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Journal of Lipid Research, Vol. 42, 1831-1840, November 2001
Copyright © 2001 by Lipid Research, Inc.

PPAR deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins

Correspondence to: Daniel Lindén, To whom correspondence should be addressed., daniel.linden{at}fysiologi.gu.se (E-mail)

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study investigates the importance of peroxisome proliferator activated receptor (PPAR) for serum apolipoprotein B (apoB) levels and hepatic secretion of apoB-containing lipoproteins. Total serum apoB and VLDL-apoB levels were higher in female PPAR-null mice compared with female wild-type mice, but no difference was seen in male mice. Furthermore, hepatic triglyceride secretion rate, determined in vivo after Triton WR1339 injection, was 2.4-fold higher in female PPAR-null mice compared with female wild-type mice, but no difference was observed in male mice. However, when fed a high fat diet, male PPAR-null mice displayed 2-fold higher serum levels of apoB and LDL cholesterol compared with male wild-type mice, but triglyceride levels were not affected. Hepatic LDL receptor protein levels were not influenced by PPAR deficiency, gender, or the fat diet. Hepatocyte cultures from female PPAR-null mice (cultured for 4 days in serum free medium) showed 2-fold higher total apoB secretion and increased secretion of apoB-48 VLDL, as well as 2.7-fold larger accumulation of VLDL-triglycerides in the medium compared with wild-type cultures.

In conclusion, PPAR-deficient female mice, but not males, display high serum apoB associated with VLDL and increased hepatic triglyceride secretion. Moreover, male PPAR-null mice show increased susceptibility to high fat diet in terms of serum apoB levels. — Lindén, D., M. Alsterholm, H. Wennbo, and J. Oscarsson. PPAR deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins. J. Lipid Res. 2001. 42: 1831–1840.

Supplementary key words: VLDL, LDL, apolipoprotein B-48, apolipoprotein B-100, triglycerides, LDL receptor, hepatocytes, dietary fat, gender, cholesterol

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Fibrates are widely used drugs in the treatment of hypertriglyceridemia and combined hyperlipidemia, which are conditions associated with type II diabetes. Fibrates belong to the structurally diverse group of peroxisome proliferators that induce peroxisome proliferation and hepatomegaly in rodents and up-regulate several genes involved in peroxisomal as well as mitochondrial ß-oxidation and microsomal -oxidation of fatty acids [as reviewed in refs. (1) (2) (3)]. Fibrates have been shown to elicit their effects through peroxisome proliferator activated receptor (PPAR) (4), a member of the steroid nuclear receptor super family. PPAR is mostly expressed in tissues that have a high ratio of fatty acid oxidation, such as liver, brown adipose tissue, skeletal muscle, heart, and kidney (5) (6). The endogenous ligands for PPAR are mono- and polyunsaturated fatty acids as well as eicosanoids (7).

The PPAR-null mouse has proved to be a valuable model for the study of the importance of PPAR for various functions in the intact animal (8). When fasted, the PPAR-null mice developed hypoglycemia, hypoketonemia, hypothermia, elevated plasma free fatty acid levels, and liver steatosis (9) (10), indicating the importance of PPAR in glucose and lipid homeostasis during fasting. The hypoglycemia was shown to be due to reduced gluconeogenesis secondary to reduced fatty acid oxidation (11). Apart from decreased gene expression of several enzymes involved in fatty acid oxidation (8) (12), PPAR-null mice have been shown to have higher serum cholesterol levels than wild-type mice (13). With age, PPAR-null mice developed elevated serum triglycerides and late onset obesity despite stabile caloric intake. The obesity and increase in plasma triglycerides were shown to be more pronounced in female PPAR-null mice, while the hepatic accumulation of triglycerides and cholesterol were more pronounced in male PPAR-null mice (14). The mechanisms for the sexually dimorphic obesity and liver steatosis are not known, but sex differences in lipoprotein metabolism may contribute.

PPAR activation by fibrates influences metabolism of both HDL and apolipoprotein B (apoB)-containing lipoproteins in rodents and man (2) (13) (15) (16). Fibrates decrease apoB-containing lipoproteins, especially large VLDL particles, but also other apoB-containing lipoproteins as well as total serum apoB levels (16). It has been suggested that an increased catabolism of VLDL and chylomicrons is important for the triglyceride-lowering effect of fibrates (1) (3). This effect of fibrates may be via both increased lipoprotein lipase expression and decreased hepatic apoC-III expression (1) (3) (13). However, a decreased production of apoB-containing lipoproteins by fibrates may also contribute (17). PPAR activation has been shown to decrease triglyceride synthesis and secretion from cultured rat hepatocytes (18) (19), indicating a decreased VLDL secretion. However, the importance of changed PPAR expression for the effects of endogenous ligands on serum apoB levels and apoB production is not known.

In humans, the liver secretes only apoB-100-containing VLDL, and apoB-48 secretion occurs exclusively from the intestine (20). VLDL secreted from the rodent liver contains either apoB-48 or apoB-100 as structural protein [as reviewed in refs. (21) and (22)]. Assembly and secretion of apoB-containing lipoproteins is mainly regulated via co- or post-translational degradation of the apoB. Correct folding of apoB and subsequent assembly of VLDL has been shown to be highly dependent on the availability of fatty acids and lipid biosynthesis (21) (22) (23) (24). Thus, PPAR expression may play a role for apoB secretion although gene expression of apoB, apoE, and microsomal triglyceride transfer protein (MTP) was not influenced by PPAR deficiency (9). The aim of the present study was to investigate the role of PPAR for apoB metabolism and VLDL secretion in vivo and in vitro. We found that lack of PPAR results in hyperapobetalipoproteinemia in female mice and in fat fed male mice. Moreover, the increased VLDL secretion in female PPAR-null mice could be reproduced in vitro, indicating that this effect of PPAR deficiency was not a result of changed flux of metabolites in vivo.

	METHODS

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The Ethics Committee of Göteborg University has approved this study. All chemicals used were from Sigma Chemical Co. (St. Louis, MO, USA) if not stated otherwise.

