Effects of Dexamethasone Administration on Hepatic Glycogen

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ABSTRACT. Adrenalectomized 24-h fasted rats lack the abil- ity to synthesize and accumulate hepatic glycogen. In addition, the ability of liver glycogen synthase ...
0013-7227/84/1152-0625$02.00/0 Endocrinology Copyright© 1984 by The Endocrine Society

Vol. 115, No. 2 Printed in U.S.A.

Effects of Dexamethasone Administration on Hepatic Glycogen Synthesis and Accumulation in Adrenalectomized Fasted Rats* RONALD N. MARGOLIS, AND RANDALL T. CURNOW Departments of Anatomy and Oncology (R.N.M.), Howard University, College of Medicine, Washington, D. C. 20059; and Departments of Internal Medicine and Pharmacology (R.T.C.), University of Virginia, School of Medicine, Charhttesville, Virginia 22908

ABSTRACT. Adrenalectomized 24-h fasted rats lack the ability to synthesize and accumulate hepatic glycogen. In addition, the ability of liver glycogen synthase to respond to acute glucose administration is muted. The defect has been localized to the level of smooth endoplasmic reticulum-associated synthase phosphatase activity which is greatly reduced in adrenalectomized fasted rat liver. In vivo exposure to dexamethasone increases hepatic glucose output and hepatic glycogen synthesis

and accumulation by 2-6 h after administration. Smooth endoplasmic reticulum synthase phosphatase activity is increased, and activation of glycogen synthase is restored. Ambient insulin concentrations are increased by steroid administration and appear to have a role in restoration of the ability to activate glycogen synthase, and consequently to restore the ability to synthesize and accumulate hepatic glycogen. {Endocrinology 115:625-629,1984)

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regulation of glycogen metabolism and the role of synthase phosphatase activity in this regulation are not known. Consequently, the present study was designed to investigate the effects of in vivo administration of glucocorticoids to ADX starved rats on hepatic glycogen synthesis and accumulation.

N ADRENALECTOMIZED (ADX) fasted rats activation of the key rate-limiting enzyme of glycogen synthesis, glycogen synthase (UDP-glucose; glucan 4-aD-glucosyltransferase, EC 2.4.1.11) is reduced, with the defect in ADX fasted liver apparently localized to the level of the synthase activating enzyme, synthase phosphatase [(UDP-glucose-a-glucan glucosyltransferase b) phosphohydrolase, EC 3.1.3.42)] (1-4). We have previously presented evidence to suggest that compartmentalization of synthase phosphatase activity with the smooth endoplasmic reticulum (SER) is an important factor in the regulation of hepatic glycogen metabolism (4, 5). Specifically, both the amount of SER and the activity of SER-associated enzymes are subject to regulation by hormonal and metabolic factors. Thus, simple refeeding of ADX fasted rats in the absence of glucocorticoids increases SER-synthase phosphatase activity and enhances hepatic glycogen synthesis and accumulation (4). Glucocorticoid administration, alone, also results in proliferation of SER (6). In spite of much current knowledge the precise mechanisms of long and short term

Materials and Methods Fifty six adult male Sprague-Dawley rats were obtained and housed in wire-mesh cages under a 12-h light, 12-h dark regimen with ad libitum access to water and food. Rats were adrenalectomized via the dorsal approach under ether anesthesia, were housed singly, and received a 0.9% NaCl solution as drinking water. All experiments with ADX rats were performed 7-14 days after surgery. Both intact and ADX rats were divided into two groups, one of which was fed. The second group was fasted for 24 h before each experiment. Intact fed, ADX fed, intact fasted 24-h, and ADX fasted 24-h rats were killed between the hours of 0800-1000 h. In addition, ADX fasted rats received 2 mg dexamethasone (DEX), ip, and were killed after 2 h or 6 h of DEX administration. Rats were killed under pentobarbital anesthesia (50 mg/kg) by making a Ushaped abdominal incision. Blood was immediately withdrawn from the inferior vena cava and placed on ice in a solution containing 5 mM EDTA and 50 mM benzamidine. Liver samples were rapidly frozen between aluminum clamps cooled to the temperature of liquid nitrogen and placed in vials and stored at -80 C. Blood samples were centrifuged in the cold to obtain plasma. Insulin and glucose concentrations were determined by RIA and the glucose oxidase method, respectively, in the Uni-

Received December 30,1983. Address requests for reprints to: Ronald N. Margolis, Ph.D., Department of Anatomy, Howard University, College of Medicine, 520 W Street, Northwest, Washington, D. C. 20059. * Supported by NIH Grants R01-AM 20787, AM-22125, RCDA AM-00471, and 5S07 RR-05361, and by grants from the Washington D. C. Affiliate of the American Diabetes Association and the Kroc Foundation.

