in supplemented William's E medium without insulin and dexamethasone and incubated with [14C]mevalonic acid lactone (0.6 µCi/ml; PerkinElmer) in the above medium for 5 h. The cells were then washed twice in supplemented William's E medium without insulin and dexamethasone and incubated in medium with or without lipid-free apolipoprotein A-I (apoA-I; 15 µg/ml; Sigma) for 18 h. Lipids from cells and media were extracted and separated by TLC as described above in Liver cholesterol. Lipid spots corresponding to free and esterified cholesterol were scraped and analyzed with a scintillation counter. Cholesterol efflux is expressed as the percentage of labeled total cholesterol transferred from cells to the medium (i.e., radioactivity in the medium ÷ radioactivity in the medium + radioactivity in the cells) after normalization to cell protein content.

Statistics. Data are expressed as means ± SE. Differences between the groups were analyzed using a nonparametric Mann-Whitney U-test, and only differences between the two genotypes are presented. To estimate the triglycerides and cholesterol content of the lipoprotein fractions obtained by FPLC analysis, areas under the curve and their deviations were calculated after a nonlinear regression curve-fitting model assuming a Gaussian distribution (GraphPad Prism version 4; GraphPad, San Diego, CA) was used. An ANOVA F-test was performed to test whether the fitted areas differed between the two genotypes. In all tests, P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Downregulation of genes involved in de novo synthesis of cholesterol in HSL-null mice. Transcriptome analysis of the liver was performed by microarray in fed HSL-null and wild-type mice that had been on a ND or HFD for 6 mo. Genes involved in the biosynthesis of lipids represented the GO category that was the most regulated in fed HSL-null mice on HFD (small P value and ROC area close to 1; Table 1). More specifically, 12 probe sets representing eight genes coding for enzymes catalyzing successive steps in de novo synthesis of cholesterol were downregulated (Table 2 and Fig. 1). This included two probe sets for HMG-CoA reductase mRNA (2.8- and 10.6-fold decrease), two probe sets for Fdps mRNA (3.2- and 3-fold decrease), and three probe sets for NADP-dependent steroid dehydrogenase-like mRNA (3.2-, 3-, and 2-fold decrease). To validate some of the results obtained in the microarray analysis, real-time quantitative PCR (RT-PCR) was performed. For this analysis, fed as well as 16-h-fasted mice were used. The decrease in mRNA levels for HMG-CoA reductase and Fdps in fed HSL-null mice on HFD was confirmed by RT-PCR (P = 0.09 and P < 0.05, respectively; Fig. 2, A and B). In addition, a reduction in mRNA levels was observed in fasted HSL-null mice on HFD for both HMG-CoA reductase (P < 0.05) and Fdps (P = 0.06). Fdps mRNA levels were also reduced in fasted HSL-null mice on ND (P < 0.05; Fig. 2B).


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  		Table 1. Top 15 most regulated GO categories out of 4,589 when comparing livers of fed HSL-null mice on HFD for 6 mo vs. wild-type after microarray analysis

 

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  		Table 2. Genes involved in cholesterol metabolism that were regulated when comparing livers of fed HSL-null mice on HFD for 6 mo vs. wild-type after microarray analysis

 

Figure 1
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  		Fig. 1. Genes involved in de novo synthesis of cholesterol that were identified as downregulated by microarray analysis in livers of fed hormone-sensitive lipase (HSL)-null mice on high-fat diet (HFD) for 6 mo. The difference in expression is given as a fold change. Several fold changes are listed for when there were many probe sets for the same gene. Underlined are the genes whose expression changes were investigated and validated by RT-PCR. Four livers were pooled for the HSL-null mice on HFD and 6 for the wild-type mice on HFD. HMG-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA reductase; PP, pyrophosphate; Mvd, mevalonate (diphospho) decarboxylase; Idi1, isopentenyl-diphosphate {delta}-isomerase; Fdps, farnesyl diphosphate synthase; Sqle, squalene epoxidase; Sc4mol, sterol-C4-methyl oxidase like; Nsdhl, NADP-dependent steroid dehydrogenase-like; Dhcr7, 7-dehydrocholesterol reductase.