Animals, diet, and serum analyses
Homozygous PPAR-null mice on pure Sv/129 genetic background and corresponding wild-type Sv/129 control mice were kindly provided by Dr. F. J. Gonzalez (National Institute of Health, Bethesda, MD, USA) and kept on the Sv/129 genetic background (8). In one high fat diet experiment, the PPAR-null mice were backcrossed to the C57BL/6 genetic background for 2 generations and littermate wild-type mice were used as controls. A PCR-based screening was used to determine the genotypes of the mice (14). In most experiments, the mice had free access to standard laboratory chow (Rat and mouse standard diet, B&K Universal Ltd, Sollentuna, Sweden). This chow contained (w/w) 2.5% fat (33% saturated fatty acids), 61% carbohydrates, including 4% fibers and 18% protein. In some experiments, the mice had free access to a high fat diet containing 57.6 energy percent (%) fat, 26.6% carbohydrates, and 15.8% protein (Bio-Serv Inc., Frenchtown, NJ, USA). The fatty acid profile of the high fat diet was as follows: oleic acid (C_18:1), 153.80 g/kg; palmitic acid (C₁₆), 93.93 g/kg; stearic acid (C₁₈), 48.39 g/kg; linoleic acid (C_18:2), 32.27 g/kg; cis-9-hexadecanoeic acid (C_16:1), 14.34 g/kg; myristic acid (C₁₄), 6.09 g/kg; eicosanoeic acid (C₂₀), 2.87 g/kg; and heptadecanoic acid (C₁₇), 1.793 g/kg. The mice were anesthetized with isofluran inhalation (Forene^®, Abbot Scandinavia AB, Sweden) and sacrificed between 0900 and 1100 h. Blood was collected through open-heart puncture and serum was assayed for triglycerides, cholesterol, and glucose as described earlier (25). Serum apoB levels were measured with an elecro-immunoassay as previously described (26). Standardization of apoB measurements was made by isolation of a narrow density cut of LDL (d = 1.030–1.055 g/ml) containing only apoB as a protein component. The protein concentration of the narrow density cut of LDL was determined by the BIO-RAD DC protein assay (BIO-RAD, Hercules, CA). The intra- and inter-assay CV for the apoB determinations were 5% and 8%, respectively. Serum ß-hydroxybutyrate was analyzed using an enzyme colorimetric assay (Sigma Chemical Co.). A rat insulin RIA with 100% cross-reactivity to mouse insulin was used for serum insulin measurements (Linco Research Inc., St. Louis, MO, USA).

Size distribution of serum lipoproteins
The size distribution profiles of lipoproteins were measured by using either fast protein liquid chromatography (FPLC) of pooled serum samples or high performance liquid chromatography system, SMART, of serum samples from individual mice. In the FPLC separation, serum from 6 to 8 mice were pooled to a total volume of 900 µl and the density adjusted to 1.215 g/ml with KBr in 0.9% NaCl. After ultracentrifugation (35,000 g, 4°C, 24 h), the total lipoprotein fraction was loaded on a 25 ml Superose 6B column (Pharmacia Upjohn, Uppsala, Sweden) using a constant flow rate of 0.35 ml/min. Triglyceride and cholesterol concentrations were determined in the collected 0.5 ml fractions. In the SMART separation, 10 µl serum sample from a single mouse was loaded on a Superose 6 PC 3.2/30 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and the chromatographic system was linked to an air segmented continuous flow system for on-line post-derivatization analysis of total cholesterol. The SMART-system was connected to a sample injector (Gina 50, Gynkotek HPLC, Germering, GmbH), and the elution buffer (0.01 M Tris, 0.03 M NaCl, pH 7.4) had the flow rate of 35 µl/min. The integrated area of the fractions was expressed in molar concentration. For both the FPLC and SMART systems, various peaks in the profiles are designated VLDL (0.98–1.006 g/ml), IDL + LDL (mentioned as LDL in the text) (1.006–1.063 g/ml), and HDL (1.063–1.215 g/ml) based on the elution profile of density fractions obtained by ultracentrifugations.

In vivo hepatic triglyceride secretion using Triton WR1339
Triglyceride secretion rate in vivo was measured by intravenous administration of Triton WR1339 (27). After a 4 h period without access to food (0700–1200 h), anesthetized mice were injected intravenously with Triton WR1339 diluted in saline (200 mg/ml) via the tail vein (500 mg/ kg body weight). Blood samples were taken before the injection (0 min) and 45 and 90 min after Triton WR1339 injection. The triglyceride accumulation was linear during this time period. Serum triglyceride levels were analyzed as described above, and micromole of triglycerides was calculated using published plasma volume in normal male (0.071 ml/g bodyweight) and female mice (0.09 ml/g body weight) (27). Hepatic triglyceride secretion rate was calculated from the slope of the curve and expressed as micromoles per hour per gram body weight.

ApoB mRNA quantification
Liver RNA was isolated with the Tri Reagent system (Sigma Chemical Co.). First strand cDNA was synthesized from total RNA using TaqMan^® reverse transcription reagents (Applied Biosystems). Real time PCR was performed with ABI Prism 7700 Sequence Detection System (Applied Biosystems) using FAM or VIC labeled fluorogenic probes. The apoB-specific primers amplified an 81 bp long fragment of apoB mRNA (nt 175-256, Acc. No. X15191.5), and the expression data were normalized against ribosomal 18S RNA (No. 4310843E, Applied Biosystems). The mean quantity of apoB and 18S in a sample were calculated from their respective standard curves, and data are expressed as apoB/18S.

Western blot analysis
Western blotting was performed using enhanced chemiluminescence (ECL) protocol (Amersham Pharmacia Biotech, Buckinghamshire, UK) and quantified using Fluor-S Multi imager and Quantity one software (BIO-RAD).

ApoB In each lane, adjacent FPLC fractions were pooled in pairs (9 µl each) and separated by SDS-PAGE. After electrophoresis, the proteins were transferred to Hybond^TM-P Polyvinylidene difluoride (PVDF) transfer membrane (Amersham Pharmacia Biotech). The membrane was blocked overnight at 4°C in PBS containing 0.1% Tween-20 (PBS-T) and 5% nonfat milk prior to incubation with polyclonal rabbit anti-rat apoB antiserum (26) diluted 1:1,000 in PBS-T. The membrane was washed and incubated with diluted (1:2,000) horseradish peroxidase-linked donkey anti-rabbit Ig (Amersham Life Science, UK) and developed.

