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Possible mechanisms by which adipocyte lipolysis is enhanced in exercise-trained rats
BBRC Biochemical and Biophysical Research Communications 295 (2002) 236–242 www.academicpress.com
Possible mechanisms by which adipocyte lipolysis is enhanced in exercise-trained rats Sachiko Nomura,a Hitomi Kawanami,a Hiroshi Ueda,a Takako Kizaki,b Hideki Ohno,b and Tetsuya Izawaa,* a
Department of Kinesiology, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, Tokyo 192-0397, Japan b Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine, Tokyo, Mitaka 181-8611, Japan Received 31 May 2002
Abstract A possible mechanism(s) behind exercise training-enhanced lipolysis was investigated in rat adipocytes. Exercise training (9 weeks; running) enhanced the activity of cAMP-dependent protein kinase (PKA) and the protein expressions of PKA subunits (catalytic, RIIa, and RIIb) in P40 fraction (sedimenting at 40,000g), but not in I40 fraction (infranatant of 40,000g) of adipocyte homogenate. The expression of PKA-anchoring protein 150 (AKAP150) in P40 fraction was greater in exercise-trained (TR) than in control (C) rats. Hormone-sensitive lipase (HSL) activities in both fractions were also greater in TR. On the other hand, stimulated lipolysis was accompanied by increased activities of HSL in P40 but not in I40 fraction. The decreases in stimulated lipolysis due to St-Ht31 were greater in TR rats. Thus, the mechanisms behind exercise training-enhanced adipocyte lipolysis could involve the increased activities of PKA and HSL with enhanced expressions of AKAP150 and some subunits of PKA, all of which may be compartmentalized within adipocytes. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Exercise; cAMP-dependent protein kinase; A-kinase-anchoring protein; Hormone-sensitive lipase; Adipocyte; Rat
It is widely accepted that the hormonal activation of adipocyte lipolysis is mediated via a cAMP-dependent process. Increased concentrations of cAMP activate cAMP-dependent protein kinase (PKA), which, in turn, leads to phosphorylation and activation of hormonesensitive lipase (HSL). Chronic exercise training enhances the lipolytic response to catecholamines in laboratory animals and in humans [1–7]. The major biochemical alteration underlying this enhanced lipolysis has been thought to occur at a site distal to cAMP production, such as at the level of HSL. Enevoldsen et al. [1] have recently shown that exercise training enhances the agonist-stimulated activities of HSL and the protein expression of HSL in the cytosolic fraction of retroperitoneal and mesenteric adipose tissue homogenates. However, accumulated evidence shows that the activation of cytosolic HSL is not always important for the breakdown of triacylglycerol [8–10]. A redistribution of
*
Corresponding author. Fax: +81-426-77-2961. E-mail address: [email protected] (T. Izawa).
HSL from cytosol to either the centrifuged pellet fractions [9] or to its substrate at the surface of the lipid droplet [10] is required for the subsequent activation of full lipolysis. Therefore, it is possible that exercise training-enhanced adipocyte lipolysis is mediated via increased activity of HSL, which is located at either the centrifuged pellet fractions or the surface of the lipid droplet. Moreover, the intracellular concentration of cAMP does not always increase due to enhanced activity of phosphodiesterase in exercise-trained rat adipocytes [6], although exercise training enhances the cAMP production events [2,7]. Neither has any increase in PKA activity been found in the cell extracts [4,5]. Thus, HSL activity may be enhanced without increases in either intracellular cAMP concentrations or cytosolic PKA activity after exercise training. This enigma might be explained by the compartmentalization of cAMP within adipocytes [11]. However, in fact, it is unlikely that a small, soluble molecule like cAMP could be restricted to a particular cytosolic compartment. It seems rational that PKA is located in specific subcellular compartments.
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 6 6 4 - 2
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A family of PKA-anchoring proteins (AKAPs) has a key role in the compartmentalization of PKA [12–15]. PKA heterotetramers are composed of a dimeric catalytic subunit and a dimeric cAMP-binding regulatory subunit type I (RIa and RIb) or type II (RIIa and RIIb). It has been postulated that, of these regulatory subunits, either RIIa or RIIb is compartmentalized through association with AKAPs [12–15]. Thereby, PKA exists in close proximity to its substrate and the interaction between RII subunits and AKAPs is required for certain intracellular signaling events. Taking all these facts together, we hypothesized that both PKA subunits and AKAPs are placed in some cellular organelles’ proximity to a signal generator (e.g., adenylate cyclase) and PKA substrate/effector proteins (e.g., HSL) in adipocytes and that exercise training could alter both PKA activity and the protein expressions of AKAPs, which are placed in some cellular organelles. Thereby, the activity of HSL, which is also compartmentalized in some cellular organelles in close proximity to lipid storage droplet, could be enhanced. Our preliminary experiments [16] and a very recent study [17] showed the existence of AKAPs in rat adipocytes and C3H/10T1/2 adipocytes, respectively. Therefore, to confirm the hypothesis, we investigated whether either PKA subunits or AKAPs are present in two subcellular fractions of centrifuged homogenate of adipocytes: the infranatant fraction (I40 fraction, supernatant of 40,000g) and the pellet fraction (P40 fraction, sedimenting at 40,000g). The effects of exercise training on subcellular distributions of either PKA subunits or HSL and the expression of AKAP proteins were then investigated. Materials and methods Animal care and exercise training program. Male Wistar rats (SLC, Japan) with an initial body weight of 90–120 g were housed two or three to a cage in a room with temperature controlled at 24 °C and a 12:12-h light–dark cycle. Food and water were available ad libitum. The animals were randomly divided into two groups, sedentary control (C, N ¼ 25) and exercise-trained (TR, N ¼ 20). The rats in the trained group were exercised on a treadmill set at a 5° incline for 5 days/week for 9 weeks according to the protocol previously reported [2–4]. Sedentary control animals were not subjected to treadmill running. Exercise-trained rats were sacrificed more than 36 h after the last exercise session. After an overnight fast, the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (5 mg/100 g body weight; Abbott, IL). The epididymal fat pads were removed and used for adipocyte isolation. All experiments conducted in this study were approved by the Animal Care Committee of Tokyo Metropolitan University Graduate School of Science. Preparation of adipocytes. Adipocytes were isolated by a modification of the method of Rodbell [18]. Fat pads were minced with scissors and placed in plastic vials in Buffer A (Krebs–Ringer bicarbonate solution buffered with 10 mM HEPES, pH 7.4, containing 5.5 mM glucose and 2% (w/v) fatty acid-free bovine serum albumin) with 200 nM adenosine and collagenase type I (1 mg/ml, Worthington Biochemical, NJ). Collagenase digestion was performed at 37 °C in a