 

Figure 2
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  		Fig. 2. RT-PCR validation of hepatic mRNA expression changes found by microarray analysis. mRNA levels of HMG-CoA reductase (A) and Fdps (B). HSL-null and wild-type mice were either fed or overnight fasted for 16 h and had been on a normal chow diet (ND) or HFD for 27–29 wk. Relative mRNA quantities were normalized by geometric averaging of 3 internal control genes [Gapdh, hypoxanthine phosphoribosyltransferase 1 (Hprt1), and ribosomal protein s29 (Rps29)]. Differences between the 2 genotypes were analyzed using nonparametric Mann-Whitney U-test; n = 4–7. AU, arbitary units.

 
HSL-null mice have a perturbed plasma lipid profile. Following the observation that several genes involved in de novo cholesterol synthesis were downregulated in HSL-null mice, the plasma levels of cholesterol, triglycerides, and NEFA were measured in refed (overnight fasting for 12 h followed by free access to food for 2 h) and 16-h-fasted mice. The refed scheme was used to synchronize the mice and to ensure that a true postprandial state was analyzed. Fasting for 16 h represents a nutritional state that should maximize the impact of HSL deficiency. Plasma cholesterol was elevated in HSL-null mice in all tested conditions: ND and HFD, refed, and fasted (P < 0.05, except for fasted mice on HFD where statistical significance was not attained; Table 3). The increase was on average 25%. Plasma triglycerides were elevated by on average 50% in refed HSL-null mice (P < 0.05, except for refed mice on HFD where statistical significance was not attained), whereas plasma triglyceride levels in the fasted state were decreased by on average 55%, regardless of dietary regimen (P < 0.001) (Table 3). Similarly, plasma NEFA were increased in refed and decreased in fasted HSL-null mice, in both dietary conditions, by on average 40 and 30%, respectively (P ≤ 0.05; Table 3).


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  		Table 3. Plasma lipid composition of HSL-null mice and wild-type littermates

 
HSL-null mice have cholesterol-enriched lipoproteins. Next, the lipid composition of the different pools of lipoproteins obtained by FPLC separation was analyzed. HDL cholesterol was increased by on average 25% for all tested conditions in HSL-null mice: ND and HFD, refed, and fasted (P ≤ 0.01; Table 4 and Fig. 3A). In addition, FPLC analysis revealed an increase of VLDL cholesterol in fed HSL-null mice on ND and on HFD and in fasted HSL-null mice on HFD (P ≤ 0.01; Table 4 and Fig. 3A). In refed HSL-null mice on ND and on HFD, increased VLDL cholesterol was associated with a similar increase in triglyceride content, i.e., an average increase of 2.2- and 2.8-fold, respectively (P ≤ 0.01), suggesting that refed HSL-null mice exhibit an expansion of their VLDL pool. However, the VLDL cholesterol of fasted HSL-null mice on HFD was doubled, whereas the triglycerides were halved (P ≤ 0.01), indicating that the VLDL composition of fasted HSL-null mice on HFD is altered (Tables 4 and 5 and Fig. 3, A and B). No changes were observed for intermediate-density lipoprotein (IDL)/LDL cholesterol under any of the tested conditions (Table 4 and Fig. 3A). However, as observed for the VLDL, refed HSL-null mice on ND and on HFD had on average a twofold increase of their IDL/LDL triglycerides (P ≤ 0.05), whereas fasted HSL-null mice on HFD presented a 0.6-fold decrease of their IDL/LDL triglycerides (P < 0 05) (Table 5 and Fig. 3B).


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  		Table 4. Cholesterol content of plasma lipoproteins of HSL-null mice and wild-type littermates

 

Figure 3
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  		Fig. 3. Lipoprotein analysis in refed (overnight fasting for 12 h followed by free access to food for 2 h) and 16-h-fasted mice on ND for 23–25 wk or HFD for 21–23 wk. A: distribution of total cholesterol after fast performance liquid chromatography (FPLC) separation of pooled plasma (n = 12–14). B: distribution of total triglycerides after FPLC separation of pooled plasma. IDL, intermediate-density lipoprotein.

