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Institute of Agricultural and Nutritional Sciences, Martin Luther University, D-06108 Halle (Saale), Germany
* To whom correspondence should be addressed. E-mail: klaus.eder{at}landw.uni-halle.de.
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ABSTRACT |
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. Furthermore, rats administered the oxidized oil had higher mRNA concentrations of Insig-1 and Insig-2a, a lower concentration of the mature SREBP-2 in the nucleus, lower mRNA concentrations of the SREBP-2 target genes 3-hydroxy-3-methylglutaryl CoA reductase and LDL receptor in their livers, and a lower concentration of cholesterol in liver, plasma, VLDL, and HDL than control rats (P < 0.05). In conclusion, this study shows that reduced cholesterol concentrations in liver and plasma of rats administered an oxidized oil were due to an inhibition of the activation of SREBP-2 by an upregulation of Insig, which in turn inhibited transcription of proteins involved in hepatic cholesterol synthesis and uptake.
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Introduction |
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and a reduced hepatic de novo fatty acid synthesis (3–5). The molecular mechanisms underlying the reduction of cholesterol concentrations in liver and plasma, however, have not yet been elucidated. Cholesterol homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP)3. SREBP belong to a large class of transcription factors containing basic helix-loop-helix-Zip domains, of which 3 isoforms have been characterized: SREBP-1a, -1c, and -2 (reviewed in 6,7). Whereas SREBP-1c, the predominant isoform in adult liver, preferentially activates genes required for fatty acid synthesis, SREBP-2 preferentially activates the LDL receptor gene and various genes required for cholesterol synthesis, such as 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase (6,7). SREBP-1a is an activator of both the cholesterol and fatty acid biosynthetic pathways, but it is present in much lower amounts in liver than the other 2 forms (8). After synthesis in membranes of the endoplasmic reticulum, SREBP form a complex with SREBP-cleavage activating protein (SCAP). When cells are depleted of sterols, SCAP escorts SREBP from the endoplasmic reticulum to the Golgi. Within the Golgi, 2 resident proteases, site-1 protease and site-2 protease, sequentially cleave the SREBP, release the amino-terminal basic helix-loop-helix-Zip-containing domain from the membrane, and allow it to translocate to the nucleus and activate transcription of their target genes. Recently, insulin-induced genes (Insig)-1 and -2 were identified as membrane proteins that reside in the endoplasmic reticulum and play a central role in the regulation of SREBP cleavage (9,10). When intracellular sterol concentrations are increased, SCAP binds to Insig, an action that prevents the translocation of the SREBP-SCAP complex from the endoplasmic reticulum to Golgi and the proteolytic activation of SREBP. As a result, the synthesis of cholesterol and fatty acids declines.
We recently observed in rats that activation of PPAR caused an upregulation of the expression of Insig-1 in the liver, which in turn inhibited proteolytic activation of SREBP-2 and lowered hepatic cholesterol synthesis and liver and plasma cholesterol concentrations (5). We and others have found that feeding an oxidized fat causes an activation of PPAR in the liver of rats or pigs and in rat fetuses (4,11–13). Therefore, we assume that oxidized fats affect cholesterol metabolism in a similar way as clofibrate did in our recent study. Our hypothesis is that the reduced concentrations of cholesterol in liver and plasma observed in rats fed an oxidized oil are mediated by an increased gene expression of Insig in the liver. An upregulation of Insig is expected to lower the concentration of the transcriptionally active SREBP-2 in the nucleus, which in turn leads to a reduced expression of its target genes involved in hepatic cholesterol synthesis (e.g. HMG-CoA reductase) and cholesterol uptake (LDL receptor) and explains reduced plasma and liver cholesterol concentrations. To proof this hypothesis, we performed an experiment with rats that were orally administered either a fresh or an oxidized oil. For an oxidized oil, we used an oil treated at a relatively low temperature over a long period, because such oils have high concentrations of primary lipid peroxidation products such as hydroxy- and hydroperoxy fatty acids, which are regarded as very potent PPAR agonists (14–16).
