![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Nutrition and Consumer Sector, Pharmacia Corporation, St. Louis, MO 63167 and * Monsanto Corporation, St. Louis, MO 63167
2To whom correspondence should be addressed. E-mail: b.ganesh.bhat{at}pharmacia.com.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
7800 cloned expressed sequences from rat were used to identify those genes that responded to dietary long chain (n-3) fatty acids. In general, dietary long chain (n-3) fatty acids exhibited statistically significant lipid-lowering effects similar to a pharmaceutical alternative, fenofibrate, but showed narrower effects on the transcription of most of the genes assayed. The transcription patterns confirmed that the expression of several key genes involved in cholesterol metabolism, fatty acid ß-oxidation and lipogenesis was affected. These analyses indicated that stearoyl-coenzyme A (
9) desaturase, a key enzyme involved in the regulation of triglyceride biosynthesis and secretion, is a potential target for nutritional intervention for hyperlipidemia and cardiovascular health. In addition these results suggested that regulation of the farnesoid X receptor may be a key nutritionally regulated mediator of serum lipid changes. A nutritional product concept based on a convenient dietary aid demonstrated comparable efficacy with less spurious gene regulation than a pharmaceutical alternative.
KEY WORDS: • (n-3) fatty acids • nutraceuticals • cardiovascular • gene transcription
The identification of mechanism-based nutraceutical products holds great promise for improving human health when diet and lifestyle approaches fail. Applying genomic technology is one way to identify targets suitable for a nutraceutical approach to improve established cardiovascular biomarkers. At the present dietary modification represents the first line of defense recommended by clinicians and dieticians for the treatment of elevated serum lipids to reduce the risk of cardiovascular disease. Numerous studies have demonstrated the beneficial effects of dietary (n-3) fatty acids for lowering serum lipids in humans and animal models (1
–4
). Dietary sources rich in (n-3) fatty acids such as fish oil have been shown to exert lipid-lowering protective effects that mediate against cardiovascular risk factors in human populations (5
). Previous studies have suggested that (n-3) fatty acids contribute to both the stimulation of fatty acid oxidation and the reduction in lipogenesis in the liver (6
,7
). However, many (n-3) fatty acid dietary studies have been performed using very high amounts of fish oil in the diets, and may not be an accurate representation of the effects of a typical human dietary intake of (n-3) fatty acids. Also although dietary (n-3) fatty acids exert cardiovascular benefits on a population level (8
), it is often difficult to realize lipid changes at the individual level without resorting to very high doses. We sought to elucidate genes whose expression was affected by nutritional modification using a docosahexaenoic acid (DHA)-rich oil from Schizochytrium sp. microalgae in a manner that correlated with beneficial lipid changes in an effort to identify "druggable" targets ideal for nutraceutical intervention.
It has been suggested that the lipid-lowering effect of (n-3) fatty acids is due in part to increased ß-oxidation, mediated via activation of the nuclear hormone receptor family member peroxisome proliferator-activated receptor (PPAR)-
3 (9
,10
). PPAR- regulated genes include those encoding enzymes involved in lipid metabolism and fatty acid ß-oxidation, such as acyl-coenzyme A (CoA) oxidase (11
), acyl-CoA thioesterase (12
), lipoprotein lipase (13
) and carnitine palmitoyl transferase 1 (14
), as well as structural proteins localized to peroxisomes. Several pharmaceutical peroxisome proliferators, including the fibrate class of compounds, have been shown to lower serum lipids via PPAR- agonism. In test rats and in patients, fibrates may exhibit undesirable side effects not seen with dietary (n-3) fatty acids.
Some studies have implicated sterol regulatory element-binding protein (SREBP)-1 and the lipogenesis pathway in (n-3) fatty acid driven lipid-lowering benefits. The reduction in lipogenesis pathway gene products caused by dietary (n-3) fatty acids has been suggested to be mediated by a decrease in mature SREBP-1 (15
). Many lipogenesis pathway proteins that have been reported to be affected by (n-3) fatty acids are regulated by SREBP-1a and -1c, including stearoyl-CoA (9) desaturase (SCD) (16
,17
), acetyl-CoA carboxylase (18
) and fatty acid synthase (19
).
DNA microarrays represent a technological intersection between biology and computers that enables gene expression analysis in tissues on a genomewide scale (20
). In an effort to identify gene products whose expression was regulated by dietary modification in a manner consistent with serum lipid remediation, the transcription profile of rat livers in response to several lipid-lowering diets was determined. A nutritional product based on a DHA-rich oil was used to identify genes whose expression pattern correlated with serum lipid concentrations. For comparative purposes fenofibrate (FF), a pharmaceutical hypolipidemic agent, and a high fat/low carbohydrate diet (HF) were also included in these studies. Analysis of the genes regulated by the treatments described and comparison with relevant phenotypic parameters implicated several potential targets for intervention in hyperlipidemia using nutraceuticals.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
All animal procedures and experiments were approved by the Institutional Animal Care Committee. Male Sprague Dawley rats (Charles Rivers Laboratories, Wilmington, MA) aged 8–10 wk were acclimated for 2 wk before treatment and then assigned to treatment groups based on body weights. All rats were housed individually in rooms set to maintain 22° ± 3°C and 40% humidity with a 12-h light/12-h dark cycle. The DHA-rich oil was derived from Schizochytrium sp. microalgae and contained 38 g of DHA/100 g. Other major fatty acid components in DHA-rich oil were 22:5 [(n-6) 16 g/100 g], 16:0 (25 g/100 g) and 14:0 (12 g/100 g). FF was obtained from Sigma Chemical Co. (St. Louis, MO). Appropriate amounts of test article were incorporated into a powdered diet by mixing. Rats were maintained on one of four dietary treatments using two diets developed to mimic fatty acid composition of typical Western-style human diets. A control group of eight rats was fed a semipurified diet (11% of energy derived from fat) referred to as the low fat (LF) diet. Diet composition was based on AIN-76A rodent diet (21
) except for fat content and source and were prepared by Research Diets, New Brunswick, NJ. This diet was formulated to mimic a standard Western-style diet with regard to the ratios of saturated, monounsaturated and polyunsaturated fatty acids, as well as the ratio of (n-6) to (n-3) fatty acids (Table 1
). A second group of eight rats were fed the LF diet containing 1.5 g of DHA-rich oil/100 g of diet. DHA content in this diet is equivalent to 0.3 g of DHA/1,000 kJ per day. The control and DHA-rich oil diets were isocaloric and contained identical amounts by weight percentage of dietary fat. A third group of rats was fed the LF diet containing 0.25 g of FF (a triglyceride-lowering agent that acts via PPAR- and is a potent peroxisome proliferator)/100 g. This represents a dosage of 200–250 mg of compound/kg of body weight per day. A fourth group of rats was fed an HF diet (37% of energy derived from fat) identical to the LF diet with respect to fatty acid composition but containing an increased overall fat content and reduced carbohydrate content (22
). Rats had ad libitum access to food and water during the study. Each rat was weighed once a week.
