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(The FASEB Journal. 2000;14:1793-1800.)
© 2000 FASEB

Decreased fatty acid synthesis due to mitochondrial uncoupling in adipose tissue

MARTIN ROSSMEISL, IVO SYROVY, FILIP BAUMRUK, PAVEL FLACHS, PETRA JANOVSKÁ and JAN KOPECKY1

Department of Adipose Tissue Biology, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

1Correspondence: Institute of Physiology, Academy of Sciences of the Czech Republic, Videská 1083, CZ - 142 20 Prague, Czech Republic. E-mail: kopecky{at}biomed.cas.cz


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis of fatty acid (FA) in adipose tissue requires cooperation of mitochondrial and cytoplasmic enzymes. Mitochondria are required for the production of ATP and they also support the formation of acetyl-CoA and NADPH in cytoplasm. Since cellular levels of all these metabolites depend on the efficiency of mitochondrial energy conversion, mitochondrial proton leak via uncoupling proteins (UCPs) could modulate FA synthesis. In 3T3-L1 adipocytes, 2,4-dinitrophenol depressed the synthesis of FA 4-fold while increasing FA oxidation 1.5-fold and the production of lactate 14-fold. Inhibition of FA synthesis in 3T3-L1 adipocytes was proportional to the decrease in mitochondrial membrane potential. FA synthesis from D-[U-14C] glucose was reduced up to fourfold by ectopic UCP1 in the white fat of transgenic aP2-Ucp1 mice, reflecting the magnitude of UCP1 expression in different fat depots and the reduction of adiposity. Transcript levels for lipogenic enzymes were lower in the white fat of the transgenic mice than in the control animals. Our results show that uncoupling of oxidative phosphorylation depresses FA synthesis in white fat. Reduction of adiposity via mitochondrial uncoupling in white fat not only reflects increased energy expenditure, but also decreased in situ lipogenesis.—Rossmeisl, M., Syrovy, I., Baumruk, F., Flachs, P., Janovská, P., Kopecky, J. Decreased fatty acid synthesis due to mitochondrial uncoupling in adipose tissue.


Key Words: UCP • lipogenesis • C57BL/6J mice • 3T3-L1 • obesity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DE NOVO fatty acid (FA) synthesis in adipocytes is an important mechanism involved in the control of fat content. This mechanism is relevant not only to rodents, but also to humans, where adipose tissue may account up to 40% of whole-body lipogenesis (1) . The biosynthesis of FA requires cooperation between mitochondrial and cytoplasmic enzymes and involves fluxes of metabolites across mitochondrial membranes (2 3 4) . Mitochondria are engaged in several pathways that are essential for FA synthesis, namely, the generation of ATP, and they also support the formation of NADPH and acetyl-CoA in cytoplasm.

That lipogenesis in white fat requires coupled mitochondria was suggested by Rognstad and Katz (2) , who studied the effect of 2,4-dinitrophenol (DNP) on glucose metabolism in epididymal fat from rats. DNP, an uncoupler of oxidative phosphorylation, acts as a proton shuttle to abolish the proton motive force and ATP synthesis in isolated mitochondria. In intact cells, DNP decreases phosphate potential and ATP-consuming processes (5) . The addition of DNP to fat fragments resulted in depressed synthesis of FA and increased production of lactate (2) . However, it has also been proposed (6) that production of ATP in adipocytes limits lipogenesis and partial uncoupling of oxidative phosphorylation enhances FA synthesis.

Because of the central role that mitochondrial proton conductance plays in the energetic state of the cell (7) , it is tempting to speculate that it may contribute to the control of FA synthesis in lipogenic tissues via mitochondrial uncoupling proteins (UCPs). UCP1, which is specifically expressed in thermogenic brown fat, functions as a regulated proton transporter (8) . Recently, two UCP1 homologues, UCP2 (9 , 10) and UCP3 (11 , 12) , have been discovered that are expressed in brown fat and in other organs of mammalians as well. Novel UCPs probably transport protons, similar to UCP1. However, the biochemical activities of these novel UCPs are not understood in detail and may differ among the UCPs (8 , 13) . UCP2 and UCP3 may play an important role in controlling basic metabolic rate and obesity (9 10 11 12 , 14 15 16 17) . An excellent review study on UCPs has been published recently (18) .