 

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  		Table 5. Triglyceride content of plasma lipoproteins of HSL-null mice and wild-type littermates

 
HSL-null mice accumulate esterified cholesterol in the liver. The observed cholesterol enrichment of lipoproteins in HSL-null mice prompted the analysis of hepatic cholesterol content. Hepatic esterified cholesterol content was unchanged in fed HSL-null mice on ND but elevated on average 2.2-fold in fed HSL-null mice on HFD (Fig. 4A) and in fasted HSL-null mice on ND and on HFD (Fig. 4B) (P = 0.056). However, the levels of free cholesterol were unchanged (Fig. 4, A and B).


Figure 4
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  		Fig. 4. Hepatic cholesterol content of HSL-null and wild-type mice. Mice were either in the fed state (A) or overnight fasted for 16 h (B) and had been on an ND or HFD for 27–29 wk. Differences between the 2 genotypes were analyzed using nonparametric Mann-Whitney U-test; n = 5. FC, free cholesterol; EC, esterified cholesterol; TC, total cholesterol. *P < 0.05.

 
HSL-null mice overexpress ABCA1 in the liver. The increase of plasma total cholesterol, HDL cholesterol, non-HDL cholesterol, and hepatic cholesterol, as well as the altered expression of hepatic HMG-CoA reductase observed in HSL-null mice, is reminiscent of the phenotype described for mice overexpressing ABCA1 in the liver (1, 19). Indeed, an elevation of hepatic ABCA1 mRNA levels in fed HSL-null mice on HFD was observed in the microarray analysis (2- and 1.8-fold increase; Table 2). Although this finding was not reproduced by RT-PCR on fed mice, hepatic ABCA1 mRNA expression was shown to be increased by RT-PCR in fasted HSL-null mice on ND and HFD by 1.4- and 1.5-fold, respectively (P < 0.05 and P < 0.01; Fig. 5A). In addition, hepatic ABCA1 protein levels were investigated by Western blot analysis (Fig. 5B). ABCA1 appeared as cleaving products ~100 kDa rather than as the expected full-length 220-kDa protein. The double band was quantified using the ImageJ software, and ABCA1 protein levels were found to be elevated in HSL-null mice vs. wild-type regardless of the dietary regimen and the nutritional state (3.3-fold increase for fed mice on ND, 15-fold increase for fed mice on HFD, 1.8-fold increase for fasted mice on ND, and 2.4-fold increase for fasted mice on HFD; Fig. 5B). Next, the mRNA levels of other transporters and receptors involved in hepatic cholesterol homeostasis were analyzed. No differences were observed for any of the groups in the expression of Abcg1, the transporter of cholesterol to lipidated apoA-I (data not shown) (46). The LDL receptor mRNA levels were decreased in fed and fasted HSL-null mice on HFD by 0.7- and 0.4-fold, respectively (P < 0.05 and P < 0.01), and trended toward a reduction in fed and fasted HSL-null mice on ND (P > 0.05) (Fig. 6A). SR-BI mRNA levels were downregulated 0.7-fold in fed HSL-null mice on ND (P < 0.05) and upregulated 1.6-fold in fasted HSL-null mice on HFD (P < 0.01) (Fig. 6B). LRP1 mRNA levels were increased 1.5-fold in fasted HSL-null mice on HFD (P = 0.052; Fig. 6C).


Figure 5
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  		Fig. 5. Hepatic expression analysis of ATP-binding cassette transporter A1 (ABCA1) in HSL-null and wild-type mice. A: gene expression analysis by RT-PCR of ABCA1. Mice were fed or overnight fasted for 16 h and had been on ND or HFD for 27–29 wk. Relative mRNA quantities were normalized by geometric averaging of 3 internal control genes (Gapdh, Hprt1, and Rps29). Differences between the 2 genotypes were analyzed using nonparametric Mann-Whitney U-test; n = 4–7. B: Western blot analysis of hepatic ABCA1 expression in 16-h-fasted HSL-null and wild-type mice on ND or HFD for 27–29 wk (n = 2 in each group). Equal amounts of protein were loaded onto the gel for comparison of HSL-null and wild-type mice. The results on the graph are expressed as %control, i.e., the corresponding wild type for a given group, i.e., ND-fed, ND-fasted, HFD-fed, or HFD-fasted group. *P < 0.05, **P < 0.01.

