Background  A 65-kD mdr1 (multi-drug resistance protein 1, P-glycoprotein)-like protein has been suggested to be the regulatory protein to the chloride channel protein 3 (ClC-3) mediating insulin granules acidification and release in mouse pancreatic beta cells. But the protein has not been deeply investigated. In this study, we identified existence of the 65-kda protein in rat islets and preliminarily explored its biological functions.
Methods  Total RNAs of rat kidneys served as positive controls, and pancreas, islets and INS-1 cells were extracted for reverse-transcript PCR (RT-PCR), respectively. The cDNAs were run with specific primers selected from the mRNA of abcb1b encoding P-glycoprotein. All PCR products were visualized in agarose gel electrophoresis and sequenced. Homogenates of rat islets and INS-1 cells were applied to SDS-PAGE. P-glycoprotein was detected by a specific monoclonal antibody, C219. Biphasic insulin release was measured in static incubations of rat islets with radioimmunology assay.
Results  Compared with positive control, expression of the P-glycoprotein mRNA segments were detected in the islets, INS-1 cells and pancreas. Sequence analysis confirmed that the PCR products were matched with mRNA of P-glycoprotein. A 65-kda protein was recognized by the antibody in the islets homogenate but not in that of INS-1 cells in Western-blotting. Instead, the homogenate of INS-1 cells contained a 160-kda protein recognized by the antibody. Insulin secretion of rat islets were stimulated by high glucose (16.7mmol/L), and showed biphasic curve during 60-minute incubation. After co-incubation with cyclosporine A (CsA), specific inhibitor to P-glycoprotein, the second phase of insulin secretion was reduced significantly while the first phase was not influenced.
Conclusions  The 65-kda protein expressed in rat islets is most likely a mini-P-glycoprotein. It may play a key role regulating biphasic insulin release.

·LogIn/LogOut

·Fulltext PDF(283K) Free

·Abstract download TXT | XML

·Articles in CMJ by TANG Yun-zhao LI Dai-qing

·Articles in PubMed by TANG YZ LI DQ

·Put into my bookshelf

·Email to Friend

·Email to author

·Visit:1619

·Download:813

·Advanced Search

·Related Articles

·Change font size:

·Cannot read some characters

Biphasic insulin release is observed not only in vivo, but also in isolated pancreatic islets and single beta cells.¹ Impairment of pancreatic beta cell secreting insulin is one of the fundamental pathogenesis in type 2 diabetes mellitus (2DM). The first phase of secretion is contributed by a small pool of granules docked on the plasma-membrane, termed as readily releasable pool; the granular membrane are fused with the plasma membrane and the cargo are released under certain stimulations, such as high blood glucose. By contrast, second-phase secretion involves the mobilization of intracellular granules to the plasma membrane to enable the distal docking and fusion steps of insulin exocytosis. The second phase depends on intragranular acidification due to the concerted action of granular V-type H⁺-ATPase and ClC-3 Cl^- channels.^2-4

Typically, ATP-binding cassette (ABC) transport proteins are highly conserved and mediating translocation of molecules across membranes, from nutrient import to toxin efflux motored by ATP hydrolysis. On the other hand, some of ABC proteins are atypical that mediate other cellular processes. Among them, the sulfonylurea receptor (SUR) is one that regulates activity of the potassium ATP channel, and particularly important in the regulation of insulin secretion from pancreatic beta cells.⁵ P-glycoprotein has been considered as a drug efflux pump resulting in the multidrug resistance of tumor cells for a long time.⁶ But recently it has been reported that a P-glycoprotein-like protein, termed as 65-kD mdr1-like protein (65-kD protein), was detected in mouse insulin granular membranes. With the patch clamp technique, it has been found that the protein could be functioned as sulfonylurea receptor and regulates insulin release. To be hypothesized, the 65-kda protein might assist the ClC-3 channel protein mediating acidification of insulin granules, similar as pancreatic zymogen granules.^2,7,8 In this study, we began with the detection of P-glycoprotein in mRNA and protein levels in rat pancreatic beta cells. Furthermore, we preliminarily investigated its role in biphasic insulin secretion.