|
Blood was collected from rats anesthetized with CO2/O2. Serum triglyceride and cholesterol concentrations were measured at 2-wk intervals and before the end of the study at 6 wk. When the anesthetized food-deprived rats were killed by exsanguination, liver samples were harvested from each rat and frozen immediately in liquid N2. The complete fatty acid profile of serum and livers was determined.
Lipid extraction and fatty acid composition.
Lipids were extracted from rat serum and liver samples using the Bligh and Dyer procedure (23
). Lipid extracts were trans-esterified with methanolic acetyl chloride, neutralized and extracted with petroleum ether/ether (9:1). Fatty acid methyl esters (FAME) were separated on a Supelco Omegawax 320 fused silica column (30 m, 0.32 mm I.D.) using a Hewlett-Packard HP6890 gas chromatograph with 7673 injector, FID and HP enhanced integrator. FAME were identified and quantified using calibration curves of authentic FAME internal standards of 17:0 and 22:0. One-tailed Student’s t tests were performed on all phenotypic data to identify significant changes.
Transcription profiling.
Total RNA was prepared from frozen livers using TriZOL reagent and protocol (GIBCO-BRL, Gaithersburg, MD). Poly(A)+ mRNA was isolated using micro poly(A)plus kits (Ambion, Austin, TX). Aliquots of 200 ng of mRNA were used to generate cyanine 3 dye (Cy3)- and Cy5-labeled cDNA probes using GEMBrite probe labeling kits (Incyte Pharmaceuticals, Palo Alto, CA). Individual treated and control samples from three rats chosen at random from each group were used to prepare Cy3-labeled cDNA probes, whereas a pool of equal proportions of all eight control sample mRNAs was used to generate Cy5-labeled cDNA probes. Labeled probes were purified away from unincorporated primer using Chromaspin TE-30 columns (Clontech, Palo Alto, CA). Probe hybridization onto Incyte RatGEM 1.0, containing 7,800 cloned rat cDNAs, was performed at Incyte Pharmaceuticals as previously described (24
). Microarray hybridization data were balanced using total average signal intensity in each channel to generate a balance coefficient. Each gene expression microarray (GEM) was evaluated with respect to five parameters: background correction, log balance coefficient, absolute average signal, differential expression ratio and differential percentage. A balanced differential expression (BDE) value was generated for every element that met minimum signal intensity, signal to background and area of coverage criteria. The BDE represents a fold change in expression, and BDE values were calculated according to Eq. 1.
 | (1) |
where P1 (probe 1) represents the signal intensity of the Cy3-labeled treated sample, and bP2 represents the signal intensity of the balanced Cy5-labeled control sample (probe 2). A fractional balanced differential expression value (fBDE), representative of a percent change in expression, was also calculated using Eq. 2.
 | (2) |
When calculating an fBDE, the balanced signal intensity in the Cy5 channel (bP2, control sample) was subtracted from the signal intensity in the Cy3 (P1, treated sample) channel. This value was then divided by the smaller of the two signal intensities. For each array, elements with signal intensities P1 + P2 < 500 (unbalanced P2 value), signal to background values P1STB + P2STB < 5 or area of array spot coverage <40% were recorded as absent values and were omitted from further consideration. Each individual treated and control versus pooled control comparison was performed twice, using the same RNA samples but different Cy-dye labeling reactions and array hybridizations performed on separate days. Values were averaged for each pair of like hybridizations.
Data analysis.
The regulation of a subset of prototypical members of three key biological pathways was considered to evaluate how faithfully these experiments reproduce previously reported gene regulation effects. These pathways included PPAR- mediated regulation of fatty acid ß-oxidation pathway genes and SREBP-mediated regulation of lipogenesis and cholesterol biosynthesis pathway genes.
To identify genes whose expression may be appropriate for modulation by a nutraceutical, we used a method to correlate gene expression with measured biological phenotypes. Fractional balanced differential expression values were calculated for each element, and, because our aim was to correlate relative changes in gene expression to relative changes in phenotype, the phenotypic data were also expressed as a fBDE value using Eq. 2. Unlike BDE values, fBDEs are continuous from -
to +. For each phenotype gene element pair, a correlation score was determined based on how well expression of the gene correlated with the phenotype using Eq. 3.
|
| (3) |
This score takes into account i) the fraction of chips that had reliable data, that is, above the quality control (QC) cutoffs described in Materials and Methods, for the gene in question and, of those, the fraction of the treated rats that displayed differential expression that was at least 70%, equivalent to a fold change of ±1.7 above or below the pooled control rats (nQC^ |fBDE|/n), ii) the absolute value of the Pearson correlation coefficient (|r|) and iii) the P-value associated with the correlation (P). On determination of a score for each gene as it related to each phenotype of interest, the genes were sorted by score. We took advantage of the control elements on each GEM that consist of empty elements, complex genomic DNA or yeast control RNA. A correlation score was calculated with respect to each phenotype for each control element as well as for every rat cDNA element. On the assumption that a good correlation would not be expected between these biological phenotypes and the control elements, only those genes that scored >20-fold higher than the highest scoring control element were considered to be relevant.
