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Endocrinology Vol. 145, No. 11 4940-4947
Copyright © 2004 by The Endocrine Society

Cooperative Activation of Lipolysis by Protein Kinase A and Protein Kinase C Pathways in 3T3-L1 Adipocytes

Katrin Fricke, Aleksandra Heitland and Erik Maronde

IPF PharmaCeuticals GmbH, D-30625 Hannover, Germany

Address all correspondence and requests for reprints to: Erik Maronde, Ph.D., IPF PharmaCeuticals GmbH, Feodor-Lynen-Strasse 31, D-30625 Hannover, Germany. E-mail: erik.maronde{at}ipf-pharmaceuticals.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we investigate the coherence of signaling pathways leading to lipolysis in 3T3-L1 adipocytes. We observe two linear signaling pathways: one well known, acting via cAMP and protein kinase A (PKA) activation, and a second one induced by phorbol 12-myristate 13-acetate treatment involving protein kinase C (PKC) and MAPK. We demonstrate that both the PKA regulatory subunits RI{alpha} and RIIß are expressed in 3T3-L1 adipocytes and are responsible for the lipolytic effect mediated via the cAMP/PKA pathway. Inhibition of the PKA pathway by the selective PKA inhibitor Rp-8-CPT-cAMPS does not impair lipolysis induced by PKC activation, and neither PD98059 nor U0126, as known MAPK kinase inhibitors, changes the level of glycerol release caused by PKA activation, indicating no cross-talk between these two pathways when only one is activated. However, when both are activated, they act synergistically on glycerol release. Additional experiments focusing on this synergy show no involvement of MAPK phosphorylation and cAMP formation. Phosphorylation of hormone-sensitive lipase is similar upon stimulation of either pathway, but we demonstrate a difference in the ability of both PKA and the PKC pathway activation to phosphorylate perilipin, which in turn may be an explanation for the different maximal lipolytic effect of both pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRIACYLGLYCEROLS STORED AS lipid droplets in white adipocytes represent large energy stores in mammals, whose energy is released from fat cells in the form of free fatty acids. Multiple hormones and receptors are involved in the cellular responses and the balance between lipogenesis and lipolysis in adipocytes (1, 2). Among others, these include lipolytic agents such as ß-adrenergic agonists and antilipolytic factors such as insulin or adenosine. The major pathway leading to lipolysis is the activation of adenylate cyclase. The resulting increase in intracellular cAMP leads to the dissociation of protein kinase A (PKA), whose free catalytic subunits phosphorylate, among other substrates, hormone-sensitive lipase (HSL), and perilipin A (3). The phosphorylation of HSL leads to enhanced hydrolytic activity, translocation from cytosol to the lipid droplet surface, and subsequent release of free fatty acids and glycerol (see references in Ref. 4).

Although it is generally accepted that lipolysis is increased mainly by activation of PKA, other signaling pathways are thought to be involved in the lipolytic response of fat cells. One such pathway can be stimulated by the phorbol ester phorbol 12-myristate 13-acetate (PMA) and involves protein kinase C (PKC). In rat white fat cell membrane preparations, such PMA treatment was shown to moderately induce adenylate cyclase activity (5), so that it may be speculated that PMA acts indirectly via adenylate cyclase activation/cAMP elevation.

ERK 1 and 2 (or p42/44 MAPK) belong to a subfamily of MAPKs, and their signaling cascade plays an important role in several physiological events such as gene expression (6, 7), proliferation (8), differentiation (9, 10), and cell homeostasis (11). There is also evidence that an increase of free fatty acid release from fat cells occurs upon stimulation of the MAPK pathway (12). Moreover, activation of this pathway in adipocytes is linked to insulin resistance (13).

Perilipin proteins located on the lipid droplet surface of fat cells are major targets for PKA phosphorylation upon ß-adrenergic stimulation. They play an important role in lipolysis and triacylglycerol storage in adipocytes (14), serving to block HSL action in unstimulated cells. Phosphorylation by PKA results in a decrease in the density of the perilipin coat on the lipid droplet surface and a subsequent attenuation of this HSL blockade. Furthermore, it was shown that the cytokine TNF-{alpha} reduces perilipin expression in adipocytes, resulting in enhanced lipolysis (1). Another possible role in preventing lipid hydrolysis by HSL was reported by Souza et al. (15), suggesting that perilipins, being redistributed from the lipid droplet surface to the cytoplasm upon stimulation with isoproterenol, prevent the binding of HSL to the lipid droplets when unphosphorylated. The prominent role of perilipin in PKA-mediated lipolysis was further substantiated by the perilipin-knockout mouse phenotype that exhibits elevated basal lipolysis and attenuated stimulated lipolytic activity (4).

