JOURNAL OF CELLULAR PHYSIOLOGY 147:487494 (1991)
Stimulation of Protein Synthesis by Internalized Insulin DAVID S. MILLER* AND DESTINY B. SYKES
laboratory of Cellular and Molecular Pharmacology, Nationdl Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
Previous studies showed that microinjected insulin stimulates transcription and translation in Stage IV Xenopus oocytes by acting at nuclear and cytoplasmic sites (Miller, D.S., 1988, 1989). The present report is concerned with the question of whether hormone, internalized from an external medium, can act on those sites to alter cell function. Both intracellular accumulation of undegraded ’251-insulin and insulin-stimulated 3’S-methionine incorporation into oocyte protein were measured. Anti-insulin antiserum and purified anti-insulin antibody were microinjected into the cytoplasm of insulin-exposed cells to determine if insulin derived from the medium acted through internal sites. In cells exposed for 2 h to 7 or 70 nM external insulin, methionine incorporation was stimulated, but intracellular hormone accumulation was minimal and microinjected antibody was without effect. In cells exposed for 24 h, methionine incorporation again increased, but now accumulation of undegraded, intracellular hormone was substantial (2.6 and 25.3 fmol with 7 and 70 nM, respectively), and microinjected anti-insulin antibody significantly reduced the insulin-stimulated component of incorporation; basal incorporation was not affected. For cells exposed to 70 nM insulin for 24 h, inhibition of the insulin-stimulated component was maximal at 39%. Thus under those conditions, about 40% of insulin’s effects were mediated by the internal sites. Together, the data show that inhibition of insulin-stimulated protein synthesis by microinjected antibody was associated with the intracellular accumulation of insulin. They indicate that when oocytes are exposed to external insulin, hormone eventually gains access to intracellular sites of action and through these stimulates translation. Control of translation appears to be shared between the internal sites and the surface receptor. In sensitive cells, insulin alters the function of virtually every organelle. However, the chain of events by which the hormone signals metabolic changes at intracellular sites remains unclear. Insulin action is initiated when hormone binds to a specific external receptor and binding induces multiple secondary events at the plasma membrane, e.g., receptor autophosphorylation, protein tyrosine kinase activation, receptor and ligand internalization, and release of second messengers (reviewed by Goldfine, 1987; Czech et al., 1988; Kahn and White, 1988; Saltiel and Cuatrecasas, 1988). Each of these in turn has been associated with one or more of insulin’s actions, but defining links between putative signalling mechanisms and final intracellular events has been difficult, especially for sites spatially removed from the plasma membrane. Experiments showing that insulin is internalized (Posner et al., 1982) and that cells possess internal receptors (Goldfine, 1981; Goldfine et al., 1985) have led to the suggestion that internal as well as surface receptors transduce the hormone’s effects. However, determining what role internalized hormone might play in the overall mechanism of insulin action has been complicated by technical problems arising primar0 1991 WILEY-LISS, INC.
ily from the small size of most cells. Many of these problems can be avoided by the use of a giant, insulinsensitive cell, the Stage IV oocyte from Xenopus laevis (-800 pM in diameter), non-aqueous microinjection and microdissection techniques, and single-cell microanalysis (Miller, 1988,1989). Recently, this experimental system was used to demonstrate that both RNA and protein synthesis increases when insulin is microinjected into oocyte cytoplasm (Miller, 1988,1989). Since such increases are found when insulin is applied directly to isolated nuclei and to droplets of undiluted cytoplasm, the hormone must be acting through internal sites (receptors?) not associated with the plasma membrane. Indeed, the nuclear and cyto lasmic sites appear to stimulate RNA and protein synt esis by local signalling mechanisms. That is, insulin increases RNA synthesis in the absence of cytoplasm, and protein synthesis in the absence of nuclear material; increases in RNA and protein synthesis can occur in the absence of plasma membrane.
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Received October 30, 1990; accepted February 27, 1991 *To whom reprint requestsicorrespondence should be addressed.
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It remained to be established whether the internal sites are activated when cells are exposed to external hormone. The present report provides evidence that this is indeed the case. We show here that when Xenopus oocytes were incubated with 7 or 70 nM external insulin, undegraded hormone accumulated in the surface membranes, cytoplasm and nucleus and methionine incorporation into oocyte protein was stimulated. When incubation times were long enough for several fmol of undegraded hormone to accumulate in the cytoplasm and nucleus, an antibody to insulin, microinjected into the cytoplasm, partially reversed the hormone-mediated stimulation of protein synthesis. Thus when cells were exposed to external insulin, hormone was internalized and internalized hormone then acted through intracellular sites to stimulate translation. MATERIALS AND METHODS Chemicals and radioisotopes Bovine pancreatic insulin (24.6 unitslmg), guinea pig preimmune serum, and anti-bovine insulin antiserum (developed in guinea pig; affinity constant 5 X lo1' limol) were obtained from Sigma Chemical Co. (St. Louis) and recombinant human insulin like growth factor-1 (IGF-1) was purchased from Collaborative Research, Inc. (Cambridge). Prior to use, sera were dialyzed a ainst intracellular buffer (below).Anti-insulin antibo y and immunoglobulins from preimmune serum were urified by Protein G se harose chromatography. Ot er unlabeled chemica s were of rea ent ade or hi her and were purchased primarily rom Kgma. 35S- ethionine (specific activity -1,000 Ci/ mmol) and 1251-insulin(monoiodinated, porcine insulin; specific activity -90 uciipg) were obtained from DuPont-New England Nuclear. Oocytes Stage IV ooc tes (800 ? 50 pm diameter) in follicles were isolated y dissection from unstimulated South African clawed toads (Xenopus Zaevis from Nasco) into an amphibian oocyte Ringer's solution (in mM: 82.5 NaC1,25 KC1,l.O CaCl,, 1.0 M Clz, 1.0 Na2HP04, 5.0 HEPES, 1.0 Na pyruvate, at p% 7.6). All procedures were carried out at 20" C. Insulin uptake, distribution, and degradation To measure insulin uptake from a physiological saline solution, oocytes were incubated in Ringer's containing 0.01% bovine serum albumin (BSA) and 1251-insulin.At the end of the incubation period, each cell was removed from the medium, washed briefly in insulin-free Ringer's, blotted, and transferred to paraffin oil as described previously (Miller, 1989).Under oil, each cell was dissected into two components, the follicle plus plasma membrane and the cytoplasm plus nucleus. Adherent cortical cytoplasm was clearly visible on membrane samples. However, light microscopic and marker enzyme analyses have shown no detectable contamination of cytoplasm-nucleus samples by plasma membrane fragments (Miller, 1989). After isolation, each cell component was transferred to a tube containing 10% trichloroacetic acid (TCA) solution and counted (gamma counter) to measure total
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label. Tubes were then centrifuged and an aliquot of the supernatant was counted to measure degraded insulin; undegraded hormone was calculated as the difference, i.e., the TCA-precipitable fraction. Sample insulin content in fmol was calculated from sample activity and medium specific activity. Microinjection Oocytes in oil were microinjected in the vegetal hemisphere with 8-15 nl of intracellular buffer solution (in mM: 10 NaC1, 125 KC1, 1.0 KH2C03, with 0.01% BSA and at pH 7.2) containing the indicated amounts of insulin, antiserum or label. Glass capillary micropipets (tip diameter < 10 pm) and a hydraulic nanopump (WPI Instruments) were used. A detailed description of the microinjection procedure is given by Miller (1988,1989).
Protein synthesis 35S-Methionineincorporation into oocyte protein was measured. Radiolabel was microinjected into cells under oil and after a suitable labelling period each cell was processed to measure label incorporation into TCA (10%) precipitable protein (Miller, 1988). Using this procedure, both the amount of label injected into each cell and the percentage of that incorporated into protein were determined. When insulin was also present in the injection solution (Fig. l), the amount of hormone injected was calculated from the total 35S-methionine counts in each cell, the activity of the injection solution
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Fig. 1. Dose response for stimulation of 35S-methionine incorporation into oocyte protein by microinjected insulin. Stage IV oocytes in oil were microinjected at time zero with 12-16 nl intracellular buffer solution containing 0.01% BSA without or with the indicated dose of insulin and 10-20 fmol 35S-methionine. One h later, cells were processed to measure label incorporation into oocyte protein as well as the amount of insulin injected (Methods). Shown are pooled data (mean SE) from 5-9 experiments; in each experiment at least 10 controls and 10 treated cells per dose were assayed. Some of these data were presented previously in tabular form (Miller, 1989). The top axis shows the amount of undegraded insulin present 30 min after microinjection, estimated from the data of Miller (1989). Although the apparent rate of intracellular insulin degradation decreases somewhat with injected dose, for simplicity's sake, the amount of hormone remaining intact was taken to be 35% of that injected.
