1 Endocrine and Diabetes Unit, Department of Molecular Medicine, Karolinska Institutet, S-171 76 Stockholm, Sweden; 2 Department of Pharmacology, Lilly Research Laboratories, Lilly Forschung GmbH, D-22419 Hamburg, Germany; and 3 Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia
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ABSTRACT |
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The
insulinotropic activity of the imidazoline derivative RX871024
was compared in pancreatic islets from nondiabetic Wistar rats and
spontaneously diabetic Goto-Kakizaki (GK) rats. RX871024 significantly
stimulated insulin secretion in islets from both animal groups. The
insulinotropic activity of RX871024 was higher than that of the
sulfonylurea glibenclamide. This difference was more pronounced in
islets from GK rats compared with Wistar rat islets. More importantly,
RX871024 substantially improved glucose sensitivity in diabetic
-cells, whereas glibenclamide stimulated insulin secretion about
twofold over a broad range of glucose concentrations in nondiabetic and
diabetic rats. RX871024 induced a faster increase in cytosolic free
Ca2+ concentration and faster inhibition of ATP-dependent
K+ channel activity in GK rat islets compared with Wistar
rat islets. RX871024 also induced a more pronounced increase in
diacylglycerol concentration in GK rat islets. These data support the
idea that imidazoline compounds can form the basis for the development
of novel drugs for treatment of type 2 diabetes, which can restore glucose sensitivity in diabetic
-cells.
diabetic rats; pancreatic islets; imidazolines; insulin secretion
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INTRODUCTION |
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ONE OF THE KEY FEATURES of type 2 diabetes is a defective insulin secretory response to glucose (5). To overcome this defect, treatment with sulfonylurea compounds, potent insulin releasers, is widely adopted (6, 8). However, a majority of patients fail to respond when sulfonylurea compounds are administered for a long period of time (8).
Previously, we have shown that the imidazoline derivative RX871024 is a potent stimulator of glucose-dependent insulin release in normal animals and insulin-producing cell lines (15, 17). The insulinotropic activity of this compound is more pronounced than that of the sulfonylurea glibenclamide (4). The stimulatory effect of RX871024 on insulin release was demonstrated to be due to both inhibition of the ATP-dependent K+ (KATP) channel and a direct effect on exocytosis (4, 15).
The spontaneously diabetic nonobese Goto-Kakizaki (GK) rat displays many of the characteristics of type 2 diabetes in humans (7). Insulin response to glucose in pancreatic islets from GK rats is severely impaired, whereas the response to nonnutrient stimuli is preserved or exaggerated (1, 2).
The aim of this study was to further explore the insulinotropic effect of RX871024 and to compare this effect with that of the sulfonylurea glibenclamide in pancreatic islets from GK rats.
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MATERIALS AND METHODS |
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Materials. The imidazoline compound RX871024 was obtained from Reckitt & Colman Pharmaceuticals (Kingston Upon Hull, UK). Fura 2-AM was from Molecular Probes, (Eugene, OR). Diacylglycerol (DAG) kinase was from Calbiochem-Novabiochem (San Diego, CA). Glibenclamide and all other reagents were obtained from Sigma Chemical (St. Louis, MO).
Isolation and incubation of islets of Langerhans. Islets were isolated from 2- to 3-mo-old male nondiabetic Wistar and diabetic GK rats (Karolinska Hospital colony) by collagenase digestion as described before (13). Islets were then maintained overnight in RPMI 1640 culture medium (Life Techologies, Paisley, UK) supplemented with 11 mM glucose, 10% fetal calf serum, 2 mM glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin. In experiments with single cells, islets were dispersed by vigorous shaking in Ca2+-free medium. The cells were then plated onto coverslips. All experiments with islets and cells from Wistar and GK rats were run in parallel in triplicate or quadruplicate from at least three different rats.