Animals
Male Wistar rats (250–300 g) were purchased from Tianjin Medical University Animal Center. They were fed with a standard pellet diet and tap water ad libitum before islet isolation. The animal experiments had been proved by animal ethical committee in Tianjin Medical University.

Drugs and chemicals
Collagenase V, CsA and bovine serum albumin (BSA) were purchased from Sigma Chemical Co (USA) and TRIZOL reagents were from Invitrogen (USA). Reverse transcription kit Taq DNA Polymeras were purchased from TaKaRa Bio (Japan). The monoclonal anti-P-glycoprotin antibody (C219) was obtained from Abcom (UK), and anti-beta-actin antibody was from Santa Cruz (USA). RIPA lysate buffer and BCA protein assay kit were purchased from Beyotime (China). Reagents for westernblot were from Bio-Rad (USA). The radioimmunology assay kits were from Linco (USA).

Islet isolation
The animal was sacrificed by CO₂. Abdominal cavity was opened to expose the pancreas and hepatopancreatic duct. The Vater′s papilla was detected and the hepatopancreatic duct on the side of duodenum was ligated with silk. The other end of the duct, near the hepatic portal, was ligated as well. A scalp acupuncture (outer diameter 0.45 mm), which was linked with a syringe containing collagenase type V solution (1 mg/ml in Hanks buffer, pH 7.4), was used to insert the hepatopancreatic duct from the side of hepatic portal. After the pancreas was expanded completely, it was taken out immediately and put into a 50 ml centrifuge tube．The digestion step was carried out in a water bath at 37°C for 23–25 minutes．The digestion was stopped by adding ice-cold Hanks buffer and stood in ice to wait sand-shaped tissues deposited on the bottom of the centrifuge tube.⁹ After three-time washes, the islets were manually picked up under stereomicroscope (S8APO, LEICA, German).

Islet viability
Islet viability was tested by simultaneous staining with acridine orange (AO) and propidium iodide (PI) under fluorescence microscope (DMI 4000B, LEICA, German). Viable cells fluoresced green from AO, and the red fluorescence from PI emanated from the nuclei of injured and dead cells.

Cell culture
Insulinoma cell line (INS-1, passage 90) was purchased from China Center for Type Culture (CCTCC, Wuhan, China). All reagents for cell culture were from GIBCO (USA). Cells were grown in RPMI 1640 containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 10 µmol/L mercaptoethanol in 5% CO₂ atmosphere at 37°C.

view in a new window

Table 1. The primers for abcb1b mRNA detection

Western blotting
Proteins from INS-1 cells and islets were extracted by RIPA lysate buffer containing 1mmol/L PMSF. The protein concentrations were determined by BCA protein assay kit. Proteins 20 µg were applied to SDS-PAGE. The trans-blotted PVDF membranes were blocked by 5% dried skim milk at room temperature for one hour with shaking. After 3 times washes with TBST (150 mmol/L NaCl, 20 mmol/L Tris-HCl, 0.05% Tween 20), the membranes were incubated with anti-beta-actin antibody (1:100) and C219 (1:20) overnight at 4°C, respectively. They were incubated with the second antibody 2 hours at room temperature after TBST washes. The bands on the membranes were visualized with HRP chemolumine- scence reagents in dark room on X-ray films (Kodak, USA).

Insulin secretion experiment
Rat islets were incubated with RPMI1640 medium (containing 10% FBS, without antibiotics) for 6 hours, then the islets were pre-incubated for 30 minutes at 37°C in Krebs-Ringer bicarbonate buffer (pH 7.4) supplemented with 10 mmol/L HEPES, 0.1% BSA, and 3.3 mmol/L glucose. After pre-incubation for 30 minutes, the buffer was changed to the one of the same composition supplemented with the glucose of two different concentration (3.3 mmol/L and 16.7 mmol/L), and the islets were incubated for 1, 5, 10, 15, 20, 30 and 60 minutes, respectively. Each incubation vial was gassed with 95% O₂ and 5% CO₂ to obtain constant pH and oxygenation. All incubations were performed at 37°C in a water bath with shaking (100/min). After incubation, an aliquot of the medium was removed for assay of insulin. The result was calculated as secretion of insulin at different time. The experiment was repeated six times with duplicated samples.

