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Endocrinology Vol. 145, No. 12 5493-5503
Copyright © 2004 by The Endocrine Society

Valproic Acid Inhibits Leptin Secretion and Reduces Leptin Messenger Ribonucleic Acid Levels in Adipocytes

Diane C. Lagace, Roger S. McLeod and Mark W. Nachtigal

Departments of Pharmacology (D.C.L., M.W.N.) and Biochemistry and Molecular Biology (R.S.M.), Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5

Address all correspondence and requests for reprints to: M. W. Nachtigal, Department of Pharmacology, 5850 College Street, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5. E-mail: mark.nachtigal{at}dal.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment of epilepsy or bipolar disorder with valproic acid (VPA) induces weight gain and increased serum levels for the satiety hormone, leptin, through an unidentified mechanism. In this study we tested the effects of VPA, a short-chain branched fatty acid (C8:0), on leptin biology and fatty acid metabolism in 3T3-L1 adipocytes. VPA significantly reduced leptin secretion in a dose-dependent manner. Because fatty acid accumulation has been hypothesized to block leptin secretion, we tested the effect of VPA on fatty acid metabolism. Using 14C-radiolabeled VPA, we found that the 14C was mainly incorporated into triacylglycerol. VPA did not alter lipogenesis from acetate, nor did it change the amount of intracellular free fatty acids available for triacylglycerol synthesis. Decreased leptin secretion was accompanied by a reduction in leptin mRNA, even though VPA treatment did not alter the protein levels for known transcription factors affecting leptin transcription including: CCAAT/enhancer binding protein-{alpha}, peroxisome proliferator-activated receptor-{gamma}, or steroid regulatory element binding protein 1a. VPA altered levels of leptin mRNA independent of de novo protein synthesis without affecting leptin mRNA degradation. This report demonstrates that VPA decreases leptin secretion and mRNA levels in adipocytes in vitro, suggesting that VPA therapy may be associated with altered leptin homeostasis contributing to weight gain in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VALPROIC ACID (VPA) is indicated for the treatment of epilepsy and bipolar disorder and in the prevention of migraine headaches. VPA has also become more widely prescribed due to several off-label indications such as in the treatment of neuropathic pain and cancer (reviewed in Refs. 1 , 2). Despite VPA being well tolerated and having a low incidence of serious side effects, one concern with VPA therapy is weight gain. A prospective study identified that 37% of female patients with epilepsy developed obesity, as defined as a body mass index (BMI) greater than 25, after 1 yr of treatment with VPA (3). Numerous retrospective and cross-sectional analyses also report that treatment with VPA is associated with a significant increase in weight ranging from 5 to 49 kg (reviewed in Refs. 4, 5, 6, 7). Studies examining VPA-induced weight gain have been conducted predominantly in adult women because VPA can induce a number of reproductive endocrine abnormalities that include hyperandrogenism, menstrual disturbances, weight gain, and/or polycystic ovaries (reviewed in Refs. 8, 9, 10). Fifty-two percent of males treated with VPA, however, also have BMI scores within the obesity category (11), and youth and adolescents treated with VPA are reported to have BMI scores over expected age norms (12, 13, 14). Similarly, a prospective double-blind comparison of the incidence and magnitude of weight gain in patients receiving VPA vs. lamotrigine monotherapy demonstrated that weight gain was greater for those patients treated with VPA and was significant within 10 wk of treatment onset (15, 16). Weight gain associated with VPA treatment is of great concern due to its physical and psychological consequences (17). Notably, obesity leads to increase risk for numerous other diseases, such as diabetes mellitus, coronary heart disease (18), and increased noncompliance with pharmacotherapy in psychiatric patients (19).

The mechanism underlying VPA-induced weight gain has not been elucidated. Age, gender, medical condition, dose and serum concentrations of VPA, and family history of body weight problems are not significantly correlated with the gain in weight associated with VPA treatment (12, 20, 21). In attempts to generate animal models of VPA-induced weight gain, VPA has been shown to induce a significant increase in body weight in female rhesus monkeys (22); however, we and others have demonstrated that VPA does not cause weight gain in rodents (23, 24, 25). The etiology of VPA-induced weight gain is most likely multifactorial because weight is the output of energy homeostasis controlled by many organs that produce and secrete a variety of appetite-regulating peptides and cytokines that act within the hypothalamus (reviewed in Ref. 26). VPA treatment in humans increases the serum level of two hormones, leptin and insulin, which are produced by the adipose tissue and pancreatic ß-cells, respectively. After VPA treatment for 1 yr, 37% of female patients with epilepsy who developed obesity had a 1.8-fold increase in fasting serum insulin and 3.4-fold increase in serum leptin levels (3). Similarly, in women receiving VPA for treatment of bipolar disorder, insulin and leptin levels were significantly elevated when compared with women receiving lithium (27). High levels of serum leptin are commonly associated with obesity and could represent a state of leptin resistance (reviewed in Refs. 26 , 28). The increase in serum leptin associated with weight gain after VPA treatment may be a consequence of the increase in adipose tissue; however, it is also possible that VPA may have a direct effect on leptin secretion from adipocytes or may alter leptin signaling and decrease negative feedback. VPA has been shown to have direct effects on hormone secretion from other endocrine cells. For example, an ex vivo study using human pancreatic islet cells has shown that VPA can directly increase insulin release (29). Moreover, VPA can also potentiate androgen production from ovarian theca cells (30).

