Masoprocol decreases rat lipolytic activity by decreasing the phosphorylation of HSL

Maya S. Gowri, Rakia K. Azhar, Fredric B. Kraemer, Gerald M. Reaven, and Salman Azhar

Stanford University School of Medicine, Stanford 94305; and Geriatric Research, Education, and Clinical Center, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Masoprocol (nordihydroguaiaretic acid), a lipoxygenase inhibitor isolated from the creosote bush, has been shown to decrease adipose tissue lipolytic activity both in vivo and in vitro. The present study was initiated to test the hypothesis that the decrease in lipolytic activity by masoprocol resulted from modulation of adipose tissue hormone-sensitive lipase (HSL) activity. The results indicate that oral administration of masoprocol to rats with fructose-induced hypertriglyceridemia significantly decreased their serum free fatty acid (FFA; P < 0.05), triglyceride (TG; P < 0.001), and insulin (P < 0.05) concentrations. In addition, isoproterenol-induced lipolytic rate and HSL activity were significantly lower (P < 0.001) in adipocytes isolated from masoprocol compared with vehicle-treated rats and was associated with a decrease in HSL protein. Incubation of masoprocol with adipocytes from chow-fed rats significantly inhibited isoproterenol-induced lipolytic activity and HSL activity, associated with a decrease in the ability of isoproterenol to phosphorylate HSL. Masoprocol had no apparent effect on adipose tissue phosphatidylinositol 3-kinase activity, but okadaic acid, a serine/threonine phosphatase inhibitor, blocked the antilipolytic effect of masoprocol. The results of these in vitro and in vivo experiments suggest that the antilipolytic activity of masoprocol is secondary to its ability to inhibit HSL phosphorylation, possibly by increasing phosphatase activity. As a consequence, masoprocol administration results in lower serum FFA and TG concentrations in hypertriglyceridemic rodents.

adipocyte; free fatty acid; triglyceride; hormone-sensitive lipase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HISTORICALLY, EXTRACTS OF the creosote bush (Larrea tridentata) have been extensively used by native healers throughout the Southwest region of North America for the treatment of type 2 diabetes. We have recently used an in vivo guided fractionation approach to identify masoprocol as the major, if not the only, compound in L. tridentata responsible for its antihyperglycemic effect. Masoprocol, also known as nordihydroguaiaretic acid, is a known lipoxygenase inhibitor (21, 34) and has been shown to lower plasma glucose concentrations and increase insulin-mediated glucose uptake in genetic models (db/db and ob/ob) of type 2 diabetes in mice (14).

In addition, rats were made insulin resistant and hyperinsulinemic by feeding a fat-enriched diet, and they were rendered hyperglycemic by injecting them with a modest amount of streptozotocin. In this situation, the insulin concentrations decline to levels similar to those in chow-fed rats and were no longer able to compensate for the insulin resistance, and hyperglycemia ensued (26). Masoprocol has been shown to effectively lower glucose concentrations in this nongenetic model of type 2 diabetes without any change in plasma insulin concentration but with decreases in free fatty acid (FFA) and triglyceride (TG) concentrations (26). In addition, masoprocol treatment has been shown to prevent hypertriglyceridemia in nondiabetic rats fed a fructose-enriched diet by significantly reducing hepatic TG secretion and increasing clearance of TG from the plasma (28).

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that catalyzes the hydrolysis of intracellular TG (lipolysis) in adipose tissue (12, 32), skeletal muscle (20), and heart (31). HSL activity is regulated by multisite phosphorylation-dephosphorylation reactions in response to hormones (1, 29). Hormones (e.g., catecholamines) and other agonists that elevate cAMP levels stimulate HSL enzymatic activity through enhanced phosphorylation catalyzed by protein kinase A (2, 5). Insulin is thought to inhibit lipolysis by inactivating HSL due to the net dephosphorylation of the enzyme protein (5, 7). Okadaic acid, a polyether fatty acid, is a very potent inhibitor of protein phosphate 1 and protein phosphatase 2A (6), two of the four major protein phosphatases in cytosol of mammalian cells that catalyze hydrolysis of phosphoserine and phosphothreonine residues (4, 30). It is cell permeable and, when added to adipocytes, mimics the action of insulin in stimulating glucose transport (6, 9) and protein kinase activity (9).

