interactions between insulin and al-adrenergic agents in the

THE JOURNAL OF BIOLOGICAL CHEMISTRY r c ) 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 10, Issue of May 25, pp. 5963-5973, 1985 Printed in U.S.A.

Interactions between Insulin and al-Adrenergic Agents in the Regulation of Glycogen Metabolism in Isolated Hepatocytes*

(Received for publication, May 14, 1984)

Andrew P. Thomas+, Angeles Martin-Requerop, and John R. Williamsonl From the Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Addition of insulin to liver cells from fed rats incubated in the absence of other hormones resulted in a 2- fold increase in glycogen synthase activity. This direct effect of insulin has been characterized and compared with the antagonism by insulin of al-adrenergic effects on glycogen metabolism. The activation of glycogen synthase by insulin developed slowly (20-25 min) and was most effective when the enzyme was partially preactivated by glucose. With glucose concentrations above 15 mM the effects of insulin and glucose were additive. In contrast to glucose, which caused inverse changes in phosphorylase and glycogen synthase activity, insulin activated glycogen synthase without af- fecting phosphorylase a. Treatment of hepatocytes with phenylephrine led to an activation of phosphorylase and inactivation of glycogen synthase, which could be partially blocked by insulin. This antagonistic effect of insulin was rapid (complete within 5 min of insulin addition) and showed an identical time course for both enzymes. The activation of glycogen synthase by insulin and inactivation by phenylephrine both resulted principally from alterations in the V,,,. Insulin added alone did not alter the basal cytosolic free Ca2+ concentration, which was 160 nM as measured with Quin 2 as an intracellular Ca” indicator. Both the magnitude and the initial rate of cytosolic free Ca2+ increase induced by phenylephrine were reduced by about 50% in cells pretreated with insulin. It is concluded that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase, whereas insulin antagonizes the effects of al-agonists by interfering with their ability to ele- vate cytosolic free Ca2+.

~

The liver is an important site for insulin action in the regulation of mammalian glucose homeostasis, but previously reported direct effects of insulin on glucose and glycogen metabolism in perfused livers or isolated hepatocytes have been rather small and inconsistent (for reviews see Refs. 1- 3). Several reports suggest that insulin alone has little effect

* This work was supported by National Institutes of Health Grants AA-05662 and AM-15120. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Recipient of a Juvenile Diabetes Foundation International Post- doctoral Fellowship. Current address: University of Bristol, Depart- ment of Biochemistry, Bristol, BS8 lTD, United Kingdom.

cil of Scientific Research, Madrid 6, Spain. 3 Current address: Institute of Gregorio Marafion, Superior Coun-

1 To whom reprint requests and correspondence should be ad- dressed.

on glycogen synthase or phosphorylase in liver cells (4-6), although there is general agreement that insulin is effective in antagonizing the action of certain glycogenolytic hormones such as glucagon and a,-adrenergic agonists (4-11). Insulin has been shown to increase the glycogen content of hepatocytes (12, 13), but where direct effects of insulin on glycogen synthase have been observed there has been some dispute as to whether phosphorylase inactivation or the presence of glucose is required for these changes to occur (14-17). Insulin increases the activity of glycogen synthase I (glucose 6-phosphate-independent form) in several other tissues, where the effects can be divided into those resulting from increased glucose transport and effects which are independent of extracellular glucose (see Refs. 17 and 18 for References). The former mechanism is very unlikely to operate in liver because insulin cannot be shown to regulate glucose transport into hepatocytes. With the recent increase in information pertain- ing to the mechanism by which skeletal muscle glycogen synthase is regulated (18-24), it has become important to define more fully the effect of insulin on the liver enzyme.

The activation of hepatic glycogenolysis by glucagon and Ca2+-dependent hormones such as al-adrenergic agents, vasopressin, and angiotensin I1 is associated with increased phosphorylase a and decreased glycogen synthase I activities (1-11, 25-29). In the case of glucagon and at-agonists (but not the other Ca2+-dependent hormones) it is widely accepted that these effects are antagonized by insulin (4-11, 14). In- sulin appears to interfere with glucagon action by reducing the elevated levels of CAMP (2, 5, 7, 27), but the mechanism by which insulin antagonizes al-effects is less clear. The activation of phosphorylase and inactivation of glycogen synthase caused by at-agonists is believed to be secondary to the increase in cytosolic free Ca2+ brought about by these agents (26-35). Since insulin does not antagonize the effects of all of the Ca2+-mobilizing hormones (5, 6, 9, 11, 36) and does not affect phosphorylase activation and glycogen synthase inactivation caused by the Ca2+ ionophore A23187 (5, 6), it is probable that insulin antagonizes al-effects at some step prior to the increase in cytosolic free Ca2+. Support for this proposal comes from the finding that insulin can partially antagonize phenylephrine-induced calcium efflux from hepatocytes and perfused livers, as well as the loss of calcium from subsequently isolated intracellular organelles (5, 6, 11). These results have led to the suggestion that insulin antagonism occurs either at the level of the al-receptor or by interfering with the effect of a second messenger which is specific for al-agonists (5, 6, 11, 37). The former proposal has been questioned because the stimulation of inositol lipid metabolism by phenylephrine does not appear to be sensitive to antagonism by insulin (37).

In the present study, the direct effects of insulin on the

5963

5964 Hormonal Regulation of Liver Glycogen Metabolism

enzymes of glycogen metabolism have been investigated and compared with the effects of glucose. In addition, we have also investigated the antagonism by insulin of a,-agonist- induced alterations of cytosolic free Ca2+ and the activities of phosphorylase and glycogen synthase. The overall purpose of this work was to characterize the mechanisms involved in these two modes of insulin action in liver.

EXPERIMENTAL PROCEDURES

Isolation and Incubation of Hepatocytes-Isolated hepatocytes were obtained from the livers of fed, male Sprague-Dawley rats of 180-220 g body weight as described previously (38).' The cells were suspended at a final concentration of 5 mg of dry weight/ml in Krebs-Ringer bicarbonate containing 2% dialyzed bovine serum albumin and unless indicated otherwise, 1.3 mM CaC12 and 15 mM glucose. For experiments where the activities of glycogen phosphorylase and glycogen synthase were determined, 2-4 ml of cell suspension were incubated at 37 "C with shaking in 25-ml plastic Erlenmeyer flasks under an atmosphere of O2/C02 (955). After a minimum of 15 min of preincubation, hormones were added as required. The incubations were subsequently terminated by centrifuging 0.5 ml of cell suspension for 5 s in an Eppendorf 3200 centrifuge. After the supernatant was removed, 0.25 ml of ice cold extraction medium was added and the samples were rapidly mixed and frozen in liquid Nz. The extraction medium was composed of 100 mM MOPS: 150 mM NaF, 20 mM EDTA, 400 mM sucrose, 5 mM dithiothreitol, 0.03% Triton X-100 at pH 7.0 (39). For most experiments triplicate incubations were carried out.

