effects of insulin, pertussis toxin and cholera toxin on protein synthesis and diacylglycerol...

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Bioscience Reports, Vol. 7, No. 7, 1987 Effects of Insulin, Pertussis Toxin and Cholera Toxin on Protein Synthesis and Diacylglycerol Production in 3T3 Fibroblasts: Evidence for a G-protein Mediated Activation of Phospholipase C in the Insulin Signal Mechanism John E. Hesketh and Gillian P. Campbell Received June 12, 1987 KEY' WORDS: insulin; protein synthesis; G-protein; diacylglycerol; phospholipase C; signa[ transduction. The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 #M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels. INTRODUCTION Insulin has effects on a wide range of cellular processes including protein synthesis and both fat and carbohydrate metabolism. Although it has been demonstrated that The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB. 533 0144.8463/87/0700-0533505.00/0 1987PlenumPublishing Corporation

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Bioscience Reports, Vol. 7, No. 7, 1987

Effects of Insulin, Pertussis Toxin and Cholera Toxin on Protein Synthesis and Diacylglycerol Production in 3T3 Fibroblasts: Evidence for a G-protein Mediated Activation of Phospholipase C in the Insulin Signal Mechanism

John E. Hesketh and Gillian P. Campbell

Received June 12, 1987

KEY' WORDS: insulin; protein synthesis; G-protein; diacylglycerol; phospholipase C; signa[ transduction.

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 #M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.

INTRODUCTION

Insulin has effects on a wide range of cellular processes including protein synthesis and both fat and carbohydrate metabolism. Although it has been demonstrated that

The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB.

533 0144.8463/87/0700-0533505.00/0 �9 1987 Plenum Publishing Corporation

534 Hesketh and Campbell

insulin binds to and activates a receptor in the cell plasma membrane (Gammeltoft and Van Obberghen, 1986), the series of membrane and/or cytoplasmic events between receptor activation and the final sites of action remain largely unknown. The insulin receptor has been found to have tyrosine kinase activity (Kasuga et al., 1982) and so a protein phosphorylation event is probably the first step in the signal pathway, but the substrate for this kinase has not been identified. Peptide mediators (Cheng and Larner, 1985), hydrolysis of membrane glycolipids (Saltiel and Cuatrecasas, 1986), and guanosine triphosphate-binding proteins (Houslay, 1986) have all been proposed as the secondary events leading to effects of insulin on fat and carbohydrate metabolism. Less attention has been paid to the mechanisms by which insulin activates protein synthesis.

Two guanosine triphosphate binding proteins (G-proteins), G~ and Gi, have been implicated in the hormonal control of adenylate cyclase activity (Gilman, 1984), but they are only two of a family of heterotrimeric GTP binding proteins and recent evidence suggests that G proteins are involved not only in the control of adenylate cyclase but also in hormone receptor signal mechanisms which require activation of phospholipase C (Cockcroft and Gomperts, 1985). G-protein dependent activation of phospholipase C stimulates the production of 1,2-diacylglycerol (DAG) and inositol triphosphate from membrane phospholipids (Cockcroft and Gomperts, 1985), and DAG in turn is presumed to activate protein kinase C (Nishizuka, 1984). The G- protein involved is similar to Gi of the adenylate cyclase system in that it is inactivated by pertussis toxin but nevertheless appears to be a distinct protein (Gilman, 1984; Cockcroft and Gomperts, 1985). The role of G-proteins in hormone and growth factor action has been widely studied using two bacterial exotoxins, cholera toxin which activates G~ by promoting dissociation of its c~ subunit and pertussis toxin which not only blocks the dissociation of the e subunit of Gi but also in some cells blocks the G- protein dependent activation of phospholipase C (Gilman, 1984; Cockcroft, 1987).

In the fasted condition, insulin stimulates protein synthesis within 30 minutes of administration in vivo (Garlick et al., 1983) and an equally rapid effect has been observed in stepped-down fibroblasts in cell culture (De Philip et al., 1979; Hesketh et al., (1986). In fibroblasts this rapid effect of insulin was shown to be independent of RNA synthesis and due to an activation of translational efficiency (Hesketh et al., 1986). The molecular mechanisms through which the signal is transduced from the insulin receptor to the ribosome are unclear, but there is evidence that activation of protein kinase C (Hesketh et al., 1986) is involved. The aim of the present work was to study the role of G-proteins and their activation of phospholipase C in the stimulation by insulin of fibroblast protein synthesis. In order to do this we have studied both the effects of pertussis and cholera toxins on rates of protein synthesis and the effects of insulin orf:~the production of DAG, the endogenous activator of protein kinase C.

