help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Miura, Y.
Right arrow Articles by Matsui, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miura, Y.
Right arrow Articles by Matsui, H.
Endocrinology Vol. 138, No. 7 2769-2775
Copyright © 1997 by The Endocrine Society

ARTICLES

Impaired Cytosolic Ca2+ Response to Glucose and Gastric Inhibitory Polypeptide in Pancreatic ß-Cells from Triphenyltin-Induced Diabetic Hamster

Yoshikazu Miura, Masakatsu Kato, Kazunori Ogino and Hisao Matsui

Department of Hygiene (Y.M., H.M.), Dokkyo University School of Medicine, Mibu, Tochigi 321–02, Japan; Department of Physiology I (M.K.), Nippon Medical School, Sendagi 1, Bunkyo, Tokyo 113 Japan; Department of Medicine (K.O.), Koshigaya Hospital, Dokkyo University School of Medicine, Koshigaya, Saitama 343, Japan

Address all correspondence and requests for reprints to: Yoshikazu Miura, Ph.D., Department of Hygiene, Dokkyo University School of Medicine, 880, Mibu, Tochigi 321–02, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oral administration of a single dose of triphenyltin compounds induces diabetes with decreased insulin secretion in rabbits and hamsters after 2–3 days without any morphological changes in pancreatic islets. In the present study, to test the possibility that the impaired insulin secretion induced by triphenyltin compounds could result from an impaired Ca2+ response in pancreatic ß-cells, we investigated the effect of triphenyltin-chloride (TPTCl) administration on the changes in the cytoplasmic Ca2+ concentration ([Ca2+]i) induced by secretagogues, such as glucose, high K+, gastric inhibitory polypeptide (GIP), and acetylcholine (ACh) in hamster pancreatic ß-cells. TPTCl administration caused partial suppression in 10 mM K+-induced rise in [Ca2+]i without suppressing the increase in [Ca2+]i evoked by 20–50 mM K+. Administration of TPTCl strongly inhibited the rises in [Ca2+]i induced by 27.8 mM glucose, 100 µM ACh in the presence of 5.5 mM glucose, and by 100 nM GIP in the presence of 5.5 mM glucose. In the ACh-induced response, TPTCl administration strongly suppressed the late sustained phase, while weakly suppressing the initial rise in [Ca2+]i. TPTCl administration significantly suppressed the rise of cAMP content in islet cells induced by 100 nM GIP with 1 mM 3-isobutyl-1-methylxanthine in the presence of 5.5 mM glucose (P < 0.01, N = 5–11). TPTCl administration also impaired the insulin secretion in islet cells induced by 27.8 mM glucose, 100 nM GIP in the presence of 5.5 mM glucose, and 100 µM ACh in the presence of 5.5 mM glucose (P < 0.05, N = 9–16). We conclude that the pathology of triphenyltin-induced diabetes in hamsters involves a defect in cellular Ca2+ response due to a reduced Ca2+-influx through voltage-gated Ca2+ channels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRIPHENYLTIN compounds, used as material for fungicides and some kind of paints (1), cause hyperglycemia and glycosuria in man (2, 3). An oral administration of triphenyltin compounds can produce diabetes in rabbits (4) and hamsters (5, 6). In such triphenyltin-induced diabetes, insulin secretion is lowered without any morphological changes in pancreatic islets (5, 7). It is also known that pancreatic islet cells from triphenyltin chloride (TPTCl)-administered hamsters showed a reduced sensitivity to glucose and to forskolin, an activator of adenylate cyclase (8), for the rise in intracellular free Ca2+ concentration ([Ca2+]i) in comparison to that by islet cells from normal animals (9). In the present studies, we further investigated the cellular mechanism of TPTCl-induced defects of insulin secretion. For this purpose, we prepared primary cultured hamster pancreatic islet cells and measured changes in [Ca2+]i induced by several secretagogues, such as glucose (10), high K+ (11), gastric inhibitory polypeptide (GIP) (12, 13), and acetylcholine (ACh) (14, 15, 16). There are two reasons why we chose to measure changes in [Ca2+]i among other indicators. One is that changes in [Ca2+]i are closely related to secretion of hormones including insulin (10, 17). Secondly, responses of individual cells can be detected by using [Ca2+]i imaging technique (18). In addition, these secretagogues, used in the present experiment, are known to raise [Ca2+]i through distinct signal transduction pathway. TPTCl administration might interfere with the ß-cell responses not only to glucose but to other secretagogues. Furthermore, we investigated how TPTCl administration causes diabetes of hamsters in cellular levels of pancreatic islets.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Triphenyltin chloride (TPTCl) was obtained from Tokyo Kasei Chemical (Tokyo, Japan), collagenase (type IV) from Worthington Biochemical Co. (Freehold, NJ), Dispase from Godo-Shusei Co. (Chiba, Japan), Ficoll 400 from Pharmacia Fine Chemicals (Uppsala, Sweden), DM-160 medium from Kyokuto Pharmaceutical Industrial Co., Ltd. (Tokyo, Japan), DMEM from Nissui Pharmaceutical Company Ltd. (Tokyo, Japan), Conray 400 (sodium iotalamate) from Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan) and 3-isobutyl-1-methylxanthine (IBMX) from Aldrich Chemical Company Inc. (Milwaukee, WI). EGTA, EDTA, and fura-2-acetoxy-methylester (fura-2/AM) were purchased from Dojindo Laboratories Co., Ltd. (Kumamoto, Japan). Rabbit antigoat IgG and peroxidase-antiperoxidase complex from Dako A/S (Glostrup, Denmark). Acetylcholine chloride (ACh) was obtained from Sigma Chemical Co. (St. Louis, MO), gastric inhibitory polypeptide (GIP) from Peptide Institute, Inc. (Osaka, Japan). Goat antihuman insulin was a generous gift from Dr. S. Tanaka of Institute for Molecular and Cellular Regulation (Gunma University, Maebashi, Japan). In this study, because high doses of secretagogues are more sensitive to the increase of insulin secretion and to the rise in [Ca2+]i than that of lower doses of stimulants, we used the dose of glucose at 27.8 mM (9, 19), ACh at 100 µM (16, 20) and GIP at 100 nM (13).

