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Originally published In Press as doi:10.1074/jbc.M504101200 on October 12, 2005

J. Biol. Chem., Vol. 280, Issue 51, 41793-41804, December 23, 2005
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Regulation of Fatty Acid Synthesis by Farnesyl Pyrophosphate*

Shubha Murthy{ddagger}1, Huaxiang Tong{ddagger}, and Raymond J. Hohl{ddagger}§

From the Departments of {ddagger}Internal Medicine and §Pharmacology, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242

Received for publication, April 14, 2005 , and in revised form, September 9, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fatty acid biosynthesis is transcriptionally regulated by liver X receptor (LXR) and its gene target, sterol regulatory element binding protein-1c (SREBP-1c). LXR activation is induced by oxysterol end products of the mevalonate pathway and is inhibited by the upstream non-sterol isoprenoid, geranylgeranyl pyrophosphate (GGPP). Whether isoprenoids play a role in regulating the transcription of genes involved in fatty acid biosynthesis is unknown. In CaCo-2 colon epithelial cells, depletion of mevalonate and its derivatives, including oxysterol ligands for LXR, increased fatty acid synthesis. Addition of mevalonate or its isoprenoid derivative, farnesyl pyrophosphate (FPP), prevented this increase. The effects of FPP were likely due to itself or its degradation products, because none of its downstream derivatives, GGPP, ubiquinone, or cholesterol, were effective. Moreover, the effects of FPP could not be accounted for by protein prenylation, because inhibition of farnesylation did not alter fatty acid synthesis in mevalonate-depleted cells incubated with the isoprenoid. Neither was fatty acid synthesis in these cells altered by inhibition of {beta}-oxidation. Mevalonate depletion increased fatty acid synthase (FAS) mRNA by transcriptional mechanisms, without increasing gene expression of other enzymes involved in fatty acid biosynthesis or of SREBP-1c. The abundance of mature SREBP-2 but not SREBP-1 was increased following mevalonate depletion. FPP prevented the increase in FAS mRNA in mevalonate-depleted cells without altering SREBP-2 activation. Thus, FPP regulates fatty acid synthesis by a mechanism that is likely independent of the SREBP pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The biosynthesis of fatty acids and cholesterol are closely related. Enzymes catalyzing the synthesis and metabolism of both lipids are coordinately regulated by the same transcription factors, the sterol regulatory element-binding proteins (SREBPs)2 and the liver X receptors (LXRs) (1–3). LXRs belong to a superfamily of nuclear hormone receptors that are ligand-activated transcription factors (3). They form obligate heterodimers with retinoid X receptors (RXRs), another member of this receptor family, and subsequently bind to specific response elements in regulatory regions of their target genes (3, 4). Targets of LXR/RXR activation include several genes involved in cholesterol metabolism (3). In addition, the nuclear receptor heterodimer also induces the transcription of genes involved in fatty acid synthesis by activating another transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c) (2). Three SREBPs have been described, SREBP-1a, SREBP-1c, and SREBP-2. SREBP-1a and SREBP-1c are products of a single gene through alternative splicing, whereas SREBP-2 is a product of separate gene (1). Although SREBP-1c activates genes involved in the synthesis of fatty acids, SREBP-2 preferentially activates genes for cholesterol synthesis and the low density lipoprotein receptor (1). Ligands for LXRs include various oxidized derivatives of cholesterol, which is an end product of the mevalonate pathway (see Fig. 1) (5–7). Depletion of cholesterol and its oxysterol derivatives decreases transcriptional activity of the LXR/RXR heterodimer (8). Addition of mevalonate and oxysterol ligands for LXRs restores transcription mediated by the nuclear receptor complex. In contrast, the upstream non-sterol isoprenoid derivative of mevalonate, geranylgeranyl pyrophosphate (GGPP), and its alcohol derivative, geranylgeraniol, inhibit the activity of the LXR/RXR heterodimer by interfering with LXR activation (8, 9). As a result, GGPP decreases transcription of an LXR target gene, the ATP-binding cassette protein A1, that is involved in cholesterol efflux from cells (9). It is not known whether other target genes of LXRs, such as enzymes involved in fatty acid biosynthesis, are similarly altered by non-sterol isoprenoid derivatives of mevalonate.

Depletion of both sterol and non-sterol derivatives of mevalonate by inhibition of HMG-CoA reductase has previously been observed to enhance fatty acid and triglyceride synthesis (10, 11). Although this was accompanied by increased expression of some of the genes involved in fatty acid synthesis, it is unclear whether the underlying mechanism involved increased activation of SREBP-1c. Because inhibition of mevalonate synthesis would limit the availability of cholesterol and its oxidized derivatives, enhanced lipogenesis under such conditions cannot be due to increased ligand-mediated activation of LXRs. In fact, under such conditions, LXR activation and transcription of SREBP-1c would be decreased. We, therefore, postulated that the effect of HMG-CoA reductase inhibition on increasing fatty acid synthesis is independent of SREBP-1c and is due to limited availability of non-sterol isoprenoid derivatives of mevalonate.

It has been suggested that isoprenoids affect cellular processes by multiple mechanisms. Isoprenoids, such as farnesyl pyrophosphate (FPP) and GGPP, are utilized for prenylating membrane-bound small GTPases, such as members of the Ras superfamily, which play critical roles in cell proliferation and cytoskeletal maintenance (12, 13). Isoprenylation of these proteins is necessary for their translocation to the plasma membrane and subsequent activation. Besides serving as substrates for post-translational modification, FPP and GGPP have been reported to regulate the transcription of some of these proteins (14). The mechanisms by which isoprenoids may directly or indirectly affect transcription of proteins are not well understood. In this respect, GGPP has been shown to inhibit the transcription of ATP-binding cassette protein A1 by acting as a direct antagonist of ligand-mediated activation of LXRs (9). However, the one or more mechanisms by which FPP could alter transcriptional events are not defined.