Desaturase activity measurement.
Hepatic 9-desaturase activity was measured as previously described (25
) using 14C-18:0 as substrate. Briefly, microsomes were isolated from frozen livers through differential centrifugation. The assay mixture contained 150 µL of buffer/cofactors (0.25 mol of sucrose, 0.15 mol of KCl, 0.04 m of NaF, 0.1 mol of sodium phosphate, pH 7.4, 1.3 mmol of ATP, 1.5 mmol of reduced glutathione, 0.06 mmol of reduced CoA, 0.33 mmol of nicotinamide, 1 g MgCl2·5H2O and 0.67 g of NADH per L) and 50 µL of rat liver microsomes (0.5 mg total protein), and 20 µL of 14C-labeled fatty acid substrate (14C-18:0) in the buffer/cofactor solution. After incubation at 37°C for 30 min, the reaction was stopped and the samples were saponified by the addition of 200 µL of 2.5 mol of KOH/L in methanol/H2O (4:1). Samples were incubated in a shaking incubator at 65°C for 1 h. After saponification, the free fatty acids were protonated by the addition of 200 µL of formic acid to each tube (final pH 3). Hexane (750 µL) was added, and the fatty acids were extracted into the hexane phase by thorough mixing. A portion from the hexane layer of each sample was spotted onto the preabsorbent loading strip of AgNO3 thin layer chromatography plates. Desaturase activity was quantified by separation of 14C-18:0 and 14C-18:1 after argentation thin layer chromatography in a solvent system consisting of chloroform/methanol/acetic acid/water (90:8:1:0.8). After chromatography, the plates were removed and air-dried, and 9-desaturase activity was qualitatively determined by autoradiography using an Instant Imager (Packard, Meriden, CT) or a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
In general, the dietary treatments had no adverse effect on the rats. FF caused a statistically significant increase in liver weight, resulting in a doubling of the liver-to-body weight ratio (Table 2
). No other gross abnormalities were noted at necropsy. Both the DHA-rich oil and FF diets caused statistically significant reductions in fasting serum triglyceride concentrations. A statistically significant serum triglyceride reduction was also demonstrated in rats fed the HF diet compared with the control LF diet (Table 2)
. In addition to lowering serum triglyceride concentrations, the treatments described resulted in statistically significant reductions in serum cholesterol concentrations.
|
Elements for which <4 of the 24 hybridizations passed QC parameters were omitted from consideration, resulting in 6,212 elements used for further analyses. Among the treatment groups, the FF group demonstrated the largest degree of gene regulation by far. Of the 6212 elements that passed QC, 287 elements (4.6%) demonstrated threefold or greater induction or repression in all three fibrate-treated rats relative to control. In contrast, only 63 (1.0%) and 2 (<<0.1%) of the elements in the DHA-rich oil and HF diet groups, respectively, showed threefold or greater changes in expression relative to control. A maximum of only 32 elements were differentially regulated (twofold or greater) on any one GEM from individual control rats relative to pooled control. These genes were either highly variable even in fairly homogeneous populations or the affected microarray elements were prone to variability. None of the elements were altered by twofold or greater on any two control versus control GEMs. In addition, none of the genes that were identified as being involved in the transcriptional response to dietary modification of serum lipids demonstrated regulation in the individual control rats.
The regulation of a subset of known genes of the PPAR- mediated fatty acid ß-oxidation pathway and SREBP-mediated regulation of lipogenesis and cholesterol biosynthesis pathways was used to assess how faithfully these experiments reproduced previously reported gene regulation effects (Table 3
). Several additional genes that encode proteins involved in lipid metabolism were also compared (Table 4
). Many of these genes may also be members of one or more of the above-mentioned three pathways. In addition, a large number of genes were regulated that have no direct involvement in the lipogenesis or fatty acid ß-oxidation pathways (Table 5
).
|
|
|
9-Desaturase activity.
As shown in Table 2
, dietary treatment of rats with DHA-rich oil and HF diets caused a significant reduction in serum triglyceride concentrations relative to the LF diet. The DHA-rich oil and HF diets also correspondingly decreased expression of SCD-1 (Table 3)
. These dietary treatments were also shown to cause reduced hepatic SCD enzyme activity, demonstrating a relationship between message concentrations and enzymatic activity (Fig. 1
A). The rats fed the DHA-rich oil showed statistically significant (P < 0.05) reductions in hepatic concentrations of 9-desaturated fatty acids 18:1 and 16:1 and arachidonic acid (20:4) (Fig. 1B)
. DHA-rich oil fed rats also showed statistically significant (P < 0.05) reductions in serum oleic [18:1 (n-9)] and arachidonic acid (20:4) and an increase in palmitic acid (16:0, a component of the DHA-rich oil) (Fig. 1C)
. In agreement with other reports in the literature (26
,27
), dietary treatment with FF resulted in increased SCD expression and activity.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
, while causing fewer extraneous gene expression changes. Serum triglyceride and cholesterol concentrations were also lower in rats fed the HF diet compared with the control LF diet. It is important to note that although the LF diet is lower in fat than the HF diet, it also has higher concentrations of carbohydrates. A high carbohydrate diet has been shown to cause an increase in serum triglyceride concentrations (28
). The reduction in triglycerides observed in the HF diet group was likely mediated by the lower concentration of dietary carbohydrates than in the control group. Although rats are useful models for triglyceride-lowering studies, the relevance to human pharmacology of cholesterol-lowering effects in rats is uncertain. Unlike humans and nonhuman primates, rats have high concentrations of circulating HDL and comparatively low concentrations of LDL, >70% and 15% of total lipoprotein, respectively (29
). For this reason it is often considered that rats are not useful models for predicting cholesterol-lowering effects. However some compounds that lower LDL in humans also lower total serum cholesterol in rats, and as such, rats have been used successfully as model systems to study cholesterol-lowering effects of some compounds (30
–33
).
