0028-3908/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press pic
Neuropharmacology Vol. 30, No. 12B, pp. 1391-1398, 1991 Printed in Great Britain. All rights reserved
DIAZEPAM BINDING INHIBITOR AND THE ENDOCRINE PANCREAS c.-G. OSTENSON,1 B. AHREN,z O. JOHANSSON,J S. KARLSSON,2 M. HILLIGES J and S. EFENDIeI 'Department of Endocrinology, Karolinska Hospital and Institute, Stockholm, Sweden, 2Department of Pharmacology and Surgery, Lunds University, Lund, Sweden and 3Department of Histology and Neurobiology, Karolinska Institute, Stockholm, Sweden Summary-Regulation of blood glucose homeostasis is complex. Its major hormonal regulators include insulin, glucagon and somatostatin from the endocrine pancreas. Secretion of these hormones is controlled predominantly by the supply of nutrients in the circulation but also by nerve signals and other peptides. Thus, it is likely that peptides, released from cells of the gut or endocrine pancreas or from peptidergic nerves, affect glucose homeostasis by modulating the secretion of insulin, glucagon and somatostatin. When searching for novel gut peptides with such effects, diazepam binding inhibitor (DBI) was isolated from the porcine small intestine. By immunocytochemistry, DBI has been demonstrated to occur not only in the gut but also in endocrine cells of the pancreatic islets, namely in the somatostatin-producing D-cells in pig and man, and in the glucagon-producing A-cells in rat. Porcine DBI (pDBI; 10- 8_10- 7 M) has been shown to suppress glucose-stimulated release of insulin from both isolated islets and perfused pancreas of the rat. Furthermore, secretion of insulin stimulated by either the sulfonylurea glibenclamide or the phosphodiesterase inhibitor 3-isobutyl-I-methylxanthine (IBMX), was inhibited by the peptide. In contrast, arginine-induced release of insulin was unaffected by pDBI. Moreover, pDBI decreased arginine-induced release of glucagon from the perfused rat pancreas, whereas release of somatostatin was unchanged. Notably, rat DBI, structurally identical with rat acyl-CoA-binding protein, has also been demonstrated to inhibit glucose-stimulated release of insulin in the rat, both in vivo and in vitro. Long-term exposure of cultured fetal rat islets to pDBI (10- 8 M) significantly decreased the synthesis of DNA in islet cells. In conclusion, DBI moderately suppresses the secretion of insulin from the B-cells, when stimulated by agents closing ATP-sensitive K + -channels (glucose, glibenclamide) or enhancing the formation of cAMP (IBMX). The mechanism of such an effect is not likely to involve binding of DBI to benzodiazepinerecognition sites of GABA-receptors, since B-cells are reportedly lacking this type of receptor. It cannot be ruled out that the peptide exerts its effect indirectly by binding factors important in regulation of secretion of insulin, due to its ability to bind amphiphilic compounds. The occurrence of DBI immunoreactivity in non-B-cells of the islets suggests that the peptide may modulate secretion of insulin locally through paracrine interaction. Key words-pancreatic islets, perfused pancreas, immunohistochemistry, insulin, glucagon, acyl-CoAbinding protein.
Regulation of blood glucose homeostasis is complex. Its major hormonal regulators include insulin and glucagon from the endocrine pancreas (Unger, Dobbs and Orci, 1978). Thus, hepatic glucose production is governed mainly by insulin and glucagon, and insulin also augments the utilization of glucose in several tissues. Secretion of pancreatic hormones is controlled predominantly by nutrients in the circulation, classical neurotransmitters of the autonomic nervous system and peptides from the gastrointestinal (GI) tract (Unger et al., 1978). In addition, islet hormone release is regulated also by peptides secreted from the neighbouring cells (paracrine effect), as well as by peptides reaching islet cells through intra-islet nerve fibres (neurocrine effect) (Ahren, Ostenson and Efendic, 1991). Modulating effects by GI peptides were proposed after the demonstration that oral glucose induced a considerably larger insulin response than the identical amount of glucose, infused intravenously, despite a
more pronounced hyperglycemia after intravenous than after oral administration of glucose (Mcintyre, Holdsworth and Turner, 1964). This observation was explained by the existence of an entero-insular axis with meal-induced release of insulin-stimulating peptide(s), incretins, from the gut. Although not decisively proved, there is evidence that glucosedependent insulinogenic polypeptide (GIP) (Creutzfeldt and Ebert, 1985) and glucagon-like peptide-l (7-36) amide (Schjoldager, Wettergren, Mortensen, Myhre, Orskov, Christiansen and Holst, 1990) are the main incretins. However, a number of the traditionally "intestinal" or "brain" peptides have also been localized to pancreatic endocrine cells or nerve fibers (Ahren et al., 1991). Thus, insulinotropic peptides, like cholecystokinin (CCK) and vasoactive intestinal polypeptide (VIP) are localized to intrapancreatic nerve fibers. Similarly, peptides which suppress the release of insulin, such as somatostatin, galanin and neuropeptide Y (NYP), have been localized both to the intestines and the
1391
1392
c.-G. {)STENSON et al.