In an effort to define the mechanism responsible for the metabolic effects of masoprocol, we have recently demonstrated that isoproterenol-induced lipolytic rate is significantly decreased when masoprocol is incubated with isolated adipocytes obtained from normal rats (8). The present studies were initiated to extend these observations and to specifically test the hypothesis that the ability of masoprocol to inhibit lipolytic activity is secondary to a direct effect on HSL.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and treatments. Male Sprague-Dawley rats obtained from Harlan Sprague-Dawley (Indianapolis, IN) were used in these studies. For in vitro studies, rats weighing ~220 g were fed Purina Rat Chow (no. 5012; St. Louis, MO) and water ad libitum and were maintained on a 12:12-h (0600-1800) light-dark cycle. On the day of the experiment, the animals were fasted for 4 h and decapitated by cervical dislocation, and epididymal fat pads were removed for preparation of adipocytes. For these studies, adipocytes were pooled from three to four animals. All experiments were performed in duplicate and were repeated at least three to six times (n = 3 or 6).

For in vivo studies, rats weighing 175-200 g were used. Rats were first maintained on a chow diet for ~1 wk and then were switched to a high-fructose diet (TD 78463; Harlan Teklad, Madison, WI) that provided 60% of total calories as fructose. After 10 days on the high-fructose diet, the degree of hypertriglyceridemia was evaluated by determining the total plasma TG levels. The hypertriglyceridemic animals (TG >250 mg/dl) were then divided into two groups (12 animals in each group, with comparable plasma TG concentrations). On day 0 of treatment, the rats were fasted for 4 h, and tail vein blood was collected for baseline measurements of serum TG, glucose, insulin, and FFA. The two groups of rats were then treated with either vehicle (Gelucire 44/14) or masoprocol at a dose of 80 mg/k body wt two times a day for 4 days, delivered by oral gavage at a volume of 2.5 ml/kg body wt. During the treatment period, rats were maintained on high-fructose diets.

After 4 days of treatment, blood was collected from the tail vein, 3 h after the last dose of vehicle or masoprocol. Serum samples were used to measure TG and glucose concentrations by enzymatic calorimetric methods (15, 33) using Sigma Diagnostic kits (St. Louis, MO). Serum insulin concentrations were measured by RIA using a Linco Rat Insulin RIA kit (St. Charles, MO). FFA concentrations were measured using the nonesterified fatty acid (NEFA) C kit by the ACS-ACOD method following the instructions of the manufacturer. Rats were killed by decapitation, and epididymal fat pads were removed quickly and used for adipocyte isolation (to measure lipolytic activity) and quantitation of HSL activity. For Western blotting of HSL protein and RNA isolation, the tissue samples were collected and frozen immediately in liquid nitrogen. The tissues were stored at -80°C until used for various measurements.

Preparation of adipocytes. Adipocytes were prepared from the epididymal fat pads by a slight modification of the procedure of Rodbell (27) as previously described (24). In brief, fat pads were minced with scissors, placed in plastic flasks in Krebs bicarbonate buffer containing 3.5% BSA, 3 mM glucose, and 1 mg collagenase/ml, and incubated for 1 h at 37°C in a gyratory water-bath shaker. The released cells were washed three times in fresh Krebs buffer with 2% albumin and allowed to separate from the infranatant by flotation. Suitable aliquots of diluted cells were taken for measurement of rates of lipolytic activity. A 100-µl aliquot of diluted cells was also fixed in a solution of 2% osmium tetroxide in collidine buffer and was counted in a coulter counter (Hialeah, FL) for determination of cell number.