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water bath shaker. After 30 min, the contents of the vials were immediately filtered and centrifuged for 1 min at 100g. The layer of floating cells was then washed three times with Buffer A. Measurement of lipolysis. Adipocytes (approx. 105 cells) were incubated in plastic vials in a total volume of 0.5 ml of Buffer A with or without indicated agents. The incubation medium contained adenosine deaminase (1 U/ml). After 30 min, a cell-free incubation medium was assayed for glycerol as an index of lipolysis. The average number of adipocytes and glycerol content were determined according to a previous study [3]. Adipocyte subcellular fraction. Adipocyte subcellular fractions were essentially prepared by the methods of Hirsh and Rosen [9]. Briefly, adipocytes were homogenized in ice-cold homogenization buffer (Buffer B or C) as described in following sections by 10 passages through a 5/8-in., 24-gauge needle attached to a syringe at 4 °C. The homogenate was centrifuged at 40,000g for 20 min at 4 °C. The resultant fat cake was carefully aspirated and the infranatant was removed. The infranatant was again centrifuged and the clear sample obtained by this procedure was used as the infranatant fraction (I40 fraction). The resultant 40,000g pellet was used for the P40 fraction. Measurements of PKA and HSL activity in two fractions. For the measurement of PKA activity, adipocytes were homogenized in icecold Buffer B (50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.25 M sucrose, 20 lM leupeptin, 20 mM NaF, and 100 lM benzamidine). Then, two subcellular fractions were prepared as described in the previous section. For the measurement of HSL activity, prior to the preparation of two subcellular fractions, adipocytes were incubated for 10 min with or without isoproterenol ð10 lMÞ plus 0.5 mM isobutylmethylxanthine in 0.15 M NaCl. The incubation was terminated by the addition of 2 ml ice-cold Buffer C (50 mM (N-morpholino)propanesulfonic acid, pH 7.0, 10 mM MgCl2 , and 1 lg=ml leupeptin). Then, adipocytes were homogenized in Buffer C and two subcellular fractions were prepared by 40,000g centrifugation. The resultant 40,000g pellet (P40 fraction) was resuspended in an ice-cold Buffer C and dispersed by needle aspiration. After centrifugation, the pellet was solubilized in a 5 mM sodium phosphate buffer (pH 7.0) containing a detergent, 0.2 % (w/v) 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (Dojindo, Japan). The suspension was incubated on ice for 10 min and centrifuged. The resultant clear infranatant below a small floating lipid residue was used for HSL activity in the P40 fraction. Measurements of HSL and PKA activity were performed according to the protocol previously reported [8]. Western blot analysis and immunoprecipitation. Adipocytes were isolated as described above and washed five times with Buffer A. The washed cells were homogenized in Buffer B. Then, the P40 and I40 fractions were prepared by 40,000g centrifugation. The samples were frozen at )80 °C until they were used for Western blot analysis. Because there was no significant difference in the protein contents per unit of cells (lg=105 cells) of the I40 and P40 fractions between the C (I40 ; 15:53 4:22; P40 ; 5:75 1:60) and TR groups (I40 ; 16:21 2:93; P40 ; 6:05 1:03), to compare exercise-trained rats with control rats, the same loading amount of proteins per unit of cells and all samples were run on the same gel (SDS–PAGE) by the method of Laemmli [19]. After electrophoresis, proteins were transferred to nitrocellulose membranes. The membranes were probed with an anti-catalytic subunit, anti-RIIb, anti-AKAP149, anti-AKAP220, anti-microtubuleassociated protein 2B (MAP2B) (Transduction Lab., KY), anti-RIa, anti-RIIa (Chemicon, CA), anti-RIb, and anti-AKAP150 (Santa Cruz, CA) antibodies. The immunoreactive bands were detected by the enhanced chemiluminescence method. Immunoprecipitations of the RII subunit were also performed. The P40 fractionations of adipocytes were incubated with antibodies to an RII subunit of protein kinase A and the immunocomplexes were then recovered by adsorption to protein A–agarose beads. The immunocomplexes were subjected to SDS– PAGE. The resolved proteins were transferred to nitrocellulose and the resultant blots were stained with antibodies to either AKAP149 or AKAP150. The membranes were scanned with Light-Capture (ATTO,
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Tokyo, Japan) and the optical density of each specific band was analyzed with a CS Analyzer (ATTO Corporation, Tokyo, Japan). Protein concentrations of the samples were determined by a commercially available kit (DC Protein Assay kit, Bio-Rad, CA). Data and statistical methods. Values represent means SE. The significance of differences between means was assessed by the Scheffe test, after analysis of variance was performed to establish that there were significant differences between the groups.
Results Adipocyte lipolysis The final mean body weight (g) was significantly less in the TR (N ¼ 20, 317:9 4:5; P < 0:05) than in the C rats (N ¼ 25, 345:8 8:4). Moreover, 100 nM isoproterenolstimulated adipocyte lipolysis (nmoles of glycerol releases/106 cells/30 min) was significantly greater in the TR (N ¼ 6, 633:5 24:9; P < 0:05) than in the C rats (N ¼ 6, 350:1 13:1). However, basal lipolysis did not differ between the C (135:1 15:8) and TR rats ð156:3 21:9Þ. The expressions of AKAPs in the P40 fraction of adipocytes First, we examined whether four types of well-known AKAPs, AKAP149, AKAP150, AKAP220, and MAP2B, were expressed in the P40 fraction of adipocyte in the C rats. As shown in Fig. 1A, the immunoreactive bands for both AKAP149 and AKAP150 in adipocytes
Fig. 1. Expression of AKAPs in the P40 fraction of rat adipocytes (A) and association of AKAP150 with RII subunits (B). Adipocytes from sedentary control rats were homogenized and the homogenate was centrifuged at 40,000g for 20 min at 4 °C. The 40,000g pellet (P40 fraction) was subjected to Western blot analysis and immunoprecipitation as described under ‘‘Materials and methods.’’ The immunoblot data are representative. Lanes 1 and 2: MAP2B. Lanes 3 and 4: AKAP220. Lanes 5 and 6: AKAP150. Lanes 7 and 8: AKAP149. Lanes 1, 3, 5, and 7: adipocytes. Lanes 2, 4, 6, and 8: positive controls, brain, testis, and brain and testis, respectively. M.W.: molecular weight (kDa).