The present study was undertaken to investigate the coherence of existing signaling pathways leading to lipolysis, using the model cell line 3T3-L1. First, we analyzed the role of the PKA isoforms for lipolysis. Second, we found that the two linear signaling pathways (i.e. cAMP/PKA phosphorylation and PMA/PKC/MAPK) do not influence one another but act synergistically when both are activated. Both pathways involve HSL phosphorylation upon lipolytic stimulation, but the latter does not lead to the phosphorylation of perilipin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and differentiation
3T3-L1 fibroblasts (American Type Culture Collection, Manassas, VA) were grown in DMEM containing 10% fetal bovine serum (FBS). Differentiation into adipocytes was initiated after reaching confluence by adding DMEM containing 10% FBS, 300 µM 8-Br-cAMP, 1 µM dexamethasone, and 1 µg/ml insulin. After 3 d, cells were transferred to DMEM containing 10% FBS and 1 µg/ml insulin. Adipocytes were used for experiments 7–9 d after the differentiation protocol was initiated.

Lipolysis
3T3-L1 adipocytes cultured in 96-well plates were transferred into fresh DMEM and treated as described in Results. Glycerol content in the incubation medium was used as an index for lipolysis and was measured using a colorimetric assay (GPO-Trinder; Sigma, Deisenhofen, Germany). Briefly, supernatant was mixed with GPO-Trinder reagent A, and optical density was measured at 540 nm. Results are either expressed as fold of control or as percent isoproterenol effect, which was used as a positive control.

Immunoblot analysis
After incubation of 3T3-L1 adipocytes with the indicated stimulants, supernatants were removed, and whole-cell extracts were prepared by adding 50 µl of lithium-dodecyl-sulfate lysis buffer (NuPAGE; Invitrogen, Karlsruhe, Germany) containing 0.05 M dithiothreitol. Lysates were sonicated three times for 3 sec each, heat denatured at 70 C for 10 min, and centrifuged at 13,000 rpm for 5 min at 4 C. Transfer, immunodetection, and normalization were done as described previously (16).

Immunoprecipitation
3T3-L1 adipocytes were lysed at 4 C in cold lysis buffer (CytoSignal, Irvine, CA) containing a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The lysates were transferred into fresh Eppendorf tubes and centrifuged at 13,000 rpm for 5 min at 4 C. Supernatants were incubated for 1 h at 4 C under constant shaking with a polyclonal rabbit antiphosphoserine antibody or with the mouse polyclonal antibody against HSL, and the immune complexes were recovered by overnight incubation with protein A/G resin (CytoSignal) at 4 C under shaking. After centrifugation at 13,000 rpm for 10 min at 4 C, pellets were washed three times with cold lysis buffer, reconstituted in lithium-dodecyl-sulfate lysis buffer, and analyzed by immunoblot.

cAMP ELISA
Intracellular cAMP was measured using a cAMP ELISA kit (IHF GmbH, Hamburg, Germany) (17). 3T3-L1 adipocytes were treated with the indicated stimulants for 30 min. Supernatants were removed, 70% ethanol was added to each well, and the cells were scraped into Eppendorf tubes. Tubes were dried under vacuum, and 100 µl of cAMP working buffer was added to each tube, followed by cAMP assay according to the supplier’s protocol.