We have previously reported that expression of UCP1 gene from aP2 gene promoter in white and brown fat of transgenic (aP2-Ucp1) mice affected fat distribution in the body and prevented development of genetic or dietary obesity (19 20 21) . Resistance to obesity resulted from genetic manipulation of white fat (21 , 22) , where it was induced by mitochondrial uncoupling in adipocytes, as documented by measurements of mitochondrial membrane potential ({Delta}{psi}m) in isolated fat cells (23) and of oxygen uptake by adipose tissue fragments (21) . Preliminary results (24) suggest that transgenic UCP1 affects body weight not only by increasing in situ FA oxidation, but also by decreasing lipogenesis.

This study was designed to analyze in detail the link between efficiency of energy conversion and the rate of FA synthesis in adipocytes and to verify the hypothesis that UCP1 could modulate adiposity via effect on FA synthesis. The effects of chemical uncouplers were studied in 3T3-L1 adipocytes differentiated in cell culture. Transgenic aP2-Ucp1 mice were used as an in vivo model of mitochondrial uncoupling induced by UCP1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Measurements of FA oxidation, FA synthesis, and lactate production in 3T3-L1 adipocytes
Cells of 3T3-L1 clonal line (25) were plated at a density of 8000 cells/cm2 (10 cm2 dish) and grown in Dulbecco’s modified Eagle’s medium at 37°C in 10% CO2 in air. The composition of the growth medium and the differentiation medium (added to confluent cells) ~4–5 days after plating were as described before (26) , except that the differentiation medium also contained 2 µM dexamethasone (present only during the first 48 h after the switch to the differentiation medium) and 100 nM 5-(4-[2-(N-methyl-N-(2-pyridyl)amino)ethoxy]benzyl)thiazolidine-2,4-dione maleic acid salt (BRL 49653; in dimethyl sulfoxide). When used for experiments (12–14 days after confluence), cultures contained 50–60% of differentiated adipocytes. Before each measurement, a complete change of the medium was performed. DNP (dissolved in 0.1% KOH) or carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP; in 70% ethanol) were also added in some experiments. FA oxidation was measured using a modification of the procedure described previously (27) during a 4 h incubation in the presence of 0.2 mM oleate, 0.4% bovine serum albumin (BSA), and 5 µCi (9, 10[n-3H] oleic acid (10.3 Ci/mmol; Amersham, Little Chalfont, U.K.)/ml. The DNA content of cells was measured fluorometrically (28) after digestion with 150 µl proteinase K solution for 15 min at 20°C (22) . FA synthesis was measured (29) by the incorporation of 14C from acetate into saponifiable FA. Cells incubated 4 h in the presence of 1 µCi/ml [2-14C]acetic acid (sodium salt, 50 mCi/mmol; Amersham) were washed twice with phosphate-buffered saline (pH 7.4) and solubilized by proteinase K (see above). Aliquots were used for DNA measurements (30 µl; see above) and incorporation of radioactivity into FA (90 µl) (30) . Lactate in the medium was measured using a coupled enzyme assay (kit 139 084 from Boehringer, Mannheim, Germany) after 24 h of culture in the presence of DNP. Lactate contributed to the medium by fetal bovine serum (around 0.2 mM) was subtracted. All samples were run in triplicate.