Inhibiting insulin secretion by CsA
Islets were incubated with RPMI1640 medium (containing 10% FBS, without antibiotics) containing 1 mg/L CsA for 6 hours.¹⁰ Then the following steps were the same to the insulin secretion experiments.

Statistical analyses
The statistical analysis was performed with SPSS11.0 statistical soft ware. The measurement data were expressed of mean ± standard error (SE). Normal distribution was tested by F test. The t test (unpaired, two tailed) was used for two-group comparison. P <0.05 was considered significant.

Islet survival
Majority of the isolated islets were stained by acridine orange and illustrated as green mass under florescence microscope. Few cells on the surface of the islets were stained by propidium iodide presenting red color (data not shown).

P-glycoprotein mRNA expression
Total RNAs of rat kidneys, islets, pancreas and INS-1 cells were analyzed by reverse-transcript PCR (RT-PCR). Three pairs of primers were designed specifically to the P-glycoprotein coding gene — abcb1b (Table 1). The targeted cDNA segments were localized in the translation region of abcb1b and covered 210 base pairs in the N-terminus and 243 base pairs in the C-terminus. Compared to kidney which has been identified to express P-glycoprotein in high level, islets, pancreas and INS-1 cells were all expressed the abcb1b gene. The expression level of islet was stronger than those of pancreas and INS cells (Figures 1 and 2). All the targeted cDNAs were analyzed by DNA sequencer and showed more than 99% homologues with abcb1b accordingly (Table 2).

view in a new window

Figure 1. PCR products electropherogram. PCR products electropherogram. The primers were MDR1① (MDR1 cDNA 3522 bp–3676 bp, C terminal). Lane 1: kidney; Lane 2: islets; Lane 3: INS-1 cells; Lane 4: pancreas; Lane 5: negative control, the primers were MDR1② (MDR1 cDNA 3434 bp－3537 bp, C terminal); Lane 6: kidney; Lane 7: islets; Lane 8: INS-1 cells; Lane 9: pancreas; Lane 10: negative control: the primers were beta-actin; Lane 11: kidney; Lane 12: islets; Lane 13: INS-1 cells; Lane 14: pancreas; Lane 15: negative control; Lane M: molecular weight.

view in a new window

Figure 2. PCR products electropherogram. PCR products electropherogram. The primers were MDR1③ (MDR1 cDNA 782 bp–991 bp, N terminal). Lane 1: kidney; Lane 2: islets; Lane 3: INS-1 cells; Lane 4: pancreas; Lane 5: negative control; the primer was beta-actin; Lane 6: kidney; Lane 7: islets; Lane 8: INS-1 cells; Lane 9: pancreas; Lane 10: negative control; Lane M: molecular weight.

view in a new window

Table 2. The sequences of the PCR products

P-glycoprotine detection
Totally 1000 rat islets and 10⁶ INS-1 cells were collected, protected by proteinase inhibiters and homogenated for protein detection. A 65 kD protein was detected in the homogenate of islets, but not in the homogenate of INS-1 cells. Instead, a 160 kD protein was recognized by the antibody in the homogenate of INS-1 cells. The 42 kD protein detected with polyclonal anti-beta-actin antibody was served as loading control (Figure 3).

view in a new window

Figure 3. Western-blotting results. Proteins were extracted by RIPA lysate buffer containing 1mmol/L PMSF, and electrophoresied on SDS-PAGE system. After tansblotting, PVDF membranes were incubated with A: anti-P-glycoprotein antibody C219 and B: anti-beta-actin antibody. Molecular weights of the visualized bands were indicated on the left.