CaCo-2 colon epithelial cells have active lipogenic pathways and thus serve as a model for examining the interaction between isoprenoids and fatty acid synthesis. Using these cells, we demonstrate that depletion of mevalonate and all its derivatives causes a transcriptional-dependent increase in fatty acid synthesis and fatty acid synthase (FAS) mRNA levels. Exogenous mevalonate and its isoprenoid derivative, FPP, prevent this increase, whereas other derivatives of mevalonate do not. Gene expression of SREBP-1c and of its other lipogenic targets, acetyl-CoA carboxylase (ACC) and ATP-citrate lyase (ACL), and the abundance of mature SREBP-1 protein remain unaltered. In contrast, the abundance of mature SREBP-2 and the expression of its lipogenic targets genes are enhanced following mevalonate depletion. FPP prevents the increase in fatty acid synthesis and FAS mRNA without altering the activation of SREBP-2. Thus, FPP regulates fatty acid synthesis by a transcriptional mechanism that is likely mediated by FAS and is independent of SREBP.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Lovastatin, mevalonolactone, isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate, geranyl pyrophosphate (GPP), FPP, farnesol (FOH), GGPP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, squalene, actinomycin D, and etomoxir were obtained from Sigma-Aldrich. Cholesterol was purchased from Steraloids (Newport, RI). Lovastatin and mevalonolactone were dissolved in 0.1N sodium hydroxide to generate their respective active open acid forms and the pH was adjusted to 7.4 with 0.1 N hydrochloric acid. FTI-277 and ubiquinone were from EMD Biosciences (San Diego, CA). [14C]Sodium acetate (56.5 mCi/mmol) was from PerkinElmer Life Sciences, and [1-14C]palmitic acid (50 mCi/mmol) was from American Radiolabeled Chemicals (St. Louis, MO). Silica gel thin layer chromatography plates (250 µm) were purchased from Analtech (Newark, DE). Monoclonal antibodies to human SREBP-1 and SREBP-2 were purchased from BD Biosciences (San Diego, CA). Monoclonal antibody to human caspase-3 and rabbit polyclonal antibody to lamin A (H102) were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody, raised against the carboxyl terminus of prelamin A, was a generous gift from Dr. Scott Kaufmann (Mayo Clinic, Rochester, MN). Anti-mouse IgG-HRP, anti-rabbit IgG-HRP, and ECL Western blotting detection reagents were purchased from Amersham Biosciences. SuperSignal West Pico chemiluminescent substrates were from Pierce. All other materials were reagent grade.

Cell Culture—CaCo-2 cells, obtained from Dr. F. J. Field (University of Iowa), were maintained at 37 °C in 5% CO2 in T-75 cm2 or T-150 cm2 flasks in Dulbecco's modified Eagle's medium (Sigma-Aldrich) containing 4.5 g/liter glucose, 1.5 mg/liter sodium bicarbonate, 1 mM sodium pyruvate, 2 mML-glutamine, and 0.1 mM non-essential amino acids and supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.1 mg/ml gentamycin. Cells were subcultured in 6-well plates at a density of 0.08 million per well. Near confluent monolayers, obtained after 5 days in culture, were incubated for 24 h in serum-free M199 containing the various treatments. Appropriate vehicle controls for every treatment were included. Serum was excluded from the incubation media to avoid the effect of exogenous sterols and fatty acids on de novo fatty acid synthesis.

Fatty Acid Synthesis—Cells were pulsed for the last 2 h of a 24-h incubation with 1 µCi/well [14C]sodium acetate. After the incubation, cells were rinsed with phosphate-buffered saline, and cellular lipids were extracted and saponified as previously described (15). Labeled sterols and free fatty acids were sequentially extracted, with 2 x 3 ml of hexane, under alkaline and acidic conditions, respectively. Incorporation of labeled acetate into each fraction was determined by liquid scintillation counting. To ensure that there was no cross-contamination of counts between the two lipid fractions, fatty acid and sterol extracts from a few separate experiments were isolated as above. Total extracts of each lipid fraction were dried under nitrogen, taken up in 100 µlof chloroform and spotted on thin layer chromatography plates together with fatty acid and cholesterol standards (Nuchek, Elysian, MN). The plates were then eluted in a solvent system of hexanes:ethyl ether:acetic acid, 75:25:1, v/v, and visualized under iodine vapor. Incorporation of labeled acetate into bands corresponding to free fatty acids and cholesterol was determined by scanning the plates using the AMBIS radioisotope detection scanner (Scanalytics, Billerica, MA). Over 95% of counts incorporated into the total sterol or fatty acid extracts were recovered in bands corresponding to the respective lipid.

{beta}-Oxidation of Palmitic Acid—Cells were collected from T-150 cm2 flasks by trypsinization and then counted. One million cells were preincubated with 10 µM etomoxir in Krebs Ringer bicarbonate buffer in flasks fitted with central wells. After 30 min 0.1 µCi of 2 µM [1-14C]palmitic acid was added to each flask, and 400 µl of hyamine hydroxide (PerkinElmer Life Sciences) was added to each central well. The total volume of the incubation mixture per flask was 1 ml. Hyamine hydroxide was used to trap released [14C]CO2. Flasks were made air-tight with non-vented caps. Following incubation for 1 h at 37°C, hyamine hydroxide was transferred to 10 ml of Emulsifier Safe liquid scintillation mixture (PerkinElmer Life Sciences), dark-adapted overnight, and counted by liquid scintillation counting.

Estimation of Relative mRNA Abundance by Real-time RT-PCR—Total RNA was extracted from cells using TRIzol reagent (Invitrogen). DNase-free RNA was reverse transcribed using SuperScript III (Invitrogen) and then subjected to real-time quantitative PCR using Sybr Green (Applied Biosystems, Foster City, CA) and the appropriate forward and reverse primers on an ABI Prism model 7000 sequence-detection system (Applied Biosystems). Results are expressed relative to control after normalizing to 18 S rRNA. Single products for each RT-PCR reaction were obtained. Primers used for the different genes examined are shown in TABLE ONE.


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  		TABLE ONE
Primers used for the different genes examined

 
Estimation of Protein Expression by Western Blotting—Following incubation with the various treatments, cells were rinsed and scraped in phosphate-buffered saline and collected by low speed centrifugation. The cell pellet was lysed in 10 mM HEPES, pH 7.4, containing 100 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM dithiothreitol, and protease and phosphatase inhibitor mixtures (Sigma). Cells were mechanically disrupted by repeated passage through a 27-gauge syringe and needle and centrifuged for 5 min at 14,000 rpm in a tabletop microcentrifuge. Equivalent amounts of the supernatant containing the solubilized proteins were separated by SDS-PAGE on 15% gels (for the detection of caspase-3) or 7.5% gels (for all other proteins) and transferred to polyvinylidene difluoride membranes. For detection of prelamin A, proteins were transferred to nitrocellulose membranes. Polyvinylidene difluoride membranes were blocked for 1 h in blocking buffer (TBS containing 5% nonfat milk and 0.05% Tween 20). Nitrocellulose membranes were blocked in TBS containing 10% nonfat dry milk and 0.05% Tween 20. Membranes were then incubated for an additional hour with anti-SREBP-1 (diluted 250-fold), anti-SREBP-2 (diluted 250-fold), anti-lamin A (diluted 200-fold), or anti-caspase-3 (diluted 100-fold) antibodies dissolved in blocking buffer containing 5% milk. For the detection of prelamin A nitrocellulose membranes were incubated with blocking buffer containing 10% milk and anti-prelamin A antibody diluted 500-fold. Membranes were washed six times for 5 min each with TBS containing 0.05% Tween 20. They were then incubated for 1 h with their respective secondary antibodies diluted 3000-fold in blocking buffer containing 5% milk. After washing six times with TBS containing 0.05% Tween 20, the membranes were exposed to HRP substrates for 5 min. SuperSignal West Pico substrate detection kit was used for the detection of SREBP proteins, and ECL detection kit was used for the other proteins. All incubations and washes were performed at 37 °C. Exposures to blue-sensitive autoradiography x-ray films (Midwest Scientific, St. Louis, MO) were between 1 and 30 min in duration. X-ray films were scanned, and the intensity of bands was determined using the image processing and analysis software, Image J (1.34s, National Institutes of Health).

