Biochemical and Molecular Actions of Nutrients

Response to Intra- and Extracellular Lipolytic Agents and HormoneSensitive Lipase Translocation Are Impaired in Adipocytes from Rats Adapted to a High-Protein, Carbohydrate-Free Diet1 Maristela P. Martins-Affe´rri, William T. L. Festuccia, Luiz C. C. Navegantes, Maria A. R. Garo´falo, Leida M. Botion, Isis C. Kettelhut, and Renato H. Migliorini2 Departments of Physiology and Biochemistry/Immunology, School of Medicine, University of Sa˜o Paulo, 14049 –900 Ribeira˜o Preto-SP, Brazil

KEY WORDS: ● hormone sensitive lipase (HSL) ● HSL content and activity ␤-adrenergic agonists ● intracellular lipolytic agents

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HSL translocation

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Adipocytes are highly specialized cells that play critical roles in energy regulation and homeostasis. Their primary and best-known role in mammals is to store energy in the form of triglycerides when energy intake exceeds energy expenditure and to release it as FFA during starvation. The white adipose tissue (WAT)3 rapidly responds to hormonal, metabolic, and neural stimuli. Studies from our laboratory showed that cold exposure in rats increases norepinephrine (NE) turnover and lipolysis in WAT, effects that are not blocked by adrenal demedullation (1). Also, an increase in NE turnover and lipolysis in WAT was observed during prolonged starvation (2). These data suggest that direct innervation of WAT by the

sympathetic nervous system plays an important role in the control of FFA mobilization. The lipolytic effects generated by catecholamines (epinephrine and norepinephrine) in white fat cells were initially defined in terms of ␤1- and/or ␤2-adrenoceptor (AR)-mediated activation. The presence of ␤1- and ␤2-ARs was clearly established in human (3–5) and nonhuman primate (6,7) fat cells. In rodents, the main lipolytic effect of catecholamines was shown to result from the activation of a third ␤-AR with a small subordinate role of the ␤1-ARs (8 –10). Since its discovery by Arch et al. (11), the ␤3-AR was cloned (12) and found to be expressed in WAT (13–15) and stimulate lipolysis with the release of fatty acids (9,16). Numerous studies reviewed by LaFontan et al. (17) indicated that the adrenergic effect on cyclic AMP production, phosphorylation and translocation of hormone-sensitive lipase (HSL) to the fat droplet and lipolysis stimulation are mediated by the control of adenylate cyclase activity by ␤1, ␤2, and ␤3 stimulatory and ␣2 inhibitory receptors linked to G proteins. The present experiments were motivated by previous findings in rats adapted to a high-protein, carbohydrate-free (HP) diet, a preparation that was used in this laboratory to investi-

1 Supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP 01/02944 –9 and 01/10050 – 8) and from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq 513296/96). 2 To whom correspondence should be addressed. E-mail: [email protected]. 3 Abbreviations used: AR, adrenoceptors; DBcAMP, dibutyryl cAMP; FSK, forskolin; HP, high-protein, carbohydrate-free; HSL, hormone sensitive lipase; IBMX, isobutylmethylxanthine; N, balanced; NE, norepinephrine; PIA, (R)-(-)N6(1-methyl-2-phenylethyl) adenosine; PMSF, phenylmethylsulfonyl fluoride; WAT, white adipose tissue.

0022-3166/04 $8.00 © 2004 American Society for Nutritional Sciences. Manuscript received 5 May 2004. Initial review completed 2 June 2004. Revision accepted 9 August 2004. 2919