Measurement of Cytosolic Free Ca2+-Changes of the cytosolic free CaZ+ concentration in isolated hepatocytes were followed by using the Ca2+-sensitive indicator Quin 2 (40, 41), essentially as described previously (35). The cells were suspended at 10 mg of dry weight/ml in buffer containing 120 mM NaC1, 20 mM HEPES, 5.4 mM KCl, 4.2 mM NaHC03, 1.2 mM KH2P04, 1.2 mM MgS04, 1.3 mM CaClZ, 15 mM glucose, and 2% dialyzed bovine serum albumin at pH 7.4 and maintained under an atmosphere of O2 (100%). Quin 2-tetraacetoxymethyl ester was then added to give 8 nmol/mg of cell dry weight. After 20 min, when maximal accumulation of Quin 2 had occurred, the cells were washed twice and resuspended, at about 3 mg of dry weight/ml in the HEPES buffer described above but with the albumin concentration reduced to 0.2%. Quin 2. Ca fluorescence and pyridine nucleotide fluorescence were measured simultaneously using the dual channel fluorometer (Johnson Research Foundation Electronic Shop) described previously (35). The excitation wavelength was 335 nm and the emission wavelengths were 490-570 nm for Quin 2. Ca and 420- 450 nm for reduced pyridine nucleotides. Using these wavelengths the fluorescence changes due to alterations in the intracellular concentrations of Quin 2. Ca complex were almost completely separated from changes in pyridine nucleotide fluorescence. Calibration of the Quin 2. Ca fluorescence in terms of cytosolic free Ca2+ concentration was carried out by sequential additions of 2.5 mM EGTA (plus Tris base to give pH 8.3) to determine the amount of extracellular Quin 2. Ca complex, followed by 0.1% Triton X-100 to release intracellular Quin 2 and give the minimum fluorescence when no Ca2+ was bound to Quin 2. Finally, 3 mM CaCll was added to obtain the maximal fluorescence when all of the Quin 2 was saturated with Ca2+. The cytosolic free Ca2+ concentration was then calculated at any inter-

' Inexplicably, some preparations of hepatocytes obtained by the normal collagenase digestion procedure appeared to be insensitive to insulin. The present study was performed using collagenase which was selected by screening different batches of collagenase for the ability of isolated hepatocytes to exhibit a stimulation of glycogen synthase activity by insulin. In addition, preparations of hepatocytes

the freshly isolated hepatocytes were incubated for 90 min in Medium which consistently displayed an insulin response were obtained when

199 (Gibco) supplemented with Eagle's minimal medium essential and nonessential amino acids, 10% fetal calf serum, and 1 pg/ml of dexamethasone. When this protocol was used the cells were washed very thoroughly before resuspension in the standard basic incubation medium.

The abbreviations used are: MOPS, 3-(N-morpho1ino)pro- panesulfonic acid; EGTA, ethylene gycol bis(Saminoethy1 ether)- N,N,N',N'-tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-l-piperaz- ineethane sulfonic acid.

mediate level of fluorescence from the minimum and maximum fluorescence levels (after subtraction of the signal due to extracellular Quin 2 and the detergent blank) as described by Tsien et al. (41).

A d y t i c a l Procedures-Phosphorylase a activity was measured at 30 "C using 30 mM glucose 1-phosphate as described by Gilboe et 01. (42). Total phosphorylase activity (phosphorylase a and b) was determined in the presence of 5 mM AMP and 10% (v/v) dimethoxy ethane (43). Glycogen synthase activity was assayed at 30 "C using the method of Thomas et al. (44). Uridine diphospho[U-'4C]glucose (0.1 pCi/ml) was used at a final concentration of 1 mM. The I-form of glycogen synthase was measured in the absence of glucose 6-phosphate while the D-form was determined in the presence of 10 mM glucose 6-phosphate. The assays were all linear for at least the 30- min period over which they were routinely carried out. Furthermore, the activity of both glycogen synthase and phosphorylase was not affected by diluting the extracts over a 10-fold range, suggesting that intracellular allosteric effectors of these enzymes were probably not carried over into the extracts at sufficient concentrations to significantly alter their activities. One unit of enzyme activity is defined as that which will convert 1 pmol of substrate to product in 1 min at 30 "C. Enzyme activities are all expressed in terms of grams of cell dry weight. Kinetic parameters of enzyme activity were determined by fitting the experimental data to the relevant equation using nonlinear least squares regression on a Hewlett-Packard 9845A computer using a BASIC program written by Dr. P. J. England, Bristol Uni- versity, United Kingdom.

Materials-L-Phenylephrine, arginine-vasopressin, and most bio- chemicals were from Sigma. Crystalline bovine insulin was a gift from Lilly and was stored as a 0.2 mM stock solution in 2 mM HCI. Quin 2-tetraacetoxymethyl ester was obtained from Behring Diagnostics and stored as a 16 mM stock solution in dimethyl sulfoxide. Uridine diph~spho[U-'~C]glucose and [U-"C]glucose 1-phosphate were from Amersham Corp.

RESULTS

DirectActiuation of Glycogen Synthase by Insulin-As noted above, there are considerable discrepancies in the literature with respect to the sensitivity of hepatic glycogen synthase to insulin (4,6,14-17). The data of Fig. lA show that in a simple bicarbonate-buffered medium containing 15 mM glucose, treatment of freshly isolated hepatocytes with insulin led to a %fold increase in the activity of glycogen synthase I (measured in the absence of glucose 6-phosphate). In these experiments the hepatocytes were preincubated with glucose for 15 min before insulin was added. This period of time was sufficient to ensure that the effects of glucose on both phosphorylase and glycogen synthase were complete before the commencement of any insulin-induced changes. Following the addition of insulin there was a delay of about 5 min before any effect could be detected, and maximal activity was ap- proached 20-25 min later. The glycogen synthase activity ratio (the activity measured in the absence of glucose 6- phosphate divided by the activity in the presence of glucose 6-phosphate) increased in parallel with glycogen synthase I (Fig. 1B). However, in all cases we found that insulin also caused a significant increase in the activity of the D-form of the enzyme (glucose 6-phosphate-dependent) and as a result of this, the activity ratio was found to be a less useful index of the insulin effect on hepatic glycogen synthase. The concentration of glucose 6-phosphate used was 10 mM which was sufficient to give maximum activation of the glycogen synthase extracted from both control and insulin-treated hepatocytes. The substrate for the enzyme, UDP-glucose, was used at a concentration of 1 mM which gave about half-maximal activity, thus allowing changes of both the S0.5 and the V,,, to be detected. The effects of insulin and other agents on the kinetic properties of glycogen synthase are described more fully below.

In most cases where insulin has been reported to activate hepatic glycogen synthase, the effects have been found to depend on the presence of glucose in the medium (Refs. 14

Hormonal Regulation of Liver Glycogen Metabolism 5965

0.4j

r,,,,,,,

i 0 5 IO 15 20 25 i0

Time(min1

I ,

FIG. 1. Time course for activation of glycogen synthase by insulin. Isolated hepatocytes were incubated in bicarbonate-buffered medium containing 2% dialyzed bovine serum albumin and 15 mM glucose. After 15 min of preincubation, insulin (20 nM) was added, and the incubations were terminated at the indicated times after insulin addition as described under “Experimental Procedures.” Panel A shows the activity of glycogen synthase measured in the absence of glucose 6-phosphate and in panel E the activity ratio expressed as the activity determined in the absence of glucose 6-phosphate divided by the activity in the presence of 10 mM glucose 6-phosphate is given. Each point is the mean f S.E. of values obtained using 4 separate preparations of hepatocytes.

and 16, but cf Ref. 17). In our experiments using hepatocytes from fed rats the effect of insulin on glycogen synthase I was very small unless the medium glucose concentration was greater than 5 mM (Fig. 2). In the absence of added glucose, insulin increased the activity of glycogen synthase by 11.8 f 3.2% (significant at p < 0.05 by paired t test). The largest per cent stimulation by insulin occurred at about 15 mM glucose and although the activation of glycogen synthase by glucose continued at least up to 50 mM glucose (see also Refs. 6 and 16), the additional increment caused by insulin was essentially constant at concentrations of glucose above 15 mM. This suggests that glucose and insulin do not act on glycogen synthase in the same manner, although it appears that glucose is necessary for the full expression of the insulin effect.

It has been suggested that the activation of glycogen synthase by glucose is a secondary result of glucose-induced inactivation of phosphorylase a (8, 27, 45). The similar sensitivities to glucose for glycogen synthase activation and phosphorylase inactivation shown in Fig. 2 are consistent with this hypothesis. However, insulin does not appear to act through such a mechanism because it is clear from the data of Fig. 2 that insulin is able to increase the activity of glycogen synthase I without causing any significant change in the activity of phosphorylase a. We have found no direct effect of insulin on phosphorylase a activity at any time point or concentration of glucose where glycogen synthase was activated.

