0013-7227/92/1304-2310%03.00/0 Endocrinology Copyright 0 1992 hy The Endocrine

Vol. 130, No. 4 Printed in U.S.A.

Society

The Regulation of Lipoprotein by Dexamethasone in Isolated JOHN

Lipase Gene Expression Rat Adipocytes”

M. ONG, ROSA B. SIMSOLO, BAHMAN SAFFARI, AND PHILIP Department of Medicine/Division of Endocrinology, Cedars-SinaiMedical Center,

A. KERN

LAXAngeles,California 90048

ABSTRACT. Lipoprotein lipaae (LPL) is an enzyme found in adipose tissue that is important in the hydrolysis of triglyceriderich lipoproteins, and in the uptake of FFA lipid into the adipocyte. To examine the effects of glucocorticoids on adipose tissue LPL, male Sprague-Dawley rats were injected with dexamethasone (1 mg/kg) every other day for 10 days, followed by measurement of LPL in epididymal adipose tissue. Compared to sham-injected controls, heparin-releasable LPL activity and LPL mass in the dexamethasone-treated rata were 44% and 62% of those in control rats, respectively. Adipocytes were prepared from the fat pads and pulse labeled with [asS]methionine, demonstrating a -decrease in the LPL synthetic rate in the treated rata to 57% of the rate in control rata. In addition, LPL mRNA was quantitated by Northern blotting, demonstrating a decrease in LPL mRNA in the dexamethasone-treated rats. A simultaneous decrease in the message for y-actin was also noted. To examine the effects of dexamethasone on LPL in vitro, adipocytes were prepared from normal rats and treated with dexamethasone for 24 h in uitro. Dexamethasone decreased

L

IPOPROTEIN triglyceride is taken up by tissues through the action of lipoprotein lipase (LPL) (1). In fat tissue, LPL is synthesized and secreted by the adipocyte and then transported to the capillary endothelium, where triglyceride hydrolysis into FFA takes place. Although adipocytes are capable of synthesizing FFA from glucose, fat cells obtain most of the lipid needed for triglyceride synthesis from FFA generated by LPL. Because of this adipose tissue lipid storage function, LPL may play a role in the development of obesity, and much research has been devoted to the regulation of LPL by hormones and other physiological events. Among the hormones known to regulate LPL activity are glucocorticoids. In studies in rats, changes in LPL have been shown to occur in parallel with circadian

heparin-releasable LPL activity in cultured adipocytes to 40 + 6% of the control value (P < 0.01). This decrease in LPL activity was accompanied by a decrease in the LPL synthetic rate using [Yqlmethionine labeling, to 33% of the control value, and no specific change in LPL turnover or secretion. In addition, dexamethasone added to adipocytes decreased LPL mRNA levels. Because the combination of insulin plus dexamethasone has been shown to yield synergistic increases in LPL in adipose tissue pieces, insulin was added to isolated adipocytes in combination with dexamethasone. Whereas insulin and dexamethasone individually had opposite effects on LPL, the combination of insulin plus dexamethasone resulted in no change in any aspect of LPL gene expression. Thus, dexamethasone resulted in a decrease in adipocyte LPL mRNA levels both when added to cultured adipocytes in vitro as well as when injected into rats. This decreased LPL mRNA level yielded corresponding changes in the LPL synthetic rate and LPL activity. (Endocrinology 130: 2310-2316,1992)

changes in serum corticosterone levels (2), while other studies demonstrated decreases in LPL activity several days after adrenalectomy (3,4). The effects of glucocorticoid injections on LPL have been variable. A single large injection of dexamethasone yielded an increase in adipose LPL within 2 h in one study (4), and prolonged injections of synthetic ACTH demonstrated no change in adipose LPL (3). In contrast, other studies demonstrated decreases in LPL activity after 3 days to 4 weeks of glucocorticoid injections in rats (5, 6). Using adipose tissue pieces, in vitro studies have examined the effects of dexamethasone alone and in combination with insulin. In both rat and human adipose tissue, dexamethasone alone had little or no effect on LPL, but the combination of dexamethasone plus insulin resulted in a synergistic increase in LPL activity (7-9), suggesting a potentiation of the effect of insulin on LPL. Although one study suggested that dexamethasone increased LPL synthesis (lo), the other studies mainly examined LPL catalytic activity and did not determine the mechanism of these hormonal effects on LPL. In this study the effects of the glucocorticoid dexa-

Received September 17,199l. Address all correspondence and requests for reprints to: Philip A. Kern, M.D., Division of Endocrinology, Becker 131, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90046. * This work was supported by a Grant-in-Aid from the American Heart Association, a Career Development Award from the Juvenile Diabetes Foundation, and NIH Grant DK-39176. This work was performed during the tenure of an Established Investigatorship from the American Heart Association. 2310

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REGULATION methasone incubation

injection

on LPL were examined

and in vitro studies

demonstrated

activity

Both the in uiuo

that dexamethasone

due to a decrease in the level of

Materials Treatment

in rat adipocytes after in vitro as well as after

with dexamethasone of rats with dexamethasone.

decreased LPL LPL mRNA.