We previously demonstrated that VPA inhibited mouse 3T3-L1 and human preadipocyte differentiation (31). Treatment with VPA during adipogenesis reduced the protein levels for several key adipocyte-specific transcription factors, including CCAAT/enhancer binding protein (C/EBP)-{alpha}, peroxisome proliferator-activated receptor (PPAR)-{gamma}, and steroid regulatory element binding protein (SREBP) 1a (reviewed in Ref. 32). The present work demonstrates that treatment with VPA in mature adipocytes significantly reduces leptin mRNA levels and secretion of the leptin protein in a dose- and time-dependent manner. These findings were paradoxical because treatment of patients with VPA is associated with increased serum leptin levels. The reduction in leptin secretion from adipocytes was not accompanied by alterations in glucose uptake or altered intracellular free fatty acid levels, which are known regulators of leptin secretion. In addition, C/EBP{alpha}, PPAR{gamma}, or SREBP1a protein levels did not change with VPA treatment, suggesting the levels of these transcription factors are not responsible for the effect of VPA on leptin expression. Evidence from experiments using actinomycin D (ActD) or cyclohexamide (CHX) show that VPA does not promote degradation of leptin mRNA; however, VPA can alter leptin transcription through an unknown mechanism independent of new protein synthesis. These results show that VPA can have direct effects on adipocytes that may contribute to altered energy balance in patients treated with VPA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and differentiation
Mouse 3T3-L1 cells were obtained from the American Type Culture Collection (Manassas, VA) and subcultured in 5% CO2 at 37 C. Cells were maintained in high-glucose DMEM (Invitrogen, Carlsbad, CA), with 10% heat-inactivated calf serum (Invitrogen) and penicillin G/streptomycin sulfate (Invitrogen). Media were changed every 2 d, and preadipocytes were maintained at less than 50% confluence. Two days after reaching confluence, preadipocytes were treated with medium to induce differentiation [MDI; DMEM, 10% fetal bovine serum (FBS; CanSera), 250 nmol/liter dexamethasone (Sigma, St. Louis, MO), 0.5 mmol/liter 3-isobutyl-1-methylxanthine (Sigma), and 100 nmol/liter human insulin (Roche Molecular Biochemicals, Indianapolis, IN)]. After 2 d in MDI, preadipocytes were cultured in DMEM containing 10% FBS, and 850 nmol/liter insulin. Subsequent medium changes occurred every second day. Valproic acid sodium salt (VPA, Sigma) was dissolved in PBS and added to media as indicated in the figure legend.

Leptin assay
Leptin concentrations were determined using a RIA for mouse/rat leptin produced by Alpco Diagnostics (catalog no.022-LEP-R61) as per the manufacturer’s instructions (Fig. 1Go, A and B). The sensitivity of this assay is 6 pg/ml and the intraassay variation is lower than 5%. Due to the discontinuation of the Alpco Diagnostics RIA kit during the course of these studies, subsequent leptin concentrations (Fig. 1CGo) were determined using the mouse/rat leptin ELISA produced by Alpco Diagnostics (catalog no. 022-LEP-E06) using undiluted samples. The sensitivity of this assay is 10 pg/ml and the intraassay variation is less than 4.4%. After the induction of differentiation with MDI, the cells were routinely cultured as described above and then serum starved for 2 h before treatment for 12 h as described in the legend for Fig. 1CGo.



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  		FIG. 1. VPA treatment decreases constitutive and insulin-induced leptin secretion but does not alter intracellular leptin levels in mouse 3T3-L1 adipocytes. A, Cells treated with VPA (0.25–5 mM) for 6–24 h in the presence of DMEM, 10% FBS, and insulin (850 nM) have a concentration- and time-dependent decrease in leptin secretion into the medium. B, Whole-cell lysates (same cells as in A) have no significant alterations in intracellular leptin levels. In A and B, data represent mean ± SEM from triplicate samples per time point per group as measured by RIA; the experiment was repeated with similar results, significantly different from UT at each time point as analyzed by post hoc comparisons based on Bonferroni (*, P < 0.05; **, P < 0.01). C, Cells were serum starved for 2 h and then treated for 12 h in the presence (+) or absence (–) of VPA and DMEM, DMEM and 10% FBS, or DMEM and 850 nM insulin. Leptin in conditioned medium was measured by ELISA. Data represent mean ± SEM from two independent experiments performed in triplicate, significantly different from UT (–) as assessed for each treatment condition as analyzed by paired t test (*, P < 0.05; **, P < 0.01).

 
Glucose uptake
Glucose uptake was determined similar to the method of Yang et al. (33). Briefly, differentiated adipocytes in six-well plates were washed twice with serum-free DMEM and serum starved for 2 h in DMEM containing 25 mM glucose and 2 mM glutamine. Medium was replaced with 1 ml Krebs-Ringer-HEPES buffer [121 mM NaCl, 4.9 mM KCl, 1.2 mM MgS04, 0.33 mM CaCl2, 12 mM HEPES (pH 7.4)] per well. Cells were incubated in the presence or absence of 100 nM insulin and presence or absence of VPA (1 mM) and labeled for the last 10 min of incubation with 2-deoxy-D-[2,6-3H]glucose (43 Ci/mmol; 1 µCi/ml). Uptake was stopped by three rapid washes with ice-cold Krebs-Ringer-HEPES buffer, cells were solubilized in 1 ml of 0.1 M NaOH, and radioactivity was measured in a 300-µl aliquot of the sample in acidified liquid scintillation fluid.

Uptake of [14C] radiolabeled VPA
Cells were treated with 10 µM VPA (1-14C) sodium salt ([14C]VPA; 55 mCi/mmol; American Radiolabeled Chemicals Inc., St. Louis, MO) in normal growth medium. After treatment cells were washed three times with PBS and then solubilized in 500 µl of 0.1 M NaOH for 1 h at room temperature. Total cellular radioactivity was determined for a 100 µl aliquot of each sample in acidified liquid scintillation fluid. Counts were normalized to total protein per sample. Protein was quantified using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). On average 0.2% of the [14C]-labeled VPA added to the medium was intracellular after 12 h of treatment.