were found to be the same as those for the position of each positive control, testis and brain. However, the observed molecular weight of AKAP149 in rat adipocytes and testis was markedly lower than 149 kDa. A shorter rat homolog of AKAP149 or its splice variant, S-AKAP84/D-AKAP1, might be detected in the present study because the anti-AKAP149 antibody used was raised against an antigen from the human cell line, HeLa cell lysate. Neither MAP2B nor AKAP220 was detected. As shown in Fig. 1B, immunoprecipitates of RII subunits of the P40 fraction with antibodies to AKAPs revealed prominent staining of AKAP150 but not AKAP149, indicating evidence of AKAP150 association with RII subunits of PKA. On the basis of these findings, the effects of exercise training on the expression of AKAP149 and AKAP150 in the P40 fraction were examined (Fig. 2). Exercise training significantly increased the protein expression of AKAP150 but not that of AKAP149. Expressions of PKA subunits in the subcellular fractions Some of the PKA subunits determined in this study were found both in the I40 and P40 fractions. A catalytic unit and two types of a regulatory unit, RIIa and RIIb, were detected in both fractions (Fig. 3). However, although a cross-reaction of anti-RIb antibody to RII (51 kDa) could be observed in both fractions, RIbspecific immunoreactive bands determined by the calculation of molecular weight were identified in the I40
Fig. 2. Effects of exercise training on the expressions of AKAP150 and AKAP149 in the P40 fraction. The expressions of AKAPs were analyzed by Western blot. Adipocytes from control (C) and exercisetrained (TR) rats were homogenized and the homogenate was centrifuged at 40,000g for 20 min at 4 °C. The 40,000g pellet (P40 fraction) was subjected to Western blot analysis as described under ‘‘Materials and methods.’’ The immunoblot data are representative (upper panel). The lower panel shows the ratio of optical density of the TR rats (N ¼ 3) to that of the C rats (N ¼ 3). Values are means SE. *P < 0:05 vs. control.
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Fig. 3. Effects of exercise training on the expressions of PKA subunits in two subcellular fractions, the P40 fraction (P) and the I40 fraction (I). Each subcellular fraction was prepared as described under ‘‘Materials and methods.’’ A-kinase subunits, a catalytic unit, and four regulatory units (RIa, RIIa, RIb, and RIIb) were measured in these two fractions. (A) shows representative immunoblots from control (C) and exercisetrained (TR) rats. The RIa subunit cannot be detected in either subcellular fraction. (B) shows the relative densities, indicating the ratio of the optical density of the TR rats (N ¼ 5) to that of the C rats (N ¼ 5). Values are means SE. *P < 0:05 vs. control. ND, not detected.
fraction but not in the P40 fraction. RIa was not detected in either fraction in the present study. A comparison of the optical density of a catalytic unit from the TR rats with that from the C rats revealed that the catalytic units tended to decrease in the I40 fraction (P ¼ 0:10) but significantly increased in the P40 fraction after exercise training. Moreover, RII subunits decreased in the I40 fraction but increased in the P40 fraction after exercise training. The RIIb subunit in the P40 fraction increased by 2-fold in the TR rats (P ¼ 0:08) with significant decrease in RIIb subunit in the I40 fraction. There was no significant effect of exercise training on the RIb subunits in the I40 fraction. PKA and HSL activities in subcellular fractions Fig. 4 shows the activities of PKA (left panels) and HSL (right panels) in both fractions from each conditioned rat. Inconsistent with the protein expressions of PKA subunits, PKA activities were observed in both fractions. The activities with or without cAMP were significantly lower in the P40 fraction than in the I40 fraction. This could be due to the lower protein expression of the catalytic and regulatory units in the P40 fraction. The activity with cAMP in the I40 fraction was significantly lower in the TR rats than in the C rats, but there was no difference in the activity without cAMP. On the other hand, the activities with or without cAMP in the P40 fraction were significantly greater in the TR rats than in the C rats. The former findings would be associated with the decreased expressions of PKA subunits (catalytic, RIIa, and RIIb) in the I40 fraction of the TR rats and the later ones with the increased expressions of these subunits in the P40 fraction of the TR rats. The HSL activities in the basal condition as well as those stimulated by isoproterenol plus methylisobutylxanthine (ISO/MIX) were significantly greater in the
Fig. 4. PKA activities (left panels)and HSL activities (right panels) in two subcellular fractions, the I40 fraction and the P40 fraction, in control (open columns) and exercise-trained (filled columns) rats. The preparation of each subcellular fraction and other experimental conditions were described under ‘‘Materials and methods.’’ Values are means SE for five separate experiments (N ¼ 5 for each group). a P < 0:05 vs. control; b P < 0:05 vs. without stimulators. w/o, Without; ISO/MIX, isoproterenol plus methylisobutylxanthine.
TR than in the C rats in both fractions. The enhanced activity in I40 fraction was in accordance with the recent finding shown by Enevoldsen et al. [1] exercise training enhances the agonist-stimulated activities of HSL in the cytosolic fraction of adipose tissue homogenates. However, even though lipolysis was negligible in the non-stimulated cell (basal), a large amount of HSL activity was found in the I40 fraction of both groups. Moreover, the stimulation of adipocytes by ISO/MIX was not accompanied by any changes in HSL activities in the I40 fraction. In contrast, in the P40 fraction, HSL activity increased along with the stimulation by agonists. The effect of St-Ht31 peptide on adipocyte lipolysis The effect of St-Ht31, a peptide that competitively displaces PKA from the AKAP complex, on adipocyte lipolysis was also examined (Fig. 5). St-Ht31 (10 and 25 lM) partially blocked 50 nM isoproterenol-stimulated lipolysis in both conditioned rats. St-Ht31 control peptide did not affect isoproterenol-stimulated lipolysis, even when the concentration of the control peptide was increased to 50 lM (data not shown). The decrease in lipolysis due to St-Ht31 was significantly greater in the TR rats than in the C rats.
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Fig. 5. Effect of St-Ht31 on 50 nM isoproterenol-stimulated adipocyte lipolysis from control (open columns, N ¼ 5) and exercise-trained rats (filled columns, N ¼ 5). Adipocytes (approx. 105 cells) were incubated with or without indicated agents. After 30 min, the cell-free incubation medium was assayed for glycerol as an index of lipolysis. The upper panel shows the absolute values of adipocyte lipolysis and the lower one shows the decreases in glycerol releases due to St-Ht31. Values are means SE for five separate experiments. a P < 0:05 vs. control; b P < 0:05 vs. without St-Ht31.
