Department of Medicine, Washington University School of Medicine, St. Louis, Missouri
Submitted 13 September 2004 ; accepted in final form 7 December 2004
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ABSTRACT |
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fatty liver; genetics of hepatic steatosis; lipogenesis; fatty acid oxidation; very low-density lipoprotein
NAFLD poses a major health problem because it may lead to steatohepatitis, cirrhosis, and liver failure. The pathophysiological mechanisms by which steatosis may progress to steatohepatitis, fibrosis, and cirrhosis remain unclear, but steatosis appears to be an essential precursor (10). Thus controlling hepatic TG levels may be important in reducing the risk of developing chronic liver disease.
Fatty liver is due to an imbalance between the availability of hepatic TG for export and the disposal system of the liver (1, 20, 36). Both genetic and environmental factors are likely to be important. The major sources of hepatic fatty acids (FA) for TG assembly are those synthesized de novo in the liver and free FAs (FFA) derived from plasma. Genes involved in de novo lipogenesis are controlled by sterol regulatory element-binding protein (SREBP)-1c (40), a mediator of both nutritional stimulus and insulin action in hepatic lipogenesis (16). Mice overexpressing SREBP-1a develop severe fatty liver due to overproduction of cholesterol and FAs (32). Elevated levels of FFA in plasma, such as those in fasting, increase delivery of FFA to the liver, which may cause excessive hepatic TG accumulation despite an accompanying increased FA oxidation (15).
The major disposal routes for hepatic FAs and TG are composed of the export of hepatic TG via VLDL (39) and the oxidation of component FAs in mitochondria, peroxisomes, and microsomes (41). Here too, both genetic and environmental factors are important. For example, the impaired capacity for FA -oxidation on fasting in peroxisome proliferator-agonist receptor (PPAR)-
null mice results in the accumulation of hepatic TG (18). A defective VLDL transport system that impairs hepatic lipid-exporting capacity also may result in fatty liver. This is seen in abetalipoproteinemia, caused by mutations in microsomal triglyceride transfer protein that is essential in the assembly of chylomicrons and VLDL particles (37), and in familial hypobetalipoproteinemia due to truncation-specifying mutations in apolipoprotein (apo)B, the indispensable structural protein in the formation and secretion of VLDL (6, 31, 35).
Thus the major genes for developing fatty liver, identified to date, are selected genes in the FA synthetic and oxidative pathways and in the VLDL export system. However, modifier genes (22, 23) as yet not identified may be contributing to the development of steatosis. This is suggested by the finding that engineered apoB gene mutation-bearing mice (e.g., apoB+/38.9 and apoB+/27.6 heterozygotes) produced on mixed C57BL/6JX129/SVJ genetic backgrounds manifest significantly elevated hepatic TG on average, but interindividual variation is high (7, 8, 31), probably reflecting the heterogeneity of the genetic backgrounds. Indeed, although groups of these mice on average contain 50% C57BL/6J and 50% 129/SVJ genes, the genetic complements of individual animals may deviate materially from the 50/50 mean (P. Yue, X. Lin, and G. Schonfeld, unpublished observation). Interindividual variation of hepatic TG levels should be significantly smaller in congenic strains bearing apoB truncation-producing mutations than in the apoB mutation-bearing mice of 50/50% C57BL/6J and 129/SVJ mixed background.
To identify potentially useful parental strains for the production of congenic mice, we performed a mouse-strain survey of liver TG levels in 10 inbred strains, the results of which form the bulk of this presentation. Significant strain-related differences in hepatic TG contents were found. To ascertain which physiological, biochemical processes may play roles in determining the differences in liver TG contents among the selected strains, experiments, including hepatic mRNA expression profiling, were performed in the strains with the highest [BALB/cByJ (BALB/c)], middle [C57BL/6J (C57BL)], and lowest [SWR/J (SWR)] hepatic TG contents.