Determination of FPP—Cells were incubated for 18 h with 25 µM lovastatin. FPP, 10 µM, was added per well for an additional hour in the continued presence of lovastatin. After the incubation cells were washed with phosphate-buffered saline, trypsinized, and collected by low speed centrifugation. Cells were then extracted with butanol/75 mM ammonium hydroxide/ethanol, 1:1.25:2.75 (v/v), and processed for determination of FPP mass by high-performance liquid chromatography as described previously (16).

Cell Viability—The number of viable and non-viable cells remaining after incubation with the treatments was counted by Trypan Blue exclusion. In addition, cell viability was also assessed by the conversion of the tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to its colored formazan product by mitochondrial dehydrogenase as described previously (17). Relative to control cells, none of the treatments at the concentrations examined, significantly altered the number of viable cells, milligrams of protein/well or mitochondrial dehydrogenase activity. To evaluate the effect of lovastatin on apoptosis, cells were incubated for 24 h with either M199 or M199 containing 10 µM lovastatin. Following incubation, cellular proteins were extracted and separated by SDS-PAGE on 15% gels and immunoblotted for caspase-3.

Statistical Analyses—Differences between treatments were assessed for statistical significance using analysis of variance followed by Tukey's t test (18).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Mevalonate Depletion on Fatty Acid Synthesis—To address the effect of mevalonate and its derivatives on fatty acid synthesis, CaCo-2 cells were depleted of mevalonate by incubation for 24 h with lovastatin. Lovastatin is a potent inhibitor of HMG-CoA reductase, the enzyme that catalyzes the conversion of HMG-CoA to mevalonate in the rate-limiting step of the mevalonate pathway (Fig. 1). During the last 2 h of incubation with lovastatin, cells were pulsed with radiolabeled acetate, and the incorporation of the precursor into fatty acids was estimated. Results are shown in Fig. 2. Lovastatin caused a dose-dependent increase in fatty acid synthesis (Fig. 2A) and, as expected, decreased the synthesis of sterols (Fig. 2B). Lovastatin did not alter cell viability, cell number or protein concentration (data not shown). Thus, results were similar whether expressed per milligram of protein or per well. Neither were these results due to changes in apoptosis. In cells incubated with either medium alone or medium supplemented with 10 µM lovastatin there was little appreciable cleavage of caspase-3, an early marker of apoptosis (Fig. 2C). In contrast, etoposide, a known inducer of apoptosis, caused breakdown of the enzyme into its active products. Lovastatin was therefore used at 10 µM in all subsequent experiments. The incorporation of labeled acetate into fatty acids was linear with increasing times of incubation with the precursor and was greater in cells incubated with lovastatin (Fig. 2D). To examine the time-dependent effects of lovastatin on fatty acid synthesis, cells were incubated for increasing times with the inhibitor, and the incorporation of labeled acetate into fatty acids was estimated. Results are shown in Fig. 3 and demonstrate that fatty acid synthesis increased significantly in cells incubated with lovastatin only after 12 h of incubation. To confirm that the effects of lovastatin were due to mevalonate depletion, fatty acid synthesis was estimated in cells incubated with lovastatin and increasing concentrations of mevalonate. In results shown in Fig. 4, mevalonate prevented the effects of lovastatin on fatty acid synthesis in a dose-dependent manner. At the highest concentration examined, there was no difference in fatty acid synthesis between cells incubated with mevalonate alone and cells incubated with mevalonate and lovastatin. At this concentration, compared with control cells, mevalonate alone caused a modest increase in fatty acid synthesis.



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  		FIGURE 1.
The mevalonate pathway. The synthesis of isoprenoids from acetyl CoA is shown. Enzymes with their respective inhibitors used in the study are shown in italicized and bold characters, respectively. FPTase, farnesyl pyrophosphate transferase; GGPTase, geranylgeranyl pyrophosphate transferase.

 



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  		FIGURE 2.
Effect of mevalonate depletion on fatty acid synthesis. Cells were incubated for 24 h with increasing concentrations of lovastatin. For the last 2 h of incubation, 1 µCi of [14C]acetate was added per well, and the incorporation of radiolabeled acetate into fatty acids (A) and total sterols (B) was determined as described under "Experimental Procedures." Results are shown as mean ± S.E. of picomoles of acetate incorporated per well. n = 3/treatment. *, p < 0.05 versus no lovastatin. C, cells were incubated for 24 h with M199 alone or medium containing 10 µM lovastatin. As a positive control cells were incubated with 50 µM etoposide (Etp). Following incubation, cells were lysed and caspase-3 cleavage detected by Western blotting. Results from one out of two representative experiments are shown. n = 3/control or lovastatin. D, cells were incubated for 24 h with M199 alone (•) or with 10 µM lovastatin ({circ}). For the last 15, 30, 60, and 120 min of the incubation cells were incubated with 1 µCi/well [14C]acetate, and the incorporation of labeled acetate into fatty acids was determined. Results from one out of two representative experiments are shown as mean ± S.E. of picomoles of acetate incorporated per well. n = 3/treatment; *, p < 0.05 versus control.