Correlation of FA synthesis and {Delta}{psi}m in 3T3-L1 adipocytes
3T3-L1 adipocytes were maintained for 4 h (in 25 cm2 dish) with or without DNP or FCCP, and FA synthesis was measured using [2-14C]acetic acid as described above. The {Delta}{psi}m was estimated in cells maintained under identical conditions (except for the absence of [2-14C]acetic acid). These cells were harvested using trypsin-EDTA solution, washed in physiological buffered saline (23) , and suspended in Krebs Ringer bicarbonate buffer (KRB) containing 5 mM glucose and various concentrations of DNP or FCCP. Estimation of {Delta}{psi}m was performed similarly as before (23) , except that a minimum of 5000 whole cells (instead of digitonin-permeabilized cells) were used for each measurement. Tetramethylrhodamine methyl ester (TMRM) was added to a final concentration of 50 nM. TMRM fluorescence (reflecting {Delta}{psi}m) was recorded using FACSort flow cytometer (Becton Dickinson, San Jose, Calif.) at 5 min intervals and expressed as mean fluorescence intensity (IFL). Fluorescence intensity increased during incubation, reaching a maximum at ~30 min (not shown). Therefore, the IFL value at 30 min was taken as a measure of {Delta}{psi}m.

Animals
Control C57BL/6J male mice and their hemizygous aP2-Ucp1 transgenic littermates were identified by Southern blot analysis (19) . The mice were maintained at 20°C with a 12 h light-dark cycle. After weaning at 4 wk of age, mice were housed 4–5 per cage and had free access to a standard chow diet (20) and water. Adult (6- to 8-month-old) animals were killed by decapitation in diethylether anesthesia. Subcutaneous (s.c.) dorsolumbar white fat (20) and epididymal fat were used for experiments. Samples for isolation of total RNA were frozen and stored in liquid nitrogen.

FA synthesis in animals and tissue fragments
Mice were injected intraperitoneally with 50–100 µl of 0.15 M NaCl containing 3H2O (35 µCi/g body weight). After 60 min at 22°C, mice were killed and the rate of FA synthesis in various tissues in vivo was measured by incorporation of 3H2O into saponifiable FA (30) . The activity of FA synthesis was expressed as dpm 3H incorporated to FA/mg tissue per hour. In other experiments, FA synthesis was measured in vitro (1) . Adipose tissue fragments (70 mg) were incubated for 2 h at 37°C (in an atmosphere of 5% CO2 and 95% O2) in total volume of 0.5 ml KRB containing 4% BSA (Serva, Cat. No. 11930), 1.1 µCi D-[U-14C] glucose (4 mCi/mmol; Amersham), 0.5 nmol nonradioactive glucose, and 40 µU insulin (Actrapid MC, Novoindustri, Denmark). 14C incorporation into saponifiable FA was estimated (30) and expressed as pmol of glucose converted to FA/g tissue per hour.

RNA analysis
Total RNA was isolated from adipose tissue and analyzed on Northern blots using a full-length cDNA probe for mouse UCP1 (21) . Hybridization with a ribosomal 18S RNA probe was used to correct for inter-sample variations. Total RNA isolated from interscapular brown fat of cold-acclimated mice served as a standard. Radioactivity was evaluated by PhosphorImager SF (Molecular Dynamics, Sunnyvale, Calif.). Semiquantification of the transcripts for acetyl-CoA carboxylase (ACC) and FA synthase (FAS) was performed by reverse transcription of RNA, followed by the polymerase chain reaction (RT-PCR), as described before (31 , 32) . Total RNA was treated with RNase-free DNase and first-strand cDNA was generated from 1 µg of RNA in a 10 µl volume by using Moloney murine leukemia virus reverse transcriptase (Top-Bio, Czech Republic) and the oligo (dT) primers. cDNA (1 µl of the reverse transcription mix) was amplified by hot-start PCR with primers specific for mouse ACC, FAS, and ß-actin (Table 1 ). A reaction cycle consisted of 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C. Four aliquots (12, 12, 8, and 8 µl, respectively) were removed at 3-cycle intervals between cycles 16–34 (log-linear phase of the reaction) and examined on 1.5% agarose gels stained with ethidium bromide. Fluorescence was evaluated by LAS-1000 (Fuji, Japan) and quantified by using AIDA 2.11 software (Raytest, Germany). Levels of the transcripts were expressed relative to that of ß-actin.