Analysis of lipid metabolism
Cells were treated with 20 µM [14C]VPA (specific activity 55 mCi/mmol) plus 980 µM VPA to bring the total concentration to 1 mM. For experiment with triacsin C, cells were simultaneously treated with radiolabeled VPA (20 µM [14C]VPA + 980 µM VPA) and triacsin C (9.6 µM) for 2 h. For control experiment with triacsin C, cells were simultaneously treated with [3H]oleate (100 mM, specific activity 4.2 dpm/pmol) and triacsin C (9.6 µM) for 2 h. Cells were rinsed twice with ice-cold PBS, scraped into 1 ml PBS with a rubber policeman, and sonicated for 1 min. Lipids were extracted by the addition of 4 ml isopropanol/3 N sulfuric acid (40:1, vol/vol), 2 ml distilled water, and 5 ml hexane. After 10 min of end-over-end mixing, the upper hexane phase was collected and the solvent was evaporated under nitrogen. The residual lipid phase was resuspended in chloroform. Samples were resolved by thin-layer chromatography (TLC) in hexane/diethyl ether/acetic acid (70:30:1, vol/vol/vol) and lipids identified using lipid standards (Sigma, 99F7817).

For analysis of lipogenesis, cells were pulse labeled with [14C]acetic acid (250 nCi/ml, Amersham Biosciences, Piscataway, NJ) for 2 h at 37 C, and incorporation into newly synthesized lipids was measured. After the labeling period, cells were rinsed twice with ice-cold PBS, scraped into 1 ml methanol/water (5:4, vol/vol) with a rubber policeman, and sonicated for 1 min. Lipids were extracted using chloroform/methanol (1:2, vol/vol) and phases separated by the addition of 0.58% NaCl. The lower organic phase was washed and extracted three times with ideal upper phase buffer (methanol/0.58% NaCl/chloroform, 45:47:3, vol/vol/vol), evaporated, and then resuspended in chloroform. Lipid standards (Sigma, 99F7817) and radiolabeled lipids were resolved by TLC in petroleum ether/diethyl ether/acetic acid (60:40:1, vol/vol/vol).

Standards were visualized with iodine vapor. Radiolabeled lipids identified by comigration with standards were scraped into vials and quantified by scintillation counting.

Analysis of fatty acids
For analysis of total fatty acids, cells were treated for 12 h with 1 mM VPA, rinsed twice with ice-cold PBS, scraped into 1 ml NaOH with a rubber policeman, and sonicated for 1 min.

Fatty acids were extracted as previously described (34).

For analysis of free fatty acids, cells were treated with 1 mM VPA for 12 h, rinsed twice with ice-cold PBS, scraped into 1 ml PBS with a rubber policeman, and sonicated for 1 min. Lipids were extracted into acidified isopropanol and then hexane as described above (Analysis of lipid metabolism). Free fatty acids were then partitioned from the hexane and separated into the aqueous phase through the addition of 1 ml of 0.1 N KOH, and mixed for 10 min. To remove residual neutral lipids, the KOH phase was reextracted with 5 ml hexane. The KOH phase was then neutralized with 50 µl glacial acetic acid, and free fatty acids were reextracted with 6 ml chloroform/methanol (2:1, vol/vol), and 50 µg internal standard (methyl heptadecanoic acid C17:0) were added to each sample.

Total or free fatty acids were passed through a sodium sulfate column to remove water and dried under nitrogen. Fatty acids were esterified by incubation with 1 ml of 20% methanol in benzene and 0.5 ml of 0.2 M (trimethylsilyl) diazomethane (Sigma) in hexane for 30 min with occasional shaking. Acetic acid was added as required to neutralize the solution and 2 ml distilled water and 2 ml petroleum ether were added to separate the methyl esters. The organic phase was collected and purified on a sodium sulfate column. Effluent samples were evaporated under nitrogen and then fractionated by gas chromatography (35). Standard mixtures of fatty acids were used to identify peaks that were quantified using the C17:0 as internal standard.

Northern blot analysis
RNA was isolated using Trizol reagent (Invitrogen). For experiments with ActD and CHX, cells were pretreated with ActD (1 µg/ml), CHX (10 µg/ml), or dimethylsulfoxide [vehicle control (1 µl/ml)] for 30 min and then cotreated in the presence or absence of VPA for 4 h before cell harvest. For experiments with trichostatin A (TSA), cells with treated with 3 nM TSA for 6 h before harvesting RNA. Twenty to thirty micrograms of total RNA were separated on a 1.5% agarose/0.67% formaldehyde gel and transferred to BrightStar Plus membrane (Ambion Inc., Austin, TX). The mouse leptin, PPAR{gamma}, C/EBP{alpha}, SREBP1a, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were labeled with [{alpha}-32P] dATP (2 µCi/ml) and blots hybridized at 42 C in ULTRAhyb buffer (Ambion Inc.). Signals were visualized by autoradiography, quantified as described previously (31), and reported as arbitrary units from phosphoimage analysis.

Western blot analysis
Cells were washed two times in ice-cold PBS, extracts were prepared using high-salt lysis buffer [10 mM Tris-Cl (pH7.4), 500 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, 1% Triton X-100, 1 x complete protease inhibitors (Roche), and 1 mM phenylmethylsulfonyl fluoride]. Cell suspensions were sonicated, clarified by centrifugation (10 min at 15,000 x g), and quantified by Bradford assay (Bio-Rad Laboratories). For SREBP1a analysis, cells were treated for 4 h before harvesting and again during lysis with 25 µg/ml of the proteasome inhibitor, N-acetyl-leucine-leucine-norleucinal (Sigma). Twenty to forty micrograms of protein per lane were used for Western blot analysis. Proteins were detected with antibodies from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) including anti-PPAR{gamma} (E-8), anti-SREBP1a (2A4), anti-C/EBP{alpha} (14AA), or rabbit polyclonal antiactin (Sigma), as previously described (31). Signal was quantified and reported as arbitrary units from densitometric analysis.