MATERIALS AND METHODS

Cell Culture

3T3 fibroblasts (Flow Laboratories, Irvine, Ayrshire, UK) were grown in Dulbecco's Minimal Eagle's medium (DMEM) supplemented with 12% foetal calf

Insulin Activates Phospholipase C 535

serum (FCS, from Gibco, UK). After subculture cells were grown for 3---5 days (medium change after 3 days) and then "stepped-down" by replacing the medium with DMEM containing only 4% serum. Cells were grown either in 35 mm plastic petri- dishes containing 2 ml medium (protein synthesis measurements) or in 100 mm dishes containing 8 mt medium (estimation of DAG production). At subculture, cells were seeded so as to achieve 5 days later cell densities of 2-4 x l0 s cells/35 mm dish and 16- 32 x 10 s cells/100 mm dish; at this density the cells were not confluent.

Measurement of Protein Synthesis

Protein synthesis was measured by the incorporation of isotopic phenylalanine ([3H]2,6 phenylalanine, 5 mCi/mmol from Amersham International, UK) into protein as described previously (Hesketh et at., 1986). At high concentration of radio-labelled precursor (Garlick et al., 1980; Smith et al., 1983) was used in order to produce isotope equilibration of the intracellular and extracellular amino-acid pools within 1 minute. The rate constant of protein synthesis, Ks, was calculated as described previously assuming that 4 % of the cell protein is phenylalanine (Garlick et al., 1980; Hesketh et at., 1986). Protein synthesis was measured over a 1 h period, immediately after addition of insulin or 1 to 2 h after addition of either toxin.

Measurement of Diacylglycerol

Cell glycerol-containing lipids were pre-labelled by incubating the cells with 4 pCi [3H]glycerol (1,3-[3H]glycerol from Amersham International, 3 mCi/mmol) for the 48 h immediately after they were stepped-down into medium containing 4 % FCS Under these conditions there was isotopic equilibration of glycerol containing tipids. Cells, in medium still containing the isotopic glycerol, were exposed to insulin for up to 5 min and lipid extraction was carried out using a modification of the method of Bligh and Dyer (1959). Cells were rinsed twice very rapidly with phosphate buffered saline and 1.6 ml methanol added to stop lipid metabolism. The cells were scraped into the methanol with a rubber policeman, transferred to a tube containing 0.8 ml chloroform and 40 gl conc. HC1 (final proportions CHC13/CH3OH/HC1 , 20:40:1) and shaken briefly. A further 2.4 ml of chloroform and 2.4 ml water were then added, the tube vortexed vigorously and then centrifuged for 1009 x 10 min at 4~ The chloroform layer was collected, evaporated over nitrogen and redissolved in 50 #1 CHC13/CH 3OH (2:1). Neutral lipids were separated by thin layer chromatography using LK5D silica gel plates (Whatman) with petroleum ether/diethylether/acetic acid (80:30:1) as the solvent phase. Standard mixtures containing 1,2 diacylglycerol, 1,3 diacylglycerol, monoglyceride, triglyceride, free fatty acids and cholesterol esters were run in parallel and areas from sample lanes which corresponded to these standards, together with the loading areas, were scraped into vials and the radioactivity measured by liquid scintillation counting. So as to allow for variation in lipid recovery, the radioactivity in 1,2-diacylglycerol was expressed as a percentage of the total recovered radioactivity.

536 Hesketh and Campbell

Chemicals

Purified pertussis toxin was obtained from PHLS Centre for Microbiology and Research, Porton Down, U K and cholera toxin from Sigma Chemical Co. Ltd. All dilutions of toxins were made in phosphate-buffered saline (pH 7.4) containing 0.5 M NaC1 (high-salt PBS). Cells were exposed to toxins for 1 h before addition of insulin and measurement of protein synthesis or D A G synthesis.