Chemicals
Laboratory reagents and cell culture medium were obtained from Sigma-Aldrich (Taufkirchen, Germany) and Invitrogen (Karlsruhe, Germany). FBS was from Biochrom (Berlin, Germany), and all cAMP analogs were from BIOLOG Life Science Institute (Bremen, Germany). PMA, isoproterenol, and forskolin were from Sigma-Aldrich, bisindolylmaleimide I, Ro-31–8220, and U0126 were from Calbiochem (Schwalbach, Germany), and PD98059 and chelerythrine were from QBiogene-Alexis (Grünberg, Germany). The final dimethylsulfoxide concentration per assay was maximally 1%, which did not show any effect in vehicle controls. Antibodies against phosphorylated MAPK (pMAPK) and total MAPK were from New England Biolabs (Frankfurt, Germany). Antibodies against PKA subunits were from BD Biosciences PharMingen Transduction Labs (Erembodegem, Belgium), and the polyclonal antibody against perilipin was purchased from Progen (Heidelberg, Germany). The polyclonal rabbit antibody against phosphoserine came from Zymed Laboratories (Zytomed, Berlin, Germany). For antibodies to HSL, a peptide based upon the rat (GenBank) HSL sequence (KDLSFKGNSEPSDSPEMC) was used to immunize mice, and the antiserum was used for immunoblot analysis at a dilution of 1:1000. Peroxidase-conjugated antibodies against mouse and rabbit were from New England Biolabs (Frankfurt, Germany), and the anti-guinea pig antibody conjugated to peroxidase was from Sigma (Deisenhofen, Germany).

Data analysis
Values are given as means ± SEM of (n) separate experiments. One-way ANOVA and Bonferroni’s multiple comparison post test were used for statistical analyses. P values ≤ 0.05 were considered statistically significant. The concentration-response curves were fitted by nonlinear regression, and EC50 values (half-maximal effective drug concentration) were calculated using the GraphPad Prism 3.03 software (GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protein expression of PKA subunits in differentiated 3T3-L1 adipocytes
Specific antibodies against PKA subunits RI({alpha}/ß), RI{alpha}, RII{alpha}, RIIß, and C{alpha}/ß were used to investigate protein expression of PKA subtypes in 3T3-L1 adipocytes. Figure 1AGo shows the PKA subunits RI{alpha}, RIIß, and C{alpha}/ß. PKA subunits RI{alpha}/ß and RII{alpha} are not detected by immunoblot analysis. A 5-h stimulation of 3T3-L1 adipocytes with 1 µM isoproterenol has no effect on PKA subunit protein levels. The cAMP analog pair 8-AHA-cAMP [8-(6-aminohexyl)amino-cAMP]/8-PIP-cAMP (8-piperidino-cAMP), which selectively activates PKA type I, strongly induces glycerol release (Fig. 1BGo). In addition, the analog pair MBC-cAMP (N6-mono-tert, butylcarbamoyl-cAMP)/Sp-5,6-DCl-cBiMPS (5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate, Sp-isomer), which selectively activates PKA type II, shows a similar result (Fig. 1BGo), indicating that both PKA subtypes expressed in 3T3-L1 adipocytes are involved in the lipolytic action.



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  		FIG. 1. PKA subunits in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were stimulated with control medium or isoproterenol (1 µM) for 5 h. Equal amounts of cell lysates were subjected to immunoblot analysis using antibodies against the PKA subunits RI, RI{alpha}, RII{alpha}, RIIß, and C{alpha}/ß as indicated. Blots were subsequently reprobed with an antibody against ß-actin to confirm equal loading of the lanes. Results are representative for at least three separate experiments. B, 3T3-L1 adipocytes were treated with different cAMP analogs of the indicated concentrations for 5 h, and glycerol release was measured. Dose-dependent treatment with 8-AHA-cAMP combined with 100 µM 8-PIP-cAMP both selectively binding to PKA type I elicited a strong lipolytic effect ({blacksquare}). Treatment with increasing concentrations of N6-MBC-cAMP combined with 5 µM Sp-5,6-DCl-cBiMPS both selectively binding to PKA type II also showed a strong lipolytic response ({square}). Data are expressed as percent of glycerol release elicited by 1 µM isoproterenol (the effect of 1 µM isoproterenol equals 100%). Data points represent the mean ± SEM (n = 9) of three experiments.

 
Effects of a cAMP analog and PMA on glycerol release of 3T3-L1 adipocytes and the role of cAMP and protein kinase A
Figure 2AGo shows that SpcAMPS (adenosine-3',5'-monophosphorothioate, Sp-isomer) induces lipolysis in 3T3-L1 adipocytes in a dose-dependent manner with an EC50 value of 140 µM. The maximal lipolytic effect is similar to that observed with forskolin (inset of Fig. 2AGo), which has an EC50 value of 0.7 µM. Interestingly, both SpcAMPS and forskolin are about twice as effective as isoproterenol, indicating that isoproterenol itself may not act exclusively via elevated cAMP levels. PMA also increases lipolysis in 3T3-L1 adipocytes in a dose-dependent manner with an EC50 value of 0.2 µM (Fig. 2BGo). However, in contrast to SpcAMPS and forskolin, the lipolytic efficacy of PMA is approximately 50% of the glycerol release induced by isoproterenol (1 µM), and basal lipolysis is approximately doubled upon PMA incubation. When PMA is applied in increasing concentrations together with different amounts of SpcAMPS (20, 60, and 100 µM), the lipolytic effect is potentiated (Fig. 2CGo).