LDL receptor In order to quantify the LDL receptor, livers were homogenized as described earlier (28) in the presence of 4 mM pefablock, 20 nM leupeptin, 15 nM pepstatin, and 0.15 nM aprotenin (Boehringer Mannheim, Germany). Proteins (30 µg/ lane) were separated and transferred as described above, but under non-reducing conditions. Primary rabbit anti-bovine LDL receptor antibodies (a generous gift from Professor Joacim Herz, Dept. of Molecular Genetics, University of Texas, Dallas, TX, USA) and secondary horseradish peroxidase-linked donkey anti-rabbit Ig were both diluted 1:2,000 in blocking buffer.

Primary hepatocyte cultures and estimation of secreted apoB and triglycerides
Hepatocytes were prepared by a non-recirculating collagenase perfusion through the portal vein of age- and sex-matched PPAR-null and wild-type mice on pure Sv/129 genetic background as described earlier (24) (29). The cells were seeded at a density of approximately 135,000 cells/cm² on laminin-rich matrigel (Collaborative Research, Medical Products, Bedford, MA, USA) coated plastic 100 mm dishes (Falcon, Plymouth, England), and plated during the first 16 to 18 h in Williams E medium supplemented as described previously (29). The medium, given the following 3 days of culture, was supplemented with 1 nM dexamethasone and 3 nM insulin (Actrapid^®, Novo Nordisk A/S, Denmark), and changed every day. The secretion of newly synthesized apoB-48 and apoB-100 into the medium was estimated by labeling the cells with a [³⁵S]methionine-cysteine mix (Amersham) for 2 h followed by a 4 h chase period using culture medium supplemented with 10 mM methionine, as described earlier (24) (30). In order to isolate the VLDL fraction of the medium, sucrose gradient ultracentrifugation was used as previously described [gradient 2, (24)]. Labeled apoB-48 and apoB-100 were isolated by immunoprecipitation as previously described (24) (30) and quantified using a Phosphoimager and Image Quant software (Molecular Dynamics, Inc., Sunnyvale, CA, USA). The obtained STORM units of apoB-48 and apoB-100 were related to the DNA content of the cells in the culture dish.

The mass of triglycerides in the VLDL fraction (d < 1.006 g/ml) of 24 h culture medium was determined as previously described (24) (31). The mass of triglycerides was related to the DNA content of the cells in the culture dish. The DNA content in each culture dish was determined according to the method of Labarca and Paigen (32).

Statistical analysis
Values are expressed as means ± SEM. In some experiments the mean value of the control group was set as 100%. Comparisons between groups in vivo were made by Student's t-test or one-way analysis of variance (ANOVA), followed by Bonferroni test. The values were transformed to logarithms when appropriate. Comparison between groups in vitro was made by Mann-Whitney U test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Serum levels of apoB, triglycerides, and cholesterol
Serum apoB levels were determined in male and female PPAR-null mice and matched wild-type controls on Sv/129 genetic background given standard laboratory chow ( Fig 1). The serum apoB levels were not different between 12-week-old male PPAR-null mice and age-matched male wild-type mice (Fig 1A). However, 12-week-old female PPAR-null mice had higher serum levels of apoB (+38%) compared with age-matched wild-type mice (Fig 1B). ApoB mRNA levels were measured by real time PCR in 12-week-old male and female mice of both genotypes. No differences in apoB mRNA levels were observed between male PPAR-null and wild-type mice (apoB/18S; KO, 10.4 ± 1.5, wt, 11.1 ± 2.2, n = 5 in each group, NS, Student's t-test) or between female PPAR-null mice and respective wild-type mice (apoB/18S; KO, 11.7 ± 2.5, wt, 9.3 ± 1.0, n = 4–5 in each group, NS, Student's t-test). Because a significant difference in body weight has been observed between female PPAR-null mice and female wild-type mice at 10 weeks of age (14), we analyzed the body fat depot weights in 12-week-old male and female mice of both genetic backgrounds. No differences in absolute or relative weights of retroperitoneal or gonadal (epididymal or parametrial) fat depots were observed between PPAR-null mice and their controls (n = 10, NS, Student's t-test, data not shown). At 8 months of age, PPAR-null mice have been shown to be more obese than their controls in terms of fat depot weights (14). Therefore, serum apoB levels were also measured in male and female PPAR-null mice at 32 weeks of age. At this age, there was still no difference in serum apoB levels between male PPAR-null mice and corresponding wild-type mice, but the female PPAR-null mice had higher serum apoB levels (+42%) than did their controls (Fig 1C and Fig D). In the same groups presented in Fig 1, serum triglyceride and cholesterol levels were analyzed ( Table 1). The serum triglyceride level was elevated in 12-week-old female PPAR-null mice (+74%) as compared with wild-type mice, and the difference was even larger at 32 weeks of age (+137%). However, no difference in serum triglyceride levels was observed between male PPAR-null and wild-type mice. In spite of a tendency toward higher serum cholesterol levels in all the PPAR-null mice compared with the respective control mice, a statistical difference was only obtained in young male mice (Table 1). Serum levels of ß-hydroxybutyrate were measured as an indication of differences in ketone production. Male PPAR-null mice had lower levels of ß-hydroxybutyrate than did wild-type males, but no difference between the genotypes was observed in female mice (Table 1). Changes in size distribution of lipoproteins were determined by FPLC analysis of total lipoproteins (d < 1.215 g/ml). Female PPAR-null mice had markedly higher levels of VLDL triglycerides and slightly higher cholesterol levels in all fractions compared with female wild-type mice ( Fig 2). Male PPAR-null mice had slightly higher levels of VLDL triglycerides and cholesterol compared with male wild-type mice (data not shown). Female PPAR-null mice had increased levels of both apoB-100 and apoB-48 in the VLDL fraction compared with female wild-type mice (Fig 2C). Male PPAR-null mice did not differ in apoB distribution compared with male wild-type mice (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 1. Serum apoB levels in female and male PPAR-null mice and corresponding wild-type mice on Sv/129 genetic background at 12 and 32 weeks of age. Serum apoB levels were determined by an electro-immunoassay as described in Methods. There were 10 mice in each group at 12 weeks of age and 5 mice in each group at 32 weeks of age. +/+, Wild-type mice; -/-, PPAR-null mice. Values are means ± SEM (* P < 0.05, Student's t-test).