In addition to showing that insulin activates glycogen synthase in a manner distinct from that of glucose and independ-

0

30

0 0 IO 20 30 40 50

[Glucose] (mM)

FIG. 2. Effects of glucose concentration on glycogen synthase and phosphorylase activity in the presence and absence of insulin. Hepatocytes were incubated as described under “Experi- mental Procedures” except that the concentration of glucose in the medium was varied as indicated and the osmolarity of the medium was maintained by reducing the NaCl content. Incubations were continued for 40 min, either in the absence of insulin (0) or with insulin present for the last 25 min (0). Panel A shows the activity of glycogen synthase I and panel E the activity of phosphorylase a measured in the same cell extracts. Each point is the mean zk S.E. of values from 3 separate experiments.

ent of changes in phosphorylase activity, the data of Fig. 2 also provide evidence against an involvement of changes in cytosolic free Ca2+ in the mechanism of insulin action. Cal- cium has been suggested to play a role in insulin action in several tissues (46-48). However, liver phosphorylase kinase is very sensitive to changes of cytosolic free Ca2+ (49), and it seems unlikely that insulin could alter the latter parameter without changing the activity of phosphorylase a. As a further approach to the question of whether the insulin effect on glycogen synthase is related to changes of the cytosolic free Ca2+, we have examined the effect of depleting intracellular calcium on the enzymes of glycogen metabolism (Table I). After incubating hepatocytes for 40 min with EGTA, total cell-associated calcium fell by about 80% and the basal activity of phosphorylase a was reduced by 50%, presumably as a result of a lowering the cytosolic free Ca2+. No effect of calcium depletion was observed on glycogen synthase I activity under these conditions and the magnitude of insulin- induced glycogen synthase activation was also unchanged. Insulin had no effect on phosphorylase activity in either the normal or the calcium-depleted cells. Thus, Ca” does not appear to mediate the effects of insulin on liver glycogen synthase. It has also been reported that extracellular Ca2+ is not required for insulin inhibition of phosphodiesterase activity (50).

In order to confirm that insulin does not alter cytosolic free Ca2+ in hepatocytes we have used the Ca2+-sensitive indicator Quin 2 (35, 40, 41). Insulin caused a marked activation of glycogen synthase within 10-15 min but no detectable alteration of cytosolic free Ca2+ was observed over a period of at


TABLE I Effects of calcium depletion on insulin activation of glycogen synthase

Isolated hepatocytes were preincubated for 15 min in bicarbonate- buffered medium containing 15 mM glucose, either in the presence of 1.3 mM calcium (+calcium) or in the absence of added calcium and with 2.5 mM EGTA added to the medium (-calcium, +EGTA). Insulin (20 nM) was then added and the incubations were terminated after a further 25 min. Other incubation and assay conditions were as indicated under "Experimental Procedures." At the end of the incubation period the cell-associated calcium measured by atomic absorption was 7.55 f 0.54 and 1.54 f 0.19 nmol/mg of cell dry weight in the presence and absence of calcium, respectively. Each value is the mean ? S.E. from 5 separate experiments.

Glycogen synthase I Phosphorylase a

unitslg dry weight Control 0.44 f 0.02 22.4 f 1.8

20 nM insulin 0.66 * 0.02" 22.2 f 0.9

Control 0.38 f 0.03 11.0 f 1.0'

20 nM insulin 0.68 f 0.05" 9.3 * 0.5'

(+calcium)

(+calcium)

(-calcium, +EGTA)

(-calcium, +EGTA) "Significantly different from the control value in the absence of

' Significantly different from the equivalent value measured in the insulin with p < 0.01.

presence of calcium with p < 0.01 calculated using Student's t test.

' "1 o r

0.6-

0.5-

$ f 0.4- H

a)-

- A

0 0.3- 545 a m I=\

B .2 0.2- - x 2 13

0.1 -

0 - 1

0 5 IO 15 20 Time (mid

FIG. 3. Time course of phosphorylase activation and glycogen synthase inactivation by phenylephrine in the presence and absence of insulin. Isolated hepatocytes were preincubated for 20 min with 15 mM glucose and then for a further 5 min in the presence (open symbols) or absence (closed symbols) of 20 nM insulin. At the end of the preincubation 1 @M phenylephrine (A, A) or H20 (0, 0) was added and the incubations were terminated at the times indicated. In panel A the activity of phosphorylase is shown and in panel B that of glycogen synthase is shown. Each point is the mean f S.E. of values obtained using 3-5 separate preparations of liver cells.

least 20 min following the addition of insulin. In four separate cell preparations the cytosolic free calcium concentration was calculated to be 158 f 12 nM in the absence of insulin and 156 f 11 nM after a 15-min treatment with insulin.

Insulin Antagonism of a,-Adrenergic Effects on Phosphory- lase and Glycogen Synthase-Although the direct effects of insulin on hepatic glycogen metabolism have been poorly defined in the past, several groups have shown that insulin is able to antagonize the glycogenolytic effects of glucagon and a,-adrenergic agonists in liver (4-11). Fig. 3A shows the time course for phosphorylase activation by the a,-adrenergic agent phenylephrine in the presence and absence of insulin. Insulin alone had no effect on phosphorylase a activity over the full 20 min of the time course. Phenylephrine (20 PM) increased phosphorylase a activity %fold within 30-60 s, and the peak activation of phosphorylase was reduced by about 50% in cells which had been pretreated with insulin for 5 min. It is interesting to note that at physiological medium Ca2+ concentrations, the maximum antagonism by insulin of the initial activation of phosphorylase by phenylephrine is never greater than 50%.3 The antagonism by insulin of a,-adrenergic phosphorylase activation was much more marked at later times, so that in insulin-treated cells phosphorylase a activity de- cayed to a level which was not significantly different from the control by 10-15 min after phenylephrine addition. Fig. 3B shows the time course of glycogen synthase inactivation by phenylephrine in hepatocytes after preincubation with and without insulin. A maximal inactivation of about 80% was achieved after a 5-min treatment with phenylephrine, which is slower than the concomitant activation of phosphorylase. The inactivation of glycogen synthase by phenylephrine was partially blocked by insulin and in common with the results obtained for phosphorylase, insulin was considerably more effective at later times. However, since insulin alone activated glycogen synthase over the period from 5-25 min after its addition (Fig. l), it is possible that the return towards basal glycogen synthase I activity, which commenced 10 min after phenylephrine addition in the presence of insulin, resulted from the direct action of insulin on this enzyme.

The data presented above show that while insulin can antagonize the effects of al-agonists on both phosphorylase and glycogen synthase, only glycogen synthase is sensitive to a direct effect of insulin. This suggests that distinct mechanisms are involved in mediating the two effects of insulin on glycogen metabolism in liver. Additional support for this proposal comes from a comparison of the time courses over which these two modes of insulin action develop. In the experiments shown in Fig. 4, isolated hepatocytes were treated for a fixed period of time with phenylephrine, and insulin was added at various times before or after the a,-agonist to obtain a time course for the onset of insulin antagonism. In all cases phenylephrine was added 2 min before the incubations were stopped, since this was the earliest time at which near maximal effects were observed for both phosphorylase and glycogen synthase. The times marked on the abscissa represent the times that insulin was present before the incubations were terminated. In the absence of insulin (zero time) the effects of phenylephrine on both enzymes were similar to those shown in Fig. 3. A significant diminution of these al-effects was observed within 2 min of insulin addition (i.e. insulin and

In cells depleted of intracellular calcium and incubated in the absence of extracellular calcium, insulin completely blocked the activation of phosphorylase by phenylephrine, in agreement with the results of Blackmore et al. ( 5 ) . Presumably insulin acts by preventing the small rise of CAMP which phenylephrine can bring about in hepatocytes incubated under these conditions (5,511.


J , 0 5 IO 15 20 25

Time after insulin addition (min 1

FIG. 4. Time course for the development of insulin antagonism of phenylephrine effects on glycogen synthase and phosphorylase. Isolated hepatocytes were preincubated for a total of 40 min, with the phenylephrine (1 p ~ ) present for the last 2 min of incubation for all of the points indicated by open circles. Insulin (20 nM) was also added to the medium at the indicated times before terminating the incubations for all of these points were terminated, except at zero time where no insulin was added and the effects result from a 2-min treatment with phenylephrine alone. The closed c i r c b indicate the basal values when neither phenylephrine nor insulin was added. Panel A shows the activity of glycogen synthase I and panel B the activity of phosphorylase u measured in the same cell extracts. All points represent the mean * S.E. of values obtained from experiments using 3 separate preparations of hepatocytes.

phenylephrine added simultaneously) and the maximum antagonism by insulin of phenylephrine-induced glycogen synthase inactivation and phosphorylase activation occurred after only 4 min of treatment with insulin. The further increase in glycogen synthase activity which occurred with prolonged insulin treatment (Fig. 4-4) was probably more related to the direct effect of this hormone as discussed above. Thus, it is clear that the antagonism of a,-effects by insulin is temporally distinct from the direct activation by insulin of glycogen synthase, the former effect being complete before the later effect on glycogen synthase is even detectable (Fig. 1 versus Fig. 4).

