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Physiol. Genomics 26: 116-124, 2006. First published April 18, 2006; doi:10.1152/physiolgenomics.00300.2005
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Received 6 December 2005; accepted in final form 12 April 2006.
Physiological Genomics 26:116-124 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Transcriptional profile reveals altered hepatic lipid and cholesterol metabolism in hyposulfatemic NaS1 null mice

Paul Anthony Dawson1, Brooke Gardiner2, Sean Grimmond2 and Daniel Markovich1

1 School of Biomedical Sciences, University of Queensland, St. Lucia, Australia
2 Institute for Molecular Bioscience, University of Queensland, St. Lucia, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sulfate plays an essential role in human growth and development, and its circulating levels are maintained by the renal Na+-SO42– cotransporter, NaS1. We previously generated a NaS1 knockout (Nas1–/–) mouse, an animal model for hyposulfatemia, that exhibits reduced growth and liver abnormalities including hepatomegaly. In this study, we investigated the hepatic gene expression profile of Nas1–/– mice using oligonucleotide microarrays. The mRNA expression levels of 92 genes with known functional roles in metabolism, cell signaling, cell defense, immune response, cell structure, transcription, or protein synthesis were increased (n = 51) or decreased (n = 41) in Nas1–/– mice when compared with Nas1+/+ mice. The most upregulated transcript levels in Nas1–/– mice were found for the sulfotransferase genes, Sult3a1 ({approx}500% increase) and Sult2a2 (100% increase), whereas the metallothionein-1 gene, Mt1, was among the most downregulated genes (70% decrease). Several genes involved in lipid and cholesterol metabolism, including Scd1, Acly, Gpam, Elov16, Acsl5, Mvd, Insig1, and Apoa4, were found to be upregulated (≥30% increase) in Nas1–/– mice. In addition, Nas1–/– mice exhibited increased levels of hepatic lipid ({approx}16% increase), serum cholesterol ({approx}20% increase), and low-density lipoprotein ({approx}100% increase) and reduced hepatic glycogen ({approx}50% decrease) levels. In conclusion, these data suggest an altered lipid and cholesterol metabolism in the hyposulfatemic Nas1–/– mouse and provide new insights into the metabolic state of the liver in Nas1–/– mice.

sulfate; gene expression; liver; transporter; slc13a1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
INORGANIC SULFATE (SO42–) is the fourth most abundant anion in mammalian plasma and is essential for numerous metabolic and cellular processes (38). The biotransformation of molecules by sulfonation is an important metabolic route in the metabolism of xenobiotics and many endogenous compounds, including hormones, neurotransmitters, and bile acids (20). In addition, sulfonation of structural components, such as proteins, lipids and glycosaminoglycans, is essential for the maintenance of normal structure and function of tissues (40).

The liver is a major site for the sulfonation of numerous compounds, due to its high sulfotransferase activities and abundant levels of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) (8). Hepatic sulfonation is an important process for decreasing the toxicity of secondary bile acids, such as lithocholate, especially during intrahepatic cholestatic conditions, including progressive familial intrahepatic cholestasis type I (PFIC1) and intrahepatic cholestasis of pregnancy (ICP) (42, 44). Defective lithocholate sulfonation activity in rhesus monkeys was shown to lead to an accumulation of lithocholic acid in the liver and hepatotoxicity (24). Hepatic sulfonation also has a significant role in the metabolism of catecholamine neurotransmitters, steroids, and iodothyronines (52). The liver also utilizes sulfate to detoxify many phenolic compounds found in food (39). In addition, sulfoconjugation is an important step in the metabolism of certain drugs, including the common analgesic acetaminophen (40). Studies in rats have shown that sulfur-deficient diets can lead to reduced blood and hepatic sulfate levels and increased acetaminophen-induced hepatotoxicity (45). Taken together, these findings underscore the importance of maintaining sufficiently high levels of sulfate for sulfonation reactions to function efficiently in the liver.

Despite the importance of sulfate in the body, plasma SO42– levels are rarely measured clinically, and little is known about the physiological consequences of hyposulfatemia. In humans, the sodium sulfate cotransporter, NaS1, is expressed primarily in the kidney (34) where it is proposed to maintain plasma SO42– levels (41). We have cloned the human and mouse NaS1 genes, designated NAS1 (34) and Nas1 (2), respectively. More recently, we generated a NaS1 null (Nas1–/–) mouse that exhibits hyposulfatemia, reduced growth, decreased serum insulin-like growth factor I (IGF-I) levels, altered serum bile acid concentrations, increased hepatic sulfotransferase activity, and hepatomegaly (16). One approach toward understanding these abnormal features in the Nas1–/– mouse is through the use of transcriptional profiles.