pancreas (Ahren et al.,1991). Hypothetically, such peptides may take part not only in the physiological modulation of the secretion of insulin but also in the pathogenesis of non-insulin-dependent diabetes mellitus, which is characterized by an impaired insulin response to glucose (Efendic, Luft and Wajngot, 1984). CHARACTERIZATION OF PORCINE DIAZEPAM BINDING INHIBITOR
In collaboration with Professor Viktor Mutt at the Department of Biochemistry, Karolinska Institute, peptide fractions, extracted and purified from porcine small intestines, were examined for their possible effects on islet hormone secretion in the perfused, isolated rat pancreas (Efendic, Nylen, Roovete and Uvnas-Wallensten, 1978). One such fraction was found to contain a peptide which significantly inhibited glucose-stimulated release of insulin (Chen, Agerberth, Gell, Andersson, Mutt, Ostenson, Efendie, Barros-Soderling, Persson and Jornvall, 1988). This peptide was shown to consist of 86 amino acid residues, with a high structural homology (>80%) with rat and human diazepam binding inhibitor. Hence, this novel peptide was designated porcine diazepam binding inhibitor (pOBI). LOCALIZATION OF pOBI TO THE PANCREATIC ISLETS
Antiserum against pOBI was raised in rabbits immunized with highly purified peptide (Johansson, Hilliges, Ostenson, Sandberg, Efendic and Mutt, 1991). This antiserum was used for immunohistochemical studies in porcine, human and rat gastrointestinal tissues. In pig and man, OBI immunoreactivity (OBI-IR) was localized to mucosal endocrine cells, as well as to nerve fibres and ganglia of the small intestines (Hoog, Sandberg and Ostenson, 1989). Surprisingly, OBI-IR was found also in distinct cells of the pancreatic islets. Consecutive semithin sections of pancreas and intestine, stained for OBI and somatostatin, showed co-localization of these two peptides to the same type of cell, the somatostatin-producing O-cell (Hoog et al., 1989). These cells constitute approximately 10% of all islet endocrine cells and are found predominantly in the periphery of the islets (Petersson, Hellerstrom and Gunnarsson, 1970). In rat islets, double-labelling experiments, using the OBI antibodies and polyclonal somatostatin or glucagon antisera, demonstrated that the cells exhibiting OBI-IR were identical with the glucagonproducing A-cells (Fig. I) (Johansson et al., 1991). The latter cells make up 20-25% of islet endocrine cells and are like the O-cells, found mainly in the periphery of the rat islet (Hellman, 1959). Thus, with immunostaining techniques, cells with OBI-IR occur in endocrine islet cells of pig, man and
rat, although the type of cell differs between rat on one side (A-cells) and pig and man on the other (O-cells). In this context, it is of interest to note that considerable amounts of OBI-IR have been found in extracts of rat pancreas, using radioimmunoassays with antisera against the octadecaneuropeptide (OON) (Ball, Burnet, Fountain, Ghatei and Bloom, 1986). Similar results were obtained also in human pancreas, with an antibody raised against homologous OBI(51-70), i.e. the fragment corresponding to rat OON, plus two additional residues at the carboxyl terminus (Ball, Ghatei, Sekiya, Krausz and Bloom, 1989). EFFECTS OF OBION THE SECRETION OF INSULIN
In the isolated, perfused rat pancreas, porcine OBI (10- 8 M) did not affect the basal release of insulin at a small (3.3 mM) concentration of glucose, while it suppressed the insulin response to 16.7mM glucose (Chen et al., 1988). The early and late phases of insulin response were inhibited by 50 and 34%, respectively (Fig. 2). In contrast, pOBI did not suppress arginine-stimulated release of insulin in the perfused rat pancreas (Ostenson, Ahren, Karlsson, Sandberg and Efendic, 1990). In isolated rat islets, insulin responses to glucose (8.3 and 16.7 mM), the sulfonylurea compound glibenclamide (2 j.tM) and the phosphodiesterase inhibitor 3-isobutyl-I-methylxanthine (IBMX; I mM), were dose-dependently inhibited by 10- 8_10- 7 M pOBI (Fig. 3) (Ostenson et al., 1990). Like in the perfused rat pancreas, the peptide did not affect arginine-stimulated release of insulin from isolated pancreatic islets (Fig. 3). The effect of rat OBI, which