OF LPL BY DEXAMETHASONE

and Methods

of rats and preparation

of

adipocytes

Male Sprague-Dawley rats, weighing 180-200 g, were purchased from Harland Laboratories (Gilmore, CA). Isolated rat adipocytes were prepared using a collagenase digestion of epididymal fat pads, as described previously (11). Cells were cultured for 24 h in medium 199 (Irvine Scientific, Santa Ana, CA) without serum in the presence or absence of the indicated

concentrations of dexamethasone. Additional experiments were performed with rata treated with dexamethasone in Go. Rats

were injected ip with either 1 mg/kg dexamethasone or saline vehicle every other day for 10 days, after which the rats were killed, and LPL was measured, as described below, in the epididymal fat pads. Previous studies with dexamethasoneinjected rats demonstrated no change in plasma glucose, but increased plasma insulin, suggesting insulin resistance (12). Measurement

of

LPL actiuity

For the measurement of LPL from whole epididymal adipose tissue, fat pieces were incubated in PBS containing 13 pg/ml heparin (Fisher Scientific Co., Pittsburgh, PA) for 30 min at 37 C. An aliquot of this buffer was then assayed as described below and is hereafter referred to as heparin-releasable (HR) LPL. The tissue was then washed, and the extractable (EXT) fraction was prepared by homogenizing the tissue in detergentcontaining buffer, as described previously (13). LPL activity was measured in the aqueous layer after centrifugation, and cell number was determined according to the method of DiGirolamo et al. (14). For isolated adipocytes, LPL activity was measured using methods previously described (15), except cells were first cultured for 24 h in the presence or absence of dexamethasone. LPL activity was determined using an emulsified [3H]triolein-containing substrate with normal human serum as a source of apolipoproteinCII(16). After incubating sample with substrate for 45 min at 37 C, the reaction was stopped and liberated [3H]FFA were measured, as described previously (17). Activity was expressed as nanoequivalents of FFA released per min/106 cells. LPL immunoreactive mass The LPL immunoreactive mass was measured using an enzyme-linked immunosorbent assay, described and characterized previously (18). Affinity-purified anti-LPL antibodies are used to detect LPL in this sandwich enzyme-linked immunosorbent assay, and purified bovine LPL is used as a standard. Samples for LPL immunoreactive mass were prepared as described above for LPL activity, except for the presence of protease inhibitors in all buffers.

2311

Pulse hbeling and immunoprecipitation

LPL synthesis and processing were studied as described previously (11, 15). After overnight culture, adipocytes were labeled with [35S]methionine for 30 min in methionine-free medium 199. Cells were then lysed and immunoprecipitated with affinity-purified anti-LPL antibody, as described previously (ll), followed by analysis on a 10% polyacrylamidesodium dodecyl sulfate gel. Previous studies have demonstrated that [35S]methionine incorporation into LPL and total protein is linear for up to 90 min, and there is no detectable secretion of labeled LPL for the first 60 min (11). To be sure that changes in methionine pool size were not altering [35S]methionine in-

corporation into LPL, the total trichloroacetic

acid (TCA)-

precipitable counts were determined in each labeled cell extract,

and the gels were loaded with equal TCA counts. However, dexamethasone treatment did not alter total [35S]methionine incorporation into protein in any consistent manner, suggesting no overall effect on protein synthesis. To study LPL processing

and secretion, pulse-chase experiments were performed. Cells were labeled for 30 min, as described above, and then chased with medium containing 30 mg/liter methionine (and no [35S]

methionine) for the times indicated in the presence of 13 c(g/ ml heparin (11). RNA extraction and Northern analysis RNA was extracted using the method of Chomczynski and Sacchi (19). Equal amounts of total RNA were resolved on a

2.2 M formaldehyde-l%

agarose gel, transferred to nylon mem-

brane, and blotted with the 32P-labeled cDNA probes for human LPL (20) and y-actin (21), as described previously (15). In some experiments total RNA was applied to a slot blot manifold and blotted with a 32P-labeled poly(dT) probe to detect poly(A) RNA.

Results To study the effects of glucocorticoids on adipose tissue LPL, rats were injected with dexamethasone every other day for 10 days, as described in Materials and Methods, followed by measurement of LPL activity and immunoreactive mass in the fat pads. As shown in Fig. 1, LPL activity and LPL mass were significantly lower in adipose tissue from dexamethasone-treated rats. HR activity and mass were reduced to 44% and 62% of those in control rats, and EXT activity and mass were reduced to 61% and 68% of control values, respectively. To examine LPL synthesis and processing, isolated adipocytes were prepared from the fat pads of the control and dexamethasone-treated rats, and cells were pulselabeled with [35S]methionine, and then chased with medium containing unlabeled methionine, as described in Materials and Methods. As shown in Fig. 2, the LPL synthetic rate (time zero, which represents the end of the pulse or the beginning of the chase) was decreased in cells from the dexamethasone-treated rats. Using laser densitometry to quantitate this and other experiments,

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REGULATION

2312

HR

n Control

OF LPL BY DEXAMETHASONE

EXT

q

DEX-Treated

* 0

The regulation of lipoprotein lipase gene expression by dexamethasone in isolated rat adipocytes.

Lipoprotein lipase (LPL) is an enzyme found in adipose tissue that is important in the hydrolysis of triglyceride rich lipoproteins, and in the uptake...
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