Insulin secretion and CsA inhibition tests
We estimated the dynamics of islets (with and without the pre-treatment of CsA) by measuring insulin secretion from islets during 1, 5, 10, 15, 20, 30 and 60 minutes of incubation in low (3.3 mmol/L) or high glucose (16.7 mmol/L) conditions and calculated the net insulin release. This provided an indication of the time course of insulin secretion. Data obtained by this protocol resemble those obtained by pancreas perfusion.^11,12 In the islets which were not pre-treated by CsA, insulin secretion in the presence of 16.7 mmol/L glucose peaked after 5 minutes (first phase) and then proceeded at a much lower rate (20%–25% of the peak; second phase). In the islets pre-treated by CsA (6 hours), the amplitude of first-phase release was not significantly reduced, but second phase secretion was reduced by 50%–60% (P <0.05; Figure 4).

view in a new window

Figure 4. The dynamics of insulin secretion: the two figures showed the biphasic insulin secretion when use the 16.7mmol/L glucose to stimulate the islet (treated with or without the CsA), insulin secretion curve (and column) from 0 to10 minutes was the first phase of the biphasic insulin secretion, after 10 minutes it was the second phase of the biphasic insulin secretion.

In brief, the beta-cell granule pools can be described as reserve, morphologically docked, readily and immediately releasable. The latter three groups named as readily releasable pool (RRP). The recruitment of insulin granules from reserve pool to RRP reflects the second phase of insulin secretion. Type 2 DM characterized as prolonged duration of high blood glucose levels has abnormalities in biphasic insulin release. Relatively little is known about the mechanisms coupling rates of high glucose metabolism to the second phase, even though the second phase is disrupted in most forms of diabetes.¹³ Barg et al^2,14 hypothesized that the insulin granular acidification was connective intimately to the insulin secretion and contribute insulin granules recruitment (priming) depending on their findings. The V-type H⁺-ATPase in insulin granular membranes fluxes H⁺ into the granular. Granular Cl^- uptake through an ion channel complex comprising of ClC-3 Cl^- channels and their regulatory proteins neutralized the electric potential to promote acidification which is crucial step for insulin priming and secretion.

The prior study showed a 65-kda mdr1 gene product (detected by mdr1 specific antibody C219) activated the Cl- channel in the membrane of pancreatic acinar zymogen granules (ZG). They concluded that the 65-kda protein promoted the ZG to acidify and be mature.⁸ Barg et al^2,14 revealed that JSB1 and C219 (antibodies to P-glycoprotein) recognized a 65-kD protein in mouse insulin granules. Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated the insulin secretion. The stimulatory action could be antagonized by the JSB1.⁷ According to the insulin granular acidification, the granular 65-kD mdr1-like protein mediated an action which initiated culminate in the activation of a granular Cl^- conductance. They speculated that the protein should be as a Cl^- channels regulatory protein.² All the experiments indicate that a relatively small size P-glycoprotein (mini-P-glycoprotein) may exist in certain types of cells. Dupuis et al¹⁵ concluded saquinavir induced expression of the multi-drug transporter mini-P-glycoprotein in human CD4 T-lymphoblastoid CEM^rev cells. Oselin et al¹⁶ found that an alternatively spliced form of the full-length P-glycoprotein in peripheral blood lymphocytes. But neither of them discussed the functions of the mini-P-glycoprotein.

Few studies have been reported to detect abcb1b gene expression in pancreatic beta cells, but not clearly.¹⁷ In our study, the expression of abcb1b gene fragments in transcriptional level was found and confirmed in rat islets, pancreas and INS-1, respectively. The quantity of PCR products of islets was significantly higher than that of pancreas. This enrichment demonstrates that the abc1b1 gene expression is remarkable in endocrine tissues compared to exocrine tissues, which have been reported to express abcb1b gene.⁸ The beta cells can be reasonable identified to express the gene combined with the results from INS-1 cells since they are 80 percent of the islet cells. A 65kDa P-glycoprotein like protein was detected in islets showed in western-blotting indicates translation of the abcb1b gene. The abcb1b gene expressed in rat islets and the product of the gene was most likely a mini-P-glycoprotein (truncated). On the other hand, the full length P-glycoprotein existed in INS-1 cells. The structure differences of the P-glycoprotein between the normal cells and the tumor cells may imply the diversity of biological functions.