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Materials and Methods |
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Preparation of the oxidized oil. The thermoxidized oil was prepared by heating sunflower oil (from a local supermarket) in an electric fryer (Saro Gastro-Products) for 25 d by 60°C. Throughout the heating process, air was continuously bubbled through the oil. The extent of lipid peroxidation was determined by assaying the peroxide value (17), concentration of TBARS (18), concentration of conjugated dienes (19), acid values (17), the percentage of total polar compounds (20), and the concentration of total carbonyls (21). The fatty acid composition of the dietary fats was determined by GC. Fats were methylated with trimethylsulfonium hydroxide (22). Fatty acid methyl esters were separated by GC using a system (HP 5890, Hewlett Packard) equipped with an automatic on-column injector, a polar capillary column (30-m FFAP, 0.53-mm i.d., Macherey and Nagel) and a flame ionization detector (23).
Sample collection. At d 6, rats received the last dose of fresh or oxidized oil and 9 g of the diet again 2 h after the beginning of the light cycle and were killed 4 h later by decapitation under light anesthesia with diethyl ether. Blood was collected into heparinized polyethylene tubes. The liver was excised. Plasma was obtained by centrifugation of the blood (1100 x g; 10 min, 4°C) and stored at –20°C. Liver samples for RNA isolation and lipid extraction were snap-frozen in liquid nitrogen and stored at –80°C.
Real-time RT-PCR analysis.
Total RNA was isolated from rat liver by TRIZOL reagent (Life Technologies) according to the manufacturer's protocol. cDNA synthesis was carried out as described (16). The messenger RNA (mRNA) expression of genes was measured by real-time detection PCR using SYBR Green I and the Rotor Gene 2000 system (Corbett Research). Real-time detection PCR was performed with 1.25 units Taq DNA polymerase, 500 µmol desoxy ribonucleotide triphosphates, and 26.7 pmol of the specific primers (Operon Biotechnologies). For determination of mRNA concentration, a threshold cycle was obtained from each amplification curve using the software RotorGene 4.6 (Corbett Research). Calculation of the relative mRNA concentration was made using the 
threshold cycle method as previously described (24). We used the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (EC1.2.1.12) for normalization. The primer sequences used for real-time detection PCR were described previously (5).
Immunoblot analysis. Nuclear extracts of rat livers were prepared from fresh tissue samples (150 mg) according to Woo et al. (25). The protein content of the samples was determined by the bicinchoninic acid assay. We purchased bicinchoninic acid reagent from Interchim. Equal amounts of proteins were pooled from 5 and 4 rats, respectively, per group and 80 µg protein per lane was separated on 10% SDS-polyacrylamide gels according to the method of Laemmli et al. (26) and electrotransferred to a nitrocellulose membrane (Pall). Polyclonal anti-SREBP-2 antibody (Abcam) was used to detect nuclear SREBP-2 using enhanced chemiluminescence reagent (GE Healthcare) and a chemiluminescence imager camera (Biostep). Signals were analyzed with the Phoretix TotalLab TL100 software. The anti-rabbit-IgG peroxidase conjugate antibody was purchased from Sigma-Aldrich.
Liver, plasma, and lipoprotein cholesterol. Rat liver lipids were extracted with a mixture of n-hexane and isopropanol (3:2, v:v) (27). Aliquots of the lipid extracts were dried and dissolved in a small volume of Triton X-100 (28). Plasma lipoproteins were separated by stepwise ultracentrifugation as described (5). Total cholesterol concentrations of liver, plasma, and lipoproteins were determined using the enzymatic reagent kit (Ecoline S+, DiaSys).
Statistical analysis. Means of treatments and control were compared by Student's t test using the Minitab Statistical software (Minitab). Values in the text are means ± SD. Means were considered significantly different at P < 0.05.
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Relative mRNA concentrations of PPAR and PPAR downstream genes in the liver.
