Insulinotropic activity of the imidazoline derivative RX871024 in the diabetic GK rat

Alexander M. Efanov1, Ioulia B. Appelskog1, Samy M. Abdel-Halim1, Akhtar Khan1, Robert Bränström1, Olof Larsson1, Claes-Göran Östenson1, Hans-Juergen Mest2, Per-Olof Berggren1, Suad Efendic1, and Sergei V. Zaitsev1,3

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -cells.

diabetic rats; pancreatic islets; imidazolines; insulin secretion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 [gamma -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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Concentration-response curves for effects of the imidazoline RX871024 on insulin secretion in pancreatic islets from nondiabetic Wistar and diabetic Goto-Kakizaki (GK) rats in the presence of 3 (open circle  Wistar,  GK) and 15 mM ( Wistar,  GK) glucose. Data are means ± SE for 16 observations from 3 independent islet preparations. Insulin secretion expressed either in absolute values (A) or as a percentage of insulin secretion at the corresponding glucose concentration and without the imidazoline derivative RX871024 (B). *P < 0.05, **P < 0.01, ***P < 0.001 vs. insulin secretion at the corresponding glucose concentration and without RX871024. dagger P < 0.05, dagger dagger P < 0.01 vs. insulin secretion at 15 mM glucose and corresponding RX871024 concentration in Wistar islets.

Insulin release in response to glucose was decreased in isolated islets from GK rats over a broad range of glucose concentrations (Fig. 2). At maximal effective concentrations of RX871024 (50 µM) and glibenclamide (2 µM), both compounds significantly increased insulin release at all glucose concentrations in Wistar and GK rats. However, RX871024 produced a larger potentiation of insulin release at all glucose concentrations in both Wistar and GK rat islets (Fig. 2).


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Fig. 2.   Effects of 50 µM RX871024 (open circle ) and 2 µM glibenclamide (black-triangle) on insulin secretion at different glucose concentrations in Wistar (A) and GK (B) rat pancreatic islets; , insulin secretion in the absence of test substances. Data are means ± SE for 14 observations from 3 independent islet preparations. *P < 0.05, **P < 0.01, ***P < 0.001 vs. insulin secretion at corresponding conditions with 3 mM glucose.

Influence of 15 mM glucose on [Ca2+]i in pancreatic beta -cells from GK and Wistar rats. In GK rat beta -cells, the increase in [Ca2+]i, after addition of 15 mM glucose, was slower compared with that in Wistar rat beta -cells (Fig. 3). One of the reasons was a delayed lag time for [Ca2+]i response (tin) (Table 1).


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Fig. 3.   Comparison of RX871024-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in single Wistar (A and C) and GK (B and D) rat pancreatic beta -cells at 15 mM glucose. Time and sequence of additions of 15 mM glucose (G) and RX871024 (RX) are shown by arrows. Fluorescence ratio (F340/F380) reflects changes in [Ca2+]i. Representative traces out of 8-11 from 4 independent cell preparations. tin and tmax, Lag time in increase in [Ca2+]i and the time of development of maximal [Ca2+]i response, respectively.


                              
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Table 1.   Time parameters for [Ca2+]i response development in GK and Wistar rat pancreatic beta -cells

Effects of RX871024 on [Ca2+]i response in pancreatic beta -cells from GK and Wistar rats. Addition of RX871024 increased [Ca2+]i in beta -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 beta -cells. The increases in [Ca2+]i in GK rat beta -cells were 114 ± 30% (n = 8), 70 ± 26% (n = 7), and 90 ± 20% (n = 9) of corresponding responses in Wistar beta -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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Fig. 4.   Comparison of RX871024-induced changes in [Ca2+]i in single Wistar (A and C) and GK (B and D) rat pancreatic beta -cells at basal glucose concentration (3 mM). Time of additions of RX871024 is shown by arrows. Representative traces out of 7-20 from 4 independent cell preparations.

Effects of RX871024 on KATP channel activity in pancreatic beta -cells from GK and Wistar rats. To examine whether the aforementioned decreased lag time for [Ca2+]i responses to RX871024 in GK rat beta -cells compared with Wistar beta -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 beta -cells compared with nondiabetic beta -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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Fig. 5.   Single ATP-dependent K+ (KATP) channel recordings in beta -cells isolated from GK and Wistar rat islets. Recordings of freshly isolated inside-out patches from Wistar (A) and GK (B) beta -cells exposed to 50 µM RX871024 and channel open probability (Popen) analysis of the corresponding recordings from Wistar (C) and GK (D) beta -cells. Popen was assessed during 1-s frames. E: mean current after addition of RX871024; F: latency between addition of the compound and channel inhibition. Single-channel kinetics in both cell types was not altered in the presence of RX (data not shown). Representative traces out of 5 from 3 independent cell preparations.

Activity of voltage-gated Ca2+ channels was recorded in beta -cells from GK and Wistar rats by use of the whole-cell configuration. RX871024 did not show any effect on L- and T-type voltage-gated Ca2+ channels in beta -cells from either animal model (data not shown).

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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Fig. 6.   Effects of RX871024 on insulin secretion in perifused Wistar (open circle ) and GK () rat pancreatic islets in the presence of 3 (A) and 15 (B) mM glucose. Time of additions of compounds is shown by arrows. Data are means ± SE for 4 independent islet preparations.

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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Fig. 7.   Effects of RX871024 on insulin secretion in Wistar (open bars) and GK (filled bars) rat electropermeabilized pancreatic islets. Data are means ± SE for 20 observations from 4 independent islet preparations. *P < 0.05, **P < 0.01, ***P < 0.001 vs. insulin secretion at 10 µM Ca2+.

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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Fig. 8.   Effects of RX871024 on glucose utilization (open bars) and glucose oxidation (filled bars) in Wistar (A) and GK (B) rat pancreatic islets. Data are means ± SE for 5 independent islet preparations.

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 beta -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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Fig. 9.   Effects of 50 µM RX871024 on diacylglycerol (DAG) concentration in Wistar (open bars) and GK (filled bars) rat islets. Data are means ± SE in arbitrary units for 9 independent islet preparations. *P < 0.05, ***P < 0.001 vs. DAG concentration under basal conditions in Wistar rat islets. dagger P < 0.05 vs. DAG concentration at 50 µM RX871024 in Wistar rat islets.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -cells from GK and Wistar rats has shown a faster [Ca2+]i response to RX871024 stimulation in diabetic compared with nondiabetic beta -cells. The faster [Ca2+]i response to RX871024 in GK rat beta -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 beta -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 282(1):E117-E124
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