Measurements of insulin release in intact pancreatic islets were performed in Krebs-Ringer bicarbonate (KRB) buffer containing (in mM): 115 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 20 NaHCO3, and 16 HEPES, pH 7.4, supplemented with 2 mg/ml bovine serum albumin (BSA) as described previously (15). Measurements of insulin release in electropermeabilized islets were performed according to a procedure described before (4, 15). Under these conditions, a buffer containing (in mM): 140 potassium glutamate, 5 NaCl, 1 MgCl2, 10 EGTA, 25 HEPES, 2 ATP, and 0.25 mg/ml BSA, pH 7.0, was used. An ATP-regenerating system consisting of 2 mM creatine phosphate and 10 U/ml creatine phosphokinase was added to the system. The actual free Ca2+ concentrations ([Ca2+]i) in the buffer were adjusted using a Ca2+-selective electrode.Measurements of cytosolic free
[Ca2+]i.
[Ca2+]i were measured with the
Ca2+-sensitive fluorescent probe fura 2-AM in single rat
pancreatic -cells, as previously described (15, 18).
Ion channel recordings. The activity of KATP channels was studied in inside-out patches, and the activity of voltage-dependent Ca2+ channels was examined in the whole cell configuration of the patch-clamp technique, as previously described (15). The volume of the perifusion chamber was 300 µl, with the perifusion rate 4 ml/min.
Glucose metabolism measurements. Glucose oxidation and glucose utilization in rat islets were analyzed according to procedures previously described (19).
Measurements of DAG concentration.
The procedure for DAG measurements was adopted from Kanoh et al.
(9). One hundred islets were incubated in KRB buffer with test compounds at 37°C for 10 min. Incubation was terminated by taking out the medium and adding 0.2 ml of 0.2% SDS and 0.75 ml of
chloroform-methanol (1:2). The cellular lipid fraction was extracted at
4°C for 1 h. The water fraction and organic fraction were
separated. The organic fraction was washed with methanol and 0.2 M NaCl
and dried. Extracted lipids were dissolved in a buffer containing (in
mM): 100 Tris · HCl, 2.5 sodium deoxycholate, and 1.25 dithiothreitol, pH 7.4. Twenty microliters of lipid solution and 20 µl of 100 mM Tris · HCl, 1 mM EGTA, and 2 µg DAG kinase, pH
7.4, were mixed. Reaction of DAG conversion to phosphatidic acid was
initiated by addition of 1.6 mM [-32P]ATP (5,000 cpm/nmol). Samples were incubated at 29°C for 30 min. Then, a
chloroform-methanol (1:2) mixture was added, and water and organic
phases were separated by centrifugation. The water layer was discarded,
and the organic layer was washed with 1% perchloric acid. The organic
phase was dried, dissolved in chloroform, and applied on thin-layer
chromatography (TLC; Silica gel 60 plates, Merck, Darmstadt,
Germany), using chloroform-acetone-methanol-acetic acid-water (50:20:10:10:5) as a developing solvent. The TLC plate with
separated lipids was submitted to autoradiography with the use of
Bio-Imaging Analyzer BAS 1000 (Fuji, Japan).
Statistical analysis. The difference of means was estimated with a t-test or analyses of variance (ANOVA), with P values corrected by the Bonferroni method using Statistica for Windows (version 5.0, StatSoft, Tulsa, OK).
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RESULTS |
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Effects of RX871024 and glibenclamide on insulin release in
batch-incubated GK and Wistar rat islets.
In GK rat islets, insulin release in response to 15 mM glucose was
significantly decreased compared with that in nondiabetic Wistar rat
islets (Fig. 1A). Addition of
RX871024 stimulated insulin release in both animals, the effect being
particularly pronounced at elevated glucose concentration. At 3 mM
glucose, 10 µM and higher concentrations of RX871024 significantly
increased insulin release. At 15 mM glucose, a significant increase in
insulin release was observed already at 1 µM RX871024 (Fig.
