INVOLVEMENT OF PROSTAGLANDINS IN THE INHIBITION
OF GROWTH HORMONE PRODUCTION IN CULTURED PITUITARY CELLS BY INSULIN A. BETTERIDGE
Biochemistry Group,
AND
M. WALLIS
of Biological Sciences, University of Sussex, Brighton, Sussex, BN1 9QG (Received 16 June 1978)
School
SUMMARY
The effect of insulin was tested on the rate of synthesis and release of growth hormone in cultured rat anterior pituitary cells. Concentrations of insulin between 10\m=-\9and 10\m=-\7 mol/l (6\p=n-\600 ng/ml or 0\m=.\15\p=n-\15mu./ml) inhibited synthesis of growth hormone; 10\m=-\8 mol insulin/1 was most effective. The effect was observed after a time-lag of at least 1 h. Insulin at concentrations between 3 \m=x\10\m=-\9mol/l and 3 \m=x\10\m=-\7mol/l also inhibited growth hormone secretion in 30 min incubations. The most effective insulin concentration in this case was 3 \m=x\10\m=-\8mol/l. Insulin (10\m=-\9\p=n-\10\m=-\7 mol/l) also decreased the intracellular content of prostaglandins E and F. The effect was rapid, reaching a maximum after 30 min. Indomethacin, an inhibitor of prostaglandin synthetase, dramatically lowered the concentration of prostaglandins in the cells within 30 min; growth hormone synthesis was also decreased, but not until after 2 h of incubation. The results suggest that an initial response to insulin treatment is a lowering of intracellular levels of prostaglandins, which may then mediate a decrease in growth hormone synthesis, after a 1\p=n-\2 h delay. INTRODUCTION
previously been shown (Betteridge «fe Wallis, 1973) that insulin has a specific stimu¬ latory effect on the synthesis of growth hormone in vitro. These experiments involved the incubation of rat hemipituitary glands with unphysiological levels of bovine insulin. Shenai & Wallis (1974) have shown that insulin at concentrations between 10~9 and 10~e mol/1 inhibits the synthesis of prolactin in vitro. It was the purpose of the present experi¬ ments to investigate the effect of more physiological doses of insulin on the synthesis and release of growth hormone, using dispersed pituitary cells in monolayer culture, and to study possible mechanisms through which insulin might act. Prostaglandins, especially those of the -series, have been shown to stimulate both the synthesis (MacLeod & Lehmeyer, 1970) and the release (Schofield, 1970) of growth hormone in vitro. They have also been implicated in the mechanism of action of luteinizing hormone releasing hormone on the release of luteinizing hormone from pituitary cells (Barden, Bergeron «fe Betteridge, 1976; Barden «fe Betteridge, 1977). The role of prostaglandins in the control of growth hormone synthesis and release by insulin has, therefore, been investigated. A preliminary report of some of this work has been published (Betteridge & Wallis, 1977). It has
MATERIALS AND METHODS
Materials bovine insulin was Crystalline (24 u./mg) provided by Burroughs Wellcome Ltd, Beckenham, Kent. Prostaglandins were provided by Upjohn Pharmaceuticals Ltd, Crawley, *
Present address: National Institute for Medical
Research, Mill Hill, London, NW7 1AA.
[5,6,8,11,12,14,15-3H]Prostaglandin E2 (PGE2,140 Ci/mmol), [5,6,8,11,12,14,153H]prostaglandin F2o( (PGF^, 130 Ci/mmol) and [4,5-3H]leucine (60 Ci/mmol) were ob¬ Sussex.
tained from the Radiochemical Centre, Amersham, Bucks. Rat growth hormone (NIAMDD-rat-GH-B-3 and NIAMDD-rat-GH-RP-1) and monkey anti-rat growth hormone (NIAMDD anti-rat-GH serum) were provided by the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, U.S.A. Sera and tissue culture media and equipment were obtained from Gibco Biocult Ltd, Scotland.
