Molecular and Cellular Endocrinology, 68 (1990) Rl-R5 Elsevier Scientific Publishers Ireland, Ltd.
MOLCEL
Rl
02208
Rapid Paper
Effect of adrenalectomy and glucocorticoid treatment on the levels of an insulin-sensitive glycosyl-phosphatidylinositol in isolated rat hepatocytes M.A. Cabello ‘, J.A. Skchez-Arias
‘, A. Liras I, J.M. Mato 2 and J.E. Feliu 1
’ Servicio de Endocrinologia Experimental, Hospital Puerta de Hierro, Universidad Aut&oma de Madrid, 28035-Madrid, and ’ Metabolismo,
Nutricih
y Hormones, Fundacih Jimhez Diaz, Instituto de Investigaciones 28040-Madrid Spain
Spain, BiomPdieas del C.S. I. C.
(Received 24 October 1989; accepted 25 October 1989)
Key work
Adrenalectomy; Glucocorticoid;
Glycosyl-phosphatidylinositol;
Insulin resistance; (Rat hepatocytes)
sumIualy Insulin resistance caused by dexamethasone administration to rats was accompanied by a marked decrease in the hepatocyte content of an insulin-sensitive glycosyl-phosphatidylinositol, as well as by a blockade of its hydrolysis in response to this hormone. In contrast, bilateral adrenalectomy provoked a significant increase of the cellular glycosyl-phosphatidylinositol levels. Under all the assayed metabolic conditions, a close direct correlation was established between the basal content of this compound and the number of insulin receptors present in the isolated hepatocytes.
Introduction Insulin was shown to promote the hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PI), localized on the outer surface of the cell membrane (Alvarez et al., 1988), with release of its polar head group (Saltiel and Cuatrecasas, 1986; Saltiel et al., 1986; Mato et al., 1987a). This polar group is an inositol phospho-oligosaccharide, of partially known chemical structure (Saltiel and Cuatrecasas, 1986; Sahiel et al., 1986; Mato et al., 1987a, b) which has been shown to mimic insulin action on high affinity CAMP-phosphodiesterase, pyruvate dehydrogenase and adenylate cyclase (Saltiel, 1987; Gottschalk and Jarret, 1988). In
Address for correspondence: J.E. Fellu, Servicio de Endocrinologia Experimental, Hospital Puerta de Hierro, San Martin de Porres 4, 28035-Madrid, Spain. 0303-7207/90/$03.50
intact adipocytes, it inhibits isoproterenol-dependent stimulation of phospholipid methyltransferase (Kelly et al., 1986) and lipolysis (Kelly et al., 1987), while in isolated rat hepatocytes it antagonizes glucagon effects on glycogen phosphorylase, pyruvate kinase and cyclic AMP levels (Alvarez et al., 1987). Moreover, the polar group was reported to copy insulin-mediated effects on the phosphorylation/dephosphorylation state of target proteins of this hormone (Alemany et al., 1987). As a consequence of these results, it has been hypothesized that the polar head group of the glycosyl-PI could participate as a second messenger in the cellular actions of insulin. It seemed interesting to investigate the levels of the glycosyl-PI, before and after insulin treatment; in hepatocytes isolated from rats with insulin resistance caused by dexamethasone treatment (Amatruda et al., 1985), and to compare them with those found in hepatocytes isolated from
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
R2
adrenalectomized animals, in which the effects of insulin are known to be more marked (Wajchenberg et al., 1964). Our results show a marked decrease in the hepatic content of the insulin-sensitive glycosyl-PI, together with a blockade of its hydrolysis in response to this hormone, in rats with glucocorticoid-induced insulin resistance. The reverse occurred in adrenalectomized animals. Materials and methods Materials. Porcine insulin was obtained from Novo Industri (Copenhagen, Denmark). Collagenase was from Boehringer Mannheim (Mannheim, F.R.G.). [U-‘4C]Glucose, porcine mono[‘251]insulin and [l-‘4C]isethionyl acetimidate were purchased from Amersham International (Amersham, U.K.). Dexamethasone phosphate (Fortecortin) was obtained from Merck Co. (Darmstadt, F.R.G.). All the remaining reagents were of analytical grade. Animals. Fed male Wistar rats (180-200 g) from our inbred colony were used. Part of the animals were submitted to bilateral adrenalectomy, or to a simulated operation, under Pentothal anesthesia (2 mg/lOO g of body weight, intravenously), 5 days before they were used; adrenalectomized rats received 0.45% NaCl solution as drinking water. Treatment of rats with glucocorticoids was carried out by administration of three doses of dexamethasone phosphate (0.2 mg/lOO g of body weight, subcutaneously) 50, 26 and 2 h before the isolation of hepatocytes. Hepatocyte isolation and cell incubations. Hepatocytes were isolated by perfusion of the liver with collagenase; cells were suspended in Krebs-Henseleit medium in the presence of 10 mM glucose and incubated as reported elsewhere (Feliu et al., 1983). After 30 min of preincubation, insulin or saline was added to cell suspensions; 3 min later, 1 ml aliquots were taken for isotopic labelling of the insulin-sensitive glycosyl-PI. Afterwards, [U-‘4C]glucose was added to the incubation medium, in order to attain a final concentration of 20 mM (0.75 pCi/pmol). 