View larger version (28K): [in this window] [in a new window]   Figure 2. Size distribution of serum lipoproteins in female PPAR-null mice and female wild-type mice. Total lipoprotein fractions (d < 1.215 g/ml) obtained from pooled serum from 8 PPAR-null and 6 wild-type mice on Sv/129 genetic background were subjected to FPLC as described in Methods. Panel A shows the distribution of triglycerides and panel B distribution of cholesterol in serum from PPAR-null mice (open circles) and wild-type mice (filled squares). VLDL, LDL, and HDL are indicated based on the elution profile of density fractions obtained by ultracentrifugations. Panel C shows the distribution of apoB-48 and apoB-100 as determined with Western blot analysis of the FPLC fractions.

View this table: [in this window] [in a new window]   Table 1. Serum levels of lipids and ß-hydroxybutyrate in male and female PPAR-null mice

In vivo hepatic triglyceride secretion rate
The hepatic triglyceride secretion rate was markedly higher in female PPAR-null mice (+136%) compared with female wild-type mice. In contrast, hepatic triglyceride secretion rate was not different between male PPAR-null mice and male wild-type mice ( Fig 3). Female wild-type mice tended to have a higher hepatic triglyceride secretion rate than did wild-type male mice (+63%), but in the absence of PPAR, female mice had markedly higher triglyceride secretion rate than did male mice (+195%).

View larger version (20K): [in this window] [in a new window]   Figure 3. In vivo hepatic triglyceride secretion rates in 32-week-old mice on Sv/129 genetic background. The triglyceride secretion rate was measured by injecting the mice with Triton WR1339 (500 mg/kg). Serum triglycerides were determined before injection (0 min) and at 45 and 90 min after Triton injection. Hepatic triglyceride secretion rate was calculated from the slope of the curve. There were 4 to 6 mice in each group, and values are means ± SEM (* P < 0.05, versus all other groups, one-way ANOVA followed by Bonferroni test).

High fat feeding of male PPAR-null mice
In order to see if hyperapobetalipoproteinemia could be induced in male PPAR-null mice by changing the diet, 9-week-old male PPAR-null and wild-type mice on Sv/129 genetic background were fed a high fat diet for 3 weeks. Fat fed male PPAR-null mice had significantly higher serum apoB levels than did fat fed male wild-type mice (+49%, P < 0.05, Student's t-test, data not shown). In addition, PPAR-null mice were back-crossed toward the C57BL/6 genetic background for two generations. The C57BL/6 strain is a common background to several obese phenotypes and highly sensitive to fat diet in terms of weight gain and hyperinsulinemia (33) (34). Using this genetic background, 13-week-old male PPAR-null mice and corresponding littermate controls were fed the high fat diet for 3 weeks. The serum levels of apoB were significantly higher (+98%) in fat fed PPAR-null mice compared with fat fed wild-type mice, whereas no difference between male PPAR-null mice and wild-type mice was observed when the mice were fed a normal diet ( Fig 4A). The high fat diet increased body weight gain to a similar degree in both PPAR-null mice and wild-type mice ( Table 2). There was a tendency toward higher liver weight in PPAR-null mice fed a high fat diet and macroscopically the livers were clearly paler as compared with the rest of the groups, indicating lipid accumulation. The weights of the epididymal and retroperitoneal fat depots were similarly increased by the high fat diet in the PPAR-null and wild-type mice (Table 2). Serum levels of insulin tended to be higher in the two groups of mice given the fat diet (P = 0.059, one-way ANOVA followed by Bonferroni test), and 20% higher serum glucose levels were observed in fat fed wild-type mice compared with wild-type mice given normal diet (Table 2). There were no significant differences in serum levels of triglycerides between the groups. The serum cholesterol levels were not significantly different between PPAR-null and wild-type mice fed the normal diet (Table 2). However, the serum cholesterol levels in the fat fed PPAR-null mice were 30% higher than in the fat-fed wild-type mice (Table 2).

View larger version (21K): [in this window] [in a new window]   Figure 4. Serum apoB levels (A) and cholesterol profile (B) in PPAR-null mice fed a high fat diet. Thirteen-week-old littermate male PPAR-null mice and wild-type mice (F2 Sv/129 x C57BL/6) were fed normal or a high fat diet for 3 weeks. Serum apoB was determined by an electro-immunoassay and the cholesterol distribution profiles were measured on individual mice by using a size exclusion high performance liquid chromatography system, SMART. The cholesterol levels were then determined in the different lipoprotein classes. There were 5 mice in each group. Values are means ± SEM (* P < 0.05 vs. wild-type normal diet, P < 0.05 vs. wild-type high fat diet, one-way ANOVA followed by Bonferroni test).

View this table: [in this window] [in a new window]   Table 2. Effects of high fat diet on tissue weights, insulin, glucose, and serum lipids in male PPAR-null mice

The size distribution of lipoproteins was measured in individual mice by using high performance liquid chromatography system, SMART, followed by determination of cholesterol levels in the different fractions (Fig 4B). VLDL cholesterol levels tended to be lower in the fat fed groups, but no significant differences were observed. The LDL cholesterol levels were significantly higher in PPAR-null mice fed either normal or high fat diet compared with the corresponding wild-type mice. HDL cholesterol levels were increased by the high fat diet in both PPAR-null and wild-type mice, but the effect of the high fat diet was not different between PPAR-null and wild-type mice (Fig 4B).

LDL receptor measurements
In order to investigate if the changed serum levels of apoB and LDL cholesterol were partly due to decreased expression of the LDL receptor (35), the LDL receptor protein expression was determined with Western blot ( Fig 5). Both the 130 kDa and the 220–240 kDa [corresponding to a dimer of the LDL receptor (36)] were quantified and added to a total expression value. The LDL receptor protein levels did not differ between female PPAR-null and wild-type mice or between fat fed male PPAR-null and wild-type mice, respectively (n = 5 in each group, NS, Student's t-test, data not shown).