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and the hormone concentration in the injection solution. RESULTS To define conditions for testing the involvement of internal sites in the action of externally applied insulin, answers to two questions were needed: (1)What levels of intracellular insulin would be ex ected to stimulate translation? 2) Under what con itions do such levels accumulate in cells exposed to external hormone? Insulin dose-response Figure 1 shows the dose-response data for microinjected insulin's effects on methionine incorporation that are the basis for an answer to the first question. Incorporation increased with increasing dose of microinjected insulin and then plateaued at a dose of about 40 fmol. Detailed analysis of these data is complicated by three time-dependent processes that inevitably follow insulin microinjection: diffusion of hormone from the injection site, rapid inactivation by degradation (Miller, 1989), and specific and nonspecific binding to cellular elements. These processes occur simultaneously and they, along with uncertainties as to insulin and water compartmentation (Horowitz and Miller, 19841, confound attempts to associate metabolic changes with bound or free insulin at any intracellular site. Of the three processes, degradation is the only one for which the dose-response data can be corrected (Miller, 1989). The top axis of Figure 1 shows undegraded, i.e., TCA-precipitable, insulin 30 min after microinjection, a time when protein synthesis has begun to increase in response to injected hormone. The corrected data indicate that about 2-3 fmol of undegraded insulin per cell significantly stimulated methionine incorporation and that about 14 fmolicell stimulated maximally. Insulin accumulation The time course of 1251-insulinuptake by oocytes was biphasic, with a rapid initial phase occurring over the first few min followed by a slower prolonged phase (Fig. 2). Dissection of these cells in oil revealed that during the initial phase label was localized to the plasma membrane and follicle, consistent with rapid binding to surface receptors; over several hours, label was also found in the cytoplasm and nucleus (Table 1). Insulin uptake partially saturated in the concentration range 70-700 nM (not shown). Two types of experiment suggest that insulin uptake occurs at least in part by a specific mechanism. First, our initial measurements of 3H-sucrose and 3H-polyethylene glycol uptake showed that fluid phase endocytosis accounted for only a small fraction of insulin accumulation. Stage IV Xenopus oocytes accumulated medium at a basal rate of 5.7 2 0.8 nlicellih. Incubation with 7 nM insulin did not change the rate, but 70 nM insulin increased it by 68% to 9.8 ? 0.6 nlicell/h (data from 5 cells in each group; significantly greater than the basal rate, P < 0.01). The value for the basal rate in Stage IV oocytes falls within the range reported for Stage IV-V oocytes (0.3-20 nlih; Wallace and Jared, 1976; Wall and Patel, 1987). A stimulation of fluid
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Fig. 2. Time course of 70 nM insulin and EGF uptake by Stage IV Xenopus oocytes. Total uptake of '2511-labeledpolypeptide was measured (Methods). Each point represents the mean -t SE for 4-5 cells.
phase endocytosis by 1 pM insulin was reported previously for Stage V oocytes by Opresko and Wiley (1987). From the measured rates of fluid phase endocytosis,we calculate that insulin accumulation by this mechanism would average 0.040 and 0.69 fmoleicellih in media with 7 and 70 nM hormone, respectively. Figure 2 shows that insulin uptake in cells exposed to 70 nM hormone avera es about 4 fmolicellih over the first to fifth hours o f t e experiment. Thus less than 20% of insulin uptake could have occurred by fluid phase endocytosis. Second, lz51-epidermalgrowth factor (EGF), a polypeptide with roughly the same molecular weight as insulin but that does not affect any metabolic function in these oocytes (Miller, 1988,1989, unpublished data), exhibited a several-fold slower rate of cellular accumulation when compared to insulin (Fig. 2). No saturation of uptake was apparent when the EGF concentration was raised from 70 nM to 700 nM (not shown). At 70 nM, EGF uptake averaged 0.7 fmolicellih over h 2 to 5, which is only 18% of insulin uptake over the same period (Fig. 2). Assuming that EGF does not affect the basal rate of fluid phase endoc osis, that process would account for about 60%of EG uptake during this period. Taken together, the insulin and EGF uptake data suggest that, during the prolonged phase, one component of the insulin uptake was specific and mediated. This may represent uptake by receptormediated endocytosis, a process that occurs in insulinsensitive somatic cells (Posner et al., 1982). 12'I-Insulin uptake and distribution data for oocytes incubated with 7 and 70 nM hormone for 2 and 24 h are shown in Table 1.As in the microinjection experiments reported previously, internalized insulin was clearly degraded, with only 10-20% of cell-associated hormone remaining precipitable by 10% TCA. Additional experiments in which undegraded intracellular insulin (24 h exposure) was measured by TCA precipitation and by binding to purified anti-insulin antibody showed that TCA precipitation can overestimate somewhat the amount of undegraded hormone (in two experiments,
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2-h Incubation 7 2.06 f 0.11(5) 70 17.25 f 1.28(5) 24-h Incubation 7 21.65 f 0.84(6) 70 160.36 k 15.03(8)
Undegraded Insulin Membrane Cytoplasm-Nucleus
Cell
*+ 0.02 0.32
0.20 k 0.04 1.87 k 0.17
0.05 f 0.03 1.21 f 0.33
4.37 ?L 0.72 37.22 f 7.14
1.79 k 0.27 13.71 f 3.28
2.58 f 0.07 25.26 6.57
0.21 3.08
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'Indicates degraded plus undegraded hormone. Data given as mean i SE for the number of cells in parentheses.
values obtained by antibody binding were 77 ? 16% and 38 2 15%of those obtained by TCA precipitation). However, this comparison also shows even that if TCA precipitation does overestimate undegraded hormone, a substantial amount of hormone that can react with anti-insulin antibod still remains in cells exposed to insulfn for 24 h (Tab e 1).Insulin degradation is known to occur in most cells studied (Duckworth, 1988) and the extent of degradation can vary greatly from cell to cell (Duckworth, 1988; Smith and Jarett, 1988; Marshall, 1985). At 2 h, most of the cell-associated,undegraded (TCAprecipitable) insulin was localized to the follicle-membrane of the oocyte, although significant accumulation was found in the cytoplasm-nucleusof cells exposed to the higher insulin concentration (Table 1). At 24 h, larger amounts of undegraded insulin were found intracellularly and about 60% of total cell-associated hormone was in the cytoplasm plus nucleus. Assuming that cytoplasm plus nucleus contains 150 nl of available water, calculated average insulin concentrations in cells exposed to 7 and 70 nM for 24 h were 17 and 170 nM, respectively. Thus in 24-h cells, average internal concentrations exceeded those in the media by a factor of about two, suggesting that insulin was bound or compartmentalized intracellularly, or that hormone was trapped by continuous internalization. Antibody microinjection Together, the dose-responsedata in Figure 1and the insulin accumulation data in Table 1 define a set of experimental conditions for testing the involvement of internal sites in insulin action. They predict that undegraded insulin levels in the cytoplasm plus nucleus of cells exposed to 7 and 70 nM hormone for 24 h should be high enough to stimulate protein synthesis by acting at the internal sites, but that levels in cells exposed for 2 h should not. In the first test of these predictions, oocytes were incubated in control (insulin-free) or insulincontaining Ringer's and then transferred to oil. In oil, cells were microinjected with intracellular buffer, preimmune serum diluted with intracellular buffer or antiserum to bovine insulin diluted with intracellular buffer. One h later, cells were injected with 35S-methionine, and after 15 min, processed to measure label incorporation into oocyte protein. Figure 3 shows that neither preimmune serum nor anti-insulin antiserum had any effect on methionine incorporation in control
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Fig. 3. Effects of anti-insulin antiserum on 3sS-methionineincorporation into oocyte protein. Oocytes were incubated in amphibian Ringer's solution containing the inhcated concentration of insulin and then transferred to oil. In oil, each cell was microinjected with 16 nl of intracellular buffer solution without or with guinea pig preimmune serum (diluted 50:l with intracellular buffer) or anti-bovine insulin antiserum similarly diluted. One h later cells were injected with 10 nl of intracellular buffer containing 12fmol of 35S-methionine; after 15 min cells were processed to measure label incorporated into TCA precipitated protein. Two-h and 24-h experiments were conducted with cells from different animals. Results are given as mean i SE for 8-10 cells. *Significantly lower than insulin-stimulated cells injected with buffer or IGG, P < 0.05.
cells; preimmune serum did not affect incorporation in cells exposed to insulin. In cells exposed to 70 nM insulin for 2 h, methionine incorporation nearly doubled (P < 0.01 vs controls), but injection of anti-insulin antiserum had no effect. In cells exposed for 24 h, 7 and 70 nM insulin increased methionine incorporation by 39 and 74%, respectively (P < 0.01), and injected antiserum significantly decreased incorporation only in the low-dose cells (P < 0.05 vs low-dose cells injected with buffer or with preimmune serum, Fig. 3). One explanation for the lack of effect of anti-insulin antiserum in cells ex osed to 70 nM insulin for 24 h is that the amount of ormone in these cells (25 fmol, Table 1) had already saturated the internal sites (Fig. 1)and that excess hormone was present intracellularly. It follows that one should be able to titrate the excess with increasing doses of antiserum and that, with a high enough dose, insulin-stimulated rnethionine incorporation should decrease.