The manually isolated islets have shown normal dynamics in insulin release which presented typical biphasic insulin secretion curve. After the islets were treated by P-glycoprotein inhibitor, CsA, the first phase of insulin secretion showed no change, but the second phase was impaired. This result is similar to the prior study, which performed in patients with hyper-glycaemic glucose clamp procedures.¹⁸ CsA affecting the insulin secretion was reported by several studies, but the underlying mechanisms have not been identified.^10,19,20 We presume that it could antagonize the functions of P-glycoprotein or mini-P-glycoprotein, thus inhibit the insulin granular acidification, which in turn impair the priming processes. The priming steps are response to the second phase of insulin secretion.²

Type 2 DM is a complex disease characterized by insulin resistance and a progressive decline in beta-cell function and mass. Current evidences suggest that beta-cell dysfunction is present early in the course of the disease. Furthermore, this dysfunction, rather than insulin resistance, is primarily responsible for the progression of 2DM. Beta-cell dysfunction can be accelerated by glucose toxicity, lipotoxicity, oxidative stress, chronic increases in inflammatory mediators and, potentially, the use of sulfonylureas.^21,22 Bacha et al²³ found that impaired glucose tolerance (IGT, pre-diabetes) had evidence of a beta-cell defect manifested in impaired first-phase insulin secretion and also declared 2DM had a more pronounced defect in both the first-phase and the second-phase insulin secretion. The two phases are linked by insulin granules priming processes, including granular acidification, physical trafficking and many coupled metabolic factors. Granular ClC-3 channel protein assisted by the mini-P-glycoprotein has a key role regulating insulin granules acidification.^2-4,7 Dysfunction of the mini P-glycoprotein may induce immature granules which can not dock onto plasma membrane properly. This long term negative effect to the readily releasable pool of insulin granules finally becomes one of the main causes to result the first phase vanished. Furthermore, the late stage of 2DM often shows absolute insufficiency of insulin secretion in the second phase.

In sum of the study, the mini-P-glycoprotein expressed in pancreatic beta cells may regulate biphasic insulin secretion, in particular second phase. Further experiments on the gene silencing and patch clamp studies will be done to elucidate the molecular mechanisms.

Acknowledgements: This study was supported by EFSD Grant Award for Collaborative Diabetes Research between China and Europe supported by Bristol-Myers-Squibb to LI Dai-qing and Tianjin Municipal Natural Science Foundation to LI Dai-qing (No. 08JCZDJC25100).

1. Barg S, Lindqvist A, Obermuller S. Granule docking and cargo release in pancreatic beta-cells. Biochem Soc Trans 2008; 36: 294-299.

2. Barg S, Huang P, Eliasson L, Nelson DJ, Obermuller S, Rorsman P, et al. Priming of insulin granules for exocytosis by granular Cl^- uptake and acidification. J Cell Sci 2001; 114: 2145-2154.

3. Li DQ, Jing X, Salehi A, Collins SC, Hoppa MB, Rosengren AH, et al. Suppression of sulfonylurea- and glucose-induced insulin secretion in vitro and in vivo in mice lacking the chloride transport protein ClC-3. Cell Metab 2009; 10: 309-315.

4. Deriv LV, Gomez EA, Jacobson DA, Wang X, Hopson JA, Liu XY, et al. The granular chloride channel ClC-3 is permissive for insulin secretion. Cell Metab 2009; 10: 316-323.

5. Burke MA, Mutharasan RK, Ardehali H. The sulfonylurea receptor, an atypical ATP-binding cassette protein, and its regulation of the KATP channel. Circ Res 2008; 102: 164-176.

6. Rees DC, Johnson E, Lewinson O. ABC transporters: the power to change. Nature Reviews Molecular Cell Bio 2009; 10: 218-227.