1A). At 15 mM glucose, the relative stimulation at any
particular concentration of RX871024 was higher in GK rat islets
compared with nondiabetic islets (Fig. 1B). At 50 µM
RX871024, insulin release in GK rats in the presence of 15 mM glucose
reached the level of 59% of the response in corresponding nondiabetics. In contrast, at 15 mM glucose alone, insulin release in
GK rat islets was only 38% of that in Wistar rat islets.
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Influence of 15 mM glucose on
[Ca2+]i in pancreatic
-cells from GK and Wistar rats.
In GK rat
-cells, the increase in [Ca2+]i,
after addition of 15 mM glucose, was slower compared with that in
Wistar rat
-cells (Fig. 3). One of the
reasons was a delayed lag time for [Ca2+]i
response (tin) (Table
1).
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Effects of RX871024 on
[Ca2+]i response in
pancreatic -cells from GK and Wistar rats.
Addition of RX871024 increased [Ca2+]i in
-cells from GK and Wistar rats. At basal glucose concentration (3 mM), an oscillatory pattern of Ca2+ response was
observed with 10 µM RX871024 (Fig.
4, A and B). At 50 µM RX871024, the increase in [Ca2+]i was
higher, and oscillations were rarely observed (Fig. 4, C and
D). The lag time for the [Ca2+]i
responses in GK animals was significantly shorter than in nondiabetics at both concentrations of RX871024 and decreased with the elevation of
RX871024 concentration from 10 to 50 µM (Table 1). In the presence of
15 mM glucose, [Ca2+]i responses to RX871024
were faster than those at basal glucose concentration in both GK and
Wistar rats (Fig. 3). Despite the pronounced differences in kinetics of
[Ca2+]i responses to RX871024 between the two
groups of animals (Table 1), there were no significant differences in
the amplitude of [Ca2+]i responses between GK
and Wistar rat
-cells. The increases in
[Ca2+]i in GK rat
-cells were 114 ± 30% (n = 8), 70 ± 26% (n = 7), and 90 ± 20% (n = 9) of corresponding responses
in Wistar
-cells at 50 µM RX871024 and 3.3 mM glucose
(n = 10), at 10 µM RX871024 and 15 mM glucose
(n = 11), and at 50 µM RX871024 and 15 mM glucose (n = 7), respectively.
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Effects of RX871024 on KATP channel activity in
pancreatic -cells from GK and Wistar rats.
To examine whether the aforementioned decreased lag time for
[Ca2+]i responses to RX871024 in GK rat
-cells compared with Wistar
-cells was due to a more efficient
regulation by the compound of the KATP channel, patch-clamp
experiments (inside-out configuration) were performed (Fig.
5, A and B).
Inhibitions of KATP channel by RX871024 tend to develop
faster in GK rat
-cells compared with nondiabetic
-cells (Fig.
5F, P = 0.11), the results being in line
with the effect of the compound on [Ca2+]i.
No difference in amplitude of RX871024-induced inhibition or
single-channel kinetics between two cell types has been observed (Fig.
5, C-E).
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Effects of RX871024 on insulin release in perifused GK and Wistar
rat islets.
To evaluate whether the faster response in
[Ca2+]i resulted in a shorter lag time for
RX871024-induced insulin secretion, perifusion experiments using
pancreatic islets from GK and Wistar rats were performed. The results
demonstrated that, in GK rat pancreatic islets, RX871024-induced
insulin release was lower than in Wistar rat islets (Fig.
6). These data were consistent with our
batch incubation experiments (Fig. 1). However, the data obtained did not reveal any difference in dynamics of the responses to RX871024 in
diabetic animals compared with nondiabetics.
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Effects of RX871024 on insulin release in electropermeabilized GK
and Wistar rat islets.
To elucidate the mechanism of glucose-dependent stimulation of insulin
secretion by RX871024 in islets from diabetic animals, experiments in
electropermeabilized GK and Wistar rat islets were performed (Fig.