 
Effect of Isoprenoids on Fatty Acid Synthesis—The above results suggest that mevalonate and/or its downstream products are involved in regulating fatty acid synthesis. Oxysterol end products of the mevalonate pathway are ligands for LXRs and can therefore stimulate the expression of LXR target genes such as SREBP-1c and genes involved in fatty acid synthesis (2, 5–7). However, because lovastatin caused a marked inhibition in the synthesis of sterols, it is unlikely that oxysterol-mediated activation of LXR was responsible for the increase in fatty acid synthesis observed in mevalonate-depleted cells. Instead, these results suggest that derivatives of mevalonate upstream of sterols are involved in fatty acid synthesis. To identify the one or more derivatives, cells were incubated for 24 h with lovastatin alone or together with each of the isoprenoid intermediates of the mevalonate pathway. These intermediates were added to cells in concentrations that are equivalent in terms of their conversion to downstream products in the pathway. Incorporation of labeled acetate into fatty acids was then determined. Results shown in Fig. 5A demonstrate that none of the immediate downstream derivatives of mevalonate, IPP, or its isomer dimethylallyl pyrophosphate, or GPP, significantly altered lovastatin-induced increase in fatty acid synthesis. In contrast, the longer 15-carbon isoprenoid, FPP, prevented the effect of lovastatin on fatty acid synthesis. Fatty acid synthesis in cells incubated with lovastatin and FPP were not significantly different from cells incubated with vehicle alone. Compared with vehicle alone FPP alone caused a modest decrease in fatty acid synthesis. Similar effects were observed in cells incubated with farnesol (FOH), the alcohol derivative of FPP. Because FPP is the precursor for the synthesis of sterols, GGPP, and ubiquinone, the effect of this sesquiterpene could have been due to conversion to any one or more of these products. However, this was found to be unlikely. Incubation with GGPP did not alter fatty acid synthesis in cells incubated with lovastatin. Addition of squalene, the sterol precursor, to mevalonate-depleted cells only modestly decreased fatty acid synthesis compared with cells incubated with lovastatin alone. Incubation with 100 µM cholesterol, a concentration significantly higher than what would be produced from 10 µM FPP, did not attenuate the effects of lovastatin. If anything, it increased fatty acid synthesis in the presence of lovastatin. Cholesterol, at a lower concentration of 5 µM that would be produced from 10 µM FPP, did not alter statin-induced increase in fatty acid synthesis (results not shown). Ubiquinone, similar to squalene, caused only a modest decrease in fatty acid synthesis in mevalonate-depleted cells. Thus the effects of FPP were either due to itself, a farnesylated protein, or its degradation products.



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  		FIGURE 3.
Time-dependent effects of mevalonate depletion on fatty acid and sterol synthesis. Cells were incubated for increasing times with M199 (•) alone or with 10 µM lovastatin ({circ}) and pulsed with radiolabeled acetate for the last 2 h of every incubation. The incorporation of acetate into fatty acids was estimated. Results from one out of two experiments are shown as mean ± S.E. of percent acetate incorporation per well relative to control at each time point. n = 3/treatment; *, p < 0.05 versus control.

 
The negative charge on pyrophosphate esters at physiological pH is thought to limit the entry of isoprenoid pyrophosphates into cells. Specificity of results obtained with incubating cells with these compounds has therefore been questioned. Recent reports, however, demonstrate that they cross the cell membrane and are metabolically active (14, 19). To confirm that isoprenoid pyrophosphates are taken up and incorporated into CaCo-2 cells, cells were preincubated for 18 h with 25 µM lovastatin and then incubated for an additional hour with 10 µM FPP. Intracellular accumulation of FPP was then estimated in these cells. Results are shown in Figs. 5B. As expected, compared with control cells, lovastatin decreased FPP mass secondary to its inhibition of mevalonate synthesis. When FPP was added to cells incubated with lovastatin, intracellular levels of FPP significantly increased by 2-fold compared with cells incubated with lovastatin alone. To evaluate whether the FPP that enters cells is incorporated into proteins, the ability of the isoprenoid to prevent the effects of lovastatin on the farnesylation of prelamin A was determined. Following farnesylation, prelamin A is cleaved at its carboxyl terminus to yield mature lamin A (20). Inhibition of farnesylation, by either inhibiting the farnesylation reaction or by limiting FPP availability, would prevent this proteolytic cleavage and leave the carboxyl terminus of prelamin A intact. Thus, an antibody directed against the carboxyl terminus of prelamin A enables its detection under conditions that inhibit its farnesylation (21). Using this antibody, the expression of prelamin A was determined in cells incubated for 24 h with 10 µM lovastatin, conditions that inhibit the synthesis of FPP. Results are shown in Fig. 5C. As expected, the expression of prelamin A was detected in cells incubated with lovastatin. When FPP was added to these cells, however, the amount of prelamin A was reduced to levels that were almost undetectable. In control cells or cells incubated with FPP alone prelamin A was undetectable. These results indicate that exogenous FPP is utilized for farnesylating prelamin A. None of the treatments altered the expression of lamin A. Taken together, the above results demonstrate that exogenous FPP is taken up, retained as such, and is functionally active in CaCo-2 cells. These results therefore validate the specificity of effects observed with FPP in these cells.



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  		FIGURE 4.
Effect of mevalonate on fatty acid synthesis. Cells were incubated for 24 h with M199 alone or with 10 µM lovastatin and/or increasing concentrations of mevalonate. During the last 2 h of the incubation cells were pulsed with labeled acetate, and the incorporation of acetate into fatty acids was determined. Results from one out of two representative experiments are shown and demonstrate mean ± S.E. of percent incorporation of acetate per well relative to control. Open bar, –lovastatin; cross-hatched bar, +lovastatin. n = 3/treatment. a, p < 0.05 versus –lovastatin for each treatment group; b, p < 0.05 versus –lovastatin-mevalonate; c, p < 0.05 versus +lovastatin-mevalonate.

 
Effect of Farnesylation on Fatty Acid Synthesis—FPP is a substrate for farnesyl transferase, which catalyzes the post-translational modification of proteins containing the conserved CAAX consensus sequence by covalent thioester linkage of FPP to the cysteine residue (22, 23). Because several of these proteins function in various signaling pathways, we questioned whether the effects of FPP were due to farnesylated protein(s). To address this, cells were incubated for 24 h with lovastatin alone or together with FPP and/or increasing concentrations of a potent peptidomimetic inhibitor of farnesylation, FTI-277. Fatty acid synthesis was then determined. Results are shown in Fig. 6A. As observed in Fig. 5, the increase in fatty acid synthesis in cells incubated with lovastatin was significantly impaired by addition of FPP. When FTI-277 was added to cells incubated with lovastatin and FPP, the inhibitory effects of FPP were not altered by FTI at any of the concentrations examined. FTI alone modestly decreased fatty acid synthesis. To verify whether FTI-277 was effective in inhibiting farnesylation of proteins, the expression of prelamin A was determined in cells incubated with the inhibitor. As with the previous experiment shown in Fig. 5, the expression of prelamin A was determined using an antibody directed against its carboxyl terminus. Results are shown in Fig. 6B, and as expected, they demonstrate that the abundance of prelamin A increased with increasing concentrations of FTI-277 indicating that the inhibitor was indeed inhibiting farnesyl pyrophosphate transferase activity. Thus, inhibition of farnesylation in mevalonate-depleted cells was unable to prevent the effects of FPP on fatty acid synthesis. It is therefore unlikely that the effect of FPP could have been due to a farnesylated protein.

