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Endocrinology Vol. 148, No. 8 4007-4014
Copyright © 2007 by The Endocrine Society

Acute and Selective Inhibition of Adipocyte Glyceroneogenesis and Cytosolic Phosphoenolpyruvate Carboxykinase by Interferon {gamma}

Wael Khazen, Emilie Distel, Martine Collinet, Valéria E. Chaves, Jean-Pierre M’Bika, Charles Chany, Ammar Achour, Chantal Benelli and Claude Forest

Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche S747 (W.K., E.D., M.C., V.E.C., C.B., C.F.) and Laboratoire des interférons et de la sarcolectine (J.-P.M., C.C., A.A.), Université Paris Descartes, Centre Universitaire des Saints-Pères, 75006 Paris, France; and Department of Physiology (V.E.C.), School of Medicine of Ribeirão Preto Avenida Bandeirantes 3900, 14.049 Ribeirão Preto, S.P. Brazil

Address all correspondence and requests for reprints to: Dr. Claude Forest, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche S747, Université Paris Descartes, Centre Universitaire des Saints-Pères, 45 rue des Saints-Pères, 75006 Paris, France. E-mail: claude.forest{at}univ-paris5.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interferon {gamma} (IFN-{gamma}) was previously shown to promote fatty acid (FA) release from adipose tissue (AT). Net lipolysis is an equilibrium between triglyceride breakdown and FA re-esterification. The latter requires activated glyceroneogenesis for glycerol-3-phosphate synthesis and increased cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), the key enzyme in this pathway. We wondered whether glyceroneogenesis and PEPCK-C would be IFN-{gamma} targets. We injected mice with IFN-{gamma}, and exposed either AT explants and isolated adipocytes from humans and mice or 3T3-F442A adipocytes to IFN-{gamma} before monitoring expression of genes involved in lipid metabolism and the metabolic consequences. We show that IFN-{gamma} induces a large increase in FA release without affecting glycerol output and decreases [1-14C]-pyruvate incorporation into lipids, thus demonstrating that FA re-esterification is reduced due to diminished glyceroneogenesis. A series of mRNA encoding proteins involved in FA metabolism remained unaffected by IFN-{gamma}, while that of PEPCK-C was rapidly and drastically lowered. IFN-{gamma} effect opposed that of the ß-agonist isoproterenol and of 8-Br-cAMP. In IFN-{gamma}-treated mice, PEPCK-C gene expression was decreased in AT, but not in liver or kidney. Thus, IFN-{gamma} exerts a tissue-specific action in rodents and humans, having glyceroneogenesis and the PEPCK-C gene as selective targets to intensify FA release from adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PARACRINE INTERACTIONS between the immune system and adipocyte metabolism have recently gained wide interest (1, 2). On the one side, it is noteworthy that adipose tissue (AT) secretes a series of cytokines, named adipokines, exerting an influence on pathophysiological processes like inflammation and cancer (3). On the other side, several cytokines such as TNF{alpha}, IL-1, and IL-6, or interferons (IFNs), appear to play important roles in the regulation of adipocyte metabolism (4, 5, 6, 7). These cytokines are secreted particularly in response to infection or inflammation (8) and mediate intercellular and intracellular communication (9).

Infection is associated with several disturbances in lipid storage and release. Although cytokines play a major role in these metabolic changes, their effects are poorly known. Of these cytokine, IFN-{gamma} is known for its specific function in immunological responses. IFN-{gamma} is synthesized and secreted by CD4 Th1 and CD8+ T lymphocytes plus natural killer cells. It induces host defense against infections. Several studies have revealed that IFN-{gamma} affects lipid metabolism and adipocyte gene expression (10, 11, 12). Grünfeld et al. demonstrated that the treatment of mice by IFN-{gamma} resulted in increased lipolysis, as reflected by an increase in plasma nonesterified fatty acids (NEFAs) (11). Furthermore, exposure of 3T3-L1 adipocytes to IFN-{gamma} caused a partial inhibition of lipoprotein lipase (LPL) activity, a reduction in fatty acid synthase mRNA level, and an increased release of NEFA to the culture medium, suggesting that this cytokine stimulated lipolysis (13, 14). Additional studies were performed with 3T3-L1 cells, showing that IFN-{gamma} affected the proportion of cells devoid of lipid droplets (5).