Insulin Antagonism of al-Adrenergic-indued Increases of Cytosolic Free Calcium-Although the direct activation of glycogen synthase by insulin is apparently independent of changes in cytosolic free Ca2+ (see above), there is considerable evidence indicating that the inactivation of glycogen synthase and activation of phosphorylase by al-agonists is secondary to an increase of cytosolic free Ca2+ (26-35). The finding that the effects of phenylephrine on glycogen synthase and phosphorylase are both antagonized by insulin with a similar time course (Fig. 4) might suggest that the mechanism of this insulin antagonism occurs at the level of cytosolic free Ca2+. Exton and co-workers have provided some indirect evidence that this is indeed the case (5, 6, 11). Fig. 5 shows the defining the role of at results of experiments where cytosolic free Ca2+ was measured using Quin "loaded hepa-

tocytes which were preincubated in the presence and absence of insulin before addition of phenylephrine or vasopressin. In cells not treated with insulin (Fig. 5A), phenylephrine caused a very rapid increase in cytosolic free Ca", which was essentially complete in 5 s. This response clearly preceded the activation of phosphorylase (maximal at 30-60 s) and inactivation of glycogen synthase (maximal at 5 min as shown in Fig. 3). A slower time course was also observed for the phenylephrine-induced reduction of cellular pyridine nucleotides (lower traces in Fig. 5, A and B) as measured simultaneously using a dual channel fluorometer (35). Insulin pretreatment decreased by about 50% both the magnitude and the initial rate of increase of the cytosolic free Ca2+ concentration caused by phenylephrine (Fig. 5B), which is consistent with the antagonism by insulin of phenylephrine-induced phosphorylase activation and glycogen synthase inactivation shown in Fig. 3.

In the experiment shown in Fig. 5 a submaximal concentration of phenylephrine was used, but similar results were also obtained at higher levels of phenylephrine (see dose response data below). When a saturating dose of vasopressin (10 nM) was added subsequent to the phenylephrine addition, the cytosolic free Ca2+ concentration increased to a peak value of about 500 nM in both the presence and absence of insulin (Fig. 5, A and B) . This is consistent with the failure of insulin to interfere with the glycogenolytic effects of vasopressin in liver (5, 6, 9, 11, 36). Further evidence that insulin does not antagonize the effects of vasopressin on calcium mobilization is given in Fig. 5, C and D, where a submaximal dose of vasopressin was used. The specificity of insulin in antagonizing only al-effects indicates that insulin must be acting at some point prior to any convergence in the Ca2+-mobilizing messenger systems for al-agonists and vasopressin, as pointed out previously (5,6, 11, 37).

In order to provide additional characterization of the mechanism by which insulin antagonizes the phenylephrine-induced elevation of cytosolic free Ca2+, the effect of insulin on the dose response to phenylephrine was examined. Fig. 6A shows data combined from several experiments where the peak cytosolic free Ca" concentration was determined in the presence and absence of insulin over a range of phenylephrine concentrations. Half-maximal effects of phenylephrine on peak Ca2+ concentrations were observed at 0.36 -+ 0.08 and 0.60 f 0.15 PM in the absence and presence of insulin, respectively (values calculated by nonlinear least squares regression to the simple noncooperative binding equation; significantly different at p < 0.05 calculated by Student's t test). When rates of cytosolic free Ca2+ increase were measured with phenylephrine as the agonist, the dose response curves were only slightly shifted towards higher phenylephrine concentrations (Fig. 6A) in contrast to previous results using vasopressin as the Ca2+-mobilizing hormone (35). This probably relates to the much smaller amounts of the second messenger involved in Ca2+ release, namely inositol, 1,4,5-trisphosphate (35, 52, 53), being formed with phenylephrine as agonist compared with vasopressin (35): Half-maximal rates of cytosolic free Caz+ increase were observed at 0.78 -+ 0.10 and 0.90 f 0.17 PM phenylephrine in the absence and presence of insulin, respectively. Thus, the major effect of insulin was to decrease the magnitude and rate of the phenylephrine-induced cytosolic free Ca2+ increase, but, in addition, it also caused a marginal decrease in sensitivity to phenylephrine.

Calcium-dependent hormones such as al-agonists and vasopressin mobilize calcium from two sources; initially an

' A. P. Thomas and J. R. Williamson, unpublished observations.


c -Insulin

0.5nM

D +Insulin

0.5 nM

U

FIG. 5. Effects of phenylephrine and vasopressin on cytosolic free calcium and pyridine nucleotide reduction in the presence and absence of insulin. The fluorescence signals due to the Quin 2.Ca complex and reduced pyridine nucleotides were measured simultaneously in Quin 2-loaded hepatocytes as described under “Experimental Procedures.” After preincubation in the absence (panels A and C) or for 10 min in the presence of 50 nM insulin (panels B and D), 0.5 pM phenylephrine (Phen; A and E) or 0.5 nM vasopressin (Vaso; C and D) was added. In panels A and B, when the calcium and pyridine nucleotide signals had reached a new elevated steady state, 10 nM vasopressin (Vaso) was added and the further changes were also measured. Insulin alone had no effect on the basal cytosolic free calcium concentration or the level of pyridine nucleotide fluorescence. Calibration of the Quin 2 fluorescence was carried out as outlined under “Experimental Procedures.” Changes of pyridine nucleotide fluorescence are expressed as a percentage of the basal fluorescence signal. The experimental traces shown are representative of results obtained with 6 different cell preparations.

intracellular calcium pool is released (26, 29-33), while subsequently changes of Ca2+ flux across the plasma membrane become more important (54-56). Since the antagonism by insulin of a,-effects is most marked at later time points (Fig. 3), it is possible that insulin acts only on the plasma membrane Ca2+ fluxes. However, the data of Fig. 7 show that insulin is also effective in antagonizing the mobilization of intracellular calcium. In these experiments excess EGTA was added to the cells shortly before phenylephrine addition so that increased cytosolic free Ca2+ must have been derived from an intracellular source. It is clear that insulin was at least as effective in antagonizing the phenylephrine-induced cytosolic free calcium increase under these conditions as it was in the presence of physiological extracellular Ca2+ concentrations (cf. Fig. 5, A and B) .

Effects of Hormones on the Kinetics of Glycogen Synthe- An examination of the effects of hormone treatment on the kinetics of glycogen synthase was carried out for two reasons. Firstly, preliminary results suggested that the kinetic changes in the liver enzyme were very different from those observed in various muscle preparations. Secondly, recent advances in defining the role of at least 6 protein kinases in phosphory- lating up to 7 distinct sites on both liver and muscle glycogen synthase (19-24,57-63) opened up the possibility of using the kinetic changes to make further deductions as to the mechanisms by which the hormones might affect glycogen synthase in liver. Fig. 8 shows the effects of varying the concentrations of UDP-glucose and glucose 6-phosphate on the activity of glycogen synthase in extracts from cells incubated under

various conditions. Glucose (15 mM) was present in the cell incubations for all of the conditions except for the one indicated as -glucose. It is clear that in the absence of glucose 6- phosphate the major effects of insulin and phenylephrine were on the Vmax of the enzyme (Fig. 8A). This is in marked contrast to the results obtained for skeletal muscle glycogen synthase where the 80 .5 for UDP-glucose was the most sensitive parameter to hormone treatment (see Refs. 18 and 19). In Fig. 8B the sensitivity to glucose 6-phosphate concentration (at saturating UDP-glucose) is shown and again it can be seen that the different treatments give different V,, values, whereas with the muscle enzyme changes of the Mo.5 for glucose 6- phosphate predominate (18,19). The persistence of the insulin effect on hepatic glycogen synthase in the presence of saturating glucose 6-phosphate concentrations indicates that con- tamination of the cell extracts with this effector cannot explain the observed insulin effect.

