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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:1219-1227 (2007)
© 2007 The Gerontological Society of America

Hypertriglyceridemia and Hepatic Steatosis in Senescence-Accelerated Mouse Associate to Changes in Lipid-Related Gene Expression

Laia Vilà, Núria Roglans, Marta Alegret, Antoni Camins, Mercè Pallàs, Rosa María Sánchez, Manuel Vázquez-Carrera and Juan Carlos Laguna

Pharmacology Unit, Department of Pharmacology and Therapeutic Chemistry, School of Pharmacy, University of Barcelona and IBUB (Institute of Biomedicine University of Barcelona), Spain.

Address correspondence to Juan Carlos Laguna, PhD, Unitat de Farmacologia, Facultat de Farmacia, Avda Diagonal 643, Barcelona 08028, Spain. E-mail: jclagunae{at}ub.edu


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Aged rodents show increasing plasma and tissue triglycerides, and reductions in liver peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and its target genes. We determined whether a similar situation is present in a model of accelerated aging, the senescence-accelerated prone (SAM-P8) mouse. Five-month-old SAM-P8 mice were hypertriglyceridemic, and exhibited hepatic steatosis and reduced fatty acid oxidation versus control 5-month-old senescence-accelerated resistant (SAM-R1) mice, with no differences in PPAR{alpha} expression and binding activity; in fact, fenofibrate administration to SAM-P8 mice induced a clear PPAR{alpha}-driven response. Complementary DNA (cDNA) microarray analysis (Affymetrix Mouse Genome 430A 2.0 GeneChip array), Western blot, and electrophoretic mobility shift assay (EMSA) experiments indicated, among other changes, a deficit in farnesoid X receptor (FXR) expression and binding activity in the livers of SAM-P8 mice with respect to SAM-R1 controls. Triglyceride accretion and a deficit in hepatic fatty acid oxidation, features of the aging process in mammals, associate to a deficit in hepatic FXR activity in the SAM-P8 mice.

THE clustering of dyslipidemia, obesity, type II diabetes, and hypertension increases the incidence of cardiovascular diseases among the elderly population (1). The prevalence of this disease cluster, referred to as the metabolic syndrome, in the U.S. adult population has increased from 6.7% among participants aged 20–29 years to 43.5% for those aged 60–69 years (2); this latter age group contains the cutoff point for advanced age (65 years) in humans (3).

Aged mammals show increasing plasma concentrations of lipids (4). This increases mainly stem from a decrease in the oxidative capacity of body tissues (5). At present, however, the mechanism underlying age-related changes in fat oxidation remain unclear.

Old rats develop metabolic alterations commonly found in aged humans. The incidence of dyslipidemia, leptin resistance-associated obesity, and a progressive failure of insulin-mediated metabolism is common to both aged rats and humans (6). Eighteen-month-old animals are considered at the cutoff point for advanced age in rats (3). We have shown that 18-month-old Sprague-Dawley rats display a set of phenotypic characteristics related to lipid metabolism that are common to humans exhibiting the metabolic syndrome. These alterations are related to a reduction in the expression and activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and several of its target genes in the livers of old animals (7). These changes have a marked effect in the responsiveness of older rats to well-known drugs affecting lipid metabolism (8–10).