Transcriptional profiling has become a valuable tool for investigating gene expression in mammalian physiology. In particular, studies of the murine hepatic transcriptome have led to the discovery of genes that are regulated by diet (19), caloric restriction (7), hormones (21), and circadian rhythm (1). The important role of the liver in sulfonation makes the hepatic transcriptome an appropriate starting place to investigate the molecular consequences of hyposulfatemia. Of particular relevance to our studies of the Nas1–/– mouse is that blood sulfate levels are reduced under certain physiological conditions, including aging (12), and also in certain neurological disorders, such as Alzheimer's, Parkinson's, motor neurone disease, and autism (27, 55). However, the etiology of hyposulfatemia in these neurological disorders is yet unknown. In addition, low dietary sulfate intake and medications, such as acetaminophen, have also been linked to reduced blood sulfate levels (29, 45). The aim of this study was to determine whether the transcriptional profile was altered in the liver of hyposulfatemic Nas1–/– mice. Our findings demonstrate changes to hepatic gene expression in Nas1–/– mice and, together with biochemical and histological analyses, suggest that hyposulfatemia may lead to a shift in energy metabolism in the liver from carbohydrate to fatty acid utilization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice.
We previously generated Nas1 knockout (Nas1–/–) mice in which the NaS1 gene was disrupted by targeted mutagenesis (16). Nas1–/– mice and wild-type Nas1+/+ littermates (controls) were housed at a constant temperature (23 ± 1°C) with a 12:12-h light-dark cycle (lights on at 0600 and off at 1800). Mice were weaned at 3 wk of age and were then fed a standard rodent chow (no. AIN93G; Glen Forrest Stockfeeders, Glen Forest, Western Australia) and water ad libitum. For food intake studies, mice were housed individually at weaning, and the amount of food consumed was measured after 1 wk, when mice reached 4 wk of age. Male mice at 4 wk of age were used for all experiments. Protocols were submitted to, and approved by, the University of Queensland Animal Ethics Committee.

Gene expression profiling.
Because circadian rhythm is known to alter hepatic gene expression (1), we isolated total RNAs from the livers of 4-wk-old male Nas1+/+ and Nas1–/– mice (n = 14 for each genotype) at 0900, using previously described methods (16). Total RNAs were amplified, labeled, and hybridized overnight at 42°C using previously described methods (10) and 22K mouse Compugen long oligonucleotide arrays from the Special Research Centre Microarray Facility, University of Queensland (Australian Research Council Centre for Functional and Applied Genomics).

Liver RNAs from Nas1+/+ and Nas1–/– mice were directly compared in quadruplicate with dye swapping incorporated into two of the hybridizations to account for bias. Arrays were scanned on an Agilent scanner 600B (Agilent Technologies), and foregound/backgroud signal intensities were quantified using Imagene5.0 (BioDiscovery). All data were normalized using a combination of the Printtiplowess and scaling between arrays algorithms from Bioconductor (linear models for microarray data; LIMMA) and statistics for microarray analysis (SMA) (50). All primary data (including images), data transformations, and methods are available via the comprehensive microarray relational database, BASE (http://kidney.scgap.org/base/index.phtml). The entire data set is also available from Gene Expression Omnibus (GEO; accession no. GSE3745). Differential expression was defined using the B statistic method (45), where both fold change and variance of signals in replicates is used to determine the likelihood that genes are truly differentially expressed. All genes with a B score >0.0 were functionally annotated via the following web-enabled tools: Clonefinder (http://microarray.imb.uq.edu.au/clonefind.html) and the Database for Annotation, Visualization, and Integrated Discovery (DAVID; http://david.niaid.nih.gov/david/version2/index.htm).