FF clearly exhibited PPAR- agonist activity (Table 3)
. Typical PPAR- regulated genes including acyl-CoA oxidase (ACOX), carnitine palmitoyl transferase (CPT1) and cytochrome P450 4A1 (CYP4A1), as well as liver fatty acid binding protein and peroxisome proliferator-induced cytosolic acyl-CoA thioesterase, were all regulated by FF. In addition, genes that encode proteins involved in peroxisome structure such as the 22-kDa integral peroxisome membrane protein (22-kDa pyridoxine monophosphate) were also highly induced by FF. PPAR- itself was not appreciably regulated by FF. The regulation of ß-oxidation pathway genes by the DHA-rich oil via PPAR- agonism was less clear. Gene knockout studies have shown that (n-3) fatty acids have modest PPAR- dependent effects on the transcription of several gene products (34
). Other studies have demonstrated that (n-3) fatty acids are weak peroxisome proliferators (35
). However, many of the PPAR- genes studied here showed little or no regulation by DHA compared with FF. Specifically, those genes encoding proteins directly involved in fatty acid ß-oxidation showed minimal regulation. Some genes that encode peroxisome structural proteins did exhibit modest induction by DHA but were typically less highly regulated by the DHA-rich oil than by FF (Table 3)
. Given the fact that DHA-rich oil exhibited similar TG lowering, this suggested that DHA-mediated reductions in serum triglycerides were not the result of PPAR- agonism.
A second major pathway potentially involved in dietary reductions in serum lipids was the lipogenesis pathway, of which many members are regulated by SREBP-1a and -1c. Studies with transgenic animal models have suggested that the liver constitutively produces SREBP-1c, which is less active than SREBP-1a (36
), but both proteins affect the expression of lipogenesis pathway genes. Polyunsaturated fatty acid regulation of lipid gene transcription, including SREBP-1, has been reviewed extensively (37
,38
). Feeding studies with large doses of fish oil have suggested that (n-3) fatty acids regulate both SREBP-1c message and mature SREBP-1 protein concentrations (16
). As with the ß-oxidation pathway, inspection of several key lipogenesis pathway genes also revealed regulation by FF, although SREBP-1 itself was not significantly regulated (Table 3)
. However, FF induced many of the lipogenesis pathway genes measured, which was not in agreement with the observed reduction in serum triglyceride concentrations in these rats. This is not unprecedented; others have reported fibrate-mediated increases in lipogenesis pathway genes (26
,27
,39
). We hypothesized that the vast increase in ß-oxidation mediated by the potent PPAR- agonism of FF may have triggered a compensatory increase in lipogenesis pathway genes. In contrast, dietary DHA-rich oil modestly down-regulated SREBP-1 itself, but also repressed expression of the lipogenesis pathway gene SCD. SCD-1, which is abundant in liver, catalyzes the desaturation of stearic acid (18:0) to produce oleic acid [18:1 (n-9)] The presence of oleic acid in the liver has a protective effect on apolipoprotein (apo) B and serves to stabilize apoB from enzymatic degradation (40
,41
). apoB is the main apolipoprotein involved in VLDL secretion. A reduction in SCD message, as seen in both the DHA-rich oil and HF groups, might result in a concomitant decrease in SCD protein and activity. Reduced SCD activity would result in lower concentrations of 18:1, thereby destabilizing apoB. This could cause a decrease in VLDL secretion, manifested as a reduction in serum triglycerides. Decreased SCD activity was confirmed in these samples, and the concentrations 9-desaturase of oleic acid (18:1) and palmitoleic acid (16:1) were significantly lower in livers from the DHA-rich oil fed rats than in control livers (Fig. 1B and C)
. Thus it seemed that dietary (n-3) fatty acids caused SREBP-1 mediated reductions in SCD message and activity, resulting in decreased serum triglyceride concentrations. The reductions in SREBP-1 also resulted in an equivocal decrease in the expression of fatty acid synthase, another lipogenesis pathway gene (Table 3)
.
In light of the DHA-mediated reductions in SREBP-1 message, a third key pathway was considered in our analyses: the cholesterol biosynthesis pathway. Both SREBP-1a and -2 have been shown to regulate genes encoding proteins involved in cholesterol biosynthesis and metabolism such as 3-hydroxy-3-methyl glutaryl CoA (HMG-CoA) reductase (42
), HMG-CoA synthase (43
) and the LDL receptor (44
). SREBP-2 has been shown to accommodate reduced concentrations of SREBP-1 in SREBP-1 knockout rats and appears to selectively activate cholesterol synthesis pathway genes rather than lipogenesis pathway genes (45
). Signal intensity of the single SREBP-2 element was low, and none of the dietary regimens resulted in clear regulation of the SREBP-2 message. However, the transcription of some SREBP-2 pathway genes was affected (Table 3)
. Although LDL receptor and HMG reductase were induced by FF, HMG synthase was not regulated under these conditions. Despite increased concentrations of LDL receptor message along with reductions in serum cholesterol, it might be expected that the LDL receptor would not have played a significant role in cholesterol lowering in rats. We therefore developed a method to correlate gene expression with phenotypic values to identify genes whose expression corresponded closely with the phenotypic observations.