30 min later, samples of cell incubations were taken for the measurement of [U-14C]glucose incorporation into glycogen (Mojena et al., 189). The insulin-sensitive glycosyl-PI was labelled with [l-‘4C]isethionyl
acetimidate and isolated by sequential TLC, as previously described (Alvarez et al., 1988). (‘251]InsuIin binding studies. Binding of mono-[ 125I]insulin to isolated rat hepatocytes was performed by a modification of the method described by Fantus et al. (1981). Hepatocytes were suspended (2 X lo6 cells/ml) in medium A, containing 50 mM Hepes and 0.2% BSA, pH 7.4; then, 250 ~1 aliquots of this cell suspension were incubated with labelled insulin (0.4 ng/ml) in the absence or in the presence of unlabelled porcine insulin (4 ng/ml to 60 pgg/ml), with agitation (120 strokes/mm), at 15 o C for 2 h. Hepatocytes were removed by centrifugation (13,000 x g for 1 min); after washing twice with medium A, the radioactivity retained in cell pellets was measured in a gamma counter. Nonspecific binding was determined by adding 60 pg/ml of unlabelled insulin, and was less than 5% of the total binding in all experiments. The total number of binding sites per cell (R,) and the affinity for insulin at low insulin concentration (K,) were derived by Scatchard analysis of the competition curves. Statistical analysis. Statistical significance of differences between values was calculated by the paired Student’s t-test. The differences were considered statistically significant when the p value was less than 0.05. Results As shown in Table 1, bilateral adrenalectomy caused a 50% increase in the amount of insulinsensitive glycosyl-PI present in liver cells, as compared to that measured in hepatocytes isolated from sham-operated animals. In contrast, dexamethasone treatment reduced by about 60% the basal levels of this compound in hepatocytes isolated from either adrenalectomized, sham-operated or normal rats. Simulated adrenalectomy did not significantly change the amount of glycosyl-PI present in the cells, when compared to normal rats. Under all the above-mentioned metabolic conditions, the basal amount of insulin-sensitive glycosyl-PI paralleled the calculated total number of insulin receptors present in the cells (Table 1). In fact, a close direct correlation could be established between these two parameters (r = 0.89; p < 0.02;
R3 TABLE 1 EFFECT OF ADRENALECTOMY AND DEXAMETHASONE TREATMENT ON THE LEVELS OF GLYCOSYL-PI AND ON THE NUMBER OF INSULIN RECEPTORS IN ISOLATED RAT HEPATOCYTES Values of glycosyl-PI are the means* SEM of different experiments; the numbers of them are indicated in parentheses. Insulin receptors are expressed by the calculated mean value of two or three experiments carried out in triplicate. Insulin receptors (number X 10 - 3/tell)
Glycosyl-PI (dpm/mg of protein) Normal rats Normal rats + dexamethasone Sham-operated Sham-operated rats + dexamethasone Adrenalectomized rats Adrenalectomized rats + dexamethasone
3,230 + 538
(7)
280
1,153+ 77* 4,022+711
(4) (5)
117 352
1,696 + 602 * (3) 6,203 + 329 * * (4)
231 376
2,429 + 989 *
270
(4)
significantly elevated (Table l), but its hydrolysis in response to insulin was also slightly increased (Fig. 2), as compared to that observed in hepatocytes obtained from sham-operated rats. This more pronounced effect of insulin was clearly observed at the lowest concentration of insulin assayed (about 25 and 8% of glycosyl-PI reduction in response to 0.1 nM insulin, respectively, for hepatocytes isolated from adrenalectomized or sham-operated rats) (Figs. 1 and 2). Treatment of the animals with dexamethasone again markedly blocked the hydrolysis of the glycosyl-PI, as well as the stimulation of glycogen synthesis in response to insulin. Discussion In man (Rizza et al., 1982) and animals (McMahon et al., 1988), glucocorticoid excess is asso-
* p -C0.05 vs. the corresponding animals not treated with the steroid. ** p -C0.05 vs. sham-operated rats.