Discussion AKAPs play a role in mediating the attachment of PKA to subcellular structures. Compartmentalization of PKA in close proximity to its specific targets may be required for controlling the specificity of cAMP-mediated signaling in cells [12–15]. Moreover, the interaction of AKAPs with PKA could also increase the rate and the magnitude of cAMP signaling to remote organelles and enzymes. For example, RII subunits anchoring by AKAP75 to the cytoskeleton in the proximity of the plasma membrane can enhance the phosphorylation of element-binding protein in response to cAMP, following c-fos transcription in the nucleus [20]. In the present study, RIIa and RIIb existed in the P40 fraction. AKAPs were also found in this fraction, and AKAP150 but not AKAP149 was co-immunoprecipitated with the RII subunit. Moreover, St-Ht31 partially blocked adipocyte lipolysis in response to isoproterenol. These data suggest
that the interaction between PKA and AKAP150, which are co-localized in the P40 fraction, is required for full activation of HSL. Increased activities of HSL in response to agonists were found in the P40 fraction but not in the I40 fraction. Moreover, in our preliminary experiment, inhibition of isoproterenol-stimulated lipolysis by papaverine (1 mM) was accompanied by an inhibition of the increase in HSL activity in the P40 fraction (unpublished observation). These results suggest that HSL compartmentalized in the P40 fraction is of importance for lipolysis. Hirsh and Rosen [9] first showed that lipolytic agonists induced a translocation of HSL from cytosol to the centrifuged pellet fractions of homogenates in 3T3-L1 adipocytes. Egan et al. [10] clearly showed that HSL shifted quantitatively from the infranatant to the floating fat-cake fraction of lipolytically stimulated rat adipocyte by Western blotting of adipocyte subcellular fractions with anti-HSL antiserum. The exact characteristics of the P40 fraction used in this study are unclear. However, the P40 fraction prepared in this study might contain the membrane surrounding a lipid droplet that was torn off by mechanical homogenization, because Egan et al. [10] demonstrated that HSL activity was observed in the membrane-rich pellet fraction obtained from mechanical homogenization of adipocytes. Thus, either the redistribution of HSL from cytosol to its substrate or the increase in the activity of HSL, which could be compartmentalized in some cellular organelles in the proximity of lipid storage droplet, would be more important for the breakdown of triacylglycerol than the activation of HSL in a cytosolic fraction. Taken all together, the present data suggest that some of the lipolytic machineries (PKA, HSL, and possibly AKAPs) could be localized within adipocytes to facilitate efficient signal transduction, resulting in subsequent full lipolysis. Planas et al. [21] suggested that the loss of responsiveness to b-agonists in RIIb knockout mice involved changes in subcellular localization of PKA holoenzymes. In this study, exercise training increased the protein expression of AKAP150 with enhanced activity and expressions of several PKA subunits (catalytic, RIIa, and RIIb) in the P40 fraction. Moreover, the decreases in lipolysis due to St-Ht31 were significantly greater in the TR than in the C rats. These data suggest that exercise training enhances the expression of AKAP150 to anchor increased regulatory units (RIIa and RIIb) in the proximity of the plasma membrane thereby increasing the magnitude of cAMP signaling to target enzymes, such as HSL. The activities of HSL significantly increased in both the I40 and P40 fractions from lipolytically stimulated adipocytes in the TR rats more than in the C rats. Thus, because HSL in the P40 fractions would be of importance for lipolysis as discussed above, the mechanisms behind exercise
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training-enhanced adipocyte lipolysis could involve the enhanced activities of HSL and PKA in the P40 fraction, with the increased expressions of PKA subunits and AKAP150. However, significant differences of lipolysis between groups were still observed after treatment of adipocytes with St-HT31. Therefore, given that a complete uptake of St-HT31 by adipocytes occurs, the partial effect of StHT31 on stimulated lipolysis indicates that the enhanced adipocyte lipolysis in exercise-trained rats cannot be completely and exclusively explained by the changes in AKAP150 and some of the PKA subunits, which are compartmentalized within adipocytes. Moreover, despite the fact that both basal and stimulated activities of HSL in the P40 fraction were significantly greater in the TR rats, the stimulated lipolysis was enhanced, but basal lipolysis was not. These enigmas might be explained by a possible change(s) in the alternative pathway for lipolysis, which is a cAMP-independent activation of HSL. Although the exact nature of the alternative pathway for lipolysis remains unclear at present, Okuda et al. [22,23] proposed the so-called ‘‘hormone-sensitive substrate theory’’ of lipolysis, in which the hormone did not act on the lipase but on the endogenous lipid substrate. Wise and Jungas [24] proposed a dual mechanism of lipolytic activation by catecholamines involving ‘‘substrate activation.’’ These studies suggest that some factor on the surface of intact lipid droplets may be necessary for the hormonal stimulation of lipolysis. In this regard, recent studies have suggested that the protein for this role is perilipin, the predominant phosphoprotein in adipocytes [25–28]. Intracellular neutral lipid droplets in adipocytes are encased in a coating of perilipins, which are phosphorylated at multiple sites by PKA in lipolytically stimulated adipocytes [26–28]. Perilipin targets intracellular neutral lipid droplets and protects triacylglycerol against hydrolysis [28]. The high levels of soluble lipase activity in adipocytes may necessitate the presence of perilipin at the surfaces of lipid droplets to protect the vast stores of triacylglycerol from hydrolysis. Thus, perilipin has been suggested to be involved in the interaction between HSL and lipid droplets and to play a major role in regulating lipolysis and the storage of triacylglycerols in adipocytes. Exercise training might also alter the amount and/or function of perilipins thereby regulating the interaction of HSL with its substrate. In summary, several lipolytic machineries, such as some subunits of PKA (catalytic, RIIa, and RIIb) and HSL activity, were detected in the P40 fraction of adipocytes. Moreover, AKAP149 and AKAP150 were also detected in the P40 fraction and AKAP150 but not AKA149 was co-immunoprecipitated with RII subunits. The experiment using St-Ht31 shows that interaction of AKAP and PKA is required for full lipolysis. Lipolytic agonists increased HSL activities in the P40 fraction but
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not in the I40 fraction. These data suggest that compartmentalization of some lipolytic machineries (PKA, HSL, and possibly AKAPs) within adipocytes is required for full lipolysis. Exercise training enhanced the protein expressions of some subunits of PKA (catalytic, RIIa, and probably RIIb) with increased activities of PKA and HSL in the P40 fraction. Furthermore, protein expression of AKAP150 and the decrease in lipolysis due to St-Ht31 were significantly greater in the TR rats. Thus, the mechanisms behind exercise trainingenhanced adipocyte lipolysis could involve the increase in the activities of HSL in the P40 fraction with the enhanced activity and expressions of PKA subunits (catalytic, RIIa, and RIIb) and AKAP150. Acknowledgment This study was supported in part by a grant-in-aid from the Japanese Ministry of Education, Science, Sports and Culture.