On completion of the correlation analysis with the serum triglyceride phenotype, no single gene could be shown to score >20-fold better than the highest scoring GEM control element using this approach, and all of the correlation scores were low. This was not surprising because the profiling results as well as data from other laboratories clearly demonstrate that fibrates mediate triglyceride lowering via PPAR- agonism, whereas our results implicated a non PPAR- dependent mechanism for (n-3) fatty acid driven triglyceride reduction. For this reason, one might not expect a single gene to correlate with triglycerides for all four diet groups. When the three fibrate-fed rats were removed from consideration and the triglyceride correlation score was recalculated, a single gene was identified whose correlation score was >10-fold higher than the highest scoring control element. That gene was SCD (data not shown). Although both the HF and DHA-rich oil diets lowered triglycerides and resulted in reductions in SCD, only the DHA-rich oil diet caused reductions in SREBP-1 message. Such modest effects on SREBP-1 expression in livers of rat deprived of food overnight by HF or DHA-rich oil diet may be because of short half-life of SREBP-1 message. Whether the reduction in the SREBP-1c target gene SCD message in the HF diet rats was a result of higher dietary fat or lower carbohydrate content relative to the control diet is unclear.
Correlation analysis was also performed using serum cholesterol values to identify genes that were highly correlative with this phenotype in all 12 rats tested in the four diet groups. The highest scoring gene was that encoding rat farnesoid X activated receptor (FXR) (Fig. 2
). FXR has been shown to be an endogenous receptor of bile acids (46
,47
) and has recently been shown to be activated by a natural product capable of lowering serum cholesterol in rats (48
). FXR represses the transcription of cholesterol 7-hydroxylase (CYP7A1) transcription in response to several specific bile acids (49
). CYP7A1 is a principal enzyme responsible for the conversion of cholesterol to bile acid. It has been demonstrated that the liver receptor homolog-1 (LRH-1) acts as a competence factor for transcription of CYP7A1 (50
). LRH-1 is responsible for both basal CYP7A1 expression and facilitation of liver X receptor (LXR)-induced CYP7A1 transcription in response to dietary cholesterol. A mechanism has been proposed whereby FXR responds to the bile acid chenodeoxycholic acid and, to a lesser degree, to lithocholic and deoxycholic acid to increase the expression of the small heterodimeric protein (SHP) (51
,52
). SHP then binds at an LRHRE element in the promoter of CYP7A1 to LRH-1, where it blocks the ability of LRH-1 to facilitate LXR-driven transcription of CYP7A1. Although CYP7A1 was not present on the microarrays and thus was not assayed in this experiment, it is not unreasonable to speculate that the decrease in FXR message caused by the DHA-rich oil could result in a loss of FXR-mediated repression of CYP7A1 expression concentrations. The resulting potential increase in CYP7A1 message relative to control could result in a reduction in serum cholesterol. LRH-1 was also not present on RatGEM 1, nor was IBABP, another FXR-regulated gene. Both LXR and SHP were present on the GEM, although neither message showed any apparent relationship with the observed serum cholesterol concentrations. SHP appeared nominally higher in some rats with lower serum cholesterol (Fig. 2)
, although the signal intensities and the increases relative to control were equivocal. A number of key genes within several relevant metabolic pathways, including fatty acid ß-oxidation, as well as lipogenesis, cholesterol biosynthesis, gluconeogenesis and xenobiotic metabolism were affected by one or more diet group (Fig. 3
). In addition to DHA, the major fatty acid, there were other fatty acids present in the DHA-rich oil. It may be possible that the lipid-lowering effects observed with this DHA-rich oil were due to other constituents of the oil, possibly in addition to the major component, DHA. Indeed, at least one component of the DHA-rich oil, 22:5 (n-6), may be a ligand for PPAR-, although in the current study we demonstrated little PPAR- agonism in the DHA-rich oil group. In fact despite similar efficacy in lipid lowering, FF caused the regulation of numerous members of several pathways, whereas the dietary DHA-rich oil affected expression of far fewer genes. The role of fibrates as inducers of peroxisomal fatty acid ß-oxidation is well established and is mediated by PPAR-induced expression of ACOX in the peroxisome (53
). Peroxisomal ß-oxidation favors the metabolism of long-chain fatty acids, whereas fatty acid ß-oxidation in the mitochondrion favors short-chain fatty acids. FF also caused changes in message concentrations of numerous genes both related to (Table 4)
and not related to (Table 5)
lipid metabolism pathways that were not seen with the DHA-rich oil or HF diets. Although the duration of the fast may have caused some gene expression changes to be obscured, many of the genes whose expression remained changed by FF after the fasting period were unaffected by the other dietary treatments. DHA concentrations in the liver (Fig. 1B)
and serum (Fig. 1C)
remained significantly elevated even after the overnight fast, suggesting that the DHA-rich oil simply caused less extraneous gene regulation than the pharmaceutical alternative. Although many fibrate-mediated effects such as hepatomegaly and hepatocarcinogenesis may not be relevant to humans, fibrates are not without side effects, as verified by the large number of gene expression changes observed in the present study.
|
|
,55
). These authors went on to demonstrate that inhibition of SCD results in reduced secretion of triglycerides in vitro (56
). Also, a mouse model with a naturally occurring disruption of the SCD gene exhibits impaired triglyceride biosynthesis (57
). Finally, studies using chicken hepatoma cells demonstrate that transfection of the SCD coding sequence increased triglyceride secretion, whereas transfection of an SCD antisense construct reduces VLDL triglyceride secretion (58
). Recent studies have implicated SCD as an important component of leptin-mediated weight loss, and crossed SCD-negative ob/ob mice had significantly lower plasma triglyceride concentrations than matched ob/ob rats with normally functioning SCD (59
). In mice with targeted disruption of SCD-1, Ntambi et al. (60
) reports that SCD-1 deficiency leads to an activation of lipid oxidation in addition to reduced triglyceride synthesis and storage. All of these studies implicate SCD as a potential target for lipid lowering and obesity. The present study confirmed that SCD can be altered nutritionally and that it maintains this expression pattern even after a period of overnight food deprivation. These factors potentially make SCD suitable for nutraceutical-mediated lipid lowering. The case for nutritionally mediated regulation of FXR for serum cholesterol lowering is less clear but remains an intriguing finding in light of recent studies (46
–48
).