Animal and animal treatment
Male Syrian hamsters, weighing 95–125 g (aged 9–10 weeks) were obtained from Experimental Animal Supply (Saitama, Japan) and housed under the controlled conditions (24 ± 2 C, 50 ± 20% relative humidity, 12-h light, 12-dark cycle). They received laboratory mouse, rat and hamster chow (MF, Oriental Yeast Company Ltd., Tokyo, Japan) and tap water ad libitum.

Hamsters were given a single oral dose of 6 mg TPTCl/100 g BW in 1 ml sesame oil suspension. Control hamsters were given the same volume of sesame oil.

Isolation of islets and dissociation of islet cells
Pancreatic islets were isolated after collagenase digestion of the pancreas (21) from fasted male hamsters 2 days after an oral administration of TPTCl. Islets were separated from the collagenase digestion by method of Ficoll-Conray gradient centrifugation (22), and individually chosen by stereoscopic microscopy in tissue culture medium (DM-160) (23), supplemented with 2% FCS. Dissociated islet cells were prepared by further digestion with dispase (0.33 mg/ml) of the isolated islets in Ca2+- and Mg2+-free HBSS as described by Takaki and Ono (24).

Identification of islet B-cells
Dissociated islet cells (105 cells/well) were plated on the 0.2% poly-L-lysine-coated glass slide and were cultured for 1 day in DMEM with 5% FCS in humidified 5% CO2-95% air. The cells were then fixed with 4% paraformaldehyde in PBS. Procedure for immunocytochemistry was described elsewhere (25). After rinsing with PBS, islet cells were incubated with a goat antihuman insulin (diluted at 1:10,000 with 50 mM PBS plus 0.3% Triton X-100) (26) for 2 days at 4 C in a humid chamber. Then cells were incubated with a rabbit antigoat IgG (diluted 1:100) for 1 h at room temperature. Cells were then incubated with peroxidase-antiperoxidase complex (diluted 1:100) for 1 h at room temperature. Then cells were incubated in 0.02% 3,3'-diaminobenzidine tetrahydrochloride and 0.01% H2O2 in 50 mM Tris HCl (pH 7.6) for 1 min at room temperature. Finally islet cells were counterstained with Mayer’s hematoxylin, dehydrated in ethanol, cleared in xylene, and coverslipped.

Measurement of intracellular free calcium in islet ß-cell
Intracellular free calcium concentration ([Ca2+]i) was measured by a modification of the method of Kato et al. (18). Dissociated islet cells (5 x 104 cells/dish) were plated onto the 0.2% poly-L-lysine-coated circular glass coverslips (13.2 mm diameter), and cultured overnight at 37 C in DMEM with 5% FCS. The cells were loaded for 30 min at 37 C in Krebs-Ringer solution buffered with bicarbonate (115 mM NaCl, 4.7 mM KCl, 2.56 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgCl2, 24 mM NaHCO3, and 20 mM HEPES, pH 7.4) (KRBH) with 1 µM fura-2/AM and 5.5 mM glucose. Calcium-free solution was prepared by replacing CaCl2 with MgCl2. High K+ solutions (10, 20, 30, and 50 mM) were prepared by replacing NaCl with KCl. The coverslips were placed in KRBH containing either 2.8 mM or 5.5 mM glucose, fixed in a hand-made chamber (fitted with a peristaltic pump for perifusion) mounted on the stage (37 C) of inverted TMD microscope (Nikon, Tokyo, Japan). Fura-2 fluorescence at 510 nm after excitation at 340 and 380 nm was detected by intensified charge-coupled device (ICCD) camera, and the ratio image was produced using an ARGUS-50 system (Hamamatsu Photonics). Alternatively, fura-2 fluorescence was detected every 1/30 sec by photomultiplier using a PI system (Nikon). The changes of [Ca2+]i in single islet cells were calculated from the ratio (R) of the fluorescence measured with excitation at 340 nm to that at 380 nm using the following equation (27):

where Kd is the dissociation constant for fura-2 (224 nM), Rmax and Rmin are the ratios for unbound and bound forms of the fura-2/Ca2+ complex, respectively, and ß is the ratio of fluorescence of fura-2 at 380 nm excitation in minimum calcium and saturating calcium. Rmax and Rmin were estimated with the fluorescence intensities of fura-2 solution (1 µM) containing 10 mM CaCl2 and 5 mM EGTA, respectively.