has been shown to be structurally identical to an acyl-Co A-binding protein (ACBP), isolated from rat liver (Knudson, Hojrup, Hansen, Hansen and Roepstorff, 1989), was also tested on isolated rat islets. In static incubation of islets, rat OBI/ACBP at 10- 8_10- 7 M significantly (P < 0.01) suppressed glucose-stimulated release of insulin. Furthermore, this form of OBI from the rat inhibited glucoseinduced insulin response from perifused rat islets (Ostenson, Knudsen Ahren, Karlsson and Efendic, unpublished). It was mainly the early phase of the secretion of insulin that was inhibited. Infusion of pOBI (25 pmol/kg/min) in rats did not modify the insulin response to a parallel infusion of glucose (Fig. 4) nor did injection of the porcine peptide (1 or 8 nmol/kg) in mice affect basal and glucose-stimulated levels of insulin in plasma (Fig. 5). However, when rat OBI/ACBP was infused in rats, at the same rate as with pOBI, a significant (P < 0.05) but modest inhibition of glucose-stimulated insulin response was noted (Ostenson et al., unpublished). Thus, when administered in vivo to the blood circulation, the effect of the peptide on the secretion of
Fig.!. Immunofluorescence micrographs of a rat pancreatic islet, after double-labelling for DBI (top) and glucagon (bottom). Note the overlap between the two peptides. The bar indicates 50 Jlm. [Reproduced with the publisher's permission from Johansson et al. (1991).]
1393
Diazepam binding inhibitor and the endocrine pancreas
insulin seemed blunted or even absent, in comparison to the in ritro effect. The previous observation, that exogenou sly applied OBI is rapidly metabolized following exposure to endo- and exopeptidases (Guidotti , Berkovich, Mukhin and Costa, 1990), may provide an explanation for the in vivo findings.
1395
4000
3000 c
'E EFFECT OF OBI ON SECRETION OF GLUCAG ON AND SOMATOSTATIN
In the perfused rat pancreas, pOBI (10- 8 M) inhibited the late phase of arginine-st imulated release of glucagon by 28% (Ostenson et al., 1990). In the same perfusions , the arginine-stimulated somatostatin response was not significantly affected by pOBI.
..... :::l :t.
5
...
1000
HOW IS OBI REGULAT ING ISLET HORMONE SECRETION?
The present knowledge ind icates a possible role for OBI in the regulation of B-cell secretion of insulin. DBI immunoreactivity has been localized to pancreatic islets- and many other organs-but the levels of DBI-IR (or ACB P-I R) in pla sma appears low (Knudsen et al., 1989). These ob servations, together with the findings that OBI inhibits stimulated secretion of insulin in vitro, but is devoid of or has a much weaker effect in rodents in vivo, make it unlikely that the peptide can exert its effects on the Bvcells by circulation as a classical hormone. Since the peptide has been localized, by immunohistochemistry, to distinct islet cell types (A-cells of rat and O-cells of pig and human pancreas), it is nevertheless possible that OBI , if secreted from these cells, modulates secretion of insulin by local, paracrine interaction with the B-cells. The mechanis m behind a depressant effect by OBI on the secretory process for insulin is unclear. It is prob ably not involving binding of the peptide to benzodiazepine recognition sites within GABA A receptors , since pancreatic B-cells do not appear to be equipped with this type of receptor (Rorsman, Berggren, Bokvist , Ericson, Mohler, Ostenson and Smith, 1989). In this context, it is of interest that OBI was able to inhibit the insulin response to glucose and glibenclamide but not to arginine . The fanner two secretagogues init iate release of insulin by closing ATP -regulated K + -channels in the B-cell plasma membrane, thereby inducing membrane depolarization and opening of voltage-dependent Ca + -channels (Arkharnmar, Nilsson, Rorsman and Berggren, 1987). The subsequent increase in the intracellular cytoplasmic calcium level is directly coupled to the exocytosis of insulin. In contrast, arginine is not affecting the K + -channels, but apparently depolarizes the B-cell membrane, due to its Own transport across the membrane in a positively charged form (Hermans, Schmeer and Henq uin, 1987). Hence, it is possible that OBI interferes with
2000
!"
