0013-7227/79/1046-1722$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society

Vol. 104, No. 6 Printed in U.S.A.

Effects of Glucocorticoids on Microsomal Membrane Synthesis in Hepatocytes from Adrenalectomized Rats* RONALD N. MARGOLIS, SANFORD A. GARFIELD, AND ROBERT R. CARDELL, JR. Department of Anatomy, University of Virginia School of Medicine, Charlottesville, Virginia 22901

ABSTRACT. Biochemical and morphological studies were performed on livers from normal, adrenalectomized (ADX), and ADX and dexamethasone (DEX)-treated rats to investigate the effects of glucocorticoids on microsomal membrane synthesis. Overnight fasted normal, ADX, and ADX rats treated 2 or 4 h with DEX received [3H]leucine and [I4C]glycerol. Livers were removed, and tissue specimens were prepared for electron microscopy and tissue fractionation. Liver microsomal subtractions were prepared and subsequently washed to produce rough and smooth microsomal membranes. Radioactivity and membrane composition were determined, and glucose-6-phosphatase activity was measured in washed microsomal membranes. Adrenalectomy caused decreased microsomal membrane synthesis. Two and 4 h of DEX administration restored microsomal membrane

W

ORK in our laboratory over the past several years (1, 2) has shown that hepatocytes from overnight fasted adrenalectomized (ADX) rats display alterations in the form and amount of smooth endoplasmic reticulum (SER) when compared to liver cells from overnight fasted normal rats. Hepatic SER in the ADX animals is highly vesiculated and the vesicles often exhibit occasional ribosomes, whereas in normal rats, the SER is tubular and devoid of ribosomes. Moreover, the amount of hepatic SER is decreased after ADX. No glycogen granules are found in hepatocytes from overnight fasted ADX rats, while they are abundant in liver cells of normal rats fasted for a similar period of time. Glucocorticoid administration to ADX rats restores the levels and form of SER in hepatocytes to almost normal. The hormone also causes rapid deposition of glycogen in the cytosome of hepatocytes. Since the SER shows a dramatic response after glucocorticoid administration, it is reasonable to search for a possible role for this cell organelle in mediating the action of the hormone on hepatic functions. One function generally recognized for the SER is participation in varReceived August 24, 1978. Address all correspondence and requests for reprints to: Dr. Ronald N. Margolis, Department of Anatomy, University of Virginia School of Medicine, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22901. * This work was supported by Grants AM-11854 and 21253 and in part by the University of Virginia Diabetes Research and Training Center (AM-22125 from the USPHS).

synthesis to normal levels. ADX also caused an alteration in composition of the microsomal membranes (reflected in decreased phospholipid:protein ratios), which was restored to normal levels by 4 h after DEX administration. The earliest effects of the hormone on membrane synthesis were observed in smooth microsomes as part of a smooth endoplasmic reticulum (SER) proliferation. These findings were supported by observations made with the electron microscope. The proliferating SER was enriched in at least one component: glucose-6-phosphatase. Although the specific relationship of SER to glucocorticoid action remains unclear, the interpretation is offered that SER proliferation and alteration in glucose-6-phosphatase distribution are component parts of the total response of the hepatocyte to glucocorticoids. (Endocrinology 104: 1722, 1979)

ious metabolic reactions. Several examples may be cited: 1) microsomal enzymes required for triglyceride synthesis in the intestinal absorptive cell (3), 2) drug-metabolizing enzymes in hepatocytes (4, 5), 3) anesthetic metabolism in liver (6), and 4) glucose-6-phosphatase (G-6-Pase; E.C. 3.1.3.9) development in livers of neonatal rats (7, 8). In the examples cited above, membranes of the SER provide a site for the location of enzymes which participate in specific metabolic reactions. In some cases, it has been shown that an inducer substance {e.g. drug) causes a proliferation of SER membranes and, concurrently, the synthesis of new enzyme molecules (5, 9). To study this problem further, we have investigated the effects of glucocorticoid administration on hepatic microsomal membrane synthesis. Accordingly, radiolabeled protein and lipid precursors were injected into overnight fasted normal, ADX, and ADX and hormonetreated rats; incorporation of these precursors into microsomal membrane proteins and lipids was measured. In addition, a specific microsomal marker enzyme, G-6Pase, was studied to determine the effects of the glucocorticoid on this key gluconeogenic enzyme. The results indicate that microsomal membrane synthesis is less in hepatocytes from ADX animals than that of microsomes from normal rats. Administration of glucocorticoid to ADX animals for 2 or 4 h results in the stimulation of the synthesis of both protein and lipid components of microsomal membranes, with the more 1722

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pronounced effect observed in SER membranes. The hormone also causes an apparent shift of the G-6-Pase enzyme from rough endoplasmic reticulum (RER) to SER. We conclude from this study that glucocorticoid administration causes synthesis of endoplasmic reticulum (ER) membranes, and the new SER membranes are enriched in at least one gluconeogenic enzyme, G-6-Pase.