Measurement of cAMP content
cAMP content was measured by a modification of the method of Nelson et al. (28). Dissociated islet cells (3 x 104 cells/dish, 20 mm) were cultured overnight at 37 C in DMEM with 5% FCS. Islet cells were washed twice with KRBH (pH 7.4) and preincubated for 30 min at 37 C in KRBH (0.4 ml) containing 1 mM IBMX, or 100 nM GIP and 1 mM IBMX in the presence of 5.5 mM glucose. The responses were stopped by addition of 0.2 ml of ice-cold trichloroacetic acid (TCA) to a final concentration of 6%. The culture plates were then shaken, left at room temperature for 15 min, and centrifuged at 7800 x g for 10 min. The supernatants were thoroughly mixed with 1.5 ml of diethyl ether, and the ether phase containing TCA was discarded. This step was repeated three times to ensure complete elimination of TCA. The extracts and cAMP standards were evaporated, added 400 µl KRBH, and assayed for cAMP by RIA kit from Yamasa Shoyu (Choshi, Japan) in which the samples and standards are succinylated.

Insulin secretion by pancreatic islet
The islets were preincubated for 90 min in DM160 medium (23) containing 5% FCS plus 10 mM glucose at 37 C in 5% CO2-95% O2. Three islets in each culture tube were then incubated for 60 min in 1 ml of Krebs-Ringer solution buffered with bicarbonate (pH 7.4) containing appropriate concentration of glucose and test substances. A portion of the medium was withdrawn at the end of the incubation and appropriately diluted for insulin assay. Insulin was measured by a double-antibody RIA kit from Eiken Chemical (Tokyo, Japan) (29). The intra- and interassay coefficients of variation were less than 10% and 12%, respectively. The minimum detectable sensitivity was 5 µU/ml, and the ED50 was 45 µU/ml.

Statistics
The experiments are illustrated as means ± SEM. Experiments were carried out with cells or islets of at least three different preparations. To assess the statistical significance of observed differences, an unpaired t test was used, and an ANOVA followed by a Dunnett’s test was used for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of TPTCl administration on a proportion of insulin containing cells
Typical insulin immunoreactive islet cells from control animals and from TPTCl-administered animals are shown in Fig. 1Go, A and B, respectively. The insulin immunoreactive cells are indicated by arrowheads. There were no detectable changes in the shape and the stainability between these two cell groups. Figure 1CGo shows a proportion of insulin immunoreactive islet cells. In islet cells from control animals, insulin immunoreactive islet cells accounted for 74.5 ± 3.1% (mean ± SEM) (n = 5; total number of islet cells, 5505). The proportion was 81.7 ± 2.8% in islet cells from TPTCl-administered animals (n = 5; total number of islet cells, 4246).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 1. Immunocytochemistry of pancreatic islet cells from normal (A) and TPTCl (6 mg/100 g BW) (B)-induced diabetic hamsters. Cells were stained with antiserum to human insulin (1:10,000). The insulin immunoreactive islet cells are indicated by arrowheads. Scale bar, 20 µm. C, Percentage of the insulin immunoreactive islet cells in control group ({square}; 74.5 ± 3.14%) (total number of islet cells, 5505) and in TPTCl-administered group ({blacksquare}; 81.7 ± 2.83%) (total number of islet cells, 4246). Data are shown as means ± SEM (n = 5).