Lipolysis is a complex mechanism controlled by many factors. A positive link between lipolysis per se, i.e. triglyceride hydrolysis, and fatty acid (FA) re-esterification is usually observed (15, 16). In fact, FA release is concomitant to the re-esterification of 30–40% of FA into triacylglycerols (17). This re-esterification process implies the availability of glycerol-3-phosphate. During fasting, the metabolic pathway that synthesizes glycerol-3-phosphate from noncarbohydrate substrates is named glyceroneogenesis (18). The key enzyme in this metabolic pathway is the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C) (19, 20, 21). Glyceroneogenesis and PEPCK-C gene expression are acutely modulated by a series of hormones and nutrients in adipocytes. The PEPCK-C gene [rodent PEPCK-C gene (Pck1)] is the primary target for cAMP inducers, polyunsaturated fatty acids, retinoic acids, and hypolipidemic drugs like thiazolidinediones (19, 22, 23, 24, 25). Conversely, glucocorticoids reduce Pck1 gene transcription and repress the effect of all inducers (21, 26, 27). There is no known posttranslational regulation of PEPCK-C activity. Therefore, all these changes in gene expression result in an immediate variation in the glyceroneogenic flux, as observed by the incorporation of 1-14C-pyruvate into triacylglycerol stores (28). Consequently, the amount of FA released by adipocytes is greatly affected without any change in glycerol output (29). Therefore, this pathway and its major enzyme, PEPCK-C, are considered as the key regulated steps for controlling net FA efflux (19).

The primary purpose of the present study was to determine whether IFN-{gamma} would affect glyceroneogenesis and the expression of enzymes involved in FA metabolism in adipocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Balb/c male mice (Elevage Janvier, Bagneux, France) were kept on a 12-h light/dark cycle at constant room temperature. At the time of the experiment, 12-wk-old mice were used per treatment. Animals were given a dose of either 0.1, 1, 10, or 25-µg/20 g body weight mouse of murine IFN-{gamma} (PreproTech, Inc., Rocky Hill, NJ) or vehicle (0.5% NaCl) via peritoneal injection at 10:00. One microgram of IFN-{gamma} corresponds to 10,000 international units. Animals were fasted for 8 h, then killed by decapitation at 1800 h. Aliquots of plasma were stored at –20 C for further NEFA (Free Fatty Acids Half Micro Test; Roche, Manheim, Germany) and glycerol (glycerol UV-method; R-Biopharm France, Saint Didier au Mont D’Or, France) determinations. Samples of AT [sc (SCAT), periepididymal (EPAT), and perirenal (PRAT)], liver, and kidney were dissected and frozen at –80 C for further analyses. The protocol for the animal studies was conducted according to the French Guidelines for the Care and Use of Experimental Animals.

Culture of explants from AT and isolation of adipocytes
Explants of SCAT were obtained either from three healthy women undergoing plastic surgery or from Balb/c male mice (see previous).

Women were aged 40.3 ± 3 yr and had a body mass index of 27.4 ± 1.9 kg/m2. None of the subjects suffered from known metabolic or malignant diseases, nor were they taking medications known to alter AT metabolism. All the patients gave informed written consent. The study was performed according to the tenets of the Helsinki protocol. SCAT specimen from the sc region was obtained within 15 min after the onset of surgery. Explants of SCAT from mice were obtained as described previously (30).

SCAT was cut in ±20-mg fragments in DMEM (GIBCO; Invitrogen Corp., Cergy-Pontoise, France) containing 12.5 mM glucose, 200 IU/ml penicillin, 50 mg·liter–1 streptomycin, and 10% fetal calf serum, after which medium was removed and replaced with fresh medium at 37 C. Explants were then preincubated for 1 h in a humidified 37 C incubator with 10% CO2 before IFN-{gamma} treatment, or not, for different periods of time.

Adipocytes from human or mice SCAT were isolated as described in Weisberg et al. (31). The pellet contained the stroma-vascular fraction of cells, whereas the floating cells were collected as the adipocyte-enriched fraction. Adipocytes were cultured for 18 h in the same medium as that used for explants, in a humidified incubator with 10% CO2. They were then exposed, or not, to IFN-{gamma} for 8 h before RNA extraction and analysis.

For all these studies, IFN-{gamma} from mouse and human sources (PreproTech, Inc.) were used respectively for mice and human explants and adipocytes.