RESULTS

The response of s tepped-down cells to insulin was rapid and there was an increase in the rate of protein synthesis within ! hour (Table 1 and Hesketh et al., 1986). Pre- incubation, for 1 hour, of cells with pertussis toxin (100 ng/ml) caused a marked inhibition of the insulin-induced stimulation of protein synthesis (Table 1) although pertussis toxin alone caused no depression of the protein synthesis rate. The inhibition by pertussis toxin was concentration dependent; typically the toxin at concentrations of 1, 10 and 100 ng/ml, reduced the % increase in Ks caused by insulin from 18.6 % to 19.0 %, 8.4 % and 2.4 % respectively; at 1 #g/ml inhibition by pertussis toxin was sub- maximal, the insulin response being reduced to only 12 %.

Table 1. Effect of pertussis toxin (100 ng/ml) and cholera toxin (1 pg/ml) on protein synthesis and its stimulation by insulin

increase in Ks compared % increase in Ks compared Additions- to control (carrier only) to cells given toxin only

Pertussis toxin 6.4 + 1.1 (12) -- Cholera toxin 25.0 ___ 1.5 (I1) -- Insulin 21.4 _ 1.8 (7) -- Insulin + pertussis toxin 11.9 + 3.3 (7) 4.0 _+ 0.9 (7)** Insulin+cholera toxin 35.7_ 9.3 (4) 9.1 ___ 1.8 (4)

Results are expressed as % increase in Ks compared to controls; % stimulation by insulin is given as increase compared to cells given toxin or toxin carrier. % stimulation by toxin or by toxin +insulin are increases compared to cells given carrier only. Results are given as means __. SEM, n in parentheses. Insulin was added to give a final concentration of 1 mU/ml. Cells were pre-treated with toxin (or carrier) for 1 hour and then given [aH]phenylalanine and insulin or saline for a second hour before measurement of protein synthesis. **, P < 0.005 (from Insulin alone) using a two-tailed "t" test.

Pre-incubation of cells with cholera toxin (1/zg/ml) for 1 hour caused an increase in protein synthesis (Table 1); there was no increase in protein synthesis during the 1 hour immediately after adding cholera toxin (results not shown). The effect of cholera toxin was concentration dependent (Fig. 1) with a maximum effect at 1/~g/ml. In the presence of cholera toxin insulin had a much reduced effect on protein synthesis; this may be due to an inhibition of the insulin response by cholera toxin or, more likely, due to additive, sub-maximal stimulation by both compounds leading to maximal stimulation.

The inhibition of the insulin response by pertussis toxin suggests that a G-protein is involved in the signal pathway between receptor activation and the ribosome. Since

Insulin Activates Phospholipase C 537

15

.E

5

~ _ . ~ t ~ ' j

0 10 100 1000 5000

Cholera toxin (ng/rnl)

Fig. 1. Stimulation of protein synthesis by cholera toxin. Cholera toxin was added to stepped-down cells and protein synthesis measured over a period 1-2 hours later. Results shown are means from 5 experiments and are expressed as ~ increase compared to controls given an equal volume of saline carrier. Bars represent SEM.

cholera toxin also interacts with G-proteins, its stimulation of protein synthesis may also be G-protein mediated and may have similar characteristics to the rapid phase of the insulin response, namely be independent of RNA synthesis and be blocked by inhibition of protein kinase C by pre-incubation of the cells with phorbol dibutyrate (Hesketh et al., 1986). Indeed, as shown in Table 2, the stimulation of protein synthesis by cholera toxin was insensitive to actinomycin D (10 gg/ml, added together with the toxin) but was inhibited by 75~o by pre-incubation with phorbol dibutyrate. Furthermore cholera toxin did not stimulate protein synthesis in the presence of pertussis toxin (Table 2). Actinomycin D alone inhibited basal protein synthesis by

Table 2. Effects of actinomycin D (10 #g/ml), pertussis toxin and phorbol dibutyrate pre-treatment on the stimulation of protein synthesis by cholera toxin

Addition ~ increase in Ks in presence of cholera toxin

Cholera (1 #g/ml) + methanol carrier Cholera (1/~g/ml) +actinomycin D

Cholera (1 #g/ml)+ ethanol carrier Cholera (1 ~g/ml + phorbol dibutyrate

Cholera (1/~g/ml) +high salt PBS Cholera (t/~g/ml)+pertussis toxin

19.5 __ 3.3 17.7 __ 1.4

26.0 + 2.4 5.8 + 3.5**

28.5 + 10.8 0ol _+ 2.1"

Results are expressed as 7oo increase of Ks in cells given cholera toxin compared to controls with other additions as designated, but with no cholera toxin added. Actinomycin D was dissolved in methanol, phorbol dibutyrate in ethanol and pertussis toxin in high-salt PBS. Results are means _+ SEM from 4 experiments. **, P < 0.02 (from stimulus due to toxin in cells given ethanol carrier), *P < 0.05 (from stimulus due to cholera toxin in cells given high-salt PBS carrier) on a two-tailed "t" test.