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  		FIG. 2. Effect of the cAMP analog SpcAMPS and PMA as well as costimulation with both agents on lipolysis in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated with SpcAMPS and PMA or both of the indicated concentrations or with forskolin for 5 h, and glycerol release was measured. A, Effect of SpcAMPS and forskolin (inset) on glycerol release. B, Effect of PMA on lipolysis. The EC50 values for SpcAMPS, PMA, and forskolin were 140, 0.2, and 0.7 µM, respectively. Results are expressed as percentages of the lipolytic response to 1 µM isoproterenol. C, Dose-response curve of glycerol release caused by PMA alone or in combination with different concentrations of SpcAMPS. Values are expressed relative to the basal lipolysis obtained from the stimulation with DMEM. Data points represent the mean ± SEM (n = 9–12) of three to four experiments. All PMA concentrations of 0.1 µM or higher had a significant effect (P ≤ 0.05) compared with 0.01 µM PMA. In addition, the combination of 0.03 µM PMA with 100 µM SpcAMPS was also significant compared with 0.01 µM PMA; *, P ≤ 0.05.

 
The selective PKA inhibitor Rp-8-CPT-cAMPS (8-para-chlor-thiophenyl-adenosine-3',5'-monophosphorothioate, Rp-isomer), which occupies cAMP-binding sites at the regulatory subunit of PKA and prevents the kinase holoenzyme from dissociating and thus from activation, was used for inhibitor studies. Rp-8-CPT-cAMPS significantly inhibits lipolysis induced by the cAMP analog SpcAMPS (100 µM), indicating involvement of PKA (Fig. 3Go), whereas lipolysis induced by PMA is not inhibited by this PKA inhibitor. Furthermore, no significant increase in intracellular cAMP was detected after stimulation of 3T3-L1 adipocytes with PMA, whereas forskolin treatment strongly elevates cAMP levels (Table 1Go). When coapplied with forskolin (1 µM), PMA does not significantly increase cAMP levels compared with the effect of forskolin alone (Table 1Go; P > 0.05). Thus, it appears that intracellular cAMP elevation and subsequent PKA activation do not participate in either PMA-induced lipolysis or in the synergy of lipolytic action between PMA and SpcAMPS observed in 3T3-L1 adipocytes.



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  		FIG. 3. Effect of a PKA inhibitor on SpcAMPS- and PMA-induced lipolysis in 3T3-L1 adipocytes. 3T3-L1 adipocytes were preincubated with or without Rp-8-CPT-cAMPS for 40 min followed by incubation with SpcAMPS (100 µM) or PMA (3 µM) for 5 h. Glycerol release was determined. SpcAMPS-induced lipolysis was significantly inhibited by Rp-8-CPT-cAMPS (300 µM), whereas the PKA inhibitor up to a concentration of 1 mM had no effect on lipolysis induced by PMA. Data points are means ± SEM (n = 9) of at least three separate experiments. *, P ≤ 0.05 compared with no inhibitor.

 

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  		TABLE 1. Effect of PMA and forskolin on intracellular cAMP levels

 
Pharmacological characterization of the PMA-induced glycerol release in 3T3-L1 adipocytes
The increase in lipolysis by PMA as shown in Fig. 2BGo was further characterized by analyzing MAPK phosphorylation and by inhibitor experiments. In parallel to stimulation of lipolysis, similar concentrations of PMA cause p42/44 MAPK phosphorylation in a dose-dependent manner, as shown by immunoblotting (Fig. 4Go, top). In contrast, stimulation of 3T3-L1 adipocytes with SpcAMPS of a lipolytic concentration (60 µM) shows no visible activation compared with stimulation with PMA (Fig. 4Go, bottom). Even higher SpcAMPS concentrations up to 1 mM do not elevate p42/44 MAPK phosphorylation (data not shown). Furthermore, when increasing concentrations of PMA are coapplied together with SpcAMPS (60 µM), no change in p42/44 MAPK phosphorylation is observed compared with the stimulation with PMA alone (Fig. 4Go, bottom). This indicates that MAPK is not involved in the synergy of the PMA- and SpcAMPS- induced lipolysis shown in Fig. 2CGo.