Relative mRNA concentration of PPAR in the liver did not differ between groups (Fig. 1). However, rats administered the oxidized oil had higher relative mRNA concentrations of the PPAR downstream genes acyl-CoA oxidase (ACO) and cytochrome P450 4A1 (Cyp4A1) than rats administered fresh oil (P < 0.05; Fig. 1).
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Discussion |
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activation. In this study, we investigated the hypothesis that oxidized fats are able to exert similar effects due to their ability to activate PPAR. Hydroxy- and hydroperoxy fatty acids, such as hydroxyoctadecadienoic and hydroperoxyoctadecadienoic acid, occurring in oxidized fats are very potent PPAR agonists (14–16). These fatty acids are produced during the early stage of lipid peroxidation. Because they are unstable and decompose at high temperatures (29), fats treated at low temperature have much higher concentrations of these primary lipid peroxidation products than fats treated at high temperatures (4). The high peroxide value and the high concentration of conjugated dienes indicate that the oxidized oil used in this study indeed had high concentrations of hydroxy- and hydroperoxy fatty acids. Due to the loss of PUFA during the heating process, the concentrations of PUFA (i.e. linoleic acid) in the oxidized oil were slightly lower than in the fresh. Although intake of PUFA can influence cholesterol metabolism, i.e. plasma cholesterol concentration (30), we assume that the small difference in the intake of PUFA cannot be the main reason for the differences in cholesterol metabolism observed in this study. This assumption is confirmed by the observation that liver and plasma cholesterol concentrations were also reduced by oxidized oils in a similar extent in recent studies in which fresh and oxidized oils were equalized for their fatty acid composition (4,31–33). So that all rats obtained the same dose of oxidized oil, we administered it orally by gavage. The oxidized and fresh oil, respectively, accounted for about 25% of total energy of the total daily feed. Because it was observed in a previous rat study that even short term application of a PPAR agonist led to the typical changes known for PPAR activation [such as upregulation of classical target genes involved in ß-oxidation and reduction of triacylglycerol concentration (16)], we decided to give the oxidized oil during a relatively short period of 6 d. The intake of oxidized fats could cause a reduction of the food intake in rats, which could in turn cause secondary effects that interact with the effect of treatment (34,35). To ensure an identical food intake in both groups of rats, we used a controlled feeding system in which each rat consumed 12 g diet/d. This amount of diet is slightly below that rats would consume ad libitum but is about 50% in excess of that necessary to meet the maintenance energy requirement (36) and therefore ensures adequate growth. The finding that rats administered the oxidized oil gained less weight during the experimental period than those administered the fresh oil even though both groups received an identical amount of diet indicates that the oxidized oil impaired the feed conversion ratio. This finding agrees with other reports that also showed that feeding of oxidized fats impairs growth of rats (37–40). We did not investigate the reason for this. Previous studies, however, have shown that oxidized fats lower the digestibility of nutrients and this may be the reason for the reduced body weight gains of the rats administered the oxidized oil observed in this study (37,41). However, daily body weight gains of the rats administered the oxidized oil were also within the normal physiological range and as these rats appeared quite normal, we assume that the oxidized oil did not cause general toxicity. Studies in rats and pigs have shown that feeding oxidized oils lowers plasma and tissue tocopherol concentrations and causes oxidative stress (4,32,34,35,42). In this study, we did not determine the vitamin E status of the animals. According to these recent studies, administration of the oxidized fat probably also lowered plasma and tissue vitamin E concentrations compared with control animals. Nevertheless, because the diet used in this study had a relatively high vitamin E concentration and because the experimental period was relatively short, we assume that the rats administered the oxidized oil had an adequate vitamin E status in spite of the vitamin E consuming effect of the oxidized oil. Therefore, it is unlikely that the results in this study were confounded by vitamin E deficiency in the rats administered the oxidized fat.