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affects lipolysis but not gene induction in white adipose tissue, J. Biol. Chem. 274 (1999) 36281–36287. H. Okuda, I. Yanagi, S. Fujii, The mechanism of in vitro stimulation of lipolysis by adrenaline, J. Biochem. (Tokyo) 59 (1966) 438–442. H. Okuda, S. Fujii, Relationship between lipase and esterase, J. Biochem. (Tokyo) 64 (1966) 377–385. L.S. Wise, R.L. Jungas, Evidence for a dual mechanism of lipolysis activation by epinephrine in rat adipose tissue, J. Biol. Chem. 253 (1978) 2624–2627. G.M. Clifford, C. Londos, F.B. Kraemer, R.G. Vernon, S.J. Yeaman, Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation of rat adipocyte, J. Biol. Chem. 275 (2000) 5011–5015. A.S. Greenberg, J.J. Egan, S.A. Wek, N.B. Garty, E.J. BlanchetteMackie, C. Londos, Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets, J. Biol. Chem. 266 (1991) 11341–11346. C. Londos, D.L. Brasaemle, J. Gruia-Gray, D.A. Servetnick, C.J. Schultz, D.M. Levin, A.R. Kimmel, Perilipin: unique proteins associated with intracellular neutral lipid droplets in adipocytes and steroidogenic cells, Biochem. Soc. Trans. 23 (1995) 611–615. J.T. Tansey, C. Sztalryd, J. Gruia-Gray, D.L. Roush, J.V. Zee, O. Gavrilova, M.L. Reitman, C.-X. Deng, C. Li, A.R. Kimmel, C. Londos, Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity, Proc. Natl. Acad. Sci. USA 98 (2001) 6494– 6499.
Possible mechanisms by which adipocyte lipolysis is enhanced in exercise-trained rats Sachiko Nomura,a Hitomi Kawanami,a Hiroshi Ueda,a Takako Kizaki,b Hideki Ohno,b and Tetsuya Izawaa,* a
Department of Kinesiology, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, Tokyo 192-0397, Japan b Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine, Tokyo, Mitaka 181-8611, Japan Received 31 May 2002
Abstract A possible mechanism(s) behind exercise training-enhanced lipolysis was investigated in rat adipocytes. Exercise training (9 weeks; running) enhanced the activity of cAMP-dependent protein kinase (PKA) and the protein expressions of PKA subunits (catalytic, RIIa, and RIIb) in P40 fraction (sedimenting at 40,000g), but not in I40 fraction (infranatant of 40,000g) of adipocyte homogenate. The expression of PKA-anchoring protein 150 (AKAP150) in P40 fraction was greater in exercise-trained (TR) than in control (C) rats. Hormone-sensitive lipase (HSL) activities in both fractions were also greater in TR. On the other hand, stimulated lipolysis was accompanied by increased activities of HSL in P40 but not in I40 fraction. The decreases in stimulated lipolysis due to St-Ht31 were greater in TR rats. Thus, the mechanisms behind exercise training-enhanced adipocyte lipolysis could involve the increased activities of PKA and HSL with enhanced expressions of AKAP150 and some subunits of PKA, all of which may be compartmentalized within adipocytes. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Exercise; cAMP-dependent protein kinase; A-kinase-anchoring protein; Hormone-sensitive lipase; Adipocyte; Rat
It is widely accepted that the hormonal activation of adipocyte lipolysis is mediated via a cAMP-dependent process. Increased concentrations of cAMP activate cAMP-dependent protein kinase (PKA), which, in turn, leads to phosphorylation and activation of hormonesensitive lipase (HSL). Chronic exercise training enhances the lipolytic response to catecholamines in laboratory animals and in humans [1–7]. The major biochemical alteration underlying this enhanced lipolysis has been thought to occur at a site distal to cAMP production, such as at the level of HSL. Enevoldsen et al. [1] have recently shown that exercise training enhances the agonist-stimulated activities of HSL and the protein expression of HSL in the cytosolic fraction of retroperitoneal and mesenteric adipose tissue homogenates. However, accumulated evidence shows that the activation of cytosolic HSL is not always important for the breakdown of triacylglycerol [8–10]. A redistribution of
*
Corresponding author. Fax: +81-426-77-2961. E-mail address: [email protected] (T. Izawa).
HSL from cytosol to either the centrifuged pellet fractions [9] or to its substrate at the surface of the lipid droplet [10] is required for the subsequent activation of full lipolysis. Therefore, it is possible that exercise training-enhanced adipocyte lipolysis is mediated via increased activity of HSL, which is located at either the centrifuged pellet fractions or the surface of the lipid droplet. Moreover, the intracellular concentration of cAMP does not always increase due to enhanced activity of phosphodiesterase in exercise-trained rat adipocytes [6], although exercise training enhances the cAMP production events [2,7]. Neither has any increase in PKA activity been found in the cell extracts [4,5]. Thus, HSL activity may be enhanced without increases in either intracellular cAMP concentrations or cytosolic PKA activity after exercise training. This enigma might be explained by the compartmentalization of cAMP within adipocytes [11]. However, in fact, it is unlikely that a small, soluble molecule like cAMP could be restricted to a particular cytosolic compartment. It seems rational that PKA is located in specific subcellular compartments.
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 6 6 4 - 2
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A family of PKA-anchoring proteins (AKAPs) has a key role in the compartmentalization of PKA [12–15]. PKA heterotetramers are composed of a dimeric catalytic subunit and a dimeric cAMP-binding regulatory subunit type I (RIa and RIb) or type II (RIIa and RIIb). It has been postulated that, of these regulatory subunits, either RIIa or RIIb is compartmentalized through association with AKAPs [12–15]. Thereby, PKA exists in close proximity to its substrate and the interaction between RII subunits and AKAPs is required for certain intracellular signaling events. Taking all these facts together, we hypothesized that both PKA subunits and AKAPs are placed in some cellular organelles’ proximity to a signal generator (e.g., adenylate cyclase) and PKA substrate/effector proteins (e.g., HSL) in adipocytes and that exercise training could alter both PKA activity and the protein expressions of AKAPs, which are placed in some cellular organelles. Thereby, the activity of HSL, which is also compartmentalized in some cellular organelles in close proximity to lipid storage droplet, could be enhanced. Our preliminary experiments [16] and a very recent study [17] showed the existence of AKAPs in rat adipocytes and C3H/10T1/2 adipocytes, respectively. Therefore, to confirm the hypothesis, we investigated whether either PKA subunits or AKAPs are present in two subcellular fractions of centrifuged homogenate of adipocytes: the infranatant fraction (I40 fraction, supernatant of 40,000g) and the pellet fraction (P40 fraction, sedimenting at 40,000g). The effects of exercise training on subcellular distributions of either PKA subunits or HSL and the expression of AKAP proteins were then investigated. Materials and methods Animal care and exercise training program. Male Wistar rats (SLC, Japan) with an initial body weight of 90–120 g were housed two or three to a cage in a room with temperature controlled at 24 °C and a 12:12-h light–dark cycle. Food and water were available ad libitum. The animals were randomly divided into two groups, sedentary control (C, N ¼ 25) and exercise-trained (TR, N ¼ 20). The rats in the trained group were exercised on a treadmill set at a 5° incline for 5 days/week for 9 weeks according to the protocol previously reported [2–4]. Sedentary control animals were not subjected to treadmill running. Exercise-trained rats were sacrificed more than 36 h after the last exercise session. After an overnight fast, the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (5 mg/100 g body weight; Abbott, IL). The epididymal fat pads were removed and used for adipocyte isolation. All experiments conducted in this study were approved by the Animal Care Committee of Tokyo Metropolitan University Graduate School of Science. Preparation of adipocytes. Adipocytes were isolated by a modification of the method of Rodbell [18]. Fat pads were minced with scissors and placed in plastic vials in Buffer A (Krebs–Ringer bicarbonate solution buffered with 10 mM HEPES, pH 7.4, containing 5.5 mM glucose and 2% (w/v) fatty acid-free bovine serum albumin) with 200 nM adenosine and collagenase type I (1 mg/ml, Worthington Biochemical, NJ). Collagenase digestion was performed at 37 °C in a