 

Figure 6
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  		Fig. 6. RT-PCR analyses in liver of HSL-null and wild-type mice. A: gene expression analysis of LDL receptor. B: scavenger receptor BI (SR-BI). C: LDL receptor-related protein 1 (Lrp1). D: stearoyl-CoA desaturase 1 (Scd1). Mice were fed or overnight fasted for 16 h and had been on ND or HFD for 27–29 wk. Relative mRNA quantities were normalized by geometric averaging of 3 internal control genes (Gapdh, Hprt1, and Rps29). Differences between the 2 genotypes were analyzed using nonparametric Mann-Whitney U-test; n = 4–7. *P < 0.05, **P < 0.01.

 
Modification of hepatic triacylglycerol unsaturated fatty acid profile in 16-h-fasted HSL-null mice. Since it has been reported that unsaturated fatty acids suppress the expression of ABCA1, we investigated the hepatic triglyceride fatty acid profile of HSL-null mice. Hepatic total triglycerides were quantified in fed and fasted mice on ND and HFD, but no differences were observed between HSL-null and wild-type mice (data not shown). However, unsaturated fatty acid composition of hepatic triglycerides was modified in fasted HSL-null mice on ND and HFD; i.e., palmitoleic (16:1), linoleic (18:2), and {gamma}-linolenic (18:3) fatty acid moieties of hepatic triglycerides were diminished in HSL-null mice, whereas oleic acid (18:1) was increased. (Supplemental Table S1; Supplemental Material for this article is available at the AJP-Endocrinology and Metabolism web site). We also measured the expression level of hepatic Scd1, a key enzyme in the biosynthesis of hepatic monounsaturated fatty acids (20). Scd1 levels were almost doubled in fasted HSL-null mice on ND and HFD (P = 0.051 and P < 0.05, respectively; Fig. 6D).

Similar apoA-I-mediated cholesterol efflux from HSL-null mouse hepatocytes and wild-type mouse hepatocytes. The next step was to assess whether the upregulation of hepatic ABCA1 at the mRNA and protein levels was accompanied by increased ABCA1 action in HSL-null mouse hepatocytes. One way to achieve this is to measure apoA-I-specific cholesterol efflux, since ABCA1 functions by adding cholesterol and phospholipids to lipid-poor apoA-I to generate nascent HDL (24, 41). Thus, hepatocytes were isolated by collagenase digestion from mice of the four experimental groups. However, hepatocytes isolated from mice fed a HFD for an extensive period of time (~10 mo) revealed very few surviving cells when analyzed by trypan blue exclusion in both wild-type and HSL-null mice (2 unsuccessful attempts/genotype were made). Consequently, the apoA-I-mediated cholesterol efflux could be analyzed only in mice fed the ND. Not only were hepatocytes from HFD-fed mice much more fragile than hepatocytes from mice fed a ND, but we also observed that hepatocytes from HSL-null mice were less robust than hepatocytes from wild-type mice, as indicated by a much lower number of surviving cells (data not shown). The increase in efflux of labeled cholesterol induced by apoA-I tended to be similar for wild-type and HSL-null mouse hepatocytes (i.e., 1.3- and 1.5-fold, respectively, and P = 0.1; Fig. 7). Moreover, labeled cholesterol efflux to apoA-I was similar in HSL-null mouse hepatocytes and wild-type mouse hepatocytes (Fig. 7).