Dispersed cell cultures Anterior pituitary glands were removed from male Sprague-Dawley rats (300-400 g) immediately after decapitation. The tissue was minced and digested with collagenase, hyaluronidase and pancreatic extract (Viokase, Gibco Biocult Ltd) as described by Vale, Grant, Amoss, Blackwell & Guillemin (1972). The dispersed cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) from Gibco Biocult Ltd containing various additions (Vale et al. 1972), to give approximately 105 cells/ml. Samples (1-5 ml) of the cell suspension were then pipetted into sterile plastic culture dishes (Falcon Plastics 3001, from Biocult Laboratories Ltd, Paisley, Scotland, or 301 V from Sterilin Ltd, Teddington, Middlesex) and incubated for 3 days at 37 °C in a humidified atmosphere of 95% air and 5% C02. During this period the viable cells stuck to the culture dishes. The medium was then changed for fresh DMEM (without sera) 2 h before the start of the experiment. In all cases three dishes were used for each treatment in each experiment. The mean values
of measurements from the three dishes were calculated and these between experiments to obtain the mean ± s.e.m. values shown.
were
then combined
Measurement of growth hormone synthesis To measure growth hormone synthesis, the medium was replaced with DMEM containing 10 µ€ [3H]leucine/ml and the cells were incubated for up to 5 h at 37 °C. The cells were then scraped from the dishes and the suspension was combined with the medium. Urea was added to the sample to saturation to solubilize the proteins and portions were sub¬ jected to polyacrylamide gel electrophoresis with 25 µg rat growth hormone (NIAMDDrat-GH-B3) as marker. The gels contained 8% acrylamide with 2-5% cross-linking (Davis, 1964). The gels were stained, after electrophoresis, with amido black and were destained in 7-5% acetic acid containing 1 mg L-leucine/ml. The most heavily stained band, which has previously been identified as growth hormone (Lewis, Cheever & VanderLaan, 1965), was excised, solubilized and counted for radio¬ activity as described by Betteridge «fe Wallis (1973). Analysis of the contents of this band using sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that 80-85% of the radioactivity co-migrated with standard rat growth hormone and this percentage was not altered by the incubation conditions used. Similarly, no other component com¬ prised more than 5% of the radioactivity when the pituitary cells were incubated under a variety of conditions. Therefore, it can be assumed that changes in the amount of radio¬ activity associated with this band are almost certainly a result of changes in the incor¬ poration of leucine into growth hormone. This method gives an estimate of the net in¬ corporation of [3H]leucine into growth hormone which is a combination of synthesis and
degradation.
ofgrowth hormone secretion To measure the secretion of growth hormone, dishes of cells were incubated for 30 min with fresh DMEM containing various concentrations of insulin. The medium was then removed and diluted (between 1 : 5 and 1 : 25) before growth hormone content was assayed with a radioimmunoassay kit obtained from NIAMDD (Bethesda, Mary¬ land, U.S.A.). The assay was performed as described in the instructions, except that the Measurement
antibody-hormone complex was precipitated with polyethylene glycol as described by Desbuquois «fe Aurbach (1971). The content of growth hormone in the samples of media was estimated from the standard curve and is expressed in terms of ng NIAMDD-GHRP-l/ml incubation medium. Measurement of intracellular prostaglandin content by radioimmunoassay To measure changes in the prostaglandin content of anterior pituitary cells, they were incubated with fresh DMEM for various periods. At the end of the incubation, the medium was removed and the cells were scraped from the dishes in 0-5 ml 0-9% NaCl solution.
The dishes were then washed with 0-5 ml ethanol. These two fractions were combined and sonicated to cause cell lysis and the prostaglandins were extracted with 2 ml ethyl acetate. This extract was dried in vacuo and the residue was resuspended for radioimmuno¬ assay (Barden «fe Betteridge, 1977). The anti-prostaglandin sera were prepared as described previously (Barden «fe Betteridge, 1977). The anti-PGE serum reacted almost equally to PGEi and PGE2. Prostaglandin Ax (PGAX) cross-reacted with 8% of the efficiency of PGE2, while PGA2, PGFl0( and PGF2a gave less than 1% cross-reaction. The anti-PGF serum reacted equally with PGFl0, and PGF2a, while PGE!, PGE2 and PGA2 cross-reacted with less than 1% of the same efficiency. Hence measurements quoted in terms of PGE are an estimate of the total amount of the -series prostaglandins (PGEX and PGE2) with some interference by PGA! if it were present in large quantities, while measurements quoted in terms of PGF are an estimate of the total amount of prostaglandins of the F series. Clearly the possibility that other material present in the pituitary gland may interact in these prostaglandin assays cannot be excluded, and for this reason the quan¬ tities of PGE and PGF determined in this work should be strictly considered as of PGElike material and PGF-like material respectively. The bound fraction was precipitated with goat anti-rabbit-y-globulin serum (Wellcome Reagents Ltd, Beckenham, Kent) and separated by filtration on cellulose membranes (Millipore Ltd, London, NW 10). Measurement of cell protein The protein contents of the dishes of cells were measured after solubilization in 0-5 NaOH using the method of Lowry, Rosebrough, Farr & Randall (1951).