n = 6). Neither bilateral adrenalectomy nor dexamethasone treatment significantly affected the estimated K, value for insulin of isolated hepatocytes which ranged between 0.3 and 0.4 x lo9M-l. As expected, insulin caused a dose-dependent hydrolysis of the glycosyl-PI, with the consequent reduction of its cellular levels. Thus, in hepatocytes isolated from sham-operated rats (Fig. l), 1 nM insulin decreased by about 20% the amount of hepatocyte glycosyl-PI. Simultaneously, insulin at all the assayed concentrations - significantly stimulated the incorporation of [U-r4C]glucose into glycogen (3.1 + 0.76 and 4.6 f 0.89 pmol of [U-14C]glucose converted into glycogen/g of cells X 30 min, respectively, for hepatocytes incubated under basal conditions or in the presence of 0.1 nM insulin; n = 3 experiments, p -c 0.01). Treatment of the rats with dexamethasone almost completely blocked the stimulatory effect of insulin on both glycosyl-PI hydrolysis and [U-14C]glucose incorporation into glycogen. In hepatocytes isolated from adrenalectomized rats, not only was the basal level of glycosyl-PI
-10
-9 ~~~[INSULIN]
-9 M
Fig. 1. Effect of insulin on glycosyl-PI levels and on the rate of glycogen synthesis, in hepatocytes isolated from sham-operated rats, treated (I) or not (0) with dexamethasone. Values are the means * SEM of three experiments.
R4
B”1 70 t O
MOLCEL
Rl
02208
Rapid Paper
Effect of adrenalectomy and glucocorticoid treatment on the levels of an insulin-sensitive glycosyl-phosphatidylinositol in isolated rat hepatocytes M.A. Cabello ‘, J.A. Skchez-Arias
‘, A. Liras I, J.M. Mato 2 and J.E. Feliu 1
’ Servicio de Endocrinologia Experimental, Hospital Puerta de Hierro, Universidad Aut&oma de Madrid, 28035-Madrid, and ’ Metabolismo,
Nutricih
y Hormones, Fundacih Jimhez Diaz, Instituto de Investigaciones 28040-Madrid Spain
Spain, BiomPdieas del C.S. I. C.
(Received 24 October 1989; accepted 25 October 1989)
Key work
Adrenalectomy; Glucocorticoid;
Glycosyl-phosphatidylinositol;
Insulin resistance; (Rat hepatocytes)
sumIualy Insulin resistance caused by dexamethasone administration to rats was accompanied by a marked decrease in the hepatocyte content of an insulin-sensitive glycosyl-phosphatidylinositol, as well as by a blockade of its hydrolysis in response to this hormone. In contrast, bilateral adrenalectomy provoked a significant increase of the cellular glycosyl-phosphatidylinositol levels. Under all the assayed metabolic conditions, a close direct correlation was established between the basal content of this compound and the number of insulin receptors present in the isolated hepatocytes.