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ABSTRACT We showed previously that rats adapted to a high-protein (70%), carbohydrate-free (HP) diet have reduced lipolytic activity. To clarify the underlying biochemical mechanisms, several metabolic processes involved in adipose tissue lipolysis were investigated. The experiments were performed in rats adapted for 15 d to an HP or a balanced diet. In agreement with previous results, microdialysis experiments showed that the concentrations of adipose tissue interstitial and arterial plasma glycerol were lower in rats adapted to the HP diet. Under nonstimulated conditions, rates of lipolysis, estimated by glycerol release to the incubation medium, were reduced in adipocytes from HP rats. Under the same conditions, there was a small, but significant (17%) reduction in the activity of hormone sensitive lipase (HSL), with no change in the content of the enzyme. Upon stimulation with isoproterenol, the percentage of the enzyme in the adipocyte cytosol translocated to the fat droplet was 20 –25% in HP rats and 40 –50% in rats fed the balanced diet. Adipocytes from HP diet–adapted rats had a significantly reduced response (⬃40%) to the lipolytic action of nonspecific (norepinephrine, epinephrine, isoproterenol) and specific (CL316,243, BRL37,344, dobutamine, clenbuterol) ␤-adrenergic agonists. Adipocytes from HP rats also had a reduced lipolytic response to the intracellular agents, dibutyryl cAMP (44%), forskolin (46%), and isobutylmethylxanthine (29%). The data suggest that the main mechanism responsible for the reduced basal and stimulated lipolysis in HP diet–adapted rats is an impairment in the intracellular process of lipolysis activation, with a deficient translocation of HSL to the fat droplet. J. Nutr. 134: 2919 –2923, 2004.

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MATERIALS AND METHODS Animals. Rats of Wistar origin were obtained from the Faculty colony, which has remained closed for ⬃50 y. Male rats initially weighing 100 –110 g were housed in suspended, wire-bottomed cages and maintained at 25 ⫾ 2°C on a 12-h light:dark cycle. Two types of purified diets, previously described in detail (20), were utilized in this study, i.e., a high-protein, carbohydrate-free (HP) diet, containing 70% protein, no carbohydrate, and 8% corn oil, and a balanced (N) diet, containing 17% protein, 66% carbohydrate, and 8% corn oil. The 2 diets were approximately isoenergetic and contained equal amounts of vitamins and minerals. The rats consumed the diets for 15 d and weighed 180 –200 g when used in the experiments, which were performed between 0800 and 1000 h. Care and treatment of experimental rats received prior institutional approval by the Ethical Committee of Sa˜o Paulo State University. Microdialysis experiments. Rats were anesthetized i.p. with thionembutal sodium (4 mg/100 g body weight) and placed on heating pads to maintain adequate temperature (37°C). A polyethylene catheter was placed in the left carotid artery to draw blood. The microdialysis probe was inserted in the right abdominal subcutaneous tissue and an equilibration period of 45 min was allowed. A catheter of single dialysis tubing (12 ⫻ 0.3 mm, Gambro, Cuprophane, 3000mol wt cutoff) was glued to nylon tubing (standardized length of 30 mm) with cyanoacrylate. After connecting the catheter inlet to a microinjection pump (Harvard), the system was perfused with 10 g/L bovine serum albumin and 1 mmol/L glucose in isotonic saline at a rate of 2.5 ␮L/min. Samples of the adipose tissue interstitial fluid and arterial plasma were collected at 20-min intervals during 1 h of experiment. In vivo probe recovery of glycerol was assessed according to the internal reference calibration technique (21), which is based on the fractional extraction of radioactivity from [14C]-glycerol added to the perfusate. Isolation of adipocytes. After cervical dislocation, the epididymal fat pads from 5–7 rats of each diet group were removed, pooled together, and disaggregated with collagenase according to the method of Rodbell (22), in a buffer containing 27 mmol/L HEPES, 137 mmol/L NaCl, 4.2 mmol/L NaHCO3, 0.4 mmol/L MgSO4 䡠 7H2O, 0.5 mmol/L MgCl2 䡠 6H2O, 0.4 mmol/L KH2PO4, 5.4 mmol/L KCl, 1.3 mmol/L CaCl2 䡠 2H2O, pH 7.4, supplemented with 10 g/L fatty acid–free albumin and 0.5 mmol/L glucose. After incubation under continuous shaking for 1 h at 37°C, the adipocytes were filtered through a 300-␮m nylon mesh and washed 3 times with the same buffer. In vitro lipolysis. Aliquots of 1 mL adipocyte suspension containing 400,000 cells were incubated with a supplement of 4.5 mmol/L glucose, in the presence of the following different adrenergic agonists: norepinephrine and epinephrine (nonselective ␣ and ␤), isoproterenol (␤-selective), phenylephrine (␣-selective), dobutamine (␤1-specific), clenbuterol (␤2-specific), BRL37,344 and CL316,243 (␤3-specific). The following intracellular-acting agents were also used: dibutyryl cAMP (DBcAMP, 1 mmol/L), forskolin (FSK, 10

