Stimulation of Insulin Release by Elevated Pressure Gradient H. ALEYASSINE AND ROBERT J. GARDINER McGill University Medical Clinic, The Montreal General Hospital, Montreal, Quebec, Canada hanced rate of insulin release caused by elevated pressure was not reversed immediately after release of pressure, but the effect was completely abolished within a period of 15 min; and 4) the stimulating effect of high glucose concentration on the rate of insulin release was completely blocked in the presence of 1 niM DNP, but the pressure-induced stimulation of insulin release was insensitive to this agent. In view of recent evidence implicating the microtubular-microfilamentous system in the secretory process of insulin, it is suggested that the application of pressure to the islet cells provides the necessary motive force to facilitate the intracellular translocation and extrusion of the (3 granules normally performed by the /3 cell contractile structures. (Endocrinology 99: 1542, 1976)

ABSTRACT. In an attempt to simulate the contractile events which presumably lead to intracellular translocation and exocytosis of the )8 granule, the effect of elevated pressure on the rate of insulin release was studied. In vitro preparations of pancreas were incubated in media of low glucose content (1.65 IHM) for several consecutive 15 min periods. The effect of pressure gradients, ranging from 0 to 45 mm Hg above atmospheric pressure, on insulin release was tested during the second period by introducing a mixture of 95% O 2 -5% CO2 into the incubation flasks. The results have shown that: 1) a rise in the pressure gradient over the gas phase was accompanied by an increase in the concentration of insulin released into the medium; 2) there was a direct relationship between the pressure gradient and the rate of insulin release; 3) the en-

I

N THE COURSE of the past few years microtubular-microfilamentous structures have been implicated in a variety of cell processes, such as cell division, cell motility, and the maintenance of cell shape (1-8). More recently a new function has been ascribed to these organelles, i.e., the intracellular translocation of cell granules to the periphery, resulting in the release of their contents to extracellular space (9-18). The inhibition of insulin release by colchicine and the occasional finding of the /3 granules in close association with microtubules led to the hypothesis that these latter organelles are involved in the secretion of insulin (9). Furthermore, studies using various chemical agents which interfere with the formation or function of microtubules confirmed the initial observations made with colchicine (19-21). There is evidence that "labile" microtubules are in dynamic equilibrium with Received February 25, 1976. This work was presented in part at the Annual Meeting of the Canadian Diabetic Association, January, 1976. Correspondence to: Dr. H. Aleyassine, University Medical Clinic, The Montreal General Hospital, 1650 Cedar Avenue, Montreal, Quebec H3G 1A4, Canada.

their basic component, and that the assembly and disassembly of microtubules is governed by a number of factors such as temperature, ionic strength, and pressure (1,2,6,22,23). Numerous in vivo and in vitro studies have, for instance, shown that microtubules in various cell types undergo dissociation when subjected to high hydrostatic presure, while the release of pressure causes the association of these organelles (1,2,6,22,23). In addition to microtubules, the presence of another contractile system, microfilaments, has been reported in the /3 cell of pancreas (24-27). Studies with antibody to actin, using immunofluorescent techniques, have revealed the occurrence of actin-like material throughout the j3 cell with preponderance at the cell periphery (28). This actin-like material is believed to be associated with the microfilamentous system (28). Recent electron microscope studies using microfilament-specific reagents also showed the relationship of insulin secretion to microfilament contraction (24,27). In the present experiments, proceeding on the assumption that the application of pressure to pancreas could result in an effect similar to contraction of the /3 cells by a