View larger version (58K): [in this window] [in a new window]   Figure 5. Hepatic LDL receptor protein levels in 32-week-old female PPAR-null mice on Sv/129 genetic background (A) and in 16-week-old male PPAR-null mice (F2 Sv129 x C57/BL/6) fed normal diet or high fat diet for 3 weeks (B). The LDL receptor protein levels were identified by Western blot. Three of five mice in each group are presented and as a negative control, liver protein preparations from an LDL receptor-deficient mouse and corresponding wild-type mouse are shown (A).

Secretion of total and VLDL-associated apoB and triglycerides from primary cultures of hepatocytes
The increased triglyceride secretion in female PPAR-null mice may be dependent on the metabolic changes in the intact animal, such as changed flux of fatty acids to the liver. In order to investigate if there is a difference in the capacity for apoB and triglyceride secretion between hepatocytes from PPAR-null and wild-type mice, primary hepatocyte cultures were used. Hepatocytes from 32-week-old female PPAR-null mice and wild-type mice on Sv/129 genetic background were prepared through collagenase perfusion. The cells were cultured for 4 days on laminin-rich matrigel in serum free medium supplemented with 1 nM dexamethasone and 3 nM insulin (24). In an initial experiment, the intracellular accumulation of labeled apoB-48 and apoB-100 was found to be similar after 1, 2, 3, and 4 h of labeling in hepatocytes from PPAR-null mice and wild-type mice. No further increase in labeling of these proteins occurred after 2 h (data not shown). Therefore, the cells were labeled for 2 h with [³⁵S]methionine-cysteine mix and chased for 4 h in excess of unlabeled methionine followed by immunoprecipitation of apoB. Neither apoB-48 nor apoB-100 could be detected in the cells after the 4 h chase period. The accumulation of labeled apoB-100 and apoB-48 in the culture medium was higher in cultures from the PPAR-null mice as compared with wild-type cells ( Fig 6A and Fig B). There was no difference in the proportion of apoB-48 of total apoB [calculated as apoB-48/(apoB-48 + apoB-100)] in the medium between PPAR-null cultures, 92.7 ± 2.6%, and wild-type cultures, 93.3 ± 2.0%. In the experiments presented, the chase medium was subjected to sucrose gradient ultracentrifugation in order to isolate the VLDL fraction (24) (Fig 6C). The accumulation of apoB-100-containing VLDL particles in the chase medium was found to be very low in both PPAR-null and wild-type hepatocyte cultures. The accumulation of apoB-48-containing VLDL particles in the chase medium was higher in cultures from PPAR-null mice compared with wild-type cells (Fig 6C). The mean individual experimental values in apoB-48-VLDL in the three experiments (wild-type; PPAR-null) were 1081; 1765, 15; 687 and 12; 1179 STORM units/µg DNA. In addition, the mass of triglycerides in the VLDL fraction of overnight culture medium (last 24 h incubation) was measured. The mass of triglycerides in the VLDL fraction of the medium was higher (+170%) in cultures from PPAR-null mice compared with the medium of wild-type cultures (Fig 6D).

View larger version (37K): [in this window] [in a new window]   Figure 6. Accumulation of apoB-100 and apoB-48 in culture medium (A, B), in the VLDL fraction (C), and VLDL triglycerides in hepatic culture medium (D) from 32-week-old female PPAR-null (n = 3–4) and wild-type mice (n = 3–4) on Sv/129 genetic background. The cells were plated with 16 nM insulin during the first 16–18 h followed by incubation with 3 nM insulin and 1 nM dexamethasone during the last 3 days of culture. Cells were labeled for 2 h with a [35S]methionine-cysteine mix, washed and cultured for 4 h in a medium containing 10 mM unlabeled methionine (chase medium). VLDL was isolated from the chase medium using sucrose gradient ultracentrifugation. Amounts of labeled apoB-100 and apoB-48 in the chase medium (total amount) (A) and in the VLDL fraction (C) were measured after immunoprecipitation and separation by SDS-PAGE as described in Methods. Representative autoradiograms are given (A, C). In the experiments, the quantified STORM units (Molecular Dynamics) of each band are divided with the DNA content in the culture dish. The data for total apoB secretion (B) is given as percentage of the increased accumulation of labeled apoB-100 and apoB-48 in the culture medium from PPAR-null cells compared with wild-type cells in three different experiments. The mass of triglycerides in VLDL fraction of 24 h culture medium (d < 1.006) was determined after extraction of total lipids as described in Methods. The result on VLDL associated triglycerides (D) is based on 4 liver perfusions of mice from each genotype. Values are means ± SEM (* P < 0.05, Mann-Whitney U test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study demonstrates that functional PPAR expression is of importance for serum apoB levels. PPAR deficiency was shown to result in higher serum levels of apoB in female mice but not in male mice, indicating that gonadal steroids and PPAR could interact in the regulation of apoB levels. Moreover, when fed a high fat diet, male PPAR-null mice displayed markedly elevated serum levels of apoB compared with male wild-type mice, indicating that the expression of PPAR also influences the effect of dietary fat on apoB levels. PPAR deficiency had no effect on LDL receptor protein level. However, the hepatic triglyceride production rate in vivo was markedly higher in female PPAR-null mice compared with female wild-type mice, whereas no difference was observed in male mice. The increased secretion of triglyceride-rich lipoproteins may therefore explain the higher apoB and VLDL triglyceride levels in females. In order to study the effect of PPAR deficiency on apoB and VLDL secretion from hepatocytes without the influence of the in vivo milieu, hepatocytes were cultured for 4 days. Secretion of total and VLDL-associated apoB and triglycerides were found to be higher in hepatic cultures from female PPAR-null mice as compared with cultures from wild-type mice. These results indicate that PPAR-deficient hepatocytes have an increased capacity for apoB and VLDL secretion.

Recently, a point mutation in the human PPAR gene (L162V) was shown to result in hyperapobetalipoproteinemia and hypercholesterolemia (37) (38) (39). Our results indicate that in addition to the effects of mutations in the PPAR gene, the level of PPAR expression might be of importance for serum levels of apoB and production of apoB-containing lipoproteins, but not LDL receptor expression. Interestingly, Tugwood et al. (40) demonstrated a great inter-individual variation in PPAR expression in human liver. Thus, individual variation in the expression of PPAR could be of importance for serum levels of apoB.