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: apo, apolipoprotein; BDE, balanced differential expression; CoA, coenzyme A; Cy3, cyanine 3 dye; Cy5, cyanine 5 dye; CYP, cytochrome P450; DHA, docosahexaenoic acid; FAME, fatty acid methyl esters; fBDE, fractional increase balanced differential expression; FF, fenofibrate; FXR, farnesoid X receptor; GEM, gene expression microarray; HF, high fat/low carbohydrate; LF, low fat; LRH-1, liver receptor homolog-1; LXR, liver X receptor; P1, probe 1, Cy3-labeled pooled control mRNA; P2, probe 2, Cy5-labeled mRNA; QC, quality control; SCD, stearoyl-CoA desaturase 1.
Manuscript received 9 May 2002. Initial review completed 4 June 2002. Revision accepted 9 October 2002.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Rambjor, G. S., Walden, A. I., Windsor, S. L. & Harris, W. S. (1996) Eicosapentaenoic acid is primarily responsible for hypotriglyceridemic effect of fish oil in humans. Lipids Suppl :S45-S49.
2. Lanzmann-Petithory, D. (2001) Alpha-linolenic acid and cardiovascular diseases. J. Nutr. Health Aging 5:179-183.[Medline]
3. Harris, W. S. & Isley, W. L. (2001) Clinical trial evidence for the cardio-protective effects of omega-3 fatty acids. Curr. Atheroscler. Rep. 3:174-179.[Medline]
4. Mori, T. A. & Beilin, L. J. (2001) Long-chain omega 3 fatty acids, blood lipids and cardiovascular risk reduction. Curr. Opin. Lipidol. 12:11-17.[Medline]
5. Nestel, P. J. (1990) Effects of n-3 fatty acids on lipid metabolism. Annu. Rev. Nutr. 10:149-167.[Medline]
6. Rustan, A., Nossen, J., Christiansen, E. & Drevon, C. (1988) Eicosapentaenoic acid reduces hepatic synthesis and secretion of triacylglycerol by decreasing the activity of acyl-coenzyme A, 1,2-diacylglycerol acyltransferase. J. Lipid Res. 29:1417-1426.[Abstract]
7. Halminski, M., Marsh, J. & Harrison, E. (1991) Differential effects of fish oil, safflower oil and palm oil on fatty acid oxidation and glycerolipid synthesis in rat liver. J. Nutr. 121:1554-1561.
8. Mantzioris, E., Cleland, L. G., Gibson, R. A., Neumann, M. A., Demasi, M. & James, M. J. (2000) Biochemical effects of a diet containing foods enriched with n-3 fatty acids. Am. J. Clin. Nutr. 72:42-48.
9. Neat, C. E., Thomassen, M. S. & Osmundsen, H. (1980) Induction of peroxisomal ß-oxidation in rat liver by high-fat diets. Biochem. J. 186:369-371.[Medline]
10. Flatmark, T., Nilsson, A., Kvannes, J., Eikhom, T. S., Fukami, M. H., Kryvi, H. & Christiansen, E. N. (1988) On the mechanism of induction of the enzyme systems for peroxisomal ß-oxidation of fatty acids in rat liver by diets rich in partially hydrogenated fish oil. Biochim. Biophys. Acta 962:122-130.[Medline]
11. Dreyer, C., Krey, G., Keller, H., Givel, F., Helftenbein, G. & Wahli, W. (1992) Control of the peroxisomal ß-oxidation pathway by a novel family of nuclear hormone receptors. Cell 68:879-887.[Medline]
12. Hunt, M. C., Nousiainen, S. E., Huttunen, M. K., Orii, K. E., Svensson, L. T. & Alexson, S. E. (1999) Peroxisome proliferator-induced long chain acyl-CoA thioesterases comprise a highly conserved novel multi-gene family involved in lipid metabolism. J. Biol. Chem. 274:34317-34326.
13. Auwerx, J., Schoonjans, K., Fruchart, J. C. & Staels, B. (1996) Transcriptional control of triglyceride metabolism, fibrates and fatty acids change the expression of the LPL and apo C-III genes by activating the nuclear receptor PPAR. Atherosclerosis 124:S29-S37.
14. Mascaro, C., Acosta, E., Ortiz, J. A., Marrero, P. F., Hegardt, F. G. & Haro, D. (1998) Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor. J Biol. Chem. 273:8560-8563.
15. Kim, H., Takahashi, M. & Ezaki, O. (1999) Fish oil feeding decreases mature sterol regulatory element-binding protein 1 (SREBP-1) by down-regulation of SREBP-1c mRNA in mouse liver. A possible mechanism for down-regulation of lipogenic enzyme mRNAs. J. Biol. Chem. 274:25892-25898.
16. Tabor, D., Kim, J. B., Spiegelman, B. M. & Edwards, P. A. (1998) Transcriptional activation of the stearoyl-CoA desaturase 2 gene by sterol regulatory element-binding protein/adipocyte determination and differentiation factor 1. J. Biol. Chem. 273:22052-22058.
17. Shimomura, I., Shimano, H., Korn, B. S., Bashmakov, Y. & Horton, J. D. (1998) Nuclear sterol regulatory element-binding proteins activate genes responsible for the entire program of unsaturated fatty acid biosynthesis in transgenic mouse liver. J. Biol. Chem. 273:35299-35306.
18. Lopez, J. M., Bennett, M. K., Sanchez, H. B., Rosenfeld, J. M. & Osborne, T. E. (1996) Sterol regulation of acetyl coenzyme A carboxylase, a mechanism for coordinate control of cellular lipid. Proc. Natl. Acad. Sci. U. S. A. 93:1049-1053.
19. Bennett, M. K., Lopez, J. M., Sanchez, H. B. & Osborne, T. F. (1995) Sterol regulation of fatty acid synthase promoter. Coordinate feedback regulation of two major lipid pathways. J. Biol. Chem. 270:25578-25583.