Figure 7
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  		Fig. 7. Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux from hepatocytes isolated from HSL-null and wild-type mice on ND. Data are expressed as %total [14C]mevalonic acid lactone-derived cholesterol efflux from cells to the medium. Values are expressed as means ± SE for 3 independent hepatocyte preparations that were analyzed in triplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that disruption of the HSL gene in the mouse results in disturbed cholesterol homeostasis. Plasma cholesterol levels were elevated in HSL-null mice due to cholesterol enrichment of HDL and VLDL. Moreover, hepatic esterified cholesterol was increased, and HMG-CoA reductase together with seven other genes coding for enzymes catalyzing successive steps in de novo synthesis of cholesterol were downregulated in HSL-null mice. An overview of the various cholesterol fluxes that were modified in the liver of HSL-null mice is depicted in Fig. 8.


Figure 8
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  		Fig. 8. Overview of the various cholesterol fluxes that were modified in the liver of HSL-null mice. Genes involved in de novo synthesis of cholesterol were downregulated in the liver of HSL-null mice. Hepatic esterified cholesterol content was elevated in HSL-null mice most likely as a result of reduced cytosolic cholesteryl ester hydrolase activity due to lack of HSL action. Buildup of esterified cholesterol in the liver may impair the SR-BI-mediated uptake of HDL-derived cholesteryl esters and thus be responsible for the elevated plasma HDL cholesterol displayed by HSL-null mice. Increased ABCA1-mediated cholesterol efflux may also contribute to the increased plasma HDL cholesterol of HSL-null mice. However, although hepatic ABCA1 expression was upregulated in HSL-null mice, it was not reflected in increased cholesterol efflux, at least not in hepatocytes isolated from mice on ND. Thin arrows indicate fluxes that were downregulated in HSL-null mice vs. wild-type mice. Thick arrows indicate fluxes that were upregulated. The dashed arrow designates a flux that needs further study.

 
Elevation of plasma levels of total cholesterol and HDL cholesterol has previously been described in the fed and 16-h-fasted state of another strain of HSL-null mice on a ND (15). However, the effect of challenging the mice with a HFD for an extensive period of time has not been investigated previously. Here we confirm the previous findings with regard to ND. Furthermore, we demonstrate that total and HDL cholesterol are elevated also in refed HSL-null mice on ND (overnight fasting for 12 h followed by 2 h of free access to food) as well as in refed and 16-h-fasted HSL-null mice that had been on a HFD for about 6 mo. The increase in HDL cholesterol in the fasted state could be due to the increased lipoprotein lipase (LPL) activity reported in muscle and white adipose tissue of fasted HSL-null mice (15), since a positive correlation between LPL activity and HDL cholesterol concentrations has been reported (23). However, it could not explain the elevation of HDL cholesterol in the fed state, since LPL activity was shown to be reduced in white adipose tissue and unchanged in skeletal muscle of fed HSL-null mice (15). Moreover, it has been reported that under normal chow feeding, only mice expressing the simian cholesteryl ester transfer protein (CETP) transgene had their HDL cholesterol levels significantly affected by LPL activity, whereas when mice were fed a high fat/high carbohydrate diet, LPL activity was correlated to HDL cholesterol levels regardless of the expression of CETP (8). Thus, several mechanisms are likely to contribute to the elevation of HDL cholesterol in HSL-null mice. Among these are mechanisms relating to the increased hepatic cholesterol ester content and the increased expression of hepatic ABCA1, as discussed further below.

It has been demonstrated that total hepatic cholesterol levels are elevated in HSL-null mice under ND conditions and following a HFD regimen for 15 wk (17). Here we confirm this observation in another strain of HSL-null mice and extend it to show that the elevation is accounted for by esterified cholesterol, whereas the levels of free cholesterol are unchanged. However, it is possible that the observed downregulation of the expression of enzymes involved in de novo synthesis of cholesterol as well as the decreased LDL receptor mRNA levels are compensatory to an initial buildup of hepatic free cholesterol that is now fully compensated. Buildup of hepatic esterified cholesterol may impair the SR-BI-mediated uptake of HDL-derived cholesteryl esters via poor clearance of the cholesteryl esters on the cytosolic side despite the fact that SR-BI is upregulated (HFD fasted) or unchanged (HFD fed and ND fasted). An increase in esterified cholesterol is consistent with our previous demonstration that cholesterol ester hydrolase activity is reduced in the liver of HSL-null mice (22). Whether HSL is expressed and enzymatically active in the liver has been debated. The data presented here suggest that HSL indeed functions as a cytosolic cholesterol ester hydrolase in the liver, although its importance in relation to the other described neutral cytosolic cholesterol ester hydrolase remains to be clarified (21, 50).