Culture and treatments of 3T3-F442A adipocytes
3T3-F442A adipoblasts were grown to confluence in DMEM containing 25 mM glucose, 200 IU/ml penicillin, 50 mg/liter streptomycin, 2 mg/liter biotin, and 4 mg/liter pantothenate supplemented with 10% newborn calf serum (Sigma-Aldrich, St. Louis, MO). Cells were cultured at 37 C in a humidified incubator with 10% CO2. At confluence, newborn calf serum was changed to fetal bovine serum (GIBCO), and 20 nM insulin (Sigma-Aldrich) was added to the medium; this medium was named "complete medium." Culture medium was changed every 2–3 d.

Once differentiated, cells were exposed, or not, to mouse IFN-{gamma} (PreproTech, Inc.), isoproterenol (Iso), or 8Br-cAMP (Sigma-Aldrich) for various times before further treatments.

RNA extraction and analysis
Adipocyte fraction was placed in Qiazol lysis reagent (QIAGEN, Courtaboeuf, France), and the stroma-vascular fraction of cells was resuspended in TRIzol (Invitrogen Corp.) for RNA extraction. The same procedure was applied to human AT.

Total RNA was extracted from 3T3-F442A cells (32) grown and differentiated as described (33) using TRIzol. For tissue samples (200 mg), extraction was performed using a RNeasy Mini Kit (QIAGEN). RNA was quantified using the Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE), and its quality was assessed by gel electrophoresis.

The first-strand cDNA was synthesized using High Capacity cDNA Archive (Applied Biosystems, Courtaboeuf, France). Quantitative RT-PCR measurements using SYBR Green (QIAGEN) were performed on an ABI Prism 7900 Sequence Detector system (Applied Biosystems). The relative mRNA levels of FA lipid binding protein (aP2), adipocyte triglyceride lipase (ATGL), glycerol-3-phosphate dehydrogenase (G3PDH), glycerol kinase (GlyK), hormone-sensitive lipase (HSL), PEPCK-C, mitochondrial PEPCK (PEPCK-M), and murine mitochondrial dicarboxylate carrier (mDIC) were estimated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Primer sequences are illustrated in Table 1Go.


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  		TABLE 1. Primer design for RT-PCR

 
Western blot
Extracts of 3T3-F442A adipocytes were isolated in HEPES buffer (pH 7.9) containing 10 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, 1 µg/ml, leupeptin, 10 µg/ml, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonylfluoride. Samples were centrifuged at 14,000 x g for 20 min at 4 C. Supernatant was removed and centrifuged for two additional steps of 20 min at 14,000 x g. Supernatants were separated by 10% SDS-PAGE, then underwent blotting. Equal transfer of proteins was confirmed by staining the nitrocellulose with Ponceau Red (0.2% wt/vol H2O diluted 99:1 in 10% trifluoroacetic acid). After blocking, blots were incubated with rabbit antirat PEPCK-C antibody (a kind gift from Professor Elmus Beale, Texas Tech University Health Sciences Center, Lubbock, TX) (34) or anti-murine [E2 subunit of pyruvate dehydrogenase (PDH-E2)] antibody (kindly given by Dr. J. G. Lindsay, University of Glasgow, Glasgow, UK), then with the horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The protein bands in the blots were detected with an enhanced chemiluminescence kit (Pierce, Interchim, France).

Metabolic studies
AT (~200 mg) was cut into pieces of about 20 mg and incubated in 60-mm dishes in a humidified atmosphere of 10% CO2 at 37 C in 1.5 ml of glucose-free DMEM containing 3% (wt/vol) FA-free BSA and 5-mmol/liter pyruvate. Three hours later, medium was changed for Krebs buffer containing 3% (wt/vol) FA-free BSA and 5-mmol/liter pyruvate plus [1-14C]-pyruvate (2.104 Bq/ml), used at an isotopic dilution of 1:250.

3T3-F442A adipocytes were cultured in six well-plates (106 cells/3.5 cm well) in complete medium for 22 h, then medium was replaced with serum-deprived, glucose-free DMEM containing 0.3% (wt/vol) FA-free BSA for 3 h. For determining pyruvate incorporation into triacylglycerol, [1-14C]-pyruvate (2.104 Bq/ml) was used at an isotopic dilution of 1:250.