Statistics
ANOVA was used to compare overall group means. A significant overall F test was followed by post hoc comparisons using the Bonferroni multiple comparison procedure. For comparison of two groups, a paired t test was used. All tests were conducted using SPSS (base system 10, SPSS Inc., Chicago, IL) with a P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VPA reduces leptin secretion
Differentiated mouse 3T3-L1 adipocytes share many similarities with human white adipose tissue (36, 37). 3T3-L1 adipocyte differentiation was confirmed by Oil Red O staining to identify triacylglycerol (TAG) droplets, as previously described (31). Adipocytes were incubated in vitro with VPA (0.25–5 mM) for 6–24 h in the presence of FBS (10%) and insulin (850 nM), and leptin secretion into the medium was measured by RIA. As expected, leptin accumulates in the medium of untreated cells over time (Fig. 1AGo). Treatment with VPA significantly decreased leptin secretion, and there was a significant effect of time and an interaction between time and treatment on leptin secretion. Post hoc analysis demonstrated that treatment with therapeutically relevant doses of VPA (0.5–1 mM) significantly inhibited leptin secretion at the earliest time point of 6 h.

The secretion of leptin can be regulated by alterations in leptin synthesis, export from the endoplasmic reticulum, or changes in the amount of preexisting pools of vesicles containing leptin (38, 39). VPA did not affect the amount of intracellular leptin (ranged from 33 to 76 pg leptin per 50 µg protein), compared with untreated samples (Fig. 1BGo). Thus decreased leptin secretion was not due to accumulation of intracellular leptin.

Cultured adipocytes can secrete leptin in either the absence or presence of leptin secretagogues, such as insulin or glucose. To determine whether VPA affected constitutive or induced leptin secretion, we measured leptin secretion from adipocytes grown in serum-free medium, medium containing 10% FBS, or medium containing 850 nM insulin (Fig. 1CGo). Basal leptin secretion was unaffected by serum but increased more than 2-fold in response to insulin. Cotreatment with VPA significantly reduced both constitutive and insulin-induced leptin secretion. Moreover, VPA attenuated leptin secretion in response to increasing doses of insulin (10–850 nM; data not shown).

VPA does not alter glucose uptake
Glucose is one of several circulating factors that is known to alter leptin secretion from adipocytes (40, 41, 42, 43). We investigated the effect of VPA on basal and insulin-sensitive (33) glucose uptake by differentiated 3T3-L1 cells. There was a significant effect of treatment, time and interaction between time and treatment on glucose uptake (Fig. 2Go). Post hoc analysis demonstrated a significant increase in glucose uptake in cells treated with insulin or insulin and VPA when compared with untreated (UT) cells at both 30 and 60 min. Treatment with VPA did not alter the uptake of glucose in either the presence or absence of insulin (no significant differences between UT and VPA or between insulin and VPA + insulin). Similar results were obtained with VPA treatment for up to 24 h and VPA treatment in the presence of increasing doses of insulin (2, 5, 10, 50, 100 nM; data not shown). These data suggest that the VPA-induced decrease in leptin secretion is not mediated by alterations in glucose uptake.



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  		FIG. 2. VPA treatment does not alter glucose uptake in the absence or presence of insulin. Uptake of 2-deoxy-D-[2,6-3H]glucose was measured in cells treated with VPA (1 mM), insulin (100 nM), or VPA and insulin for 0.01–60 min. At 30 and 60 min, there was a significant increase in glucose uptake in insulin treated vs. UT cells. There was also a significant increase in glucose uptake in VPA + insulin vs. VPA-treated cells. Data represent mean ± SEM from triplicate samples; experiment was repeated three times with similar results, significantly different from UT at each time point as analyzed by post hoc comparisons based on Bonferroni (*, P < 0.05; **, P < 0.01).

 
VPA incorporation into adipocyte lipid pools
Saturated fatty acids with chain lengths of more than seven carbons and a variety of long-chain mono- and polyunsaturated fatty acids (PUFAs) can attenuate leptin secretion (44, 45, 46). The mechanism of fatty acid suppression of leptin secretion remains unknown but is suggested to be due to alterations in lipid metabolism or levels of intracellular free fatty acids (44, 45, 46). Because VPA is an eight-carbon branched chain fatty acid, we sought to determine whether VPA treatment had an effect on lipid metabolism and/or levels of intracellular free fatty acids.

VPA has been shown to be actively transported via a carrier-mediated system, such as the protein-coupled monocarboxylic acid transporter, in a human trophoblast cell line and across the rat blood-brain barrier (47, 48). There are currently no published studies examining VPA uptake by adipocytes; therefore, we used [14C]-labeled VPA to measure uptake into adipocytes. VPA uptake was linear for up to approximately 30 min after treatment and then reached a plateau at 90 min (Fig. 3AGo). Levels continued to increase up to 12 h, most likely due to metabolism of VPA and incorporation of the [14C] into intracellular lipids. Data represented at time 0 are from cells in which [14C]-labeled VPA was added and immediately removed and shows the level of radioactivity that nonspecifically adhered to the cell surface.



