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Thomas B. Miller, Jr., Alice K. Garnache, John Cruz, R. Kirk McPherson, and Charles .... and were suspended in minima1 essential medium containing Earle's.
Vol. 261, No. 2, Issue of January 15, pp. 785-790 1986 Printed in S.A .

THEJOURNALOF BIOLOGICAL CHEMISTRY

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0 1986 hy The American Society of Biological Chemists, Inc

Regulation of Glycogen Metabolismin Primary Cultures of Rat Hepatocytes RESTORATION OF ACUTE EFFECTS OF INSULIN AND GLUCOSE IN CELLS FROM DIABETIC RATS* (Received for publication, July 19, 1985)

Thomas B. Miller, Jr., Alice K. Garnache, John Cruz,R. Kirk McPherson, and CharlesWolleben From the Department of Biochemistry, University of Massachusetts Medical School, Worcester,Massachusetts 01605

Defects in the deposition of glycogen and the regu- synthase are missing or reduced in perfused livers (22-25) lation of glycogen synthesis in the livers of severely and hepatocytes (12, 18, 19) isolated from diabetic rats. The insulin-deficient rats can be reversed, in uiuo, within defect in activation of glycogen synthase in livers of alloxan hours of insulin administration. Using primary cul- and streptozotocin diabetic rats canbe repaired by injection tures of hepatocytes isolated from normal and diabetic of insulin, in uiuo, supporting the contention that thedefect rats in a serum-free chemically defined medium, the is due to insulin deficiency rather than a hepatotoxic effect present study addresses the chronic action of insulin to of alloxan or streptozotocin (20, 24-27). Studies carried out facilitate the direct effects of insulin and glucose on in perfused rat liver (24-26), isolated rat hepatocytes(18-20), the short term regulation of the enzymes controlling and primary culturesof rat hepatocytes(19) have shown that glycogen metabolism. Primary cultures were maintained in the presenceof insulin, triiodothyronine, and loss of synthase activation in the diabetic rat isaccompanied by loss of synthase phosphatase activity, and that restoration cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10“ M insulin didnot acutely activate glyco- of synthase activation with insulin is associated with a resgen synthase over a period of 15 min or l h, whereas toration of synthase phosphatase activity. Since the activityof glycogen synthase is partially dependinsulin acutely activated synthase in cultures of normal of phosphorylation (28), it has been assumed hepatocytes. By day 3 in hepatocytes isolated from ent upon its state alloxan diabetic rats, insulin effected an approximate that acute regulation of the enzyme is mediated through the 30%increase in per cent synthaseI within 15 min as action of effectors on specific kinases and/or phosphatases. was also the case for normal cells. The acute effect of In aprevious study(19), we demonstratedthat glycogen independent of synthesis in primary cultures of hepatocytes from diabetic insulin on synthaseactivationwas changes in phosphorylase a. Whereas glycogen syn- rats could be restoredtocontrol valueswhencells were thase phosphatase activity could not be shown to be maintained in the presence of insulin, triiodothyronine, and acutely affectedby insulin, the total activitydiabetic in cortisol for a period of 3 days, concomitant with the partial cells was restored to normal control values over the3- restoration of glycogen synthase phosphatase activity. In the day cultureperiod. present study, it is demonstrated that primary cultures of The acuteeffect of 30 mM glucose to activateglyconormal rat hepatocytes are acutelyresponsive to synthase gen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes iso- activation by glucose and insulin and that short term activalated from either alloxan or spontaneously diabetic tion of glycogen synthase by insulinand glucose can be of diabetic hepatocyteswhen the (BB/W) rats. After 3 days in culture, glucose produced restored in primary cultures chemicallydefined a 50%increase in glycogen synthase activity duringa cells are maintained in this serum-free 10-min period under the sameconditions. These stud- medium. However, theacute effect of insulintoactivate with an assayies clearly demonstrate that insulin acts in a chronic glycogen synthase appears not to be associated activity. manner in concert with thyroidhormones and steroids able stable change in synthase phosphatase to facilitate acute regulationof hepatic glycogen synEXPERIMENTALPROCEDURES thesis by both insulin andglucose.