The ovarian and retroperitoneal adipose tissue depot weights were similar between the genotypes in both males and females at 12 weeks of age. Thus, the increased serum levels of apoB and triglycerides seen in female PPAR-null mice were probably not primarily due to obesity and associated insulin resistance. The gender differences in serum apoB and hepatic triglyceride secretion rate might be due to different actions of the sex hormones. Female rats have been shown to have a higher VLDL secretory rate than male rats (41) (42), and our results indicate that a difference between the sexes in VLDL secretion rate may exist also in mice. Furthermore, it has been shown that gonadectomy of female rats decreases triglyceride secretion, while estrogen replacement therapy increases triglyceride secretion (42). Testosterone may have the opposite effect (43). Thus, it is concluded from the present results that lack of PPAR unmasks a gender-specific influence on VLDL secretion. A cross-talk between PPAR and estrogen receptors has been suggested and may be involved in the regulation of apoB and triglyceride secretion (44) (45). In this context, it is interesting to note that livers from PPAR-null females accumulate less triglyceride than male mice (14), which may at least partly be explained by the larger export of triglycerides from the liver of female PPAR-null mice. Furthermore, female PPAR-null mice have been shown to be less susceptible to the action of etomoxir than male PPAR-null mice (46). Etomoxir inhibits mitochondrial ß-oxidation and results in severe hypoglycemia and accumulation of fatty acids in liver and heart, causing death among male, but not to the same extent among female, PPAR-null mice given this drug (46). Our results indicate that a possible cause of better tolerance against etomoxir treatment among female PPAR-null mice could be a larger export of fatty acids from the liver as VLDL particles. Our finding that female PPAR-null mice had higher serum levels of ß-hydroxybutyrate than male PPAR-null mice indicates that female PPAR-null mice also have a larger capacity for ß-oxidation than male PPAR-null mice. In line with our study, it has been reported that male PPAR-null mice have lower ß-hydroxybutyrate levels than wild-type males in the fed state (9).

The 4 day cultures of hepatocytes indicated that the secretion of apoB and VLDL triglycerides is higher from female PPAR-deficient cells independent of the flux of metabolites in vivo. The secretion of both apoB-48 and apoB-100 was increased to a similar degree from PPAR-null hepatocytes, indicating that there was no effect on editing of apoB mRNA. The low total secretion of apoB-100 explains the very low and nearly undetectable apoB-100-VLDL secretion. We were therefore not able to show if the apoB-100-VLDL secretion was influenced. The increased VLDL triglyceride accumulation in the medium from PPAR-null hepatocytes is in line with the increased apoB-48-VLDL secretion and strengthens the importance of PPAR for normal apoB-VLDL secretion from hepatocytes.

We tested the effect of high fat diet in mouse on pure Sv129 background and in mice on Sv/129 x C57BL/6 genetic background because different mouse strains have been found to respond differently to a high fat diet (33) (34). The response to a high fat diet on serum apoB levels was similar between the genotypes, but the magnitude of the increase in serum apoB differed. This finding indicates that the genetic background of the mice does not determine the effect of PPAR deficiency on serum apoB levels after fat feeding. We also showed that the increased total serum cholesterol after fat feeding of PPAR-null mice was due to an increased LDL cholesterol level because LDL, but not VLDL or HDL, cholesterol differed between the PPAR-null mice and wild-type mice after fat feeding. In a recent paper by Tordjman et al., fat fed male PPAR-null mice on an apoE-deficient background showed an increased hepatic triglyceride secretion rate and unchanged VLDL clearance compared with fat fed wild-type mice on apoE-deficient background (47). Our data extends these findings by showing that PPAR-deficient mice are more sensitive to high fat diet in terms of serum apoB and LDL cholesterol levels in the presence of apoE expression.

We showed that variation in the expression of PPAR does not influence hepatic LDL receptor expression in males or females or during manipulation of the dietary intake of fat. These results indicate that changed expression of the LDL receptor is not responsible for the effect of PPAR deficiency on serum apoB levels. In line with our results, male PPAR-null mice have been shown to have similar LDL receptor mRNA levels as wild-type mice (48). Moreover, fibrate treatment of rat hepatocytes did not influence LDL receptor mRNA levels (49).

In summary, these results indicate that PPAR expression is of large importance for serum levels of apoB. The increase in serum levels of apoB was associated with increased VLDL triglyceride levels in female mice on ordinary chow and associated with increased LDL cholesterol levels in fat fed male mice. Hepatocytes from female PPAR-deficient mice showed higher apoB and VLDL secretion, indicating that the increased secretion of triglycerides in female mice lacking PPAR is not a result of increased flux of fatty acids to the liver.

	ACKNOWLEDGMENTS

We thank Professor F. J. Gonzalez at NIH, Bethesda, for providing the PPAR-null mice and Professor Joacim Herz at the University of Texas, Dallas, for providing LDL receptor antibodies. In addition, we thank Dr. Lennart Svensson and Anette Bergström at AstraZeneca R&D, Mölndal, Sweden, and Dr. Jan Borén and Christofer Flood at Wallenberg Laboratory, Sahlgrenska University Hospital, Göteborg, Sweden, for excellent technical assistance. This work was supported by grant 8269 from the Swedish Medical Research Council, the Novo Nordisk Foundation, Åke Wibergs Foundation, Magnus Bergvalls Foundation, King Gustav V and Queen Victoria's Foundation and AstraZeneca R&D.

Manuscript received May 7, 2001; and in revised form July 10, 2001

Abbreviations: apoB, apolipoprotein B; apoB-48, apolipoprotein B-48; apoB-100, apolipoprotein B-100; FPLC, fast protein liquid chromatography; MTP, microsomal triglyceride transfer protein; PPAR, peroxisome proliferator activated receptor

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

1. Schoonjans, K., Staels, B., Auwerx, J. 1996. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J. Lipid Res. 37:907-925[Abstract].

2. Fruchart, J. C., Duriez, P., Staels, B. 1999. Peroxisome proliferator-activated receptor-alpha activators regulate genes governing lipoprotein metabolism, vascular inflammation and atherosclerosis. Curr. Opin. Lipidol. 10:245-257[Medline].