␮mol/L) or isobutylmethylxanthine (IBMX, 0.1 mmol/L). The rate of lipolysis was estimated by the release of glycerol to the incubation medium and was expressed as ␮mol/(400,000 cells 䡠 h). The concentration of glycerol was determined enzymatically (23). Western blot for HSL protein analysis. Adipocytes were sonicated in a buffer containing 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF) and 20 mmol/L HEPES, pH 7.4, using a cell disrupter (Virsonic model 150), 3 times for 20 s. The homogenates were centrifuged (13,000 ⫻ g for 5 min) and the fat-free supernatant below the triglyceride layer was collected and centrifuged (150,000 ⫻ g for 1 h) to prepare a cytosolic extract. The extracts were assayed for protein by a bicinchoninic acid protein assay kit (Pierce Chemical), diluted to equal protein concentrations with 2-fold concentrated Laemmli sample buffer (24), and heated for 5 min at 95°C. The extracts were resolved on SDS-PAGE (8% gel) and then electrophoretically transferred to nitrocellulose. The nitrocellulose membranes (0.45 ␮m) were blocked with 50 g/L milk, probed with the antisera against HSL (an 84-kDa protein), and developed with goat anti-rabbit IgG coupled to alkaline phosphatase (25). The blots were quantified by densitometry on Image Quant software and expressed as arbitrary units/400,000 cells. Measurement of total HSL activity. Fat cells were isolated as described above except that the buffer contained 20 g/L albumin and 5 mmol/L glucose. Packed cells (200 ␮L) were homogenized in a plastic tube with 450 ␮L of buffer [50 mmol/L Tris-HCl, 250 mmol/L sucrose, 1 mmol/L EDTA, 2 mg/L leupeptin, 1 ␮mol/L okadaic acid, pH 7.0 (26)]. The homogenate was centrifuged at 5500 ⫻ g for 10 min at 4°C; after the addition of 100 ␮L of ethyl ether to the fat layer, the centrifuge tube was shaken for 30 s by hand and centrifuged at 1200 ⫻ g for 5 min at 4°C (26). The upper ether layer was aspirated, and an aliquot of the supernatant was used as the enzyme solution for the HSL assay. Total HSL activity was determined in the presence of 14 C-triolein as previously described (27). Incubation was carried out for 1 h at 37°C. The [14C]oleic acid released was measured by the method of Belfrage and Vaughan (28). A 1 mL-aliquot of the upper aqueous phase was used for liquid scintillation counting. Results were expressed as nmol/(400,000 cells 䡠 h). Translocation of HSL. Measurement of HSL translocation was done as described by Clifford et al. (29) with some modifications. Briefly, aliquots of adipocyte suspension (1 mL) containing ⬃400,000 cells were washed and incubated with the albumin free Krebs-Ringer/ HEPES buffer containing either 1 ␮mol/L of (R)-(-)N6-(1-methyl-2phenylethyl) adenosine (PIA) or 1 ␮mol/L of isoproterenol in the presence of adenosine deaminase (1000 U/L) for 5 min at 37°C. Then the samples were transferred to an ice bath and permitted to rise when the buffer was withdrawn. The cells were homogenized in 400 ␮L of buffer containing 250 mmol/L sucrose, 0.2 mol/L Tris-Maleate, 1 mol/L MgCl2, 1 mmol/L EGTA, 0.1 mmol/L PMSF, 1 mg/L leupeptin, 10 mmol/L MG 115 (proteasome inhibitor), and 20 mmol/L HEPES. The homogenates were centrifuged at 13,000 ⫻ g at 4°C for 15 min and the cytosolic fraction was aspirated from below the solidified fat cake and destined to SDS-PAGE. The fat cake fraction was centrifuged again and any contaminating cytosol aspirated and discarded. The fat cake was warmed to room temperature, 100 ␮L of HEPES buffer containing 100 g/L SDS was added, and the solution was placed on a vortex mixer. After centrifugation at 13,000 ⫻ g at 4°C for 15 min, the fat cake protein extract was aspirated from below the floating fat layer for SDS-PAGE. Statistical methods. Data are expressed as means ⫾ SEM. Twoway ANOVA and a subsequent Newman-Keuls multiple range test were used to compare the effects of doses and types of the different lipolytic agents. Student’s t test was used for comparisons between means. P ⬍ 0.05 was taken as criterion of significance.