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Initial experiments showed that raising the amount of preimmune serum injected much above that used in Figure 3 inhibited protein synthesis. To circumvent this problem, a partially purified anti-insulin antibody was pre ared (binding capacity about 8 fmol insulin per ng anti ody) and used in microinjection experiments with insulin-exposed cells. These were similar in design to the experiment shown in Figure 3. That is, cells were exposed to insulin in Ringer’s, transferred to oil and injected with buffer, purified immunoglobulins (IGG),or purified anti-insulin antibody; after 1h, cells were injected with 35s-methionine for labelling of synthesized protein. Figure 4 shows the results of one such experiment. Microinjection of 6 ng of IGG from preimmune serum caused no inhibition of methionine incorporation in controls or in insulin-stimulated cells; likewise, microinjection of 6 ng of anti-insulin antibody had no effect on incorporation in control cells. However, in cells exposed to 70 nM insulin for 24 h, microinjected antibody significantly reduced methionine incorporation. In this experiment, 24 h exposure to 70 nM insulin more than doubled methionine incorporation and about 50% of the insulin stimulated component of incorporation was blocked by microinjected antibody. In contrast, the same dose of antibody had no effect on methionine incorporation in oocytes exposed to 70 nM insulin for only 2 h (Fig. 4). Figure 5 shows the combined results of several experiments in which 0.8-9.0 ng of purified IGG or purified anti-insulin antibody were injected into control cells and into cells exposed to 70 nM insulin for 24 h. Clearly, over the dose range studied, control cells were not affected by IGG or antibody microinjection; nor were insulin-exposed cells affected by IGG microinjection. Methionine incorporation was only reduced
when anti-insulin antibody was microinjected into cells exposed to 70 nM insulin. No inhibition was found with an antibody dose of 0.8 ngicell. Inhibition increased over the range of 2.5-6 ngicell and then plateaued at 2530% of total methionine incorporation (Fig. 5). If we consider only the insulin-stimulated component of incorporation, injection of 6-7.5 ng antibody inhibited on average by 39 f 4% (n = 3 experiments). Previous experiments have shown that both external IGF-1 and insulin stimulate protein synthesis with nearly identical dose-response functions in Stage IV Xenopus oocytes (Miller, 1989). An important control for the above antibody injection experiments would be to determine the effects of microinjected anti-insulin antibody on cells stimulated by IGF-1 rather than insulin. In two experiments, 24 h exposure to 70 nM IGF-1 stimulated methionine incorporation by 46 2 8 and 118 ? 12%.Using the same protocol as in Fi ure 4, microinjection of 6 ng of anti-insulin antibody ad no effect on methionine incorporation in these IGF-1 stimulated oocytes (15 min incorporation values in antibody-injected cells were 98 & 7 and 110 ? 7% of buffer injected IGF-1 cells). Thus antibody inhibited specifically that component of protein synthesis stimulated by insulin. DISCUSSION Previous studies from this laboratory have shown that protein synthesis in a giant, insulin-sensitive cell, the Stage IV Xenopus oocyte, is stimulated by external insulin acting at surface receptors and by microinjected, intracellular insulin acting at internal sites (Miller, 1988,1989). These microinjection experiments established the existence of functionally important internal sites through which insulin could control translation. However, for those sites to be of physiological significance, they must be activated when cells are exposed to external insulin. That is, a biologically 30 active form of the hormone must get from the medium 24 HOUR 2 HOUR into the cells and then to the sites. The resent study is B+ focused on the question of whether t is sequence of MlCROlWECTlON < events actually occurs in the oocyte. Together, the data BUFFER 20 show that extracellular insulin does indeed gain access to internal sites and through them stimulates transla0 z tion. Y To detect the action of internalized insulin at the ANTBODY z 0 10 internal sites, we microinjected an antibody to the E hormone into the cytoplasm of oocytes that had been Y exposed to external insulin. Antibody injection signifdl icantly reduced the insulin-dependent component of 0 protein synthesis (measured as methionine incorporaNONE 70nM NONE 7OnM tion). The basal component of incorporation was not INSULIN EXPOSURE affected. This inhibition by microinjected antibody did Fig. 4. Effect of microinjected anti-insulin antibod on methionine not occur in all insulin-stimulated cells; rather, it was incorporation in control and insulin-stimulated (24-E exposure to 70 found only in those cells that had accumulated more nM hormone) oocytes. The antibody was purified from anti-bovine than 2 fmol of undegraded (TCA-precipitable) insulin insulin antiserum by protein G sepharose chromatography and di- in the cytoplasma and nucleus. Thus in cells exposed to luted in intracellular buffer solution. The binding capacity of the dose of purified antibody was about five times that of the dose of anti- external insulin for short times, little undegraded insulin antiserum used in Figure 3. Cells were incubated for 2 or 24 insulin accumulated internally and, although methioh in Ringer’s with 0 or 70 nM insulin and then transferred to oil for nine incorporation was stimulated, microinjected antiantibody injection (6 ng of protein in 12 nl). One h later, they were body was without effect. With longer exposure times, injected with 10 nl of intracellular buffer containing 35S-methionine; 15 min later, cells were processed to measure label incorporation into insulin accumulation increased, incorporation was protein. Data given as mean -t SE for 8-10 cells. *Significantlylower again stimulated, and now microinjected antibody sigthan insulin-stimulated cells injected with buffer or IGG,P < 0.05. nificantly reduced the insulin-stimulated component of
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Fig. 5 . Dose response for inhibition of methionine inco oration by microinjected, purified anti-insulin antibody. Cells were incubated for 24 h in Ringer’s wigout (controli or with 70 nM insulin and then transferred to oil for antibody or IGG injection (above). After 1h, they were injected a second time with 10 nl of intracellular buffer containing 35S-methionine; 15 min later, cells were processed to measure label incorporation into protein. Data from 7 ex eriments have been pooled to produce this figure. Each point gives the mean per cent change caused pby microinjection of antibody or IGG protein with the baseline being buffer-injected cells from the same animal (8-10 cells per treatment, thus 16-20 cells per point). When shown, error bars indicate t 1SE. For the sake of clarity, error bars have been omitted from data for control cells and insulin cells injected with IGG. Cells in these groups showed only small effects of microinjection; the effects were rarely statistically significant and were not dose-dependent. *Significantly different from cells injected with buffer, P < 0.05.
incorporation. The inhibition caused by a low dose of microinjected antibody (antiserum) was abolished in cells that had accumulated an excess of hormone and inhibition became apparent again when the amount of antibody injected was increased further (for cells exposed to 70 nM insulin for 24 h, compare the effects of antiserum injection and purified antibody injection in Figs. 3 and 4).Finally, when methionine incorporation was stimulated by IGF-1, microinjected anti-insulin antibody was without effect. Two observations indicate that the inhibitory effects of microinjected antibody cannot be explained by leakage to the cell surface and subsequent removal of hormone from surface receptors. First, in insulin-stimulated cells that had not accumulated hormone intracellularly, injected antibody was without effect (2-h cells in Figs. 3,4). If leakage had occurred in these cells, one would expect to have seen a reduction in the insulin-dependent component of synthesis. Second, in cells exposed to 70 nm hormone for 24 h, the inhibitory effects of microinjected, purified antibody were circumscribed (Fig. 5). If inhibition was due t o antibody leakage, incorporation would have continued to decrease with increasing dose. Having ruled out leakage, the most likely explanation for the present results is that microinjected antibody reduced the insulin-stimulated component of protein synthesis by sequestering internalized hormone and thus removing it from intracellular sites involved in the control of translation.