The calculated kinetic parameters for glycogen synthase extracted from hepatocytes which had been preincubated with insulin or phenylephrine or in the absence of glucose are shown in Tables I1 and 111. Glucose (15 mM) increased the Vmax of the enzyme by about 3-fold. Superimposed on this effect of glucose, insulin increased the V,. by 64% and phenylephrine decreased it by 74% (Table 11). The effect of phenylephrine on the Vmax could be partially antagonized by a brief pre-exposure to insulin. Phenylephrine had no effect on the for UDP-glucose but insulin caused a small decrease. None of the treatments affected the Hill coefficient, which was not significantly different from 1. When saturating


[Phenylephrine] (pM) FIG. 6. Dose response to phenylephrine for the magnitude

and rate of cytosolic free calcium increases in the presence and absence of insulin. Changes in the cytosolic free calcium concentration brought about by the addition of different concentrations of phenylephrine to Quin 2-loaded hepatocytes were measured exactly as described under “Experimental Procedures” and the legend to Fig. 5. Panel A shows the peak cytosolic free calcium concentration achieved for each dose of phenylephrine and in panel B the rate of cytosolic free calcium increases is shown (measured at half of the peak increase). Closed symbols indicate the changes which occurred in the absence of insulin and open symbols show the results obtained when cells had been preincubated for 10 min with 50 nM insulin. Each point is the mean f S.E. of values obtained using 4 separate preparations of hepatocytes.

-Insulin

250

“‘j 150 J

+Insulin IB

Phen I

4

FIG. 7 . Effects of phenylephrine and insulin on cytosolic free calcium in the absence of extracellular calcium. Quin 2- loaded hepatocytes were preincubated for 10 min in the absence (panel A ) or presence (panel B ) of 50 nM insulin. An excess of EGTA (2.5 mM) was added to chelate extracellular free calcium 5 min before addition of 0.5 p~ phenylephrine (Phen). Apart from a small blank caused by the removal of calcium bound to extracellular Quin 2, EGTA addition had little effect on the Quin 2 or pyridine nucleotide fluorescence signals and no detectable decrease in basal cytosolic free calcium was observed during the period of the experiment. Other conditions were as described under “Experimental Procedures” and the legend to Fig. 5. Similar traces to those shown here were obtained using 3 other preparations of cells.

glucose 6-phosphate was included in the assay medium, there was a very large increase in the VmaX and a substantial decrease in the SO, of glycogen synthase from control (+15 mM glucose) hepatocytes.

Table I11 shows the effect of increasing concentrations of glucose 6-phosphate on the activity of glycogen synthase measured at saturating (10 mM) UDP-glucose. In this case the data were fitted to a cooperative activation equation with an additional variable parameter to account for the enzyme activity measured in the absence of glucose 6-phosphate (see legend to Table 111). As expected, the calculated activities of glycogen synthase at zero glucose 6-phosphate ( VO) were similar to the V,, values calculated in Table 11. The maximal activation due to glucose 6-phosphate ( VG~P) was similar for all the cell treatments, with only phenylephrine causing a significant, but rather small, decrease. The M0.5 for glucose 6- phosphate was unaffected by insulin but did increase slightly with phenylephrine and in the absence of glucose. The Hill coefficient ( n ) was again not significantly different from 1 under any of the conditions tested. Similar results to those shown in Table I11 were obtained when the UDP-glucose concentration was reduced to 1 mM (not shown). These kinetic data show that for liver glycogen synthase, the -glucose 6-phosphate/+glucose 6-phosphate activity ratio may be an inappropriate expression of hormone-induced changes in enzyme activity since glucose 6-phosphate apparently causes a fixed increment in glycogen synthase activity, which is relatively independent of the V,,, of the enzyme measured in the absence of this activator.

DISCUSSION

The data presented here show that the antagonism by insulin of al-adrenergic activation of phosphorylase and inactivation of glycogen synthase is a rapid event which is maximal after just 4 min of pre-exposure of hepatocytes to insulin. This antagonistic effect of insulin can be explained entirely by a reduction in the elevated cytosolic free Ca2+ concentration resulting from al-agonist treatment. A some- what slower (20-25 min) effect of insulin which occurs in the absence of other hormones is the activation of glycogen synthase. The direct activation of glycogen synthase by insulin in isolated hepatocytes is not associated with any change in the cytosolic free Ca2+ and can occur in the absence of an alteration of phosphorylase a activity. It has previously been proposed that much of the acute hormonal control of glycogen synthase in liver may be secondary to changes in the proportions of phosphorylase a and b (16, 27, 45), but the present data suggest that an additional mechanism must play a role in the activation of glycogen synthase by insulin,

Direct Activation of Glycogen Synthase by Insulin-In agreement with the results of Witters and Avruch (16), we have found that the activation of glycogen synthase by insulin in hepatocytes requires the presence of glucose in the incubation medium for full expression of the insulin effect. An increased activity of glycogen synthase induced by insulin has also been observed in perfused livers from fed rats (14), while the effect of insulin on glycogen synthase in hepatocytes prepared from starved rats and incubated in the absence of substrates reported by Ciudad et al. (17) was rather small. In the present study using hepatocytes from fed rats, we also found that insulin was relatively ineffective as an activator of glycogen synthase in the absence of glucose. However, with an optimal concentration of about 15 mM glucose, the activity of glycogen synthase could be increased up to 2-fold after incubation with insulin. The finding of Katz et al. (64) that other physiological substrates can increase the sensitivity of hepatic glycogen synthase to glucose might suggest that insulin would be effective at lower glucose concentrations in vivo. The reason why a number of investigators have observed either small or no direct effects of insulin on glucose production or glycogen


A 18 - Insulin

Insulin .

-Glucose I p - - n ,Phen I I I

" d 2 4 6 8 1 0 0 2 4 6 8 1 0 [UDP-glucose] (mM) [Glucose 6-phosphate] (mM)

FIG. 8. Hormone effects on the kinetic properties of glycogen synthase. Isolated hepatocytes were incubated for 40 min in the absence of glucose (m, - Glucose), or in the presence of 15 mM glucose with no other additions (0, Control), with 20 nM insulin for the last 25 min (A, Insulin), or with 1 PM phenylephrine for the last 5 min (V, Phen). The effect of varying the concentrations of UDP-glucose (panel A ) or glucose 6-phosphate (panel B ) on the activity of glycogen synthase extracted from these cells is shown. In panel A no glucose 6-phosphate was added except for (0, +G6P) where 10 mM glucose 6-phosphate was added to the extracts from control cells. The UDP-glucose concentration used for the data given in panel B was 10 mM. Each point is the mean of values obtained from duplicate cell incubations using 3 separate cell preparations and a different set of 3 cell preparations was used for panels A and B. The curves were fitted to the data by nonlinear least squares regression using the equations given in the legends to Table I1 (for panel A ) and Table 111 (for panel B).