RESULTS Microdialysis experiments. In both experimental groups, adipose tissue interstitial levels of glycerol were significantly higher than arterial plasma glycerol. HP diet–adapted rats had a significant decrease in the concentration of interstitial (33%) and arterial plasma (40%) glycerol compared with controls fed the balanced diet (Fig. 1). The difference between

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gate the nutritional control of energy metabolism. Due to a marked increase in hepatic gluconeogenesis, rats adapted to the HP diet have normal blood glucose levels that are very resistant to prolonged starvation (18). In relation to lipid metabolism, these rats have a reduced mobilization of FFA in vivo that is accompanied by a reduction in epididymal adipose tissue lipolysis in vitro, estimated by the release of glycerol and FFA into the incubation medium (19). The present experiments were designed to investigate the biochemical mechanisms underlying the reduction in adipose tissue lipolysis induced by adaptation to the HP diet. To this end, we examined, in addition to the changes in the interstitial and plasma levels of glycerol in microdialysis experiments, the changes induced by the HP diet on the following: 1) the basal lipolytic activity of adipocytes, the content and activity of the HSL, and the translocation of the enzyme to the fat droplet; and 2) the response of adipocytes to nonselective and selective ␣-and ␤-adrenergic agonists and to intracellular lipolytic agents.

REDUCED LIPOLYSIS IN RATS FED A HIGH-PROTEIN DIET

interstitial and arterial plasma glycerol concentrations in adipose tissue was also significantly decreased in HP rats by 38% (Fig. 1). Basal lipolysis. Adaptation to the HP diet reduced the adipocyte basal (nonstimulated) rate of lipolysis 40 –50% [0.061 ⫾ 0.005 ␮mol glycerol/(400,000 cells 䡠 h), n ⫽ 4 – 8 experiments, P ⬍ 0.05)], compared with controls fed the balanced diet (0.102 ⫾ 0.010). Under the same conditions, the content of HSL did not differ in adipocytes from HP diet–adapted and control rats, but adaptation to the HP diet reduced adipocyte total HSL activity by 17% (Fig. 2). The process of translocation of the enzyme from the cytosol to the fat droplet was reduced in HP adipocytes (Fig. 3). Upon stimulation of adipocytes from rats fed the balanced diet with isoproterenol, 40 –50% of HSL in the cytosol translocated to the fat droplet, whereas in HP rats adipocytes, only 20 –25% of the enzyme was translocated. Response to lipolytic agents. A comparison of the lipolytic effects of NE, epinephrine, isoproterenol, or phenylephrine (nonspecific agonists) (Fig. 4A) shows that maximal lipolytic responses to adrenergic stimuli were markedly reduced (⬃50%) in HP rats. In HP-fed and control rats. the rank order of potency was: isoproterenol ⬎ NE ⬎ epinephrine ⬎⬎ phenylephrine. HP diet–fed rats had a marked reduction in maximal lipolytic response to the specific adrenergic agonists (dobutamine, clenbuterol, BRL37,344 or CL316,243) similar to that observed with the nonspecific agents (Fig. 4B). The rank order of potency was CL316,243 ⬎ BRL37,344 ⬎ dobutamine ⬎ clenbuterol for both groups. The lipolytic response to intracellular lipolytic agents (DBcAMP and FSK) in adipocytes from HP diet–fed rats was reduced to 44 and 46%, respectively, of that in adipocytes from rats fed the control diet (Fig. 5). The lipolytic response to IBMX was also reduced, but less markedly (29%).