If rotein synthesis in Xenopus oocytes is affected by ins& acting at two classes of spatially separate sites (surface and internal), how is overall control distributed between the sites? The answer to this question clearly depends on the history of the cell. Consider an experiment in which oocytes are exposed to 70 nM external insulin. Significant increases in rates of protein synthesis are seen within 15-30 min (Miller, unpublished data). However, over the first 2 h, such increases are signalled solely by hormone interactions with the surface receptors. Support for this conclusion comes from the present experiments where microinjected anti-insulin antibody had no effect on methionine incorporation in cells exposed to 70 nM insulin for 2 h (Fig. 4)and from previous experiments where the effects of external and microinjected insulin were found to be additive (Miller, 1988,1989). During the period between 2 and 24 h, the internal sites do come into play. In fact, the present data show that after 24-h exposure to 70 nM insulin, oocytes accumulated more than enough intracellular hormone to saturate those sites. Because of that, the dose response curve for injected antibody was tri- hasic (Fig. 5), with an initial section over which antibo y had no effect (titration of excess insulin), a middle section in which inhibition of incorporation increased with inected dose, and a final plateau. On the plateau, inhiition was less than complete. On average about 40% of the insulin-dependent component of incorporation was
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inhibited by microinjected antibody. This represents the portion of insulin’s effects on translation mediated by the internal sites. Clearly, under these conditions, control of translation is nearly equally shared between the surface receptor and the internal sites. At present it is not clear what the distribution of control might be in cells exposed to different insulin concentrations or the same concentration over much longer times, e.g., in vivo. The finding that external insulin can affect translation through internal sites of action implies that Xenous oocytes possess an integrated transport and signal[ng system that provides information about the cell’s external environment to internal control points using the hormone itself to convey the message. At a minimum, mechanisms are required to 1)transport insulin across the plasma membrane, 2) deliver it to the internal sites, 3) recognize insulin at the sites, and 4) transduce the signal to the protein synthetic machinery. We currently possess some information about the beginning (transport) and end (effect on translation) of this chain of events, but very little about the intermediate links. Initial studies of 1251-insulinuptake by Stage IV Xenopus oocytes indicate that twd types of transport mechanism are involved, one nonspecific and unsaturable, fluid phase endocytosis, and the other specific and saturable, probably receptor mediated endocytosis (present study). Although the latter is the major route of hormone entry, it is not yet clear which route provides the insulin that acts at the internal sites. This is an im ortant point, because it determines the extent to whic insulin action at internal sites might be ultimately dependent on normal interactions with the surface receptor. It also bears importantly on the next ste , i.e., does delivery to internal sites involve simple di!f usion, vesicular transport or, in analogy with steroid hormones, specific cytoplasmic carrier proteins? With regard to the internal sites of insulin action themselves, little is currently known about functionally important interactions between insulin and intracellular elements in any cell. In the Stage IV Xeno us oocyte, the present and previous studies (Mi ler, 1988,1989)show that intracellular sites through which insulin stimulates translation are accessible to both microinjected hormone and anti-insulin antibody. Since insulin and the antibody would not be expected to readily penetrate intracellular membranes, the sites are probably not within membrane-bounded structures, e.g., endocytotic vesicles. Thus it is likely that hormone that eventually reaches the internal sites must either have esca ed from the endocytic compartment in an biologica ly active form or never have entered it in the first place. It is clear from previous oocyte microinjection experiments that insulin can stimulate protein synthesis in isolated cytoplasm samples (Miller, 1989) and RNA synthesis in isolated nuclei (Miller, 1988). Thus both cytoplasmic and nuclear sites could be involved. However, the nature of these internal sites and their hormone binding pro erties remain unknown. In contrast, available data or somatic cells show that insulin can bind t o internal sites, some of which resemble the surface receptor (reviewed by Goldfine, 1981)and some
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of which do not (Peralta Soler et al., 1989; Thompson et al., 1989). However, neither class of site has been linked directly to any specific action of the hormone. Clearly, insulin is not the only regulatory polypeptide that is internalized and that binds s ecifically to intracellular elements. Many polypeptiI fe hormones and growth factors have long been known to exhibit similar behavior (reviewed by Burwen and Jones, 1987). Importantly, recent microinjection studies now provide direct evidence that two such polypeptides, gamma-interferon and tumor necrosis factor-alpha, may act through intracellular mechanisms (Smith et al., 1990a,1990b). Whether intracellular sites actually play a role in the overall action of those polypeptides remains to be determined. For insulin and the other regulatory polypeptides, action at internal sites may support or fine-tune signals originating at surface membrane receptors, thus contributing to the long-term control of transcription and translation. Understanding the physiological mechanisms that deliver active polypeptides to internal sites and the specific roles those sites play in homeostasis, growth and differentiation, and in pathological states, e.g., insulin resistance, is a long-term goal of future studies.
ACKNOWLEDGMENTS We thank Dr. K. Oliver for purifying anti-insulin antibody, and Drs. D. Armstrong, J.B. Pritchard, and J.W. Putney for helpful discussions.
LITERATURE CITED Burwen, S.J. and Jones, A.L. (1987) The association of polypeptide hormones and growth factors with the nuclei of target cells. Trends Biochem. Sci., I2t159-162. Czech, M.P., Karlund, J.K., Yagaloff, K.A., Bradford, A.P., and Lewis, R.E. (1988) Insulin receptor signalling. Activation of multiple serine kinases. J. Biol. Chem., 263:11017-11020. Duckworth, W.C. (1988) Insulin degradation: Mechanisms, products and significance. Endocrine Rev., 9.319345. Goldfine, LD. (1981)Interaction of insulin, polypeptide hormones and growth factors with intracellular membranes. Biochim. Biophys. Acta, 650t53-67. Goldfine, I.D. (1987) The insulin receptor: Molecular biology and transmembrane signalling. Endocr. Rev., 8:235-255. Goldfine, I.D., Purrello: F., Vigneri, R., and Clawson, G.A. (1985) Insulin and the regulation of isolated nuclei and nuclear subfractions: Potential relationship to mRNA metabolism. DiabetesiMetab. Rev., I:119-137. Horowitz, S.B., and Miller, D.S. (1984) Solvent properties of ground substance studied by cryomicrodissection and intracellular reference-phase techniques. J . Cell Biol., 99:172s179s. Kahn, C.R., and White, M.F. (1988) The insulin receptor and the molecular mechanism of insulin action. J . Clin. Invest., 82:11511156. Marshall, S., (1985) Kinetics of insulin receptor internalization and recycling in adipocytes. Shunting of receptors to a degradative pathway by inhibitors of recycling. J. Biol. Chem., 260:4136-4144. Miller, D.S. (1988) Stimulation of RNA and protein synthesis by intracellular insulin. Science 240:506509. Miller, D.S. (1989) Stimulation of protein synthesis in Stage IV Xenopus oocytes by microinjected insulin. J. Biol. Chem., 264:i0438-10446. Opresko, L.K., and Wiley, H.S. (1987) Receptor-mediated endocytosis in Xenopus oocytes 11. Evidence for two novel mechanisms of hormonal regulation. J. Biol. Chem., 262:4116-4123. Peralta Soler, A., Thompson, K.A., Smith, R.A., and Jarett, L. (1989) Immunological demonstration of the accumulation of insulin. but not insulin receptors, in nuclei of insulin-treated cells. Roc. Natl. Acad. Sci., USA, 86.6640-6644.
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Posner, B.I., Kahn, M.N., and Bergeron, J.J. (1982) Endocytosis of peptide hormones and other ligands. Endocr. Rev., 3:280-298. Saltiel, A.R., and Cuatrecasas, P. (1988) In search of a second messenger for insulin. Am. J. Phyiol., 225:Cl-Cll. Smith, R.M., and Jarett, L. (1988) Receptor-mediated endocytosis and intracellular processing of insulin: Ultrastructural and biochemical evidence for cell-specific heterogeneity and distinction from nonhormonal ligands. Lab. Invest., 58:613-629. Smith, M.R., Muegge, K., Keller, J.R., Kung, H.-S., Young, H.A. and Durum, S.K. (1990a) Direct evidence for an intracellular role for IFN-gamma. Microinjection of human IFN-gamma induces Ia expression on murine macrophages. J . Immunol.. 144:1777-1782. Smith, M.R., Munger, W.E., Kung, H . 3 , Takacs, L., andDurum, S.K.
(1990b) Direct evidence for an intracellular role for tumor necrosis factor-alpha. Microinjection of tumor necrosis factor kills target cells. J. Immunol. 144:162-169. Thompson, K.A., Soler, A.P., Smith, R.W., and Jarett, L. (1989) Intranuclear localization of insulin in rat hepatoma cells: insulin/ matrix association. Eur. J.Cell Biol., 50:442446. Wall, D.A., and Patel, S.(1987) The intracellular fate of vitellogenin in Xenopus oocytes is determined by its extracellular concentration during endocytosis. J. Biol. Chem., 262:14779-14789. Wallace, R.A.: and Jared, D.W. (1976) Protein incorporation by isolated amphibian oocytes V. Specificity for vitellogenin incorpcration. J. Cell Biol., 69:345-351.