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  		Table 1. Sequences of PCR primers

Statistics
A two- or three-way analysis of variance (ANOVA) with post hoc multiple comparisons was used as described before (20) . Statistical significance was evaluated using Student’s t test. All the comparisons were judged to be significant at P<0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of chemical uncouplers on metabolic fluxes and {Delta}{psi}m in 3T3-L1 adipocytes
To characterize in detail the effect of mitochondrial uncoupling on metabolism of adipocytes, experiments were performed on 3T3-L1 adipocytes differentiated in cell culture (Fig. 1 ). Adipocytes were maintained for 4–24 h in the presence of different concentrations of DNP. FA oxidation and lactate production increased with increasing concentrations of DNP (2 , 33) , whereas activity of FA synthesis became inhibited. All three metabolic parameters were affected at similar concentrations of the uncoupler, with 300 µM DNP exerting 80–90% effect. Most pronounced was the effect on lactate production, which was stimulated ~14-fold by DNP. Maximum inhibitory effect of DNP (at 700 µM concentration) on FA synthesis was ~4-fold, whereas oxidation of FA was stimulated only ~1.5-fold under the same experimental conditions (Fig. 1) . It is apparent that decreased ATP production uncoupled by DNP from substrate (FA) oxidation in mitochondria resulted in stimulation of glucose utilization by adipocytes (33) . Moreover, these experiments support the notion (2 , 34) that mitochondrial uncoupling decreases FA synthesis in adipocytes.



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  		Figure 1. Effect of a chemical uncoupler on metabolic fluxes in 3T3-L1 adipocytes. Adipocytes were incubated for 4 h in a cell culture dish with or without different concentrations of DNP for measurements of FA oxidation (pmol oleate oxidized/dish per hour) or FA synthesis (pmol acetate x 10 converted to FA/dish per hour), or for 24 h for measurements of lactate production (nmol lactate produced/dish per hour). DNA measurements confirmed that no cells had been lost during the procedure (see Materials and Methods). Values are means ± SE of three independent experiments.

The in vitro model of 3T3-L1 adipocytes allowed us to analyze directly the link between mitochondrial energetics and FA synthesis. In a separate set of experiments, 3T3-L1 adipocytes differentiated in cell culture were incubated with uncouplers (DNP or FCCP), as above, and FA synthesis was evaluated in parallel with estimation of the {Delta}{psi}m in whole cells (Fig. 2 ). The latter type of measurement was performed by flow cytometry using a potential-sensitive fluorescent dye, TMRM. Under these conditions, TMRM fluorescence intensity (IFL) reflects {Delta}{psi}m (23) . FA synthesis decreased with increasing concentrations (100–600 µM) of DNP, as in the previous experiment (Fig. 1) . The lowest activity was observed in the presence of 50 µM FCCP (Fig. 2) . Addition of the uncoupler also resulted in a decrease of IFL values. Positive correlation was found between decreased FA synthesis and decreased IFL value, indicating a close link between the magnitude of {Delta}{psi}m and activity of FA synthesis in adipocytes.



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  		Figure 2. Correlation between {Delta}{psi}m and FA synthesis in 3T3-L1 adipocytes. A) 100 µM DNP; B) 300 µM DNP; C) 600 µM DNP; D) 50 µM FCCP. Values are expressed in % of the values estimated in the absence of the chemical uncouplers (Control). Values are means ± SE of four independent experiments.

Gene expression in adipose tissue of control and aP2-Ucp1 transgenic mice
In further experiments, aP2-Ucp1 transgenic mice were used as a model of mitochondrial uncoupling induced in vivo by ectopic UCP1 in white fat. As observed before (19 , 20) , the aP2-Ucp1 transgene affected differentially the size of various fat depots (see legend to Table 2 ). Thus, the weight of s.c. white fat was twofold lower in transgenic compared with control mice and the size of epididymal fat was marginally increased (19 , 20) .