Measurement of rates of lipolytic activity. Rate of adipocyte lipolysis was determined using an established procedure of this laboratory (24). Aliquots of diluted cells (~1 × 105 cells/ml) were placed in plastic vials and preincubated in the presence or absence of 50 µM masoprocol in 1 ml of Krebs buffer containing 2% albumin at 37°C for 60 min, with continuous shaking at 40 counts/min. Subsequently, cells were incubated with or without (-)-isoproterenol (3 nM) or 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP, 0.5 mM) for 60 min at 37°C. In some instances, cells were also incubated with insulin (400 pM) for 60 min at 37°C with isoproterenol. At the end of the incubation, the cells were centrifuged, and the infranatant was collected for the quantification of glycerol and FFA, as described below. To further establish the specificity of masoprocol, the effect of esculetin, another specific inhibitor of lipoxygenase (18), was evaluated. Aliquots of adipocytes were preincubated with 4 or 40 µM esculetin for 60 min at 37°C and then were incubated with 3 nM isoproterenol for 60 min at 37°C to measure rates of lipolytic activity. For some other studies, adipocytes were also preincubated with 500 nM okadaic acid, a serine/threonine protein phosphatase inhibitor (6), or 1 µM wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor (19), for 15 min at 37°C before additional incubation with masoprocol and/or isoproterenol, as described above. Glycerol concentration in the infranatant was measured by an enzymatic method (23) using the TG kit. FFA concentration in the infranatant was measured using the NEFA C kit by the ACS-ACOD method.

To determine if masoprocol treatment could reduce lipolytic activity in studies performed ex vivo, the adipocytes were isolated from epididymal fat pads obtained from fructose-fed rats treated with vehicle or masoprocol. Adipocytes were incubated with isoproterenol (100 nM) for 60 min at 37°C. At the end of the incubation, the cells were centrifuged, and the infranatant was collected for the measurement of glycerol and FFA.

HSL activity. To assess the HSL protein and activity levels, adipose tissues from animals treated with masoprocol or vehicle or isolated adipocytes were homogenized using a Polytron in 50 mM Tris · HCl buffer (pH 7), 250 mM sucrose, and 5 µM EDTA. The homogenates were sequentially centrifuged at 1,500 (10 min) and 43,000 g (15 min) at 4°C (17). The clear supernatants (43,000 g) were used for measurement of HSL activity and HSL protein content by Western blotting. Protein content of supernatants was determined by a modification (22) of the technique of Lowry et al.

HSL activity was assayed as neutral cholesteryl esterase by following the release of [1-14C]oleic acid from cholesteryl [1-14C]oleate as described by Nakamura et al. (17) with minor modifications. The incubation medium in a final volume of 200 µl contained 100 nM potassium phosphate buffer, pH 7.4, 0.025% BSA, 1.25 nmol cholesteryl [1-14C]oleate (~3 × 104 dpm) added in 4 µl acetone, and 10 µg supernatant. After incubation (10 min), the reaction was terminated (10) by addition of 1 ml of borate/carbonate buffer (0.1 M, pH 10.5) followed by 3 ml of chloroform-methanol-heptane (1.39:1.28:1). The reaction tubes were vortexed vigorously for 1 min and centrifuged (1,500 g for 20 min at 10°C), and the released [1-14C]oleate in the aqueous phase was determined by a scintillation counter. The results are expressed as picomoles [14C]oleate released per minute per milligram protein.

Western blotting of HSL protein. An aliquot of adipose supernatant (25 µg protein) was mixed with equal volumes of 2× sample-loading buffer [4.6% (wt/vol) SDS, 16% (wt/vol) sucrose, 10% (vol/vol) beta -mercaptoethanol, and 0.1 M Tris · HCl, pH 6.8], heated at 95°C for 5 min, and cooled to room temperature. The sample was subjected to electrophoresis on a 7% SDS-polyacrylamide gel (11). After electrophoretic separation, proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) using standard techniques. The membranes were blocked in PBS-T buffer [pH 7.4; 10 mM sodium phosphate, 0.15 M NaCl, 2.5 mM MgCl2, 0.1% Tween 20, and 5% (wt/vol) nonfat dried milk] for 1 h at 37°C. After being washed, the membranes were incubated with rabbit anti-HSL IgG (10) in blocking solution with agitation at 4°C overnight. Subsequently, the membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit IgG. The signals corresponding to HSL were detected using the enhanced chemiluminescence system (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. The Western blots were scanned by a densitometer.