that these rats have lower levels of plasma FFA (18). The reduced lipolytic activity was accompanied by a relatively small (17%) decrease in total hormone-sensitive lipase activity (Fig. 2) with no change in the tissue content of the enzyme (Fig. 2), suggesting that the adaptation of the HP diet resulted in an increased proportion of the enzyme in the dephosphorylated form. Notwithstanding the markedly decreased response of adipocytes from HP-adapted rats to ␤-adrenergic agonists, the rank order of drug potencies (␤3 ⬎ ␤2 ⬎ ␤1) was similar to that of control adipocytes. In addition to reinforcing data from the literature indicating that lipolysis in rats is maintained mainly by ␤3-AR (30,31) which are also highly resistant to desensitization (30,32), these findings suggest that the composition of ␤-receptors in the adipocyte membrane is not affected by the HP diet. In fact, from the data of the present work, the possibility cannot be excluded that the reduced lipolytic response of adipocytes from HP-adapted rats was due to an impairment of the intracellular process of activation of lipolysis, with no alteration in receptor function. Indeed, the reduction in the lipolytic response of HP-adipocytes, calculated as the percentage of control values, obtained with ␤-agonists was similar (⬃40%, Fig. 4 A and B) to that obtained with intracellular-acting agents, DBcAMP and FSK (Fig. 5). Also, the shape of the curves obtained with catecholamines (Fig. 4A) in HP and control adipocytes, especially the almost parallel increase to maximal response, suggests that the apparent affinity of the receptors for the adrenergic agents was not affected. However, further studies, including receptor-binding experiments, are required to establish this hypothesis. Whatever the participation of membrane receptors, the finding in the present work of a deficient isoproterenol-stimulated translocation of the HSL present in cytosol to the fat droplet (Fig. 3) is clear evidence of the impairment of the intracellular process of lipolysis activation in adipocytes from HP rats. It is widely accepted that the action of lipolytic hormones is mediated by the so-called cAMP cascade. Hormones activate adenylate cyclase, thus increasing cAMP formation. Cyclic AMP then promotes lipolytic activity by activating cAMPdependent protein kinase, which phosphorylates HSL, resulting in hydrolysis of stored triacylglycerols, [reviewed in (33)]. According to this view, the increase in the catalytic activity of the enzyme, induced by its phosphorylation, is essential for the acti-

DISCUSSION The present data show that, under nonstimulated conditions, adipose tissue lipolytic activity, assessed in situ by microdialysis (Fig. 1), and in vitro with isolated adipocytes, is reduced in rats adapted to a high-protein, carbohydrate-free diet. These findings are in agreement with previous studies with fragments of adipose tissue (19) and with the verification

FIGURE 2 Effect of adaptation to an HP diet on rat adipocyte content and activity of HSL. Data are means ⫾ SEM, n ⫽ 8. *P ⬍ 0.05 vs. N (balanced) diet. Top: representative Western blots of adipocyte HSL from N and HP diet-adapted rats.

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FIGURE 1 Effect of adaptation to an HP diet on rat arterial plasma and interstitial glycerol concentrations. Data are means ⫾ SEM, n ⫽ 10. *P ⬍ 0.05 vs. N (balanced) diet.