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  		Table 2. Quantification of gene expression in white fat depots of control and transgenic micea

The aP2-Ucp1 transgene induced ectopic expression of UCP1 gene in white fat depots of adult mice, whereas no expression could be detected in control animals (Fig. 3 and refs 19 , 21 ). The levels of UCP1 transcript in s.c. white fat were significantly (~twofold) higher than in epididymal fat (Fig. 3 and Table 2 ). The expression of UCP1 in the white fat of transgenic mice was at least one order of magnitude lower than that in interscapular brown fat of control, cold-acclimated mice (Table 2 and refs 19 , 20 ). Expression of the genes for ACC and FAS, cytoplasmic enzymes engaged in the synthesis of C2 moiety of FA, was significantly depressed by the transgene in both white fat depots studied (Fig. 3 and Table 2 ).



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  		Figure 3. Expression of various genes in s.c. (Sc-WF) and epididymal (Epid-WF) white fat of control (+/+) or transgenic (tg/+) mice. UCP1 transcript was detected using Northern blots; expression of ACC, FAS, and ß-actin was evaluated by RT-PCR (inverted gray scale).

Effect of transgenic UCP1 on FA synthesis in adipose tissue
FA synthesis in white fat depots of control and transgenic mice was evaluated (Fig. 4 ) in vivo by measuring incorporation of injected 3H2O into saponifiable FA and in vitro as the incorporation of 14C into FA extracted from tissue fragments incubated in the presence of D-[U-14C] glucose and insulin (see Materials and Methods). 3H incorporation was similarly high in both s.c. and epididymal white fat of control mice (Fig. 4 , upper part), which was ~5- to 10-fold lower compared to that in brown fat or liver and ~4-fold higher than that in skeletal muscle (not shown). 3H incorporation in both white fat depots was decreased to a similar extent by the transgene (~1.6- to 1.8-fold; Fig. 4 , upper part). This suppression was found only in adipose tissue and not in other tissues (not shown), reflecting the fat-specific expression of UCP1 from the aP2 gene promoter contained in the transgene (19 , 35) .



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  		Figure 4. Synthesis of FA in s.c. (Sc-WF) and epididymal (Epid-WF) white fat of control (empty bars) or transgenic (solid bars) mice. Incorporation of radioactivity into saponifiable FA was measured using 3H2O in vivo (n=6; upper part) or D-[U-14C] glucose in vitro (n=15; lower part). Activities are expressed as dpm 3H incorporated to FA/mg tissue/h or as pmol glucose converted to FA/g tissue/h, respectively (see Materials and Methods). Values are means ± SE. Asterisks indicate statistically significant differences between genotypes. The difference between s.c. and epididymal fat of control animals in vitro was also significant; other differences were not.

The in vitro experiments (Fig. 4 , lower part) confirmed the inhibition of FA synthesis by ectopic UCP1 in white fat. However, the inhibition was much more pronounced in s.c. fat (~3.8-fold difference between the genotypes) than in epididymal fat (1.5-fold difference; not significant, P=0.08). The differential effect of the transgene on FA synthesis in s.c. and epididymal fat is in accordance with the relatively higher expression of the aP2-Ucp1 transgene in the former tissue (Fig. 3 and Table 2 ). The results also suggest that the in vitro measurements of the incorporation of 14C from D-[U-14C] glucose better reveal the link between mitochondrial energetics and FA synthesis than does the estimation of lipogenesis in whole animals injected by 3H2O (see Discussion).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mechanism of decreased lipogenesis
This report demonstrates that uncoupling of oxidative phosphorylation, while reducing adiposity in white fat, depresses in situ FA synthesis. Experiments on adipocytes differentiated in cell culture confirmed previous findings on the effect of DNP in adipose tissue (2 , 34) and showed a strong correlation between decreased FA synthesis and decreased {Delta}{psi}m. The experiments on aP2-Ucp1 mice document the ability of UCP1 to decrease FA synthesis in white fat in vivo while excluding possible side-effects of chemical uncouplers on cell metabolism.