Stimulation of Protein Synthesis by Internalized Insulin DAVID S. MILLER* AND DESTINY B. SYKES
laboratory of Cellular and Molecular Pharmacology, Nationdl Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
Previous studies showed that microinjected insulin stimulates transcription and translation in Stage IV Xenopus oocytes by acting at nuclear and cytoplasmic sites (Miller, D.S., 1988, 1989). The present report is concerned with the question of whether hormone, internalized from an external medium, can act on those sites to alter cell function. Both intracellular accumulation of undegraded ’251-insulin and insulin-stimulated 3’S-methionine incorporation into oocyte protein were measured. Anti-insulin antiserum and purified anti-insulin antibody were microinjected into the cytoplasm of insulin-exposed cells to determine if insulin derived from the medium acted through internal sites. In cells exposed for 2 h to 7 or 70 nM external insulin, methionine incorporation was stimulated, but intracellular hormone accumulation was minimal and microinjected antibody was without effect. In cells exposed for 24 h, methionine incorporation again increased, but now accumulation of undegraded, intracellular hormone was substantial (2.6 and 25.3 fmol with 7 and 70 nM, respectively), and microinjected anti-insulin antibody significantly reduced the insulin-stimulated component of incorporation; basal incorporation was not affected. For cells exposed to 70 nM insulin for 24 h, inhibition of the insulin-stimulated component was maximal at 39%. Thus under those conditions, about 40% of insulin’s effects were mediated by the internal sites. Together, the data show that inhibition of insulin-stimulated protein synthesis by microinjected antibody was associated with the intracellular accumulation of insulin. They indicate that when oocytes are exposed to external insulin, hormone eventually gains access to intracellular sites of action and through these stimulates translation. Control of translation appears to be shared between the internal sites and the surface receptor. In sensitive cells, insulin alters the function of virtually every organelle. However, the chain of events by which the hormone signals metabolic changes at intracellular sites remains unclear. Insulin action is initiated when hormone binds to a specific external receptor and binding induces multiple secondary events at the plasma membrane, e.g., receptor autophosphorylation, protein tyrosine kinase activation, receptor and ligand internalization, and release of second messengers (reviewed by Goldfine, 1987; Czech et al., 1988; Kahn and White, 1988; Saltiel and Cuatrecasas, 1988). Each of these in turn has been associated with one or more of insulin’s actions, but defining links between putative signalling mechanisms and final intracellular events has been difficult, especially for sites spatially removed from the plasma membrane. Experiments showing that insulin is internalized (Posner et al., 1982) and that cells possess internal receptors (Goldfine, 1981; Goldfine et al., 1985) have led to the suggestion that internal as well as surface receptors transduce the hormone’s effects. However, determining what role internalized hormone might play in the overall mechanism of insulin action has been complicated by technical problems arising primar0 1991 WILEY-LISS, INC.
ily from the small size of most cells. Many of these problems can be avoided by the use of a giant, insulinsensitive cell, the Stage IV oocyte from Xenopus laevis (-800 pM in diameter), non-aqueous microinjection and microdissection techniques, and single-cell microanalysis (Miller, 1988,1989). Recently, this experimental system was used to demonstrate that both RNA and protein synthesis increases when insulin is microinjected into oocyte cytoplasm (Miller, 1988,1989). Since such increases are found when insulin is applied directly to isolated nuclei and to droplets of undiluted cytoplasm, the hormone must be acting through internal sites (receptors?) not associated with the plasma membrane. Indeed, the nuclear and cyto lasmic sites appear to stimulate RNA and protein synt esis by local signalling mechanisms. That is, insulin increases RNA synthesis in the absence of cytoplasm, and protein synthesis in the absence of nuclear material; increases in RNA and protein synthesis can occur in the absence of plasma membrane.
K
Received October 30, 1990; accepted February 27, 1991 *To whom reprint requestsicorrespondence should be addressed.
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MILLER AND SYKES
It remained to be established whether the internal sites are activated when cells are exposed to external hormone. The present report provides evidence that this is indeed the case. We show here that when Xenopus oocytes were incubated with 7 or 70 nM external insulin, undegraded hormone accumulated in the surface membranes, cytoplasm and nucleus and methionine incorporation into oocyte protein was stimulated. When incubation times were long enough for several fmol of undegraded hormone to accumulate in the cytoplasm and nucleus, an antibody to insulin, microinjected into the cytoplasm, partially reversed the hormone-mediated stimulation of protein synthesis. Thus when cells were exposed to external insulin, hormone was internalized and internalized hormone then acted through intracellular sites to stimulate translation. MATERIALS AND METHODS Chemicals and radioisotopes Bovine pancreatic insulin (24.6 unitslmg), guinea pig preimmune serum, and anti-bovine insulin antiserum (developed in guinea pig; affinity constant 5 X lo1' limol) were obtained from Sigma Chemical Co. (St. Louis) and recombinant human insulin like growth factor-1 (IGF-1) was purchased from Collaborative Research, Inc. (Cambridge). Prior to use, sera were dialyzed a ainst intracellular buffer (below).Anti-insulin antibo y and immunoglobulins from preimmune serum were urified by Protein G se harose chromatography. Ot er unlabeled chemica s were of rea ent ade or hi her and were purchased primarily rom Kgma. 35S- ethionine (specific activity -1,000 Ci/ mmol) and 1251-insulin(monoiodinated, porcine insulin; specific activity -90 uciipg) were obtained from DuPont-New England Nuclear. Oocytes Stage IV ooc tes (800 ? 50 pm diameter) in follicles were isolated y dissection from unstimulated South African clawed toads (Xenopus Zaevis from Nasco) into an amphibian oocyte Ringer's solution (in mM: 82.5 NaC1,25 KC1,l.O CaCl,, 1.0 M Clz, 1.0 Na2HP04, 5.0 HEPES, 1.0 Na pyruvate, at p% 7.6). All procedures were carried out at 20" C. Insulin uptake, distribution, and degradation To measure insulin uptake from a physiological saline solution, oocytes were incubated in Ringer's containing 0.01% bovine serum albumin (BSA) and 1251-insulin.At the end of the incubation period, each cell was removed from the medium, washed briefly in insulin-free Ringer's, blotted, and transferred to paraffin oil as described previously (Miller, 1989).Under oil, each cell was dissected into two components, the follicle plus plasma membrane and the cytoplasm plus nucleus. Adherent cortical cytoplasm was clearly visible on membrane samples. However, light microscopic and marker enzyme analyses have shown no detectable contamination of cytoplasm-nucleus samples by plasma membrane fragments (Miller, 1989). After isolation, each cell component was transferred to a tube containing 10% trichloroacetic acid (TCA) solution and counted (gamma counter) to measure total
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label. Tubes were then centrifuged and an aliquot of the supernatant was counted to measure degraded insulin; undegraded hormone was calculated as the difference, i.e., the TCA-precipitable fraction. Sample insulin content in fmol was calculated from sample activity and medium specific activity. Microinjection Oocytes in oil were microinjected in the vegetal hemisphere with 8-15 nl of intracellular buffer solution (in mM: 10 NaC1, 125 KC1, 1.0 KH2C03, with 0.01% BSA and at pH 7.2) containing the indicated amounts of insulin, antiserum or label. Glass capillary micropipets (tip diameter < 10 pm) and a hydraulic nanopump (WPI Instruments) were used. A detailed description of the microinjection procedure is given by Miller (1988,1989).
Protein synthesis 35S-Methionineincorporation into oocyte protein was measured. Radiolabel was microinjected into cells under oil and after a suitable labelling period each cell was processed to measure label incorporation into TCA (10%) precipitable protein (Miller, 1988). Using this procedure, both the amount of label injected into each cell and the percentage of that incorporated into protein were determined. When insulin was also present in the injection solution (Fig. l), the amount of hormone injected was calculated from the total 35S-methionine counts in each cell, the activity of the injection solution
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Fig. 1. Dose response for stimulation of 35S-methionine incorporation into oocyte protein by microinjected insulin. Stage IV oocytes in oil were microinjected at time zero with 12-16 nl intracellular buffer solution containing 0.01% BSA without or with the indicated dose of insulin and 10-20 fmol 35S-methionine. One h later, cells were processed to measure label incorporation into oocyte protein as well as the amount of insulin injected (Methods). Shown are pooled data (mean SE) from 5-9 experiments; in each experiment at least 10 controls and 10 treated cells per dose were assayed. Some of these data were presented previously in tabular form (Miller, 1989). The top axis shows the amount of undegraded insulin present 30 min after microinjection, estimated from the data of Miller (1989). Although the apparent rate of intracellular insulin degradation decreases somewhat with injected dose, for simplicity's sake, the amount of hormone remaining intact was taken to be 35% of that injected.