 
Insulin secretagogues-induced changes in [Ca2+]i in single islet cells from TPTCl-administered hamster
When hamster islet cells were perifused with a medium containing 2.8 mM glucose and 2.5 mM Ca2+, basal [Ca2+]i was 43.0 ± 1.36 nM in the control cells (n = 141) and 42.6 ± 1.13 nM in the cells (n = 213) from TPTCl-administered animals. Application of 27.8 mM glucose did not elicit the rise in [Ca2+]i in some cultured islet cells from the control animals and TPTCl-administered animals. As shown in Fig. 1CGo, cultured islet cells contained 20–25% of nonislet ß cells. In the present experiments, islet cells with a {Delta} [Ca2+]i ([Ca2+]i with stimulus subtracted by the basal [Ca2+]i) less than 10 nM after stimulus were defined as nonresponsive cells and were eliminated from analysis. Nonresponding islet cells after stimulation with 27.8 mM glucose were seen in 22.6% of the control islet cells and in 51.6% of the cells from TPTCl-administered animals. Figure 2AGo shows the temporal patterns of 27.8 mM glucose-induced rise in [Ca2+]i in the control responding islet cells and in the responding cells from TPTCl-administered animals. Addition of 27.8 mM glucose elicited a sustained rise in [Ca2+]i with maximum value of 128.3 ± 8.9 nM in the control cells (n = 109) and 83.2 ± 3.6 nM (n = 103) in the cells from TPTCl-administered animals. The maximum value in {Delta} [Ca2+]i evoked by 27.8 mM in islet cells ranged from 10.8 nM to 438 nM with a median of 48.8 nM in the control (n = 109), whereas that in the cells (n = 103) from TPTCl-administered animals ranged from 10.1 nM to 156.7 nM with a median of 28.4 nM (Fig. 2BGo).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 2. Inhibitory effects of TPTCl administration on the glucose-induced rise in [Ca2+]i of single islet cells. A, The cells were preincubated with a buffer containing 2.8 mM glucose for about 3 min before a [Ca2+]i imaging started. These traces are correspond to the mean responses (± SEM) in nine responsive islet cells of control (—) and in 10 cells from TPTCl-administered animals (---). B, Population distribution of glucose-induced response derived from [Ca2+]i imaging experiments. Data are the number of {Delta} [Ca2+]i values (nM) at each level indicated, plotted as a percentage of the total number of the responsive cells recorded for each group. Those with a {Delta} [Ca2+]i less than 10 nM were defined as nonresponsive cells and were eliminated from the analysis. Data were obtained from 109 cells from control animals ({square}) and from 103 cells from TPTCl-administered animals ({blacksquare}).

 
Control cells responded to high K+ with a rapid initial rise in [Ca2+]i followed by a relatively sustained rise with lower amplitude (Fig. 3Go, A and B), whereas cells from TPTCl-administered animals responded to the same stimulus without the initial prominent rise in [Ca2+]i (Fig. 3BGo). In the case of 10 mM K+, both initial and sustained phases were lowered in TPTCl-administered group (Fig. 3AGo), whereas in that of 30 mM K+ the late phase was rather higher in TPTCl-administered group than control group as shown in Fig 3BGo. When K+ concentration returned to 4.8 mM, [Ca2+]i reverted to the basal level. Figure 4Go shows the effects of TPTCl administration on the maximum rise in [Ca2+]i over basal level induced by various concentration of K+ ions (10, 20, 30, and 50 mM). TPTCl administration significantly decreased 10 mM K+-induced rise in [Ca2+]i in single islet cells (P < 0.05, n = 100–140) without affecting the initial rise in [Ca2+]i induced by 20, 30, and 50 mM K+. However, TPTCl administration augmented the late sustained phase in [Ca2+]i by 20–50 mM K+.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 3. Effects of TPTCl administration on the rise in [Ca2+]i induced by depolarization with 10 mM K+ (A) or 30 mM K+ ions (B) in the presence of 5.5 mM glucose. The cells were preincubated with a buffer containing 5.5 mM glucose for 3 min before [Ca2+]i imaging started. These traces are correspond to the mean responses (± SEM) in 9–10 responsive islet cells of the control (—) and in 7–8 cells from TPTCl-administered animals (---).

 


View larger version (12K):
[in this window]
[in a new window]
  		Figure 4. The effects of TPTCl administration on the maximum rise in [Ca2+]i over basal levels in single islet cells after depolarization of plasma membrane by various concentrations of extracellular K+ ions (10, 20, 30, and 50 mM) in the presence of 5.5 mM glucose. Data are expressed as means ± SEM (nM) of {Delta} [Ca2+]i. ({square}), control (n = 53–492); ({blacksquare}), TPTCl administration (n = 32–392). **, P < 0.01 vs. control.

 
Addition of 100 nM GIP did not elicit the rise in [Ca2+]i in 28.2% of the control islet cells (n = 184) and in 52.0% of the cells (n = 198) from TPTCl-administered animals. Application of 100 nM GIP with 5.5 mM glucose produced a gradual increase in [Ca2+]i followed by a slow decline of [Ca2+]i in the control (Fig. 5AGo). The response was suppressed in the cells from TPTCl-administered animals (Fig. 5AGo). GIP-induced maximum rise in {Delta} [Ca2+]i in islet cells ranged from 10.1 nM to 600 nM with a median of 38.4 nM in the control cells (n = 132), while that in the cells (n = 95) from TPTCl-administered animals ranged from 10 nM to 237 nM with a median of 27.6 nM (Fig. 5BGo).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 5. Inhibitory effects of TPTCl administration on GIP (100 nM)-induced changes in [Ca2+]i in single islet cells. A, The cells were preincubated with a buffer containing 5.5 mM glucose for about 3 min before [Ca2+]i imaging started. These traces are correspond to the mean responses (± SEM) in nine responsive islet cells of control (—) and in 8 cells from TPTCl-administered animals (---). B, Population distribution of GIP-induced response derived from [Ca2+]i imaging experiments. Data are the number of {Delta} [Ca2+]i values (nM) at each level indicated, plotted as a percentage of the total number of the responses recorded from each group. Those with a {Delta} [Ca2+]i less than 10 nM were defined as nonresponsive cells and were eliminated from the analysis. Data were obtained from 132 cells from control animals ({square}) and from 95 cells from TPTCl-administered animals ({blacksquare}).