Quantitative RT-PCR.
To validate our microarray data, we examined the mRNA levels of 12 genes that were found to be increased (Sult3a1, Tgtp, Sult2a2, Acly, Igfbp2, Gpam, and Scd1) or decreased (Chst7, Bteb1, G0s2, Kap, and Mt1). Total RNA (2 µg) was reverse transcribed by use of random hexamers and an Omniscript RT kit (Qiagen) as recommended by the manufacturer. PCR was performed in quadruplicate with 5 µl of cDNA (from 25 ng of RNA) and 10 µl of master mix containing Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) and 200 nM forward and reverse primers (Table 1) in a Rotor-Gene 3000 thermal cycler (Corbett Research, Sydney, Australia). The thermal cycling protocol was as follows: 50°C for 2 min; 94°C for 2 min; and 45 cycles of 94°C for 1 s, 60°C for 10 s, and 72°C for 15 s. Relative mRNA levels of each gene were normalized to those of ribosomal RNA. Amplification specificity was confirmed by melting curve analysis and agarose gel electrophoresis.


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  		Table 1. Primers used for PCR-amplification

 
Analysis of sulfate anion transporter-1 mRNA.
Using Northern blot and RT-PCR analyses, we measured the mRNA levels of a liver sulfate transporter, sulfate anion transporter-1 (sat-1) (35). Total RNA (10 µg) was separated on a 1% agarose formaldehyde gel in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer and transferred to Hybond-XL nylon membranes (Amersham Pharmacia). The blots were probed with a 1.4-kb sat-1 cDNA and a 250-bp mouse ß-actin cDNA probe. For RT-PCR, total RNA (2 µg) was reverse transcribed by use of random hexamers and Moloney murine leukemia virus RT (Progen, Brisbane, Queensland, Australia) as recommended by the manufacturer. Primers used to amplify sat-1 and ß-actin cDNAs are shown in Table 1. PCR products, in the linear phase of amplification, and Northern blot signals were analyzed by densitometry (Scion Image Beta 4.0.2).

Histological analysis.
Liver samples were dissected into {approx}50 volumes of 10% buffered formalin and fixed for 3 days. To detect neutral lipids, 6-µm-thick sections were stained in Oil Red O and then counterstained with hematoxylin. Periodic acid Schiff (PAS) staining was used for the detection of glycogen. All sections were examined by light microscopy at 40x magnification.

Measurement of liver lipid and glycogen levels.
Total liver lipids were extracted, using previously described methods (23). Lipid content was calculated from the weight of the dried lipids (mg lipid/mg liver). Liver glycogen content was quantitated by using a colorimetric assay (37). Unpaired Student's t-tests were used to analyze lipid and glycogen data.

Blood analysis.
Whole blood triglycerides, glucose, total cholesterol, LDL, HDL, and VLDL were measured with a Cholestech LDX Analyzer (Cholestech, Hayward, CA). Unpaired Student's t-tests were used to analyze all blood data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hepatic transcriptional profile of Nas1–/– mice.
We recently reported physiological abnormalities in Nas1–/– mice, including growth retardation, reduced serum IGF-I levels, hepatomegaly, increased sulfotransferase activity, and altered serum bile acid levels (16). These findings led us to study the hepatic gene expression profile of Nas1–/– and Nas1+/+ mice using cDNA microarrays, which revealed transcriptional differences in the Nas1–/– mice. Because numerous studies have shown that small changes (<2-fold) in gene expression can be important in critical biological pathways (51), we have listed all genes that showed significant changes, as assessed statistically at B >0.0 (Tables 2 and 3). Similar total numbers of genes were up- (n = 66) and downregulated (n = 64) in Nas1–/– livers compared with Nas1+/+ livers. Of the 130 genes that were differentially transcribed, 38 were not annotated for molecular function using the Clonefinder and DAVID databases and are therefore not discussed hereafter.


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  		Table 2. Genes upregulated in Nas1–/– liver

 

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  		Table 3. Genes downregulated in Nas1–/– liver

 
Validation of microarray data by quantitative RT-PCR.
Quantitative RT-PCR (QRT-PCR) was used to verify changes in transcript levels for a selected subset of 12 genes, representing different functional categories with increased (Sult3a1, Tgtp, Sult2a2, Acly, Igfbp2, Gpam, and Scd1) or decreased (Chst7, Bteb1, G0s2, Kap, and Mt1) transcriptional levels. QRT-PCR data (Fig. 1) showed that the expression levels of these 12 genes were similar (R2 = 0.9601) to those obtained with gene arrays.