The finding of increased mRNA concentrations of the typical PPAR downstream genes ACO and Cyp4A1 (43) in the liver and increased liver masses indeed indicates that the oxidized oil caused an activation of PPAR in the liver of the rats. This indication agrees with recent studies in rats and pigs and in rat fetes, which also showed that intake of oxidized fats leads to an activation of PPAR in the liver (3,4,12,44,45). Activation of PPAR by the oxidized oil may be due to the presence of hydroxy- and hydroperoxy fatty and cyclic fatty acids, all of which have been shown to be potent PPAR activators (14–16,46). We recently showed that the effect of oxidized fats on activation of PPAR is independent of the dietary vitamin E concentration (4). The finding that oxidized fats also exert a PPAR-activating effect at very high dietary vitamin E concentrations (which suppress the induction of oxidative stress) indicates that activation of PPAR is not caused by oxidative stress but by lipid peroxidation products present in the oxidized oil.
This study shows for the first time, to our knowledge, that administration of an oxidized oil upregulates the gene expression of Insig-1 and Insig-2a in the liver. Because Insig are able to retain the SCAP-SREBP-complex within the endoplasmic reticulum, thus inhibiting the proteolytic activation of SREBP in the Golgi (9,10), this event is probably the reason for the lower concentration of the mature SREBP-2 in the nucleus, which in turn leads to a reduced transcription of HMG-CoA reductase, the rate-limiting enzyme of de novo synthesis of cholesterol, and LDL receptor. Reduced cholesterol concentrations in liver and plasma, therefore, are likely the result of a reduction of hepatic cholesterol synthesis. Reduced hepatic cholesterol concentrations, moreover, may be in part due to a reduced uptake of LDL into liver cells. Besides nuclear concentrations of SREBP-2, mRNA concentration of SREBP-2 was also reduced in the liver of rats administered the oxidized oil. Because SREBP-2 contains a sterol-regulatory element in its enhancer/promoter region and thus the nuclear form can activate its own gene in an autoregulatory loop (47), this reduction is probably the effect of the reduced nuclear SREBP-2 concentration. In previous experiments with Fao cells treated with the PPAR agonist WY 14643, we demonstrated that the decreased SREBP-2 mRNA concentration did not precede the decrease of its nuclear form, indicating that it is due, rather, to increased expression of Insig (5).
Considering that similar effects were observed in the liver of rats treated with the synthetic PPAR agonist clofibrate and in rat hepatoma cells treated with the more potent and selective PPAR agonist WY 14,643 (5), we propose that the oxidized oil upregulated Insig in the liver of rats by PPAR activation. A functional PPAR response element that is regulated by PPAR
has already been identified in the human Insig-1 gene (48). Analysis of the 5' flanking region of rat Insig-1 using the PPAR response element consensus sequence from literature revealed 2 putative PPAR response elements at positions –592 and –1181 upstream of the transcription start site of the reported cDNA. The functionality of these PPAR response elements should be examined in future experiments.
We have recently observed that troglitazone, a synthetic PPAR agonist, also lowers the mature SREBP-2 concentration and inhibits cholesterol synthesis in HepG2, a human hepatoma cell line (49). Because oxidized fatty acids are also able to bind to and activate PPAR (50,51), we cannot exclude the possibility that the oxidized oil induced the effects observed in this study by activating PPAR, whose expression in the liver is, however, much lower than that of PPAR (52). The expression of Insig is also regulated by insulin. Insig-1 is upregulated by insulin, an effect caused by the insulin-induced stimulation of SREBP-1c gene transcription (53,54), which in turn leads to increased transcription of Insig-1 that is an obligatory SREBP target gene (9). In contrast, the Insig-2a transcript in the liver is strongly repressed by insulin. Thus, during fasting and feeding, Insig-1 and Insig-2a are regulated reciprocally (55). It has been shown that dietary oxidized frying oil lowers postprandial plasma concentration of insulin and induces glucose intolerance in rats and mice (56). As reduced plasma insulin concentrations would be expected to lower gene expression of Insig-1, it is unlikely that the upregulation of Insig-1 in the liver of rats administered the oxidized oil was mediated by insulin. Whether or not the observed upregulation of Insig-2a in the liver of rats treated with oxidized oil is mediated by reduced insulin concentrations or by PPAR activation remains unclear. In Fao cells treated with the PPAR agonist WY 14,643, mRNA concentration of Insig-2a was also increased, indicating that PPAR activation may also play a role in upregulation of Insig-2a.