Effect of {beta}-Oxidation on Fatty Acid Synthesis—FOH has been observed to increase the transcription of peroxisome proliferator-activated receptor target genes involved in fatty acid oxidation (24). FPP and FOH are inter-convertible, and FOH has been previously demonstrated to reverse the effects of mevalonate depletion on Ras isoprenylation (25, 26). This is consistent with the observation in the present study that FOH, similar to FPP, inhibited fatty acid synthesis in mevalonate-depleted cells. As the synthesis and oxidative degradation of fatty acids are reciprocally related, it is possible that FPP decreased fatty acid synthesis in mevalonate-depleted cells by increasing its oxidation. To examine this, cells were incubated with lovastatin and/or FPP in the presence or absence of etomoxir, a known inhibitor of carnitine palmitoyl transferase-1 and of mitochondrial {beta}-oxidation of fatty acids (27, 28). Fatty acid synthesis was then estimated. Results are shown in Fig. 7. As observed previously the increase in fatty acid synthesis in mevalonate-depleted cells was inhibited by FPP (Fig. 7, A and B, left panel). Addition of etomoxir to cells incubated with lovastatin and FPP did not prevent the inhibitory effects of FPP on fatty acid synthesis (Fig. 7B). Etomoxir, by itself or in the presence of lovastatin, modestly decreased fatty acid synthesis by ~25% (Fig. 7A). The efficacy of etomoxir in inhibiting {beta}-oxidation of fatty acids was assessed by measuring the release of [14C]CO2 from [1-14C]palmitic acid. After 1 h of incubation with etomoxir the release of labeled carbon dioxide was inhibited by ~60% (3.5 ± 0.48 versus 1.34 ± 0.05 pmol of [14C]CO2/million cells). These results suggest that the effects of FPP were not due to increased {beta}-oxidation of fatty acids.



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  		FIGURE 5.
Effects of intermediates of the mevalonate pathway on fatty acid synthesis. A, cells were incubated for 24 h with 0.1% ethanol and 0.3% methanol in the presence or absence of 10 µM lovastatin and/or 30 µM IPP, 30 µM dimethylallyl pyrophosphate, 10 µM GPP, 10 µM FPP, 10 µM GGPP, 5 µM squalene, 100 µM cholesterol, or 50 µM ubiquinone. Radiolabeled acetate was added to cells for the last 2 h of the incubation, and its incorporation into fatty acids was determined. Results from one out of at least two experiments are shown as mean ± S.E. of percent incorporation of acetate per well relative to control. Open bar, –lovastatin; cross-hatched bar, +lovastatin. n = 3/treatment. a, p < 0.05 versus –lovastatin control for each isoprenoid; b, p < 0.05 versus vehicle-lovastatin; c, p < 0.05 versus vehicle+lovastatin. B, cells were preincubated for 18 h with M199 alone or with 25 µM lovastatin (Lov) before being exposed to 10 µM FPP for 1 h. Following incubation, cells were washed, and lipids were extracted and processed for estimating FPP mass as described under "Experimental Procedures." Results from one out of three representative experiments are shown as mean ± S.E. of picomoles of FPP/well. n = 3/treatment. *, p < 0.05 versus Control; **, p < 0.05 versus lovastatin. C, cells were incubated for 24 h with 0.3% methanol in the presence or absence or 10 µM lovastatin (Lov), 10 µM FPP or both lovastatin and FPP together. Following incubation, cellular proteins were extracted, separated by SDS-PAGE, and transferred to polyvinylidene difluoride or nitrocellulose membranes for the detection, by Western blotting, of lamin A and prelamin A, respectively. Results from one out of two representative experiments are shown. n = 3/treatment.

 
Effect of Mevalonate Depletion on mRNA Abundance of Genes Involved in Fatty Acid Synthesis—Ligand-mediated activation of LXR would not be expected in statin-incubated cells deficient in cholesterol and its oxidized derivatives. Increased fatty acid synthesis in these cells is therefore unlikely to be due to increased expression of SREBP-1c and its lipogenic targets. To examine whether this was true, cells were incubated for 24 h with lovastatin alone or together with mevalonate or FPP. Total RNA was extracted, and the mRNA abundance of ACL, ACC, FAS, and SREBP-1c was determined by quantitative real-time RT-PCR. Results shown in Fig. 8 are normalized to 18 S rRNA and are expressed relative to control values. Of all the genes that were examined, only FAS mRNA was significantly increased by mevalonate depletion (Fig. 8A). Compared with control cells, mRNA levels of FAS increased 2-fold following mevalonate depletion. When mevalonate or FPP was added together with lovastatin, the abundance of FAS mRNA decreased to levels that were not different from control values (Fig. 8, A and B). Mevalonate or FPP alone did not significantly alter the expression of this gene. SREBP-1c mRNA levels were modestly decreased following mevalonate depletion and remained suppressed even after supplementation with exogenous mevalonate.



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  		FIGURE 6.
Effect of inhibition of farnesylation on fatty acid synthesis. A, cells were incubated for 24 h with 10µM lovastatin alone or together with 10µM FPP and/or increasing concentrations of FTI-277, an inhibitor of farnesyl pyrophosphate transferase. Control cells received 0.02% Me2SO and 0.3% methanol. Radiolabeled acetate was added to cells for the last 2 h of the incubation, and fatty acid synthesis was estimated as described. Results from one out of three representative experiments are shown as mean ± S.E. of percent incorporation of acetate per well relative to control. n = 2/treatment; *, p < 0.05 versus Control. B, cells were incubated for 24 h with FTI-277 at the same concentrations as indicated in A. Control cells received 0.02% Me2SO alone. As a positive control cells were incubated for 24 h with 25 µM lovastatin (Lov). Following incubation, cellular proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and the abundance of prelamin A was determined by Western blotting as described under "Experimental Procedures." Results from one out of three representative experiments are shown. n = 2/treatment.

 
The selective increase in FAS but not in ACC and ACL gene expression and the lack of increase in SREBP-1c mRNA suggests that SREBP-1c was not activated in mevalonate-depleted cells. To verify whether this was associated with unaltered levels of the active form of the SREBP-1 protein, the expression of both the precursor 125-kDa protein and of the mature cleaved product (65 kDa) was estimated in cells incubated with lovastatin in the presence or absence of mevalonate or FPP. The results are shown in Fig. 9 (A and C). Compared with control cells, in cells incubated with lovastatin the amount of the precursor protein was significantly decreased (Fig. 9, A and C). Addition of mevalonate, but not FPP, together with lovastatin prevented the inhibitory effects of lovastatin. When added alone, mevalonate and FPP had very modest effects on the abundance of the precursor protein. In contrast to the precursor protein, abundance of the mature protein was significantly less (Fig. 9A). It was either very modestly increased (Fig. 9C, bottom left panel) or remained unaltered (Fig. 9C, bottom right panel) by mevalonate depletion. Addition of either mevalonate or FPP to mevalonate-depleted cells did not alter the amount of mature protein. Similarly, neither mevalonate nor FPP alone affected the abundance of the mature protein. These results demonstrate that the precursor SREBP-1 protein is more sensitive to mevalonate depletion than the mature protein. It is therefore unlikely that depletion of mevalonate significantly affects the amount of active SREBP-1 protein. This is consistent with the lack of increase in mRNA levels of the SREBP-1c itself and its gene targets, ACC and ACL. Thus, it is unlikely that the regulation of fatty acid synthesis in mevalonate-depleted cells is dependent on SREBP-1c activation and instead is likely due to a selective increase in the expression of the FAS gene.