We have investigated whether the above-mentioned age-related changes in metabolism were exhibited by another experimental model of aging, the senescence-accelerated mouse (SAM). The SAM model was established through phenotypic selection, based on the degree of senescence, the life span, and the age-associated pathologic phenotypes, from a common genetic pool of AKR/J strain mice. The senescence-accelerated resistant (SAM-R) series (substrains SAM-R1, 4, and 5) exhibit normal aging characteristics, whereas the senescence-accelerated prone (SAM-P) series (substrains SAM-P1–3 and 6–11) display accelerated aging including loss of skin glossiness, increased skin coarseness, hair loss, periophthalmic lesions, and increased lordokyphosis of the spine (11). In this study, we used 5-month-old SAM-P8 mice, which are characterized by both accelerated aging and neuronal dysfunctions (12) appearing from age 2 to 8 months. Life span of SAM-P8 mice ranges from 10 to 17 months, whereas SAM-R1 mice life span ranges from 19 to 21 months, showing normal aging with an increased propensity to nonthymic lymphoma, histiocytic sarcoma, and ovarian cyst. Our data demonstrate that 5-month-old SAM-P8 mice, similar to 18-month-old Sprague-Dawley rats, exhibit hypertriglyceridemia and hepatic steatosis, both related to a marked reduction in hepatic fatty acid ß-oxidation, compared to SAM-R1 controls. Nevertheless, complementary DNA (cDNA) microarray, Western blot, and binding activity data indicate that this metabolic disturbance is related to a deficit in farnesoid X receptor (FXR), rather than to a deficit in PPAR{alpha} activity.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals and Experimental Design
Five male SAM-P8 and five male SAM-R1 mice were bred under standard conditions with free access to food and water. After 5 months, mice were killed under pentobarbitone anesthesia at 10 AM. Ten male SAM-P8 mice aged 5 months were randomly assigned to two treatment groups: a control group (SAM-P8) fed a standard diet, and a fenofibrate group (FFB), fed standard diet supplemented with fenofibrate (500 mg FFB/kg/day). After 7 days, the animals were killed as describe above. Animal handling and disposal were performed in accordance with law 5/1995, 21 July, from the Generalitat de Catalunya.

Sample Preparations
Blood was collected in 5% EDTA tubes; plasma was obtained by centrifugation and stored at –80°C until needed. Livers were excised and perfused in 0.9% NaCl. Liver tissue (150 mg) from each animal was homogenized in a 150 mM NaCl, 1 mM dithiothreitol, 30 mM EDTA, 50 mM KH2PO4, pH 7.4, buffer to obtain the postnuclear supernatant fraction. Liver tissue (10–100 mg) was immediately frozen in liquid N2 and stored at –80°C until used for total RNA extraction. Two additional samples (150 mg) were stored at –80°C for quantifying liver lipids and obtaining the nuclear protein extracts. Nuclear extracts were isolated using the Helenius method (13). The protein concentration of each fraction was determined by the Bradford method (14).

Lipids, Glucose, and Insulin Determination
Plasma triglyceride, nonesterified fatty acids (NEFA), total cholesterol, and glucose concentrations were measured by using the colorimetric tests triglyceride L-type, NEFAC, Chol-H L-type, and Glucose kit, respectively, from Wako Chemicals (Neuss, West Germany). Plasma insulin concentration was determined using the Rat Insulin RIA kit (RPA547) from Amersham Biosciences Europe (Freiburg, Germany). Liver lipids were extracted and measured as described previously, using the homogenate fraction (15).

Enzyme Assays
Hepatic fatty acid ß-oxidation activity was determined as described previously (16), using 30 µg of postnuclear supernatant from each sample.

RNA Preparation and Semiquantitative Reverse Transcription–Polymerase Chain Reaction
Total RNA was isolated from liver tissue by using TRIZOL (Invitrogen, Carlsbad, CA), purified by using an RNeasy kit (Qiagen, Valencia, CA), quantified by spectrometry at 260 nm, and its purity was assessed by the absorbance ratio at 260–280 nm. RNA integrity was assessed by 1% agarose gel electrophoresis.

The relative levels of specific messenger RNAs (mRNAs) were assessed by reverse transcription–polymerase chain reaction (RT–PCR), basically as described previously (8). The sequences of the sense and antisense primers used for amplification, the cycle numbers, and the expected size were as follows: PPAR{alpha}, 5'-GGCTCGGAGGGCTCTGTCATC-3' and 5'-ACATGCACTGGCAGCAGTGGA-3' (21 cycles; 645 bp), liver–carnitine palmitoyltransferase-I (L-CPT-I), 5'-TATGTGAGGATGCTGCTT-3' and 5'-CTCGGAGAGCTAAGCTTG-3' (28 cycles; 629 bp), acyl-coenzyme A (CoA) oxidase (ACO), 5'-GGCATCGCAGACCCTGAAGAA-3' and 5'-GCAGTGGTTTCCAAGCCTCGA-3' (18 cycles; 205 bp). Adenosyl phosphoribosyltransferase (APRT), 5'-GCCTCTTGGCCAGTCACCTGA-3' and 5'-CCAGGCTCACACACTCCACCA-3' (21 cycles; 339 bp) was used as an internal control and coamplified with target sequences in the same tube. The mRNA levels were always expressed as the ratio to APRT mRNA levels.