m-
RESULTS
Effects of various insulin concentrations on growth hormone synthesis To investigate the effect of low concentrations of insulin on the synthesis of growth hor¬ mone, dishes of dispersed cells were incubated for 5 h with [3H]leucine and various con¬ centrations of insulin, ranging from 5 IO-10 mol/1 (3 ng/ml) to 10-7 mol/1 (600 ng/ml). All concentrations of insulin tested from 2 IO-9 mol/1 to higher concentrations signifi¬ cantly (P< 0-02) inhibited synthesis of growth hormone (Fig. 1). The most effective concen¬ tration was 10-8 mol/1 (78% of control). Above 10-8 mol/1, the effect was found to diminish with increasing concentration up to 10-7 mol/1. It has previously been shown (Betteridge & Wallis, 1973) that 10-6 mol insulin/1 has no significant effect on growth hormone synthesis, whereas higher concentrations are stimulatory. This is true whether pituitary glands or dispersed cells are used (A. Betteridge «fe M. Wallis, unpublished observations). The results cannot be a consequence of an effect of insulin on the number of cells attached to the culture dishes, since the protein contents of the dishes were measured after incubation and there was less than 5% variation between dishes in any one
experiment.
Time-course of inhibition of growth hormone synthesis To determine the time course of the effect of insulin, dishes of dispersed cells were incu¬ bated with [3H]leucine and 10~8 mol insulin/1 for various periods. Figure 2 shows that
there was no significant difference between the rates of synthesis of growth hormone, in the presence or absence of insulin, after 1 or 2 h of incubation. After 3 h, however, insulin significantly (P
OF GROWTH HORMONE PRODUCTION IN CULTURED PITUITARY CELLS BY INSULIN A. BETTERIDGE
Biochemistry Group,
AND
M. WALLIS
of Biological Sciences, University of Sussex, Brighton, Sussex, BN1 9QG (Received 16 June 1978)
School
SUMMARY
The effect of insulin was tested on the rate of synthesis and release of growth hormone in cultured rat anterior pituitary cells. Concentrations of insulin between 10\m=-\9and 10\m=-\7 mol/l (6\p=n-\600 ng/ml or 0\m=.\15\p=n-\15mu./ml) inhibited synthesis of growth hormone; 10\m=-\8 mol insulin/1 was most effective. The effect was observed after a time-lag of at least 1 h. Insulin at concentrations between 3 \m=x\10\m=-\9mol/l and 3 \m=x\10\m=-\7mol/l also inhibited growth hormone secretion in 30 min incubations. The most effective insulin concentration in this case was 3 \m=x\10\m=-\8mol/l. Insulin (10\m=-\9\p=n-\10\m=-\7 mol/l) also decreased the intracellular content of prostaglandins E and F. The effect was rapid, reaching a maximum after 30 min. Indomethacin, an inhibitor of prostaglandin synthetase, dramatically lowered the concentration of prostaglandins in the cells within 30 min; growth hormone synthesis was also decreased, but not until after 2 h of incubation. The results suggest that an initial response to insulin treatment is a lowering of intracellular levels of prostaglandins, which may then mediate a decrease in growth hormone synthesis, after a 1\p=n-\2 h delay. INTRODUCTION
previously been shown (Betteridge «fe Wallis, 1973) that insulin has a specific stimu¬ latory effect on the synthesis of growth hormone in vitro. These experiments involved the incubation of rat hemipituitary glands with unphysiological levels of bovine insulin. Shenai & Wallis (1974) have shown that insulin at concentrations between 10~9 and 10~e mol/1 inhibits the synthesis of prolactin in vitro. It was the purpose of the present experi¬ ments to investigate the effect of more physiological doses of insulin on the synthesis and release of growth hormone, using dispersed pituitary cells in monolayer culture, and to study possible mechanisms through which insulin might act. Prostaglandins, especially those of the -series, have been shown to stimulate both the synthesis (MacLeod & Lehmeyer, 1970) and the release (Schofield, 1970) of growth hormone in vitro. They have also been implicated in the mechanism of action of luteinizing hormone releasing hormone on the release of luteinizing hormone from pituitary cells (Barden, Bergeron «fe Betteridge, 1976; Barden «fe Betteridge, 1977). The role of prostaglandins in the control of growth hormone synthesis and release by insulin has, therefore, been investigated. A preliminary report of some of this work has been published (Betteridge & Wallis, 1977). It has
MATERIALS AND METHODS
Materials bovine insulin was Crystalline (24 u./mg) provided by Burroughs Wellcome Ltd, Beckenham, Kent. Prostaglandins were provided by Upjohn Pharmaceuticals Ltd, Crawley, *
Present address: National Institute for Medical
Research, Mill Hill, London, NW7 1AA.