7). There was a clear stimulation of
insulin release by RX871024 in nondiabetic islets, which was similar to
that described previously (4, 15). RX871024 stimulated
insulin release in electropermeabilized islets from GK rats too (Fig.
7). There were no significant differences in the effects of RX871024
between the two groups of animals.
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Effects of RX871024 on glucose oxidation and glucose
utilization in islets from GK and Wistar rats.
To examine whether RX871024 affects glucose metabolism in GK and
Wistar rats, the effect of the compound on glucose oxidation and
glucose utilization was studied (Fig. 8).
The obtained data confirmed our previous studies showing that the rates
of glucose oxidation and glucose utilization are higher in GK rat
islets (11). However, neither 10 nor 50 µM RX871024
influenced glucose oxidation or glucose utilization in the two groups
of animals.
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Effects of RX871024 on DAG concentration in GK and Wistar rat
islets.
DAG is an example of a second messenger that plays an important role in
the process of insulin secretion in the pancreatic -cell (10,
12). Hence, effects of RX871024 on DAG formation were examined
in pancreatic islets from nondiabetic and diabetic rats. At 3 mM
glucose, 50 µM RX871024 induced a 30% increase in DAG concentration
in Wistar rat islets (Fig. 9). Basal DAG
concentration in GK islets was 50% higher than in Wistar rat islets.
RX871024 at 50 µM produced a nearly 100% DAG increase over basal
level in GK rat islets. The increase in DAG concentration produced in GK rat islets was significantly higher than that in Wistar rat islets.
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DISCUSSION |
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The aim of this study was to evaluate the effect of the imidazoline compound RX871024 on insulin release in pancreatic islets from GK rats, an animal model of type 2 diabetes, which demonstrates a severe impairment in insulin release in response to glucose both in vivo and in vitro (1, 7, 11). Effects of RX871024 were compared with those of the classic sulfonylurea compound glibenclamide. We have confirmed our previous findings obtained in the islets from normoglycemic Wistar rats, namely that the imidazoline compound RX871024 was more effective in promoting islet insulin release compared with the sulfonylurea glibenclamide (4). In islets from diabetic GK rats, RX871024 was also a more potent insulinotropic agent than glibenclamide.
An important finding of this study was that, in GK rat islets, RX871024 induced a progressive rise in stimulation of insulin release in response to glucose when the concentration of glucose was increased from 3 to 15 mM. What, then, can constitute the mechanisms behind the pronounced effect of RX871024 on glucose-induced insulin release in GK rats? We addressed this question by studying effects of RX871024 on [Ca2+]i, insulin exocytosis, glucose metabolism, and DAG concentration in rat pancreatic islets.
The [Ca2+]i response to 15 mM glucose was
delayed in GK rat islets compared with nondiabetic rat islets. This
observation is in line with our previous study demonstrating a delayed
[Ca2+]i response to high glucose in GK rat
islets compared with that in Wistar rat islets (16).
However, comparison of [Ca2+]i responses to
RX871024 in pancreatic -cells from GK and Wistar rats has shown a
faster [Ca2+]i response to RX871024
stimulation in diabetic compared with nondiabetic
-cells. The faster
[Ca2+]i response to RX871024 in GK rat
-cells might be explained by the faster blockade of KATP
channel activity with RX871024 in diabetic rats. However, we have not
observed significant changes in the dynamics of RX871024-induced
insulin release in islet perifusion experiments between the two groups
of animals.
Comparative studies of the effects of RX871024 on insulin release in electropermeabilized GK and Wistar rat islets under Ca2+-clamped conditions and in the presence of high ATP concentration showed no significant differences between the two groups of animals. Hence, under these conditions, the ability of RX871024 to activate the insulin exocytotic machinery was the same in GK and Wistar rats. Likewise, RX871024 did not affect glucose metabolism in islets from GK or Wistar rats. This indicates that the higher potentiation of glucose-induced insulin secretion by RX871024 in GK rat islets cannot be attributed to a direct effect of RX871024 on glucose metabolism.