TABLE I1 Effects of hormone treatment on the sensitivity of glycogen synthase

to UDP-glucose Glycogen synthase was extracted from hepatocytes incubated as

described in the legend to Fig. 8. Glucose (15 mM) was present in all of the incubations except that marked Zero glucose, and insulin was added for the last 25 min when present alone, or for 5 min before phenylephrine when used in combination with this agonist. Phenyi- ephrine was added 5 min before termination of the incubations. Glycogen synthase was assayed as described under "Experimental Procedures" in the absence of glucose 6-phosphate (except that 10 mM glucose 6-phosphate was added for the condition marked + G6P) and the UDP-glucose concentration was varied over the range 0.25- 25 mM. All of the data for each condition from three separate experiments (only one experiment for insulin plus phenylephrine together) were combined and fitted by nonlinear least squares regression to the equation:

u = V,, 1 + (So.~/[UDP-glucoseJ)"

V,. SO, n

unitslg dry weight mM Control 1.00 f 0.04 1.02 f 0.10 1.05 f 0.11 20 nM insulin 1.64 f 0.05" 0.71 f 0.06" 1.17 2 0.12 1 PM phenylephrine 0.26 f 0.02" 1.02 f 0.02 1.10 2 0.02 Zero glucose 0.38 f 0.02" 1.31 2 0.27 0.93 2 0.14 Control + G6P 2.66 f 0.07" 0.34 2 0.02" 1.16 f 0.13 20 nM insulin + 1 PM 0.57 f 0.12 1.01 f 0.21 1.09 2 0.19

phenylephrine Significantly different from the equivalent control value withp <

0.05.

synthase activity in perfused livers or isolated hepatocytes (e.g. Refs. 1, 4, 6, 14, and 65-67) can be ascribed to a variety of factors. Changes of net glucose production or glycogen content are poor parameters for measuring direct insulin effects because of the relatively slow onset of the effect on glycogen synthase, the absence of an effect on phosphorylase, and complications arising from increases of the glucose concentration in the medium (65-67). Other contributing factors, particularly in the earlier studies (for review, see Ref. l), relate to use of high insulin concentrations in preparations

insufficiently free of glucagon, omission of sufficient substrates from the medium (e.g. when livers from fasted animals were used), and short incubation periods with insulin. A further complicating factor with isolated hepatocytes is that some preparations of collagenase produce cells that exhibit no insulin effect, possibly by causing a disruption of the membrane signalling mechanism subsequent to insulin binding.'

Hers and co-workers (27, 45, 68, 69) have shown that in liver, glucose activates a protein phosphatase which converts phosphorylase a to phosphorylase b, and that when phosphorylase a levels fall sufficiently low this increased phosphatase activity is able to activate glycogen synthase. The inverse sensitivities to glucose of phosphorylase and glycogen synthase observed here are consistent with this mechanism, although there does not appear to be a threshold below which phosphorylase a activity must fall before glycogen synthase is affected by increased phosphatase activity, as was previously suggested (15, 16, 27, 45). It has been proposed that insulin may activate hepatic glycogen synthase in a similar manner to glucose (15). However, such a mechanism cannot explain the activation caused by insulin in the present study because the effects of insulin and glucose on glycogen synthase were essentially additive, and unlike glucose, insulin did not alter the activity of phosphorylase a under conditions where it was effective in activating glycogen synthase. In isolated hepatocytes most other workers have also found that phosphorylase is insensitive to insulin (Refs. 4-6 but cf. Ref. 16), and it seems probable that decreases in hepatic phosphorylase a activity brought about by insulin administration in vivo (15) could be due to indirect effects on the levels or effectiveness of other circulating hormones.

In common with the situation in skeletal muscle (18, 23, 24), the activation by insulin of hepatic glycogen synthase probably results from a decrease in the phosphorylation state of at least one of the four or more phosphorylation sites (62, 63). This could result from an increase in the activity of a protein phosphatase or a decrease in the activity of a protein kinase, but in either case the phosphatase or kinase must

Hormonal Regulation of Liver Glycogen Metabolism TABLE I11

Effect of hormone treatment on the sensitivity of glycogen synthase to activation by glucose 6-phosphate Isolated hepatocytes were incubated exactly as described in the legends to Fig. 8 and Table 11. Glycogen synthase

was subsequently assayed with UDP-glucose at 10 mM and glucose 6-phosphate varied from 0-10 mM. The values shown were calculated by fitting the combined data from 3 separate experiments to the equation:

5971

where v is the measured velocity, [G6P] is the concentration of glucose 6-phosphate added, VO is the calculated velocity in the absence of glucose 6-phosphate, VGGP is the calculated maximum activation by glucose 6-phosphate, Mo.5 is the concentration of glucose 6-phosphate giving half-maximal activation, and n is the Hill coefficient.

VD VWP VGSP + vo MD.~ n unitslg d r y weight mM

Control 1.22 f 0.11 2.38 f 0.37 3.60 1.37 f 0.52 0.94 f 0.23 20 nM insulin 1.74 f 0.03" 2.37 f 0.12 4.11 1.41 f 0.18 0.90 k 0.07 1 PM phenylephrine 0.39 f 0.02" 1.82 f 0.08" 2.21 2.02 f 0.18" 1.13 f 0.08 Zero glucose 0.66 f 0.04" 2.47 +- 0.19 3.16 2.07 f 0.37 0.95 f 0.10 ~

Significantly different from the equivalent control value with p < 0.05.

show specificity for glycogen synthase over phosphorylase. An inhibition of glycogen synthase kinase 3 has been proposed to mediate the effect of insulin in skeletal muscle (23), and this enzyme has recently been purified from rat and rabbit livers (62, 63). Another potential site of insulin action is a glycogen synthase-specific phosphatase which could be stim- ulated by the inactivation of an inhibitor protein analogous to that found in muscle (70) or by the direct action of the insulin "mediator" as suggested by Larner et al. (71). Although insulin has been proposed to act through changes of cytosolic free ea'+ (46-48), our results using both phosphorylase a activity as an indirect indicator and Quin 2 as a direct indicator of cytosolic free ca'+ clearly show that insulin alone does not alter the basal concentration of calcium in the cytosol of rat liver cells.

The role of glucose in the activation by insulin of hepatic glycogen synthase is unclear. It is possible that glucose changes the phosphorylation state of a specific site on glycogen synthase, which then permits insulin-induced changes in the phosphorylation of a separate site to be fully expressed in terms of altered enzyme activity. Some support for a syner- gistic effect of one phosphorylation site in allowing the phosphorylation of a second site to influence enzyme activity has been obtained using purified rabbit liver glycogen synthase (63).

Insulin Antagonism of al-Adrenergic Effects-The activation of phosphorylase and inactivation of glycogen synthase caused by al-adrenergic agents, vasopressin and angiotensin 11, in liver are believed to result from an increase of the cytosolic free Ca'+ concentration (26-35). This increase of the cytosolic free ea'+ is secondary to the breakdown of phosphatidylinositol 4,5-bisphosphate in the plasma membrane and formation of diacylglycerol and inositol 1,4,5-trisphosphate (35, 39, 54, 72, 73). Based on its ability to release ea'+ from the endoplasmic reticulum of permeabilized hepatocytes, inositol 1,4,5-trisphosphate has been proposed to be the second messenger for calcium mobilization (35, 52-54). While the elevation of cytosolic free Ca" presumably causes an increase in phosphorylase a activity by stimulating phosphorylase kinase (27-29), the mechanism of glycogen synthase inhibition by the ea'+-mobilizing hormones is less clear. The ea'+/ calmodulin-dependent glycogen synthase kinase, which has been purified from rabbit liver (58-60), is an obvious candi- date for mediating the effects of Caz+ on glycogen synthase. However, although this kinase phosphorylates the liver enzyme with some degree of inactivation (62, 63), experiments with crude liver extracts have suggested that ea'+ inactivation

of glycogen synthase occurs primarily through increased phosphorylase a levels with the consequent inhibition of glycogen synthase phosphatase (61), as suggested by Hers (27).

Phorbol esters, which stimulate protein kinase C, inactivate glycogen synthase in isolated hepatocytes (74) and since ea2+- mobilizing hormones can increase the levels of diacylglycerol (a physiological activator of protein kinase C) in liver cells (39), it may be that this represents a ea'+-independent mechanism of action for these hormones (75). However, there is some question as to how the phorbol esters affect glycogen synthase in the intact liver cell because phosphorylation of the purified enzyme by protein kinase C has been reported to have no effect on its catalytic activity (62), although other studies have shown a decrease in the -glucose 6-P/+glucose 6-P activity ratio concomitant with increased protein kinase C-mediated phosphorylation of liver glycogen synthase (76). The relative importance of the Ca'+-dependent and ea'+- independent mechanisms for inactivation of glycogen synthase by al-adrenergic agents in the intact cell remains to be established.