Arrays
Six total RNA pools were prepared, three for SAM-P8 mice and three for SAM-R1 mice. Each pool was prepared with the same amount of RNA from two different SAM mice. The RNA pools were used in tandem with microarray technology to analyze gene expression profile (Progenika Biopharma S.A.). cDNA was prepared by reverse transcription, whereas biotinylated RNA was prepared according to the Affymetrix protocol (Affymetrix, Santa Clara, CA). Labeled cRNA was purified using the GeneChip Sample Cleanup Module from Affymetrix, fragmented, and hybridized to an Affymetrix Mouse Genome 430A 2.0 GeneChip array. All six hybridized arrays were considered high enough quality for further analytical use based on the presence of spike controls as well as the 3'/5' sequence ratio of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Array Data Processing and Analysis
GeneChip arrays were washed and scanned using a GeneArray Scanner, and quantified according to Affymetrix standard procedures to obtain a signal intensity for every gene in each array. Using Affymetrix software GCOS 1.1, the mean intensity of each array was calculated, with the arrays linearly scaled to an average expression level of 100. Signals were normalized by using GeneSpring 7.1 software by dividing each gene by the median of its measurements in each sample.

To identify those genes that varied significantly in SAM-P8 versus SAM-R1 mice, two methods were applied: (i) a comparison algorithm using the GCOS 1.1 software (pairwise comparisons [nine in total] were performed, and only sequences overexpressed or repressed in at least 7 of the 9 comparisons were selected), and (ii) a parametric analysis of variance (ANOVA) test, carried out using the GeneSpring 7.1 software, whereby only the changes with a p value ≤.05 were considered relevant. By applying these methods, two different lists of genes were generated; only those genes appearing in both lists were regarded as differentially expressed.

Real-Time RT-PCR
To confirm the expression patterns of upregulated or downregulated genes, we chose several genes for further analysis by using relative quantitative real-time RT–PCR in a 96-well format. cDNA from the same RNA samples used in microarray experiments was synthesized as described previously (8). Real-time RT–PCR was performed by the Perkin-Elmer ABI Prism 7700 sequence detection system, with the TaqMan Universal PCR Master Mix, PCR primers, and TaqMan probes obtained from TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). Primers for GAPDH were amplified in parallel with the genes of interest. All samples were run in triplicate. Sequence detector software (S.D.S. 1.9.1) was used for data analysis. A threshold cycle value (CT) was obtained for each amplification plot, and after normalization by the reference gene GAPDH, the relative expression ratio for each gene was calculated, based on the difference between the mean CT of a sample and the corresponding control value.

Electrophoretic Mobility Shift Assays
The consensus oligonucleotide for the farnesoid-X response element (FXRE; 5'-GATCTCAAGAGGTCATTGACCTTTTTG-3') and the peroxisome proliferator response element (PPRE) L-CPT-I probe (5'-AGTACGGGCATGGAGCAAAGAGCT-3'; nucleotides –266 to –290 of the rat L-CPT-I gene) were obtained from annealing single-stranded complementary oligonucleotides. Electrophoretic mobility shift assays (EMSAs) were performed exactly as described previously (17). Antibodies against PPAR{alpha}, retinoic-X-receptor {alpha} (RXR{alpha}), FXR, and octamer motif-1 transcription factor (Oct-1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Western Blot Analysis
Thirty micrograms of liver crude nuclear extract were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After transfer, membranes were then incubated with the primary polyclonal antibody raised against PPAR{alpha} (dilution 1:1000) and FXR (dilution 1:500) in Tris-buffered saline (TBS)-0.1% Tween-20 with 5% nonfat milk at 4°C overnight. After several washes, they were incubated with horseradish peroxidase (HRP)-conjugated antirabbit immunoglobulin G (IgG; 1:3000 dilution). Detection was achieved using the enhanced chemiluminescence (ECL) kit for HRP (Amersham Biosciences). To confirm the uniformity of protein loading in each lane, the blots were stained with Ponceau S (18). The size of detected proteins was estimated using protein molecular mass standards (Invitrogen, Life Technologies). All antibodies were obtained from Santa Cruz Technologies.