Two hours later, AT fragments and 3T3-F442A cells were rinsed and frozen in liquid nitrogen before lipid extraction according the simplified method of Bligh and Dyer (35). The subsequent [1-14C]-pyruvate incorporation was estimated by counting the radioactivity associated with the fragments. The incubation medium (2 h) was stored at –20 C for further NEFA (Free Fatty Acids Half Micro Test) and glycerol (glycerol UV-method) determinations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo and in vitro acute and selective action of IFN-{gamma} on AT
When administered to mice, IFN-{gamma} rapidly induced an increase in serum NEFAs (11). It was then suggested that AT lipolysis was enhanced. Knowing the role played by FA re-esterification in the whole lipolytic process, we decided to examine the relative proportion of blood NEFA vs. glycerol 8 h after IFN-{gamma} administration to Balb/c mice, at the concentration of 25 µg/20 g body weight mouse previously demonstrated as maximally efficient (5, 11). As shown in Fig. 1AGo, IFN-{gamma} induced almost a 50% increase in plasma NEFA, while glycerol concentration was unaffected, leading to an increase in the NEFA to glycerol ratio from 1.95–2.78. Such a result indicated to us that glyceroneogenesis could be the IFN-{gamma} target. Therefore, we monitored IFN-{gamma} action on the expression of Pck1, which encodes PEPCK-C, the key glyceroneogenic enzyme. We injected various amounts of IFN-{gamma}, from 0.1–25 µg, to mice, and monitored Pck1 mRNA in SCAT, EPAT, and PRAT 8 h later, using real-time RT-PCR. IFN-{gamma} induced a concentration-dependent decrease in Pck1 mRNA, with a 30–34% inhibition at the lower amount used (0.1 µg/mouse) and a maximal inhibition of 68, 81, and 83% in respectively SCAT, EPAT, and PRAT when the highest amount was injected (25 µg/mouse) (Fig. 1BGo). Although PEPCK-C is also expressed in liver and kidney, IFN-{gamma} injected at 25 µg/mouse did not affect Pck1 expression in these tissues, showing some tissue-specific action of the cytokine. We focused on SCAT and assessed the expression of a panel of genes encoding enzymes involved in FA metabolism, plus the PEPCK-M and mDIC. mDIC was highly expressed in adipocytes, and a proposed role in glyceroneogenesis was suggested (36, 37). IFN-{gamma} displayed some selectivity of action on Pck1 because mRNAs for PEPCK-M, aP2, GlyK, G3PDH, LPL, HSL, and ATGL remained unchanged (Fig. 1CGo). In addition, mDIC mRNA was also reduced, although to a lower extent (40%), than Pck1.


Figure 1
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  		FIG. 1. Effect of IFN{gamma} on gene expression and blood NEFA vs. glycerol concentrations in mice. Balb/c mice were treated, or not, for 8 h with IFN-{gamma} injected ip at various doses from 0.1–25 µg/20 g mouse, then sc, periepididymal (EP), and perirenal (PR) ATs plus liver and kidney were collected. A, Glycerol and NEFA concentrations were monitored in the plasma of mice treated, or not, with 25 µg IFN-{gamma}. B and C, Total RNA was extracted and used for mRNA determination by real-time RT-PCR. Results are expressed in percentage (%) mRNA relative to control (Ctl) taken as 100% for each gene. Each value represents the mean ± SEM of data obtained from six mice with three different samples taken for each mouse. *, P < 0.05 vs. control. **, P < 0.01 vs. control. ***, P < 0.001 vs. control.

 
IFN-{gamma} effect can be either direct on AT or indirect. To address this issue, we exposed cultured explants from SCAT from mice, isolated adipocytes, and differentiated adipocytes from the mouse 3T3-F442A cell line to IFN-{gamma} for 8 h before determining Pck1 mRNA levels. A large 60–70% decrease in Pck1 mRNA was observed whatever the ex vivo/in vitro model used (Fig. 2Go). Therefore, IFN-{gamma} effect is direct.


Figure 2
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  		FIG. 2. Effect of IFN-{gamma} on PEPCK-C mRNA in mouse SCAT, isolated adipocyte from SCAT and 3T3-F442A adipocyte. Subcutaneous AT explants from Balb/c mice, isolated adipocytes from these explants, and 9-d post-confluent 3T3-F442A adipocytes were treated, or not, with 50 ng/ml IFN-{gamma} for 8 h before RNA extraction and evaluation of PEPCK-C mRNA concentration by real-time RT-PCR. Results are expressed in percentage (%) mRNA relative to control (Ctl) taken as 100% for each tissue. Each value represents the mean ± SEM of triplicate determinations from three different series of explants or cells. ***, P < 0.001 vs. control.