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SHORT TERM REGULATION OF GLYCOGEN METABOLISM

versity of Virginia Endocrine Core Lab. Glycogen concentrations in aliquots of liver were determined as acid-hydrolyzed glucose by the method of Dubois et al. (7). Glycogen synthase was assayed in 8000 X g extracts by the filter paper method of Thomas et al. (8), as described in previous publications (4, 9). Glycogen phosphorylase activity was assayed in the 8000 X g extract by the low-high substrate method of Tan and Nuttall (10), as described (4, 9). Glycogen synthase phosphatase activity was assayed in a two-stage assay using partially purified dog skeletal muscle glycogen synthase b as substrate (4, 5, 9). This assay measured the enzymatic conversion of synthase from the inactive (b) form to the active (a) form. Synthase b was prepared from dog skeletal muscle by the method of Takeda et al. (11). The synthase phosphatase activity measured both in soluble and SER fractions derived from the 8000 X g extract is presented as units (/imol/min • ml)/g wet wt after correction for tissue glycogen content (12), with 1 U equal to 1 fimol synthase b converted to synthase a per min. Phosphorylase phosphatase activity was assayed in analogous fashion, with exogenous dialyzed rabbit skeletal muscle phosphorylase a as substrate. In this case, aliquots were removed from the phosphorylase phosphatase reaction tubes at various times and placed in phosphorylase a tubes. Thus, enzymatic conversion with time of phosphorylase a (active) to 6 (inactive) forms was measured. Phosphorylase phosphatase activity is presented as units per g wet wt, with 1 U equal to 1 /imol phosphorylase a converted to b per min/ml. All enzyme assays were carried out at 30 C, and were linear with respect to time, temperature, substrate, and enzyme concentrations. Liver samples were subfractionated by the method of Dallner et al (13), as described by Margolis et al. (4-6, 9). The soluble fraction consisted of the high speed supernatant remaining above the microsomal fractions. Characterization of the contents and composition of subfractions obtained via this procedure is described in previous publications in this series (5, 14). Protein concentrations were determined by the method of Lowry et al. (15). Significance of differences between means was analyzed using Student's t test and analysis of variance with multiple range test, where appropriate.

Results A fasting mediated decrease in plasma insulin concentration was observed in both intact and ADX rats (Table 1). Plasma glucose concentrations were also diminished by fasting, less so in intact animals fasted for 24 h than for ADX rats fasted for 24 h (93 mg/dl vs. 63 mg/dl). ADX, per se, resulted in decreased amounts of liver glycogen (20.8 mg/g wet wt vs. 9.9 mg/g wet wt, P < 0.01, for intact vs. ADX fed rats), and after a 24-h fast hepatic glycogen was reduced to barely detectable levels in ADX rats (Table 1). Administration of the synthetic glucocorticoid DEX to ADX fasted rats resulted in increased plasma insulin levels and increased plasma glucose concentrations after both 2 and 6 h of hormone administration. Hepatic glycogen levels were also increased in ADX fasted rats by DEX administration, increasing from 1

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TABLE 1. Hormonal and metabolite parameters

Normal fed Normal fast 24 n ADX fed ADX fast 24 h ADX fast 24 h + DEX 2 h ADX fast 24 h + DEX 6 h

Glucose (mg/dl)

Glycogen (mg/g wet wt)

Insulin 0iU/ml) 30 (6) ± 4 11 (3) ± 3°

130 (6) ± 8 20.8 (6) ±3.4 93 (3) ± 3 " 3.4 (3) ±0.8°

25 (6) ± 4 3 (10) ± 0.5c 11 (11) ± 4d-e

114 (6) ± 5 63 (18) ± 4C 101 (11) ± 6e

9.9 (6) ±0.5 1.0 (18) ± 0.2c 3.0 (11) ± 0.7c-e

11 (12) ± 2d-e

136 (12) ± 8e

6.9 (12) ± 1.1**

Values expressed are means ± SEM of (N) determinations. P< 0.01 vs. Normal fed. b P < 0.05 us. Normal fed.

a

c

P