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water bath shaker. After 30 min, the contents of the vials were immediately filtered and centrifuged for 1 min at 100g. The layer of floating cells was then washed three times with Buffer A. Measurement of lipolysis. Adipocytes (approx. 105 cells) were incubated in plastic vials in a total volume of 0.5 ml of Buffer A with or without indicated agents. The incubation medium contained adenosine deaminase (1 U/ml). After 30 min, a cell-free incubation medium was assayed for glycerol as an index of lipolysis. The average number of adipocytes and glycerol content were determined according to a previous study [3]. Adipocyte subcellular fraction. Adipocyte subcellular fractions were essentially prepared by the methods of Hirsh and Rosen [9]. Briefly, adipocytes were homogenized in ice-cold homogenization buffer (Buffer B or C) as described in following sections by 10 passages through a 5/8-in., 24-gauge needle attached to a syringe at 4 °C. The homogenate was centrifuged at 40,000g for 20 min at 4 °C. The resultant fat cake was carefully aspirated and the infranatant was removed. The infranatant was again centrifuged and the clear sample obtained by this procedure was used as the infranatant fraction (I40 fraction). The resultant 40,000g pellet was used for the P40 fraction. Measurements of PKA and HSL activity in two fractions. For the measurement of PKA activity, adipocytes were homogenized in icecold Buffer B (50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.25 M sucrose, 20 lM leupeptin, 20 mM NaF, and 100 lM benzamidine). Then, two subcellular fractions were prepared as described in the previous section. For the measurement of HSL activity, prior to the preparation of two subcellular fractions, adipocytes were incubated for 10 min with or without isoproterenol ð10 lMÞ plus 0.5 mM isobutylmethylxanthine in 0.15 M NaCl. The incubation was terminated by the addition of 2 ml ice-cold Buffer C (50 mM (N-morpholino)propanesulfonic acid, pH 7.0, 10 mM MgCl2 , and 1 lg=ml leupeptin). Then, adipocytes were homogenized in Buffer C and two subcellular fractions were prepared by 40,000g centrifugation. The resultant 40,000g pellet (P40 fraction) was resuspended in an ice-cold Buffer C and dispersed by needle aspiration. After centrifugation, the pellet was solubilized in a 5 mM sodium phosphate buffer (pH 7.0) containing a detergent, 0.2 % (w/v) 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (Dojindo, Japan). The suspension was incubated on ice for 10 min and centrifuged. The resultant clear infranatant below a small floating lipid residue was used for HSL activity in the P40 fraction. Measurements of HSL and PKA activity were performed according to the protocol previously reported [8]. Western blot analysis and immunoprecipitation. Adipocytes were isolated as described above and washed five times with Buffer A. The washed cells were homogenized in Buffer B. Then, the P40 and I40 fractions were prepared by 40,000g centrifugation. The samples were frozen at )80 °C until they were used for Western blot analysis. Because there was no significant difference in the protein contents per unit of cells (lg=105 cells) of the I40 and P40 fractions between the C (I40 ; 15:53 4:22; P40 ; 5:75 1:60) and TR groups (I40 ; 16:21 2:93; P40 ; 6:05 1:03), to compare exercise-trained rats with control rats, the same loading amount of proteins per unit of cells and all samples were run on the same gel (SDS–PAGE) by the method of Laemmli [19]. After electrophoresis, proteins were transferred to nitrocellulose membranes. The membranes were probed with an anti-catalytic subunit, anti-RIIb, anti-AKAP149, anti-AKAP220, anti-microtubuleassociated protein 2B (MAP2B) (Transduction Lab., KY), anti-RIa, anti-RIIa (Chemicon, CA), anti-RIb, and anti-AKAP150 (Santa Cruz, CA) antibodies. The immunoreactive bands were detected by the enhanced chemiluminescence method. Immunoprecipitations of the RII subunit were also performed. The P40 fractionations of adipocytes were incubated with antibodies to an RII subunit of protein kinase A and the immunocomplexes were then recovered by adsorption to protein A–agarose beads. The immunocomplexes were subjected to SDS– PAGE. The resolved proteins were transferred to nitrocellulose and the resultant blots were stained with antibodies to either AKAP149 or AKAP150. The membranes were scanned with Light-Capture (ATTO,
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Tokyo, Japan) and the optical density of each specific band was analyzed with a CS Analyzer (ATTO Corporation, Tokyo, Japan). Protein concentrations of the samples were determined by a commercially available kit (DC Protein Assay kit, Bio-Rad, CA). Data and statistical methods. Values represent means SE. The significance of differences between means was assessed by the Scheffe test, after analysis of variance was performed to establish that there were significant differences between the groups.
Results Adipocyte lipolysis The final mean body weight (g) was significantly less in the TR (N ¼ 20, 317:9 4:5; P < 0:05) than in the C rats (N ¼ 25, 345:8 8:4). Moreover, 100 nM isoproterenolstimulated adipocyte lipolysis (nmoles of glycerol releases/106 cells/30 min) was significantly greater in the TR (N ¼ 6, 633:5 24:9; P < 0:05) than in the C rats (N ¼ 6, 350:1 13:1). However, basal lipolysis did not differ between the C (135:1 15:8) and TR rats ð156:3 21:9Þ. The expressions of AKAPs in the P40 fraction of adipocytes First, we examined whether four types of well-known AKAPs, AKAP149, AKAP150, AKAP220, and MAP2B, were expressed in the P40 fraction of adipocyte in the C rats. As shown in Fig. 1A, the immunoreactive bands for both AKAP149 and AKAP150 in adipocytes
Fig. 1. Expression of AKAPs in the P40 fraction of rat adipocytes (A) and association of AKAP150 with RII subunits (B). Adipocytes from sedentary control rats were homogenized and the homogenate was centrifuged at 40,000g for 20 min at 4 °C. The 40,000g pellet (P40 fraction) was subjected to Western blot analysis and immunoprecipitation as described under ‘‘Materials and methods.’’ The immunoblot data are representative. Lanes 1 and 2: MAP2B. Lanes 3 and 4: AKAP220. Lanes 5 and 6: AKAP150. Lanes 7 and 8: AKAP149. Lanes 1, 3, 5, and 7: adipocytes. Lanes 2, 4, 6, and 8: positive controls, brain, testis, and brain and testis, respectively. M.W.: molecular weight (kDa).