0 60 c
50
E ..... 0 Do
. ....r•
40
C
OJ
30
e
..
20
n=8
III
10
0
i
-20
-10
0
i
10
20
30
40
50
Min
Fig. 2. Glucose-stimulated release of insulin (top) and somatostatin (bottom) from the isolated, perfused rat pancreas. The continuous curves (. -. ) show hormonal responses in control experiments, during administration of 16.7 mM glucose (open ba r). The dashed curves (0--0) illustrate hormonal responses when porcine OBI (10- 8 M) was added 10 min prior to and during administration of glucose (- 10 to 40 min) . Means ± SEM of eight exper iments. [Reproduced with the publisher's permission from Chen et al. ( 1988).)
the regulation of K + permeability in the B-cell and thereby of the secretion of insulin . Alternatively OBI, due to its ability to bind amphiphilic compounds (Knudsen et al., 1989), may exert its effect indirectly by binding factors, important in the regulation of the secretion of insulin. It is also conceivable that O BI acts on the synthesis or action of cyclic AMP, since both glucose an d glibenclamide are reported to markedly increase cAMP in isolated rat islets (Grill and Cerasi, 1978), while arginine is without such an effect or induces only a small cAMP response (Charles, Lawecki, Steiner an d Grodsky, 1976; Grill, 1980). The secretion of insulin induced by IBMX, which potently
c.-G. OSTENSON et al.
1396
3
16.7m'" glucose
20m'" ~lrginin.
211'" gllb,ncl,mld'
lmM ISMX
25
.
20 s: .... 'ij ]! .... ::> ::L
It
15
...
Neuropharmacology Vol. 30, No. 12B, pp. 1391-1398, 1991 Printed in Great Britain. All rights reserved
DIAZEPAM BINDING INHIBITOR AND THE ENDOCRINE PANCREAS c.-G. OSTENSON,1 B. AHREN,z O. JOHANSSON,J S. KARLSSON,2 M. HILLIGES J and S. EFENDIeI 'Department of Endocrinology, Karolinska Hospital and Institute, Stockholm, Sweden, 2Department of Pharmacology and Surgery, Lunds University, Lund, Sweden and 3Department of Histology and Neurobiology, Karolinska Institute, Stockholm, Sweden Summary-Regulation of blood glucose homeostasis is complex. Its major hormonal regulators include insulin, glucagon and somatostatin from the endocrine pancreas. Secretion of these hormones is controlled predominantly by the supply of nutrients in the circulation but also by nerve signals and other peptides. Thus, it is likely that peptides, released from cells of the gut or endocrine pancreas or from peptidergic nerves, affect glucose homeostasis by modulating the secretion of insulin, glucagon and somatostatin. When searching for novel gut peptides with such effects, diazepam binding inhibitor (DBI) was isolated from the porcine small intestine. By immunocytochemistry, DBI has been demonstrated to occur not only in the gut but also in endocrine cells of the pancreatic islets, namely in the somatostatin-producing D-cells in pig and man, and in the glucagon-producing A-cells in rat. Porcine DBI (pDBI; 10- 8_10- 7 M) has been shown to suppress glucose-stimulated release of insulin from both isolated islets and perfused pancreas of the rat. Furthermore, secretion of insulin stimulated by either the sulfonylurea glibenclamide or the phosphodiesterase inhibitor 3-isobutyl-I-methylxanthine (IBMX), was inhibited by the peptide. In contrast, arginine-induced release of insulin was unaffected by pDBI. Moreover, pDBI decreased arginine-induced release of glucagon from the perfused rat pancreas, whereas release of somatostatin was unchanged. Notably, rat DBI, structurally identical with rat acyl-CoA-binding protein, has also been demonstrated to inhibit glucose-stimulated release of insulin in the rat, both in vivo and in vitro. Long-term exposure of cultured fetal rat islets to pDBI (10- 8 M) significantly decreased the synthesis of DNA in islet cells. In conclusion, DBI moderately suppresses the secretion of insulin from the B-cells, when stimulated by agents closing ATP-sensitive K + -channels (glucose, glibenclamide) or enhancing the formation of cAMP (IBMX). The mechanism of such an effect is not likely to involve binding of DBI to benzodiazepinerecognition sites of GABA-receptors, since B-cells are reportedly lacking this type of receptor. It cannot be ruled out that the peptide exerts its effect indirectly by binding factors important in regulation of secretion of insulin, due to its ability to bind amphiphilic compounds. The occurrence of DBI immunoreactivity in non-B-cells of the islets suggests that the peptide may modulate secretion of insulin locally through paracrine interaction. Key words-pancreatic islets, perfused pancreas, immunohistochemistry, insulin, glucagon, acyl-CoAbinding protein.