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MATERIALS AND METHODS |
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Ten inbred strains of mice (6 wk old), AKR/J (AKR), BALB/c, C3H/HeJ (C3H), C57BL, C57BL/6ByJ (C57BLBY), DBA/1LacJ (DBA), 129/SVJ, NZB/B1NJ (NZB), PL/J (PL), and SWR, of both genders were purchased from Jackson Laboratory (Bar Harbor, ME). AKR males were not studied because only AKR females were available from the supplier. Mice were housed in a room maintained at 24°C with a 12:12-h light-dark cycle (6:00 AM to 6:00 PM). All mice were given Purina mouse chow 5053 containing 4.5% fat, 20.0% protein, and 54.8% carbohydrate (LabDiet). Food was removed in the beginning of the light cycle, and mice were fasted for 46 h, except in the fasting experiment where times are indicated. Mice were 1112 wk of age when killed. All animal procedures were performed in accordance with guidelines of Washington University's Animal Studies Committee and approved by the IACUC of Washington University.
Analytical Procedures
Body fat (%) was assessed on anesthetized living mice by dual-energy X-ray absorptiometry with the use of a small animal densitometer (Lunar) (3). Plasma samples were assayed for triglycerides, total cholesterol, and free cholesterol as described (7, 19). Lipids were extracted from liver and assayed for TG (7). Hepatic TG levels were somehow lower in the three strains compared with their counterparts during the survey carried out at 46 h of fasting, probably due to the use of a different TG analysis kit from a supplier, which systematically did yield lower hepatic TG levels when used on the surveyed livers (data not shown). The same kit was used for all of the fasting experiment. Cellular protein contents were determined as described (7). Hepatic TG contents were expressed as milligrams of lipid per gram of protein. Plasma FFA were analyzed by an enzymatic method (Wako). Blood glucose was determined using a B-glucose analyzer (Hemocue, Ängelholm, Sweden). The Core Laboratory of the General Clinical Research Center at Washington University analyzed plasma -hydroxybutyrate (BHB) concentration. Mice were weighed and killed. Blood was obtained from the inferior vena cava. Livers were excised, washed in cold PBS, weighed, cut into pieces that were then frozen in liquid nitrogen, and stored at 80°C until further analysis.
Glucose Tolerance Tests
After mice were fasted for 5 h, mice received an intraperitoneal injection of 10% D-glucose (0.75 g/kg body wt) for glucose tolerance. Blood (10 µl) was drawn from the tail vein at 0, 30, 60, and 120 min and assayed for glucose.
In Vivo Measurement of FA Synthesis
Mice were injected with [1-14C]acetate (0.25 Ci/ml) at 25 µCi/30 g body wt and killed after 1.0 h. Liver (300 mg) was excised and digested in potassium hydroxide. After extraction of the nonsaponifiable lipids with petroleum ether, the sample solution containing the saponified lipids was acidified with sulfuric acid and FAs were extracted with petroleum ether as described (11). The radioactivity in total FAs was determined by scintillation counting. The FA synthesis rates are reported as disintegrations per hour per milligram of liver.
Determination of In Vivo Hepatic TG Secretion Rates
Hepatic production of VLDL-TG was measured after injection (intravenous) of Triton WR1339 (500 mg/kg body wt) on mice fasted for 4 h (7). Tail vein blood samples were taken at the specified times after injection for TG measurement and measured as described above.
Determination of FA Oxidation in Primary Cultures of Hepatocytes
Primary mouse hepatocytes were isolated as described (7). Cells (1.0 x 106) were cultured in a flask for 2 h in 4 ml DMEM containing 10% FBS, and medium was then changed to DMEM containing 5% FBS. After an overnight culture, cells were washed three times with PBS. To determine FA oxidation, 14C-labeled palmitate (0.25 µCi/ml) was added to the medium. At the end of 2-h incubation, a portion of the cell medium (250 µl) was removed to determine acid-soluble products (ASP) as described (13). A septum and center well were then fitted into the flask. Sodium hydroxide (2 N, 0.25 ml) was injected onto the filter paper placed in the center well to capture CO2. Hydrochloric acid (6 N, 2 ml) was injected into the medium to release 14CO2. Both ASP and 14CO2 were counted. Two separate flasks treated in the same way without the 14C-labeled palmitate were used for assay of cellular protein. Total FA oxidation activity was obtained by adding the counts of 14CO2 and ASP and expressed as DPM per hour per microgram of cellular protein.