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INTERNALIZED INSULIN AND PROTEIN SYNTHESIS
and the hormone concentration in the injection solution. RESULTS To define conditions for testing the involvement of internal sites in the action of externally applied insulin, answers to two questions were needed: (1)What levels of intracellular insulin would be ex ected to stimulate translation? 2) Under what con itions do such levels accumulate in cells exposed to external hormone? Insulin dose-response Figure 1 shows the dose-response data for microinjected insulin's effects on methionine incorporation that are the basis for an answer to the first question. Incorporation increased with increasing dose of microinjected insulin and then plateaued at a dose of about 40 fmol. Detailed analysis of these data is complicated by three time-dependent processes that inevitably follow insulin microinjection: diffusion of hormone from the injection site, rapid inactivation by degradation (Miller, 1989), and specific and nonspecific binding to cellular elements. These processes occur simultaneously and they, along with uncertainties as to insulin and water compartmentation (Horowitz and Miller, 19841, confound attempts to associate metabolic changes with bound or free insulin at any intracellular site. Of the three processes, degradation is the only one for which the dose-response data can be corrected (Miller, 1989). The top axis of Figure 1 shows undegraded, i.e., TCA-precipitable, insulin 30 min after microinjection, a time when protein synthesis has begun to increase in response to injected hormone. The corrected data indicate that about 2-3 fmol of undegraded insulin per cell significantly stimulated methionine incorporation and that about 14 fmolicell stimulated maximally. Insulin accumulation The time course of 1251-insulinuptake by oocytes was biphasic, with a rapid initial phase occurring over the first few min followed by a slower prolonged phase (Fig. 2). Dissection of these cells in oil revealed that during the initial phase label was localized to the plasma membrane and follicle, consistent with rapid binding to surface receptors; over several hours, label was also found in the cytoplasm and nucleus (Table 1). Insulin uptake partially saturated in the concentration range 70-700 nM (not shown). Two types of experiment suggest that insulin uptake occurs at least in part by a specific mechanism. First, our initial measurements of 3H-sucrose and 3H-polyethylene glycol uptake showed that fluid phase endocytosis accounted for only a small fraction of insulin accumulation. Stage IV Xenopus oocytes accumulated medium at a basal rate of 5.7 2 0.8 nlicellih. Incubation with 7 nM insulin did not change the rate, but 70 nM insulin increased it by 68% to 9.8 ? 0.6 nlicell/h (data from 5 cells in each group; significantly greater than the basal rate, P < 0.01). The value for the basal rate in Stage IV oocytes falls within the range reported for Stage IV-V oocytes (0.3-20 nlih; Wallace and Jared, 1976; Wall and Patel, 1987). A stimulation of fluid
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Fig. 2. Time course of 70 nM insulin and EGF uptake by Stage IV Xenopus oocytes. Total uptake of '2511-labeledpolypeptide was measured (Methods). Each point represents the mean -t SE for 4-5 cells.
phase endocytosis by 1 pM insulin was reported previously for Stage V oocytes by Opresko and Wiley (1987). From the measured rates of fluid phase endocytosis,we calculate that insulin accumulation by this mechanism would average 0.040 and 0.69 fmoleicellih in media with 7 and 70 nM hormone, respectively. Figure 2 shows that insulin uptake in cells exposed to 70 nM hormone avera es about 4 fmolicellih over the first to fifth hours o f t e experiment. Thus less than 20% of insulin uptake could have occurred by fluid phase endocytosis. Second, lz51-epidermalgrowth factor (EGF), a polypeptide with roughly the same molecular weight as insulin but that does not affect any metabolic function in these oocytes (Miller, 1988,1989, unpublished data), exhibited a several-fold slower rate of cellular accumulation when compared to insulin (Fig. 2). No saturation of uptake was apparent when the EGF concentration was raised from 70 nM to 700 nM (not shown). At 70 nM, EGF uptake averaged 0.7 fmolicellih over h 2 to 5, which is only 18% of insulin uptake over the same period (Fig. 2). Assuming that EGF does not affect the basal rate of fluid phase endoc osis, that process would account for about 60%of EG uptake during this period. Taken together, the insulin and EGF uptake data suggest that, during the prolonged phase, one component of the insulin uptake was specific and mediated. This may represent uptake by receptormediated endocytosis, a process that occurs in insulinsensitive somatic cells (Posner et al., 1982). 12'I-Insulin uptake and distribution data for oocytes incubated with 7 and 70 nM hormone for 2 and 24 h are shown in Table 1.As in the microinjection experiments reported previously, internalized insulin was clearly degraded, with only 10-20% of cell-associated hormone remaining precipitable by 10% TCA. Additional experiments in which undegraded intracellular insulin (24 h exposure) was measured by TCA precipitation and by binding to purified anti-insulin antibody showed that TCA precipitation can overestimate somewhat the amount of undegraded hormone (in two experiments,
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(nW
Total Uptake' Cell
2-h Incubation 7 2.06 f 0.11(5) 70 17.25 f 1.28(5) 24-h Incubation 7 21.65 f 0.84(6) 70 160.36 k 15.03(8)
Undegraded Insulin Membrane Cytoplasm-Nucleus
Cell
*+ 0.02 0.32
0.20 k 0.04 1.87 k 0.17
0.05 f 0.03 1.21 f 0.33
4.37 ?L 0.72 37.22 f 7.14
1.79 k 0.27 13.71 f 3.28
2.58 f 0.07 25.26 6.57
0.21 3.08
+
'Indicates degraded plus undegraded hormone. Data given as mean i SE for the number of cells in parentheses.
values obtained by antibody binding were 77 ? 16% and 38 2 15%of those obtained by TCA precipitation). However, this comparison also shows even that if TCA precipitation does overestimate undegraded hormone, a substantial amount of hormone that can react with anti-insulin antibod still remains in cells exposed to insulfn for 24 h (Tab e 1).Insulin degradation is known to occur in most cells studied (Duckworth, 1988) and the extent of degradation can vary greatly from cell to cell (Duckworth, 1988; Smith and Jarett, 1988; Marshall, 1985). At 2 h, most of the cell-associated,undegraded (TCAprecipitable) insulin was localized to the follicle-membrane of the oocyte, although significant accumulation was found in the cytoplasm-nucleusof cells exposed to the higher insulin concentration (Table 1). At 24 h, larger amounts of undegraded insulin were found intracellularly and about 60% of total cell-associated hormone was in the cytoplasm plus nucleus. Assuming that cytoplasm plus nucleus contains 150 nl of available water, calculated average insulin concentrations in cells exposed to 7 and 70 nM for 24 h were 17 and 170 nM, respectively. Thus in 24-h cells, average internal concentrations exceeded those in the media by a factor of about two, suggesting that insulin was bound or compartmentalized intracellularly, or that hormone was trapped by continuous internalization. Antibody microinjection Together, the dose-responsedata in Figure 1and the insulin accumulation data in Table 1 define a set of experimental conditions for testing the involvement of internal sites in insulin action. They predict that undegraded insulin levels in the cytoplasm plus nucleus of cells exposed to 7 and 70 nM hormone for 24 h should be high enough to stimulate protein synthesis by acting at the internal sites, but that levels in cells exposed for 2 h should not. In the first test of these predictions, oocytes were incubated in control (insulin-free) or insulincontaining Ringer's and then transferred to oil. In oil, cells were microinjected with intracellular buffer, preimmune serum diluted with intracellular buffer or antiserum to bovine insulin diluted with intracellular buffer. One h later, cells were injected with 35S-methionine, and after 15 min, processed to measure label incorporation into oocyte protein. Figure 3 shows that neither preimmune serum nor anti-insulin antiserum had any effect on methionine incorporation in control
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Fig. 3. Effects of anti-insulin antiserum on 3sS-methionineincorporation into oocyte protein. Oocytes were incubated in amphibian Ringer's solution containing the inhcated concentration of insulin and then transferred to oil. In oil, each cell was microinjected with 16 nl of intracellular buffer solution without or with guinea pig preimmune serum (diluted 50:l with intracellular buffer) or anti-bovine insulin antiserum similarly diluted. One h later cells were injected with 10 nl of intracellular buffer containing 12fmol of 35S-methionine; after 15 min cells were processed to measure label incorporated into TCA precipitated protein. Two-h and 24-h experiments were conducted with cells from different animals. Results are given as mean i SE for 8-10 cells. *Significantly lower than insulin-stimulated cells injected with buffer or IGG, P < 0.05.
cells; preimmune serum did not affect incorporation in cells exposed to insulin. In cells exposed to 70 nM insulin for 2 h, methionine incorporation nearly doubled (P < 0.01 vs controls), but injection of anti-insulin antiserum had no effect. In cells exposed for 24 h, 7 and 70 nM insulin increased methionine incorporation by 39 and 74%, respectively (P < 0.01), and injected antiserum significantly decreased incorporation only in the low-dose cells (P < 0.05 vs low-dose cells injected with buffer or with preimmune serum, Fig. 3). One explanation for the lack of effect of anti-insulin antiserum in cells ex osed to 70 nM insulin for 24 h is that the amount of ormone in these cells (25 fmol, Table 1) had already saturated the internal sites (Fig. 1)and that excess hormone was present intracellularly. It follows that one should be able to titrate the excess with increasing doses of antiserum and that, with a high enough dose, insulin-stimulated rnethionine incorporation should decrease.