were found to be the same as those for the position of each positive control, testis and brain. However, the observed molecular weight of AKAP149 in rat adipocytes and testis was markedly lower than 149 kDa. A shorter rat homolog of AKAP149 or its splice variant, S-AKAP84/D-AKAP1, might be detected in the present study because the anti-AKAP149 antibody used was raised against an antigen from the human cell line, HeLa cell lysate. Neither MAP2B nor AKAP220 was detected. As shown in Fig. 1B, immunoprecipitates of RII subunits of the P40 fraction with antibodies to AKAPs revealed prominent staining of AKAP150 but not AKAP149, indicating evidence of AKAP150 association with RII subunits of PKA. On the basis of these findings, the effects of exercise training on the expression of AKAP149 and AKAP150 in the P40 fraction were examined (Fig. 2). Exercise training significantly increased the protein expression of AKAP150 but not that of AKAP149. Expressions of PKA subunits in the subcellular fractions Some of the PKA subunits determined in this study were found both in the I40 and P40 fractions. A catalytic unit and two types of a regulatory unit, RIIa and RIIb, were detected in both fractions (Fig. 3). However, although a cross-reaction of anti-RIb antibody to RII (51 kDa) could be observed in both fractions, RIbspecific immunoreactive bands determined by the calculation of molecular weight were identified in the I40
Fig. 2. Effects of exercise training on the expressions of AKAP150 and AKAP149 in the P40 fraction. The expressions of AKAPs were analyzed by Western blot. Adipocytes from control (C) and exercisetrained (TR) rats were homogenized and the homogenate was centrifuged at 40,000g for 20 min at 4 °C. The 40,000g pellet (P40 fraction) was subjected to Western blot analysis as described under ‘‘Materials and methods.’’ The immunoblot data are representative (upper panel). The lower panel shows the ratio of optical density of the TR rats (N ¼ 3) to that of the C rats (N ¼ 3). Values are means SE. *P < 0:05 vs. control.
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Fig. 3. Effects of exercise training on the expressions of PKA subunits in two subcellular fractions, the P40 fraction (P) and the I40 fraction (I). Each subcellular fraction was prepared as described under ‘‘Materials and methods.’’ A-kinase subunits, a catalytic unit, and four regulatory units (RIa, RIIa, RIb, and RIIb) were measured in these two fractions. (A) shows representative immunoblots from control (C) and exercisetrained (TR) rats. The RIa subunit cannot be detected in either subcellular fraction. (B) shows the relative densities, indicating the ratio of the optical density of the TR rats (N ¼ 5) to that of the C rats (N ¼ 5). Values are means SE. *P < 0:05 vs. control. ND, not detected.
fraction but not in the P40 fraction. RIa was not detected in either fraction in the present study. A comparison of the optical density of a catalytic unit from the TR rats with that from the C rats revealed that the catalytic units tended to decrease in the I40 fraction (P ¼ 0:10) but significantly increased in the P40 fraction after exercise training. Moreover, RII subunits decreased in the I40 fraction but increased in the P40 fraction after exercise training. The RIIb subunit in the P40 fraction increased by 2-fold in the TR rats (P ¼ 0:08) with significant decrease in RIIb subunit in the I40 fraction. There was no significant effect of exercise training on the RIb subunits in the I40 fraction. PKA and HSL activities in subcellular fractions Fig. 4 shows the activities of PKA (left panels) and HSL (right panels) in both fractions from each conditioned rat. Inconsistent with the protein expressions of PKA subunits, PKA activities were observed in both fractions. The activities with or without cAMP were significantly lower in the P40 fraction than in the I40 fraction. This could be due to the lower protein expression of the catalytic and regulatory units in the P40 fraction. The activity with cAMP in the I40 fraction was significantly lower in the TR rats than in the C rats, but there was no difference in the activity without cAMP. On the other hand, the activities with or without cAMP in the P40 fraction were significantly greater in the TR rats than in the C rats. The former findings would be associated with the decreased expressions of PKA subunits (catalytic, RIIa, and RIIb) in the I40 fraction of the TR rats and the later ones with the increased expressions of these subunits in the P40 fraction of the TR rats. The HSL activities in the basal condition as well as those stimulated by isoproterenol plus methylisobutylxanthine (ISO/MIX) were significantly greater in the
Fig. 4. PKA activities (left panels)and HSL activities (right panels) in two subcellular fractions, the I40 fraction and the P40 fraction, in control (open columns) and exercise-trained (filled columns) rats. The preparation of each subcellular fraction and other experimental conditions were described under ‘‘Materials and methods.’’ Values are means SE for five separate experiments (N ¼ 5 for each group). a P < 0:05 vs. control; b P < 0:05 vs. without stimulators. w/o, Without; ISO/MIX, isoproterenol plus methylisobutylxanthine.
TR than in the C rats in both fractions. The enhanced activity in I40 fraction was in accordance with the recent finding shown by Enevoldsen et al. [1] exercise training enhances the agonist-stimulated activities of HSL in the cytosolic fraction of adipose tissue homogenates. However, even though lipolysis was negligible in the non-stimulated cell (basal), a large amount of HSL activity was found in the I40 fraction of both groups. Moreover, the stimulation of adipocytes by ISO/MIX was not accompanied by any changes in HSL activities in the I40 fraction. In contrast, in the P40 fraction, HSL activity increased along with the stimulation by agonists. The effect of St-Ht31 peptide on adipocyte lipolysis The effect of St-Ht31, a peptide that competitively displaces PKA from the AKAP complex, on adipocyte lipolysis was also examined (Fig. 5). St-Ht31 (10 and 25 lM) partially blocked 50 nM isoproterenol-stimulated lipolysis in both conditioned rats. St-Ht31 control peptide did not affect isoproterenol-stimulated lipolysis, even when the concentration of the control peptide was increased to 50 lM (data not shown). The decrease in lipolysis due to St-Ht31 was significantly greater in the TR rats than in the C rats.
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Fig. 5. Effect of St-Ht31 on 50 nM isoproterenol-stimulated adipocyte lipolysis from control (open columns, N ¼ 5) and exercise-trained rats (filled columns, N ¼ 5). Adipocytes (approx. 105 cells) were incubated with or without indicated agents. After 30 min, the cell-free incubation medium was assayed for glycerol as an index of lipolysis. The upper panel shows the absolute values of adipocyte lipolysis and the lower one shows the decreases in glycerol releases due to St-Ht31. Values are means SE for five separate experiments. a P < 0:05 vs. control; b P < 0:05 vs. without St-Ht31.