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  		FIG. 4. Effects of PMA and SpcAMPS on MAPK phosphorylation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were stimulated with PMA of the indicated concentrations alone (top) or in combination with SpcAMPS (60 µM; bottom) for 30 min, extracted, subjected to SDS-PAGE, immunoblotted, and probed with antibodies against pMAPK and MAPK. The positions of the pMAPK and MAPK bands are indicated. The upper and lower bands represent p44 and p42 MAPK, respectively. The dose-dependent MAPK phosphorylation caused by PMA was not affected by the coapplication of SpcAMPS. Results shown are representative for three independent experiments.

 
Preincubation of 3T3-L1 adipocytes with different PKC inhibitors leads to an inhibition of PMA-induced glycerol release for bisindolylmaleimide I and Ro-31–8220. However, the PKC inhibitor chelerythrine is not able to inhibit lipolysis induced by PMA in 3T3-L1 adipocytes in concentrations up to 3 µM (Fig. 5AGo).



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  		FIG. 5. Effects of PKC and MEK inhibitors on PMA- and SpcAMPS-induced lipolysis and effect of PKC inhibitors on MAPK phosphorylation in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were preincubated with or without PKC inhibitor for 40 min and then incubated with PMA (3 µM) for 5 h. Glycerol release was measured. Ro-31–8220 (1 µM) and bisindolylmaleimide I (3 µM) inhibited PMA-induced lipolysis, whereas chelerythrine had no significant effect. Data points are means ± SEM (n = 9) of three independent experiments. *, P ≤ 0.05 compared with no inhibitor. B, 3T3-L1 adipocytes were preincubated with or without inhibitor of the same concentrations as used in A for 40 min, followed by an incubation with control medium or PMA (3 µM) for 30 min. Cell lysates were subjected to immunoblot analysis using antibodies against pMAPK and total MAPK, which band positions are indicated. Results shown are representative for two to three experiments performed in duplicate. C and D, 3T3-L1 adipocytes were preincubated with or without either PD98059 or U0126 for 40 min followed by a 5-h stimulation with PMA (3 µM) (C) or SpcAMPS (100 µM) (D). Glycerol release was determined. Concentrations of 30 and 100 µM PD98059 inhibited lipolysis induced by PMA; 1 µM U0126 also inhibited PMA-induced lipolysis. Neither MEK inhibitor significantly decreased SpcAMPS-induced lipolysis. Data points are means ± SEM (n = 9) of three separate experiments. *, P ≤ 0.05 compared with no inhibitor.

 
To find out whether the lipolytic effect of PMA is mediated by MAPK phosphorylation, the ability of PKC inhibitors to reduce MAPK phosphorylation was investigated by immunoblot analysis. Figure 5BGo shows that bisindolylmaleimide I has the strongest inhibitory effect on MAPK phosphorylation induced by stimulation with PMA. Ro-31–8220 also reduces the amount of pMAPK signal, although more weakly than bisindolylmaleimide I. After preincubation with chelerythrine, MAPK phosphorylation levels do not significantly change. These results are consistent with the data on lipolysis after incubation with PKC inhibitors.

As depicted in Fig. 5CGo, the MAPK kinase (MEK) inhibitors PD98059 and U0126 inhibit PMA-induced lipolysis. This indicates an involvement of MAPK phosphorylation in glycerol release induced by PMA. In contrast, neither PD98059 nor U0126 significantly inhibits lipolysis induced by SpcAMPS (Fig. 5DGo; P ≥ 0.05), which is consistent with our observation that SpcAMPS does not induce MAPK phosphorylation (Fig. 4Go).