Immunoprecipitation and detection of HSL phosphorylation. Anti-phosphoserine antibody was used to detect HSL phosphorylation. Aliquots of adipocyte extracts (200 µg protein) were first incubated with 5 µg of polyclonal anti-HSL in 2× immunoprecipitation buffer (2% Triton X-100, 300 mM NaCl, 20 mM Tris, pH 7.4, 2 mM EDTA, 2 mM EGTA, 4 mM sodium vanadate, 4.0 mM phenylmethylsulfonyl fluoride, and 1.0% Nonidet P-40) in a final volume of 1 ml for 2-3 h at 4°C. After incubation, protein A/G plus-agarose conjugate (50 µl) was added to each sample, and the tubes were vortexed and incubated with agitation overnight at 4°C. Immunoprecipitates were collected by centrifugation at 2,500 rpm for 5 min at 4°C. The pellets were carefully washed four times with 1× immunoprecipitation buffer as before. The pellets were resuspended in 25 µl of 2× electrophoresis buffer, boiled for 5 min, and centrifuged for 5 min at 2,500 rpm. The released supernatants were subjected to SDS-PAGE followed by Western blotting, as described above with some minor modifications. The primary antibody used for detecting HSL phosphorylation was rabbit anti-phosphoserine IgG at a dilution of 1:100.

RNA preparation. Approximately 1-2 g of adipose tissue, cut into small pieces, was homogenized in 5 ml of denaturing solution [4 M guanidium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 M beta -mercaptoethanol (added before use)] at room temperature using a Polytron (setting 7 for 30 s). The solution was then extracted two times with an equal volume of chloroform to delipidate the sample (13). The aqueous phase was transferred to a polypropylene tube, and 0.1 ml of 2 M sodium acetate, pH 4, 1 ml of water-saturated phenol, and 0.2 ml of chloroform-isoamyl (49:1) mixture were sequentially added per 1 ml of aqueous phase. The RNA was then purified according to the procedure described by Chomczynski and Sacchi (3), dissolved in diethyl pyrocarbonate (DEPC)-treated water, and stored at -70°C.

Construction of HSL and 18S rRNA cDNA probes. A 470-bp Apa I-Pst I (position 687-1156) fragment from rat HSL cDNA was subcloned into the Apa I-Pst I sites of pBluescript KSII+ (pBS KSII+; Stratagene, La Jolla, CA). A 274-bp Dra II-Tha I (position 134-408) fragment of 18S ribosomal RNA cDNA (25) was subcloned into the Dra II-EcoR V sites of pBS KSII+. The plasmids were linearized with appropriate restriction endonucleases (Apa I for HSL and BamH I for 18S ribosomal RNA), and 3'-ends of the linearized HSL plasmids were filled using Klenow fragment, extracted two times with phenol and two times with chloroform, precipitated in ethanol, and redissolved in DEPC-treated water. The linearized plasmids were used for riboprobe synthesis.

Preparation of riboprobes. The antisense [32P]cRNA probes were synthesized using [alpha -32P]rCTP and the appropriate T3 or T7 RNA polymerase according to instructions supplied by the Strategene's in vitro transcription kit. Because of their high liability, the riboprobes were always freshly prepared before hybridization.