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  		FIGURE 7.
Effect of inhibition of {beta}-oxidation on fatty acid synthesis. Cells were incubated for 24 h with 10 µM lovastatin alone and/or 10 µM FPP in the presence or absence of 10 µM etomoxir (ETO), an inhibitor of mitochondrial {beta}-oxidation. Control cells were incubated with 0.1% ethanol and 0.3% methanol. During the last 2 h of incubation, cells were incubated with radiolabeled acetate, and its incorporation into fatty acids was estimated. Results from one of two representative experiments are shown as mean ± S.E. of picomoles of acetate incorporated/well (A) and as percent incorporation of acetate per well relative to control (B, left panel) or ETO (B, right panel). Open bar, –lovastatin; cross-hatched bar, +lovastatin. n = 3/treatment. a, p < 0.05 versus –lovastatin for each treatment; b, p < 0.05 versus vehicle-lovastatin; c, p < 0.05 versus vehicle+lovastatin.

 
Effect of Mevalonate Depletion on SREBP-2 Activation—Recently, the FAS gene was shown to be one of several targets of SREBP-2 (29). SREBP-2 would be expected to be activated under conditions of sterol deficiency. In animals fed a cholesterol depletion diet, activation of SREBP-2 was associated with increased fatty acid synthesis (30). Thus, it is possible that mevalonate depletion increases fatty acid synthesis by activating SREBP-2, and addition of FPP, a precursor for the synthesis of sterols, restores fatty acid synthesis and FAS mRNA by decreasing SREBP-2 activation. To address this possibility SREBP-2 protein expression was estimated in cells incubated for 24 h with lovastatin in the presence or absence of mevalonate or FPP. Cells were lysed, and the precursor and mature forms of SREBP-2 protein were determined by immunoblotting. Results are shown in Fig. 9 (B and D). The abundance of the precursor protein was significantly less than that of the mature protein (Fig. 9B). Mevalonate depletion decreased the amount of precursor protein in the presence of methanol, the vehicle in which FPP was delivered to cells (Fig. 9, B (bottom panel) and D (top right panel)). FPP did not prevent this decrease. In comparison to control cells, FPP alone did not alter the amount of precursor protein. The expression of precursor SREBP-2 did not appear to change with lovastatin, mevalonate, or both in the absence of methanol (Fig. 9, B (top panel) and D (top left panel)). In contrast to the precursor protein, the amount of mature SREBP-2 was dramatically increased following mevalonate depletion (Fig. 9, B and D (bottom panels)). Addition of mevalonate, but not FPP, prevented this increase. The amount of mature SREBP-2 was not significantly different between cells incubated with FPP and lovastatin and cells incubated with lovastatin alone. Moreover, the amount of mature protein in cells incubated with lovastatin and FPP remained significantly greater than the amount of protein in control cells. Neither mevalonate nor FPP alone altered the abundance of the mature protein. Thus, FPP does not prevent the increase in amount of mature active SREBP-2 in mevalonate-depleted cells.



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  		FIGURE 8.
Effects of mevalonate depletion and FPP on mRNA levels of SREBP-1c and enzymes involved in fatty acid synthesis. A, cells were incubated for 24 h with 10 µM lovastatin (Lov) alone or together with 5 mM mevalonate (Mev). B, another set of cells was incubated with vehicle alone (0.3% methanol) or with 10 µM Lov and/or 10 µM FPP. Total RNA was extracted and mRNA levels of SREBP-1c, ACC, ACL, and FAS estimated by quantitative real-time RT-PCR. Results from one of three representative experiments were normalized to 18 S rRNA levels and expressed as mean ± S.E. of arbitrary units relative to control. n = 3/treatment. *, p < 0.05 versus control; **, p < 0.05 versus lovastatin alone.

 
These results suggest that FPP does not interfere with the increased transcription of SREBP-2 target genes in mevalonate-depleted cells. To verify whether this was true the mRNA levels of four SREBP-2 target genes, HMG-CoA synthase, HMG-CoA reductase, FPP synthase, and squalene synthase were examined in cells incubated with lovastatin alone or in the presence of mevalonate or FPP. The results are shown in Fig. 10 (A–D). As expected, mRNA levels of all four gene targets of SREBP-2 were increased in cells incubated with lovastatin consistent with increase in SREBP-2 activation in sterol-depleted cells. Addition of mevalonate, the precursor for sterols, prevented this increase (Fig. 10, A–D (top panels)). Addition of FPP, however, did not alter the effects of lovastatin on HMG-CoA synthase, HMG-CoA reductase, and squalene synthase and only modestly attenuated the effects of lovastatin on FPP synthase mRNA levels (Fig. 10, A–D (bottom panels)). In other experiments FPP synthase mRNA levels, despite being attenuated by ~20% in mevalonate-depleted cells by the addition of FPP, remained elevated at values at least 2-fold greater than control (data not shown). These results are consistent with the effects of FPP on SREBP-2 protein expression in mevalonate-depleted cells. Thus, SREBP-2 activation is probably not involved in FPP-mediated inhibition of fatty acid synthesis and FAS mRNA in mevalonate-depleted cells.