[5,6,8,11,12,14,15-3H]Prostaglandin E2 (PGE2,140 Ci/mmol), [5,6,8,11,12,14,153H]prostaglandin F2o( (PGF^, 130 Ci/mmol) and [4,5-3H]leucine (60 Ci/mmol) were ob¬ Sussex.
tained from the Radiochemical Centre, Amersham, Bucks. Rat growth hormone (NIAMDD-rat-GH-B-3 and NIAMDD-rat-GH-RP-1) and monkey anti-rat growth hormone (NIAMDD anti-rat-GH serum) were provided by the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, U.S.A. Sera and tissue culture media and equipment were obtained from Gibco Biocult Ltd, Scotland.
Dispersed cell cultures Anterior pituitary glands were removed from male Sprague-Dawley rats (300-400 g) immediately after decapitation. The tissue was minced and digested with collagenase, hyaluronidase and pancreatic extract (Viokase, Gibco Biocult Ltd) as described by Vale, Grant, Amoss, Blackwell & Guillemin (1972). The dispersed cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) from Gibco Biocult Ltd containing various additions (Vale et al. 1972), to give approximately 105 cells/ml. Samples (1-5 ml) of the cell suspension were then pipetted into sterile plastic culture dishes (Falcon Plastics 3001, from Biocult Laboratories Ltd, Paisley, Scotland, or 301 V from Sterilin Ltd, Teddington, Middlesex) and incubated for 3 days at 37 °C in a humidified atmosphere of 95% air and 5% C02. During this period the viable cells stuck to the culture dishes. The medium was then changed for fresh DMEM (without sera) 2 h before the start of the experiment. In all cases three dishes were used for each treatment in each experiment. The mean values
of measurements from the three dishes were calculated and these between experiments to obtain the mean ± s.e.m. values shown.
were
then combined
Measurement of growth hormone synthesis To measure growth hormone synthesis, the medium was replaced with DMEM containing 10 µ€ [3H]leucine/ml and the cells were incubated for up to 5 h at 37 °C. The cells were then scraped from the dishes and the suspension was combined with the medium. Urea was added to the sample to saturation to solubilize the proteins and portions were sub¬ jected to polyacrylamide gel electrophoresis with 25 µg rat growth hormone (NIAMDDrat-GH-B3) as marker. The gels contained 8% acrylamide with 2-5% cross-linking (Davis, 1964). The gels were stained, after electrophoresis, with amido black and were destained in 7-5% acetic acid containing 1 mg L-leucine/ml. The most heavily stained band, which has previously been identified as growth hormone (Lewis, Cheever & VanderLaan, 1965), was excised, solubilized and counted for radio¬ activity as described by Betteridge «fe Wallis (1973). Analysis of the contents of this band using sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that 80-85% of the radioactivity co-migrated with standard rat growth hormone and this percentage was not altered by the incubation conditions used. Similarly, no other component com¬ prised more than 5% of the radioactivity when the pituitary cells were incubated under a variety of conditions. Therefore, it can be assumed that changes in the amount of radio¬ activity associated with this band are almost certainly a result of changes in the incor¬ poration of leucine into growth hormone. This method gives an estimate of the net in¬ corporation of [3H]leucine into growth hormone which is a combination of synthesis and
degradation.