Regulation of blood glucose homeostasis is complex. Its major hormonal regulators include insulin and glucagon from the endocrine pancreas (Unger, Dobbs and Orci, 1978). Thus, hepatic glucose production is governed mainly by insulin and glucagon, and insulin also augments the utilization of glucose in several tissues. Secretion of pancreatic hormones is controlled predominantly by nutrients in the circulation, classical neurotransmitters of the autonomic nervous system and peptides from the gastrointestinal (GI) tract (Unger et al., 1978). In addition, islet hormone release is regulated also by peptides secreted from the neighbouring cells (paracrine effect), as well as by peptides reaching islet cells through intra-islet nerve fibres (neurocrine effect) (Ahren, Ostenson and Efendic, 1991). Modulating effects by GI peptides were proposed after the demonstration that oral glucose induced a considerably larger insulin response than the identical amount of glucose, infused intravenously, despite a
more pronounced hyperglycemia after intravenous than after oral administration of glucose (Mcintyre, Holdsworth and Turner, 1964). This observation was explained by the existence of an entero-insular axis with meal-induced release of insulin-stimulating peptide(s), incretins, from the gut. Although not decisively proved, there is evidence that glucosedependent insulinogenic polypeptide (GIP) (Creutzfeldt and Ebert, 1985) and glucagon-like peptide-l (7-36) amide (Schjoldager, Wettergren, Mortensen, Myhre, Orskov, Christiansen and Holst, 1990) are the main incretins. However, a number of the traditionally "intestinal" or "brain" peptides have also been localized to pancreatic endocrine cells or nerve fibers (Ahren et al., 1991). Thus, insulinotropic peptides, like cholecystokinin (CCK) and vasoactive intestinal polypeptide (VIP) are localized to intrapancreatic nerve fibers. Similarly, peptides which suppress the release of insulin, such as somatostatin, galanin and neuropeptide Y (NYP), have been localized both to the intestines and the
1391
1392
c.-G. {)STENSON et al.
pancreas (Ahren et al.,1991). Hypothetically, such peptides may take part not only in the physiological modulation of the secretion of insulin but also in the pathogenesis of non-insulin-dependent diabetes mellitus, which is characterized by an impaired insulin response to glucose (Efendic, Luft and Wajngot, 1984). CHARACTERIZATION OF PORCINE DIAZEPAM BINDING INHIBITOR
In collaboration with Professor Viktor Mutt at the Department of Biochemistry, Karolinska Institute, peptide fractions, extracted and purified from porcine small intestines, were examined for their possible effects on islet hormone secretion in the perfused, isolated rat pancreas (Efendic, Nylen, Roovete and Uvnas-Wallensten, 1978). One such fraction was found to contain a peptide which significantly inhibited glucose-stimulated release of insulin (Chen, Agerberth, Gell, Andersson, Mutt, Ostenson, Efendie, Barros-Soderling, Persson and Jornvall, 1988). This peptide was shown to consist of 86 amino acid residues, with a high structural homology (>80%) with rat and human diazepam binding inhibitor. Hence, this novel peptide was designated porcine diazepam binding inhibitor (pOBI). LOCALIZATION OF pOBI TO THE PANCREATIC ISLETS