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Initial experiments showed that raising the amount of preimmune serum injected much above that used in Figure 3 inhibited protein synthesis. To circumvent this problem, a partially purified anti-insulin antibody was pre ared (binding capacity about 8 fmol insulin per ng anti ody) and used in microinjection experiments with insulin-exposed cells. These were similar in design to the experiment shown in Figure 3. That is, cells were exposed to insulin in Ringer’s, transferred to oil and injected with buffer, purified immunoglobulins (IGG),or purified anti-insulin antibody; after 1h, cells were injected with 35s-methionine for labelling of synthesized protein. Figure 4 shows the results of one such experiment. Microinjection of 6 ng of IGG from preimmune serum caused no inhibition of methionine incorporation in controls or in insulin-stimulated cells; likewise, microinjection of 6 ng of anti-insulin antibody had no effect on incorporation in control cells. However, in cells exposed to 70 nM insulin for 24 h, microinjected antibody significantly reduced methionine incorporation. In this experiment, 24 h exposure to 70 nM insulin more than doubled methionine incorporation and about 50% of the insulin stimulated component of incorporation was blocked by microinjected antibody. In contrast, the same dose of antibody had no effect on methionine incorporation in oocytes exposed to 70 nM insulin for only 2 h (Fig. 4). Figure 5 shows the combined results of several experiments in which 0.8-9.0 ng of purified IGG or purified anti-insulin antibody were injected into control cells and into cells exposed to 70 nM insulin for 24 h. Clearly, over the dose range studied, control cells were not affected by IGG or antibody microinjection; nor were insulin-exposed cells affected by IGG microinjection. Methionine incorporation was only reduced
when anti-insulin antibody was microinjected into cells exposed to 70 nM insulin. No inhibition was found with an antibody dose of 0.8 ngicell. Inhibition increased over the range of 2.5-6 ngicell and then plateaued at 2530% of total methionine incorporation (Fig. 5). If we consider only the insulin-stimulated component of incorporation, injection of 6-7.5 ng antibody inhibited on average by 39 f 4% (n = 3 experiments). Previous experiments have shown that both external IGF-1 and insulin stimulate protein synthesis with nearly identical dose-response functions in Stage IV Xenopus oocytes (Miller, 1989). An important control for the above antibody injection experiments would be to determine the effects of microinjected anti-insulin antibody on cells stimulated by IGF-1 rather than insulin. In two experiments, 24 h exposure to 70 nM IGF-1 stimulated methionine incorporation by 46 2 8 and 118 ? 12%.Using the same protocol as in Fi ure 4, microinjection of 6 ng of anti-insulin antibody ad no effect on methionine incorporation in these IGF-1 stimulated oocytes (15 min incorporation values in antibody-injected cells were 98 & 7 and 110 ? 7% of buffer injected IGF-1 cells). Thus antibody inhibited specifically that component of protein synthesis stimulated by insulin. DISCUSSION Previous studies from this laboratory have shown that protein synthesis in a giant, insulin-sensitive cell, the Stage IV Xenopus oocyte, is stimulated by external insulin acting at surface receptors and by microinjected, intracellular insulin acting at internal sites (Miller, 1988,1989). These microinjection experiments established the existence of functionally important internal sites through which insulin could control translation. However, for those sites to be of physiological significance, they must be activated when cells are exposed to external insulin. That is, a biologically 30 active form of the hormone must get from the medium 24 HOUR 2 HOUR into the cells and then to the sites. The resent study is B+ focused on the question of whether t is sequence of MlCROlWECTlON < events actually occurs in the oocyte. Together, the data BUFFER 20 show that extracellular insulin does indeed gain access to internal sites and through them stimulates transla0 z tion. Y To detect the action of internalized insulin at the ANTBODY z 0 10 internal sites, we microinjected an antibody to the E hormone into the cytoplasm of oocytes that had been Y exposed to external insulin. Antibody injection signifdl icantly reduced the insulin-dependent component of 0 protein synthesis (measured as methionine incorporaNONE 70nM NONE 7OnM tion). The basal component of incorporation was not INSULIN EXPOSURE affected. This inhibition by microinjected antibody did Fig. 4. Effect of microinjected anti-insulin antibod on methionine not occur in all insulin-stimulated cells; rather, it was incorporation in control and insulin-stimulated (24-E exposure to 70 found only in those cells that had accumulated more nM hormone) oocytes. The antibody was purified from anti-bovine than 2 fmol of undegraded (TCA-precipitable) insulin insulin antiserum by protein G sepharose chromatography and di- in the cytoplasma and nucleus. Thus in cells exposed to luted in intracellular buffer solution. The binding capacity of the dose of purified antibody was about five times that of the dose of anti- external insulin for short times, little undegraded insulin antiserum used in Figure 3. Cells were incubated for 2 or 24 insulin accumulated internally and, although methioh in Ringer’s with 0 or 70 nM insulin and then transferred to oil for nine incorporation was stimulated, microinjected antiantibody injection (6 ng of protein in 12 nl). One h later, they were body was without effect. With longer exposure times, injected with 10 nl of intracellular buffer containing 35S-methionine; 15 min later, cells were processed to measure label incorporation into insulin accumulation increased, incorporation was protein. Data given as mean -t SE for 8-10 cells. *Significantlylower again stimulated, and now microinjected antibody sigthan insulin-stimulated cells injected with buffer or IGG,P < 0.05. nificantly reduced the insulin-stimulated component of
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Fig. 5 . Dose response for inhibition of methionine inco oration by microinjected, purified anti-insulin antibody. Cells were incubated for 24 h in Ringer’s wigout (controli or with 70 nM insulin and then transferred to oil for antibody or IGG injection (above). After 1h, they were injected a second time with 10 nl of intracellular buffer containing 35S-methionine; 15 min later, cells were processed to measure label incorporation into protein. Data from 7 ex eriments have been pooled to produce this figure. Each point gives the mean per cent change caused pby microinjection of antibody or IGG protein with the baseline being buffer-injected cells from the same animal (8-10 cells per treatment, thus 16-20 cells per point). When shown, error bars indicate t 1SE. For the sake of clarity, error bars have been omitted from data for control cells and insulin cells injected with IGG. Cells in these groups showed only small effects of microinjection; the effects were rarely statistically significant and were not dose-dependent. *Significantly different from cells injected with buffer, P < 0.05.
incorporation. The inhibition caused by a low dose of microinjected antibody (antiserum) was abolished in cells that had accumulated an excess of hormone and inhibition became apparent again when the amount of antibody injected was increased further (for cells exposed to 70 nM insulin for 24 h, compare the effects of antiserum injection and purified antibody injection in Figs. 3 and 4).Finally, when methionine incorporation was stimulated by IGF-1, microinjected anti-insulin antibody was without effect. Two observations indicate that the inhibitory effects of microinjected antibody cannot be explained by leakage to the cell surface and subsequent removal of hormone from surface receptors. First, in insulin-stimulated cells that had not accumulated hormone intracellularly, injected antibody was without effect (2-h cells in Figs. 3,4). If leakage had occurred in these cells, one would expect to have seen a reduction in the insulin-dependent component of synthesis. Second, in cells exposed to 70 nm hormone for 24 h, the inhibitory effects of microinjected, purified antibody were circumscribed (Fig. 5). If inhibition was due t o antibody leakage, incorporation would have continued to decrease with increasing dose. Having ruled out leakage, the most likely explanation for the present results is that microinjected antibody reduced the insulin-stimulated component of protein synthesis by sequestering internalized hormone and thus removing it from intracellular sites involved in the control of translation.