Figure 1
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  		Fig. 1. Comparison of quantitative RT-PCR data with microarray data. The mean log2 expression ratios (Nas1–/–:Nas1+/+) are shown for genes upregulated (Sult3a1, Tgtp, Sult2a2, Acly, Igfbp2, Gpam, and Scd1) or downregulated (Chst7, Bteb1, G0s2, Kap, and Mt1) in Nas1–/– mice compared with Nas1+/+ mice. RNA was isolated from the livers of 14 Nas1–/– mice and 14 Nas1+/+ littermates, and 4 replicates of a mixed RNA pool were evaluated.

 
Hepatic lipid and glycogen data.
Hepatic lipid levels were assayed to determine whether the altered lipogenic transcriptional profiles of Nas1–/– mice were associated with changes in lipid content. Staining mouse livers with Oil Red O demonstrated increased lipid deposition in Nas1–/– mice (Fig. 2A) compared with Nas1+/+ mice (Fig. 2B). In addition, decreased glycogen content, as indicated by PAS staining, was observed in the Nas1–/– livers (Fig. 2C) compared with livers of Nas1+/+ mice (Fig. 2D). Quantification of liver lipid content (Fig. 2E) revealed an {approx}16% increase in Nas1–/– mice (7.7 ± 0.5%, n = 7; P = 0.0001) compared with Nas1+/+ mice (6.6 ± 0.2%, n = 7), further confirming the fatty liver phenotype. The hepatic glycogen content in Nas1–/– mice (0.80 ± 0.17 µg/mg protein, n = 7; P = 0.025) determined by colorimetric analysis was {approx}50% of the glycogen level in Nas1+/+ mice (1.50 ± 0.22 µg/mg protein, n = 7) (Fig. 2F). These changes in hepatic lipid and glycogen content found in 4-wk-old Nas1–/– mice prompted us to compare the food intake of Nas1+/+ and Nas1–/– mice from 3 to 4 wk of age. Food intake in Nas1–/– mice (11.8 ± 1.6 g, n = 7) was not significantly different (P = 0.63) from that of Nas1+/+ mice (13.0 ± 1.5 g, n = 5).


Figure 2
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  		Fig. 2. Fatty liver and reduced hepatic glycogen content in Nas1–/– mice. Oil red O staining (A and B) and periodic acid Schiff staining (C and D) of liver sections from Nas1–/– and Nas1+/+ mice. Bar = 50 µm. Lipid (E) and glycogen (F) content in liver from Nas1–/– and Nas1+/+ mice. Each bar represents the mean ± SD of measurements from 7 Nas1–/– and 7 Nas1+/+ mice. **P = 0.0001 and *P = 0.025 compared with Nas1+/+ mice.

 
Blood lipid and glucose data.
Because hepatic lipid and glycogen levels of Nas1–/– mice were significantly different from that of their Nas1+/+ littermates, we measured blood lipid and glucose parameters in the Nas1–/– and Nas1+/+ mice (Table 4). No significant differences were found in whole blood HDL, VLDL, triglycerides, or glucose, but significant increases in blood concentrations of LDL (100%, P = 0.006), total cholesterol (TC) ({approx}20%, P = 0.008), and the TC-to-HDL ratio (>30%, P = 0.037) were observed in Nas1–/– mice when compared with Nas1+/+ littermates.


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  		Table 4. Blood profile for Nas1+/+ and Nas1–/– mice

 
Sulfate transporter mRNA levels in Nas1–/– mice.
We examined the mRNA levels of three sulfate transporters: diastrophic dysplasia sulfate transporter (dtdst), downregulated in adenoma (dra), and sat-1 (26, 35, 49). Using microarray analyses, we observed no changes (B <0) in dtdst and dra mRNA expression in Nas1–/– mice. Because the sat-1 cDNA was not present on the gene array used in this study, we used Northern hybridization (Fig. 3A) and RT-PCR (data not shown) to determine sat-1 mRNA expression in the livers of Nas1–/– mice. sat-1 mRNA levels were increased in Nas1–/– ({approx}280%) and Nas1+/– ({approx}120%) mice compared with Nas1+/+ littermates (Fig. 3B).


Figure 3
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  		Fig. 3. Analysis of anion transporter-1 (sat-1) mRNA. A: Northern analysis of RNA from liver of Nas1+/+, Nas1+/–, and Nas1–/– mice. RNA was hybridized sequentially with 32P-labeled sat-1 cDNA (top) and ß-actin cDNA (bottom). B: densitometric analysis of the Northern blot signals in A, representative of 3 independent experiments with similar data.