Antiserum against pOBI was raised in rabbits immunized with highly purified peptide (Johansson, Hilliges, Ostenson, Sandberg, Efendic and Mutt, 1991). This antiserum was used for immunohistochemical studies in porcine, human and rat gastrointestinal tissues. In pig and man, OBI immunoreactivity (OBI-IR) was localized to mucosal endocrine cells, as well as to nerve fibres and ganglia of the small intestines (Hoog, Sandberg and Ostenson, 1989). Surprisingly, OBI-IR was found also in distinct cells of the pancreatic islets. Consecutive semithin sections of pancreas and intestine, stained for OBI and somatostatin, showed co-localization of these two peptides to the same type of cell, the somatostatin-producing O-cell (Hoog et al., 1989). These cells constitute approximately 10% of all islet endocrine cells and are found predominantly in the periphery of the islets (Petersson, Hellerstrom and Gunnarsson, 1970). In rat islets, double-labelling experiments, using the OBI antibodies and polyclonal somatostatin or glucagon antisera, demonstrated that the cells exhibiting OBI-IR were identical with the glucagonproducing A-cells (Fig. I) (Johansson et al., 1991). The latter cells make up 20-25% of islet endocrine cells and are like the O-cells, found mainly in the periphery of the rat islet (Hellman, 1959). Thus, with immunostaining techniques, cells with OBI-IR occur in endocrine islet cells of pig, man and
rat, although the type of cell differs between rat on one side (A-cells) and pig and man on the other (O-cells). In this context, it is of interest to note that considerable amounts of OBI-IR have been found in extracts of rat pancreas, using radioimmunoassays with antisera against the octadecaneuropeptide (OON) (Ball, Burnet, Fountain, Ghatei and Bloom, 1986). Similar results were obtained also in human pancreas, with an antibody raised against homologous OBI(51-70), i.e. the fragment corresponding to rat OON, plus two additional residues at the carboxyl terminus (Ball, Ghatei, Sekiya, Krausz and Bloom, 1989). EFFECTS OF OBION THE SECRETION OF INSULIN
In the isolated, perfused rat pancreas, porcine OBI (10- 8 M) did not affect the basal release of insulin at a small (3.3 mM) concentration of glucose, while it suppressed the insulin response to 16.7mM glucose (Chen et al., 1988). The early and late phases of insulin response were inhibited by 50 and 34%, respectively (Fig. 2). In contrast, pOBI did not suppress arginine-stimulated release of insulin in the perfused rat pancreas (Ostenson, Ahren, Karlsson, Sandberg and Efendic, 1990). In isolated rat islets, insulin responses to glucose (8.3 and 16.7 mM), the sulfonylurea compound glibenclamide (2 j.tM) and the phosphodiesterase inhibitor 3-isobutyl-I-methylxanthine (IBMX; I mM), were dose-dependently inhibited by 10- 8_10- 7 M pOBI (Fig. 3) (Ostenson et al., 1990). Like in the perfused rat pancreas, the peptide did not affect arginine-stimulated release of insulin from isolated pancreatic islets (Fig. 3). The effect of rat OBI, which has been shown to be structurally identical to an acyl-Co A-binding protein (ACBP), isolated from rat liver (Knudson, Hojrup, Hansen, Hansen and Roepstorff, 1989), was also tested on isolated rat islets. In static incubation of islets, rat OBI/ACBP at 10- 8_10- 7 M significantly (P < 0.01) suppressed glucose-stimulated release of insulin. Furthermore, this form of OBI from the rat inhibited glucoseinduced insulin response from perifused rat islets (Ostenson, Knudsen Ahren, Karlsson and Efendic, unpublished). It was mainly the early phase of the secretion of insulin that was inhibited. Infusion of pOBI (25 pmol/kg/min) in rats did not modify the insulin response to a parallel infusion of glucose (Fig. 4) nor did injection of the porcine peptide (1 or 8 nmol/kg) in mice affect basal and glucose-stimulated levels of insulin in plasma (Fig. 5). However, when rat OBI/ACBP was infused in rats, at the same rate as with pOBI, a significant (P < 0.05) but modest inhibition of glucose-stimulated insulin response was noted (Ostenson et al., unpublished). Thus, when administered in vivo to the blood circulation, the effect of the peptide on the secretion of
Fig.!. Immunofluorescence micrographs of a rat pancreatic islet, after double-labelling for DBI (top) and glucagon (bottom). Note the overlap between the two peptides. The bar indicates 50 Jlm. [Reproduced with the publisher's permission from Johansson et al. (1991).]