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  		FIG. 3. Uptake of [14C]VPA into adipocytes and incorporation of [14C] label into TAG. A, Adipocytes were treated with [14C]VPA for 1–720 min, and uptake of [14C] label was measured and normalized against intracellular protein content (counts per minute per microgram protein). B, Autoradiograph of a representative TLC plate demonstrating the presence of the [14C] label in different lipid pools including TAG, fatty acids, cholesterol, DAG, and PL 6 and 12 h after treatment with 1 mM [14C]VPA. Lipids standards were visualized with iodine vapor. C, Quantification of the percent of [14C] label that was incorporated into TAG after treatment with [14C]VPA for 1, 6, 12, and 24 h. Data represent mean ± SEM from triplicate samples; experiment was repeated three times with similar results.

 
Previous results in a neuronal cell line suggested that VPA is incorporated into phospholipids and neutral lipids (49). After [14C]-labeled VPA treatment of adipocytes, on average 74.6 ± 2.3% of the total cellular radioactivity was within the organic phase, with the remaining 25.3 ± 2.3% in the aqueous phase. Within the organic phase, the [14C] label was incorporated mainly into TAG and the general lipid pools of diacylglycerol (DAG), cholesterol, and phospholipids (PL) in a time-dependent manner (Fig. 3BGo). The incorporation of [14C] label into free cholesterol suggests that VPA is catabolized and broken down into acetate, which is required for cholesterol biosynthesis. More than 75% of the [14C] label in the cellular organic phase was incorporated into TAG by 6 h (Fig. 3CGo).

To be incorporated into TAG, long-chain fatty acids are normally activated by acyl-coenzyme A synthetase (ACS) and subsequently metabolized. Previous studies found that VPA was activated into valproyl-coenzyme A and subsequently undergoes complete ß-oxidation (50, 51). To determine whether VPA was activated by ACS in adipocytes, we cotreated cells with VPA and triacsin C, a known inhibitor of ACS1 and ACS4 (52) that blocks TAG synthesis (53). Cotreatment inhibited the [14C]-label incorporation into TAG by 57% (2568 ± 142 vs. 1109 ± 154 cpm/µg protein). Similar results were obtained using [3H]-labeled oleate as a control for radiolabel incorporation into TAG (58% reduction). These findings support the notion that VPA undergoes activation by ACS.

To identify whether [14C]VPA was catabolized and the [14C]-labeled acetate incorporated into the fatty acid side chain of TAG or whether the activated VPA was incorporated directly as a fatty acid, we identified the fatty acids on the glycerol backbone of TAG after VPA treatment. UT and VPA treated cells had similar long-chain fatty acids including C14:1, C16:0, C16:2, C18:0, and C18:1. VPA was not one of the TAG side chains. These data show that [14C]VPA was likely activated, metabolized to acetyl-coenzyme A, and incorporated into naturally occurring fatty acids within TAG. These results strongly suggest that the ability of VPA to decrease leptin secretion is not due to VPA altering TAG composition.

An increase in lipolysis is one mechanism suggested to cause a decrease in leptin secretion from adipocytes (46). Lipolysis occurs through the metabolism of TAG into free fatty acids, which are transported into the mitochondria to undergo ß-oxidation. Carnitine is involved in the transport of activated long-chain fatty acids through the inner mitochondria membrane to gain access to the enzymes responsible for ß-oxidation. VPA treatment in patients is known to cause carnitine deficiency (54, 55, 56, 57). Decreased carnitine levels result in accumulation of intracellular free fatty acids due to decreased mitochondrial transport of long chain fatty acids. This led us to hypothesize that VPA may decrease leptin secretion through increasing intracellular levels of free fatty acids. As shown in Fig. 4AGo, intracellular free fatty acids were measured after VPA treatment. Compared with UT cells, cells treated with VPA had similar levels of intracellular free fatty acids. In yeast VPA has been shown to significantly reduce phospholipid biosynthesis (58). Therefore, in addition to examining levels of free fatty acids, we also examined whether VPA altered the rate of lipid synthesis in adipocytes. Cells were pulse labeled with [14C]-acetic acid, which is subsequently metabolized into acetyl coenzyme A and incorporated into lipids. VPA did not alter the synthesis of TAG, free fatty acids, DAG, cholesterol, or PLs (Fig. 4BGo). Therefore, the ability of VPA to cause a decrease in leptin secretion is not associated with changes in lipid synthesis or the amount of intracellular free fatty acids.



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  		FIG. 4. VPA treatment does not alter the level of intracellular free fatty acids or lipogenesis. A, Total intracellular free fatty acids (C14:0, C16:0, C18:0, C18:1, C20:0) in adipocytes that were UT or treated with 1 mM VPA for 12 h. Free fatty acids were quantified using gas liquid chromatography and normalized against intracellular protein content (microgram free fatty acid per milligram protein). Data represent mean ± SEM from triplicate samples; experiment was repeated two times with similar results; no significant differences were observed by ANOVA analysis. B, De novo lipid synthesis of TAG, PLs, and DAG was quantified in cells treated with 1 mM VPA for 0–12 h and then pulse labeled with [14C] acetic acid. Data represent mean ± SEM from triplicate samples; experiment was repeated three times with similar results; no significant differences were observed using ANOVA analysis.

 
VPA reduces leptin mRNA
Because VPA did not affect lipid metabolism, we sought to identify other mechanisms that could explain the observed decrease in leptin levels. Northern blot analysis was conducted to determine whether VPA treatment altered leptin mRNA levels. Leptin mRNA in mature adipocytes was reduced by treatment with VPA (Fig. 5Go, A and B). Both between treatment groups and over time, there was a significant change in relative leptin mRNA levels (Fig. 5BGo). Post hoc tests indicated a significant reduction in leptin mRNA at 6 and 12 h after treatment with 1 mM VPA, compared with UT and at all time points (3, 6, and 12 h) after treatment with 5 mM VPA, compared with UT.