It has been suggested that insulin may act at the level of the a,-receptor ( l l ) , but we have previously shown that at least some of the effects of phenylephrine (acting through al- receptors) on inositol lipid metabolism were insensitive to insulin (37). From the present work it seems unlikely that a major effect of insulin could be to alter the binding affinity of agonists for the cyl-receptor, because insulin did not mark- edly shift the dose response curves for phenylephrine-induced cytosolic free ea2+ increase but instead decreased the maximal response to phenylephrine. Nevertheless, it is clear that insulin must antagonize the effects of al-adrenergic agents at some step prior to ea'+ mobilization. Possible mechanisms to account for the insulin antagonism of the elevation of cytosolic free Ca" mediated by al-adrenergic agents must take into account the following findings. Of the ea2+-mobilizing hormones, only the effects of a,-agonists are sensitive to antagonism by insulin (5, 6,9, 11,36), insulin does not affect al-agonist-induced decrease of phosphatidylinositol content or turnover (37), the al-agonist-induced peak increase of cytosolic free ea'+ (in the presence and absence of external ea'+) is only partially inhibited, and the duration of the increase of the cytosolic free ea'+ in the presence of external ea'+ is diminished. Further studies are required to elucidate whether insulin acts at the transducing step between al- receptor occupancy and activation of the plasma membrane phosphodiesterase responsible for polyphosphoinositide breakdown. Other possibilities include an alteration of the


relative affinity of the phosphodiesterase for its inositol lipid substrates (phosphatidylinositol, phosphatidylinositol 4- phosphate, and phosphatidylinositol 4,5-bisphosphate), thereby changing the proportions of diacylglycerol and inositol 1,4,5-trisphosphate that are produced (77), or an activation of the inositol trisphosphate phosphatase. In addition, the present evidence suggests that insulin causes an inhibition of the secondary increased permeability of the plasma membrane to Ca2+; this effect may also be linked to alterations of inositol lipid metabolism.

Effects of Hormones on the Kinetic Properties of Hepatic Glycogen Synthuse-The effects of pretreating hepatocytes with insulin, phenylephrine, and glucose on the kinetics of subsequently extracted glycogen synthase have been examined in the present study. Based on work in other tissues, the effects which we observed on the kinetics of liver glycogen synthase presumably represent alterations in the phosphorylation state of the enzyme rather than effects of non- covalently bound effectors, and some evidence for this comes from our finding that dilution of the extracts did not influence enzyme activity. It has recently been shown that liver glycogen synthase is susceptible to multisite phosphorylation by kinases similar to those which act on the muscle enzyme (62, 63). However, the phosphorylated peptides obtained by cyan- ogen bromide digestion of liver and muscle glycogen synthase are different, and it appears from peptide mapping that there may be differences in the primary sequences of these two enzymes (62,63). Differences between the glycogen synthases of liver and muscle are also suggested by our findings that hormones and glucose predominantly alter the V,, of the liver enzyme, while in muscle the s0.5 for UDP-glucose and the M0.5 for glucose 6-phosphate are the parameters affected by hormones and their related changes in phosphorylation state (18, 19). Camici et al. (63) recently showed that phosphorylation of purified liver glycogen synthase by purified glycogen synthase kinase 3 or CAMP-dependent protein kinase also caused a change in the Vmax of this enzyme.

It has been noted previously that glucose increases the activity of both the I (-glucose 6-phosphate)- and the D (+glucose 6-phosphate)-forms of liver glycogen synthase (6, 64). The present data show that the activation of both forms by a submaximal concentration of glucose (15 mM) can be explained entirely by an increase in the VmaX of the enzyme, while the additional increment in activity caused by glucose 6-phosphate is the same for glycogen synthase extracted from cells incubated with or without glucose. A similar fixed increment of activation by glucose 6-phosphate was observed with glycogen synthase which had either been activated by pretreating the cells with insulin or inactivated by phenylephrine treatment. Thus, it appears that glucose 6-phosphate is almost equally effective in activating the a and the b forms of glycogen synthase from rat liver. The activity ratio (+glucose 6-phos- phatel-glucose 6-phosphate) which is routinely used to ex- press changes in the phosphorylation state of the muscle enzyme is consequently not a reliable index of similar changes in liver glycogen synthase.

In the absence of glucose 6-phosphate, glycogen synthase extracted from hepatocytes incubated with 15 mM glucose had a V,,, of about 1 unit/g of dry weight and an sO.5 for UDP-glucose of 1 mM. Insulin increased the Vmax to 1.65 and phenylephrine decreased it to 0.26 unit/g of dry weight. In a previous study (17) using hepatocytes from starved rats incubated without substrates, no effect of insulin on the Vmax

was observed and instead insulin caused a small (about 20%) decrease in the s0.5 for UDP-glucose and the Mo.5 for glucose 6-phosphate. However, in that study the basal s0.5 of glycogen

synthase was 20-fold higher than that calculated under our conditions, and it seems possible that the enzyme was in a very different form, perhaps resulting from changes in the phosphorylation at sites involved in long term regulation by nutritional state rather than those due to the acute effects of hormones which we have observed. In addition to showing that both insulin and phenylephrine bring about their major effects on glycogen synthase by altering its V,, in hepatocytes from fed rats, our kinetic studies also identified some differences in the effects of these two agonists which indicate that they do not act in an entirely opposite manner. Insulin slightly lowered the for UDP-glucose while phenylephrine was without affect on this parameter and, conversely, phenylephrine increased the M0.5 for glucose 6-phosphate but insulin produced no change. These results might suggest that insulin and phenylephrine cause changes at different phosphorylation sites on liver glycogen synthase. This proposal is consistent with the data discussed above, indicating that different protein kinases and/or phosphatases probably mediate the effects of insulin and al-agonists on glycogen synthase. However, a full understanding of the complex hormonal control of hepatic glycogen synthase must await a complete characterization of all of the phosphorylation sites and their associated kinases and phosphatases.

1. 2. 3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16. 17.

18.

19. 20.

21.

22.

23.

24.

REFERENCES Exton, J. H., and Park, C. R. (1972) Handb. Physwl. 7,437-455 Hems, D. A., and Whitton, P. D. (1980) Physiol. Rev. 60, 1-50 Jungas, R. L. (1981) in Handbook of Diabetes Mellitus (Brownley,

M., ed) Vol. 2, pp. 151-195, Garland STPM Press, New York Massague, J., and Guinovant, J. J. (1977) FEBS Lett. 82, 317- 320

Blackmore, P. F., Assimacopoulos-Jeannet, F., Chan, T. M., and Exton, J. H. (1979) J. Bwl. Chem. 254,2828-2834

Strickland, W. G., Blackmore, P. F., and Exton, J. H. (1980) Diabetes 29,617-622

Williams, T. F., Exton, J. H., Friedmann, N., and Park, C. R. (1971) Am. J. Physiol. 221, 1645-1651

Van de Werve, G., Hue, L., and Hers, H . 4 . (1977) Bwchem. J.

Ma, G. V., Gove, C. D., and Hems, D. A. (1978) Biochem. J. 174,

Massague, J., and Guinovant, J. J. (1978) Biochim. Biophys. Acta

Dehaye, J. P., Hughes, B. P., Blackmore, P. F., and Exton, J. H.

Johnson, M. E. M., Das, N. M., Butcher, F. R., and Fain, J. N.

Beynen, A. C., and Geelen, M. J. H. (1981) Horm. Metab. Res.

Miller, T. B., Jr., and Larner, J. (1973) J. Biol. Chem. 248,3483-

Van de Werve, G., Stalmans, W., and Hers, H. G. (1977) Biochem.

Witters, L. A., and Avruch, J. (1978) Biochemistry 17,406-410 Ciudad, C. J., Bosch, F., and Guinovant, J. J. (1981) FEBS Lett.

Larner, J., Lawrence, J. C., Walkenbach, R. J., Roach, P. J., Hazen, R. J., and Huang, L. C. (1978) Adu. Cyclic Nucleotide Res. 9,425-439

162, 135-142

761-768

643,269-272

(1981) Biochem. J. 194,949-956

(1972) J. Biol. Chem. 247, 3229-3235

13,376-378

3488

J. 162,143-146

129,123-126

Roach, P. J. (1981) Curr. Top. Cell. Regul. 20, 45-105 Cohen, P., Yellowlees, D., Aitken, A., Donella-Deana, A., Hem-

mings, B. A., and Parker, P. J. (1982) Eur. J. Biochem. 124,

Picton, C., Aitken, A., Bilham, T., and Cohen, P. (1982) Eur. J.