538

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Hesketh and Campbell

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150

.~ 1~176 l "

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~ 50 e e

[

i = i i i i J =

0 40 80 120 300

T ime (sec)

Fig. 2. 1,2-Diacylglycerol synthesis following administration of insulin. Insulin (1 mU/ml) was added to stepped-down cells in the absence (0) or presence (�9 of pertussis toxin (100ng/ml). Lipid analyses were carried out on extracts from cells incubated with insulin for various time periods (10 sec-5 rain). DAG synthesis is expressed as the radioactivity in DAG as a proportion of that recovered in total phospholipids and glycerides. Results (means _+ SEM) are from 4 experiments. *P < 0.05, **P < 0.001, significantly different from value at same time point but with pertussis toxin added.

24 % whilst phorbol dibutyrate pre-treatment had little effect (7 ~ stimulation) on the basal rate.

DAG synthesis was studied in stepped-down cells which had been pre-incubated with [3H]glycerol to label the membrane phospholipids. Addition of insulin to such cells caused a rapid (10-30 secs) and transient increase in D A G production as measured by the proportion of extracted radioactivity recovered as D A G (Fig. 2). Incubation of cells for 1 hour with pertussis toxin (100 ng/ml) prior to addition of insulin totally prevented the insulin-induced increase in D A G production (Fig. 2).

Addition of exogenous DAG (10 pM 1-oleoyl-2-acetyl glycerol) to stepped-down cells caused a rapid stimulation of protein synthesis; over the periods 30-60 min, 60- 90 min and 90-120 min after addition of DAG protein synthesis was stimulated by 14.5 _+ 6.6 (sere, n = 3), 40.4% ___ 16.9 and 15.9 % _+ 7.0 (n -- 4) respectively.

DISCUSSION

Addition of insulin to stepped-down cells led to a rapid and transient increase in synthesis of DAG, the product of phospholipase C action on membrane phospholipids. The observation that the insulin-induced rise in DAG production was inhibited by pre-treatment of the cells with pertussis toxin strongly suggests that the activation of phospholipase C by insulin is G-protein mediated. The time-course of the response is similar to that observed with other activators in several cell types such as platelets (Siess et al., 1983; Rittenhouse, 1984) and macrophages (Holian, 1986). Insulin has been found previously to elevate the D A G concentration in myocytes for up to 60 minutes but the reason for the stable response in this case is not known (Farese et al., 1985). The present work shows that insulin increases D A G synthesis and, moreover, that it does so under conditions where it also produces a biological response, namely a stimulation of protein synthesis.

Insulin Activates Phospholipase C 539

The insulin-induced increase in protein synthesis in stepped-down 3T3 cells consists of two components, a rapid response within 1 hour which is independent of RNA synthesis and a slower response which is dependent on the production of new RNA (Hesketh et al., 1986). The rapid stimulation of protein synthesis by insulin, due to ribosome activation was inhibited by pertussis toxin, suggesting that a G protein- dependent step is involved in the signal pathway for ribosome activation. The concentration of toxin which inhibited protein synthesis was similar to that which inhibited G-protein related events in other cells (Heyworth e~ al., 1986; Cockcroft, 1987) and, as observed also by Schlondorff et al. (1986), there was an optimal concentration above which the inhibitory effect of the toxin was reduced. Pertussis toxin has been found in other cells to inhibit both the Gp protein that activates phospholipase C and Gi, the inhibitory G-protein associated with adenylate cyclase (Gilman, 1984; Cockcroft, 1987). The inhibition of the insulin-induced rise in DAG by pertussis toxin (Fig. 2) is consistent with the hypothesis that it is phospholipase C activation, and not changes in cAMP levels, which is the G-protein dependent step in the signal mechanism leading to ribosome activation. Further support for this point of view is provided by the observed stimulation of protein synthesis by exogenous DAG.