Short-chain Acyl-CoA Dehydrogenase Mutation Assay
A PCR assay was used to distinguish between the wild-type and mutant alleles for the structural gene of short-chain acyl-CoA dehydrogenase (SCAD), which employs oligonucleotide primers that flank a 278-bp deletion in the mutant allele to produce PCR products of 870 and 592 bp for the wild-type and mutant alleles for SCAD, respectively (38).
Responses of Liver TG Levels to Fasting
To determine whether there was a differential susceptibility of the fasting raising effect on liver TG levels, male mice of BALB/c, C57BL, and SWR were killed at 0, 6, and 14 h of fasting. Liver TG contents, plasma FFA, and BHB concentrations were determined as described in Analytical Procedures. A separate kit (Thermo Electron, Melbourne, Australia) was used to determine hepatic TG levels, because Wako discontinued its TG kit used in this study. The latter kit, although yielding precise results, deviated systematically downward from the Wako kit, yielding somewhat lower values for the fasting study (see RESULTS).
mRNA Expression Profiling
Dual-channel microarray analysis was performed on total liver RNA pooled from each strain (male, n = 5 for each strain). Extracted RNA was further purified using RNeasy spin columns (Qiagen, Valencia, CA) following the manufacturer's protocol. Purified RNA was quantitated by ultraviolet absorbance at 260 and 280 nm and assessed qualitatively using Bioanalyzer 2100 (Agilent, Palo Alto, CA). Three comparison pairs (BALB/c vs. C57BL, BALB/c vs. SWR, C57BL vs. SWR) were set up comparing against each pair of two strains of mice. In each pair, 5 µg of purified total RNA were converted to cDNA that was labeled either with Cy3 or Cy5, which were hybridized to mouse Oligo Array (Sigma 65-mer Probe Set) containing 21,676 transcripts. In "dye-flip" experiments with the same pair of comparison, the dye labeling was reversed. Oligonucleotide array analysis was performed by the Genomic Core Facility of Digestive Diseases Research Core Center at Washington University in St. Louis, MO. All protocols were performed as recommended by Genisphere (Hatfield, PA).
Array images were scanned on an Axon scanner. Array data were extracted and analyzed using GenePix Pro 4.1 software from Axon Instruments. Further analysis was performed by using BRB-Array Tools (version 3.1, http://linus.nci.nih.gov/BRB-ArrayTools.html) according to the instructions provided. Differential gene expression between two of the three strains (BALB/c, SWR, and C57BL) was done by using class comparison expression analysis.
Quantitative Real-Time RT-PCR
Total RNA from male BALB/c, C57BL, and SWR each was pooled and digested with RNase-free DNase, followed by purification with RNeasy Mini Kit (Qiagen). mRNAs of interest were quantified by fluorescence RT-PCR with an Applied Biosystems GeneAmp 5700 sequence-detection system using SYBR Green dye binding to the PCR product (5). Primer Express 2.0 software was used to design amplimers for both genes of interest and GAPDH, the latter of which was used for normalization in RT-PCR.
Statistics
All statistic analyses were conducted using SAS (version 9, SAS Institute, Cary, NC). Data are expressed as means ± SD. ANOVA (PROC GLM) followed by either Duncan's multiple tests or t-tests were performed for comparisons between treatments as appropriate with an overall -level of 0.01 or 0.05 as indicated. Pearson's correlation was performed using SAS Proc CORR using data from all 10 strains. The total variance (Vtotal) in the general linear model was partitioned into variance due to "environment" (Venv) and variance due to strain or genetic effects (Vstrain), where Vstrain/Vtotal gave an estimate of Vtotal explained by strain effect, an indication of heritability (12).