We have, however, found a significantly increased islet DAG level in GK
rats under basal conditions. These data are similar to a previous
observation that muscles from GK rats have also increased levels of DAG
(3). The increased level of DAG in GK rat islets may
probably be explained by increased de novo synthesis of DAG from
glucose under conditions of persistent hyperglycemia, as has been
reported from other tissues (14). It remains to be
identified whether increased DAG levels can be responsible for the
deterioration in -cell functions in diabetic animals as has been
suggested for other tissues where elevated DAG levels may be involved
in adverse effects of hyperglycemia (14).
In GK rat islets, the magnitude of the DAG response to RX871024 was almost threefold higher compared with that in nondiabetic islets. The increased DAG response in GK rat islets to RX871024 may shed light on the mechanism by which RX871024 exerts this effect. Thus it may be speculated that RX871024 works as a positive modulator of DAG de novo synthesis. In this case, increased flux of glucose to DAG in GK rats and positive modulation of this pathway with RX871024 would give a synergistic increase in DAG. Alternatively, RX871024 may inhibit DAG conversion/degradation processes in the cell. Higher DAG synthesis and lower DAG conversion would give a high rise in DAG concentration in diabetic islets when treated with RX871024.
In conclusion, we have confirmed that the imidazoline compound RX871024 exhibits glucose-dependent insulinotropic activity in normal rats (15). In diabetic GK rat islets, RX871024 elicits pronounced insulinotropic effects and restores glucose sensitivity. The restoration of glucose sensitivity seen in GK rat islets could, at least in part, be accounted for by the higher RX871024-induced DAG elevation. These observations favor the idea that this class of imidazoline compounds may form the basis of a novel principle for treatment of type 2 diabetes.
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ACKNOWLEDGEMENTS |
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This study was supported by Eli Lilly & Co., the Swedish Medical Research Council (Grants 72X-00034, 72X-09890, 72XS-12708), the Royal Swedish Academy of Sciences, the Swedish Diabetes Association, the Nordic Insulin Foundation Committee, the Nordic Insulin Foundation, the Berth von Kantzows Foundation, and Funds of the Karolinska Institutet.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. V. Zaitsev, Dept. of Molecular Medicine, Karolinska Hospital, L3, S-171 76, Stockholm, Sweden (E-mail: Sergei.Zaitsev{at}enk.ks.se).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.000031.2001
Received 29 January 2001; accepted in final form 11 September 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abdel-Halim, SM,
Guenifi A,
Efendic S,
and
Östenson CG.
Both somatostatin and insulin responses to glucose are impaired in the perfused pancreas of the spontaneously noninsulin-dependent diabetic GK (Goto-Kakizaki) rats.
Acta Physiol Scand
148:
219-226,
1993[ISI][Medline].
2.
Abdel-Halim, SM,
Guenifi A,
Khan A,
Larsson O,
Berggren PO,
Östenson CG,
and
Efendic S.
Impaired coupling of glucose signal to the exocytotic machinery in diabetic GK rats: a defect ameliorated by cAMP.
Diabetes
45:
934-940,
1996[Abstract].
3.
Avignon, A,
Yamada K,
Zhou X,
Spencer B,
Cardona O,
Saba-Siddique S,
Galloway L,
Standaert ML,
and
Farese RV.
Chronic activation of protein kinase C in soleus muscles and other tissues of insulin-resistant type II diabetic Goto-Kakizaki (GK), obese/aged, and obese/Zucker rats. A mechanism for inhibiting glycogen synthesis.
Diabetes
45:
1396-1404,
1996[Abstract].
4.
Efanov, AM,
Zaitsev SV,
Efanova IB,
Östenson CG,
Berggren PO,
and
Efendic S.
Signaling and sites of interaction for imidazoline and sulfonylurea in the stimulation of insulin release.
Am J Physiol Endocrinol Metab
274:
E751-E757,
1998
5.
Efendic, S,
Kindmark H,
and
Berggren PO.