FA synthesis, which requires (2 3 4) production of extramitochondrial acetyl-CoA, proceeds via ‘pyruvate cycle’, i.e., conversion of pyruvate into oxalacetate and citrate in mitochondria, transport of citrate to the cytoplasm, and cleavage to acetyl-CoA and oxalacetate. Cytosolic oxalacetate is reduced to malate by NADH, and the malate is decarboxylated to pyruvate with generation of NADPH (in a reaction catalyzed by malic enzyme). Pyruvate returns to the mitochondrion, where it serves to reform citrate. Operation of the cycle not only provides acetyl units for FA synthesis, but also contributes about half the NADPH required, with the rest supplied by the pentose cycle (2) . For each molecule of acetate incorporated into long-chain FA, two molecules of NADPH are necessary in the reaction catalyzed by FA synthase. Mitochondrial production of ATP is required for synthesis of oxalacetate by carboxylation of pyruvate and subsequent synthesis of citrate in mitochondria and for FA synthesis and esterification in cytoplasm (2 3 4) .

A 4- to 5-fold reduction of FA synthesis was detected in both adipocytes incubated with uncoupler and in fragments of s.c. white fat of transgenic mice, whereas substrate oxidation increased only ~1.5-fold (Fig. 1 and ref 21 , respectively). Inhibition of FA synthesis by mitochondrial uncoupling probably results from limited availability of intramitochondrial ATP for the carboxylation of pyruvate (2 , 3) whereas ATP level in cytoplasm remains relatively high (2 , 33) . Activity of pyruvate carboxylase is threefold greater in adipose tissue than in liver mitochondria, and the ATP:ADP ratio directly affects the activity of the enzyme (3 , 4) . This suggests an important regulatory role of the enzyme in lipogenesis in white fat. The inhibition of pyruvate carboxylation probably slows down FA synthesis as a result of limited supply of acetyl units (2 , 3) .

When measured in tissue fragments (as the incorporation of D-[U-14C] glucose in the presence of insulin) but not in vivo (in mice injected by 3H2O), FA synthesis in s.c. fat was more sensitive to inhibition by the transgene than that in epididymal fat. The levels of UCP1 transcript in transgenic mice were significantly higher in s.c. than in epididymal fat, in accordance with the fat depot-specific difference in the activity of the aP2 gene promoter (35) . Therefore, inhibition of FA synthesis in tissue fragments correlated with the levels of UCP1 transcript in different depots. The results support the link between ectopic UCP1 and FA synthesis in white fat, providing that the metabolic flux through the pathway of FA synthesis is higher under the in vitro conditions compared to in vivo measurements (36) and therefore relatively more sensitive to the alteration of mitochondrial functions. Moreover, systemic control of lipid metabolism in the animal may counteract and equalize the effect of UCP1 in various fat depots. However, it is likely that FA synthesis in various fat depots is also differentially affected by the transgene in vivo and the difference is too small to be detected. Also, the steady-state levels of the transcripts for lipogenic enzymes (ACC and FAS) were similarly depressed in both white fat depots of the transgenic mice. The differential effect of the transgene on FA synthesis in vivo may explain, at least in part, why the size of s.c. white fat in transgenic mice is reduced whereas that of epididymal fat is not (19 , 20) . Low accumulation of s.c. and not epididymal fat counteracts development of obesity in the transgenic mice (19 , 20) .

Biological significance
FA synthesis in white fat is inhibited by starvation (37 38 39) and by high-fat diets (40) and potentiated by glucose, high-sugar meals, and insulin. These effects result in part from the regulation of ACC activity (40) by insulin and glucagon. However, starvation in both humans (41 , 42) and mice (J. Kopecky et al., unpublished data), and high-fat diets in mice (9 , 15) also up-regulate UCP2 in white fat. Thus, the same factors that negatively affect lipogenesis in white fat would also up-regulate the expression of UCP2. This suggests that UCP2 may be involved in physiological control of FA synthesis in white fat.