mRNA quantitation by RNase protection assay. The HSL mRNA levels were determined using a sensitive RNase protection assay. Aliquots of adipose tissue RNA or control tRNA (10 µg) were dried under vacuum and redissolved in 30 µl of hybridization buffer containing 104 cpm of probe [i.e., the radiolabeled HSL riboprobe or the 18S rRNA riboprobe that was used as an internal standard for quantification]. The mixture was incubated for 5 min at 85°C to denature RNA and was then rapidly transferred to a hybridization temperature of 42°C for incubation overnight (~16-18 h). The unprotected probe was hydrolyzed by digestion with 40 µg/ml RNase A and 2 µg/ml RNase T1 for 1 h at 30°C. The RNase digestion was terminated by the addition of proteinase K (50 µg) and SDS (2 mg) and incubation for 15 min at 37°C followed by phenol-chloroform extraction. The protected RNA-RNA hybrids were ethanol precipitated in the presence of yeast tRNA, and the pellets were dissolved in 15 µl of RNA loading buffer and heated for 5 min at 85°C. The protected fragments were separated on 6% acrylamide-urea denaturing gels. After electrophoresis, gels were exposed to Kodak XAR-5 film at -70°C with intensifying screens. For quantitation, the films were analyzed by densitometry. The data are expressed as a ratio of the HSL signal to that of 18S rRNA to correct for differences in loading the small amounts of RNA. In our studies, the levels of 18S rRNA remained constant in both vehicle- and masoprocol-treated rats.

Statistical analysis. Statistical significance of differences between the experimental groups was compared by ANOVA, and results are expressed as means ± SE.

Reagents. Masoprocol was obtained from either Sigma (St. Louis, MO) or Western Engineering and Research (El Paso, TX). Each batch of masoprocol was tested for glucose uptake in rat adipocytes to make sure that no difference existed between two sources or lots. Gelucire 44/14 was obtained from Gattefosse (Westwood, NJ). Type I collagenase was purchased from Worthington Biochemical (Freehold, NJ). BSA, (-)-isoproterenol, goat anti-rabbit IgG, TG kits (catalog nos. 339-10 and 320-10), and a Glucose Trinder Kit (catalog no. 315-100) were supplied by Sigma Chemical. The NEFA C kit (code no. 994-75409) was the product of Wako Chemicals (Richmond, VA). Esculetin, okadaic acid, and wortmannin were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Cholesteryl [1-14C]oleate was purchased from American Radiolabeled Chemicals (St. Louis, MO). [alpha -32P]CTP (specific activity 29.6 TBq/mmol; 800 Ci/mmol) was supplied by Du Pont (NEN Research Products, Boston, MA). Anti-phosphoserine antibody was obtained from Zymed (South San Francisco, CA). Protein A/G PLUS-Agarose was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In vivo studies. Table 1 compares the effects of vehicle and masoprocol administration on fructose-induced changes in body weight, serum glucose, TG, FFA, and insulin concentrations. Body and liver weight were similar in the two groups of rats, as were the serum glucose concentrations. In contrast, serum FFA, TG, and insulin concentrations were significantly lower after masoprocol treatment but did not change in vehicle-treated rats.

                              
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Table 1.   Effect of masoprocol treatment on serum glucose, insulin, triglyceride, and FFA concentrations in fructose-fed rats

Ex vivo studies. The prolipolytic effects of isoproterenol (100 nM) on adipocytes isolated from fructose-fed rats treated with vehicle or masoprocol are shown in Fig. 1. These results demonstrate that release of both glycerol (P < 0.002) and FFA (P < 0.02) by adipocytes from masoprocol-treated rat is decreased compared with adipocytes from rats receiving vehicle alone. HSL activity in adipocytes isolated from rats treated with vehicle or masoprocol is shown in Fig. 2A. The significant decrease (P < 0.001) in HSL activity in adipocytes from masoprocol-treated rats was associated with a concomitant fall in HSL protein, as determined by Western blot analysis (Fig. 2B).