Activators of protein kinase C, such as phorbol myristate acetate (Hesketh et al., 1986) or exogenous diacylglycerol, have been found to stimulate protein synthesis in 3T3 cells and, therefore, the increase in endogenous DAG by insulin would be expected both to activate protein kinase C and to increase ribosomal activity. The observed increase in DAG coupled with the effect of exogenous DAG on protein synthesis and the sensitivity of the insulin-induced increase in protein synthesis to protein kinase C depletion (Hesketh et al., 1986) point to protein kinase C activation as being an important step in the mechanism of insulin action.

It has been suggested that both cholera toxin and insulin act on membrane phosphodiesterasethrough a specific G protein, Gins (Heyworth et al., 1985; Houslay, 1986), and evidence for this is provided both by the inability of insulin to further stimulate the enzyme in the presence of cholera toxin and by the inhibition of the cholera toxin effect by pertussis toxin. Comparable effects were observed in the present experiments where (1) cholera toxin-induced stimulation of protein synthesis was inhibited by pertussis toxin and (2) insulin had a reduced effect in the presence of cholera toxin. It is possible therefore that the activation of phospholipase C also occurs through such an insulin-specific G-protein. Alternatively, insulin may activate different signal pathways through distinct G-proteins. It is not known how insulin receptor activation is linked to changes in G-protein activity, but one possibility is that the G-protein(s) is a substrate for the receptor kinase. Indeed, while this manuscript was in preparation, the purified insulin receptor kinase was reported to phosphorylate purified brain Gi/Go (O'Brien et al., 1987).

In conclusion, the present results show that an important biological response of ceils to insulin, namely activation of ribosome activity, is blocked by an agent which interferes with G-proteins. These results, together with changes in DAG synthesis and the previously observed effect of protein kinase C depletion (Hesketh et al., 1986), suggest that the signal cascade that occurs following insulin receptor activation includes a G-protein mediated activation of phospholipase C which increases synthesis of DAG and this in turn activates protein kinase C (Fig. 3). This hypothesis is

540 Hesketh and Campbell

insulin "~t insulin receptor

~ phorbo! esters

\

./ oyto0,aso 73 i

Increase in r ibosome activity

Fig. 3. Hypothesis: The signal mechanisms involved in the stimulation of ribosomal activity by insulin. DAG, 1,2-diacylglycerol; IP3-inositol triphosphates.

suppor ted by observations that cholera toxin, which also interacts with G proteins stimulates protein synthesis by a mechanism which is insulin-like in that it is independent of R N A synthesis but blocked by protein kinase C depletion. G-proteins appear therefore to mediate at least two actions of insulin, the control of c A M P levels (Houslay, 1986) and the activation of phosphol ipase C and thus the levels of two second messengers, D A G and presumably inositol t r iphosphate. The generat ion of these three messengers th rough receptor G-protein coupled effector systems would allow insulin to affect a variety of cell processes and can account for some of its diverse effects.

A C K N O W L E D G E M E N T S

We are grateful to Miss Nicola Hami l ton for excellent technical assistance, Dr P. J. Reeds for encouragement and discussion and Dr K. W. J. Wahle for advice on lipid analysis.

R E F E R E N C E S

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18:4812-4817. Farese, R. V., Davis, J. S., Barnes, D. E., Standoert, M. L., Babischkin, J. S., Hock, R., Rosic, N. K. and

Pellet, R. J. (1985). Biochem. J. 231:269-278. Gammeltoft, S. and Van Obberghen, E. (1986). Biochem. J. 235:1-11. Garlick, P. J., McNurlan, M. A. and Preedy, V. R. (1980). Biochem. J. 192:719-723. Garlick, P. J., Fern, M. and Preedy, V. R. (1983). Biochem. J. 210:669-676. Gilman, A. G. (1984). Cell 36:577-579. Hesketh, J. E., Campbell, G. P. and Reeds, P. J. (1986). Biosci. Rep. 6:797-804. Heyworth, C. M., Whetton, A. D., Wong, S., Martin, B. R. and Houslay, M. (1985). Biochem. J. 228:593-

603, Heyworth, C. M., Grey, A.-M., Wilson, S. R., Hanski, E. and Houslay, M. (1986). Biochem. J. 235:145-149.

Insulin Activates Phospholipase C 541

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