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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 atPreparation 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.
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) -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
-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
[-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). [
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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).
|
|
|
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 · 105
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.
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anthonsen, MW,
Ronnstrand L,
Wernstedt C,
Degerman E,
and
Holm C.
Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro.
J Biol Chem
273:
215-221,
1998
2.
Carey, GB.
Mechanisms regulating adipocyte lipolysis.
Adv Exp Med Biol
441:
157-170,
1998[ISI][Medline].
3.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
4.
Cohen, P.
The structure and regulation of protein phosphatases.
Annu Rev Biochem
58:
453-508,
1989[ISI][Medline].
5.
Cohen, P.
Signal integration at the level of protein kinases, protein phosphatases and their substrates.
TIBS
17:
408-314,
1992[Medline].
6.
Cohen, P,
Holmes CFB,
and
Tsukitani Y.
Okadaic acid: a new probe for the study of cellular regulation.
TIBS
15:
98-102,
1990[Medline].
7.
Degerman, E,
Landstrgm TR,
Wijkander J,
Holst LS,
Ahmad F,
Belfrage P,
and
Manganiello V.
Phosphorylation and activation of hormone-sensitive adipocyte phosphodiesterase type 3B.
Methods
14:
43-53,
1998[ISI][Medline].
8.
Gowri, MS,
Reaven GM,
and
Azhar S.
Effect of masoprocol on glucose transport and lipolysis by isolated rat adipocytes.
Metabolism
48:
411-414,
1999[ISI][Medline].
9.
Haystead, TAJ,
Weiel JE,
Lichfield DW,
Tsukitani Y,
Fischer EH,
and
Krebs EG.
Okadaic acid mimics the action of insulin in stimulating protein kinase activity in isolated adipocytes: the role of protein phosphatase 2A in attenuation of the signal.
J Biol Chem
265:
16571-16580,
1990
10.
Kraemer, FB,
Patel S,
Saedi MS,
and
Sztalryd C.
Detection of hormone sensitive lipase in various tissues. I. Expression of an HSL/bacterial fusion protein and generation of anti-HSL antibodies.
J Lipid Res
34:
663-671,
1993[Abstract].
11.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
12.
Langin, D,
Holm C,
and
Lafontan M.
Adipocyte hormone-sensitive lipase: a major regulator of lipid metabolism.
Proc Natl Acad Sci USA
55:
93-109,
1996.
13.
Louveau, I,
Chaudhri S,
and
Etherton TD.
An improved method for isolating RNA from porcine adipose tissue.
Anal Biochem
196:
308-310,
1991[ISI][Medline].
14.
Luo, J,
Chuang T,
Cheung J,
Quan J,
Tsai J,
Sullivan C,
Hector RF,
Reed MJ,
Meszaros K,
King SR,
Carlson TJ,
and
Reaven GM.
Masoprocol (nordihydroguaiaretic acid): a new antihyperglycemic agent isolated from the creosote bush (Larrea tridentata).
Eur J Pharmacol
346:
77-79,
1998[ISI][Medline].
15.
McGowan, MW,
Artiss JD,
Strandbergh DR,
and
Zak B.
A peroxidase-coupled method for the calorimetric determination of serum triglycerides (Abstract).
Clin Chem
29:
538,
1983
16.
Medicherla, S,
Azhar S,
Cooper A,
and
Reaven E.
Regulation of cholesterol responsive genes in ovary cells: impact of cholesterol delivery systems.
Biochemistry
35:
6243-6250,
1996[ISI][Medline].
17.
Nakamura, K,
Inoue Y,
Watanabe N,
and
Tomita T.
Studies on cholesterol esterase in rat adipose tissue: comparison of substrates and regulation of the activity.
Biochim Biophys Acta
963:
320-328,
1988[ISI][Medline].
18.
Neichi, T,
Koshihara Y,
and
Murota S-I.
Inhibitory effect of Esculetin on 5-lipoxygenase and leukotriene biosynthesis.
Biochim Biophys Acta
753:
130-132,
1983[ISI][Medline].
19.
Okada, T,
Kawano Y,
Sakakibara T,
Hazeki O,
and
Ui M.
Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes: studies with a selective inhibitor wortmannin.
J Biol Chem
269:
3568-3573,
1994
20.
Oscai, LB,
Essig DA,
and
Palmer WK.
Lipase regulation of muscle triglyceride hydrolysis.
J Appl Physiol
69:
1571-1577,
1990
21.
Papadogiannakis, N,
and
Barbieri B.
Lipoxygenase inhibitors counteract protein kinase C mediated events in human T lymphocyte proliferation.
Int J Immunopharmacol
19:
263-275,
1997[ISI][Medline].
22.
Peterson, GL.
A simplification of the protein assay method of Lowry et al. which is more generally applicable.
Anal Biochem
83:
346-356,
1977[ISI][Medline].
23.
Pinter, JK,
Hayashi JA,
and
Watson JA.
Enzymatic assay of glycerol, dihydroxyacetone and glyceraldehyde (Abstract).
Arch Biochem Biophys
121:
404,
1967[ISI][Medline].
24.
Reaven, GM,
Chang H,
Hoffman BB,
and
Azhar S.
Resistance to insulin-stimulated glucose uptake in adipocytes isolated from spontaneously hypertensive rats.
Diabetes
38:
1155-1160,
1989[Abstract].
25.
Reaven, E,
Tsai L,
Spicher M,
Shilo L,
Philip M,
Cooper AD,
and
Azhar S.
Enhanced expression of granulosa cell low density lipoprotein receptor activity in response to in vitro culture conditions.
J Cell Physiol
161:
449-462,
1994[ISI][Medline].
26.
Reed, MJ,
Meszaros K,
Entes LJ,
Claypool MD,
Pinkett JG,
Brignetti D,
Luo J,
Khandwala A,
and
Reaven GM.
Effects of masoprocol on carbohydrate and lipid metabolism in a rat model of type II diabetes.
Diabetologia
42:
102-106,
1999[ISI][Medline].
27.
Rodbell, M.
Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis.
J Biol Chem
239:
375-380,
1964
28.
Scribner, KA,
Gadbois TM,
and
Reaven GM.
Masoprocol lowers serum triglyceride concentrations in rats with fructose-induced hypertriglyceridemia (Abstract).
J Investig Med
46:
130,
1998.
29.
Shaen, WJ,
Patel S,
Natu V,
and
Kraemer FB.
Mutational analysis of structural features of rat hormone-sensitive lipase.
Biochemistry
37:
8973-8979,
1998[ISI][Medline].
30.
Shenolikar, SS,
and
Nairn AC.
Protein phosphatases: recent progress.
Adv Cyc Nucl Pro Phos Res
23:
1-121,
1990.
31.
Small, CA,
Garton AJ,
and
Yeaman SJ.
The presence and role of hormone-sensitive lipase in heart muscle.
Biochem J
258:
67-72,
1989[ISI][Medline].
32.
Stralfors, P,
Olsson H,
and
Belfrage P.
Hormone sensitive lipase.
In: The Enzymes, edited by Boyer PD,
and Krebs EG.. Orlando, FL: Academic, 1987, vol. 18, p. 147-177.
33.
Trinder, P.
Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor (Abstract).
Ann Clin Biochem
6:
24,
1969.
34.
Yasumoto, K,
Yamamoto A,
and
Mitsuda H.
Effect of phenolic antioxidants on lipoxygenase reaction.
Agric Biol Chem
34:
1162-1168,
1970[ISI].