 
In the presence of extracellular Ca2+ (2.5 mM), 100 µM ACh elicited a biphasic response in [Ca2+]i. Addition of 100 µM ACh did not elicit the initial rise in [Ca2+]i in 25% of the control islet cells (n = 48) and in 36.6% of the cells (n = 150) from TPTCl-administered animals. Initial phase responsive islet cells elicited a late phase of [Ca2+]i. in 48.7% islet cells of the control (n = 39) and 21.0% of cells (n = 95) from TPTCl-administered animals. Ach-induced both phases are suppressed by 50% in the cells from TPTCl-administered animals (Fig. 6AGo). Removal of Ca2+ from extracellular solution lowered the ACh-induced initial phase and almost completely abolished the late phase. In this case, there was no suppression in the amplitude of the initial phase by TPTCl-administerion, whereas a significant suppression was observed in a late phase (P < 0.05, n = 9) (Fig. 6BGo).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. The effects of TPTCl administration on acetylcholine (100 µM)-induced rise in [Ca2+]i in single islet cells in the presence of (A) or in the absence of extracellular Ca2+ (B). The cells were preincubated with a buffer containing 5.5 mM glucose for about 3 min before [Ca2+]i imaging started. These traces are correspond to the mean responses (± SEM) in nine responsive islet cells of control (—) and in 7–9 cells from TPTCl-administered animals (---).

 
Effects of TPTCl administration on GIP-induced cyclic AMP content in islet cells
Figure 7Go shows the cAMP content in islet cells induced by 100 nM GIP with 1 mM IBMX or 1 mM IBMX alone in the presence of 5.5 mM glucose. TPTCl administration significantly reduced the cellular cAMP content induced by 100 nM GIP with 1 mM IBMX (P < 0.01, n = 5–11). TPTCl administration did not affect cAMP content in islet cells induced by 1 mM IBMX alone.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 7. Inhibitory effect of TPTCl administration on cAMP content in cultured single islet cells stimulated by 100 nM GIP in the presence of 5.5 mM glucose. The dissociated islet cells (3 x 104 cells) cultured overnight in DMEM containing 5% FCS and 5 mM glucose. They were incubated with 1 mM IBMX alone or 100 nM GIP plus 1 mM IBMX for 30 min at 37 C with 5.5 mM glucose. Data are expressed as the means ± SEM. ({square}), control (N = 8–11); ({blacksquare}), TPTCl administration (N = 5–7). **, P < 0.01 vs. control.

 
Effects of TPTCl administration on secretagogues-induced insulin secretion in pancreatic islets
Figure 8Go shows the insulin secretion from the pancreatic islet stimulated by 100 nM GIP, 100 µM ACh or glucose (2.8, 5.5, or 27.8 mM). In control islets, 27.8 mM glucose was significantly increased insulin secretion (P < 0.05, n = 9–16) (Fig. 8AGo). Insulin secretion by 100 nM GIP and 100 µM ACh with 5.5 mM glucose was also increased (P < 0.05, respectively, n = 9–16) (Fig. 8BGo). In islets from TPTCl-administered animals, glucose (27.8 mM)-induced insulin secretion were significantly reduced (P < 0.05, n = 9–16) (Fig. 8AGo). In the presence of 5.5 mM glucose, the insulin release induced by both 100 nM GIP and 100 µM ACh were also impaired to the basal level in the islets from TPTCl-administered hamsters (P < 0.05, respectively, n = 9–16) (Fig. 8BGo).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 8. The effects of TPTCl administration on the insulin secretion from islets induced by glucose (2.8, 5.5, 27.8 mM), 100 nM GIP, or 100 µM ACh in the presence of 5.5 mM glucose. Islets were preincubated in DMEM containing 5% FCS and 10 mM glucose for 90 min at 37 C. Three islets in each tube were incubated for 60 min in 1 ml of KRB (pH 7.4) containing glucose (2.8, 5.5, 27.8 mM), or 100 nM GIP, and 100 µM ACh in the presence of 5.5 mM glucose. Data are expressed as the means ± SEM. ({square}), control (N = 9–16); ({blacksquare}), TPTCl administration (N = 13–16). *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study has shown that TPTCl administration suppresses the rise in [Ca2+]i and insulin secretion by several insulin secretagogues in hamster pancreatic islet cells.