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  		FIG. 5. VPA treatment decreases leptin mRNA levels. A, Northern blot analysis of leptin mRNA shows decreased levels after 3 h treatment with VPA (1, 5 mM); each treatment is shown in triplicate. GAPDH is shown as a loading control. B, Quantification of leptin mRNA levels normalized against GAPDH levels. Data represent mean ± SEM from triplicate sample collected in two independent experiments, significantly different from UT at each time point as analyzed by post hoc comparisons based on Bonferroni (*, P < 0.05; **, P < 0.01). C, Northern blot analysis demonstrates treatment with 3 nM TSA for 6 h did not alter leptin mRNA levels. Data represent the ratio of leptin/GAPDH (± SEM) from duplicate samples collected in two independent experiments for untreated cells and is not significantly different for TSA-treated cells.

 
We have shown that histone deacetylase (HDAC) inhibition by VPA can indirectly suppress gene expression during adipogenesis (31). There is no literature examining the effects of HDAC inhibitors on leptin secretion or leptin mRNA levels in mature adipocytes. We found that acute treatment of adipocytes with the commonly used HDAC inhibitor TSA did not alter leptin mRNA levels (Fig. 5CGo), making it unlikely that VPA reduces leptin transcription through its HDAC inhibitory properties.

VPA does not alter C/EBP{alpha}, PPAR{gamma}, and SREBP1a mRNA or protein levels
We previously demonstrated that daily treatment with 1 mM VPA throughout adipogenesis results in decreased mRNA and protein levels for PPAR{gamma} and SREBP1a, two transcription factors that are required for adipocyte differentiation (31). Transcription of the leptin gene is regulated by C/EBP{alpha}, PPAR{gamma}, and SREBP1; thus, we tested whether acute VPA treatment (≤12 h) altered the level of these transcription factors in mature adipocytes. Post hoc analysis indicated that VPA treatment caused a significant decrease in PPAR{gamma} and SREBP1a mRNA levels at 3 h, without altering C/EBP{alpha} mRNA levels (Fig. 6Go, A and B). However, at 6 and 12 h, the amount of PPAR{gamma}, SREBP1a, and C/EBP{alpha} mRNA levels were not significantly different (Fig. 6Go, A and B). The reduction in PPAR{gamma} mRNA levels was not accompanied by changes in the amount of either PPAR{gamma}1 or PPAR{gamma}2 protein (Fig. 7Go, A and C). After detection of PPAR{gamma}, Western blots were stripped and reprobed with an antibody against C/EBP{alpha}, which detects both the 42- and 30-kDa alternative translation products. In agreement with the C/EBP{alpha} mRNA data, there was no detectable alteration in the amount of 30- or 42-kDa C/EBP{alpha} protein after treatment with VPA (Fig. 7Go, A and C). Furthermore, there was no statistically significant change in the amount of the precursor (125 kDa) or cleaved (68 kDa) forms of SREBP1a protein after 1 mM VPA (Fig. 7Go, B and C); however, there was a nonsignificant decrease at 8 and 12 h. Therefore, the decrease in leptin secretion and mRNA levels, after treatment with 1 mM VPA, was not associated with significant decreases in C/EBP{alpha}, PPAR{gamma}, and SREBP1a protein levels. There are numerous transcription factors that can regulate leptin promoter activity (45, 59, 60, 61), raising the possibility that VPA may alter the production, activity, or stability of these transcription factors ultimately resulting in decreased leptin mRNA levels.



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  		FIG. 6. VPA treatment reduces PPAR{gamma} and SREBP1a mRNA levels after 3 h but does not alter C/EBP{alpha} mRNA levels. A, Northern blot analysis of leptin (as shown in Fig. 5AGo), PPAR{gamma}, SREBP1a, and C/EBP{alpha} mRNA levels in adipocytes after treatment for 3 h with 1 mM VPA. Each treatment is shown in triplicate, and GAPDH is shown as a loading control. B, Quantification of mRNA levels for PPAR{gamma}, SREBP1a, and C/EBP{alpha} normalized against GAPDH levels. Data represent mean ± SEM from triplicate samples collected in two independent experiments, significantly different from UT at each time point as analyzed by post hoc comparisons based on Bonferroni (*, P < 0.05).

 


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  		FIG. 7. VPA treatment does not alter PPAR{gamma}, C/EBP{alpha}, or SREBP1a protein levels. A, Western analysis to detect PPAR{gamma}1 and PPAR{gamma}2 and p30 and p41 C/EBP{alpha} from adipocyte whole-cell protein extracts treated (1–12 h) with 1 mM VPA. B, Cells were pretreated with, and protein samples isolated in the presence of, the proteosome inhibitor N-acetyl-leucine-leucine-norleucinal (ALLN), and Western analysis to detect cleaved SREBP1a (C) and the precursor SREBP1a (P) protein. C, Quantification of protein levels for PPAR{gamma}, SREBP1a, and C/EBP{alpha} as normalized against actin levels. Data represent mean ± SEM from a minimum of three independent experiments; no significant differences were observed using ANOVA analysis.

 
VPA does not enhance leptin mRNA degradation
Studies have suggested that leptin expression can be controlled by both transcriptional and posttranscriptional regulation (62). To determine whether VPA altered leptin mRNA levels through increasing degradation, we measured leptin mRNA levels from cells cotreated with VPA (1 mM) and the RNA synthesis inhibitor ActD. The half-life of leptin mRNA in 3T3-L1 cells is reported to be approximately 2–4 h (63, 64), and we observed greater than 50% reduction in leptin mRNA levels 4 h after treatment with ActD (Fig. 8Go, A and C). There was no significant difference in the cells treated with ActD or VPA + ActD, suggesting that VPA does not alter leptin mRNA stability (Fig. 8Go, A and C). We also found that GAPDH mRNA levels were decreased in adipocytes after ActD treatment; therefore, the 18S rRNA is shown to assess loading (Fig. 8AGo). VPA does not affect leptin mRNA degradation, suggesting that VPA can affect leptin gene transcription.



