Roles of sulfonylurea receptor 1 and multidrug resistance protein 1 in modulating insulin secretion in human insulinoma

Cheng-Jiang Li, Hua-Li Zhou, Jun Li, Hong-Tian Yao, Rong Su and Wen-Peng Li

Hangzhou, China

Original Article / Pancreas

Roles of sulfonylurea receptor 1 and multidrug resistance protein 1 in modulating insulin secretion in human insulinoma

Cheng-Jiang Li, Hua-Li Zhou, Jun Li, Hong-Tian Yao, Rong Su and Wen-Peng Li

Hangzhou, China

BACKGROUND:Sulfonylurea receptor 1 (SUR1) and multidrug resistance protein 1 (MRP1) are two prominent members of multidrug resistance proteins associated with insulin secretion. The aims of this study were to investigate their expression in insulinomas and their sole and synergistic effects in modulating abnormal insulin secretion.

METHODS:Fasting glucose, insulin and C-peptide were measured in 11 insulinoma patients and 11 healthy controls. Prolonged oral glucose tolerance tests were performed in 6 insulinoma patients. Insulin content, SUR1 and MRP1 were detected in 11 insulinoma patients by immunohistochemistry. SUR1 and MRP1 were also detected in 6 insulinoma patients by immunofluorescence.

RESULTS:Insulinoma patients presented the typical demonstrations of Whipple's triad. Fasting glucose of each insulinoma patient was lower than 2.8 mmol/L, and simultaneous insulin and C-peptide were increased in insulinoma patients. Prolonged oral glucose tolerance tests showed that insulin secretion in insulinoma patients were also stimulated by high glucose. Immunohistochemistry and immunofluorescence staining showed that SUR1 increased, but MRP1 decreased in insulinoma compared with the adjacent islets.

CONCLUSIONS:The hypersecretion of insulin in insulinomas might be, at least partially, due to the enrichment of SUR1. In contrast, MRP1, which is down-regulated in insulinomas, might reflect a negative feedback in insulin secretion.

(Hepatobiliary Pancreat Dis Int 2011; 10: 88-94)

sulfonylurea receptor 1; multidrug resistance protein 1; ATP-binding cassette transporters; insulinoma; insulin secretion

Introduction

Insulinomas are rare neuroendocrine tumors of pancreatic islet cells that can produce and secrete insulin. In contrast to normally differentiated betacells, insulinoma cells continue to secrete insulin and proinsulin at low blood glucose without fine regulation,[1]which results in hypoglycemia clinically. ATP-sensitive potassium (KATP) channels play crucial roles in insulin secretion. Sulfonylurea receptor 1 (SUR1) and multidrug resistance protein 1 (MRP1) are two prominent members of ATP-binding cassette transporters, which are associated with multidrug resistance in diabetes mellitus.[2]They are associated with insulin secretion via modulating KATP channels.[2,3]However, the underlying mechanisms of SUR1 and MRP1 in the autonomous insulin hypersecretion by insulinoma remain to be clarified.

SUR1 (ATP-binding cassette transporter C8, ABCC8) is a subunit of the pancreatic beta-cell KATP channels and plays a key role in the regulation of glucose-induced insulin secretion.[2-4]Mutations of SUR1 are associated with persistent hyperinsulinemic hypoglycemia of infancy as well as type 2 diabetes of adults.[5-7]MRP1 (ATP-binding cassette transporter C1, ABCC1) is also a regulator of insulin secretion related to KATP channel.[2,3,8]However, the role of SUR1 and MRP1 in insulin secretion by insulinoma, and whether they might influence insulin secretion synergistically warrant further investigation. The aim of this study was, therefore, to investigate the alteration of these two important multidrug resistance proteins in insulinoma of human subjects, which might help to understand theunderlying mechanisms whereby insulin secretion by insulinoma is unexhausted.

Methods

Recruitment of subjects

Eleven cases of insulinoma inpatients were recruited from Department of Endocrinology at the First Affiliated Hospital of Zhejiang University School of Medicine. The diagnosis of insulinoma was made according to the following criteria: the demonstration of Whipple's triad (i.e., biochemical hypoglycemia <2.8 mmol/L, symptoms consistent with hypoglycemia and reversal with carbohydrate replacement) and inadequately suppressed insulin levels (>3 μIU/ml). Computed tomography or magnetic resonance imaging was performed for the localization of insulinomas before operation. There were no related familial history, and the patients with insulinomas were also considered sporadic. Prolonged oral glucose tolerance tests (OGTTs) were performed in 6 insulinoma patients who could be intolerant to fasting for 8-10 hours. Blood samples were taken after an overnight fast. Fasting glucose was determined on a HITACH 7170 bio-chemicalautoanalyzer (Tokyo, Japan). Insulin and C-peptide were measured by direct chemiluminescence immunoassay using commercially available kits on the ADVIA Centaur®XP Immunoassay System (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA). Insulin release index was calculated according to the following formula: insulin (μIU/ml)/glucose (mg/dl). Fasting glucose, insulin and C-peptide were also measured in 11 age- and gender-matched healthy volunteers recruited from the community.