HSL phosphorylation after PMA and SpcAMPS exposure in 3T3-L1 adipocytes
Because the phosphorylation of HSL at serine residues is thought to be the limiting step of HSL activation and lipolysis, we studied the effects of PMA and SpcAMPS on HSL serine phosphorylation rate at concentrations known to stimulate lipolysis in 3T3-L1 adipocytes. Both PMA and SpcAMPS induce the serine phosphorylation of HSL. As shown in Fig. 6AGo, immunoprecipitates obtained with antiphosphoserine antibody on cell lysates from cells stimulated with PMA and SpcAMPS show that both stimulants induce HSL serine phosphorylation compared with control. This serine phosphorylation of HSL was confirmed by using an antibody directed against HSL for immunoprecipitation and subsequent immunoblot analysis with an antibody against phosphoserine (Fig. 6 BGo). Costimulation of 3T3-L1 adipocytes with PMA and SpcAMPS (3 and 100 µM, respectively) leads to approximately the same induction of serine phosphorylation of HSL as the incubation with PMA alone. Immunoprecipitation using the antibody against HSL results in two bands of which the larger one corresponds to mouse HSL. The smaller band, possibly a degradation product of HSL, shows a molecular mass of approximately 32 kDa and the same induction of phosphorylation at serine by PMA and SpcAMPS compared with control as described above. Immunoprecipitation using the antibody directed against phosphoserine detected only the smaller band displayed in Fig. 6Go.



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  		FIG. 6. PMA- and SpcAMPS-dependent stimulation of HSL phosphorylation in 3T3-L1 adipocytes. Cells were exposed to 3 µM PMA, 1 mM SpcAMPS, or PMA and SpcAMPS (3 and 100 µM, respectively) for 15 min. A, HSL phosphorylation profile was analyzed by immunoprecipitation using an antibody directed against phosphoserine followed by an immunoblot analysis using an antibody directed against HSL. B, Adipocytes were incubated with PMA, SpcAMPS, or both as described above, and immunoprecipitates were obtained using an antibody directed against HSL. Immunoblot analysis was performed using the antibody against phosphoserine. Probing of immunoblots with the antibody against HSL was used to assess the level of HSL in each lane. IP, Immunoprecipitation; IB, immunoblot.

 
Effect of PMA and SpcAMPS on perilipin phosphorylation in 3T3-L1 adipocytes
Perilipin, like HSL, is described to be phosphorylated in response to PKA activation and is involved in the lipolytic reaction of fat cells. To examine the effect of SpcAMPS and PMA on phosphorylation of perilipin in differentiated 3T3-L1 adipocytes, we used a guinea pig polyclonal antiserum against perilipin. As depicted in Fig. 7Go, 100 µM and 1 mM SpcAMPS induce the characteristic shift in the migration of perilipin, whereas no such electrophoretic shift is observed after stimulation with 3 µM PMA.



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  		FIG. 7. Effect of PMA and SpcAMPS on the electrophoretic shift of perilipin. 3T3-L1 adipocytes were serum depleted overnight and then incubated with 3 µM PMA, DMEM, 1 mM SpcAMPS, or 0.1 mM SpcAMPS for 30 min. Equal amounts of cell lysates were subjected to immunoblot analysis. Blots were probed with an antibody against perilipin. The image of immunodetected perilipin with the characteristic phosphorylation-dependent molecular weight shift is representative for at least three (PMA and 100 µM SpcAMPS) and two (1 mM SpcAMPS) experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study was conducted to compare the different signaling pathways leading to lipolysis in 3T3-L1 adipocytes and to elucidate which signaling steps are essential for triglyceride release.

The major pathway leading to lipolysis involves activation of cAMP-dependent PKA, which in turn activates other substrates such as HSL and perilipin. Eukaryotic PKA exists as two different isozymes (type I and type II). In a deactivated status, both types form a tetrameric complex consisting of two regulatory and two catalytic subunits, each of the former offering two cooperative binding sites A and B for the activator cAMP. After binding of cAMP to the regulatory subunits, the holoenzyme complex dissociates, releasing free catalytic subunits that then are able to phosphorylate suitable substrates (see references in Ref. 18). It has been shown that RIIß is the principal PKA regulatory subunit in adipose tissue (19). However, in this study, we first describe that regulatory PKA subunit RIIß as well as RI{alpha} are expressed in 3T3-L1 adipocytes. Using different pairs of cAMP analogs that complement each other for the selective activation of either PKA type I or PKA type II (18) clearly shows that both of these PKA subtypes participate in the lipolytic action involving the known PKA pathway. Besides that, we did not detect any change in protein levels of PKA subunits after 5-h stimulation with isoproterenol, which is consistent with the data described for the PKA subunits in the rodent pineal organ (20), but different from the human endometrium, where RI{alpha} disappears after prolonged treatment with relaxin or cAMP analogs (21).

In our studies, cAMP elevation using the phosphodiesterase-resistant cAMP analog SpcAMPS (22) induced a significant dose-dependent increase in lipolysis, which was inhibited by the selective PKA inhibitor Rp-8-CPT-cAMPS (23), demonstrating the involvement of PKA in SpcAMPS-induced lipolysis via intracellular cAMP elevation.

Whereas the hormonal regulation of lipolysis via the cAMP/PKA pathway is well established, the role of the PKC/MAPK pathway in terms of its physiological relevance is not fully understood. On the one hand, MEK/ERK activation during the early stage of differentiation promotes adipogenesis (7), but on the other hand, MEK/ERK activation in differentiated adipocytes leads to insulin resistance (13) and increased lipolysis (12). Adipocytokines, such as TNF-{alpha}, IL-6, or IL-8 activate ERK downstream of their receptors (24, 25, 26), which raises evidence that ERK activation may play a critical role in the adipocytokine-derived lipolytic signal and insulin resistance in adipocytes.

According to our data, lipolysis induced by PMA is not linked to the PKA pathway in 3T3-L1 adipocytes. The phorbol ester PMA is a diacylglycerol analog and activator of PKC (27), which can activate the kinase MEK (28). Other cell-permeable diacylglycerols have been shown to induce lipolysis in 3T3-L1 adipocytes via activation of the MAPK/ERK pathway (12), and PMA also induces lipolysis in adipose tissue in situ (29). However, deMazancourt et al. (30) detected no change in glycerol release by PMA in isolated rat fat cells. Candidate PKC isoforms involved are the novel isoforms {delta} and {epsilon}, as well as the atypical isoforms {lambda} and {iota}, which are highly expressed in differentiated 3T3-L1 adipocytes (31) (our own unpublished data). In the present investigation, we first demonstrate the dose dependence in glycerol release upon stimulation of 3T3-L1 adipocytes with PMA. In terms of the signaling events participating, we show that the PKC inhibitors bisindolylmaleimide I and Ro-31–8220 reduce PMA-induced MAPK phosphorylation and lipolysis. However, so far we have no explanation for our finding that the PKC inhibitor chelerythrine does not have an inhibitory effect on PMA-induced glycerol release and MAPK activation, although this fact supports the need to inhibit MAPK phosphorylation to be able to inhibit PMA-induced lipolysis. We furthermore show that the highly specific MEK inhibitors PD98059 and U0126 reduce lipolytic stimulation by PMA. Both PD98059 and U0126 block ERK activation, but via different mechanisms. Whereas PD98059 prevents MEK activation by Raf (32), U0126 directly protects ERK from being phosphorylated by MEK (33).

In 3T3-L1 adipocytes, we show that activation of both the PKA and the PKC pathway leads to HSL phosphorylation at serine residues to a similar degree. These data are consistent with the identification of a phosphorylation site on HSL as a target of MAPK/ERK by using a tamoxifen-regulatable Raf system (12). Because it was shown that ERK-mediated phosphorylation of rat HSL leads to an increase in HSL hydrolytic activity, we conclude that HSL phosphorylation by PMA shown in murine adipocytes used in this study is also caused by MAPK phosphorylation, which subsequently increases glycerol release. Our findings that immunoprecipitation using the antibody directed against phosphoserine obtained only a 32-kDa band, but no band corresponding to full-size mouse HSL might be explained by competition of serine-phosphorylated proteins that bind to the phosphoserine antibody with different efficacy.

SpcAMPS-dependent lipolysis is associated with an increase in perilipin phosphorylation similar to that of isoproterenol and natriuretic peptides described earlier in human primary adipocytes (34), whereas PMA did not induce any shift in perilipin migration, indicating a lack of perilipin phosphorylation upon PKC and/or MEK activation in 3T3-L1 adipocytes. These data are consistent with studies in adrenal cells, in which activation of PKC by PMA did not affect the redistribution of perilipin onto the lipid droplet surface (35). However, phosphorylation of perilipin is necessary for the translocation of HSL from the cytoplasm to the lipid droplet surface upon lipolytic stimulation to achieve maximal glycerol release (36). Because of the lack of perilipin phosphorylation by PMA treatment and our finding that the HSL phosphorylation does not correlate with the lipolysis rate of PMA and SpcAMPS, we consider it likely that translocation of HSL may be impaired, resulting in the observed moderate lipolytic effect of PKC stimulation, because the increase in lipolysis induced by PMA has only approximately 50% of the magnitude seen with ß-adrenergic stimulation by isoproterenol, compared with the dramatic increase in glycerol release up to approximately 200% after activation of the PKA pathway by cAMP analogs.