Introduction Insulin was shown to promote the hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PI), localized on the outer surface of the cell membrane (Alvarez et al., 1988), with release of its polar head group (Saltiel and Cuatrecasas, 1986; Saltiel et al., 1986; Mato et al., 1987a). This polar group is an inositol phospho-oligosaccharide, of partially known chemical structure (Saltiel and Cuatrecasas, 1986; Sahiel et al., 1986; Mato et al., 1987a, b) which has been shown to mimic insulin action on high affinity CAMP-phosphodiesterase, pyruvate dehydrogenase and adenylate cyclase (Saltiel, 1987; Gottschalk and Jarret, 1988). In
Address for correspondence: J.E. Fellu, Servicio de Endocrinologia Experimental, Hospital Puerta de Hierro, San Martin de Porres 4, 28035-Madrid, Spain. 0303-7207/90/$03.50
intact adipocytes, it inhibits isoproterenol-dependent stimulation of phospholipid methyltransferase (Kelly et al., 1986) and lipolysis (Kelly et al., 1987), while in isolated rat hepatocytes it antagonizes glucagon effects on glycogen phosphorylase, pyruvate kinase and cyclic AMP levels (Alvarez et al., 1987). Moreover, the polar group was reported to copy insulin-mediated effects on the phosphorylation/dephosphorylation state of target proteins of this hormone (Alemany et al., 1987). As a consequence of these results, it has been hypothesized that the polar head group of the glycosyl-PI could participate as a second messenger in the cellular actions of insulin. It seemed interesting to investigate the levels of the glycosyl-PI, before and after insulin treatment; in hepatocytes isolated from rats with insulin resistance caused by dexamethasone treatment (Amatruda et al., 1985), and to compare them with those found in hepatocytes isolated from
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
R2
adrenalectomized animals, in which the effects of insulin are known to be more marked (Wajchenberg et al., 1964). Our results show a marked decrease in the hepatic content of the insulin-sensitive glycosyl-PI, together with a blockade of its hydrolysis in response to this hormone, in rats with glucocorticoid-induced insulin resistance. The reverse occurred in adrenalectomized animals. Materials and methods Materials. Porcine insulin was obtained from Novo Industri (Copenhagen, Denmark). Collagenase was from Boehringer Mannheim (Mannheim, F.R.G.). [U-‘4C]Glucose, porcine mono[‘251]insulin and [l-‘4C]isethionyl acetimidate were purchased from Amersham International (Amersham, U.K.). Dexamethasone phosphate (Fortecortin) was obtained from Merck Co. (Darmstadt, F.R.G.). All the remaining reagents were of analytical grade. Animals. Fed male Wistar rats (180-200 g) from our inbred colony were used. Part of the animals were submitted to bilateral adrenalectomy, or to a simulated operation, under Pentothal anesthesia (2 mg/lOO g of body weight, intravenously), 5 days before they were used; adrenalectomized rats received 0.45% NaCl solution as drinking water. Treatment of rats with glucocorticoids was carried out by administration of three doses of dexamethasone phosphate (0.2 mg/lOO g of body weight, subcutaneously) 50, 26 and 2 h before the isolation of hepatocytes. Hepatocyte isolation and cell incubations. Hepatocytes were isolated by perfusion of the liver with collagenase; cells were suspended in Krebs-Henseleit medium in the presence of 10 mM glucose and incubated as reported elsewhere (Feliu et al., 1983). After 30 min of preincubation, insulin or saline was added to cell suspensions; 3 min later, 1 ml aliquots were taken for isotopic labelling of the insulin-sensitive glycosyl-PI. Afterwards, [U-‘4C]glucose was added to the incubation medium, in order to attain a final concentration of 20 mM (0.75 pCi/pmol). 30 min later, samples of cell incubations were taken for the measurement of [U-14C]glucose incorporation into glycogen (Mojena et al., 189). The insulin-sensitive glycosyl-PI was labelled with [l-‘4C]isethionyl