 
Mechanism of IFN-{gamma} action in 3T3-F442A adipocytes
The time course of IFN-{gamma} action on Pck1 mRNA in 3T3-F442A adipocytes is shown in Fig. 3AGo. IFN-{gamma} induced a time-dependent reduction in mRNA, with a maximum of 80% decrease, reached at 5 h, which remained approximately constant up to 43 h later. We chose an 8-h treatment time to determine the concentration dependence of the effect. Half-maximum effect was reached at about 50 ng/ml (Fig. 3BGo), in agreement with what was shown earlier for 3T3-L1 cells (12). Similar to what was observed in vivo (Fig. 1CGo), analysis of gene selectivity revealed that only Pck1 and mDIC mRNA amounts were reduced by IFN-{gamma} in 3T3-F442A adipocytes (Fig. 3CGo). A 65% reduction in Pck1 mRNA was monitored, while mDIC mRNA showed a 40% decrease.


Figure 3
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  		FIG. 3. Effect of IFN-{gamma} on gene expression in 3T3-F442A adipocytes. Nine-day post-confluent 3T3-F442A adipocytes were treated, or not, with IFN-{gamma}, Iso, 8-br-cAMP, or the various combinations indicated in D, before RNA extraction and analysis by real-time RT-PCR. For experiments reported in A, C, and D, 50 ng/ml IFN-{gamma} was used. For experiments reported in B, C, and D, a treatment time of 8 h was used. C and D, Results are expressed in percentage (%) mRNA relative to control (Ctl) taken as 100% for each gene. Each value represents the mean ± SEM of triplicate determinations from three different series of cells. *, P < 0.05 vs. control. ***, P < 0.001 vs. control. &, P < 0.05 vs. Iso- and 8-Br-cAMP-treated cells.

 
The ß-agonist Iso and the cAMP analog 8-Br-cAMP were previously shown as good inducers of Pck1 transcription and mRNA stabilization in 3T3-F442A adipocytes (26). As expected, both agents stimulated PEPCK-C transcript amount by 2.8- to 5-fold in 8-h treated adipocytes (Fig. 3DGo). Interestingly, IFN-{gamma} inhibited these inductions either partly (Iso 1 µM) or totally (Iso 25 nM and 8-Br-cAMP 100 µM). Therefore, IFN-{gamma} opposes cAMP-induced expression of Pck1.

PEPCK-C protein amount was also reduced by IFN-{gamma} from 30% at 8 h to about 70% at 24 and 48 h, as seen by Western blot experiments (Fig. 4AGo). In addition, IFN-{gamma} inhibited Iso induction in PEPCK-C (Fig. 4BGo). In both cases, the PDH-E2 control protein amount was unaffected. We then checked whether glyceroneogenesis was the target for IFN-{gamma}. To address this issue, we preincubated 3T3-F442A adipocytes with either IFN-{gamma}, Iso, or both for 22 h, then placed cells under starving conditions, i.e. in glucose-deprived medium, for 3 h before incubating cells with [1-14C]-pyruvate for 2 h. Incorporation of radiolabeled pyruvate into the neutral lipid fraction was then evaluated. As expected from previous experiments, Iso stimulated 1-[1-14C]-pyruvate incorporation by 40%, while IFN-{gamma} reduced incorporation 2.8-fold and opposed Iso induction (Fig. 4CGo). Therefore, glyceroneogenesis is strongly diminished by IFN-{gamma}.


Figure 4
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  		FIG. 4. Effect of IFN-{gamma} on PEPCK-C amount, glyceroneogenesis, and FA release in 3T3-F442A adipocytes. Nine-day post-confluent 3T3-F442A adipocytes were treated, or not, with 50 ng/ml IFN-{gamma} and/or 25 nM Iso. Cytosolic protein extracts from cells treated for 8, 24, and 48 h (A) or 20 h (B) were isolated then submitted to Western blot with either anti-PEPCK-C or anti-PDH-E2 antibody. Autoradiograms of representative Western blots from three separate experiments with similar results are shown. C, Cells were treated with IFN-{gamma} and/or Iso for 22 h, then used for pyruvate incorporation into triacylglycerols as described in Materials and Methods. D, NEFA concentration was monitored in the same Krebs medium as that used for pyruvate incorporation, i.e. during 2 h. For C and D, each value represents the mean ± SEM of triplicate determinations from three different series of cells. *, P < 0.05 vs. control (Ctl). ***, P < 0.001 vs. control.