ofgrowth hormone secretion To measure the secretion of growth hormone, dishes of cells were incubated for 30 min with fresh DMEM containing various concentrations of insulin. The medium was then removed and diluted (between 1 : 5 and 1 : 25) before growth hormone content was assayed with a radioimmunoassay kit obtained from NIAMDD (Bethesda, Mary¬ land, U.S.A.). The assay was performed as described in the instructions, except that the Measurement
antibody-hormone complex was precipitated with polyethylene glycol as described by Desbuquois «fe Aurbach (1971). The content of growth hormone in the samples of media was estimated from the standard curve and is expressed in terms of ng NIAMDD-GHRP-l/ml incubation medium. Measurement of intracellular prostaglandin content by radioimmunoassay To measure changes in the prostaglandin content of anterior pituitary cells, they were incubated with fresh DMEM for various periods. At the end of the incubation, the medium was removed and the cells were scraped from the dishes in 0-5 ml 0-9% NaCl solution.
The dishes were then washed with 0-5 ml ethanol. These two fractions were combined and sonicated to cause cell lysis and the prostaglandins were extracted with 2 ml ethyl acetate. This extract was dried in vacuo and the residue was resuspended for radioimmuno¬ assay (Barden «fe Betteridge, 1977). The anti-prostaglandin sera were prepared as described previously (Barden «fe Betteridge, 1977). The anti-PGE serum reacted almost equally to PGEi and PGE2. Prostaglandin Ax (PGAX) cross-reacted with 8% of the efficiency of PGE2, while PGA2, PGFl0( and PGF2a gave less than 1% cross-reaction. The anti-PGF serum reacted equally with PGFl0, and PGF2a, while PGE!, PGE2 and PGA2 cross-reacted with less than 1% of the same efficiency. Hence measurements quoted in terms of PGE are an estimate of the total amount of the -series prostaglandins (PGEX and PGE2) with some interference by PGA! if it were present in large quantities, while measurements quoted in terms of PGF are an estimate of the total amount of prostaglandins of the F series. Clearly the possibility that other material present in the pituitary gland may interact in these prostaglandin assays cannot be excluded, and for this reason the quan¬ tities of PGE and PGF determined in this work should be strictly considered as of PGElike material and PGF-like material respectively. The bound fraction was precipitated with goat anti-rabbit-y-globulin serum (Wellcome Reagents Ltd, Beckenham, Kent) and separated by filtration on cellulose membranes (Millipore Ltd, London, NW 10). Measurement of cell protein The protein contents of the dishes of cells were measured after solubilization in 0-5 NaOH using the method of Lowry, Rosebrough, Farr & Randall (1951).
m-
RESULTS
Effects of various insulin concentrations on growth hormone synthesis To investigate the effect of low concentrations of insulin on the synthesis of growth hor¬ mone, dishes of dispersed cells were incubated for 5 h with [3H]leucine and various con¬ centrations of insulin, ranging from 5 IO-10 mol/1 (3 ng/ml) to 10-7 mol/1 (600 ng/ml). All concentrations of insulin tested from 2 IO-9 mol/1 to higher concentrations signifi¬ cantly (P< 0-02) inhibited synthesis of growth hormone (Fig. 1). The most effective concen¬ tration was 10-8 mol/1 (78% of control). Above 10-8 mol/1, the effect was found to diminish with increasing concentration up to 10-7 mol/1. It has previously been shown (Betteridge & Wallis, 1973) that 10-6 mol insulin/1 has no significant effect on growth hormone synthesis, whereas higher concentrations are stimulatory. This is true whether pituitary glands or dispersed cells are used (A. Betteridge «fe M. Wallis, unpublished observations). The results cannot be a consequence of an effect of insulin on the number of cells attached to the culture dishes, since the protein contents of the dishes were measured after incubation and there was less than 5% variation between dishes in any one
experiment.
Time-course of inhibition of growth hormone synthesis To determine the time course of the effect of insulin, dishes of dispersed cells were incu¬ bated with [3H]leucine and 10~8 mol insulin/1 for various periods. Figure 2 shows that
there was no significant difference between the rates of synthesis of growth hormone, in the presence or absence of insulin, after 1 or 2 h of incubation. After 3 h, however, insulin significantly (P