Effect of Inhibition of Transcription on Fatty Acid Synthesis—In addition to transcriptional regulation, the activity and steady-state mRNA levels of enzymes involved in fatty acid synthesis may be regulated post-transcriptionally (31, 32). To address whether the increase in fatty acid synthesis and FAS mRNA in mevalonate-depleted cells could have occurred by post-transcriptional mechanisms, cells were incubated with lovastatin in the presence or absence of the general inhibitor of transcription, actinomycin D. Fatty acid synthesis and FAS mRNA abundance were then estimated. Inhibition of transcription prevented the increase in fatty acid synthesis (Fig. 11A) and FAS mRNA (Fig. 11B) observed in mevalonate-depleted cells without altering cell viability. Thus, mevalonate depletion likely enhances fatty acid synthesis and FAS mRNA by transcriptional mechanisms.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In cells depleted of mevalonate and all of its derivatives, including oxysterols, fatty acid synthesis was enhanced. This occurred by a mechanism that was likely independent of SREBP-1c activation, because neither gene expression of SREBP-1c itself nor of its targets, ACC and ACL, was increased in mevalonate-depleted cells. In fact, compared with control cells, in cells incubated with lovastatin there was a modest decrease in SREBP-1c mRNA. This was accompanied by decreased levels of SREBP-1 precursor protein levels. The amount of the mature protein, however, remained unaltered suggesting a lack of increase in SREBP-1 activation. The difference in response of the mature and precursor protein to mevalonate depletion is likely due to the differential regulatory mechanisms of SREBP-1 activation. Both transcriptional and post-transcriptional regulation of SREBP proteins has been described (1). Lack of cholesterol availability in mevalonate-depleted cells would inhibit oxysterol-mediated activation of LXR and LXR-dependent gene transcription of SREBP-1c. This would lead to decreased synthesis of SREBP-1c protein. This is consistent with a lower amount of precursor SREBP-1 protein in mevalonate-depleted cells. As considerably more precursor than mature protein was detected, mevalonate depletion decreased total SREBP-1 protein levels as well (data not shown). In contrast, post-transcriptional mechanisms increase the amount of mature active SREBP-1 protein under conditions of sterol deficiency. Depletion of mevalonate and sterols would promote sterol-sensitive SREBP cleavage-activating protein-mediated escort of SREBP-1 from the endoplasmic reticulum to the Golgi where cleavage of the precursor SREBP-1 protein to its mature isoform occurs. While the amount of mature protein did not increase in cells incubated with lovastatin, the relative amount of precursor protein that was cleaved to its mature isoform was significantly greater in these cells as compared with control cells. Thus, post-transcriptional regulation was likely responsible for maintaining the amount of mature protein despite decreased SREBP-1 precursor and total protein levels in cells depleted of mevalonate. The lack of increase in mature SREBP-1 protein in mevalonate-depleted cells compared with control cells was consistent with the inability of mevalonate depletion to cause a consistent increase in gene expression of all the lipogenic targets of SREBP-1c examined. Only FAS mRNA levels were increased in cells incubated with lovastatin, whereas mRNA levels of other enzymes, ACC and ACL, remained unaltered. In a previous study incubation with a concentration of a statin, 5-fold greater than was used in the present study, significantly decreased SREBP-1c mRNA levels as well as SREBP-1 precursor and mature protein levels (33). Our results, although following the same trend as this previous study, were less dramatic, and this is probably due to the differences in concentrations of statins used. It is possible that, at these high concentrations, statins would suppress SREBP-1c gene transcription and protein synthesis enough to decrease the amount of mature protein as well. Even if high doses of statin were to similarly affect CaCo-2 cells and decrease SREBP-1c activation, it is unlikely that they would decrease fatty acid synthesis as well. In results not shown, fatty acid synthesis, compared with control cells, was increased in cells incubated with lovastatin at concentrations 10-fold greater that what we report in the present study.



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  		FIGURE 9.
Effects of mevalonate depletion and FPP on SREBP activation. Cells were incubated for 24 h with 10 µM lovastatin (Lov) alone or together with 5 mM mevalonate (Mev) (top panels). Another set of cells was incubated with 0.3% methanol alone or with 10 µM Lov and/or 10 µM FPP (bottom panels). Cells were lysed and precursor and mature SREBP-1 (A) and SREBP-2 (B) proteins were separated by SDS-PAGE and estimated by Western blotting. Representative autoradiograms from one out of two experiments are shown. Intensity of bands corresponding to the precursor and mature SREBP-1 (C) and SREBP-2 (D) proteins are shown as mean ± S.E. densities relative to control. *, p < 0.05 versus control; **, p < 0.05 versus lovastatin alone. n = 3/treatment.

 
Unlike other enzymes in the fatty acid biosynthetic pathway, such as ACC, which are regulated both transcriptionally and post-transcriptionally, FAS is primarily regulated at the level of gene transcription (31, 32). The induction in FAS mRNA in refed animals was inhibited by an inhibitor of gene transcription and was shown to be due to increased promoter activity of the FAS gene (32). Consistent with this observation, we observed that the increase in FAS mRNA and fatty acid synthesis following mevalonate depletion was prevented in the presence of an inhibitor of gene transcription. The FAS gene is under tight transcriptional control by several hormonal and nutritional factors many of which recruit SREBP-1c for mediating their effects (31, 32). However, regulation of fatty acid synthesis and of FAS gene transcription also occurs independently of SREBP-1 (15, 30, 34, 35). Fatty acid synthesis was still maintained, albeit at levels lower than control, in cells defective in SREBP processing suggesting regulation by factors other than SREBP-1 (34). Similarly, involvement of an SREBP-1-independent mechanism was also demonstrated in the nutritional regulation of the FAS gene. SREBP-1 could only partially account for the increase in FAS gene transcription in fasted rats fed a high glucose diet (35). In the intestinal epithelial cells, an influx of saturated, monounsaturated, and polyunsaturated fatty acids all suppressed de novo fatty acid synthesis but only the polyunsaturated fatty acids decreased SREBP-1 protein and SREBP-1c mRNA levels (15). Similarly, dramatic changes in cholesterol flux in the intestine altered fatty acid synthesis by a mechanism that was independent of SREBP-1c (30). It was suggested that regulation of fatty acid synthesis following changes in cholesterol flux involved SREBP-2. This notion is supported by the observation that the FAS gene is a target for SREBP-2 (29). Because SREBP-2 is activated under conditions of sterol deficiency, we examined whether the selective increase in FAS mRNA in cells depleted of mevalonate could have been due to increased activation of SREBP-2. This possibility, however, was ruled out, because FPP, which prevented the increase in fatty acid synthesis and FAS mRNA in statin-treated cells, did not alter the increase in gene expression of SREBP-2 targets, HMG-CoA synthase, HMG-CoA reductase, and squalene synthase. The increase in FPP synthase mRNA in mevalonate-depleted cells was only modestly attenuated by the addition of FPP. Moreover, FPP was unable to prevent the increase in mature SREBP-2 observed in cells treated with lovastatin. This is surprising given that FPP is a precursor for the synthesis of sterols and should therefore have caused a sterol-dependent decrease in the activation of SREBP-2 and the expression of its target genes. However, it is possible that at the concentration used, 10 µM, FPP was not sufficiently converted to sterols. This is consistent with the inability of cholesterol to mimic the effects of FPP on fatty acid synthesis in mevalonate-depleted cells. These results strongly suggest an SREBP- and sterol-independent mechanism of regulation by FPP of fatty acid synthesis in mevalonate-depleted cells.



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  		FIGURE 10.
Effects of mevalonate depletion and FPP on mRNA levels of HMG-CoA synthase, HMG-CoA reductase, FPP synthase, and squalene synthase. Cells were incubated for 24 h with 10 µM lovastatin (Lov) alone or together with 5 mM mevalonate (Mev). Another set of cells was incubated with 0.3% methanol alone or with 10 µM lovastatin and/or 10 µM FPP. Total RNA was extracted and mRNA levels of HMG-CoA synthase (A), HMG-CoA reductase (B), FPP synthase (C), and squalene synthase (D) were estimated by quantitative, real-time RT-PCR. Results from one of three representative experiments were normalized to 18 S rRNA levels and expressed as mean ± S.E. of arbitrary units relative to control. n = 3. *, p < 0.05 versus control; **, p < 0.05 versus lovastatin alone.