A role for hepatic ABCA1 in modulating plasma HDL cholesterol level has been established (1, 37), and the overall phenotype of HSL-null mice, characterized by an elevation of plasma total and HDL cholesterol as well as an increase in hepatic cholesterol content, is reminiscent of the phenotype of mouse models overexpressing ABCA1 specifically in the liver (1, 19). A similar level of hepatic overexpression of ABCA1 occurred: an average of 1.7-fold increase in ABCA1 mRNA for HSL-null mice vs. a twofold increase in ABCA1 mRNA for the model described by Joyce et al. (19) vs. a 1.6- to 1.7-fold increase in ABCA1 protein for the model described by Basso et al. (1). HSL-null mice with decreased HMG-CoA reductase and LDL receptor mRNA levels and elevation of both VLDL cholesterol and LRP1 exhibit more similarities to the Joyce et al. (19) model than to the Basso et al. (1) model. One difference, however, is that HSL-null mice show no cholesterol enrichment of the LDL pool. An explanation for this could be that the LDL receptor is totally absent in the Joyce et al. (19) mouse model, whereas LDL receptor gene expression level is only reduced in the HSL-null mouse model.

Following the realization that the phenotype of HSL-null mice resembles that of ABCA1 transgenic mice, the next obvious question concerned the link between inactivation of the HSL gene and upregulation of hepatic ABCA1. Although the expression of ABCA1 has been reported to be regulated by the liver X receptor/retinoid X receptor heterodimer with cholesterol-derived oxysterols as the physiological ligand, free cholesterol content was not changed in the liver of HSL-null mice, and the hepatic mRNA levels of apoE, a liver X receptor-regulated gene, did not differ between any of the investigated groups (data not shown). Unsaturated fatty acids have also been reported to regulate ABCA1 expression via suppression of ABCA1 transcription (38, 39) and via increased degradation of ABCA1 protein through its phosphorylation by protein kinase C{delta}, which is activated by diacylglyceride subspecies enriched in unsaturated fatty acids (43–45). In the fasted state, the fatty acid composition of hepatic triglycerides, which principally reflects plasma-derived fatty acids, i.e., HSL action in adipose tissue (12), was changed mainly toward a reduction in the unsaturated fatty acid content. This change is in accordance with the reported selective mobilization of fatty acids from a lipid emulsion by HSL according to chain length and degree of unsaturation (29). Additionally, the composition of NEFA released after in vitro lipolysis from white adipose tissue was altered in another strain of HSL-null mice fed a ND (14). Overall, decreased delivery of unsaturated fatty acids to the liver, as a result of lack of HSL action in white adipose tissue, might be at least one mechanism whereby hepatic ABCA1 expression is increased in HSL-null mice.

ABCA1-dependent cholesterol efflux from primary hepatocytes was measured to evaluate the consequences of the upregulation of hepatic ABCA1 expression in HSL-null mice. The ex vivo study showed no differences in ABCA1-mediated cholesterol efflux from HSL-null mouse hepatocytes compared with wild-type mouse hepatocytes. Thus, the increased expression of hepatic ABCA1 in HSL-null mice had no major effect on cholesterol efflux. This indicates that hepatic accumulation of cholesteryl esters due to lack of action of hepatic HSL is the predominant mechanism behind the disturbed cholesterol metabolism displayed by the HSL-null mice. Further support for this notion comes from a very recent study that was published during the preparation of this article (33). Nonetheless, the possibility remains that the situation is different in the HFD-fed mice, an experimental condition that could not be evaluated due to the poor survival of hepatocytes from long-term HFD-fed mice.