 
The incubation medium was used for NEFA determination. As expected from the opposite effect exerted by Iso and IFN-{gamma} on glyceroneogenesis, NEFA concentration was dampened 1.6-fold by Iso, whereas IFN-{gamma} opposed this effect (Fig. 4DGo).

Effect of IFN-{gamma} in explants and isolated adipocytes from human AT
We used SCAT explants from humans undergoing plastic surgery and adipocytes isolated from these explants to show that IFN-{gamma} reduced human PEPCK-C gene (PCK1) mRNA in a time-dependent manner with respectively a 70% and 50% decrease at 24 h (Fig. 5Go, A and B). A concentration-dependent response of AT explants was observed with a maximum reached at 50 ng/ml IFN-{gamma} (Fig. 5CGo). Here again, like with 3T3-F442A adipocytes, striking gene selectivity was detected with PCK1 mRNA being the sole gene affected by IFN-{gamma} in AT explants and in isolated adipocytes (Fig. 5Go, D and E), although mDIC mRNA was not monitored here. As expected, IFN-{gamma} produced a 50% reduction in the incorporation of radiolabeled pyruvate into neutral lipids from AT explants, resulting in an almost 2-fold increase in NEFA release to the medium (Fig. 5Go, F and G). Therefore, IFN-{gamma} acts similarly in human and mouse AT.


Figure 5
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  		FIG. 5. Effect of IFN-{gamma} in human SCAT and isolated adipocyte. Experiments with SCAT explants from humans (A, C, D, F, and G) and with adipocytes isolated from these explants (B and E) were designed as described in the legends for Figs. 1–4GoGoGoGo. Each value represents the mean ± SEM of triplicate determinations from three different patients, except for B, which reports results of triplicate determinations from two patients. *, P < 0.05 vs. control (Ctl). ***, P < 0.001 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several recent studies point to FA re-esterification in adipocytes as the key regulated pathway for FA release during lipolysis. In a recent and elegant review article, Hanson et al. (38) suggested that mammals might have difficulties controlling lipolysis, i.e. triacylglycerol hydrolysis, to the extent necessary to ensure the exact amount of energy needed by an individual. The lipolytic process could induce release of more FA than is essential for immediate energetic needs. Therefore, it is reasonable to consider that cells reconvert part of these FA back to triacylglycerol stores, allowing the fine-tuning of the whole process. In agreement with this viewpoint, adipocyte glyceroneogenesis and PEPCK-C have been described as acutely regulated by fasting/refeeding, and by a series of hormones, nutrients, and hypolipidemic drugs known to affect lipid metabolism like ß-agonists, glucocorticoids, retinoic acids, glucose, Fas, and thiazolidinediones (17, 19, 22). Of interest was the discovery that all these effects are rapid, and show some adipocyte-specific and gene-selective action on Pck1 expression (17, 22, 25).

Although the inhibitory effect of IFN-{gamma} action on adipocyte differentiation was described earlier by Floyd and Stephens (10), and Waite et al. (12), the mechanism of IFN-{gamma} action on FA metabolism in mature adipocytes had remained unresolved. In the present study, we demonstrate that IFN-{gamma} exerts an adipocyte-specific and gene-selective effect, inducing a rapid and extensive decrease in PEPCK-C expression and in glyceroneogenesis, both in rodents and humans, therefore largely augmenting FA release. Of interest is the demonstration that mDIC gene expression is also reduced by IFN-{gamma}. mDIC is a rate-limiting factor for the mitochondrial respiratory chain. It was previously suggested by Das et al. (36) and Lin et al. (37) that mDIC could be involved in glyceroneogenesis. Our results are in agreement with such a hypothesis.