Immunohistochemistry staining

The patients were subjected to surgical operations, and the samples of insulinomas were fixed in 4% formalin and embedded in paraffin. Hematoxylin-eosin staining and immunohistochemistry for insulin, SUR1 and MRP1 were conducted in 11 patients. Sections were generally pretreated and incubated with the respective primary antibody at room temperature for 2 hours. The primary antibodies of mouse monoclonal anti-insulin (Fitzgerald Industries International, Concord, MA, USA), rabbit polyclonal anti-SUR1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit polyclonal anti-MRP1 (ABBIOTEC, San Diego, CA, USA) were diluted with Trisbuffered saline (TBS). The sections were then incubated with anti-mouse or anti-rabbit ENVISIONTM+System, HRP Labeled Polymer (DAKO, Carpinteria, CA, USA) as secondary antibody at room temperature for 30 minutes. Between all steps, the sections were rinsed three times with TBS. Diaminobenzidine (Sigma Chemical Co., Louis, MO, USA) was used for color development and hematoxylin was used to counterstain the specimen. The specificity of immunohistochemistry was confirmed by omitting the primary antibodies. Quantitative analysis was made using Image-Pro® Plus, and the results were expressed as the integrated option density within unit area.

Immunofluorescence staining

The fresh tissues of 6 insulinoma patients were stored at -80 ℃ for immunofluorescence staining. The cryosections of insulinoma and the adjacent normal pancreatic tissues (10 μm-thick) were fixed for 10 minutes in acetone at 4 ℃. The sections were then washed in phosphate buffered saline (PBS) three times for 15 minutes and blocked for 1 hour in PBS with 5% bovine serum albumin (BSA). The sections were incubated overnight at 4 ℃ in the respective primary antibody (rabbit polyclonal anti-SUR1 and anti-MRP1), and recovered to the room temperature. After 3 washes in PBS the sections were then incubated for 1 hour at room temperature with a diluted goat anti-rabbit Alexa Fluor 555-conjugated IgG (Jackson ImmunoResearch, West Grove, PA, USA). After 3 washes in PBS, several drops of 1∶1000 diluted DAPI (BIOMOL Research Laboratories, Plymouth, USA) were added to the sections to stain the cell nuclei blue. The stained sections were imaged on an Olympus IX81 epifluorescence microscope (Tokyo, Japan) using 555 nm illumination for Alexa Fluor and 390 nm illumination for DAPI. One section was used as a negative control (no primary antibody). Quantitative analysis was made using Image-Pro® Plus, and the results were expressed as the integrated option density within unit area.

Results

Clinical features

The clinical features of the subjects are shown in Table 1.Age and gender were matched between the healthy controls and insulinoma patients. The chief complaints ofinsulinoma patients were consistent with hypoglycemia symptoms such as dizziness, anxiety, tremulousness, pallor, palpitations, sweating, nausea, hunger, weakness, drowsiness, fatigue, visual disturbance, confusion, seizures and coma (Table 2), and they could be reversed immediately by the intake of sugars. Fasting glucose of each insulinoma patient was lower than 2.8 mmol/L, simultaneous insulin and C-peptide were much higher in the insulinoma patients, and as a result, insulin release index was significantly higher in the insulinoma patients compared with the healthy controls. Prolonged OGTT showed that insulin secretion in the insulinoma patients was also stimulated by high glucose level (Fig. 1).

Table 1. The clinical features of the subjects

Table 2. The clinical manifestations of the insulinoma patients

Fig. 1. Prolonged OGTT performed in insulinoma patients. Prolonged OGTT performed in insulinoma patients who could be intolerant of fasting for 8-10 hours. After fasting for 8-10 hours, 75 g glucose was taken orally, and blood was withdrawn at various time points. Blood glucose, insulin and C-peptide were measured. Data were expressed as mean±SEM (n=6).