acetimidate and isolated by sequential TLC, as previously described (Alvarez et al., 1988). (‘251]InsuIin binding studies. Binding of mono-[ 125I]insulin to isolated rat hepatocytes was performed by a modification of the method described by Fantus et al. (1981). Hepatocytes were suspended (2 X lo6 cells/ml) in medium A, containing 50 mM Hepes and 0.2% BSA, pH 7.4; then, 250 ~1 aliquots of this cell suspension were incubated with labelled insulin (0.4 ng/ml) in the absence or in the presence of unlabelled porcine insulin (4 ng/ml to 60 pgg/ml), with agitation (120 strokes/mm), at 15 o C for 2 h. Hepatocytes were removed by centrifugation (13,000 x g for 1 min); after washing twice with medium A, the radioactivity retained in cell pellets was measured in a gamma counter. Nonspecific binding was determined by adding 60 pg/ml of unlabelled insulin, and was less than 5% of the total binding in all experiments. The total number of binding sites per cell (R,) and the affinity for insulin at low insulin concentration (K,) were derived by Scatchard analysis of the competition curves. Statistical analysis. Statistical significance of differences between values was calculated by the paired Student’s t-test. The differences were considered statistically significant when the p value was less than 0.05. Results As shown in Table 1, bilateral adrenalectomy caused a 50% increase in the amount of insulinsensitive glycosyl-PI present in liver cells, as compared to that measured in hepatocytes isolated from sham-operated animals. In contrast, dexamethasone treatment reduced by about 60% the basal levels of this compound in hepatocytes isolated from either adrenalectomized, sham-operated or normal rats. Simulated adrenalectomy did not significantly change the amount of glycosyl-PI present in the cells, when compared to normal rats. Under all the above-mentioned metabolic conditions, the basal amount of insulin-sensitive glycosyl-PI paralleled the calculated total number of insulin receptors present in the cells (Table 1). In fact, a close direct correlation could be established between these two parameters (r = 0.89; p < 0.02;
R3 TABLE 1 EFFECT OF ADRENALECTOMY AND DEXAMETHASONE TREATMENT ON THE LEVELS OF GLYCOSYL-PI AND ON THE NUMBER OF INSULIN RECEPTORS IN ISOLATED RAT HEPATOCYTES Values of glycosyl-PI are the means* SEM of different experiments; the numbers of them are indicated in parentheses. Insulin receptors are expressed by the calculated mean value of two or three experiments carried out in triplicate. Insulin receptors (number X 10 - 3/tell)
Glycosyl-PI (dpm/mg of protein) Normal rats Normal rats + dexamethasone Sham-operated Sham-operated rats + dexamethasone Adrenalectomized rats Adrenalectomized rats + dexamethasone
3,230 + 538
(7)
280
1,153+ 77* 4,022+711
(4) (5)
117 352
1,696 + 602 * (3) 6,203 + 329 * * (4)
231 376
2,429 + 989 *
270
(4)
significantly elevated (Table l), but its hydrolysis in response to insulin was also slightly increased (Fig. 2), as compared to that observed in hepatocytes obtained from sham-operated rats. This more pronounced effect of insulin was clearly observed at the lowest concentration of insulin assayed (about 25 and 8% of glycosyl-PI reduction in response to 0.1 nM insulin, respectively, for hepatocytes isolated from adrenalectomized or sham-operated rats) (Figs. 1 and 2). Treatment of the animals with dexamethasone again markedly blocked the hydrolysis of the glycosyl-PI, as well as the stimulation of glycogen synthesis in response to insulin. Discussion In man (Rizza et al., 1982) and animals (McMahon et al., 1988), glucocorticoid excess is asso-
* p -C0.05 vs. the corresponding animals not treated with the steroid. ** p -C0.05 vs. sham-operated rats.
n = 6). Neither bilateral adrenalectomy nor dexamethasone treatment significantly affected the estimated K, value for insulin of isolated hepatocytes which ranged between 0.3 and 0.4 x lo9M-l. As expected, insulin caused a dose-dependent hydrolysis of the glycosyl-PI, with the consequent reduction of its cellular levels. Thus, in hepatocytes isolated from sham-operated rats (Fig. l), 1 nM insulin decreased by about 20% the amount of hepatocyte glycosyl-PI. Simultaneously, insulin at all the assayed concentrations - significantly stimulated the incorporation of [U-r4C]glucose into glycogen (3.1 + 0.76 and 4.6 f 0.89 pmol of [U-14C]glucose converted into glycogen/g of cells X 30 min, respectively, for hepatocytes incubated under basal conditions or in the presence of 0.1 nM insulin; n = 3 experiments, p -c 0.01). Treatment of the rats with dexamethasone almost completely blocked the stimulatory effect of insulin on both glycosyl-PI hydrolysis and [U-14C]glucose incorporation into glycogen. In hepatocytes isolated from adrenalectomized rats, not only was the basal level of glycosyl-PI
-10
-9 ~~~[INSULIN]
-9 M
Fig. 1. Effect of insulin on glycosyl-PI levels and on the rate of glycogen synthesis, in hepatocytes isolated from sham-operated rats, treated (I) or not (0) with dexamethasone. Values are the means * SEM of three experiments.
R4
B”1 70 t O