It has been previously demonstrated that triphenyltin compounds induce reversible diabetes with decreased insulin secretion in rabbits (4) and hamsters (5, 6). TPTCl-administered hamsters showed reduced sensitivity to glucose for the insulin secretion from pancreatic islets (5). However, TPTCl administration caused no change in morphology of pancreas (6, 7) or in the insulin contents (5) and in population of ß-cells in the islet cells (Fig. 1Go). These results indicate that TPTCl-induced diabetes is due to inhibitory effects on the process of insulin secretion from the hamster pancreatic islet.

It is now widely accepted that the glucose metabolism generates coupling factors such as ATP that close KATP-channels and trigger plasma membrane depolarization. This is followed by an influx of Ca2+ through voltage-dependent Ca2+ channels, which triggers exocytosis (11, 30). Glucose metabolism also generates other second messengers such as cAMP (17, 31). TPTCl-administered hamsters showed a reduced sensitivity to glucose and to forskolin, an activator of adenylate cyclase, for the rise in [Ca2+]i in comparison to that of control cells (9). In the present study, TPTCl administration also impaired the rise in [Ca2+]i by stimulation of glucose in islet cells. In a cell-free system, TPTCl does not affect the fluorescence intensity of 340 nm and 380 nm excitation in fura-2 by Ca2+-containing or Ca2+-free buffer (32). These results suggest that TPTCl administration has an inhibitory effect on the rise in [Ca2+]i in islet cells in response to glucose.

Excess K+ ions depolarize the pancreatic islet ß-cells and open the voltage-dependent (L-type) calcium channels (11). It is also known that pancreatic islet cells possess at least two types of voltage-dependent Ca2+ channels (19, 33); one is dihydropyridine sensitive L-type Ca2+ channel, and the other is dehydopyridine-insensitive non-L-type Ca2+ channel. To evaluate the effects of TPTCl administration on Ca2+-influx through voltage-dependent calcium channels in pancreatic islet cells, we tested the changes of [Ca2+]i induced by high K+ in the islet cells. TPTCl administration caused partial suppression of 10 mM K+-induced rise in [Ca2+]i of islet cells without suppressing the rise in [Ca2+]i induced by 20 mM or higher concentration of K+ in islet cells. Therefore, these results suggest that TPTCl administration is unlikely to suppress the high threshold Ca2+ channels (L-type), although TPTCl administration might suppress the low threshold Ca2+ channels (non L-type). We reported that TPTCl, in vitro, was a potent inhibitor of membrane potential change and Ca2+-signal transduction in N-formyl-methionyl-leucyl-phenylalanine stimulation, in association with superoxide production in neutrophils (32). Taken these together, TPTCl administration may suppress indirectly the Ca2+-influx through voltage-dependent L-type calcium channel by affecting the closure of an ATP-sensitive K+ channel in islet cells induced by stimulus.

cAMP-induced insulin release are mediated through an increase in [Ca2+]i (34, 8). GIP increases cAMP levels in pancreatic islets by activating adenylate cyclase (9, 35), thereby increasing Ca2+-influx and facilitating insulin secretion in the presence of glucose (13). In this study, in the presence of glucose, addition of GIP did not elicit the rise in [Ca2+]i in 52.0% of the cells from TPTCl-administered animals. TPTCl administration suppressed the rise in [Ca2+]i in islet cells stimulated by GIP. TPTCl administration also inhibited GIP-induced insulin secretion from the islet and impaired rise in cAMP content in islet cells. Because TPTCl administration reduced the rise in [Ca2+]i induced by forskolin, TPTCl administration may suppress the GIP-induced activation of adenylate cyclase in pancreatic ß-cells.

Acethylcholine has been shown to facilitate insulin secretion by mobilizing Ca2+ from intracellular store(s) via phosphatidyl inositol (PI) turnover (15, 36, 37, 38) and subsequent increase of voltage-dependent Ca2+-influx (39, 20). In the absence of extracellular Ca2+, there were no difference between ACh-induced initial phase of [Ca2+]i in islet cells from TPTCl administration and that of control, indicating that TPTCl administration did not alter the PI turnover and the subsequent Ca2+-mobilization from intracellular stores in pancreatic ß-cells. In the presence of glucose and extracellular Ca2+, TPTCl administration impaired ACh-induced rise in [Ca2+]i and insulin secretion in islet cells. The depolarizing effect of ACh is accompanied by an increase in the membrane permeability for Na+ (39). Furthermore, the muscarinic receptors (M3) have been shown to couple to Na+ channels in islet ß-cells (40). These suggest that TPTCl administration inhibits the influx of Ca2+ through voltage-dependent Ca2+ channels by affecting a Na+-dependent depolarization.

We conclude that the pathology of triphenyltin-induced diabetes in hamsters that is associated with impaired insulin secretion involves a defect in cellular Ca2+ response due to a reduced Ca2+-influx through voltage-gated Ca2+-channels. Recently our colleagues indicated that the tin concentration in the pancreas of TPTCl-administered hamsters were distinctly higher than that of the control hamsters (7) and of the TPTCl-administered rats (41). The close correlation between the tin concentration in the pancreas and the plasma glucose levels (41) raises the possibility that diabetogenic action of TPTCl depends upon the amount of tin compounds in the pancreas.