Interestingly, both the PKA and the PKC pathway do not influence one another when selectively activated. SpcAMPS does not induce MAPK phosphorylation in concentrations that show a strong lipolytic effect, and PKA-induced lipolysis alone cannot be inhibited by MEK inhibitors. These results differ from the published data of others (12), who detected MAPK phosphorylation after stimulation with 10 µM isoproterenol. In contrast to these studies, we used a cAMP analog, which does not require any interaction with receptors to selectively activate PKA. This should exclude most other receptor-mediated effects besides PKA activation. In line with our data, forskolin, an adenylate cyclase activator, also failed to induce MAPK phosphorylation (data not shown). Furthermore, lipolysis via the ERK pathway is not decreased by the selective PKA inhibitor Rp-8-CPT-cAMPS, and PMA alone does not increase cAMP formation in 3T3-L1 adipocytes. However, when both pathways are activated, they act synergistically in terms of lipolysis, yet it remains to be determined which mechanisms are involved in this synergy. First, the dose-dependent phosphorylation of MAPK induced by PMA is not changed by coapplication of SpcAMPS. Second, stimulation of both pathways via forskolin and PMA leads to the same cAMP formation caused by forskolin alone, and third, HSL phosphorylation by both PMA and SpcAMPS together shows a similar extent to that when PMA is applied alone. This leads us to the conclusion that neither MAPK phosphorylation nor cAMP formation nor HSL phosphorylation participates in the synergy concerning lipolysis. The synergy may be caused by low to moderate perilipin phosphorylation via the PKA pathway (as induced by, e.g. low concentrations of SpcAMPS), which increases the availability of substrate for HSL. This configuration does not occur when only the PKC/MAPK pathway is activated. In that sense, SpcAMPS works as a gate for the limited action of PMA alone. A similar synergism between PKA and PKC pathways has been described before, e.g. in HUH-7 hepatoma cells (37). There, expression of a reporter gene construct using luciferase driven by the human per1-promoter was also potentiated by coapplication of PMA and SpcAMPS, but neither ERK nor cAMP response element-binding protein (CREB) phosphorylation explained the synergism.

It has been shown in other cell lines that calcium- and p38-dependent pathways (38, 39) are inducible by PMA. However, we did not observe calcium influx after PMA application in 3T3-L1 adipocytes (data not shown). Thus, other mediators for PMA-induced potentiation of the PKA-mediated increase in lipolysis besides perilipin remain to be determined and may include p38 as well as other signaling pathway components.

In summary, we show that the well-known cAMP/PKA pathway acts via the PKA regulatory subunits RI{alpha} and RIIß in terms of lipolysis in 3T3-L1 adipocytes. The PKC/MAPK-dependent pathway that also exists in these cells shows a synergistic effect with the former in terms of the lipolytic reaction. This synergistic effect is caused neither by MAPK phosphorylation nor by cAMP formation or phosphorylation of HSL at serine residues. Possibly, the portion of perilipin phosphorylation, which is achieved by a moderate stimulation of the PKA pathway, but not by stimulation of the PKC/MAPK pathway, enhances the lipolytic action of the latter in a far more than additive manner.


   Acknowledgments
 
The expert technical assistance in peptide synthesis of Jessica Babierowski is gratefully acknowledged.


   Footnotes
 
Abbreviations: FBS, Fetal bovine serum; HSL, hormone-sensitive lipase; MEK, MAPK kinase; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; pMAPK, phosphorylated MAPK; Rp-8-CPT-cAMPS, 8-para-chlor-thiophenyl-adenosine-3'5'-monophosphorothioate, Rp-isomer; SpcAMPS, adenosine-3',5'-monophosphorothioate, Sp-isomer.

Received June 25, 2004.

Accepted for publication July 20, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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