Discussion AKAPs play a role in mediating the attachment of PKA to subcellular structures. Compartmentalization of PKA in close proximity to its specific targets may be required for controlling the specificity of cAMP-mediated signaling in cells [12–15]. Moreover, the interaction of AKAPs with PKA could also increase the rate and the magnitude of cAMP signaling to remote organelles and enzymes. For example, RII subunits anchoring by AKAP75 to the cytoskeleton in the proximity of the plasma membrane can enhance the phosphorylation of element-binding protein in response to cAMP, following c-fos transcription in the nucleus [20]. In the present study, RIIa and RIIb existed in the P40 fraction. AKAPs were also found in this fraction, and AKAP150 but not AKAP149 was co-immunoprecipitated with the RII subunit. Moreover, St-Ht31 partially blocked adipocyte lipolysis in response to isoproterenol. These data suggest
that the interaction between PKA and AKAP150, which are co-localized in the P40 fraction, is required for full activation of HSL. Increased activities of HSL in response to agonists were found in the P40 fraction but not in the I40 fraction. Moreover, in our preliminary experiment, inhibition of isoproterenol-stimulated lipolysis by papaverine (1 mM) was accompanied by an inhibition of the increase in HSL activity in the P40 fraction (unpublished observation). These results suggest that HSL compartmentalized in the P40 fraction is of importance for lipolysis. Hirsh and Rosen [9] first showed that lipolytic agonists induced a translocation of HSL from cytosol to the centrifuged pellet fractions of homogenates in 3T3-L1 adipocytes. Egan et al. [10] clearly showed that HSL shifted quantitatively from the infranatant to the floating fat-cake fraction of lipolytically stimulated rat adipocyte by Western blotting of adipocyte subcellular fractions with anti-HSL antiserum. The exact characteristics of the P40 fraction used in this study are unclear. However, the P40 fraction prepared in this study might contain the membrane surrounding a lipid droplet that was torn off by mechanical homogenization, because Egan et al. [10] demonstrated that HSL activity was observed in the membrane-rich pellet fraction obtained from mechanical homogenization of adipocytes. Thus, either the redistribution of HSL from cytosol to its substrate or the increase in the activity of HSL, which could be compartmentalized in some cellular organelles in the proximity of lipid storage droplet, would be more important for the breakdown of triacylglycerol than the activation of HSL in a cytosolic fraction. Taken all together, the present data suggest that some of the lipolytic machineries (PKA, HSL, and possibly AKAPs) could be localized within adipocytes to facilitate efficient signal transduction, resulting in subsequent full lipolysis. Planas et al. [21] suggested that the loss of responsiveness to b-agonists in RIIb knockout mice involved changes in subcellular localization of PKA holoenzymes. In this study, exercise training increased the protein expression of AKAP150 with enhanced activity and expressions of several PKA subunits (catalytic, RIIa, and RIIb) in the P40 fraction. Moreover, the decreases in lipolysis due to St-Ht31 were significantly greater in the TR than in the C rats. These data suggest that exercise training enhances the expression of AKAP150 to anchor increased regulatory units (RIIa and RIIb) in the proximity of the plasma membrane thereby increasing the magnitude of cAMP signaling to target enzymes, such as HSL. The activities of HSL significantly increased in both the I40 and P40 fractions from lipolytically stimulated adipocytes in the TR rats more than in the C rats. Thus, because HSL in the P40 fractions would be of importance for lipolysis as discussed above, the mechanisms behind exercise
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training-enhanced adipocyte lipolysis could involve the enhanced activities of HSL and PKA in the P40 fraction, with the increased expressions of PKA subunits and AKAP150. However, significant differences of lipolysis between groups were still observed after treatment of adipocytes with St-HT31. Therefore, given that a complete uptake of St-HT31 by adipocytes occurs, the partial effect of StHT31 on stimulated lipolysis indicates that the enhanced adipocyte lipolysis in exercise-trained rats cannot be completely and exclusively explained by the changes in AKAP150 and some of the PKA subunits, which are compartmentalized within adipocytes. Moreover, despite the fact that both basal and stimulated activities of HSL in the P40 fraction were significantly greater in the TR rats, the stimulated lipolysis was enhanced, but basal lipolysis was not. These enigmas might be explained by a possible change(s) in the alternative pathway for lipolysis, which is a cAMP-independent activation of HSL. Although the exact nature of the alternative pathway for lipolysis remains unclear at present, Okuda et al. [22,23] proposed the so-called ‘‘hormone-sensitive substrate theory’’ of lipolysis, in which the hormone did not act on the lipase but on the endogenous lipid substrate. Wise and Jungas [24] proposed a dual mechanism of lipolytic activation by catecholamines involving ‘‘substrate activation.’’ These studies suggest that some factor on the surface of intact lipid droplets may be necessary for the hormonal stimulation of lipolysis. In this regard, recent studies have suggested that the protein for this role is perilipin, the predominant phosphoprotein in adipocytes [25–28]. Intracellular neutral lipid droplets in adipocytes are encased in a coating of perilipins, which are phosphorylated at multiple sites by PKA in lipolytically stimulated adipocytes [26–28]. Perilipin targets intracellular neutral lipid droplets and protects triacylglycerol against hydrolysis [28]. The high levels of soluble lipase activity in adipocytes may necessitate the presence of perilipin at the surfaces of lipid droplets to protect the vast stores of triacylglycerol from hydrolysis. Thus, perilipin has been suggested to be involved in the interaction between HSL and lipid droplets and to play a major role in regulating lipolysis and the storage of triacylglycerols in adipocytes. Exercise training might also alter the amount and/or function of perilipins thereby regulating the interaction of HSL with its substrate. In summary, several lipolytic machineries, such as some subunits of PKA (catalytic, RIIa, and RIIb) and HSL activity, were detected in the P40 fraction of adipocytes. Moreover, AKAP149 and AKAP150 were also detected in the P40 fraction and AKAP150 but not AKA149 was co-immunoprecipitated with RII subunits. The experiment using St-Ht31 shows that interaction of AKAP and PKA is required for full lipolysis. Lipolytic agonists increased HSL activities in the P40 fraction but
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not in the I40 fraction. These data suggest that compartmentalization of some lipolytic machineries (PKA, HSL, and possibly AKAPs) within adipocytes is required for full lipolysis. Exercise training enhanced the protein expressions of some subunits of PKA (catalytic, RIIa, and probably RIIb) with increased activities of PKA and HSL in the P40 fraction. Furthermore, protein expression of AKAP150 and the decrease in lipolysis due to St-Ht31 were significantly greater in the TR rats. Thus, the mechanisms behind exercise trainingenhanced adipocyte lipolysis could involve the increase in the activities of HSL in the P40 fraction with the enhanced activity and expressions of PKA subunits (catalytic, RIIa, and RIIb) and AKAP150. Acknowledgment This study was supported in part by a grant-in-aid from the Japanese Ministry of Education, Science, Sports and Culture.
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