If rotein synthesis in Xenopus oocytes is affected by ins& acting at two classes of spatially separate sites (surface and internal), how is overall control distributed between the sites? The answer to this question clearly depends on the history of the cell. Consider an experiment in which oocytes are exposed to 70 nM external insulin. Significant increases in rates of protein synthesis are seen within 15-30 min (Miller, unpublished data). However, over the first 2 h, such increases are signalled solely by hormone interactions with the surface receptors. Support for this conclusion comes from the present experiments where microinjected anti-insulin antibody had no effect on methionine incorporation in cells exposed to 70 nM insulin for 2 h (Fig. 4)and from previous experiments where the effects of external and microinjected insulin were found to be additive (Miller, 1988,1989). During the period between 2 and 24 h, the internal sites do come into play. In fact, the present data show that after 24-h exposure to 70 nM insulin, oocytes accumulated more than enough intracellular hormone to saturate those sites. Because of that, the dose response curve for injected antibody was tri- hasic (Fig. 5), with an initial section over which antibo y had no effect (titration of excess insulin), a middle section in which inhibition of incorporation increased with inected dose, and a final plateau. On the plateau, inhiition was less than complete. On average about 40% of the insulin-dependent component of incorporation was
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inhibited by microinjected antibody. This represents the portion of insulin’s effects on translation mediated by the internal sites. Clearly, under these conditions, control of translation is nearly equally shared between the surface receptor and the internal sites. At present it is not clear what the distribution of control might be in cells exposed to different insulin concentrations or the same concentration over much longer times, e.g., in vivo. The finding that external insulin can affect translation through internal sites of action implies that Xenous oocytes possess an integrated transport and signal[ng system that provides information about the cell’s external environment to internal control points using the hormone itself to convey the message. At a minimum, mechanisms are required to 1)transport insulin across the plasma membrane, 2) deliver it to the internal sites, 3) recognize insulin at the sites, and 4) transduce the signal to the protein synthetic machinery. We currently possess some information about the beginning (transport) and end (effect on translation) of this chain of events, but very little about the intermediate links. Initial studies of 1251-insulinuptake by Stage IV Xenopus oocytes indicate that twd types of transport mechanism are involved, one nonspecific and unsaturable, fluid phase endocytosis, and the other specific and saturable, probably receptor mediated endocytosis (present study). Although the latter is the major route of hormone entry, it is not yet clear which route provides the insulin that acts at the internal sites. This is an im ortant point, because it determines the extent to whic insulin action at internal sites might be ultimately dependent on normal interactions with the surface receptor. It also bears importantly on the next ste , i.e., does delivery to internal sites involve simple di!f usion, vesicular transport or, in analogy with steroid hormones, specific cytoplasmic carrier proteins? With regard to the internal sites of insulin action themselves, little is currently known about functionally important interactions between insulin and intracellular elements in any cell. In the Stage IV Xeno us oocyte, the present and previous studies (Mi ler, 1988,1989)show that intracellular sites through which insulin stimulates translation are accessible to both microinjected hormone and anti-insulin antibody. Since insulin and the antibody would not be expected to readily penetrate intracellular membranes, the sites are probably not within membrane-bounded structures, e.g., endocytotic vesicles. Thus it is likely that hormone that eventually reaches the internal sites must either have esca ed from the endocytic compartment in an biologica ly active form or never have entered it in the first place. It is clear from previous oocyte microinjection experiments that insulin can stimulate protein synthesis in isolated cytoplasm samples (Miller, 1989) and RNA synthesis in isolated nuclei (Miller, 1988). Thus both cytoplasmic and nuclear sites could be involved. However, the nature of these internal sites and their hormone binding pro erties remain unknown. In contrast, available data or somatic cells show that insulin can bind t o internal sites, some of which resemble the surface receptor (reviewed by Goldfine, 1981)and some
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of which do not (Peralta Soler et al., 1989; Thompson et al., 1989). However, neither class of site has been linked directly to any specific action of the hormone. Clearly, insulin is not the only regulatory polypeptide that is internalized and that binds s ecifically to intracellular elements. Many polypeptiI fe hormones and growth factors have long been known to exhibit similar behavior (reviewed by Burwen and Jones, 1987). Importantly, recent microinjection studies now provide direct evidence that two such polypeptides, gamma-interferon and tumor necrosis factor-alpha, may act through intracellular mechanisms (Smith et al., 1990a,1990b). Whether intracellular sites actually play a role in the overall action of those polypeptides remains to be determined. For insulin and the other regulatory polypeptides, action at internal sites may support or fine-tune signals originating at surface membrane receptors, thus contributing to the long-term control of transcription and translation. Understanding the physiological mechanisms that deliver active polypeptides to internal sites and the specific roles those sites play in homeostasis, growth and differentiation, and in pathological states, e.g., insulin resistance, is a long-term goal of future studies.
ACKNOWLEDGMENTS We thank Dr. K. Oliver for purifying anti-insulin antibody, and Drs. D. Armstrong, J.B. Pritchard, and J.W. Putney for helpful discussions.
LITERATURE CITED Burwen, S.J. and Jones, A.L. (1987) The association of polypeptide hormones and growth factors with the nuclei of target cells. Trends Biochem. Sci., I2t159-162. Czech, M.P., Karlund, J.K., Yagaloff, K.A., Bradford, A.P., and Lewis, R.E. (1988) Insulin receptor signalling. Activation of multiple serine kinases. J. Biol. Chem., 263:11017-11020. Duckworth, W.C. (1988) Insulin degradation: Mechanisms, products and significance. Endocrine Rev., 9.319345. Goldfine, LD. (1981)Interaction of insulin, polypeptide hormones and growth factors with intracellular membranes. Biochim. Biophys. Acta, 650t53-67. Goldfine, I.D. (1987) The insulin receptor: Molecular biology and transmembrane signalling. Endocr. Rev., 8:235-255. Goldfine, I.D., Purrello: F., Vigneri, R., and Clawson, G.A. (1985) Insulin and the regulation of isolated nuclei and nuclear subfractions: Potential relationship to mRNA metabolism. DiabetesiMetab. Rev., I:119-137. Horowitz, S.B., and Miller, D.S. (1984) Solvent properties of ground substance studied by cryomicrodissection and intracellular reference-phase techniques. J . Cell Biol., 99:172s179s. Kahn, C.R., and White, M.F. (1988) The insulin receptor and the molecular mechanism of insulin action. J . Clin. Invest., 82:11511156. Marshall, S., (1985) Kinetics of insulin receptor internalization and recycling in adipocytes. Shunting of receptors to a degradative pathway by inhibitors of recycling. J. Biol. Chem., 260:4136-4144. Miller, D.S. (1988) Stimulation of RNA and protein synthesis by intracellular insulin. Science 240:506509. Miller, D.S. (1989) Stimulation of protein synthesis in Stage IV Xenopus oocytes by microinjected insulin. J. Biol. Chem., 264:i0438-10446. Opresko, L.K., and Wiley, H.S. (1987) Receptor-mediated endocytosis in Xenopus oocytes 11. Evidence for two novel mechanisms of hormonal regulation. J. Biol. Chem., 262:4116-4123. Peralta Soler, A., Thompson, K.A., Smith, R.A., and Jarett, L. (1989) Immunological demonstration of the accumulation of insulin. but not insulin receptors, in nuclei of insulin-treated cells. Roc. Natl. Acad. Sci., USA, 86.6640-6644.
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Posner, B.I., Kahn, M.N., and Bergeron, J.J. (1982) Endocytosis of peptide hormones and other ligands. Endocr. Rev., 3:280-298. Saltiel, A.R., and Cuatrecasas, P. (1988) In search of a second messenger for insulin. Am. J. Phyiol., 225:Cl-Cll. Smith, R.M., and Jarett, L. (1988) Receptor-mediated endocytosis and intracellular processing of insulin: Ultrastructural and biochemical evidence for cell-specific heterogeneity and distinction from nonhormonal ligands. Lab. Invest., 58:613-629. Smith, M.R., Muegge, K., Keller, J.R., Kung, H.-S., Young, H.A. and Durum, S.K. (1990a) Direct evidence for an intracellular role for IFN-gamma. Microinjection of human IFN-gamma induces Ia expression on murine macrophages. J . Immunol.. 144:1777-1782. Smith, M.R., Munger, W.E., Kung, H . 3 , Takacs, L., andDurum, S.K.
(1990b) Direct evidence for an intracellular role for tumor necrosis factor-alpha. Microinjection of tumor necrosis factor kills target cells. J. Immunol. 144:162-169. Thompson, K.A., Soler, A.P., Smith, R.W., and Jarett, L. (1989) Intranuclear localization of insulin in rat hepatoma cells: insulin/ matrix association. Eur. J.Cell Biol., 50:442446. Wall, D.A., and Patel, S.(1987) The intracellular fate of vitellogenin in Xenopus oocytes is determined by its extracellular concentration during endocytosis. J. Biol. Chem., 262:14779-14789. Wallace, R.A.: and Jared, D.W. (1976) Protein incorporation by isolated amphibian oocytes V. Specificity for vitellogenin incorpcration. J. Cell Biol., 69:345-351.