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FIGURE 3 Effect of adaptation to an HP diet on the isoproterenol stimulated translocation of rat adipocyte HSL. Adipocytes from rats adapted to an N or HP diet were incubated with 1 ␮mol/L of PIA or 1 ␮mol/L of ISO and the cytosolic fraction and fat cake were prepared as indicated in Materials and Methods. Data are means ⫾ SEM, n ⫽ 6, *P ⬍ 0.05 vs. PIA; †P ⬍ 0.05 vs. N diet. Top: representative Western blots of adipocyte HSL.

showed that this discrepancy is due in part to a stimulationinduced migration of HSL toward the lipid droplet (35) and changes in the droplet surface, thus providing the lipase access to its substrate (34). A protein, perilipin A, was shown to participate in this process, as indicated by the finding that it can protect neutral lipids within droplets from hydrolysis. It has been hypothesized that, upon phosphorylation, perilipin A would allow access to the lipid droplet, thereby permitting HSL interaction with its substrates (34). Recent studies showed that lipolytic agents, including catecholamines, cAMP, and FSK activate lipolysis without affecting the concentration or the activity of HSL, but inducing HSL translocation from the cytosol to the fat droplet (36). Thus, the critical event in the lipolytical activation of adipocytes seems not to be the increase in catalytic activity of HSL, but rather the translocation of the enzyme to its substrate at the surface of lipid droplets. This view is supported by the verification in the present study of an impairment of the process of translocation in a physiologic situation in which adipocyte lipolysis was reduced. We previously measured the diameters of adipocytes from HP diet–fed and control, normally fed rats (19). Notwithstanding a similar distribution, with no significant differences

FIGURE 4 Effect of different concentrations of nonspecific (A) or specific (B) adrenergic agents on the rate of lipolysis in adipocytes from rats adapted to an HP or N diet. Data are means ⫾ SEM, n ⫽ 4 – 8. *P ⬍ 0.05 vs. N diet.

FIGURE 5 Effect of DBcAMP (1 mmol/L), FSK (10 ␮mol/L) or IBMX (0.1 mmol/L) on the rate of lipolysis in adipocytes from rats adapted to an HP or N diet. Data are means ⫾ SEM, n ⫽ 4. *P ⬍ 0.05 vs. N diet.

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vation of lipolysis. Recent evidence, however, indicates that the process of lipolysis activation is more complex. It was shown that phosphorylation of HSL in vitro produces a small (⬃2-fold) increase in activity against emulsified substrates, compared with a very large (⬃50-fold) increase in triacylglycerol hydrolysis induced by stimulation of intact adipocytes (34). Further studies

REDUCED LIPOLYSIS IN RATS FED A HIGH-PROTEIN DIET

ACKNOWLEDGMENTS We are very grateful to Dr. Allan Green for the HSL antibody. We are indebted to Elza Aparecida Filippin, Neusa Maria Zanon Resano, Carlos Eduardo da Silva, and Victor Diaz Galba´n for technical assistance.

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in minimal or maximal values, as well as in the most prevalent (modal) values, the calculated average diameter was ⬃16% lower in adipocytes from rats adapted to the HP diet. It seems highly unlikely that this difference in size contributed to the changes observed here in adipocyte lipolysis, especially in the response to extra- and intracellular agents. In fact, the only reason for raising the possibility of such a contribution is the existing consensus that in rats (37), but not in humans (38,39), there is a direct correlation between basal lipolysis and adipocyte size. In contrast, the relation between lipolytic responsiveness to extracellular agents is controversial, with direct (40), inverse (41), or no correlation (42) being reported. In summary, adipocytes from rats adapted to the HP diet had a decreased rate of lipolysis under nonstimulated conditions, which was accompanied by a small reduction in the activity of HSL, with no change in the content of the enzyme. Upon stimulation with isoproterenol, the percentage of enzyme present in the cytosol that translocated to the fat droplet was lower in HP diet–adapted rats. Adipocytes from HP rats also had a reduced response to lipolytic agents. The data suggest that the main mechanism responsible for the reduced basal and stimulated lipolysis in HP diet–adapted rats is an impairment in the intracellular process of lipolysis activation, with a deficient translocation of HSL to the fat droplet.

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