Statistical Analysis
The results are expressed as the mean of n values ± standard deviation. Plasma samples were assayed in duplicate. Significant differences were established by the unpaired t test (V2.03; GraphPad Software, San Diego, CA). When appropriate, a nonparametric test was performed (Mann–Whitney U test). The level of statistical significance was set at p <.05.


   RESULTS
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
SAM-P8 Mice Are Hypertriglyceridemic and Exhibit Liver Steatosis and a Deficit in Hepatic Fatty Acid Oxidation
Five-month-old SAM-P8 mice had a 68% higher concentration of plasma triglycerides, an 86% higher concentration of liver triglycerides, and a 42% reduction in the hepatic fatty acid ß-oxidation activity. In addition, they were heavier than controls (19.5% increase in body weight), while having an ingestion of pelleted diet similar to SAM-R1 mice (6.4 ± 1.2 vs 6.3 ± 0.7 g/day, respectively). No other aspects related to plasma or liver tissue were found to differ significantly between SAM-P8 and SAM-R1 mice (Table 1).


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  		Table 1. Body Weight, Plasma, and Hepatic Tissue Analytes and Hepatic ß-Oxidation Activity.

 
SAM-P8 Mice Show no Sign of Altered Hepatic PPAR{alpha} Activity
Phenotypic changes presented in SAM-P8 mice (i.e., hypertriglyceridemia, hepatic accretion of triglycerides, and reduction in liver fatty acid catabolism), were strikingly similar to those presented by 18-month-old male Sprague-Dawley rats, as we have previously reported (8). As those changes in the latter were related to a marked reduction in hepatic PPAR{alpha} activity, we investigated whether a similar situation was present in SAM-P8 mice. The levels of specific mRNAs corresponding to PPAR{alpha} target genes, such as PPAR{alpha} itself (19), L-CPT-I and ACO (20), remained unchanged in liver samples from SAM-P8 mice, when compared to control SAM-R1 mice (Figure 1). Furthermore, hepatic PPAR{alpha} protein levels also remained unchanged between SAM-P8 and SAM-R1 mice (Figure 1). We assayed the binding of PPAR{alpha} present in liver nuclear extracts to an oligonucleotide containing the specific PPRE found in the promoter of L-CPT-I, obtaining two specific bands (I and II), which were completely competed in the presence of an excess of unlabeled oligonucleotide (Figure 2A). Both bands contained RXR{alpha}, as they were either supershifted (band II) or disappeared (band I) in the presence of a specific RXR{alpha} antibody. Band I also contained PPAR{alpha}, as shown by its complete disappearance in the presence of a PPAR{alpha} antibody (Figure 2A). When comparing the intensity of bands I and II between hepatic samples from SAM-R1 and SAM-P8 mice, we noted a marked decrease in the intensity of band II, but observed no change in band I intensity, which contained the RXR{alpha}-PPAR{alpha} heterodimer (Figure 2B). We have no explanation for the reduction in band II intensity, but our data indicate that the proteins involved in the formation of band II are not determinant in L-CPT-I expression, given that there was no difference in L-CPT-I mRNA levels between SAM-R1 and SAM-P8 samples.


Figure 01
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  		Figure 1. Relative levels of peroxisome proliferator-activated receptor {alpha} (PPAR {alpha}) messenger RNA (mRNA) (A), and protein (B), and liver–carnitine palmitoyltransferase-I (L-CPT-I) (C) and acyl-coenzyme A (CoA) oxidase (ACO) (D) mRNAs in samples from senescence-accelerated resistant (SAM-R1) (white bar) and senescence-accelerated prone (SAM-P8) (black bar) mice. Each bar represents the mean ± standard deviation (SD) of values from five animals

 

Figure 02
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  		Figure 2. A, Electrophoretic mobility shift assay (EMSA) showing the binding of senescence-accelerated resistant (SAM-R1) mouse hepatic nuclear extracts (NE) to a liver–carnitine palmitoyltransferase-I (L-CPT-I) peroxisome proliferator response element (PPRE) oligonucleotide, forming at least two specific bands (bands I and II). Oct-1 antibody was used to demonstrate that the changes in the intensity of shifted bands produced by the specific peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and retinoic-X-receptor {alpha} (RXR{alpha}) antibodies were not due to unspecific interference by the presence of immunoglobulin proteins in the incubation medium. B, Representative EMSA autoradiography showing the specific bands I and II formed with hepatic NEs from four SAM-R1 and four senescence-accelerated prone (SAM-P8) mice