3. Staels, B., Dallongeville, J., Auwerx, J., Schoonjans, K., Leitersdorf, E., Fruchart, J. C. 1998. Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation. 98:2088-2093[Abstract/Free Full Text].

4. Issemann, I., Green, S. 1990. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature. 347:645-650[Medline].

5. Kliewer, S. A., Forman, B. M., Blumberg, B., Ong, E. S., Borgmeyer, U., Mangelsdorf, D. J., Umesono, K., Evans, R. M. 1994. Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc. Natl. Acad. Sci. USA. 91:7355-7359[Abstract/Free Full Text].

6. Braissant, O., Foufelle, F., Scotto, C., Dauca, M., Wahli, W. 1996. Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-, -ß, and - in the adult rat. Endocrinology. 137:354-366[Abstract].

7. Kliewer, S. A., Sundseth, S. S., Jones, S. A., Brown, P. J., Wisely, G. B., Koble, C. S., Devchand, P., Wahli, W., Willson, T. M., Lenhard, J. M., Lehmann, J. M. 1997. Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors and . Proc. Natl. Acad. Sci. USA. 94:4318-4323[Abstract/Free Full Text].

8. Lee, S. S., Pineau, T., Drago, J., Lee, E. J., Owens, J. W., Kroetz, D. L., Fernandez-Salguero, P. M., Westphal, H., Gonzalez, F. J. 1995. Targeted disruption of the isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol. Cell. Biol. 15:3012-3022[Abstract].

9. Kersten, S., Seydoux, J., Peters, J. M., Gonzalez, F. J., Desvergne, B., Wahli, W. 1999. Peroxisome proliferator-activated receptor mediates the adaptive response to fasting. J. Clin. Invest. 103:1489-1498[Medline].

10. Leone, T. C., Weinheimer, C. J., Kelly, D. P. 1999. A critical role for the peroxisome proliferator-activated receptor (PPAR) in the cellular fasting response: the PPAR-null mouse as a model of fatty acid oxidation disorders. Proc. Natl. Acad. Sci. USA. 96:7473-7478[Abstract/Free Full Text].

11. Le May, C., Pineau, T., Bigot, K., Kohl, C., Girard, J., Pégorier, J-P. 2000. Reduced hepatic fatty acid oxidation in fasting PPAR-null mice is due to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression. FEBS Lett. 475:163-166[Medline].

12. Aoyama, T., Peters, J. M., Iritani, N., Nakajima, T., Furihata, K., Hashimoto, T., Gonzalez, F. J. 1998. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor (PPAR). J. Biol. Chem. 273:5678-5684[Abstract/Free Full Text].

13. Peters, J. M., Hennuyer, N., Staels, B., Fruchart, J. C., Fievet, C., Gonzalez, F. J., Auwerx, J. 1997. Alterations in lipoprotein metabolism in peroxisome proliferator-activated receptor -deficient mice. J. Biol. Chem. 272:27307-27312[Abstract/Free Full Text].

14. Costet, P., Legendre, C., Moré, J., Edgar, A., Galtier, P., Pineau, T. 1998. Peroxisome proliferator-activated receptor -isoform deficiency leads to progressive dyslipidemia with sexually dimorphic obesity and steatosis. J. Biol. Chem. 273:29577-29585[Abstract/Free Full Text].

15. Vu-Dac, N., Schoonjans, K., Kosykh, V., Dallongeville, J., Fruchart, J. C., Staels, B., Auwerx, J. 1995. Fibrates increase human apolipoprotein A-II expression through activation of the peroxisome proliferator-activated receptor. J. Clin. Invest. 96:741-750.

16. Milosavljevic, D., Griglio, S., Le Naour, G., Chapman, M. J. 2001. Preferential reduction of very low density lipoprotein-1 particle number by fenofibrate in type IIB hyperlipidemia: consequences for lipid accumulation in human monocyte-derived macrophages. Atherosclerosis. 155:251-260[Medline].

17. Kesäniemi, Y. A., Grundy, S. M. 1984. Influence of gemfibrozil and clofibrate on metabolism of cholesterol and plasma triglycerides in man. J. Am. Med. Assoc. 251:2241-2246[Abstract].

18. Lamb, R. G., Koch, J. C., Bush, S. R. 1993. An enzymatic explanation of the differential effects of oleate and gemfibrozil on cultured hepatocyte triacylglycerol and phosphatidylcholine biosynthesis and secretion. Biochim. Biophys. Acta. 1165:299-305[Medline].

19. Skrede, S., Bremer, J., Berge, R. K., Rustan, A. C. 1994. Stimulation of fatty acid oxidation by a 3-thia fatty acid reduces triacylglycerol secretion in cultured rat hepatocytes. J. Lipid Res. 35:1395-1404[Abstract].

20. Glickman, R. M., Rogers, M., Glickman, J. N. 1986. Apolipoprotein B synthesis by human liver and intestine in vitro. Proc. Natl. Acad. Sci. USA. 83:5296-5300[Abstract/Free Full Text].

21. Gibbons, G. F. 1990. Assembly and secretion of hepatic very-low-density lipoprotein. Biochem. J. 268:1-13[Medline].

22. Olofsson, S. O., Asp, L., Boren, J. 1999. The assembly and secretion of apolipoprotein B-containing lipoproteins. Curr. Opin. Lipidol. 10:341-346[Medline].

23. Ginsberg, H. N. 1995. Synthesis and secretion of apolipoprotein B from cultured liver cells. Curr. Opin. Lipidol. 6:275-280[Medline].

24. Lindén, D., Sjöberg, A., Asp, L., Carlsson, L., Oscarsson, J. 2000. Direct effects of growth hormone on production and secretion of apolipoprotein B from rat hepatocytes. Am. J. Physiol. Endocrinol. Metab. 279:E1335-E1346[Abstract/Free Full Text].

25. Sjöberg, A., Oscarsson, J., Olofsson, S. O., Edén, S. 1994. Insulin-like growth factor-I and growth hormone have different effects on serum lipoproteins and secretion of lipoproteins from cultured rat hepatocytes. Endocrinology. 135:1415-1421[Abstract].