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  		FIG. 8. VPA treatment did not affect leptin mRNA degradation but reduced leptin mRNA levels in the presence of the protein synthesis inhibition, CHX. A, Treatment with the RNA synthesis inhibitor, ActD, for 4 h resulted in decreased leptin mRNA levels. Cotreatment of ActD + VPA did not alter leptin mRNA levels when compared with ActD treatment alone. ActD treatment decreased GAPDH levels; thus, ribosomal 18S RNA is shown as a loading control. B, Treatment with CHX for 4 h does not alter leptin mRNA levels. VPA decreased leptin mRNA in the presence of CHX when compared with CHX treatment alone. C, Quantification of leptin mRNA levels normalized against GAPDH mRNA. Data represent mean ± SEM from three independent experiments, significantly different from UT as analyzed by ANOVA (*, P < 0.05).

 
VPA reduces leptin mRNA in the presence of CHX
VPA has been demonstrated to activate a variety of intracellular signaling pathways. Leptin mRNA levels may be reduced by VPA treatment through posttranslational effects on transcriptional machinery. CHX is a commonly used inhibitor of protein synthesis. Cells treated with CHX (10 µg/ml) have similar levels of leptin mRNA, compared with untreated cells (Fig. 8Go, B and C). Pretreatment with CHX followed by cotreatment with CHX and VPA produced a significant reduction in leptin mRNA levels, compared with VPA treatment alone (Fig. 8Go, B and C). These results show that the VPA-induced decrease in leptin mRNA is independent of de novo protein synthesis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A significant proportion of patients receiving VPA for a variety of disorders experience weight gain. Attempts to elucidate the mechanisms for this side effect led us to the paradoxical discovery that VPA can inhibit adipogenesis (31). Because adipocyte hypertrophy contributes to weight gain, we examined the effect of VPA treatment on mature adipocytes. Whereas VPA did not affect lipid metabolism or glucose uptake, we found that VPA decreased leptin secretion and mRNA levels in mature adipocytes. Acute treatment with VPA inhibited both constitutive and insulin-induced leptin secretion and significantly reduced leptin mRNA in a dose- and time-dependent manner. Whereas our results cannot explain the mechanism for how patients gain weight, we provide preclinical data demonstrating that VPA can have direct effects on mature adipocytes that may contribute to a disruption in energy balance.

VPA effects on leptin secretion are similar to those of saturated fatty acids (>C8) as well as long-chain monounsaturated fatty acids and PUFAs, which have been demonstrated to inhibit leptin secretion (46). It has been argued that lipids reduce leptin secretion and mRNA levels in both fully differentiated 3T3-L1 cells and primary cultures of rat adipocytes because of an accumulation of intracellular free fatty acids (44, 65). The ability of fatty acids to suppress leptin secretion does not appear to depend on fatty acid breakdown (mitochondrial fatty acid oxidation) because inhibitors of oxidation were unable to reverse the effects of fatty acids on leptin secretion (46). These experiments indirectly suggested that increased free fatty acid levels correlated with decreased leptin mRNA levels and/or protein secretion. By contrast, our results demonstrate that the VPA-induced decrease in leptin secretion is not due to altered intracellular free fatty acid levels.

Additionally, there is evidence indicating that some, but not all, long-chain fatty acids inhibit leptin production by altering leptin gene transcription (45, 46, 66). We found that VPA reduced leptin mRNA levels as early as 3 h after treatment and that this was not due to enhanced leptin mRNA degradation. In addition, PUFAs decrease leptin promoter reporter activity in BeWo cells (45). Using a leptin promoter reporter construct transfected into BeWo cells, we found that VPA increased leptin reporter activity; however, VPA also increased promoterless reporter activity (data not shown). VPA produces nonspecific effects on reporter activity in numerous reporter gene assays, likely due to the HDAC inhibitory properties of VPA (31, 67, 68). Our results suggest that the ability of VPA to decrease leptin mRNA levels is not due to the HDAC inhibitory properties of VPA because TSA did not alter leptin mRNA levels.

To further evaluate the effects of VPA on leptin transcription, we examined whether VPA affected the production of transcription factors that are known regulators of the leptin promoter, including C/EBP{alpha}, PPAR{gamma}, and SREBP1a (59, 60, 69, 70). We found that VPA did not alter the level of C/EBP{alpha} mRNA or protein; however, C/EBP{alpha} activity can be modulated by posttranslational mechanisms such as phosphorylation or protein-protein interactions (71, 72, 73, 74). Future work is required to examine whether VPA can alter C/EBP{alpha} activity. PPAR{gamma} protein levels or SREBP1a processing was not affected by acute VPA treatment. Synthetic ligands of PPAR{gamma}, such as antidiabetic thiazolidinedione agents, or endogenous ligands, such as prostaglandins (75), decrease leptin secretion and mRNA levels in 3T3-L1 cells (76) and rodent (77) and human adipocyte tissue (78). Many fatty acids are PPAR{gamma} ligands and whereas some investigators suggest that VPA is a PPAR{gamma} ligand (79, 80), we have demonstrated that VPA is not a PPAR{gamma} ligand (31). Therefore, it is un-likely that VPA decreases leptin mRNA levels through a direct effect on PPAR{gamma} expression or activity. SREBP1a is a basic-helix-loop-helix transcription factor that transactivates the leptin promoter (81). Reseland et al. (45) proposed that PUFAs decreased leptin secretion by inhibiting SREBP processing because activation of SREBP1 protein processing through cholesterol starvation prevented the decrease in leptin mRNA by PUFAs. We found that VPA reduced SREBP1a mRNA levels at 3 h but not at later time points. This change in mRNA levels did not result in statistically significant alterations in SREBP1a protein levels or processing into its bioactive form. We observed a nonstatistically significant decrease of cleaved SREBP1a at 8 and 12 h, raising the possibility that VPA may affect SREBP1a activity. However, alterations in leptin secretion occurred as early as 6 h after VPA treatment, suggesting that the putative VPA effects on SREBP1a processing are not solely responsible for alterations in leptin production and secretion. These results demonstrate that VPA effects on leptin production are not dependent on alterations in protein levels for C/EBP{alpha}, PPAR{gamma}, or SREBP1a.