Materials and Methods Animals Young adult male rats of the Wistar strain (100-150 g) were ADX under ether anesthesia 4-8 days before each experiment. ADX animals were provided with 0.9% saline as drinking water, maintained on a 12-h light, 12-h dark cycle, and allowed to eat ad libitum. Normal rats which served as controls were housed as above but were provided with tap water. Animals were fasted for 20 h and were divided into the following groups: 1) 36 normal rats, 2) 28 ADX rats which received no further treatment, 3) 30 ADX. rats injected ip with dexamethasone (DEX; 2 mg/100 g BW) 2 h before sacrifice, and 4) 30 ADX rats given DEX (2 mg/100 g BW) 4 h before sacrifice.

FIG. 1. Electron micrograph of section taken from high speed pellet of rough microsomes. Note ribosomes bound to outer (cytoplasmic) surface of vesicles derived from RER (X58.400). See text for details.

Subcellular fractionation and preparation of microsomal membranes Animals were sacrificed by decapitation. Their livers were carefully removed to avoid excessive connective tissue contamination, cut into pieces of appropriate size, and placed in cold 0.25 M sucrose. The liver specimens were blotted dry, weighed, frozen in either dry ice-acetone or liquid nitrogen, and stored in vials at —80 C for up to 3 weeks. Samples were thawed and homogenized in cold 0.25 M sucrose to make a 20% (wt/vol) homogenate. The homogenate was fractionated by a modified Dallner (10) procedure to produce rough and smooth microsomes. Briefly, the homogenate was centrifuged twice at 10,000 x g for 20 min, which caused mitochondria, lysosomes, and other cellular debris to form a pellet. The postmitochondrial supernatant was collected and brought to 15 mM CsCl by adding sufficient 1 M CsCl. The postmitochondrial supernatant was layered on top of 15 ml 1.3 M sucrose-15 mM CsCl in an SW 27 centrifuge tube and centrifuged at 105,000 X g for 4 h in a Beckman L5-50 centrifuge (Beckman Instrument Co., Palo Alto, CA). A band, which consisted primarily of smooth rnicrosomes, appeared at the 0.25-1.3 M sucrose interface. A pellet of rough microsomes formed beneath the 1.3 M sucrose. The smooth microsomal band was drawn off, diluted with distilled water, and centrifuged in an SW 41 rotor at 225,000 X g for 1 h. The rough microsomal pellet was resuspended in 0.25 M sucrose and centrifuged at 225,000 X g for 30 min. Morphological and biochemical analyses indicated that the fractions were greater than 95% pure, i.e. the pellet consisted primarily of RER (Fig. 1) and the band consisted primarily of SER (Fig. 2). Adsorbed protein, vesicular contents, and ribosomes were removed from the microsomes by washing in high ionic strength-high pH buffer (0.15 M Tris, pH 8.0), sonicating in distilled water, and washing in the high ionic strength-high pH buffer (4, 11-13).

FIG. 2. Electron micrograph of section taken from high speed pellet of smooth microsomes. Smooth-surfaced vesicles are predominantly derived from SER (X58.400). See text for details.

Electron microscopy Samples of liver were prepared for electron microscopy by methods routinely used in this laboratory (2), which involved exposing the left lateral lobe of the liver, rapidly removing a sample of tissue, and placing it in a drop of 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3). The tissue was cut into small pieces about 1 mm3 in size and placed in a vial containing the glutaraldehyde fixative. After 2 h of fixation at room temperature, the tissue was rinsed in cacodylate buffer (0.1 M, with 10% sucrose) and postfixed in 1% osmium tetroxide (in 0.1 M phosphate buffer). The tissue was then dehydrated in a graded series of alcohol and embedded in Epon (14). Ultrathin sections were stained with uranyl acetate and lead citrate (15, 16) and examined in a Phillips EM-300 electron microscope (Philips Electronic Instruments, Inc., Mount Vernon, NY). Labeled precursors [3H]Leucine (10 juCi/100 g) and [I4C]glycerol (10 juCi/100 g) were used to measure protein and lipid synthesis, respectively. These precursors rapidly enter hepatocytes after an ip injection