20. King, H. C. & Sinha, A. A. (2001) Gene expression analysis by DNA microarrays: promise and pitfalls. J. Am. Med. Assoc. 286:2280-2288.
21. American Institute of Nutrition (1980) AIN second report of the AIN Ad Hoc Committee on Standards for Nutritional Studies. J. Nutr. 110:1726-1729.
22. Hansen-Petrik, M. B., McEntee, M. F., Johnson, B. T., Obukowicz, M. & Whelan, J. (2000) Highly unsaturated (n-3) fatty acids, but not -linolenic, conjugated linoleic or -linolenic acid, reduce tumorigenesis in ApcMin/+ mice. J. Nutr. 130:2434-2443.
23. Bligh, E. G. & Dyer, W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-937.
24. Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P. O. & Davis, R. W. (1996) Parallel human genome analysis, microarray-based expression monitoring of 1000 genes. Proc. Natl Acad. Sci. U. S. A. 93:10614-10619.
25. Obukowicz, M. G., Welsch, D. J., Salsgiver, W. J., Martin-Berger, C. L., Chinn, K. S., Duffin, K. L., Raz, A. & Needleman, P. (1998) Novel, selective delta-6 or delta-5 fatty acid desaturase inhibitors as anti-inflammatory agents in mice. J. Pharmacol. Exp. Ther. 287:157-166.
26. Alegret, M., Cerqueda, E., Ferrando, R., Vazquez, M., Sanchez, R. M., Adzet, T., Merlos, M. & Laguna, J. C. (1995) Selective modification of rat hepatic microsomal fatty acid chain elongation and desaturation by fibrates, relationship with peroxisome proliferation. Br. J. Pharmacol. 114:1351-1358.[Medline]
27. Mille, C. W. & Ntambi, J. M. (1996) Peroxisome proliferators induce mouse liver stearoyl-CoA desaturase 1 gene expression. Proc. Natl. Acad. Sci. U. S. A. 93:9443-9448.
28. Ullmann, D., Connor, W. E., Hatcher, L. F., Connor, S. L. & Flavell, D. P. (1991) Will a high-carbohydrate, low-fat diet lower plasma lipids and lipoproteins without producing hypertriglyceridemia?. Arterioscler. Thromb. 11:1059-1067.
29. Kris-Etherton, P. M. & Dietschy, J. (1997) Design criteria for studies examining individual fatty acid effects on cardiovascular disease risk factors: human and animal studies. Am. J. Clin. Nutr. 65:1590S-1596S.
30. Windler, E. E. T., Kovanen, P. T., Choa, Y.-S., Brown, M. S., Havel, R. J. & Goldstein, J. L. (1980) The estradiol-stimulated lipoprotein receptor of rat liver. J. Biol. Chem. 255:10464-10471.
31. Black, L. J., Sato, M., Rowley, E. R., Magee, D. E., Bekele, A., Williams, D. C., Cullinan, G. J., Bendele, R., Kauffman, R. F., Bensch, W. R., Frolik, C. A., Termine, J. D. & Bryant, H. U. (1994) Raloxifene (LY139481) prevents bone loss and reduced serum cholesterol without causing uterine hypertrophy. J. Clin. Invest. 93:63-69.
32. Lundeen, S., Carver, J., McKean, M.-L. & Winneker, R. (1997) Characterization of the ovariectomized rat model for the evaluation of estrogen effects on plasma cholesterol levels. Endocrinology 138:1552-1558.
33. Poplawski, J., Lozowicka, B., Dubis, A. T., Lachowska, B., Witkowski, S., Siluk, D., Petrusewicz, J., Kaliszan, R., Cybulski, J., Strzalkowska, M. & Chilmonczyk, Z. (2000) Synthesis and hypolipidemic and antiplatelet activities of alpha-asarone isomers in humans (in vitro), mice (in vivo), and rats (in vivo). J. Med. Chem. 43:3671-3676.[Medline]
34. Ren, B., Thelen, A., Peters, J., Gonzalez, F. & Jump, D. (1997) Polyunsaturated fatty acid suppression of hepatic fatty acid synthase and S14 gene expression does not require peroxisome proliferator-activated receptor alpha. J. Biol. Chem. 272:26827-26832.
35. Wysynski, A. M., Baldwin, L. A., Leonard, D. A. & Calabrese, E. J. (1993) Interactive potential of omega-3 fatty acids with clofibrate or DEHP on hepatic peroxisome proliferation in male Wistar rats. Hum. Exp. Toxicol. 12:337-340.[Medline]
36. Shimano, H., Horton, J. D., Shimomura, I., Hammer, R. E., Brown, M. & Goldstein, J. L. (1997) Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J. Clin. Invest. 99:846-854.[Medline]
37. Clarke, S. D. (2001) Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve metabolic syndrome. J. Nutr. 131:1129-1132.
38. Jump, D. B. & Clarke, S. D. (1999) Regulation of gene expression by dietary fat. Annu. Rev. Nutr. 19:63-90.[Medline]
39. Karbowska, J., Kochan, Z., Zelewski, L. & Swierczynski, J. (1999) Tissue-specific effect of clofibrate on rat lipogenic enzyme gene expression. Eur. J. Pharmacol. 370:329-336.[Medline]
40. Dashti, N. & Wolfbauer, G. (1987) Secretion of lipids, apolipoproteins, and lipoproteins by human hepatoma cell line, HepG2, effects of oleic acid and insulin. J. Lipid Res. 28:423-436.[Abstract]
41. Moberly, J. B., Cole, T. G., Alpers, D. H. & Schonfeld, G. (1990) Oleic acid stimulation of apolipoprotein B secretion from HepG2 and Caco-2 cells occurs post-transcriptionally. Biochim. Biophys. Acta 1042:70-80.[Medline]
42. Kawabe, Y., Honda, M., Wada, Y., Yazaki, Y., Suzuki, T., Ohba, Y., Nabata, H., Endo, A., Matsumoto, A., Itakura, H. & Kodama, T. (1994) Sterol mediated regulation of SREBP-1a, 1b, 1c and SREBP-2 in cultured human cells. Biochem. Biophys. Res. Commun. 202:1460-1467.[Medline]
43. Inoue, J., Sato, R. & Maeda, M. (1998) Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase. J. Biochem. (Tokyo) 123:1191-1198.