How does VPA decrease leptin mRNA production and protein secretion? Our results rule out several possibilities including increased levels of intracellular fatty acids and increased degradation of leptin mRNA. Whereas VPA reduced secreted leptin levels, we found that VPA did not alter the intracellular level of leptin, likely due to a small intracellular pool. These results suggest that VPA does not affect secretion because VPA treatment does not increase intracellular leptin levels; rather, the reduction in leptin mRNA is responsible for reduced leptin protein production. Experiments with CHX showed that leptin mRNA levels decreased in response to VPA independent of new protein synthesis, suggesting that VPA affects preexisting cellular components and may affect posttranslational activation of factors such as C/EBP{alpha}. VPA can also affect a variety of cell signaling pathways including, and not limited to, increased {gamma}-aminobutyric acid (GABA) signaling and activation of MAPK pathways and can affect the expression of a variety of intracellular signaling molecules such as protein kinase C (reviewed in Refs. 82 , 83). We hypothesize that the ability of VPA to alter intracellular signaling pathways may be one mechanism through which VPA can affect leptin mRNA expression.

Patients treated with VPA have significant weight gain that is often associated with increased serum leptin levels. Increased serum leptin levels are associated with human obesity, and some have suggested that obesity is a state of leptin resistance (reviewed in Refs. 26 , 28). In patients receiving VPA, it is unclear whether increased leptin levels are due to a direct effect of VPA on leptin production in adipocytes, increased serum leptin is a consequence of VPA increasing adipocyte number or size, or VPA enhances patient appetite. Our results suggest that the increase in leptin levels are not due to a direct effect of VPA on adipocytes because in vitro VPA paradoxically inhibited adipogenesis (31), decreased leptin secretion and mRNA levels, and did not alter adipocyte size (indirectly determined by quantification of TAG biosynthesis). Compared with control subjects with similar BMI scores, patients receiving VPA did not have significantly elevated leptin levels, suggesting that increased leptin levels are due to increased patient adiposity (11). To assess whether VPA has similar effects on leptin biosynthesis in vivo, it will be necessary for future studies to examine leptin production by adipocytes obtained from patients receiving VPA.

It is possible that the inhibition of leptin secretion by VPA acutely induces enhanced appetite in patients, resulting in enhanced adiposity and an increase in leptin secretion. A number of studies have noted that patients who gain weight during VPA treatment report appetite stimulation, increased thirst, and quenching with calorie-rich beverages (13, 20, 84). In contrast, one study reported that VPA treatment was not associated with an increase in total caloric intake (85), yet it is well known that it is difficult to get an accurate measurement of food intake from humans (reviewed in Ref. 26). This increase in appetite in patients receiving VPA treatment has been suggested to result from enhanced GABA transmission within the hypothalamic axis of the central nervous system. Yet as recently reviewed by van den Pol (86), the role of GABA and glutamate in regulating energy balance has yet to be determined. It is unlikely that the ability of VPA to induce weight gain is solely due to increased GABA transmission because other anticonvulsants that increase GABA, including tiagabine, are not reported to induce weight gain (87). To determine whether an acute reduction in serum leptin levels by VPA correlates with an increase in appetite, clinical studies are required that measure leptin levels immediately after VPA treatment. Such studies may then begin to elucidate whether VPA can cause an initial decrease in leptin secretion that is followed by a subsequent increase in adiposity and serum leptin levels.


   Acknowledgments
 
We thank Drs. T. Shepherd, Y. Fu, N. Ridgway, and T. A. Lagace for advice and reviewing the manuscript; E. J. Campbell and D. L. Currie for technical assistance; and the laboratories of Drs. J. Goldstein, G. S. Hotamisligil, O. MacDougald, and B. Spiegelman for cDNA plasmids.


   Footnotes
 
This work was supported by the Canadian Psychiatric Research Foundation. D.C.L. is supported by a Canadian Institutes of Health Research Doctoral Award, and M.W.N. is a Research Scientist of the Canadian Cancer Society through an award from the National Cancer Institute of Canada.

Abbreviations: ACS, Acyl-coenzyme A synthetase; ActD, actinomycin D; BMI, body mass index; C/EBP, CCAAT/enhancer binding protein; CHX, cyclohexamide; DAG, diacylglycerol; FBS, fetal bovine serum; GABA, {gamma}-aminobutyric acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; MDI, medium to induce differentiation; PL, phospholipid; PPAR, peroxisome proliferator-activated receptor; PUFA, polyunsaturated fatty acid; SREBP, steroid regulatory element binding protein; TAG, triacylglycerol; TLC, thin-layer chromatography; TSA, trichostatin A; UT, untreated; VPA, valproic acid.

Received July 9, 2004.

Accepted for publication August 17, 2004.


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 Introduction
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 Results
 Discussion
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