Parker, P. J., Embi, N., Caudwell, F. B., and Cohen, P. (1982)

Parker, P. J., Caudwell, F. B., and Cohen, P. (1983) Eur. J.

Lawrence, J. C., Jr., Hiken, J. F., DePaoli-Roach, A. A., and

21-35

Biochem. 124,37-45

Eur. J. Biochem. 124,47-55

Bwchem. 130,227-234

Roach, P. J. (1983) J. Biol. Chem. 258,10710-10719

Hormonal Regulation of L1 25. Hue, L., Feliu, J. E., and Hers, H . 4 . (1978) Biochem. J. 176 ,

26. Blackmore, P. F., Hughes, B. P., Shuman, E. A., and Exton, J.

27. Hers, H. G. (1976) Annu. Reu. Biochem. 4 5 , 167-189 28. Exton, J. H. (1980) Am. J. Physiol. 2 3 8 , E3-El2 29. Williamson, J . R., Cooper, R. H., and Hoek, J. B. (1981) Biochim.

30. Blackmore, P. F., Brumley, F. T., Marks, J. L., and Exton, J. H.

31. Blackmore, P. F., Dehaye, J.-P., and Exton, J. H. (1979) J. Biol.

32. Murphy, E., Coll, K., Rich, T. L., and Williamson, J. R. (1980)

33. Joseph, S. K., and Williamson, J. R. (1983) J. Biol. Chem. 258,

34. Charest, R., Blackmore, P. F., Berthon, B., and Exton, J. H.

35. Thomas, A. P., Alexander, J., and Williamson, J. R. (1984) J.

36. Whitton, P. D., Rodrigues, L. M., and Hems, D. A. (1978)

37. Thomas, A. P., and Williamson, J. R. (1983) J. Biol. Chem. 258 ,

38. Meijer, A. J., Gimpel, J. A., Deleeuw, G. A., Tager, J. M., and Williamson, J. R. (1975) J. Biof. Chem. 2 5 0 , 7728-7738

39. Thomas, A. P., Marks, J. S., Coll, K. E., and Williamson, J. R. (1983) J. Biol. Chem. 258,5716-5725

40. Tsien, R. Y . (1980) Biochemistry 19,2396-2404 41. Tsien, R. Y. , Pozzan, T., and Rink, T. J. (1982) J. Cell Biol. 94,

42. Gilboe, D. P., Larson, K. L., and Nuttall, F. Q. (1972) Anal.

43. Uhing, R. J., Janski, A. M., and Graves, D. J. (1979) J . Bwl.

44. Thomas, J . A., Schlender, K. K., and Larner, J. (1968) Aml .

45. Hue, L., Bontemps, F., and Hers, H. G. (1975) Biochem. J. 152,

46. Kissebah, A. H., Clarke, P., Vydelingum, N., Hope-Gill, H., Tulloch, B., and Fraser, T. R. (1975) Eur. J. Clin. Invest. 5, 339-349

47. Hope-Gill, H. F., Kissebah, A. H., Clarke, P., Vydelingum, N., Tulloch, B., and Fraser, T. R. (1976) Horm. Metab. Res. 8, 184-190

48. Clausen, T., and Martin, B. R. (1977) Bwchem. J. 164,251-255 49. Chrisman, T. D., Vandenheede, J. R., Khandelwal, R. L., Gella,

F. J., Upton, J. D., and Krebs, E. G. (1980) Adv. Enzyme Regul.

50. Hobson, C. H., Upton, J. D., Loten, E. G., and Rennie, P. I. C.

791-797

H. (1982) J. Biol. Chem. 257,190-197

Bwphys. Acta 639,243-295

(1978) J. Biol. Chem. 253,4851-4858

Chem. 254,6945-6950

J. Biol. Chem. 255,6600-6608

10425-10432

(1983) J. Biol. Chem. 258,8769-8773

Bwl. Chem. 259,5574-5584

Biochem. J. 176,893-898

1411-1414

325-334

Bwchem. 47,20-27

Chem. 254,3166-3169

Biochem. 2 5 , 486-499

105-114

18, 145-159

(1980) J. Cycfic Nucleotide Res. 6 , 179-188

iuer Glycogen Metabolism 5973 51. Chan, T. M., and Exton, J. H. (1977) J. Biol. Chem. 252,8645-

8651 52. Joseph, S. K., Thomas, A. P., Williams, R. J., Imine, R. F., and

Williamson, J. R. (1984) J. Bwl. Chem. 259,3077-3081 53. Streb, H., Imine, R. F., Berridge, M. J., and Schulz, I. (1983)

Nature 306,67-69 54. Williamson, J. R., Cooper, R. H., Joseph, S. K., and Thomas, A.

P. (1985) Am. J. Physwl. 2 4 8 , C203-C216 55. DeWitt, L. M., and Putney, J. W. (1983) FEBS Lett. 160 , 259-

263 56. Reinhart, P. H., Taylor, W. M., and Bygrave, F. L. (1984)

Bwchem. J. 223, 1-13 57. Jett, M. F., and Soderling, T. R. (1979) J. Biol. Chem. 254,6739-

6745 58. Payne, M. E., and Soderling, T. R. (1980) J. Biol. Chem. 2 5 5 ,

59. Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J. (1982) J. Biol.

60. Payne, M. E., Schworer, C. M., and Soderling, T. R. (1983) J.

61. Strickland, W. G., Imazu, M., Chrisman, T. D., and Exton, J. H.

62. Imazu, M., Strickland, W. G., Chrisman, T., and Exton, J. H.

63. Camici, M., Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J.

64. Katz, J., Golden, S., and Wals, P. A. (1979) Biochem. J. 180,

65. Haft, D. E. (1968) Diabetes 17 , 244-250 66. Seglen, P. 0. (1973) FEBS Lett. 36,309-312 67. Walker, P. R. (1977) Biochim. Biophys. Acta 496 , 255-263 68. Stalmans, W., DeWulf, H., Lederer, B., and Hers, H. G. (1970)

69. Stalmans, W., DeWulf, H., and Hers, H. G. (1971) Eur. J.

70. Foulkes, J. G., Jefferson, L. S., and Cohen, P. (1980) FEBS Lett.

71. Larner, J., Galasko, G., Cheng, K., DePaoli-Roach, A. A., Huang, L., Daggy, P., and Kellogg, J. (1979) Science (Wash. D. C . )

72. Michell, R. H., Kirk, C. J., Jones, L. M., Downes, C. P., and Creba, J. A. (1981) Philos. Trans. R. SOC. hnd. B Biol. Sci.

73. Creba, J. A., Downes, C. P., Hawkins, P. T., Brewster, G., Michell, R. H., and Kirk, C. J. (1983) Biochem. J. 212,733-747

74. Roach, P. J., and Goldman, M. (1983) Proc. Natl. Acad. Sci. U.

75. Garrison, J. C., Johnsen, D. E., and Campanile, C. P. (1984) J.

76. Ahmad, Z., Lee, F.-T., DePaoli-Roach, A., and Roach, P. J. (1984)

77. Wilson, D. B., Bross, T. E., Hofmann, S. L., and Majerus, P. W.

8054-8056

Chem. 257,8348-8355

Bwl. Chem. 258,2376-2382

(1983) J. Biol. Chem. 258,5490-5497

(1984) J. Biol. Chem. 259 , 1813-1821

(1984) J. Biol. Chem. 259, 2466-2473

389-402

Eur. J. Biochem. 15,9-12

Bwchem. 18,582-587

112,21-24

206,1408-1410

296, 123- 138

S. A. 80,7170-7172

Biol. Chem. 259 , 3283-3292

J. Biol. Chem. 259,8743-8747

(1984) J. Bwl. Chem. 259,11718-11724

interactions between insulin and al-adrenergic agents in the

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