26. Oscarsson, J., Olofsson, S. O., Bondjers, G., Edén, S. 1989. Differential effects of continuous versus intermittent administration of growth hormone to hypophysectomized female rats on serum lipoproteins and their apoproteins. Endocrinology. 125:1638-1649[Abstract].

27. Li, X., Catalina, F., Grundy, S. M., Patel, S. 1996. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48- relative to B-100-containing lipoproteins. J. Lipid. Res. 37:210-220[Abstract].

28. Hedin, L., Gaddy-Kurten, D., Kurten, R., DeWitt, D. L., Smith, W. L., Richards, J. S. 1987. Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and evidence for hormonal induction preceding ovulation. Endocrinology. 121:722-731[Abstract].

29. Carlsson, L., Nilsson, I., Oscarsson, J. 1998. Hormonal regulation of liver fatty acid-binding protein in vivo and in vitro: effects of growth hormone and insulin. Endocrinology. 139:2699-2709[Abstract/Free Full Text].

30. Sjöberg, A., Oscarsson, J., Boström, K., Innerarity, T. L., Edén, S., Olofsson, S. O. 1992. Effects of growth hormone on apolipoprotein-B (apoB) messenger ribonucleic acid editing, and apoB 48 and apoB 100 synthesis and secretion in the rat liver. Endocrinology. 130:3356-3364[Abstract].

31. Bligh, E. G., Dyer, W. J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917.

32. Labarca, C., Paigen, K. 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344-352[Medline].

33. Rebuffé-Scrive, M., Surwit, R., Feinglos, M., Kuhn, C., Rodin, J. 1993. Regional fat distribution and metabolism in a new mouse model (C57BL/6J) of non-insulin-dependent diabetes mellitus. Metabolism. 42:1405-1409[Medline].

34. Surwit, R. S., Feinglos, M. N., Rodin, J., Sutherland, A., Petro, A. E., Opara, E. C., Kuhn, C. M., Rebuffé-Scrive, M. 1995. Differential effects of fat and sucrose on the development of obesity and diabetes in C57BL/6J and A/J mice. Metabolism. 44:645-651[Medline].

35. Brown, M. S., Goldstein, J. L. 1986. A receptor-mediated pathway for cholesterol homeostasis. Science. 232:34-47[Free Full Text].

36. van Driel, I. R., Davis, C. G., Goldstein, J. L., Brown, M. S. 1987. Self-association of the low density lipoprotein receptor mediated by the cytoplasmic domain. J. Biol. Chem. 262:16127-16134[Abstract/Free Full Text].

37. Vohl, M-C., Lepage, P., Gaudet, D., Brewer, C. G., Bétard, C., Perron, P., Houde, G., Cellier, C., Faith, J. M., J-P. Després, K., Morgan,, Hudson, T. J. 2000. Molecular scanning of the human PPAR gene: association of the L162V mutation with hyperapobetalipoproteinemia. J. Lipid Res. 41:945-952[Abstract/Free Full Text].

38. Flavell, D. M., Pineda Torra, I., Jamshidi, Y., Evans, D., Diamond, J. R., Elkeles, R. S., Bujac, S. R., Miller, G., Talmud, P. J., Staels, B., Humphries, S. E. 2000. Variation in the PPAR gene is associated with altered function in vitro and plasma lipid concentrations in Type II diabetic subjects. Diabetologia. 43:673-680[Medline].

39. Lacquemant, C., Lepretre, F., Pineda Torra, I., Manraj, M., Charpentier, G., Ruiz, J., Staels, B., Froguel, P. 2000. Mutation screening of the PPAR gene in type 2 diabetes associated with coronary heart disease. Diabetes Metab. 26:393-401[Medline].

40. Tugwood, J. D., Aldridge, T. C., Lambe, K. G., Macdonald, N., Woodyatt, N. J. 1996. Peroxisome proliferator-activated receptors: structures and function. Ann. NY. Acad. Sci. 804:252-265[Abstract].

41. Patsch, W., Kim, K., Wiest, W., Schonfeld, G. 1980. Effects of sex hormones on rat lipoproteins. Endocrinology. 107:1085-1094[Medline].

42. Watkins, M. L., Fizette, N., Heimberg, M. 1972. Sexual influences on hepatic secretion of triglyceride. Biochim. Biophys. Acta. 280:82-85[Medline].

43. Elam, M. B., Umstot, E. S., Andersen, R. N., Solomon, S. S., Heimberg, M. 1987. Deprivation and repletion of androgen in vivo modifies triacylglycerol synthesis by rat hepatocytes. Biochim. Biophys. Acta. 921:531-540[Medline].

44. Keller, H., Givel, F., Perroud, M., Wahli, W. 1995. Signaling cross-talk between peroxisome proliferator-activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements. Mol. Endocrinol. 9:794-804[Abstract].

45. Nunez, S. B., Medin, J. A., Braissant, O., Kemp, L., Wahli, W., Ozato, K., Segars, J. H. 1997. Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor. Mol. Cell. Endocrinol. 127:27-40[Medline].

46. Djouadi, F., Weinheimer, C. J., Saffitz, J. E., Pitchford, C., Bastin, J., Gonzalez, F. J., Kelly, D. P. 1998. A gender-related defect in lipid metabolism and glucose homeostasis in peroxisome proliferator-activated receptor -deficient mice. J. Clin. Invest. 102:1083-1091[Medline].

47. Tordjman, K., Bernal-Mizrachi, C., Zemany, L., Weng, S., Feng, C., Zhang, F., Leone, T. C., Coleman, T., Kelly, D. P., Semenkovich, C. F. 2001. PPAR deficiency reduces insulin resistance and atherosclerosis in apoE-null mice. J. Clin. Invest. 107:1025-1034[Medline].

48. Patel, D. D., Knight, B. L., Wiggins, D., Humphreys, S. M., Gibbons, G. F. 2001. Disturbances in the normal regulation of SREBP-sensitive genes in PPAR-deficient mice. J. Lipid. Res. 42:328-337[Abstract/Free Full Text].

49. Raspe, E., Madsen, L., Lefebvre, A. M., Leitersdorf, I., Gelman, L., Peinado-Onsurbe, J., Dallongeville, J., Fruchart, J. C., Berge, R., Staels, B. 1999. Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPAR activation. J. Lipid Res. 40:2099-2110[Abstract/Free Full Text].

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