44. Briggs, M. R., Yokoyama, C., Wang, X., Brown, M. S. & Goldstein, J. L. (1993) Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. I. Identification of the protein and delineation of its target nucleotide sequence. J. Biol. Chem. 268:14490-14496.
45. Shimano, H., Shimomura, I., Hammer, R. E., Herz, J., Goldstein, J. L., Brown, M. S. & Horton, J. D. (1997) Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene. J. Clin. Invest. 100:2115-2124.[Medline]
46. Makishima, M., Okamoto, A. Y., Repa, J. J., Tu, H., Learned, R. M., Luk, A., Hull, M. V., Lustig, K. D., Mangelsdorf, D. J. & Shan, B. (1999) Identification of a nuclear receptor for bile acids. Science (Washington, D. C.) 284:1362-1365.
47. Parks, D. J., Blanchard, S. G., Bledsoe, R. K., Chandra, G., Consler, T. G., Kliewer, S. A., Stimmel, J. B., Willson, T. M., Zavacki, A. M., Moore, D. D. & Lehmann, J. M. (1999) Bile acids: natural ligands for an orphan nuclear receptor. Science (Washington, D. C.) 284:1365-1368.
48. Urizar, N. L., Liverman, A. B., Dodds, D. T., Silva, F. V., Ordentlich, P., Yan, Y., Gonzalez, F. J., Heyman, R. A., Mangelsdorf, D. J. & Moore, D. D. (2002) A natural product that lowers cholesterol as an antagonist ligand for FXR. Science (Washington, D. C.) 296:1703-1706.
49. Chiang, J., Kimmel, R., Weinberger, C. & Stroup, D. (2000) Farnesoid X receptor responds to bile acids and represses cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription. J. Biol. Chem. 275:10918-10924.
50. Nitta, M., Ku, S., Brown, C., Okamoto, A. Y. & Shan, B. (1999) CPF, an orphan nuclear receptor that regulates liver-specific expression of the human cholesterol 7alpha-hydroxylase gene. Proc. Natl. Acad. Sci. U. S. A. 96:6660-6665.
51. Lu, T. T., Makishima, M., Repa, J. J., Schoonjans, K., Kerr, T. A., Auwerx, J. & Mangelsdorf, D. J. (2000) Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol. Cell 6:507-515.[Medline]
52. Goodwin, B., Jones, S. A., Price, R. R., Watson, M. A., McKee, D. D., Moore, L. B., Galardi, C., Wilson, J. G., Lewis, M. C., Roth, M. E., Maloney, P. R., Willson, T. M. & Kliewer, S. A. A. (2000) Regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol. Cell 6:517-526.[Medline]
53. Tugwood, J. D., Issemann, I., Anderson, R. G., Bundell, K. R., McPheat, W. L. & Green, S. (1992) The mouse peroxisome proliferator activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl CoA oxidase gene. EMBO J 11:433-439.[Medline]
54. Legrand, P., Mallard, J., Bernard-Griffiths, M. A., Douaire, M. & Lemarchal, P. (1987) Hepatic lipogenesis in genetically lean and fat chickens. In vitro studies. Comp. Biochem. Physiol. 87B:789-792.
55. Legrand, P. & Hermier, D. (1992) Hepatic delta 9 desaturation and plasma VLDL level in genetically lean and fat chickens. Int. J. Obes. 16:289-294.
56. Legrand, P., Catheline, D., Fichot, M. C. & Lemarchal, P. (1997) Inhibiting delta9-desaturase activity impairs triacylglycerol secretion in cultured chicken hepatocytes. J. Nutr. 127:249-256.
57. Miyazaki, M., Kim, Y. C., Gray-Keller, M. P., Attie, A. D. & Ntambi, J. M. (2000) The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1. J. Biol. Chem. 275:30132-30138.
58. Diot, C., Lefevre, P., Herve, C., Belloir, B., Narce, M., Damon, M., Poisson, J. P., Mallard, J. & Douaire, M. (2000) Stearoyl-CoA desaturase 1 coding sequences and antisense RNA affect lipid secretion in transfected chicken LMH hepatoma cells. Arch. Biochem. Biophys. 380:243-250.[Medline]
59. Cohen, P., Miyazaki, M., Socci, N. D., Hagge-Greenberg, A., Liedtke, W., Soukas, A. A., Sharma, R., Hudgins, L. C., Ntambi, J. M. & Friedman, J. M. (2002) Role for stearoyl-CoA desaturase-1 in leptin-mediated weight loss. Science (Washington, D. C.) 297:240-243.
60. Ntambi, J. M., Miyazaki, M., Stoehr, J. P., Lan, H., Kendziorski, C. M., Yandell, B. S., Song, Y., Cohen, P., Friedman, J. M. & Attie, A. D. (2002) Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity. Proc. Natl. Acad. Sci. U S A. 99:11482-11486.
This article has been cited by other articles:
|
|
 |
|
  D. M. Mutch, W. Wahli, and G. Williamson Nutrigenomics and nutrigenetics: the emerging faces of nutrition FASEB J, October 1, 2005; 19(12): 1602 - 1616. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Perez-Granados, M. P. Vaquero, and M. P. Navarro Comparative Effects of the Fat Extracted from Raw and Fried Sardines Upon Rat Growth and Zinc Bioavailability Food Science and Technology International, August 1, 2003; 9(4): 285 - 293. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||