 



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  		FIGURE 11.
Effects of actinomycin D on fatty acid synthesis and FAS mRNA. Cells were incubated for 24 h with 10 µM lovastatin alone or together with 4 µM actinomycin D (Act D) in 0.05% ethanol. Control cells received vehicle alone. At the end of the incubation, fatty acid synthesis (A) and FAS mRNA (B) were estimated as described in Figs. 2 and 8, respectively. Results from one out of two representative experiments are shown as mean ± S.E. Open bar, –lovastatin; cross-hatched bar, +lovastatin. n = 3/treatment. *, p < 0.05 versus control.

 
Besides SRE binding sites, other regions in the FAS promoter confer transcriptional regulation. LXR has been shown to bind directly to its response elements located in the promoter region of the FAS gene and activate transcription of the gene even in the absence of SREBP-1c (36). If LXRs were mediating the effects of mevalonate depletion on fatty acid synthesis it would be unlikely that it was acting in its ligand-bound state. Transcriptional activity of LXR can be modulated in the absence of its ligand by altering its interaction with co-repressors of transcription; in the presence of excess repressors basal transcriptional activity of LXR is reduced (37). One of the transcriptional repressors of LXR is the small heterodimer partner (SHP) and was found to interact with LXR even in the absence of its ligand (38). SHP is a target gene of the nuclear receptor for bile acids, FXR, which is activated by high concentrations of FOH (39–41). Because FXR is abundantly expressed in the intestine (42), we postulated that activation of FXR by FPP in CaCo-2 cells would increase expression of SHP, which in turn, would repress LXR-mediated activation of its target genes. This notion is consistent with the recent observation that activation of FXR decreases lipogenesis via increased SHP transcription and SHP-mediated repression of LXR activity (43). This possibility, however, was found to be unlikely, because we were unable to observe an increase in SHP mRNA levels following incubation of mevalonate-depleted cells with FPP (results not shown). Furthermore, FPP and FOH were used at much lower concentrations in the present study than what were initially shown to activate FXR (39). Although these results do not rule out the possibility that LXR, in its basal unliganded state, could mediate the effects of mevalonate depletion on fatty acid synthesis, they do not support a mechanism involving SHP.

A unique site in the FAS promoter has been identified to mediate the stimulatory effects of glucose independently of insulin (44). This site, termed the carbohydrate response element, is located upstream of the insulin response sequence and includes a CCAAT box that binds CCAAT enhancer-binding protein (C/EBP). Interestingly, statins have been shown to augment the transcription of CCAAT-containing genes, such as, tumor necrosis factor-{alpha}, interleukin-12p40, and cyclooxygenase-2, in a (C/EBP)-dependent manner (45, 46). Furthermore, similar to the results obtained in the present study, the effects of statins on interleukin-12p40 could be reversed by mevalonate and isoprenoids but not by sterols (45). Because C/EBP is expressed in the intestine (47) and has been shown to promote lipogenesis by increasing the transcription of FAS (48), it is possible that the stimulation of fatty acid synthesis in cells incubated with lovastatin was due to enhanced C/EBP-mediated induction in FAS gene transcription. The mechanism(s) by which FPP and/or its metabolites regulate the expression of this transcription factor, its binding to promoter elements in the FAS gene, or its interaction with repressors or activators of transcription need to be elucidated.

FOH and, to a lesser extent, geranylgeraniol have been shown to induce the activity of peroxisome proliferator-activated receptor and the transcription of peroxisome proliferator-activated receptor-responsive genes involved in fatty acid {beta}-oxidation (24). Because fatty acid oxidation and synthesis are coordinately regulated in a reciprocal manner, FPP could have prevented the increase in fatty acid synthesis in statin-treated cells by increasing its oxidation. This possibility was ruled out, because inhibition of mitochondrial {beta}-oxidation of fatty acids did not alter the effects of FPP in mevalonate-depleted cells. This observation is not entirely unexpected. We have previously observed that fatty acids are not significantly oxidized in the gut (49). Unlike the liver, {beta}-oxidation of fatty acids in the intestine is not a significant metabolic pathway for fatty acids and therefore does not likely account for changes in fatty acid synthesis in this organ.

Although FPP is a precursor of sterols, its effects on fatty acid synthesis were not mediated by sterols. Neither were its effects due its other downstream derivatives, GGPP and ubiquinone, or due to farnesylated proteins. Instead, results from the present study suggest that the effects of FPP were due to the isoprenoid itself, its alcohol derivative, FOH, or its degradation products. A previous study demonstrated that FOH suppresses triglyceride synthesis in the liver (50). It was suggested that the effects of the isoprenoid were due to its dicarboxylic acid degradation products (50, 51). Whether FPP is degraded in non-hepatic tissues is unknown. Future studies are underway to examine the intracellular metabolism of FPP in the intestine and to further explore possible mechanisms by which the isoprenoid and/or its metabolites regulate fatty acid synthesis.

Results from the present study demonstrate that, besides sterols, the mevalonate pathway also provides another product, the upstream non-sterol isoprenoid, FPP, that has regulatory effects on fatty acid synthesis. To the best of our knowledge, the effects of this isoprenoid on fatty acid synthesis have not been reported so far. We demonstrate that, in contrast to sterols, FPP regulates fatty acid synthesis by a mechanism that is likely independent of SREBP but one that involves transcription of the FAS gene. A systematic study of the FAS promoter will enable the identification of one or more regions that are regulated by FPP and possibly of transcription factors that mediate the effects of the isoprenoid. We believe that results presented in the present study form the basis for such future studies. Our results suggest that fatty acid synthesis may be differentially regulated by sterol and non-sterol products of the mevalonate pathway and that flux through the pathway could be an important factor in the regulation of lipogenesis.


   FOOTNOTES
 
* This work was supported by the Roy J. Carver Charitable Trust as a Research Program of Excellence and the Roland W. Holden Family Program for Experimental Cancer Therapeutics. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Internal Medicine, 5219 MERF, 375 Newton Road, University of Iowa, Iowa City, IA 52242. Tel.: 319-335-8272; Fax: 319-335-8891; E-mail: murthys{at}mail.medicine.uiowa.edu.

2 The abbreviations used are: SREBP, sterol regulatory element-binding protein; LXR, liver X receptor; ACC, acetyl-Coenzyme A carboxylase; ACL, ATP-citrate lyase; FAS, fatty acid synthase; FOH, farnesol; FPP, farnesyl pyrophosphate; FTI, farnesyl pyrophosphate transferase inhibitor; FXR, farnesoid X receptor; GPP, geranyl pyrophosphate; geranylgeraniol, geranylgeraniol; GGPP, geranylgeranyl pyrophosphate; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; IPP, isopentenyl pyrophosphate; RXR, retinoid X receptor; C/EBP, CCAAT enhancer-binding protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Scott Kaufmann (Mayo Clinic, Rochester, MN) for his generous gift of the prelamin A antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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