7. Barg S, Renström E, Berggren PO, Bertorello A, Bokvist K, Braun M, et al. The stimulatory action of tolbutamide on Ca²⁺-dependent exocytosis in pancreatic b cells is mediated by a 65-kDa mdr-like P-glycoprotein. Proc Natl Acad Sci 1999; 96: 5539-5544.

8. Thévenod F. Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins. Am J Physiol Cell Physiol 2002; 283: C651-C672.

9. Macgregor RR, Williams SJ, Tong PY, Kover K, Moore WV, Stehno-Bittel L. Small rat islets are superior to large islets in in vitro function and in transplantation outcomes. Am J Physiol Endocrinol Metab 2006; 290: e771-e779.

10. Ajabnoor MA, EI-Naggar MM, Elayat AA, Abdulrafee A. Functional and morphological study of cultured pancreatic islets treated with cyclosporine. Life Sci 2007; 80: 345-355.

11. Jing X, Li DQ, Charlotta S, Salehi A, Surve VV, Caballero J, et al. CaV2.3 calcium channels control second-phase insulin release. J Clin Invest 2005; 115: 146-154 .

12. Schulla V, Renström E, Feil R, Feil S, Franklin I, Gjinovci A, et al. Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca²⁺ channel null mice. EMBO J 2003; 22: 3844-3854.

13. Geng XH, Li LH, Bottino R, Balamurugan AN, Bertera S, Densmore E, et al. Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATP channels. Am J Physiol Endocrinol Metab 2007; 293: e293-e301.

14. Speidel D, Salehi A, Obermulleller S, Lundquist I, Brose N, Renstrom E, et al. CAPS1 and CAPS2 regulate stability and recruitment of insulin granules in mouse pancreatic beta cells. Cell Metab 2008; 7: 57-67.

15. Dupuis ML, Flego M, Molinari A, Cianfriglia M. Saquinavir induces stable and functional expression of the multidrug transporter P-glycoprotein in human CD4 T-lymphoblastoid CEMrev cells. HIV Med 2003; 4: 338-345.

16. Oselin K, Nowakowski-Gashaw I, Mrozikiewicz PM, Wolbergs D, Pähkla R, Roots I. Quantitative determination of MDR1 mRNA expression in peripheral blood lymphocytes: a possible role of genetic polymorphisms in the MDR1 gene. Eur J Clin Invest 2003; 33: 261-267.

17. Bani D, Brandi ML, Axiotis CA, Bani-Sacchi T. Detection of P-glycoprotein on endothelial and endocrine cells of the human pancreatic islets by C494 monoclonal antibody. Histochemistry 1992; 98: 207-209.

18. Hjelmasaeth J, Hagen LT, Asberg A, Midtvedt K, Storset O, Halvorsen CE, et al. The impact of short-term ciclosporin A treatment on insulin secretion and insulin sensitivity in man. Nephrol Dial Transplant 2007; 22: 1743-1749.

19. Song HK, Han DH, Song JH, Ghee JY, Piao SG, Kim SH, et al. Influence of sirolimus on cyclosporine-induced pancreas islet dysfunction in rats. Am J Transpl 2009; 9: 2024-2033.

20. Penfornis A, Kury-Paulin S. Immunosuppressive drug-induced diabetes. Diabetes Metab 2006; 32: 539-546.

21. Tibaldi J. Preserving insulin secretion in type 2 diabetes mellitus. Expert review of endocrinology and metabolism 2008; 3: 147-159.

22. Guillausseau PJ, Meas T, Virally M, Laloi-Michelin M, Médeau V, Kevorkian JP. Abnormalities in insulin secretion in type 2 diabetes mellitus. Diabetes and Metabolism 2008; 34: s43-s48.

23. Bacha F, Gungor N, Lee S, Arslanian SA. In vivo insulin sensitivity and secretion in obese youth: what are the differences between normal glucose tolerance, impaired glucose tolerance, and type 2 diabetes? Diabetes Care 2009; 32: 100-105.