 
Genes involved in sulfate and amino acid metabolism.
Our transcriptional profiling showed no changes (B <0) in genes involved in amino acid metabolism (Plac1, Oat, Mod1, Pep4, Slc6a14, Ggtla1, Hdc, Srr, Hmgcl, Agxt, Shmt1, Slc6a6, B3gnt1, and B3gat1), including the sulfhydryl-containing amino acids methionine (Mat1a, Bhmt, Mthfr, Cbs, and Impa1) and cysteine (Cdo1, Gclm, Gclc, Got1, Got2, and Scly). In addition, no change (B <0) was found for genes involved in sulfate synthesis from sulfhydryl-containing amino acids (Cdo1, Got1, and Got2), the hydrolysis of sulfate esters (Sts, Galns, Arsb, Ids, Sgsh, and Arsa) or synthesis of the sulfate donor, PAPS (Papss1 and Papss2). Furthermore, we found no change (B <0) in the mRNA levels of genes involved in the sulfonation of proteoglycans (Hs6st2, H26st3, Hs6st1, Hs3st1, Hs2st1, and Hs3st3b1) and glutathione synthesis (Gss, Ggt1, Slco1b2, and Slco2a1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we demonstrated an altered hepatic gene expression profile in hyposulfatemic Nas1–/– mice. These transcriptional changes were associated with increased levels of hepatic lipid, serum cholesterol, and LDL; reduced hepatic glycogen levels; and no changes in blood HDL, VLDL, triglycerides, glucose, and food intake. Because NaS1 mRNA is not expressed in the liver (2), we propose that the altered transcriptional profile of Nas1–/– mice is most likely due to reduced sulfate availability in the liver, as a consequence of hyposulfatemia found in Nas1–/– mice (16).

Nas1–/– mice exhibit fatty liver and reduced hepatic glycogen levels.
Several genes with increased mRNA levels in the liver of Nas1–/– mice have a functional association with fatty acid metabolism (Gpam, Acsl5, Acly, Scd1, Elov16, and Apoa4), whereas the expression of genes linked to glycolysis (Hk2, Aldh1a1), gluconeogenesis (Pck1), and carbohydrate metabolism (Chst7) was suppressed. These findings suggest a shift in energy metabolism from carbohydrate to fatty acid utilization in Nas1–/– mice. This shift in hepatic metabolism may be relevant when considering the etiology of reduced growth observed in the Nas1–/– mice (16). Interestingly, Nas1–/– mice exhibit reduced serum IGF-I levels (16), and here we demonstrate that their transcriptional profile parallels those found in mouse models with decreased growth hormone (GH)/insulin/IGF-I signaling (4, 47). Both the Snell (Pitdw/dwJ) dwarf and growth hormone receptor gene-disrupted (GHR–/–) mice exhibit changes in the expression of genes linked to sulfate metabolism and an altered lipogenic and cholesterologenic transcriptional profile that would favor fatty acid utilization in the liver. Thus the transcriptional changes found in Nas1–/– mice may share common molecular mechanisms with Pitdw/dwJ and GHR–/– mice, through a diminished GH/IGF-I signaling pathway. In addition, metallothionein-null (Mt1–/–) mice exhibit fatty liver and reduced hepatic glycogen levels (14), which is consistent with the decrease in hepatic Mt1 mRNA levels observed in Nas1–/– mice (Table 3).

The increased hepatic lipid level and low glycogen content in Nas1–/– mice could also be explained by reduced nutrition. Both caloric restriction (CR) and chronic malnutrition causesglycogenolysis and mobilization of fatty acids to the liver, where they are oxidized by mitochondrial ß-oxidation, peroxisomal ß-oxidation, and cytochrome-catalyzed microsomal {omega}-oxidation pathways, leading to the export of ketone bodies out of the liver to serve as fuels for other tissues (6, 28). This is consistent with Nas1–/– mice having increased levels of Cyp2b13 mRNA, a cytochrome p-450 that oxidizes fatty acids and is transcriptionally induced by CR (18) and ketone bodies (56). The present study demonstrated that food intake of Nas1–/– mice was similar to that of Nas1+/+ mice; future studies can be aimed at determining whether Nas1–/– mice have altered nutritional absorption and amino acid breakdown, as previously shown for malnutrition (reviewed in Ref. 11). However, our transcriptional profiling showed no changes in genes involved in amino acid metabolism, including the sulfhydryl-containing amino acids methionine and cysteine, suggesting that amino acid metabolism may not be altered in Nas1–/– mice. Nonetheless, the present study suggests that SO42– may be an important factor in the modulation of hepatic lipid and glycogen levels.