The key enzyme responsible forthe incorporationof glucose moieties into a pre-existing glycogen chain is glycogen synthase (1).Acute or short term activationof glycogen synthase (conversion of synthase from a less active to a more active form) by insulin and glucose has been shown to be a direct action of these effectors using isolatedperfused rat livers (25) and isolated dispersedrat hepatocyte preparations (6-21). Several studies have demonstrated that the acute effects of insulin and/or glucose on glycogen synthesis and/or glycogen * This investigation was supported by Research Grant AM18269 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adoertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Animals-Fed male rats of the Sprague-Dawley strain (caesareanderived albino outbred from Charles Rivers Breeding Laboratories) weighing 150-200 g were used in the fed state. Diabetes was induced by injection of alloxan (60 mg/kg) into the tail vein and diagnosed by a maximum positive urine glucose and positive urine ketones using Ames Keto-Diastix. Diabetic animals were used 3-5 days after injection of alloxan. Spontaneously diabetic rats from the BioBreeding/ Worcester (BB/W) colony were a generous gift from Dr. Authur Like of the University of Massachusetts Medical School and were used 2 days after withdrawal from insulin maintenance. Male New Zealand White rabbits were used for purification of skeletal muscle synthase (29). Preparation of Hepatocytesfor Primary Culture-Hepatocytes were prepared for primary culture by isolated liver perfusion in the presence of collagenase as previously described (19). In summary, livers were physically isolated from rats during the perfusion procedure 5% using aseptic conditions and perfused with oxygenated (95% 02, COS) calcium-free Krebs-Henseleit bicarbonate buffer containing 0.025% collagenase, but without redbloodcells. After perfusion,

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hepatocytes were further dissociated in a shaker bath for an additional 10 min in buffer containing 142 mhl NaCI, 6.7 mM KCI, and 10 mM HEPES' buffer at pH 7.4.All buffers were sterile and, in addition, contained penicillin (100 units/ml), streptomycin (100 rg/ml), and gentamycin (22 gg/ml) to retard microbial growth. Final cell suspensions were filtered through a Bellco Collector Tissue Sieve (50-mesh screen, 280-bm pore size) into 50-ml sterile centrifuge tubes. Filtered suspensions were centrifuged at 50 X g using the above buffer and repeated twice to remove nonviable hepatocytes and parenchymal cells. The final packed cell pellets contained 68-70 million cells/ml and were suspended in minima1 essential medium containing Earle's salts and 10% fetal bovine serum (Gibco). Viable cell counts were performed using trypan blue prior to seeding. Procedures Used for Primary Culture of Hepatocytes-Primary cultures were started by seeding 3 X IO6 viable cells onto 60-mm plastic tissue culture dishes which had been precoated with rat tail collagen. After plating for 4 h at 37 "C in the presence of humidified 95% air, 5% COS, the medium was aspirated, the plates rinsed, and the medium replaced with a chemically defined serum-free medium containing minimal essential medium with Earle's salts, penicillin, streptomycin, gentamycin, 0.3% crystalline albumin, five times the physiological amino acid concentrations for fed rats (30), 50 pg/ml cortisol, 50 ng/ml triiodothyronine, and either or lo-' M insulin. All subsequent incubations were carried out at37 "C in the presence of humidified air andcarbon dioxide, as described above. Acute effects of insulin and glucosewere determined after 1, 2, and 3 days of primary culture. After the shortterm incubations were carried out to test the effects of insulin and glucose, cells were harvested by transferring the culture dishes to ice, aspirating the medium, and washing the dishes twice with ice-cold buffer containing 5 mM Tris, 2 mM EDTA, and 20% glycerol at pH 7. After rinsing, 0.5 ml of the above buffer was added to each dish, whichwas immediately frozen by floating on liquid nitrogen and stored at -70 "C until analyses were carried out. Analyses-For enzymatic analysis, frozen dishes containing cells plus 0.5 ml of 5 mM Tris, 2 mM EDTA, and 20% glycerol were allowed to thaw on ice. Immediately after thawing, cells were scraped from the dishes using a rubber policeman, and cell suspensions were homogenized for 12 s at *A speed on a Polytron homogenizer, all a t 5 "C. All enzymatic assays were carried out on whole homogenates. Glycogen synthase was assayed without and with added glucose 6phosphate as described by Thomas et al. (31), except that assay time was extended to 30 min. Although the data are notshown, synthase assays were linear for up to 1 h.Glycogen phosphorylase a was assayed in the presence of 0.5 mM caffeine (32), and total phosphorylase was assayed in the presence of 3 mM AMP, all according to the method described by Gilboe et al. (33). Glycogen synthase phosphatase was assayed using purified rabbit skeletal muscle synthase D as substrate for D to I conversion over a 10-min incubation period a t 30 "C. Phosphatase assays were terminated by assaying aliquots for synthase in the absence of glucose 6-phosphate as previously described (25). All analyses on cells in primary culture were based on DNA determination by the method of Burton (34). Materials-Nucleotides, alloxan, triiodothyronine, hydrocortisone hemisuccinate, crystalline albumin, glucose 1-phosphate, glucose 6phosphate, and uridine diphosphoglucosewere purchased from Sigma. Clostridium histolyticum collagenase was purchased from Worthington. [14C]Uridinediphosphoglucose and glucose 1-phosphate were purchased from ICN Radiochemicals. Monocomponent insulin was provided by NOVO. Cell culture dishes were purchased from NUNC. All other chemicals and reagents were of the highest quality available. Data computation and Expression-Data are expressed as mean + 1 S.E. on either side of the mean. Statistical significance was determined by Student's t test. Graphs were prepared using an Apple IIe Computer and Apple Imagewriter Printer in conjunction with Analytical Graphics and Paper Graphics software from Scientific Software Products, Inc.