General pathological findings

Fig. 2. Morphology and insulin content of insulinomas (original magnification ×200). Hematoxylin-eosin shows that outside the lesion of insulinoma, islets have a normal appearance (A). For those islets in the insulinoma patients with a long duration, the islets became small and irregular (C). In insulinomas, the normal pancreatic tissue disappears, and tumoral endocrine cells are organized in nests scattered throughout a fibro-vascular stroma (E). Immunohistochemistry for insulin shows that insulinomas are almost exclusively composed of β-cells, with a weak and variable insulin staining (F). Outside the lesion, insulin staining is much more intense within the islets (B and D).

Fig. 3. SUR1 and MRP1 measured by immunohistochemistry staining. The staining for SUR1 is homogeneous in the whole cytoplasm of endocrine cells, it is stronger in insulinoma (B, original magnification ×200) compared with the adjacent islets (A, original magnification ×200). The staining for MRP1 is also homogeneous in the whole cytoplasm of endocrine cells, it is weaker in insulinoma (D, original magnification ×100) compared with the adjacent islets (C, original magnification ×100). Quantitative analysis is made (E), data are expressed as mean± SEM (n=11), paired t test was performed. *: P<0.05, compared with the adjacent normal islets.

Insulinomas by hematoxylin-eosin staining showed the disappearance of the normal pancreatic tissue (Fig. 2E). Tumoral endocrine cells were organized in nests scattered throughout a fibro-vascular stroma. There were several islets outside the lesion, which had a normal appearance (Fig. 2A). For those with a long duration of the disease, the adjacent normal islets became small and irregular (Fig. 2C). Immunohistochemistry for insulin showed that insulinomas were almost exclusively composed of beta-cells, with a weak and variable insulin staining (Fig. 2F). Outside the lesion, insulin staining was much more intense within the adjacent islets (Fig. 2B and D).

Expression of SUR1 and MRP1

Fig. 4. SUR1, MRP1 measured by immunofluorescence staining (A-D, original magnification ×100). The staining for SUR1 is homogeneous in the whole cytoplasm of endocrine cells, this staining is stronger in insulinoma (B) compared with the adjacent islets (A). The staining for MRP1 is homogeneous in the whole cytoplasm of endocrine cells, this staining is weaker in insulinoma (D) compared with the adjacent islets (C). Red indicates fluorescence of Alexa Fluor 555, and blue indicates staining the cell nuclei by DAPI. Quantitative analysis is made (E), data are expressed as mean±SEM (n=6), paired t test was performed; *: P<0.05, compared with the adjacent normal islets.

Immunohistochemistry showed that SUR1 expression significantly increased in insulinomas compared with the adjacent islets (Fig. 3A and B), which was also confirmed by immunofluorescence staining (Fig. 4A and B). In contrast, MRP1 expression significantly decreased in insulinomas compared with the adjacent islets (Fig. 3C and D), which was also confirmed by immunofluorescence staining (Fig. 4C and D). The quantitative analysis (Fig. 3E and 4E) showed that the alteration of SUR1 and MRP1 was statistically significant (P<0.05).

Discussion

Insulinoma presents a variety of hypoglycemia symptoms including adrenergic, cholinergic and neuroglycopenic,[1]and they are easily mistaken as cardiovascular disorders, hysteria or neurological deficits. Hence there are missed cases frequently, and in this study, the longest duration of the disease was 10 years. Because recurrenthypoglycemia may lead to brain damage, it has crucial clinical relevance for the early diagnosis and treatment of insulinomas. In the present study, about two-thirds of the tumors were larger than 2 cm in diameter. However it was difficult to be localize small tumors less than 2 cm in diameter, and blind distal pancreatomies were not recommended.[1]Insulin levels were inadequately suppressed in the insulinoma patients at fasting status; however, prolonged OGTT tests showed that the insulin peak time and insulin increase were within normal ranges. It is of great interest to explore the underlying mechanisms of insulin hypersecretion by insulinoma, which might help to find interesting aspects in the timely diagnosis of insulinoma or to develop a drug for insulinomas that fail to be localized.

SUR1 is an important MRP that is associated with abnormal insulin secretion. The normal insulin secretion by beta-cells is controlled by a KATP channel, which is a hetero-octameric complex composed of four SUR1 subunits and four Kir6.2 subunits.[9-13]SUR1 plays a crucial role in modulating insulin secretion, and the mutations of SUR1 have been found to be associated with various conditions of inappropriate insulin secretion.[14-16]In this study, we found that SUR1 expression increased in insulinomas compared with the adjacent islets, which was consistent with the previous findings by Sempoux et al.[7]These data suggested that the hypersecretion of insulin by insulinoma might be, at least partially, due to the overexpression of SUR1.