Nas1–/– mice exhibit increased serum LDL and TC levels.
The findings of increased blood TC and LDL levels in Nas1–/– mice could be associated with an upregulation of genes involved in cholesterol biosynthesis (Mvd) and metabolism (Insig1). Several mouse models of hypercholesterolemia [e.g., Ldlr knockout (Ldlr–/–) and apolipoprotein B (apoaB) transgenic mice] have been generated previously (5, 30), but to our knowledge, the Nas1–/– mouse is the first reported animal model of hyposulfatemia that is linked to hypercholesterolemia. The chow-fed Ldlr–/– and apoaB mice have little atherosclerosis and exhibit slightly elevated LDL and TC levels, similar to the levels found in Nas1–/– mice. Although gross histological analysis did not reveal any signs of atherosclerosis in Nas1–/– mice (16), it would be interesting to feed a high-fat diet to Nas1–/– mice, previously shown to cause more pronounced hypercholesterolemia and atherosclerotic lesions in the Ldlr–/– mice (31). Several metabolic pathways influence blood cholesterol homeostasis, including hepatic bile acid metabolism (48). Because Nas1–/– mice have increased serum bile acid levels (16), we examined the hepatic mRNA levels of several genes with key roles in bile acid metabolism. However, our transcriptional profiling showed no changes in genes involved in bile acid synthesis (Cyp7a1, Cyp27, and LXRa) or transport (Bsep, mdr2, Ntcp, and Oatp-1), previously shown to be maintaining serum bile acids levels (43). Because bile acids act as signaling molecules in the transcriptional regulation of numerous metabolic pathways, including cholesterol and lipid metabolism (reviewed in Ref. 17), we need to consider the possibility that the increased serum bile acid levels found in Nas1–/– mice (16) may be contributing to the altered hepatic transcriptional profile of Nas1–/– mice. However, we found no changes in the mRNA level of genes involved in bile acid sensing, such as the hepatocyte nuclear factor-4 (Hnf4a) and farnesoid X receptor (Nr1h4). In addition, the lack of similarities between the Nas1–/– hepatic transcriptional profile and that obtained from the livers of mice fed a cholate- and cholesterol-enriched diet (54), or ursodeoxycholic acid-treated rat hepatocytes (9), suggests that the altered mRNA profile of Nas1–/– mice may be due to entities other than increased serum bile acid levels (16) and mild hypercholesterolemia (Table 4) found in Nas1–/– mice.

Nas1–/– mice exhibit altered sulfotransferase mRNA levels.
Three members of the sulfotransferase gene family with functional links to steroid metabolism were up- (Sult3a1 and Sult2a2) or downregulated (Sult1e1) in livers of Nas1–/– mice. Both Sult3a1 and Sult2a2 can sulfonate a variety of steroids and sterols, including dehydroepiandrosterone (DHEA) (3). The serum concentration of DHEA-sulfate (DHEA-S) is normally 300–500 times higher than that of DHEA (33), suggesting that DHEA-S forms a circulating reservoir. However, only the unconjugated steroid hormone DHEA has growth-promoting activity, and changes in the activity of DHEA sulfotransferases have been implicated in pathophysiological disorders of growth (32). Nas1–/– mice exhibit reduced growth (16), and future studies of steroid hormone levels in these mice will determine the consequences of increased Sult3a1 and Sult2a2 mRNA levels on DHEA metabolism. In addition, the strong induction of Sult3a1, which also catalyzes the sulfate conjugation of p-nitrophenol, is consistent with our previous findings of elevated hepatic sulfotransferase activity (16). However, unlike the upregulation of Sult3a1 and Sult2a2, we observed reduced mRNA levels of Sult1e1, suggesting that members of the sulfotransferase family may be differentially regulated at the transcriptional level in Nas1–/– mice. Sult1e1 catalyzes the sulfonation of ß-estradiol and estrone and has an important role in mammalian reproductive physiology (53). Disruption of the mouse Sult1e1 gene results in reproductive abnormalities of female mice as soon as they reach sexual maturity, whereas male sult1e1 null mice develop testicular abnormalities and a decreased fertility later in adulthood (46). Interestingly, female Nas1–/– mice have reduced fertility at sexual maturity, which was not seen in male Nas1–/– mice at 2–6 mo of age (16). However, the fertility of male Nas1–/– mice aged >6 mo is yet to be determined. Also downregulated was AKr1c18, a hydroxysteroid dehydrogenase that is essential in estrogen biosynthesis. The solute carrier organic anion transporter, Slco1b2, which mediates the transport of steroids from the liver, including DHEA-S and estrone sulfate, was also suppressed in Nas1–/– mice. In addition, the Cyp3a25 microsomal cytochrome, known to be involved in steroid oxidation, was transcriptionally downregulated in Nas1–/– mice. Taken together, these data show an altered transcriptional profile of genes linked to steroid metabolism, which may be relevant when considering the etiology of reduced growth and fertility in Nas1–/– mice.