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Fig. 1.   Isoproterenol-induced glycerol (A) and free fatty acid (FFA; B) release by adipocytes isolated from fructose-fed rats treated with either vehicle or masoprocol. Isolated adipocytes were incubated with or without isoproterenol (100 nM) for 60 min at 37°C. After incubation, the media were collected and assayed for released glycerol and FFA. The basal rate of lipolysis in adipocytes from fructose-fed rats in the absence of added isoproterenol was 715 ± 8 nmol · 10-5 cells · h-1. In vivo administration of masoprocol did not alter basal lipolysis (787 ± 20 nmol · 10-5 cells · h-1). The results represent means ± SE of 9 individual measurements in each group.



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Fig. 2.   Effects of masoprocol treatment on hormone-sensitive lipase (HSL) activity and protein content in adipose tissue extracts from fructose-fed rats. A: HSL activity. Groups of 12 fructose-fed animals were treated with either vehicle or masoprocol as described in Animals and treatments. The freshly prepared adipose tissue extracts were used for the measurement of HSL activity as described in MATERIALS AND METHODS. The results represent means ± SE of 12 individual rats in each group. B: Western blot analysis of HSL protein in adipose tissue from fructose-fed rats treated with either vehicle or masoprocol. The experimental details were the same as described in MATERIALS AND METHODS except 25 µg of protein were loaded in each lane. The four lanes under each vehicle and masoprocol treatment represent four individual animals.

Figure 3A shows the results obtained using a highly sensitive RNase protection assay to assess the levels of HSL mRNA in the adipose tissues of rats treated with vehicle or masoprocol. As a control, the expression of the stable marker 18S rRNA was also examined. The results presented in Fig. 3B (expressed as a ratio of HSL to 18S rRNA) demonstrate that expression of HSL mRNA was not significantly altered after exposure of rats to masoprocol and suggest the possibility that masoprocol modulates HSL activity posttranscriptionally.


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Fig. 3.   A: analysis of adipose tissue HSL mRNA expression by RNase protection assay. Total RNA was isolated from adipose tissue of fructose-fed individual animals treated with either vehicle (lanes 1-3) or masoprocol (lanes 4-6). Aliquots of 10 µg RNA from various samples were subjected to RNase protection assay using 32P-labeled HSL and 18S rRNA riboprobes. Other details were the same as described in MATERIALS AND METHODS. The protected fragments were resolved on 6% acrylamide-urea denaturing gels. After electrophoresis, gels were exposed to Kodak XAR-5 films at -70°C with intensifying screens. For quantitation, the signals from the films were analyzed by densitometry. C, tRNA control; P, probe. B: densitometric quantification of the bands shown in A. HSL mRNA is normalized to 18S rRNA as in A. Data represent experiments performed on 2 different occasions; each set includes 3 animals/vehicle and masoprocol-treated group.

In vitro studies. The next series of experiments were performed on adipocytes obtained from chow-fed rats, with masoprocol being added in vitro. The inhibitory effect of masoprocol and insulin on glycerol and FFA released during isoproterenol-induced lipolysis is shown in Fig. 4. It is clear from Fig. 4, A and B, that adipocytes pretreated with 50 µM masoprocol had a significant reduction in both glycerol (83 ± 3 vs. 165 ± 14 nmol · 10-5 cells · h-1; P < 0.01) and FFA (180 ± 30 vs. 440 ± 50 neq · 10-5 cells · h-1; P < 0.02) release compared with adipocytes incubated with isoproterenol alone. It can also be seen that this effect was comparable to the inhibition produced by 400 pM insulin. Masoprocol also significantly (P < 0.05) reduced 8 CPT-cAMP-induced lipolysis as shown in Fig. 5.


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Fig. 4.   Comparison of the ability of masoprocol and insulin to inhibit isoproterenol-induced lipolytic activity in adipocytes isolated from chow-fed rats. Isolated adipocytes were incubated with isoproterenol (ISO, 3 nM) with or without masoprocol (50 µM) or insulin (400 pM), and the amounts of glycerol and FFA released were quantified. Results represent means ± SE of 6 individual experiments done in duplicate on pooled adipocytes. The basal rate of lipolysis in adipocytes in the absence of isoproterenol was 66 ± 13 nmol · 10-5 cells · h-1. Pretreatment of adipocytes with masoprocol did not have any effect on the basal rate of lipolysis (65 ± 14 nmol · 10-5 cells · h-1).