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and immediately enter the protein and lipid precursor pools within the cell (17). Glycerol is quickly phosphorylated in preparation for phospholipid (PLP) synthesis (17). Although exchange of [I4C[glycerol-labeled PLPs occurs between the subcellular fractions during the fractionation procedures, this exchange appears to be minimal (18). Animals from each experimental group were injected ip with labeled precursors, and the rats were killed at intervals of 5,10, 15, 30, 60, and 120 min after injection of isotope. Aliquots of washed microsomes were placed in cold 10% trichloroacetic acid (TCA), and the acid-insoluble material was precipitated onto nitrocellulose filters (pore size, 0.45 /xm). The filters were washed sequentially with 10% TCA, 5% TCA, and 95% ethanol, dried under a tungsten lamp, and placed in a scintillation vial containing 10 ml Filter-Solv (Beckman). Samples were counted in a Beckman LS-8000 scintillation counter and corrected for channel spillover and quenching using external standardization and automatic quench compensation. Chemical and enzymatic determinations Protein was determined by the method of Lowry et al. (19) using bovine serum albumin as a standard. PLP was extracted by the method of Folch et al. (20), and PLP-phosphorus was determined by the methods of Bartlett (21) and Fiske and SubbaRow (22). RNA was measured by the orcinol reaction (23). G-6-Pase was assayed by a modified method of Freedland and Harper (24) using 0.06 M glucose-6-phosphate as a substrate. Statistics Student's t test was used to determine the significance of differences between means. Values of P > 0.05 were deemed NS.

Endo • 1979 Vol 104 • No 6

and each cell showed increased quantities of glycogen (Fig. 5). Often hepatocytes showed several glycogen areas within the cytosome. In centrilobular hepatocytes, glycogen granules were dispersed throughout the glycogen areas and SER was abundant between the glycogen particles. In periportal cells, the glycogen accumulated in dense masses, with the tubules and vesicles of SER restricted to the edges of the glycogen masses. [zH]Leucine incorporation into microsomal membranes [3H]Leucine was rapidly incorporated into membrane proteins of both rough and smooth microsomes (Fig. 6). The rate of incorporation was linear for the first 15 min in both membrane fractions and then declined by 30 min to a plateau level observed for the duration of the experiment (120 min). After adrenalectomy, the rate and level of incorporation of [3H]leucine into both rough and smooth microsomal membranes was decreased to 52% and 44% of normal, respectively (P < 0.02 and 0.05; Fig. 6). After 2 h of hormone administration, there was a slight increase in 3H-labeled protein in rough microsomal membranes (17% increase; P = NS) and a marked increase in incorporation in smooth microsomes (125%; P < 0.05) when compared with incorporation into microsomal membranes from ADX rats not treated with glucocorticoid. After 4 h of hormone administration, the pattern of 3H incorporation was similar to that after 2 h. Again, incorporation of labeled protein was greatest in smooth microsomal membranes, with peak incorporation occurring 30 min after injection of the label.

Results Electron microscopy

f14CJGlycerol incorporation into microsomal membranes

Hepatocytes from normal overnight fasted rats showed sparse amounts of glycogen and moderate amounts of SER (1), with more SER in centrilobular than in periportal hepatocytes. In addition, regular parallel stacks of RER were more evident in centrilobular hepatocytes (1). In contrast, hepatocytes from overnight fasted ADX animals exhibited no glycogen granules and a severely diminished SER content (Fig. 3) (2). The RER appeared less often as stacks of parallel cisternae, and frequently the cisternae of RER were dilated, with fewer ribosomes appearing on the membranes. By 2 h after a single administration of DEX to a fasted ADX animal, glycogen granules appeared in the cytosome of many, but not all, hepatocytes (Fig. 4). Associated with the newly deposited glycogen granules were tubules and vesicles of SER. The RER also assumed a structural arrangement similar to that seen in hepatocytes of normal rats. At 4 h after hormone administration, more cells contained glycogen

[14C]Glycerol was incorporated at an initially rapid rate into both rough and smooth microsomal membranes (Fig. 7). A pattern resembling that of [3H]leucine incorporation was noted, with peak levels of 14C-labeled PLP at 15 min, followed by a decline to a plateau that continued for the remainder of the experiment (120 min; Fig. 7). After adrenalectomy, both the rate and amount of incorporation of [14C]glycerol into membrane PLP were reduced in rough and smooth microsomes (P < 0.05). Two hours after glucocorticoid administration to ADX rats, the incorporation of [14C]glycerol into membrane PLP was increased in both membrane fractions (P < 0.0001-0.001), with peak levels occurring 30 min after injection of the label. Four hours after DEX administration, there was a broad increase in [14C]glycerol incorporation in both membrane fractions (P < 0.01-0.001) when compared with incorporation into microsomal membranes prepared from ADX rats.

