Brief Notes and Comments
Physiologic Role of Somatostatin Insulin Release from Rat Islets Treated by Somatostatin Antiserum Hiroshi Taniguchi, M.D., Masafumi Utsumi, M.D., Masami Hasegawa, M.D., Tetsuo Kobayashi, M.D., Youzo Watanabe, M.D., Keiji Murakami, M.D., Michio Seki, M.D., Akimitsu Tsutou, M.D., Hiroyuki Makimura, M.D., Masahiro Sakoda, M.D., and Shigeaki Baba, M.D., Kobe, Japan
SUMMARY
In order to clarify the physiologic role of somatostatin in insulin release, rat pancreatic islets treated by somatostatin antiserum were incubated in media containing various concentrations of glucose. Insulin release from antiserum-treated islets was significantly elevated above that from nontreated ones at 3.3 and 8.3 mM glucose, while the former was not different from the latter at 16.7 mM glucose. It is suggested that somatostatin plays an important role in the regulation of insulin release in the physiologic range of glucose concentration. DI\BETES 26:700-02, July, 1977.
The growth-hormone release-inhibiting hormone (somatostatin) has been demonstrated by radioimmunologic assay 1 to occur not only in the hypothalamus but also in the pancreas, stomach, duodenum, and upper portion of jejunum. It has also been detected with immunohistochemical techniques2 in the thyroid gland as well as in the aforementioned organs. On the other hand, somatostatin has been generally accepted as inhibitor of the secretion of several hormones, such as GH, TSH, 3 insulin, glucagon, 4 and gastrin, 5 some enzymes such as amylase, chymotrypsin, trypsin, 6 and pepsin 7 and gastric acid, 5 7 basally and after stimulation. However, its From the Second Department of Internal Medicine, Kobe University School of Medicine, 7-chome, Kusunoki-cho, Ikuta-ku, Kobe 650, Japan. Accepted for publication January 17, 1977. 700
role in the control of hormone secretion has not been satisfactorily proved. The present investigation of the role of somatostatin in the physiologic regulation of insulin release was, therefore, performed with the use of the antiserum to somatostatin. METHODS AND MATERIALS
Antiserum to somatostatin (cyclized form, Protein Research Foundation, Osaka, Japan) was produced in rabbits according to the method of Arimura et al. 8 The antiserum used in this study was confirmed by courtesy of Dr. Arimura to have a binding rate with somatostatin of 51.3 per cent at 1:70 dilution by radioimmunoassay (personal communication). Our precise method was described elsewhere.9 The pancreatic islets were isolated with collagenase (Boehringer Mannheim Corp. Ltd., Germany (according to the method of Lacy and Kostianovsky10 from Wistar male rats 250-270 gm. in weight, fed ad libitum. Krebs-Henseleit bicarbonate buffer adjusted to pH 7.4 (KHBB) containing 3.3 mM glucose was utilized instead of Hanks' solution for disruption of the acinar tissue. Five islets were incubated in 0.4 ml. of basic medium supplemented with 3.3 mM glucose and 0.1 ml. of somatostatin antiserum for 60 minutes at 37° C. under the gas phase of 95 per cent O2-5 per cent CO2, subsequently being rinsed three times in the basic medium and transferred to a vial containing 0.5 JULY, 1977
HIROSHI TANIGUCHI, M.D., AND ASSOCIATES
ml. of new basic medium supplemented with 3.3, 8.3, or 16.7 mM glucose as final concentration. The basic medium comprised KHBB and 0.5 per cent bovine serum albumin (fraction V, Armour Pharmaceutical Co.) as final concentration. The last incubation was conducted for 60 minutes at the same condition as the first in an incubator (Taiyo incubator M - 1 N type, Taiyo Kagakukogyo Co., Tokyo, Japan) at 80 strokes per minute. For the control study, normal rabbit serum was applied instead of the somatostatin antiserum and otherwise the incubation procedure was similar to that done in the experimental study. The glucose concentrations of the first incubation medium supplemented with somatostatin antiserum and with normal rabbit serum were determined by the glucose-oxidase method to be 3.6 and 3.7 mM, respectively. Insulin release in the medium at the last incubation was radioimmunoassayed with rat inslin (donated by Dr. R. J. Hosley, Lilly Research Laboratories) as standard. The results were analysed by Student's /-test. RESULTS Insulin output from the somatostatin-pretreated islets was 1.60 ± 0.21 (n = 5) and 3.93 ± 0.19 ng./islet/60 min. (n = 7) at 3.3 and 8.3 mM glucose, respectively. These values were significantly higher than their controls', i.e., 0.61 ± 0.13 (n = 5) and 3.24 ± 0.01 ng./islet/60 min. (n = 7) at 3-3 and 8.3 mM glucose, respectively. There was no significant difference in insulin release induced by 16.7 mM glucose between the antiserum-pretreated and the control groups—i.e., 7.52 ± 0.41 (n = 7) and 6.96 ± 0.51 ng./islet/60 min. (n = 7), respectively. Expressed as per cent against the value released from the islets without prior treatment of antiserum, which was shown as 100 per cent, insulin released from the islets treated by somatostatin antiserum at 3.3 and 8.3 mM glucose was 306.8 ± 64.3 per cent (n = 5) and 121.1 ± 4.9 per cent (n = 7) respectively, both of which were significantly elevated over the values from the nontreated islets. The former at 16.7 mM glucose, however, was 109.0 ± 5.9 per cent (n = 7), which was not different from the insulin output from the control islets (figure 1).
the hypothalamopituitary system have been reported with the use of the method of passive immunization of somatostatin antiserum in rats. 11 " 14 It has been demonstrated that GH and TSH responses are exaggerated after treatment not only at the basal level but also under stress conditions, such as ether, cold exposure, swimming, and TRH loading. But there have been no studies on the insulin secretion from pancreatic islets treated by somatostatin antibody, from which the physiologic role of somatostatin in insulin release would be elicited. In the present investigation, somatostatin antiserum was not applied to the incubation medium because of its possible contamination with various factors that might affect insulin release directly and prevent determining the role of somatostatin per se on glucose-induced insulin release. In consideration of the rather slow progress of antigen-antibody reaction, islets were incubated with the antiserum for as long as 60 minutes during the first incubation period, in which the binding of the antiserum to the pancreatic D-cells has been confirmed in our previous histochemical observations.9 The present study showed almost similar observations to those found in the secretion of GH and TRH by other authors 1 1 " 1 4 —that is, the somatostatin antiserum-treated islets released much more insulin than the nontreated ones ac glucose concentrations as low as 3.3 and 8.3 mM. However, the insulin release did not differ between both groups at higher glucose concentration as 16.7 mM. Considering the absence or presence of a very small, if any, amount of somatostatin in the last incubation medium because of the Somatostatin Antiserum Control
i R I(#/#) 100'
300
11
150
200
100
100
50
Glucose
3.3
P