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Fig. 5.   Effect of masoprocol on 8-(4-chlorophenylthio)-cAMP (8 CPT-cAMP)-induced lipolytic activity in adipocytes isolated from chow-fed rats. The experimental details were as described in Fig. 4 except isoproterenol was replaced with 05 mM 8 CPT-cAMP. The results are means ± SE of 5 individual experiments performed in duplicate on pooled adipocytes.

The effects of masoprocol and esculetin on isoproterenol-induced lipolytic activity in isolated adipocytes were also examined. As expected, pretreatment with 50 µM masoprocol significantly (P < 0.01) inhibited lipolytic activity, whereas esculetin (4 and 40 µM) did not (data not shown). The lipolytic effect of okadaic acid, a serine/threonine phosphatase inhibitor, in the presence or absence of masoprocol is shown in Fig. 6. Okadaic acid (500 nM) significantly enhanced isoproterenol-induced lipolytic activity (P < 0.05) by isolated adipocytes. Moreover, okadaic acid decreased the antilipolytic effect of masoprocol. Masoprocol (50 µM) inhibited isoproterenol-induced lipolytic activity by ~40%, but, in the presence of okadaic acid, masoprocol inhibited isoproterenol-induced lipolytic activity by only 5%. Wortmannin, a specific inhibitor of PI 3-kinase, did not affect the ability of masoprocol to inhibit isoproterenol-induced lipolytic activity (data not shown).


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Fig. 6.   Relationship between masoprocol and okadaic acid (OKA) modulation of isoproterenol-induced lipolytic activity in adipocytes isolated from chow-fed rats. The experimental conditions to measure lipolytic activity were as described in Fig. 4 and MATERIALS AND METHODS. The concentrations of isoproterenol, masoprocol, and okadaic acid used were 3 nM, 50 µM, and 500 nM, respectively. The results represent means ± SE of 3 individual experiments done in duplicate on pooled adipocytes.

Figure 7 shows the in vitro inhibitory actions of masoprocol on isoproterenol-induced HSL activity in isolated adipocytes. In the presence of 3 nM isoproterenol, HSL activity was reduced by ~40% when adipocytes were preincubated with 50 µM masoprocol. Evidence that masoprocol decreases HSL activity by changing the phosphorylated state of HSL is presented in Fig. 8. In this experiment, an anti-phosphoserine antibody was used to detect the phosphorylation of HSL. The results clearly show that HSL protein immunoprecipitated from adipocytes treated with masoprocol had a decreased phosphoserine band.


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Fig. 7.   Effect of in vitro addition of masoprocol on isoproterenol-induced stimulation of HSL activity in adipocytes isolated from chow-fed rats. Isolated adipocytes were preincubated with or without masoprocol for 60 min at 37°C, followed by a 60-min incubation with isoproterenol. The adipocyte samples were homogenized, and cellular extracts were employed for the measurement of HSL activity. Results represent means ± SE of 6 individual experiments done in duplicate on pooled adipocytes.



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Fig. 8.    In vitro effects of masoprocol on isoproterenol-stimulated phosphorylation of HSL protein. Adipocytes were sequentially treated with or without masoprocol followed by isoproterenol. Suitable aliquots of various cytosolic fractions (200 µg protein) were subjected to HSL immunoprecipitation with rabbit anti-HSL as described in MATERIALS AND METHODS. In each case, all of the immunoprecipitated HSL pellet was subjected to SDS-PAGE followed by Western blotting using the anti-phosphoserine and enhanced chemiluminescence detection system. The extent of HSL phosphorylation was determined by densitometric scanning of phospho (P)-HSL band. Under similar experimental conditions, the total amount of HSL protein (phosphorylated + nonphosphorylated HSL) was also determined using rabbit anti-rat HSL. Lane 1, adipocytes treated with isoproterenol alone; lane 2, adipocytes treated with masoprocol and isoproterenol.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study was initiated to test the hypothesis that masoprocol inhibits the activity of HSL and that this action accounts for its ability to decrease the rate of adipocyte lipolysis and lower plasma FFA, TG, and insulin concentrations. The results presented not only provide strong support for this view, but they define a molecular mechanism to explain how masoprocol inhibits lipolytic activity.