Further studies are necessary to elucidate the precise mechanism of impaired insulin secretion with respect to the reduced Ca2+-influx in relation to the action of tin compounds.


   Acknowledgments
 
The authors wish to thank Dr. K. Kasai, Dr. S. Matsuzaki, and Dr. K. Koibuchi for helpful advice. We also wish to thank Dr. H. Maesawa and Dr. M. Iizuka for the measurement of cytosolic Ca2+.

Received December 6, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. World Health Organization 1980 Tin and organotin compounds: a preliminary review. In: Environmental Health Criteria, Geneva, 15:16–40
  2. Zuckerman JJ, Reisdorf RP, Ellis III HV, Wilkinson RR 1978 Organotins in biology and the environment. In: Brinckman FE, Bellama JM (eds) Organometal and organometalloides: occurrence and fate in the environment ACS symposium series, No 82. American Chemical Society, Washington DC, p 388–424
  3. Boyer IJ 1989 Toxicity of dibutyltin, tributyltin and other organotin compounds to humans and to experimental animals. Toxicology 55:253–298[CrossRef][Medline]
  4. Manabe S, Wada O 1981 Triphenyltin fluoride (TPTF) as a diabetogenic agent: TPTF induces diabetic lipemia by inhibiting insulin secretion from morphologically intact rabbit B-cell. Diabetes 30:1013–1021[Abstract]
  5. Miura Y, Matsui H 1987 The effects of triphenyltin on insulin release from pancreatic islets in hamsters. J Japan Diab Soc 30:895–900
  6. Matsui H, Wada O, Manabe S, Ushijima Y, Fujikura T 1984 Species difference in sensitivity to the diabetogenic action of triphenyltin hydroxide. Experientia 40:377–378[Medline]
  7. Ogino K, Inukai T, Ohhira S, Matsui H, Takemura Y 1996 The mechanism of triphenyltin-induced glucose tolerance disorder in hamsters. Dokkyo J Med Sci 23:101–110
  8. Rajan AS, Hill RS, Boyd lll AE 1982 Effect of rise in cAMP levels on Ca2+ influx through voltage-dependent Ca2+ channels in HIT cells: second-messenger synarchy in ß-cells. Diabetes 38:874–880[Abstract]
  9. Miura Y, Matsui H 1991 Triphenyltin-chloride-induced diabetes in hamsters: inhibition of stimulus-induced changes of cytosolic free calcium on insulin secretion in isolated pancreatic islet cells. In: Goto Y, Kanazawa Y (eds) Lessons from animal diabetes. The Tokyo Workshop 1990, Smith-Gorden, London, pp 75–80
  10. Wolheim CB, Sharp GWG 1981 Regulation of insulin release by calcium. Physiol Rev 61:914–973[Free Full Text]
  11. Detimary P, Gilon P, Nenquin M, Henquin J-C 1994 Two sites of glucose control of insulin release with distinct dependence on the energy state in pancreatic B-cells. Biochem J 297:455–461
  12. Karup T 1988 Immunoreactive gastric inhibitory polypeptide. Endocr Rev 9:122–134[Medline]
  13. Lu M, Wheeler MB, Leng X-H, Boyd lll AE 1993 The role of the free cytosolic calcium level in ß-cell signal transduction by gastric inhibitory polypeptide and glucagon-like peptide I (7–37). Endocrinology 132:94–100[Abstract]
  14. Hermans MP, Henquin J-C 1989 Relative importance of extracellular and intracellular Ca2+ for acetylcholine stimulation of insulin release in mouse islets. Diabetes 38:198–204[Abstract]
  15. Biden TJ, Peter-Reisch B, Schlegel W, Wollheim CB 1987 Ca2+-mediated generation of inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakisphosphate in pancreatic islets. Studies with K+, glucose, and carbamylcholine. J Biol Chem 262:3567–3571[Abstract/Free Full Text]
  16. Wang J, Baimbridge KD, Brown JC 1992 Glucose- and acetylcholine-induced increase in intracellular free Ca2+ in subpopulations of individual rat pancreatic ß-cells. Endocrinology 131:146–152[Abstract]
  17. Prentki M, Wolheim CB 1984 Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles. Experientia 40:1052–1060[CrossRef][Medline]
  18. Kato M, Hoyland J, Sikdar SK, Mason WT 1992 Imaging of intracellular calcium in rat anterior pituitary cells in response to growth hormone releasing factor. J Physiol 447:171–189[Abstract/Free Full Text]
  19. Komatsu M, Yokokawa N, Takeda T, Nagasawa Y, Aizawa T, Yamada T 1989 Pharmacological characterization of the voltage-dependent calcium channel of pancreatic B-celll. Endocrinology 125:2008–2014[Abstract]
  20. Gilon P, Nenquin M, Henquin J-C 1995 Muscarinic stimulation exerts both stimulatory and inhibitory effects on the concentration of cytoplasmic Ca2+ in the electrically excitable pancreatic B-cell. Biochem J 311:259–267