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PRESSURE-INDUCED INSULIN RELEASE

microtiibular-microfilamentous system, the rate of insulin release was studied under the influence of various pressure gradients. However, the gradients used here were much below the levels which promote dissociation of microtubules. Materials and Methods Male Wistar rats, weighing 250-300 g, were used in all experiments. Under ether anesthesia the pancreas was removed, cut into small pieces, and distributed among several flasks each containing 5 ml Krebs bicarbonate solution (29) supplemented with 0.2% bovine serum albumin and equilibrated with 95% O 2 -5% CO2, pH 7.4. Glucose was present in the medium at a final concentration of 1.65 mM. To establish a steady rate of release, all flasks were preincubated for 15 min at 37 C. Subsequently the contents of each flask were incubated for several consecutive periods of 15 min. At the end of each period, samples of incubation medium were removed for insulin measurement, and the tissues were transferred to flasks containing fresh medium for further incubation. The effect of pressure gradients or of a high glucose concentration on the rate of insulin release was tested during the second period of incubation. Insulin concentration of the medium was measured by the radioimmunoassay technique (30). A standard solution of rat insulin was used as a reference. Anti-insulin serum was prepared by immunizing guinea pigs according to the method of Moloney and Coval (31). Iodinated porcine insulin (I25I) was purchased from Nuclear International Corporation, Burlington, Mass., USA. The rate of insulin release was expressed as mU insulin/g pancreas/15 min. Differences in mean rates of release were compared by Student's t test.

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rubber bulb. When the desired pressure in the bag was established, the needle connecting the gas cylinder and the flask was exchanged for a needle and tube connection to one incubation flask, or by multiple secondary connecting tubes to several incubation flasks. When the effect of various pressure gradients was to be studied simultaneously, several sphygmomanometers were used attached individually to the incubation flasks via an intermediate flask. The pressure used in the present experiments did not exceed 45 mm Hg; however, the danger of shattering may be avoided by using plastic flasks if higher pressures are to be used.

Results

Effect of various pressure gradients on the rate of insulin release Pieces of rat pancreas were incubated for two consecutive periods of 15 min at 37 C in media of low glucose content (30 mg per 100 ml). The effect of various levels of pressure was tested during the second period of incubation. As may be seen from Fig. 2, there was no significant change during the second period in the rate of insulin release without additional pressure over the gas phase. However, application of pressure by introducing a mixture of 95% O2-5% CO2 into the incubation flasks, resulted in a substantial rise in the rate of insulin release. A correlation was observed between the level of pressure applied and the rate of insulin released into the medium; the highest effect was observed at 45 mm Hg, the maximum pressure level tested in these studies.

Reversibility of pressure effect on insulin release A sphygmomanometer, as commonly used in Since the increased rate of insulin release hospitals, was employed as a pressure device. observed with elevated pressure could be Communication between the sphygmomanomthe result of a deleterious effect on the /3 eter bag and a gas cylinder (95% O 2 -5% CO2) cells, it was considered of importance to was established via a 25 ml plastic flask covered by a rubber sealing cap (Fig. 1). Using two assess the reversibility of this phenomenon. extension tubings, each attached at one end to For this purpose, pieces of pancreas were an injection needle, the gas flow was directed incubated for four consecutive periods of 15 from the cylinder to the flask, and from the flask min. The effect of pressure (40 mm Hg) to the sphygmomanometer, after removing its was tested only during the second period.

Pressure device

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ALEYASSINE AND GARDINER

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Endo • 1976 Vol 99 • No 6

FIG. 1. Pressure device. 1) Gas cylinder (95% O 2 -5% CO2); 2) Extension tubing to direct the gas flow from the cylinder to the intermediate flask; 3) Intermediate flask; 4) Extension tubing to direct the gasflowfrom the intermediate flask to the sphygmomanometer bag; 5) Sphygmomanometer bag; 6) Manometer gauge; 7) Incubation flasks connected via intermediate flask to sphygmomanometer bag to pressurize the tissue during incubation—one flask (control) remains unpressurized.

In parallel experiments we have also examined the effect of a high glucose concentration (300 mg per 100 ml) during the second period in the absence of additional pressure. The results are shown in Table 1. In control experiments a steady rate of release was observed when incubations were carried out under basal conditions (30 mg per 100 ml) dextrose and no additional pressure). Application of pressure during the second period caused a marked increase in the rate of insulin release. It was of particular

interest to note that the rate of release remained elevated during the third period ("washout period"), despite the release of the pressure. However, with a further period of incubation (fourth period), the insulin release returned to a level comparable to its basal rate (first period). Similar observations were made when a high concentration of glucose was used as a stimulus. These results appear to indicate that both pressure and high concentrations of glucose are capable of inducing changes in the

10 1st period 2nd (test) period

6-

4-

2-

mmHg-

FIG. 2. Mean (±SE) insulin release from pieces of rat pancreas incubated for two periods of 15 min in medium of low glucose content (1.65 HIM), at 37 C. During the first period, all incubations were carried out under atmospheric pressure. The effects of various pressure gradients on the rate of insulin release were tested at the second period. There are 8 observations for each level of pressure.

0

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PRESSURE-INDUCED INSULIN RELEASE

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TABLE 1. Reversibility of pressure effect on insulin release

Addition

First period

Second (test) period

Third period

Fourth period

None (control) Pressure (40 mm Hg) Dextrose (16.5 mM)

1.72 ± 0.42 1.68 ± 0.35 1.83 ± 0.44

1.56 ± 0.36 7.15 ± 1.22 10.84 ± 1.40

1.62 ± 0.31 6.22 ± 1.04 8.20 ± 0.91

1.50 ± 0.34 1.57 ± 0.48 1.54 ± 0.39

Mean (± SE) insulin release from pieces of rat pancreas incubated for four 15 min periods. The effect of pressure or of a high glucose concentration was tested during the second period. Results are expressed as milliunits insulin per gram wet weight pancreas per 15 min. There were 12 observations for each experiment.

(3 cells which persist for some time after removal of stimuli (pressure or glucose). However, the effect of both agents were reversible after a 15 min period of "washout."

ATP supply. The inhibition of glucoseevoked insulin release by DNP has previously been well documented (32,33).

Effect of DNP on pressure-induced insulin release Stimulation of insulin release by increased pressure over the gas phase raised the question whether the pressure acts as such (physical action), or whether its stimulatory effect on insulin release is mediated via an increased oxygen uptake which in turn would accelerate metabolic processes in the /3 cells. To gain information about this question, the effect of 2,4-dinitrophenol (DNP), an uncoupling agent of oxidative phosphorylation, on the rate of pressureinduced insulin release was examined. As is shown in Table 2, a range of concentrations of DNP failed to reverse the stimulating effect of 40 mm Hg pressure. However, DNP at these concentrations proved to be a potent inhibitor of glucose-stimulated insulin release, indicating that this latter process, unlike the former, is dependent on

The results of the present studies have shown that the exposure of in vitro preparations of pancreas to elevated pressure gradients causes an increase in the rate of insulin release in the absence of other stimuli. Although a clear explanation for this phenomenon is not yet available, we propose that the stimulatory effect of pressure is the result of physical action contracting the /3 cell. This hypothesis is based on numerous observations which implicate the /8 cell's contractile structure, the microtubular-microfilamentous system, in the secretory process of insulin. One example is the case with cytochalasins, a group of mold metabolites. The exposure of various cell types to these agents has been generally associated with contraction of micron1 laments, reduction of cell volume, and increased intracellular translocation of hormone, and enzyme-containing granules to the cell

Discussion

TABLE 2. Effect of DNP on pressure-induced insulin release Addition None (control) Pressure (40 mm Hg) Pressure + DNP (5 x 10" 4 M) Pressure + DNP (1 x 10" 3 M) Pressure + DNP (5 x 10" 3 M)

Dextrose (16.5 mM) Dextrose + DNP (1 x 10" 3 M)

First period 1.73 1.81 1.62 1.76 1.94 1.83 1.79

± 0.32 ± 0.37 ± 0.35 ± 0.40 ± 0.51 ± 0.47 ± 0.49

Second (test) period 1.84 d:0.37 6.86 d; 1.04 6.47 dt 1.11 7.05 dt 1.23 7.10 dt 1.33 11.43 dt 1.42 2.42 :tO.63

Third period

Fourth period

1.96 dt0.40 5.18 dE 1.12 5.30 dtO.97 5.83 dt 1.17 5.67 dt 1.19 8.97: t 1.20 2.46:t 0.67

1.85 dt 0.41 1.92 dt0.56 1.79 dtO.62 1.60 dtO.39 1.80 dt0.51 1.62: tO.57 1.83: tO.54

Mean (± SE) insulin release from pieces of rat pancreas incubated for four periods of 15 min. The effects of various concentrations of DNP in combination with high pressure or with high glucose were tested during the second period. Results are expressed as milliunits insulin per gram wet weight pancreas per 15 min. Eight observations were made for each experiment.

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Endo • 1976 Vol 99 • No 6

ALEYASSINE AND GARDINER

periphery (7,8,15,27). More specifically, in perfused pancreas, as well as in isolated islets, cytochalasin B caused contraction of the /3 cell microfilaments along with stimulation of insulin release (24,26,27). These effects have been ascribed to biophysical, rather than chemical changes in the /3 cell (27). The stimulating effect of pressure on insulin release reported here would not appear to be due to depolymerization of microtubules, since dissociation of these organelles requires pressure levels much greater than those used in the present experiments (1,2,6,22,23). Furthermore, microtubule depolymerization would be expected to cause inhibition of insulin release, as was seen in studies using microtubule poisons (9,19-21). A point of particular interest in the present studies is the failure of DNP to alter the rate of insulin release induced by pressure. In the muscle cell, an energy-capturing arrangement is used that can lead to a mechanical phenomenon, contraction. In the case of pressure-induced insulin release no special transduction device seems necessary since the chemomechanical transduction process has been bypassed by direct provision of motive force in the form of pressure. This would explain why inhibition of ATP regeneration by DNP cannot alter the rate of pressure-induced insulin release. The stimulation of insulin release by various agents is generally regarded as a multistage process. If it is assumed that the involvement of the contractile structure of the (3 cell represents the ultimate stage by providing the motive force for the intracellular translocation and exocytosis of the j8 granules, the compression of the j3 cell by application of pressure should result in the same effect. It is possible that in addition to /3 cell contraction, elevated pressure also induces changes in j8 cell membrane permeability. Such a possibility is consistent with the results obtained using a group of antibiotics acting as ion-transporting carriers across

lipid barriers. One of these ionophores, X-537A, has been shown to stimulate insulin release at low glucose concentrations (34). It is interesting to note that the stimulatory effect of X-537A was not affected by the presence of DNP in the incubation medium. Furthermore, the insulin secretagogic action of the ionophore was not influenced by either 3-0-methylglucose, or adrenergic blocking agents (34). Addition of metabolic inhibitors or omission of calcium, which normally reduce the secretory response to glucose, paradoxically increased the stimulation of insulin release by X-537A. On the basis of these observations, it has been suggested that mechanisms other than emiocytosis, such as intracellular dissolution of the (3 granules or increased plasma cell permeability, may be responsible for the insulin releasing effect of the ionophore (34). In conclusion, the results of the present experiments appear to indicate that elevated pressure may be used as a new approach in studying the secretory process of insulin. As far as physiologic implication of pressure in regulating insulin release is concerned, these in vitro studies offer little for constructive speculation. References 1. Inoue, S., and H. SatoJ Gen Physiol 50: 259, 1967. 2. Tilney, L. G., In Reinert, J., and H. Ursprung (eds.), Origin and Continuity of Cell Organelles, SpringerVerlag, New York, 1971, p. 385. 3. Wilson, L., Fed Proc 33: 151, 1974. 4. Bryan, J., Fed Proc 33: 152, 1974. 5. Wilson, L., J. R. Bamburg, S. B. Mizel, L. M. Grisham, and K. M. Creswell, Fed Proc 33: 158, 1974. 6. Borisy, G. G., J. B. Olmsted, J. M. Marcum, and C. Allen, Fed Proc 33: 167, 1974. 7. Miranda, A. F., G. C. Godman, A. D. Deitch, and S. W. TanenbaumJ Cell Biol 61: 481, 1974. 8. Miranda, A. F., G. C. Godman, and S. W. TanenbaumJ Cell Biol 62: 406, 1974. 9. Lacy, P. E., S. L. Howell, D. A. Young, and C. J. Fink, Nature 219: 1177, 1968. 10. Poisner, A. M., and J. Bernstein, J Pharmacol Exp Ther 177: 102, 1971.