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RESULTS |
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Liver TG levels, averaged on both genders, varied over a sixfold range among the strains, from 49.7 ± 5.5 mg/g protein (SWR) to 316.5 ± 25.8 (BALB/c; Fig. 1, A and B). Liver TG contents showed similar strain-related differences across both genders. The presence of the SCAD mutation was confirmed in both BALB/c males and females (data not shown), but it was not detected in any of the other strains (data not shown).
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Body weights ranged from 25.3 ± 2.7 g in BALB/c males to 20.8 ± 2.8 g in SWR males. Among females, AKR were heaviest (26.8 ± 1.7 g), and PL were the lightest (24.1 ± 2.0 g; Table 1). Body fat contents were highest in AKR females (males were not available), followed by PL of both genders (Table 1). BALB/c and SWR males and NZB females were among the lowest. With respect to liver-to-body weight ratio, BALB/c males and females ranked at the top, and 129/SVJ ranked at the bottom (Table 1).
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Plasma TG concentrations varied approximately threefold, ranging from 59.2 ± 16.6 (C57BLBY) to 173.8 ± 12.4 mg/dl (BALB/c) for males and from 61.1 ± 17.5 (C57BL) to 203.8 ± 22.7 mg/dl (PL) in females (Table 2). Plasma TC ranged 2.5-fold, from 70.6 ± 5.0 (C57BLBY) to 153.1 ± 4.7 mg/dl (NZB) for males and from 63.3 ± 7.7 (C57BLBY) to 164.8 ± 10.1 mg/dl (C3H) in females (Table 2). The range of concentrations for plasma-free cholesterol was greater in males (4.3-fold difference ranging from 6.1 ± 1.0 mg/dl for BALB/c to 26.2 ± 1.1 mg/dl for NZB) than in females (2.6-fold difference ranging from 9.3 ± 1.0 mg/dl for BALB/c to 23.8 ± 2.0 mg/dl for NZB; Table 2).
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Plasma BHB concentrations were alike in males (although male SWR had the highest level numerically) but differed among strains in females (Table 3). SWR had the highest concentration, whereas PL had the lowest. Significant differences in glucose concentration existed among the strains of both genders. The C57BL strain had the highest plasma glucose concentrations for both males and females (Table 3).
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Many of the parameters reported above have been shown to correlate with liver fat in humans (31). Therefore, a series of correlations was sought in an attempt to identify correlates of hepatic fat in mice. Correlations between body weight and hepatic TG were significant only in males (data not shown). Correlations between body fat and hepatic TG were significant only in females. Hepatic TG level was significantly and positively correlated with liver-to-body weight ratio for both males and females (Table 4). Hepatic TG was positively correlated with plasma TG only in males. No significant correlations existed between hepatic TG and TC (Table 4). However, hepatic TG level was negatively correlated with plasma-free cholesterol level for both genders (Table 4). No significant correlations existed between hepatic TG levels and plasma BHB concentrations (Table 4). Thus univariate analysis did not identify any consistent covariates of hepatic TG except for the liver-to-body weight ratio.
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Physiological/Biochemical Studies on Male BALB/c, C57BL, and SWR
Hepatic lipogenesis, TG secretion, and FA oxidation.
In vivo rates of hepatic lipogenesis, measured by [1-14C]-acetate incorporation were similar in BALB/c and C57BL, but smaller in SWR (Fig. 2A). Rates of liver TG secretion, quantified by Triton WR-1339 injection, were similar in BALB/c and C57BL and lower in SWR (Fig. 2B). FA -oxidation rates, determined in primary hepatic cell cultures, were similar in BALB/c and C57BL and higher in SWR (Fig. 2C). These data suggest that SWR livers synthesize FAs at a lesser rate and oxidize them at a faster rate than the other two strains, leaving less TG for hepatic accumulation. Clearly, low levels of hepatic TG in SWR are not due to enhanced export from the liver.