  21. Lancy PE, Kostianovsky M 1967 Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16:35–39[Medline]
  22. Okeda T, Ono J, Takaki R, Todo S 1979 Simple method for the collection of pancreatic islets by the use of Ficoll-Conray gradient. Endocrinol Jpn 26:495–499[Medline]
  23. Katsuta H, Takaoka T 1978 Protein- and lipid-free synthetic media for the cultivation of mammalian cells in tissue culture. In: Katsuta H (eds) Nutritional requirements of cultured cells. Japan Scientific Press, Tokyo, pp 257–275
  24. Takaki R, Ono J 1985 Preparation and culture of isolated islets and dissociated islet cells from rodent pancreas. J Tissue Cult Methods 9:61–66[CrossRef]
  25. Watanabe K, Koibuchi N, Ohtake H, Yamaoka S 1993 Circadian rhythms of vasopressin release in primary cultures of rat suprachiasmatic nucleus. Brain Res 624:115–120[CrossRef][Medline]
  26. Tanaka S, Kurabuchi S, Mochida H, Kato T, Takahashi S, Watanabe T, Nakayama K 1996 Immunocytochemical localization of prohormone convertases PC1/PC3 and PC2 in rat pancreatic islets. Arch Histol Cytol 59:261–271[Medline]
  27. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450[Abstract/Free Full Text]
  28. Nelson TY, Gaines Kl Rajan AS, Berg M, Boyd AE III 1987 Increased cytosolic calcium: a signal for sulfonylurea-stimulated insulin release from beta cells. J Biol Chem 262:2608–2612[Abstract/Free Full Text]
  29. Morgan CR, Lazarow A 1963 Immunoassay of insulin: two antibody system, plasma insulin levels of normal, subdiabetic and diabetic rats. Diabetes 12:115–126
  30. Arhammar P, Nilsson T, Rorsman P, Berggren P-O 1987 Inhibition of ATP-regulated K+ channels precedes depolarization-induced increase in cytoplasmic free Ca2+ concentration in pancreatic ß-cells. J Biol Chem 262:5448–5454[Abstract/Free Full Text]
  31. Malaisse WJ, Malaisse-Lagae F 1984 The role of cyclic AMP in insulin release. Experientia 40:1068–1075[CrossRef][Medline]
  32. Miura Y, Matsui H 1991 Inhibitory effects of phenyltin compounds on stimulus-induced changes in cytosolic free calcium and plasma membrane potential of human neutrophils. Arch Toxicol 65:562–569[Medline]
  33. Satin LS, Cook DL 1988 Evidence for two calcium currents in insulin-secreting cells. Pflügers Arch 411:401–409[CrossRef][Medline]
  34. Prentki M, Matschinsky FM 1987 Ca2+, cAMP, and phospholipide-derived messengers in coupling mechanisms of insulin secretion. Physiol Rev 67:1185–1248[Free Full Text]
  35. Goke R, Fehmann H-C, Goke B 1991 Glucagon-like peptide-l (7–36) amide is a new incretin/enterogastrone candidate. Eur J Clin Invest 21:135–144[Medline]
  36. Best L, Malaisse WJ 1984 Nutrient and hormone-neurotransmitter stimuli induce hydrolysis of polyphosphoinositides in rat pancreatic islets. Endocrinology 115:1814–1820[Abstract]
  37. Wollheim CB, Biden TJ 1986 Second messenger function of inositol 1,4,5-trisphosphate. Early changes in inositol phosphates, cytosolic Ca2+, and insulin release in carbamylcholine-stimulated RINm5F cells. J Biol Chem 261:8314–8319[Abstract/Free Full Text]
  38. Turk J, Gross RW, Ramanadham S 1993 Amplification of insulin secretion by lipid messengers. Diabetes 42:367–374[Abstract]
  39. Henquin J-C, Garcia MC, Bozem M, Hermans MP, Nenquin M 1988 Muscarinic control of pancreatic B cell function involves sodium-dependent depolarization and calcium influx. Endocrinology 122:2134–2142[Abstract]
  40. Miura Y, Gilon P, Henquin J-C 1996 Muscarinic stimulation increases Na+ entry in pancreatic B-cells by a mechanism other than the emptying of intracellular Ca2+ pools. Biochem Biophys Res Commun 224:67–73[CrossRef][Medline]
  41. Ohhira S, Matsui H 1996 Comparative study of the metabolism of triphenyltin in hamsters and rats after a single oral treatment with triphenyltin chloride. Toxicol Lett 85:3–8[Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
Y. Miura and H. Matsui
Glucagon-like peptide-1 induces a cAMP-dependent increase of [Na+]i associated with insulin secretion in pancreatic {beta}-cells
Am J Physiol Endocrinol Metab, November 1, 2003; 285(5): E1001 - E1009.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Miura, Y.
Right arrow Articles by Matsui, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Miura, Y.
Right arrow Articles by Matsui, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals