BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

Transcription

1 Vol. 46, No. 4, November 1998 Pages GLUCOSE- AND PHORBOL ESTER-INDUCED INSULIN SECRETION IN HUMAN INSULINOMA CELLS -ASSOCIATION WITH PROTEIN KINASE C ACTIVATION- Atsushi Miura, Tatsuo Ishizuka, Satomi Itaya, Masayoshi Ishizawa, Yoshinori Kanoh, Mika Kimura, Kazuo Kajita and Keigo Yasuda Third Department of Internal Medicine, Gifu University School of Medicine, Gifu , Japan Received July 7, 1998 SUMMARY. This study examined the effect of glucose and tetradecanoylphorbol-13-acetate (TPA) on insulin secretion in isolated human insulinoma cells. In addition, we analyzed conventional PKCa and [3 activation in the membrane fractions, respectively. Treatment with 5 mm and 20 mm glucose for 5 min and 20 min resulted in 6-7-fold increases in insulin secretion, and treatment with 1 ~tm TPA for 5 min also resulted in 3-fold increases in insulin secretion from the basal level. Immunoblot analysis of membrane fractions showed increases in PKCa and ~ immunoreactivities after treatment with 5 mm, 20 mm glucose and 1 p2vi TPA. Translocations of PKCa after treatment with glucose and TPA were greater than those of PKC~ in membrane fractions. These results suggest that TPA independently provokes insulin secretion v/a PKC activation and that PKCa and I~ activation may be involved in insulin secretion in human insulinoma cells. Key words: PKC, Human insulinoma cells, Insulin secretion, TPA, Glucose INTRODUCTION Activation of protein kinase C (PKC) has been demonstrated to be an important mechanism for transduction of signals generated upon external stimulation of cells by hormones, neurotransmitters, and growth factors (1). The expression of PKC in rat pancreatic islets (2) suggests that PKC activation is involved in glucose-induced insulin secretion (3,4), although PKC has also been described to be not involved in glucose-induced insulin secretion in mouse ~-cells (5). The abbreviations are: PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol- 13-acetate; H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine /98/ /0 Copyright by Academic Press Australia, All rights of reproduction in any form reserved.

2 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL In addition, 12-O-tetradecanoylphorbol-13-acetate (TPA), an established activator of PKC (6), induces a glucose-independent insulin secretion (7,8). On the other hand, insulinoma, an adenoma of the islets of Langerhans, secretes excessive insulin. Therefore, to clarify the mechanism of insulin secretion in these tumors, we prepared isolated human insulinoma cells and studied the effect of glucose and TPA on insulin secretion and PKC activation. In this study, we examined the effect of 5 mm glucose, 20 mm glucose and 1 ~M TPA on insulin secretion and PKCa and ~ activation in isolated insulinoma cells. We reported here that 1) insulin secretion was found under the condition of TPA stimulation as well as high and low concentrations of glucose, and 2) both glucose- and TPA-induced PKCa and ~ translocations were observed in the membrane fraction, and 3) PKCa activation was greater than PKC~ in human insulinoma cells. MATERIALS AND METHODS Subject A 23-year-old woman consulted Gifu Prefectural Gifu Hospital because of cloudiness of consciousness. Computed tomographic (CT) scanning of the brain was performed, but there was no abnormal finding. Laboratory tests were performed. Hyperinsulinemic hypoglycemia was noted during a spontaneous episode of unconsciousness. She was admitted, and abdominal CT scanning and angiography detected an insulinoma in the pancreas body. The detailed results of laboratory examinations are shown in Table 1. The insulinoma was resected, and the pathological findings were consistent with a typical insulin producing islet cell adenoma. Materials Collagenase (type 4) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma (St. Louis, MO). Porcine insulin was obtained from Novo- Nordisk (Copenhagen, Denmark). Anti-PKCa and ~ antibodies were purchased from GIBCO BRL (Gaithersburg, MD). All other chemicals were of reagent grade. Methods The resected tumor was cut into small pieces in Krebs-Ringer phosphate (KRP) buffer (ph 7.4, 127 mm NaC1, 12.3 mm NaHzPO4, 5.1 mm KC1, 1.3 mm MgSO4, 1.4 mm CaC12, 3% bovine serum albumin (BSA),. 2.5 mm glucose). The pieces were incubated in 2 mg/ml collagenase at 37~ for 30 min. Cells were passed through silk mesh, then washed three times with glucose-free KRP buffer 740

3 Table 1. Laboratory findings of a patient with insulinoma fasting plasma glucose 38 mg/dl immunoreactive insulin 16 ~tu/ml IRI/glucose = 0.42 > 0.4 proinsulin 71.3 pmol/1 C-peptide 3.0 ng/ml ACTH 26 pg/ml GH 0.4 ng/ml glucagon 50 ng/ml containing 1% BSA. Isolated cells were resuspended (4 106 cells/ml) in glucosefree KRP buffer, and divided into plastic tubes in order to estimate the time course of insulin secretion and PKC immunoreactivity. Following preincubation at 37% for 30 min, cells were stimulated with either 5 mm glucose, 20 mm glucose or 1 ~M TPA for 5 min and 20 min at 37~ The reaction was terminated bythe addition of ice-cold buffer A (20 mm Tris/HC1, ph 7.4, 0.1 mm phenylmethylsulfonyl fluoride, 0.25 mm sucrose, 2 mm ethylene glycol bis (~-aminoethylether)-n,n,n',n'- tetraacetic acid, 2 mm 2-mercaptoethanol, 20 ~tg/ml leupeptin). To determine the insulin secretion, each concentration of insulin in supernatant was measured with a 1~I RIA kit (Phadeseph Insulin RIA, Pharmasia Diagnostics, Uppsala, Sweden). Cell suspensions were washed and homogenized with ice-cold buffer A. The resultant homogenates were centrifuged at 105,000 g for 60 min. The pellet was incubated and sonicated in ice-cold buffer A containing 1% Triton-X 100, and then centrifuged at 105,000 g for 60 min to obtain the solubilized membrane fraction. The protein concentration in each fraction was determined. Immunoreactive PKC was measured as follows; twenty ~tg of each sample was subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane (NC). NC was incubated with anti-pkca or antibody, and subjected to an ECL (Enhanced Chemiluminescence) Western blotting detection system (Amersham, Buckinghamshire, UK) according to the manufacturer's protocol. These results were scanned and analyzed with a laser densitometer (UltroScan XL, Pharmacia LKB Biotechnology, Tokyo). RESULTS AND DISCUSSION A tight relationship between insulin secretion and PKC translocation was observed in human insulinoma cells. In this study, insulin secretion was increased to 737% and 684% from the basal level (100%) after stimulation with both 5 mm and 20 mm glucose for 5 min, respectively. TPA-induced insulin secretion was increased to 337% for 5 min from the basal level (100%) (Fig. 1). Previously, several investigators have reported that diacylglycerol-pkc signaling might link to insulin secretion based on studies using rat islets (3,4,7,8). However, there is no report 741

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 100o 8 8oo r 200- I o o 1'o l's 20 Time (min) Fig. 1. Glucose- or TPA-induced insulin secretion. Insulin secretory responses of human insulinoma cells stimulated with either 5 mm glucose (O), 20 mm glucose (A) or 1 ~M TPA (m) for 5 min and 20 min. describing the regulation of glucose-induced insulin secretion by PKC activation in human insulinoma cells. On the contrary, TPA alone did not induce insulin secretion in rat islets (9). As shown in Fig. 1, TPA alone provoked insulin secretion to 337% from the basal level. These results correspond to previous reports that TPA stimulates insulin secretion in the absence of glucose in rat islets (7,8). On the other hand, membrane-associated PKCa and ~ immunoreactivities were increased at 5 min and 20 min time-dependently during stimulation with 5 ram, 20 mm glucose and 1 ~tm TPA (Fig. 2). PKCa immunoreactivities after stimulation with glucose and TPA were apparently higher than PKC~ as shown in Fig. 2. Densitometric analysis indicated that 5 mm glucose-induced increases in PKCa and ~ were highest among glucose- and TPA-mduced PKC activation (Fig. 3). Recently, several studies suggest that the biphasic insulin release from islets may be generated by an interaction of ATP-sensitive K channel-dependent and - independent glucose actions (5,10). Closure ofatp-sensitive K channels by glucose leads to depolarization of the membrane, opening of the voltage-dependent calcium channels, and calcium influx. Thus, it is likely that elevation of cytosolic free calcium influx may activate conventional PKCs (Fig. 2) (11,12). According to several investigators, insulin secretion by ATP-sensitive K channel-independent glucose 742

5 PKC a 80 kda PKC /3! I CON min 5 mm Glu 20 rnm Glu TPA Fig. 2. Translocation of membrane-associated PKCa and B. Insulinoma cells were treated with either 5 mm glucose, 20 mm glucose or 1 ~tm TPA for 5 min and 20 rain. Western blot analysis was performed as described under Materials and Methods. Upper panel, PKCa; lower panel, PKCB. action is coupled to PKC activation (10,13). This activation might be involved in Ca2 PKCs. Accordingly, we should also have studied whether novel and atypical PKCs could be activated by glucose stimulation. In fact, Ito et ai. found that PKCB2 was identified in the B-cells of rat pancreatic islets making it a possible candidate for involvement in glucose-induced insulin secretion, although the B 2 isoform could not be detected in cultured RINr cells, a rat insulinoma cell line (14). In contrast, it was reported that expression of PKCa was found in the B-cells of rat pancreatic islets, and that glucose-induced insulin secretion is mediated by PKCa (11,15). Moreover, Knutson eta]. identified expression of five isoforms (a,b,b,e,~) of PKC in the insulinoma-derived B-cells (16). However, these findings on PKC expression in B-cells are still controversial. There have been no reports on PKC activation in human tumor cells. We have to remember that the insulin secretion mechanism of islet tumor cells may be different from that of control B-cells. Interestingly, PKCa and B were clearly identified in the membrane fraction of insulinoma cells after stimulation with glucose and TPA as shown in Fig. 2. Actually, we failed to detect any decrease of cytosohc PKC~ and B (data not shown), probably, due to the big difference in the cytosol and membrane protein ratio (protein content of cytosol/membrane ratio; > 4.18). PKC~ translocation was markedly larger than PKCB (Fig. 3). These results 743

6 CON 5mM Glu 5' 5mM Glu 20' 20mN Glu 5' 20mM Glu 20' TPA S' TPA 20' CON 5mM glu 5' 5mM Glu 20' 20mM Glu 5' 20ran Glu 20' TPA S' TPA 20' m i I I I I I I000 PKCa immunoreactivity (%of control) m 0 I O PKC/] irnrnunoreactivity (% of control) Fig. 3. Quantitation of membrane-associated PKCa and ~. Each intensity was analyzed with a laser densitometer. Upper graphs, PKCa; lower graphs, PKC~. suggest that PKCa activation rather than PKC~ may contribute to glucose-induced insulin secretion in human insulinoma cells. Although we did not ascertain whether PKC inhibitor could suppress glucose- and TPA-induced insulin secretion, previous reports have suggested that PKC inhibitors, polymyxin B (17) and H-7 (8), inhibit glucose- and TPA-stimulated insulin secretion in rat islets. In conclusion, glucose stimulates insulin secretion like TPA in human insulinoma cells. Furthermore, we examined the translocation of PKCa and ~. Further studies will be required to clarify the insulin secretion mechanism in human islets. ACKNOWLEDGMENTS The authors thank Dr. Masanori Murayama and Shoko Andoh (Internal Medicine, Gifu Prefectural Gifu Hospital) for providing the resected insulinoma. 744

7 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL REFERENCES 1. Nishizuka, Y. (1988) Nature 334, Tanigawa, K., Kuzuya, H., Imura, H., Taniguchi, H., Baba, S., Takai, Y., and Nishizuka, Y. (1982) FEBS Lett. 138, Ganesan, S., Calle, R., Zawalich, K., Smallwood, J. I., Zawalich, W., and Rasmussen, H. (1990) Proc. Natl. Acad. Sci. USA 87, Zawalich, W. S., Zawalich, K. C., Ganesan, S., Calle, R., and Rasmussen, H. (1991) Biochem. J. 278, Gembal, M., Detimary, P., Gilon, P., Gao, Z. Y., and Henquin, J. C. (1993) J. Clin. Invest. 91, Castagna, M., Takai, Y., Kitabuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 257, Malaisse, W. J., Sener, A., Herchuelz, A., Carpinelli, A. R., Poloczek, P., Winand, J., and Castagna, M. (1980) Cancer Res. 40, Niki, I., Tamagawa, J., Niki, H., Niki, A., Koide, T., and Sakamoto, N. (1988) Acta. Endocrinol. (Copenh) 118, Virji, M. A. G., Steffes, M. W., and Estensen, R. D. (1978) Endocrinology 102, Aizawa, T., Sato, u Ishihara, F., Taguchi, N., Komatsu, M., Suzuki, N., Hashizume, K., and Yamada, T. (1994) Am. J. Physiol. 266, C622-C Ganesan, S., Calle, R., Zawalich, K., Greenawalt, K., Zawalich, W., Shulman, G. L, and Rasmussen, H. (1992) J. Cell Biol. 119, Deeney, J. T., Cunningham, B. A., Chheda, S., Bokvist, K., Juntti-Berggren, L., Lam, K., Korchak, H. M., Corkey, B. E., and Berggren, P. O. (1996) J. Biol. Chem. 271, Taguchi, N., Aizawa, T., Sato, Y., Ishihara, F., and Hashizume, K. (1995) Endocrinology 136, Ito, A., Saito, N., Taniguchi, H., Chiba, T., Kikkawa, U., Saitoh, Y., and Tanaka, C. (1989) Diabetes 38, Onoda, K., Hagiwara, M., Hachiya, T., Usuda, N., Nagata, T., and Hidaka, H. (1990) Endocrinology 126, Knutson, K. L., and Hoeing, M. (1994) Endocrinology 135, Stutchfield, J., Jones, P. M., and Howell, S. L. (1986) Biochem. Biophys. Res. Commun. 136,