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In the fed state, BHB levels were highest in SWR. With fasting, levels rose in all strains, but rises were greatest in SWR and least in BALB/c. These data are compatible with a greater capacity by livers of SWR to adapt to fasting-induced FFA mobilization by most effectively enhancing FA oxidation among the three strains, thus limiting hepatic TG accumulation.
Glucose tolerance. Responses to the glucose tolerance test were similar between male and female; thus results were combined for both genders. Glucose levels were highest in C57BL in response to glucose loading (Fig. 3). Levels were similar in BALB/c and SWR.
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DISCUSSION |
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In an attempt to identify metabolic covariates of liver TG, we performed a series of correlation analyses. Only liver-to-body weight ratios were significantly and positively correlated with liver TG in both genders. The other parameters of body weight, body fatness and plasma metabolites such as glucose, BHB, plasma TG were not consistently correlated across strains and genders (Table 4). Thus the parameters of adiposity or insulin action that correlate with liver fat in humans (31, 34) appear not to be correlated in mice, for reasons that are not clear. The negative correlation of hepatic TG with plasma-free cholesterol remains unexplained.
To understand some of the physiological/biochemical processes underlying the strain-related differences in hepatic TG, we studied mice representing the highest (BALB/c), middle range (C57BL), and the lowest (SWR) levels of hepatic TG. Males were used because they were more readily available, and we saw similar strain differences in hepatic TG in both genders. Glucose tolerance, reflecting insulin action, was studied in the 4- to 6-h fasted state. Processes contributing to hepatic TG levels such as hepatic FA synthesis and oxidation and hepatic TG transport were studied (7, 9, 15, 20) in the fed state. Finally, responses of hepatic TG to 6 and 14 h of fasting were compared with the fed state. We attempt to explain the differences among the three strains in terms of the relative rates of the above processes we studied.
SWR showed significantly lower rates of hepatic lipogenesis (Fig. 2A) and TG secretion (Fig. 2B) and higher rates of FA oxidation (Fig. 2C) than both the BALB/c and C57BL strains, whereas these parameters were similar in BALB/c and C57BL. These data suggest that SWR may maintain lower hepatic TG levels than the other two strains by presenting smaller TG loads to the VLDL export system. Clearly, because hepatic TG export was low, liver TG levels were not low because export was increased. SWR livers also have more efficient capacities to resist the hepatic TG-raising effects of fasting (Table 5). They appear to accomplish this by keeping plasma FFA levels low, which may indicate relatively low rates of lipolysis in adipose tissue and less delivery of FFA substrate to hepatic TG production, and BHB levels high, which may indicate efficient rates of ketogenesis. Both processes would tend to reduce the amounts of hepatic TG loads available for export via the VLDL system during fasting, compared with BALB/c and C57BL strains.
Glucose levels were highest in C57BL in the survey (Table 3) and during the glucose tolerance test (Fig. 3). The relatively high expression level of the SCD-1 gene in C57BL (Tables 6 and7) is compatible with the relatively high rates of hepatic FA synthesis and the relative glucose intolerance of C57BL. These data suggest that although livers of C57BL may produce higher amounts of hepatic TG (certainly relative to SWR), the presence of adequate VLDL-TG export rates and FA oxidation rates was able to restrain hepatic TG contents. It is possible that the accumulation of hepatic TG over time, in C57BL, may further contribute to the increasing glucose intolerance with age (4, 26), but high levels of hepatic TG do not explain the absence of glucose intolerance in BALB/c. It is possible that not all causes of fatty liver produce insulin resistance.