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FIG. 3. Low power electron micrograph of centrilobular hepatocyte from overnight fasted ADX rat. RER is scattered throughout the cytoplasm along with elements of SER. Note the absence of glycogen granules from cytoplasm. Nucleus (N), mitochondria (M), microbodies (Mi), and lysosomes (Ly) are identified (x 13,000).

PLPiprotein ratios To study relative changes in the composition of hepatic rough and smooth microsomal membranes from control, ADX, and ADX and hormone-treated rats, protein and PLP concentrations from both membrane fractions were

determined and a PLPiprotein ratio was calculated. The values presented (Table 1) were obtained from washed microsomal membranes, with most nonmembranous material {e.g. ribosomes, serum albumin, and serum lipoproteins) removed (4,11-13). After adrenalectomy, the protein and PLP contents of

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Endo • 1979 Vol 104 • No 6

FIG. 4. Low power electron micrograph of centrilobular hepatocyte from overnight fasted ADX rat treated 2 h with DEX. RER is found in familiar stacked arrays. Numerous elements of SER are observed in close proximity to deposits of glycogen granules (Gl; x 13,000). Inset, High power electron micrograph of centrilobular hepatocyte from overnight fasted ADX rat treated 2 h with DEX. Note elements of SER in close proximity to glycogen particles (X31.847).

both rough and smooth microsomal membranes were reduced compared to that of washed microsomal membranes from normal rat livers (Table 1). The reduced concentrations of protein and PLP were reflected in significantly altered PLP:protein ratios (P < 0.02). After 2 h of treatment with DEX, the membrane protein content of both rough and smooth microsomes increased

relative to that of ADX membranes, with PLP:protein ratios near normal for smooth membranes and intermediate between ADX and normal for rough membranes. Four hours after hormone administration, the protein and PLP concentrations of both membrane fractions were significantly increased over ADX levels (P < 0.01) and were at or above levels obtained in the livers of

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FIG. 5. Lower power electron micrograph of centrilobular hepatocyte from overnight fasted ADX rat treated 4 h with DEX. Several glycogen areas within the cytoplasm are evident. Interspersed with the glycogen granules are elements of SER. RER is found as parallel stacks (x26,500).

normal animals. This was reflected in PLP:protein ratios that were no different from those of membranes from normal rats. G-6-Pase activity in rough and smooth microsomes G-6-Pase specific activities in washed rough and smooth microsomal membranes were similar in normal

and ADX rat livers (Fig. 8), with activity in the rough membranes being greater than that in smooth membranes (P < 0.05). After 2 h of DEX administration, enzyme specific activity was reduced in rough microsomes and elevated in smooth microsomes compared with enzyme activity in ADX rats. There was also no difference between the activity in the two membrane fractions. By 4 h after hormone administration, G-6-Pase

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Time (min) after Injection of 3H-Leucine (lOuCi/lOOg) 3

FIG. 6. Incorporation of [ H]leucine into washed microsomal membrane proteins of overnight fasted normal, ADX, and ADX and hormone-treated rats. Membranes were prepared and assayed as described in the text. Preliminary studies indicated that incorporation of label into total cellular (homogenate) protein (or lipid) was not affected by any of the treatment conditions (data not shown), a) Rough microsomal membranes. Peak incorporation (951 cpm/mg protein) in normal rats occurred at 15 min after injection of label (•— - - — • ) . ADX rats reached a peak at 15 min (499 cpm/mg protein) (A- -A). Two hours after DEX, a peak (705 cpm/mg protein) occurred at 30 min (O O), and at 4 h, there was a broad peak and extended plateau from 15-120 min (~700 cpm/mg protein; • — • ) . b) Smooth microsomal membranes. Peak incorporation occurred at 15 min in normal (934 cpm/mg protein) and ADX (519 cpm/mg protein) rats. Two hours of DEX administration to ADX rats resulted in a peak at 30 min (1110 cpm/mg protein) and another peak at 30 min after 4 h of DEX administration (970 cpm/mg protein).

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Effects of glucocorticoids on microsomal membrane synthesis in hepatocytes from adrenalectomized rats.

0013-7227/79/1046-1722$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society Vol. 104, No. 6 Printed in U.S.A. Effects of Glucocorticoids...
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