 
Eighteen-month-old Sprague-Dawley rats are resistant to the hypolipidemic effect of fibrate drugs, a well-known class of PPAR{alpha} agonists (8). To confirm that SAM-P8 mice had no impairment in their hepatic PPAR{alpha} system, we treated them with a fibrate drug, fenofibrate (500 mg/kg/day, 7 days), and evaluated the following markers of PPAR{alpha} activity in rodents: the percentage of liver weight to body weight (LW/BW); plasma triglyceride, liver tissue triglyceride, and cholesterol concentrations; level of L-CPT-I–specific mRNA; and the hepatic activity of the fatty acid ß-oxidation system (8,21). Fenofibrate administration increased by 2.1-, 2.4-, and 2.5-fold the LW/BW, the liver fatty acid ß-oxidation activity, and the specific mRNA for L-CPT-I, respectively, and decreased by 54%, 50%, and 42% the concentration of plasma triglycerides, liver triglycerides, and cholesterol, respectively, confirming that PPAR{alpha} was fully functional in the livers of SAM-P8 mice (Table 2).


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  		Table 2. Plasma and Hepatic Analytes in FFB-Treated Mice.

 
SAM-P8 Mice Exhibit a Significant Proportion of Differentially Expressed Genes Related to Lipid Metabolism
Livers from 5-month-old SAM-P8 mice presented 89 differentially expressed genes vis-à-vis control SAM-R1 mice; 52 were upregulated (58%) and 37 were downregulated (42%). A significant proportion (26%) was related to lipid metabolism, with 10 genes upregulated (11%) and 13 genes downregulated (15%) in SAM-P8 mice (Tables 3 and 4, respectively).


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  		Table 3. Differentially Upregulated Genes Related to Lipid Metabolism in Livers of 5-Month-Old SAM-P8 Versus SAM-R1 Mice.

 

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  		Table 4. Differentially Downregulated Genes Related to Lipid Metabolism in Livers of 5-Month-Old SAM-P8 Versus SAM-R1 Mice.

 
To validate our results, several genes were selected for analysis by real-time PCR on the basis of their fold-change and/or biological relevance. Comparative results between real-time PCR and array data are shown in Figure 3.


Figure 03
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  		Figure 3. Validation of differentially expressed genes in hepatic senescence-accelerated prone (SAM-P8) mice samples compared to control senescence-accelerated resistant (SAM-R1) samples. Comparison of results obtained by Affymetrix array (black bars) and real-time polymerase chain reaction (PCR) (Taqman) (white bars). Values are expressed as percentage of increase or decrease in gene expression compared to the corresponding control. SHP, small heterodimerization partner; FXR, farnesoid X receptor; STARD5, StAR-related lipid transfer (START) domain containing 5; mCAR, mouse constitutive androstane receptor; ECH, enoy-CoA hydratase; ELOvl3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; INSIG2, insulin-induced gene 2; ADRP, adipose differentiation-related protein (adipophilin)

 
Of the three upregulated genes selected, namely adipose differentiation-related protein or adipophilin (ADRP), elongation of very long-chain fatty acids (ElovI3), and insulin-induced gene 2 (INSIG2), real-time PCR data proved practically identical to the array data: ADRP (2.3- and 2.1-fold change, respectively); ElovI3 (8.8- and 8.3-fold change, respectively); upregulation of INSIG2, however, was not confirmed (0.8- and 5.7-fold change, respectively). Of the five downregulated genes selected, namely mouse constitutive androstane receptor (mCAR), enoyl-CoA hydratase, FXR, StAR-related lipid transfer domain (StARD5), and syndecan-1, fold change values obtained in the array assay were confirmed by real-time PCR data.