Transcriptional changes associated with immune response and cell defense.
The liver is an essential part of the immune system, producing defensin and acute-phase proteins (25). Several defensin (Defcr2, Defcr9, Defcr6, and Defb6), heat shock (Hspa5, Hspb1, and Hspa8), immune response (Gbp2 and Xbp1), and acute-phase response (Itih3) genes were upregulated in Nas1–/– mice (Table 2). Similar transcriptional responses were reported during acute systemic inflammation (13). However, Nas1–/– mice showed no evidence of liver inflammation in hematoxylin- and eosin-stained sections (16), suggesting an injury in extrahepatic tissues. During acute stress, hepatocyte proliferation and steatosis is stimulated (15), which is consistent with our findings of hepatomegaly (16) and fatty liver (Fig. 2) in Nas1–/– mice. It is also consistent with the upregulation of several genes linked to protein synthesis (Eef1g, Yars, Rpl8, and Gfm) and cell growth (Igfbp2, Igtp, and Tgtp) (Table 2).

Increased hepatic sat-1 mRNA levels in Nas1–/– mice.
Previously, we cloned the mouse sulfate anion transporter-1 gene (sat-1), which is strongly expressed in the liver where it is proposed to play a role in bile acid metabolism (35). In the present study, we showed increased sat-1 mRNA levels in the livers of Nas1–/– and Nas1+/– mice, which was most likely a compensatory response to reduced serum sulfate levels in Nas1–/– (0.22 mM) and Nas1+/– (0.56 mM) mice (16). We have characterized the mouse sat-1 gene promoter to have numerous and diverse transcription factor-binding sites (36), including a functional T3 response element (35). To date, no studies have examined the effect of hyposulfatemia on thyroid hormone levels, but the lack of similarities between the Nas1–/– hepatic transcriptional profile and that obtained from T3-treated and hypothyroid mice (22) may argue against a change in thyroid hormone levels in Nas1–/– mice. Our data here suggest that sat-1 mRNA expression (Fig. 3) is sensitive to changes in blood sulfate levels, as observed in Nas1+/– mice with an {approx}50% reduction in serum sulfate levels (16). Alternatively, the mRNA levels of two other sulfate transporters, dtdst (slc26a2) and dra (slc26a3), were not changed, indicating that they may not be sensitive to altered blood sulfate levels. In addition, no change was found for genes involved in sulfate metabolism, sulfonation of proteoglycans, or the metabolism of sulfur-containing amino acids, including cysteine, methionine, and glutathione. Taken together, the transcriptional changes in sat-1 and sulfotransferases (Sult3a1 and Sult2a2) are most likely due to low hepatic sulfate levels as a consequence of hyposulfatemia.

In conclusion, this study has revealed that NaS1 sulfate transporter deficiency leads to an altered hepatic transcriptional profile, increased hepatic lipid and blood cholesterol levels, and decreased hepatic glycogen content. These findings suggest that sulfate may play important, and previously undescribed, roles in the modulation of energy metabolism in the liver and in blood cholesterol homeostasis. The significance of hyposulfatemia on hepatic metabolism and blood cholesterol levels in humans awaits further investigation.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Australia National Health and Medical Research Council (NHMRC) and the Australian Research Council. S. Grimmond is a recipient of an NHMRC career development award. The array reagents were provided by the Australian Cancer Research Foundation DNA microarray initiative.


   ACKNOWLEDGMENTS
 
We thank Drs. T. Walker and H. Cooper for advice on QRT-PCR and P. Addison and K. A. Nguyen for technical assistance.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. Markovich, School of Biomedical Sciences, Univ. of Queensland, St. Lucia, QLD 4072, Australia (e-mail: d.markovich{at}uq.edu.au).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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