saline. To each dish was added 4 ml of insulin-free culture medium, and dishes were incubated for 1 h. The 1-h incubation period was included to remove as much of the residual insulin as possible which might have carried over from the maintenance medium. After 1 h without insulin, insulin was added to half of the dishes at a final concentration of M, and dishes were further incubated prior to harvesting and analyses. Fig. 1, a-c shows the results of the acute effects of insulin on glycogen synthase activity after 15,30, and 60 min

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FIG. 1. Acute effects of insulin on glycogen synthase activity in primary cuItures of hepatocytes. Preparation of hepatocytes and experimental protocols were described in the text and under "Experimental Procedures." Data are expressed as percentage synthase Z or as (synthase assayed in the absence of added glucose 6phosphate/synthase assayed in the presence of added glucose 6phosphate) X 100. Means represent an n of 8-12 determinations. a-c represent data obtained after 1, 2, and 3 days of maintenance in primary culture, respectively. Open bars represent hepatocytes isoRESULTS lated from normal ratsand darkened bars represent hepatocytes isolted from diabetic rats. C represents control data at 15, 30, or 60 For experiments determining short term effects of insulin, min while Z represents data obtained after incubation with insulin cells were maintained with culture medium containing 1 nM for 15, 30, or 60 min. Zero time values for norma1,cells were 41.6 f insulin. After 1,2, or 3 days in primary culture, culture dishes 2.0, 49.3 -+ 1.8, and 45.1 f 2.8 for days 1, 2, and 3, respectively. Zero were aspirated and washed twice with phosphate-buffered time values for diabetic cells were 15.0 + 1.1, 31.8 -t 1.5, and 29.6 -+ 1.7 for days 1, 2, and 3, respectively. Zero time values were not The abbreviation used is: HEPES, 4-(2-hydroxyethyl)-l-pipera- different than 15-min values in cells incubated acutely without insulin. zineethanesulfonic acid.

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in normal and diabetic hepatocytes in primary culture for 1, 2, and 3 days. On day 1 (Fig. la), insulin had essentially no effect on the percentage of active synthase at any time period tested in hepatocytes isolated from severely insulin-deficient rats. Furthermore, the percentage of synthase in the active form was significantly reduced below that seen in normal hepatocytes, as hasbeen previously reported using this same primaryculturesystem (19). Inhepatocytesisolated from (p < 0.001) effect of normalrats, a smallbutsignificant insulin was observed at the60-min time period, but not at 15 or 30 min. nay 2 Day 3 On day 2 in hepatocytes from normal rats(Fig. l b ) , insulin Days Cultured produced a slight but significant (p < 0.05) increase in synFIG. 2. Total glycogen synthase activity i d primary hepathase activity after 15 min. After 30 min, insulin produced a 27% increase in synthase activity in normal hepatocytes when tocytes cultured for 2 and 3 days. Protocols, conditions, and compared to the30-min control(p < 0.001), and after60 min, definitions were the same as described in Fig. 1. Data for total synthase activity did not change during the short term incubation insulin effected a 44% increase in synthase activity over the periods. Each mean represents an n of 24-36 determintions. Data are control value (p < 0.001). These data are similar to that expressed as picomoles of glucosefrom uridine diphosphoglucose reported in primary cultures of hepatocytes after 2 days of incorporated into glycogen/min/Fg DNA assayed in the presence of maintenance in the presenceof 5% calf serum by Nakamura added glucose 6-phosphate. In all cases for normal uersus diabetic hepatocytes, p < 0.001. et at. (35). By day 2 in the hepatocytes from diabetic rats, insulin facilitated significant increases in synthase activity after 30 and 60 min incubation, but not at15 min. Likewise, O the control synthase ativities in the diabetic hepatocytes were 180 significantlyelevated over the day 1 values. The decrease \ observed in control synthaseI at 30 and 60 min is most likely C due to the diminishing effects of residual insulin left over E 120 \ from the long term maintenance with insulin. VI By day 3 of maintenance of hepatocytes from normal and ,, 60.0 diabetic rats (Fig. IC), the short term effect of insulin t o E a activate glycogen synthase was observed at the earliest time 0.00 periods tested, and the effect was sustained over the entire 1 2 3 I 2 3 60-min exposure t o insulin. In normal hepatocytes, insulin Days cultured produced a 30% increase in synthase activity a t 15 min (p < FIG. 3. Effects of insulin on glycogen synthase phosphatase 0.05), a 40%increase in synthase activity at 30 min0, < 0.01), activity in primary cultures of hepatocytes. Protocols, condiand a 57% increase in synthase activityat 60 min (p < 0.001). tions, and definitions were the same as described for Fig. 1. Means In diabetic hepatocytes, insulin produced a 35% increase at represent pooled data for 15, 30, and 60 min for cells cultured for 1, 15 min (p < 0.05), a 38% increase at 30 min (p