Expression of UCP2 in white adipose tissue is relatively high (9 , 10) whereas that of UCP1 is very low in both rodents (32 , 43 44 45 46) and humans (16) . UCP3 is normally absent (47) . However, both UCP1 (43 44 45 46) and UCP3 (47) can be induced in white fat depots by pharmacological treatments that reduce adiposity. Whether these effects reflect transdifferentiation of white adipocytes or recruitment of brown fat precursor cells remains an important question (35 , 43 , 46) . Our findings suggest that UCP1 has a significant role in the regulation of lipogenesis in brown fat. Indeed, short-term (2 h) cold exposure, which increases adrenergic stimulation and activates UCP1-mediated proton transport via FA released from intracellular triglycerides (8 , 13) , results in a threefold reduction in FA synthesis in brown fat (38) . Norepinephrine or exogenous FA inhibited synthesis of FA in isolated brown adipocytes (48) . Therefore, activation of UCP1 in brown fat during acute cold stress probably results not only in thermogenesis, but also in depression of FA synthesis. However, prolonged cold exposure is accompanied by the increase in FA synthesis and increased ACC and FAS activities (38) .

The high lipogenic rate in adipose tissue may contribute to development of obesity (49) and may explain the high rate of relapse in patients treated by caloric restriction (32) . Compared with lean controls, morbidly obese subjects displayed lower levels of both UCP1 (16) and UCP2 (17) transcripts in intraabdominal fat, and UCP2 expression remained low subsequent to weight-reducing surgery (17) . Induction of UCP2 by a high-fat diet correlated with obesity resistance in different strains of mice (9 , 15) . Lipogenesis in adipose tissue has not been measured in these studies. However, in rats with hyperleptinemia induced by adenovirus transfer of the leptin gene, body fat is depleted whereas expression of UCP1 and UCP2 in white adipose tissue is up-regulated, FA oxidation is increased, and expression of genes for enzymes engaged in lipogenesis (like ACC and FAS) is profoundly suppressed (32) . In aP2-Ucp1 mice, resistance against obesity was also accompanied by down-regulation of genes for lipogenic enzymes (ACC and FAS) and by increased in situ oxidation of FA in adipose tissue, which would suggest that the depression of lipogenesis in white fat in both models of obesity resistance results from up-regulation of UCP1 and/or UCP2. The changes in the expression of genes for lipogenic enzymes probably reflect decreased metabolic flux through the pathway of FA synthesis. Tumor necrosis factor {alpha} also down-regulates lipogenic genes and prevents their induction by insulin in adipocytes (50) . It may be hypothesized that all these effects result from the inhibition of mitochondrial energy conversion by tumor necrosis factor {alpha} (51) .

In summary, our results suggest a new link between mitochondrial UCPs and regulation of body weight, showing that lower efficiency of mitochondrial energy conversion in fat cells may decrease adiposity not only due to increased energy expenditure, but also by depression of in situ lipogenesis. The measurement of FA synthesis is a new way for detecting the activity of UCPs in adipose tissue. Because the oxidative capacity of white fat is relatively low, the link between mitochondrial energetics and FA synthesis may be a superior target for treatment strategies for obesity.


   ACKNOWLEDGMENTS
 
This work was supported by the Grant Agency of Academy of Sciences of the Czech Republic (grant No. A5011710), the Grant Agency of the Czech Republic (grant No. 311/99/0196), the Howard Hughes Medical Institute (grant No. 75195–541001), and COST-918. We thank Dr. H. Green (Harvard Medical School, Boston, Mass.) for 3T3-L1 cell line, SmithKline Beecham Pharmaceuticals (Collegeville, Pa.) for BRL 49653, Prof. B. D. Nelson (University of Stockholm, Sweden) and S. Sustková for critical reading of the manuscript, J. Bémová and I. Mertelíková for technical assistance, and Prof. K. Garlid (Oregon Graduated Institute, Portland, Oreg.) for useful discussions.

Received for publication November 11, 1999. Revision received February 15, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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