SUR1 is also the best known candidate gene of the congenital persistent hyperinsulinemic hypoglycemia of infancy.[17-21]Strikingly, the alteration of SUR1 is not only responsible for the oversecretion of insulin, but also associated with insufficient insulin secretion. Recently, SUR1 mutations have also to be linked to the pathogenesis of adult diabetes[22-24]or neonatal diabetes.[25-29]The distinct SUR1 peptides may, therefore, plays unique roles in modulating insulin secretion.[30-32]SUR1 have been the targets of several important anti-diabetic drugs, especially sulphanylureas including glimepiride,[33-35]and the effective treatment with sulfonylureas in diabetic patients has been associated with SUR1 mutations.[36]Accordingly, further investigation on the underlying mechanisms of unexhausted insulin secretion mediated by SUR1 and other functional proteins might contribute to setting novel targets to treat the abnormal secretion of insulin such as insulinomas or diabetes millitus.

MRP1 is another MRP, which has also been identified in human pancreatic beta-cells, and associated with insulin secretion.[2,3]Since MRP1 serves as the regulator of Kir6.2 protein, the essential composition of KATP channel for insulin secretion, MRP1 and SUR1 may play a synergistic effect in inducing insulin secretion.[2,8]Previously it was unclear about the alteration of MRP1 in insulinoma of human subjects. In contrast to SUR1, MRP1 expression was decreased in insulinomas compared with the adjacent islets, suggesting that MRP1 did not play a crucial role in mediating the high insulin secretion by insulinomas.

The down-regulation of MRP1 in insulinomas might reflect an adaptive mechanism controlling the excessive secretion of insulin due to the enriched SUR1. It is not feasible to obtain normal tissues of islets from healthy volunteers, so it was uncertain in our study whether the expression of MRP1 in the adjacent normal islets was significantly altered in the insulinoma patients. However, MRP1 did express well in the adjacent normal islets. Thus the response of MRP1 in the loop of negative feedback seemed to be limited in the local over-secretary tumor cells. However, this feedback was obviously very limited to prevent hypoglycemia in these insulinoma patients. Dysregulation of insulin secretion in children with congenital hyperinsulinism might shift to the late development of diabetes;[5,37,38]however, insulin secretion by insulinomas was progressively aggressive and seemed to be never exhausted. Further investigation in vitro or in animal models is needed to be focused on how these multidrug resistance proteins or other criminal proteins modulate insulin secretion, which might be helpful to the diagnosis and treatment of insulinomas. It is good for developing novel anti-diabetic target by using the characteristics of active insulin secretion by insulinomas.

In conclusion, SUR1 and MRP1 are two multidrug resistance proteins that are involved in a variety of abnormal insulin secretion conditions. The enrichment of SUR1 in insulinomas might, at least partially, account for the over secretion of insulin. In contrast, MRP1, which is down-regulated, seems to be involved in rescuing the hypernomic insulin secretion, but insufficiently. Functional studies of these proteins might provide insights into the physiological and pathophysiological changes in insulinoma, which might help to treat insulinomas by efficiently inhibiting the high secretion, or to set up new targets to treat diabetes by using the trait of unexhausted secretion if it could be regulated appropriately.

Acknowledgements

We acknowledge the following colleagues at the First Affiliated Hospital, Zhejiang University School of Medicine for their generous help in completing this study: Dr. Ming-Zhi Xu for her help and suggestions. Dr. Hai-Yang Xie from Key Laboratory of Multi-organ Transplantation of Ministry of Public Health for his technical assistant, Dr. Shao-Hua Shi, Hua Guo, Xiao-FengTang from Department of Hepatobiliary and Pancreatic Surgery for collecting the insulinoma samples.

Funding:None.

Ethical approval:Not needed.

Contributors:ZHL proposed the study. LCJ and ZHL wrote the first draft. ZHL analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. ZHL is the guarantor.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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September 30, 2010

Accepted after revision December 31, 2010

Author Affiliations: Department of Endocrinology (Li CJ, Zhou HL and Li WP), Department of Pathology (Li J and Yao HT), and Key Laboratory of Multi-organ Transplantation of Ministry of Public Health (Su R), First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China

Hua-Li Zhou, MD, Department of Endocrinology, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Tel: 86-571-87236866; Fax: 86-571-87236874; Email: lhlzhou@ hotmail.com)

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