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PRESSURE-INDUCED INSULIN RELEASE 11. Williams, J. A., and J. Wolff, Proc Natl Acad Sci USA 67: 1901, 1970. 12. Wolff, J., and J. A. Williams, Recent Prog Horm Bes 29: 229, 1973. 13. Kraicer, J., and J. V. Milligan, Endocrinology 89: 408, 1971. 14. Gautvik, K. M., R. F. Hoyt, Jr., and A. H. Tashjian, J r . J Cell Physiol 82: 401, 1973. 15. Hawkins, D . J Immunol 110: 294, 1973. 16. Davies, A., R. Fox, M. Polyzonis, A. Allison, and A. Haswell, Lab Invest 28: 16, 1973. 17. Henson, P., and A. Oades, J Immunol 110: 290, 1973. 18. Zurier, R. B., S. Hoffstein, and G. Weissmann, Proc Natl Acad Sci USA 70: 844, 1973. 19. Malaisse-Lagae, F., M. H. Greider, W. J. Malaisse, and P. E. Lacy,; Cell Biol 49: 530, 1971. 20. Lacy, P. E., M. M. Walker, and C. J. Fink, Diabetes 21: 987, 1972. 21. Van Obberghen, E., G. Somers, G. Devis, M. Ravazzola, F. Malaisse-Lagae, L. Orci, and W. J. Malaisse, Endocrinology 95: 1518, 1974.

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22. Kennedy, J. R., and A. M. Zimmerman, J Cell Biol 47: 568, 1970. 23. Salmon, E. D., Science 189: 884, 1975. 24. Orci, L., K. H. Gabbay, and W. J. Malaisse, Science 175: 1128, 1972. 25. Lacy, P. E., N. J. Klein, and C. J. Fink, Endocrinology 92: 1458, 1973. 26. Van Obberghen, E., G. Somers, G. Devis, G. D. Vaughan, F. Malaisse-Lagae, L. Orci, and W. J. Malaisse, J Clin Invest 52: 1041, 1973. 27. Van Obberghen, G. Somers, C. Devis, M. Ravazzola, F. Malaisse-Lagae, L. Orci, and W. J. Malaisse, Diabetes 24: 892, 1975. 28. Gabbiani, G., F. Malaisse-Lagae, B. Blondel, and L. Orci, Endocrinology 95: 1630, 1974. 29. Krebs, H., Biochim Biophys Ada 4: 249, 1950. 30. Herbert, V., K.-S. Lau, C. W. Gottlieb, and S. J. BleicherJ Clin Endocrinol Metab 25: 1375, 1965. 31. Moloney, P. J., and M. Coval, Biochem J 59: 179, 1955. 32. Coore, H. G., and P. J. Randle, Biochem J 93: 66, 1964. 33. Aleyassine, H., Endocrinology 87: 84, 1970. 34. Hellman, B., Biochem Biophys Ada 399:157,1975.

Distribution of Lubrol-PX Dr. Melvin Blecher, Department of Biochemistry, Georgetown University School of Medicine, Washington, D.C. 20007, has some [3H]LubroI-PX available for free distribution to any interested scientist. Lubrol-PX is a non-ionic detergent widely used to solubilize membrane components. The compound was tritiated for Dr. Blecher by New England Nuclear by the Wilzbach silicic acid column chromatography. It runs as a single spot on silica gel thin layers. The material is available in tracer concentrations in ethanolic solution. Recipients will have to provide tlieir own unlabeled material to create their required mass concentrations. Those interested should communicate tlieir needs directly to Dr. Blecher and should provide their Nuclear Regulatory Agency license number.

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Stimulation of insulin release by elevated pressure gradient.

Stimulation of Insulin Release by Elevated Pressure Gradient H. ALEYASSINE AND ROBERT J. GARDINER McGill University Medical Clinic, The Montreal Gener...
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