1393
Diazepam binding inhibitor and the endocrine pancreas
insulin seemed blunted or even absent, in comparison to the in ritro effect. The previous observation, that exogenou sly applied OBI is rapidly metabolized following exposure to endo- and exopeptidases (Guidotti , Berkovich, Mukhin and Costa, 1990), may provide an explanation for the in vivo findings.
1395
4000
3000 c
'E EFFECT OF OBI ON SECRETION OF GLUCAG ON AND SOMATOSTATIN
In the perfused rat pancreas, pOBI (10- 8 M) inhibited the late phase of arginine-st imulated release of glucagon by 28% (Ostenson et al., 1990). In the same perfusions , the arginine-stimulated somatostatin response was not significantly affected by pOBI.
..... :::l :t.
5
...
1000
HOW IS OBI REGULAT ING ISLET HORMONE SECRETION?
The present knowledge ind icates a possible role for OBI in the regulation of B-cell secretion of insulin. DBI immunoreactivity has been localized to pancreatic islets- and many other organs-but the levels of DBI-IR (or ACB P-I R) in pla sma appears low (Knudsen et al., 1989). These ob servations, together with the findings that OBI inhibits stimulated secretion of insulin in vitro, but is devoid of or has a much weaker effect in rodents in vivo, make it unlikely that the peptide can exert its effects on the Bvcells by circulation as a classical hormone. Since the peptide has been localized, by immunohistochemistry, to distinct islet cell types (A-cells of rat and O-cells of pig and human pancreas), it is nevertheless possible that OBI , if secreted from these cells, modulates secretion of insulin by local, paracrine interaction with the B-cells. The mechanis m behind a depressant effect by OBI on the secretory process for insulin is unclear. It is prob ably not involving binding of the peptide to benzodiazepine recognition sites within GABA A receptors , since pancreatic B-cells do not appear to be equipped with this type of receptor (Rorsman, Berggren, Bokvist , Ericson, Mohler, Ostenson and Smith, 1989). In this context, it is of interest that OBI was able to inhibit the insulin response to glucose and glibenclamide but not to arginine . The fanner two secretagogues init iate release of insulin by closing ATP -regulated K + -channels in the B-cell plasma membrane, thereby inducing membrane depolarization and opening of voltage-dependent Ca + -channels (Arkharnmar, Nilsson, Rorsman and Berggren, 1987). The subsequent increase in the intracellular cytoplasmic calcium level is directly coupled to the exocytosis of insulin. In contrast, arginine is not affecting the K + -channels, but apparently depolarizes the B-cell membrane, due to its Own transport across the membrane in a positively charged form (Hermans, Schmeer and Henq uin, 1987). Hence, it is possible that OBI interferes with
2000
!"
0 60 c
50
E ..... 0 Do
. ....r•
40
C
OJ
30
e
..
20
n=8
III
10
0
i
-20
-10
0
i
10
20
30
40
50
Min
Fig. 2. Glucose-stimulated release of insulin (top) and somatostatin (bottom) from the isolated, perfused rat pancreas. The continuous curves (. -. ) show hormonal responses in control experiments, during administration of 16.7 mM glucose (open ba r). The dashed curves (0--0) illustrate hormonal responses when porcine OBI (10- 8 M) was added 10 min prior to and during administration of glucose (- 10 to 40 min) . Means ± SEM of eight exper iments. [Reproduced with the publisher's permission from Chen et al. ( 1988).)
the regulation of K + permeability in the B-cell and thereby of the secretion of insulin . Alternatively OBI, due to its ability to bind amphiphilic compounds (Knudsen et al., 1989), may exert its effect indirectly by binding factors, important in the regulation of the secretion of insulin. It is also conceivable that O BI acts on the synthesis or action of cyclic AMP, since both glucose an d glibenclamide are reported to markedly increase cAMP in isolated rat islets (Grill and Cerasi, 1978), while arginine is without such an effect or induces only a small cAMP response (Charles, Lawecki, Steiner an d Grodsky, 1976; Grill, 1980). The secretion of insulin induced by IBMX, which potently
c.-G. OSTENSON et al.
1396
3
16.7m'" glucose
20m'" ~lrginin.
211'" gllb,ncl,mld'
lmM ISMX
25
.
20 s: .... 'ij ]! .... ::> ::L
It
15
...