Mechanisms involved in the regulation of the insulin secretory process.
J Intern Med
229:
9-22,
1991[ISI][Medline].
6.
Gerich, JE.
Oral hypoglycemic agents.
N Engl J Med
321:
1231-1245,
1989[ISI][Medline].
7.
Goto, Y,
Kakizaki M,
and
Mazaki N.
Spontaneous diabetes produced by selective breeding of normal Wistar rats.
Proc Jpn Acad
51:
80-85,
1975.
8.
Groop, LC.
Sulfonylureas in NIDDM.
Diabetes Care
15:
737-754,
1992[Abstract].
9.
Kanoh, H,
Kondoh H,
and
Ono T.
Diacylglycerol kinase from pig brain. Purification and phospholipid dependencies.
J Biol Chem
258:
1767-1774,
1983
10.
Malaisse, WJ,
Dunlop ME,
Mathias PC,
Malaisse-Lagae F,
and
Sener A.
Stimulation of protein kinase C and insulin release by 1-oleoyl-2-acetyl-glycerol.
Eur J Biochem
149:
23-27,
1985[Abstract].
11.
Stenson, CG,
Khan A,
Abdel-Halim SM,
Guenifi A,
Suzuki K,
Goto Y,
and
Efendic S.
Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat.
Diabetologia
36:
3-8,
1993[ISI][Medline].
12.
Peter-Riesch, B,
Fathi M,
Schlegel W,
and
Wollheim CB.
Glucose and carbachol generate 1,2-diacylglycerols by different mechanisms in pancreatic islets.
J Clin Invest
81:
1154-1161,
1988[ISI][Medline].
13.
Sutton, R,
Peters M,
McShane P,
Gray DWR,
and
Morris PJ.
Isolation of rat pancreatic islets by ductal injection of collagenase.
Transplantation
42:
689-691,
1986[ISI][Medline].
14.
Xia, P,
Inoguchi T,
Kern TS,
Engerman RL,
Oates PJ,
and
King GL.
Characterization of the mechanism for the chronic activation of diacylglycerol-protein kinase C pathway in diabetes and hypergalactosemia.
Diabetes
43:
1122-1129,
1994[Abstract].
15.
Zaitsev, SV,
Efanov AM,
Efanova IB,
Larsson O,
Östenson CG,
Gold G,
Berggren PO,
and
Efendic S.
Imidazoline compounds stimulate insulin release by inhibition of KATP channels and interaction with exocytotic machinery.
Diabetes
45:
1610-1618,
1996[Abstract].
16.
Zaitsev, SV,
Efanova IB,
Östenson CG,
Efendic S,
and
Berggren PO.
Delayed Ca2+ response to glucose in diabetic GK rat.
Biochem Biophys Res Commun
239:
129-133,
1997[ISI][Medline].
17.
Zaitsev, SV,
Efanov AM,
Raap A,
Efanova IB,
Schloos J,
Steckel-Hamann B,
Larsson O,
Östenson CG,
Berggren PO,
Mest HJ,
and
Efendic S.
Three modes of action of imidazoline compound RX871024 in pancreatic -cell: blocking of K+-channels, mobilization of Ca2+ from endoplasmic reticulum and interaction with exocytotic machinery.
Ann NY Acad Sci
881:
241-253,
1999
18.
Zaitsev, SV,
Efendic S,
Arkhammar P,
Bertorello AM,
and
Berggren PO.
Dissociation between changes in cytoplasmic free Ca2+ concentration and insulin secretion as evidenced from measurements in mouse single pancreatic islets.
Proc Natl Acad Sci USA
92:
9712-9716,
1995[Abstract].
19.
Zong-Chao, L,
Efendic S,
Wibom R,
Abdel-Halim SM,
Östenson CG,
Landau BB,
and
Khan A.
Glucose metabolism in Goto-Kakizaki rat islets.
Endocrinology
139:
2670-2675,
1998