Decreased FXR Protein and Binding Activity in Livers of 5-Month-Old SAM-P8 Mice
As FXR activity lowers plasma triglyceride levels and hepatic triglyceride accretion (22,23), we were interested in determining whether the decreased FXR-specific mRNA levels detected in the livers of SAM-P8 mice (see Figure 3) were related to a decreased FXR protein expression and binding activity. FXR protein was significantly reduced (30%) in liver samples from SAM-P8 mice (Figure 4). Liver nuclear extracts from SAM-R1 mice incubated in the presence of a specific FXRE oligonucleotide produced at least two specific retardation bands (bands I and II) that disappeared in the presence of an excess of unlabeled FXRE oligonucleotide. Band I also contained FXR protein, as a supershifted band (IC, see Figure 4B) appeared when the nuclear extracts were coincubated with a specific FXR antibody. When we compared the retarded band pattern between liver nuclear extracts from SAM-R1 and SAM-P8 mice (Figure 4C), bands I and II proved almost undetectable in retardation assays performed with SAM-P8 mice samples, in agreement with a reduction of FXR expression and activity in these animals. Furthermore, the specific mRNA for syndecan-1 and SHP (small heterodimerization partner), two genes the expression of which is under FXR control, were also significantly reduced in SAM-P8 livers (see Figure 3).


Figure 04
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  		Figure 4. A, Bar graph showing the level of farnesoid X receptor (FXR) protein in hepatic samples from senescence-accelerated resistant (SAM-R1) (white bar), and senescence-accelerated prone (SAM-P8) (black bar) mice. Each bar represents the mean ± standard deviation (SD) of values from five animals. Left: Representative Western blot shows the peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) bands corresponding to three different SAM-R1 and SAM-P8 mice. The amount of protein loaded was confirmed by the Bradford method, and the uniformity of protein loading in each lane was assessed by staining the blots with Ponceau S. B, Electrophoretic mobility shift assay (EMSA) showing the binding of SAM-R1 hepatic nuclear extracts (NE) to an farnesoid X response element (FXRE) oligonucleotide, forming at least two specific bands (bands I and II). Oct-1 antibody was used to demonstrate that the changes in the intensity of shifted bands produced by the specific FXR antibody was not due to unspecific interference by the presence of immunoglobulin proteins in the incubation medium. C, Representative EMSA autoradiography showing the specific bands I and II formed with hepatic NEs from four SAM-R1 and four SAM-P8 mice

 

   DISCUSSION
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
A progressive increase in plasma and liver tissue concentrations of triglycerides appears to be a phenotypic trait of physiological aging in both humans and rodents (1,5,7). Our present data indicate that triglyceride accretion is also common to an experimental model of accelerated senescence, the SAM-P8 mouse, pointing to lipid metabolic deterioration as a key factor inherent to senescence. The accumulation of triglycerides in the liver tissues of SAM-P8 mice was accompanied by a 2-fold increase in the expression of adipophilin, an ubiquitously expressed lipid storage droplet-associated protein (24), further confirming that an active process of lipid accumulation was occurring in the livers of the senescence-prone mice. Similar to what we have reported previously for 18-month-old Sprague-Dawley rats (7,8), these lipid changes were present in SAM-P8 mice without any alteration in plasma glucose and insulin concentrations, and in the glucose to insulin ratio, indicating that the deterioration of liver lipid metabolism precedes and is probably the cause of the altered glucose metabolism and insulin resistance that appears with aging (3).

The increased burden of triglycerides in SAM-P8 mice probably originates from two processes. The first involves decreased clearance of circulating lipoproteins by hepatic cells. Liver samples of SAM-P8 mice showed a 50% reduction in syndecan-1 expression, a heparin sulfate proteoglycan highly expressed in the liver that binds to lipoprotein lipase and hepatic lipase, thereby contributing to the hepatic clearance of lipoprotein remnants (25). The second process is an increased production of liver triglycerides, driven by a significant decrease in fatty acid catabolism in senescence-prone mice. Most likely, the increased expression in the livers of SAM-P8 mice of ElovI3, an enzyme that adds two carbon units to fatty acid acyl chains (26), is related to the increased availability of fatty acids, substrate of ElovL3 activity, resulting from a reduction in their oxidative metabolism.

The lipid metabolic phenotype exhibited by 18-month-old Sprague-Dawley rats was clearly related to a marked reduction in PPAR{alpha} expression and activity (7). In the aged C57BL/6 mouse, changes in liver lipid metabolism are also associated with a decreased PPAR{alpha} activity (27). This was not the case for the lipid changes exhibited by the SAM-P8 mouse. Neither the expression nor the binding activity of PPAR{alpha} was significantly altered in the liver samples from SAM-P8 mice. Moreover, in contrast with the reported resistance of 18-month-old Sprague-Dawley rats to the hypolipidemic effects of fibrates, a well-known class of PPAR{alpha} agonists (8), SAM-P8 mice were responsive to the administration of fenofibrate, suggesting the absence of any liver PPAR{alpha} deficit in senescence-prone mice. Although the enoyl-CoA hydratase activity is not a rate-limiting step in fatty acid ß-oxidation (28), the marked reduction (80%) of its expression in the livers of SAM-P8 mice could explain the decrease in fatty acid oxidative activity. Nevertheless, livers of fenofibrate-treated SAM-P8 mice exhibited no change in enoyl-CoA hydratase expression (data not shown), despite showing a 2.4-fold increase in fatty acid oxidation vis-à-vis untreated SAM-P8 animals. This finding indicates that the remaining enoyl-CoA hydratase present in the livers of senescence-prone mice was sufficient to drive fatty acid oxidation activity. The lack of responsiveness of enoyl-CoA hydratase to fenofibrate could be related to the decreased expression (75%) of mCAR in the livers of SAM-P8 mice, given that the gene coding for this enzyme has a PPRE in its promoter region. Among other activities, mCAR binds to the PPRE present in the enoyl-CoA gene and controls its expression (29).

Besides PPAR{alpha}, several transcription factors participate in the control of fatty acid and lipid liver metabolism, including, e.g., hepatic nuclear factor 4, liver X receptor, RXR, and FXR (30). FXR has a strong influence on triglyceride metabolism; its activation has a pronounced hypotriglyceridemic effect, not only accelerating the plasma triglyceride-rich lipoprotein clearance via the increased expression of genes such as apo CII and syndecan-1, but also increasing the activity of the liver fatty acid ß-oxidation system (17,23,30,31). Our results indicate that, in SAM-P8 mice, the accumulation of plasma and liver triglycerides associate with a reduction in the expression (mRNA and protein) and binding activity of FXR. Although decreased binding in gel retardation assays is not a direct proof of reduced trans-activating activity, the decreased expression of syndecan-1 and SHP, two FXR-target genes, and FXR itself [when activated, FXR also promotes its own expression (32)], agrees with a deficit in FXR transcriptional activity. Bile acids are the endogenous activators of FXR (33); we detected a 50% reduction in StARD5 expression in the livers of SAM-R8 mice, a mitochondrial cholesterol transporter that controls the acidic pathway activity of bile acid synthesis (34), suggesting a possible bile acid deficit in these animals. Although we have no data on the actual liver content of bile acids in SAM-P8 mice, the lack of an appropriate bile acid level could explain the reduction in the hepatic expression and activity of FXR in senescence-prone mice.

Summary
Our data indicate that, although triglyceride accretion and a deficit in hepatic fatty acid oxidation appear to be common phenotypic features of the aging process in mammals, several possible age-related mechanisms are responsible for these traits, namely, a deficit in PPAR{alpha} expression and activity such as occurs in 18-month-old Sprague-Dawley rats, or a similar deficit in FXR expression and activity in SAM-P8 mice. It remains to be determined if the derangement of a common link, further down in the signaling pathway controlling fatty acid oxidation, is responsible for the age-related alteration of both lipid-related transcription factors.


   Acknowledgments
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by grants from FPCNL (Fundació Privada Catalana de Nutrició i Lípids), MCYT (Ministerio de Ciencia y Tecnología; SAF2004-03045), Fundación Ramón Areces, and European Community FEDER (Fondo Europeo de Desarrollo Regional) funds. Laia Vila was supported by a Research and Teaching Grant from the University of Barcelona.

We have been nominated as a Consolidated Research Group by the Generalitat de Catalunya (SGR05-00833), with no financial aid whatsoever.


   Footnotes
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 Discussion
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Decision Editor: Huber R. Warner, PhD

Received February 26, 2007

Accepted June 26, 2007


   References
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 Results
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