The results of this study disagree with a recent study that investigated the effect of a moderately oxidized fat on triacylglycerol and cholesterol metabolism in pigs (45). In that study, the oxidized fat caused a moderate activation of PPAR but did not alter expression of genes involved in cholesterol metabolism, including SREBP-2, Insig, HMG-CoA reductase, and LDL receptor. That study and our study may disagree because of at least 2 reasons. First, the animal model used, pigs, belong to the group of nonproliferating species and have a lower expression of PPAR in the liver and a much weaker response of many genes to PPAR activation than rats, which belong to the group of proliferating species (57). Second, the fat used in the recent study performed with pigs was, according to concentrations of lipid peroxidation products, less oxidized than the fat used in this study. In the pig study, we used a mildly oxidized fat in which concentrations of peroxides (4-fold), conjugated dienes (4-fold), carbonyls (10-fold), and thiobarbituric acid reactive substances (30-fold) were only moderately increased compared with the fresh control fat. The oxidized fat used in this study had much higher concentrations of lipid peroxidation products, particularly of primary lipid peroxidation products, than that used in the pig study.
Although this study in rats shows that oxidized fats influence cholesterol metabolism via an upregulation of Insig, an effect probably mediated by activation of PPAR, it remains to be investigated whether such an effect also occurs in humans. With respect to expression and activation of PPAR, humans behave similarly to pigs. Humans and pigs have a similar expression of PPAR in the liver that is, however, 90% lower than in rats (58). Accordingly, upregulation of PPAR target genes in the liver by PPAR agonists is much weaker in pigs and humans than in rats (59,60). Therefore, it is expected that effects of oxidized fats on cholesterol metabolism, mediated by PPAR activation, in humans are weaker than those in rats observed in this study.
The fat used in this study prepared by heating at a relatively low temperature over a long period does not directly reflect the oxidized fats in human nutrition that originate predominantly from deep frying of foods. However, we have recently shown that fats produced under deep frying conditions lower liver and plasma cholesterol concentrations in rats to a similar extent as fats heated at a low temperature over a long period such as that used in this study (61). Moreover, it has been shown that fats prepared by deep frying are able to activate PPAR in the liver of rats (3,44). Therefore, it is likely that deep-fried fats influence the cholesterol metabolism in a similar way as fats prepared at a lower temperature for a longer period.
In conclusion, this study shows that oxidized oils are able to affect the activation of SREBP-2 by an upregulation of Insig-1 and Insig-2a in the liver of rats, which in turn lowers transcription of genes involved in cholesterol synthesis and uptake. This provides an explanation for reduced concentrations of cholesterol in liver and plasma observed in rats in this and recent studies. Although we assume that these effects are triggered by activation of PPAR, this must be proven in future studies.
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FOOTNOTES |
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2 Author disclosures: A. Koch, B. König, J. Spielmann, A. Leitner, G. I. Stangl, and K. Eder, no conflicts of interest.
3 Abbreviations used: ACO, acyl-CoA oxidase; Cyp4A1, cytochrome P450 4A1; HMG-CoA, 3-hydroxy-3-methylglutaryl CoA; Insig, insulin-induced gene; mRNA, messenger RNA; SCAP, SREBP-cleavage activating protein; SREBP, sterol regulatory element-binding protein.
Manuscript received 9 May 2007. Initial review completed 1 June 2007. Revision accepted 26 June 2007.
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68 kDa) in the liver of rats treated with fresh or oxidized oil was determined by western blot (A). Liver nuclear extracts of 5 and 4 rats, respectively, from each group were pooled. Relative mRNA concentrations of SREBP-2, HMG-CoA reductase, and LDL receptor in the liver of rats treated with fresh or oxidized oil (B). Values are means ± SD, n = 9. *Significantly different from rats treated with fresh oil, P < 0.05.