SCAD is a mitochondrial enzyme that catalyzes the first reaction in the -oxidation of short-chain FAs. BALB/c mice bear a spontaneous deletion in SCAD gene and develop fatty liver after 18 h of fasting (28, 29). However, it is worth noting that in the fed state, whereas BALB/c had considerably higher hepatic TG levels than C57BL, the two strains had similar rates of hepatic FA synthesis, hepatic TG secretion, and similar concentrations of plasma FFA and BHB. Similar rates of long-chain FA
-oxidation, as measured by using 14C-labeled palmitate as the substrate, were also found between C57BL and BALB/c. This raises questions as to whether the SCAD mutation is sufficient to explain the higher hepatic TG contents in BALB/c in the fed state.
It is possible that static measurements of plasma concentrations do not adequately reflect the deficiency in the action of SCAD. Plasma levels of FFA are determined by relative rates of lipolysis in adipose tissue (input) and rates of uptake by the liver, muscle, and other organs (output) (25). Plasma BHB concentrations are determined by relative rates of ketogenesis in liver (input) and ketone body uptake by brain and other organs (output) (21). It is impossible from the present experiments to determine whether the similar plasma levels of the two metabolites in fed BALB/c and C57BL reflect similar rates in input or output. It is conceivable that the rates of input and output differ in the two strains, but the balance between the two rates resulted in similar plasma levels. For example, if rates of lipolysis and tissue uptake are both higher in BALB/c, FFA levels in plasma could remain similar to C57BL levels, but the higher hepatic uptake of FFA in BALB/c could result in higher levels of hepatic TG, not explainable by the SCAD defect alone.
The fasting experiment appeared to support an FA -oxidation defect in BALB/c mice (the least rise in plasma BHB levels in response to fasting). However, this reflected more of a defect in the fasting situation, consistent with the development of fatty liver on 18 h of fasting in this strain (29).
One way to settle these issues would be to examine another BALB/c strain with intact SCAD function. We have studied another BALB/c strain available from Jackson Laboratories: the BALB/cJ strain. Hepatic TG contents of male BALB/cJ in fact are lower, resembling those of SWR. However, the two BALB/c strains also differ in at least 43 of 300 microsatellite markers genotyped across the genome (http://www.cidr.jhml.edu/mouse/mouse.html). Thus, at this time, it is not possible unequivocally to ascribe the excess hepatic TG in the fed state to the SCAD deficiency alone. It remains to be investigated whether any other factor(s) besides SCAD mutation causes fatty liver in this mouse strain.
We performed a gene expression survey expecting to find additional explanations for the interstrain differences in hepatic TG contents. Most of the differences in hepatic gene expression remain to be explained. However, as noted, the relative overexpression of the SCD-1 transcript in C57BL was useful in explaining the contribution of presumed increased palmitate desaturation to oleate to the higher hepatic TG levels in C57BL (Tables 6 and 7) (24). It is not clear whether high hepatic FA synthesis with lower SCD1 mRNA level in BALB/c results from a lower oxidation rate of newly synthesized FA. MT1 has been linked to the regulation of energy balance in mice, because mice deficient in both MT1 and MT2 were obese (2). SWR had the highest level of expression in MT1 when compared with both C57BL and BALB/c, consistent with the lowest body weight of SWR in the three strains. Mouse hepatic SELENBP1 was decreased in response to peroxisome proliferators such as ciprofibrate (14). The lowest expression of hepatic SELENBP1 in SWR relative to both C57BL and BALB/c may be related to the highest hepatic FA oxidation in SWR. Another consideration is that differential gene expression may be the consequence, rather than the cause of differences in liver TG levels. For example, hepatic TG accumulation may raise the level of reactive oxygen products in liver (1) stimulating alterations in the expression levels of mRNAs of acute phase reactant molecules.
In conclusion, results of these studies have demonstrated a significant genetic contribution to hepatic TG contents in inbred mice. They also helped identify potential parental strains for construction of congenic strains bearing apoB truncations. Breeding is in progress.
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FOOTNOTES |
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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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REFERENCES |
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