In addition to HDL cholesterol, VLDL cholesterol was also increased in refed HSL-null mice on ND and HFD as well as in fasted HSL-null mice on HFD. In refed HSL-null mice, plasma NEFA were increased, providing the liver with more substrate for triglyceride synthesis. Thus, the increase in VLDL cholesterol was associated with a similar elevation of VLDL triglycerides, indicating that the VLDL pool was increased. In fasted mice on HFD, the lack of expression of HSL in the white adipose tissue was reflected in a reduction of plasma NEFA by 45%, associated with a lowering in VLDL triglycerides by 55%, although the VLDL cholesterol content was doubled. This suggests that the lipid composition of VLDL is altered in fasted HSL-null mice on HFD.

IDL/LDL cholesterol was unchanged in HSL-null mice regardless of nutritional state or feeding condition. In fasted HSL-null mice on HFD, an elevation of IDL/LDL cholesterol would have been expected as a result of the cholesterol enrichment of the VLDL. Since mice lack plasma CETP activity, excess cholesterol present in IDL/LDL could not have been removed through this mechanism. SR-BI and LRP1, the expression of which were upregulated 1.6- and 1.5-fold, respectively, in fasted HSL-null mice on HFD, could be responsible for the removal of excess IDL/LDL cholesterol. It is indeed established that SR-BI mediates the selective uptake of cholesteryl ester and other lipids both from HDL and LDL, whereas LRP is involved in the uptake of cholesterol-enriched apoB lipoproteins (19, 35, 36). However, as stated above, the uptake of cholesteryl esters via SR-BI might be impaired due to the accumulation of cholesteryl esters on the cytosolic side. Therefore, cholesteryl esters may preferentially be taken up through LRP, which relies on endocytosis and subsequent hydrolysis of cholesteryl esters by the lysosomal acid lipase.

The relevance of the results of the present study for human physiology is difficult to evaluate for several reasons. First, there are major differences in lipid and lipoprotein metabolism between mice and humans. In particular, high HDL/LDL ratios and lack of CETP are major contributors to the low susceptibility to atherosclerosis of mice compared with humans. Second, the type of gene inactivation employed in this study, i.e., total whole body inactivation from early embryogenesis and onward, is extreme and may induce compensatory changes that obscure the interpretation of the phenotype. A human counterpart to the experimental model employed in this study does not exist because total HSL deficiency has not been described in humans. Noteworthy, however, is that low HSL activity has been reported in familial combined hyperlipidemia (30), and a substitution (C-60G) in the HSL promoter that may have a role in determining serum cholesterol levels in familial combined hyperlipidemia families has been described (57). In addition, an HSL amino acid polymorphism that is correlated to mildly elevated levels of serum total cholesterol in Japanese subjects has been reported (34). Future studies, including targeted and moderate knockdown experiments in adult mice as well as studies in humans addressing the lipoprotein composition in relation to HSL activity, will have to be performed to establish the relevance of the current findings for cholesterol homeostasis in humans.

In conclusion, our results show that plasma cholesterol is elevated in HSL-null mice due to cholesterol enrichment of VLDL and HDL. It is likely that several mechanisms contribute to the altered cholesterol homeostasis in HSL-null mice, of which we have described two, i.e., increased ABCA1 expression and hepatic accumulation of cholesteryl esters due to lack of action of hepatic HSL, where the latter appears to be more important than the former.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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Financial support was provided by the Swedish Research Council (project no. 112-84 to C. Holm), the Swedish Diabetes Association, and the following foundations: Novo Nordisk, A. Påhlsson, Torsten, and Ragnar Söderberg. C. Fernandez was supported by the Swedish Research School in Genomics and Bioinformatics. M. Krogh was supported by the Swedish Foundation for Strategic Research and the Knut and Alice Wallenberg Foundation through the Swegene consortium and the Strategic Science Foundation CREATE Health Centre.


   ACKNOWLEDGMENTS
 
We thank Ann-Helen Thorén-Fischer and Elin Björk for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Fernandez, Dept. of Experimental Medical Science, Lund University, BMC C11, SE-221 84 Lund, Sweden (e-mail: celine.fernandez{at}med.lu.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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