At the simplest level, the studies of fructose-induced hypertriglyceridemia demonstrate that administration of masoprocol lowers plasma FFA and TG concentrations in nondiabetic rats, similar to earlier results demonstrating the same phenomenon in rats with an experimental form of type 2 diabetes (26). However, the two situations differ with regard to the effect of masoprocol administration on insulin and glucose concentrations. In both instances, there is evidence based on the serum insulin measurements that insulin sensitivity was enhanced after masoprocol administration. In the fructose-fed rat, the physiological response of the pancreatic beta -cell to enhanced insulin sensitivity would be to secrete less insulin, leading to lower insulin but unchanged glucose concentrations, as shown in Table 1. In contrast, the masoprocol-induced increase in insulin sensitivity in the fat-fed/streptozotocin rat model of type 2 diabetes would lead to increased glucose disposal and a fall in glucose concentration. However, because these animals remain hyperglycemic, the pancreatic beta -cell continues to secrete as much insulin as before. In addition to its effect on enhancing insulin sensitivity, the results presented indicated that isoproterenol-induced stimulation of adipocytes isolated from masoprocol-treated rats was associated with significantly less FFA and glycerol release. Finally, HSL activity was significantly lower in adipose tissue from masoprocol-treated rats, associated with a decrease in HSL protein.

The results of the in vivo administration of masoprocol raised the possibility that the antilipolytic effect of masoprocol was mediated via its ability to inhibit phosphorylation of HSL, and this conclusion received strong support from the in vitro effects of masoprocol on isolated adipocytes. More specifically, the results of the experiments with okadaic acid (a specific serine/threonine phosphatase inhibitor) and wortmannin (an inhibitor of PI 3-kinase) indicated that the antilipolytic effect of masoprocol was likely due to its stimulation of a serine/threonine phosphatase and not by stimulation of PI 3-kinase. The antilipolytic effect of masoprocol on isolated adipocytes was associated with a fall in HSL activity, and, by using as anti-phosphoserine antibody, we were able to show that this loss in activity was associated with a decrease in the phosphorylated state of HSL. This confirms that masoprocol may be stimulating a serine/threonine phosphatase via a second messenger pathway and may be causing dephosphorylation of HSL. Furthermore, the current in vivo and in vitro data suggest that masoprocol regulates HSL activity by a dual mechanism: a short-term effect on the phosphorylation state of HSL and a long-term effect on the HSL protein content.

Although a well-known lipoxygenase inhibitor, the profound metabolic effects of masoprocol only recently became apparent (8, 14, 26). The possibility that these effects may not be mediated by the lipoxygenase pathway must be considered, given the observation that esculetin, another lipoxygenase inhibitor, had no antilipolytic activity. In any event, the results of the present experiments confirm the ability of masoprocol to inhibit lipolysis in vitro (8), demonstrate that similar changes are seen after administration of masoprocol to rats with fructose-induced hypertriglyceridemia, and provide substantiative evidence that the metabolic effects of masoprocol are secondary to its ability to decrease HSL activity. Finally, these data, coupled with previous results (14, 26) showing that masoprocol can enhance insulin sensitivity and lower plasma FFA, glucose, and TG concentrations in hyperglycemic and/or hypertriglyceridemic rats, suggest that this may be a future therapeutic target of great interest.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46942 and DK-49705 and by the Office of Research and Development, Medical Research Service, Dept. of Veterans Affairs.


    FOOTNOTES

Address for reprint requests and other correspondence: G. M. Reaven, 213 E. Grand Ave., South San Francisco, CA 94080.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 19 July 1999; accepted in final form 13 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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