A lipolytic role of IFN-{gamma} was previously described in mice (11). However, in this study glycerol release was not monitored, and the potential effect of IFN-{gamma} on glyceroneogenesis was not discussed. We show here that when mice are treated with IFN-{gamma}, blood FA is increased with no variation in glycerol concentration. This selective action of IFN-{gamma} on PEPCK-C expression and other potential glyceroneogenic genes, like mDIC, is in contrast to what was reported with other cytokines or with lipopolysaccharide (LPS) (8, 39). LPS treatment of mice for 16 h resulted in an extensive reduction in the expression of a large series of genes, particularly those encoding enzymes of glucose and lipid metabolism, including PEPCK-C (39). A series of class II nuclear receptor genes are also decreased in response to LPS. Similar results were obtained when 3T3-L1 adipocytes were exposed to TNF{alpha} for 24 h, resulting in increased lipolysis (7, 13). The difference in the broad effect of TNF{alpha} compared with the restricted action of IFN-{gamma} points to distinct mechanisms between both cytokines. When 3T3-F442A adipocytes are placed in conditions of stimulated Pck1 expression, i.e. Iso treatment, IFN-{gamma} inhibits Iso-induced increase in PEPCK-C mRNA, suggesting that IFN-{gamma} interferes with cAMP signaling. This long-term (8 h) effect of Iso is in contrast with the minute lipolytic action of this ß-agonist, which results in FA and glycerol output (40). Iso-induction of PEPCK-C leads to increased glyceroneogenesis and decreased FA release, both processes being counteracted by IFN-{gamma}.

What could be the pathophysiological relevance of this IFN-{gamma} effect? AT and lymph nodes are anatomically in close contact (41). In lymph nodes, T lymphocytes are activated by antigens. One of the consequences of such an activation process is a large increase in cell size (42). Activated lymphocytes are then induced to proliferate (43). Both phenomena require the supply of FAs that are used to participate in the formation of membrane phospholipids (44, 45). Newly incorporated FAs could change membrane fluidity, and modulate cell mobility and signaling (43). In compliance with this viewpoint, variations in the FA composition of membrane phospholipids can be observed as early as 4 h after exposure of T lymphocytes to an antigen (46). The hypothesis that T lymphocyte proliferation necessitates a local increase in FA, the origin of which is the immediately adjacent AT, has been nicely proposed by Pond and Mattacks (47). Our results argue for IFN-{gamma} being the inducing agent, targeting adipocyte PEPCK-C and glyceroneogenesis, for FA release. In addition, the immunomodulatory action of polyunsaturated FA has been described, demonstrating the existence of a bidirectional signaling between T lymphocytes and adipocytes (48). Finally, the infiltration of lymphoid cells in AT has been observed (49). The number of T cells appears to be increased in obesity (50). Thus, we can postulate that the IFN-{gamma}-induced overproduction of FA by adipocytes under such circumstances could result in an increase in plasma FA level, therefore participating in the occurrence of an insulin-resistant state often linked to obesity.


   Acknowledgments
 
We thank Dr. L. Benelli (Plastic Surgery Unit, Hartman Clinic, Paris, France) for his helpful involvement with human studies and P. Juraver (Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche S747) for animal handling.


   Footnotes
 
This work was supported by the Institut National de la Recherche Médicale, the Université René Descartes, and by a grant from the Association de Langue Française pour l’Etude du Diabète et des Maladies Métaboliques-GlaxoSmithKline (to C.F.). W.K. is the recipient of a fellowship from the Centre français pour l’accueil et les échanges internationaux. V.E.C. is the recipient of a fellowship from the Brazilian Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 10, 2007

Abbreviations: aP2, Fatty acid lipid binding protein; AT, adipose tissue; ATGL, adipocyte triglyceride lipase; EPAT, periepididymal adipose tissue; FA, fatty acid; G3PDH, glycerol-3-phosphate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GlyK, glycerol kinase; HSL, hormone-sensitive lipase; IFN-{gamma}, interferon {gamma}; Iso, isoproterenol; LPL, lipoprotein lipase; LPS, lipopolysaccharide; mDIC, murine mitochondrial dicarboxylate carrier; NEFA, nonesterified fatty acid; Pck1, rodent cytosolic phosphoenolpyruvate carboxykinase gene; PDH-E2, E2 subunit of pyruvate dehydrogenase; PEPCK-C, cytosolic phosphoenolpyruvate carboxykinase; PEPCK-M, mitochondrial cytosolic phosphoenolpyruvate; PRAT, perirenal adipose tissue; SCAT, sc adipose tissue.

Received September 14, 2006.

Accepted for publication May 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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