Original Article
The Presence of Human Chorionic Gonadotropin/Luteinizing Hormone Receptors in Pancreatic b-Cells
Reproductive Sciences 1-8 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719115570910 rs.sagepub.com
Jai Parkash, PhD1,2, Zhenmin Lei, MD, PhD3, and C. V. Rao, PhD4
Abstract We investigated the possible presence of functional human chorionic gonadotropin (hCG)/luteinizing hormone (LH) receptors in b-cells of pancreas, using a combination of techniques on hCG/LH receptor knockout mice, immortalized rat insulinoma cells, and human pancreatic islets. The results showed the presence of receptors and their activation resulted in a dose-dependent increase in glucose-induced release of insulin. These findings place hCG and LH among the regulators of insulin release with potential implications for insulin-level changes during the periods of altered hCG and LH secretion. Keywords hCG/LH receptors, b-cells of pancreas, colocalization, insulin release, glucose
Introduction Human placenta and anterior pituitary gland secrete human chorionic gonadotropin (hCG) and luteinizing hormone (LH), respectively.1 They are structural and functional homologous that bind to the same G-protein-coupled cell surface receptors in target cells.2,3 For a long time, gonadal cells were thought to be the only targets of hCG/LH actions. However, this has been changed by the data published from around the world during the last 25 years to include many nongonadal tissues.4 The activation of nongonadal receptors results in tissue and cell-specific effects.4 In view of these advances, we considered it possible due to seemingly close relationship between hCG/LH and insulin levels and the role of insulin in reproduction5-7 that b-cells of pancreas could contain hCG/LH receptors and their activation may influence glucoseinduced release of insulin. The present studies investigated these possibilities by using pancreas of hCG/LH receptor knockout mice, immortalized rat b-cells and human pancreatic islets. The results showed that b-cells of pancreas do contain hCG/LH receptors and their activation results in a dose-dependent increase in glucose-induced release of insulin. These findings should place hCG and LH among the regulators of insulin release, which would require the reevaluation of insulin level changes during the periods of altered hCG and LH secretion.
Materials and Methods hCG/LH Receptor Knockout Mice These animals have previously been generated by homologous DNA recombination technology and extensively characterized.8 Briefly, the homozygous animals exhibited expected phenotypes
from the lack of hCG/LH receptors in gonadal as well as in nongonadal tissues.8-13 Pancreas and ovaries were harvested for the present studies from adult females of all 3 genotypes and frozen until used.
Immortalized Pancreatic b-Cells and Human Pancreatic Islets Rat insulinoma cells (RINr 1046-38, abbreviated as RIN), which are insulin-producing b-cells, were kindly provided by Dr Bruce Chertow (previously at the Veterans Administration Medical Center, Huntington, West Virginia). Human islets, isolated from cadavers of nondiabetic human subjects, were obtained from the National Disease Research Interchange, Philadelphia, Pennsylvania. Exemption 4 approval was obtained from the Institutional Review Board of Florida International University for their use. 1 Department of Environmental and Occupational Health, Robert Stempel College of Public Health and Social Work, Florida International University, Miami, FL, USA 2 Keiser University, Fort Myers, FL, USA 3 Department of Obstetrics and Gynecology, University of Louisville Medical Center, Louisville, KY, USA 4 Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, USA
Corresponding Author: C. V. Rao, Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199, USA. Email: [email protected]
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Figure 1. Immunocytochemistry for hCG/LH receptors in pancreas of hCG/LH receptor knockout mice. White arrows point out islets. Mag 40. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
Cell and Islets Culture RIN cells were grown in 5% CO2 and 95% air incubator at 37 C in 6-well culture plates or in T 75 culture dishes in RPMI-1640 supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, and 100 mg/mL of streptomycin (Invitrogen Corporation, Grand Island, New York). The human islets were maintained under culture conditions in 6-well plates in a 5% CO2 incubator at 37 C in CMRL culture medium containing 15% fetal calf serum and 100 units/mL of penicillin and 100 mg/mL of streptomycin.
Reverse Transcription-Polymerase Chain Reaction RNA preparation. The total RNA was isolated from RIN cells using TRIzol reagent (Invitrogen, Carlsbad, California) and its concentrations determined by absorbance at 260 nm, using a Biotek Synergy HT 96-well plate reader. The purity was 2
checked by electrophoresis in 1.5% agarose gels. The reverse transcription was carried out using the kits and instructions from Applied Biosystems (Foster City, California ), BioRad CFX96 Real-TimeSystem (Hercules, California ), and SsoFast Probes Supermix (Bio-Rad). A total of 100 ng of complementary DNA (cDNA) was used per reaction. The polymerase chain reaction (PCR) sample consisted of 10 mL containing cDNA, 5 mL SsoFast EvaGreen Supermix (BioRad, cat #1725200), and 1 mL 200 nmol/L probes (LHR and b-actin) from IDT Technologies (Coralville, Iowa). PCR amplification protocol was set for 1 min at 95 C, followed by 44 cycles at 95 C for 5 seconds, 60 C for 25 seconds. The data was analyzed by using the BioRad CFX Manager Software. The Primers were Synthesized by IDT Technologies (Coralville, Iowa) and their Sequences were: Rat b actin (forward): 50 -AGGGTGTAGAGTGTTTGCAGTCGT-30 ;
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(reverse): 50 -AACGCCTGACTCCCAATACTGTGT-30 Rat LHR (forward): 50 -TGTATGCGATCTTCACGAAGGCGT-30 (reverse): 50 -TGCACTGTGGACAACTTCAGGGTA-30 No cDNA, only primer set, cDNA but no primer set and water were used for controls. The amplified products were analyzed by electrophoresis in 1.5% agarose gels.
Western Blotting The cell lysates were prepared using RIPA buffer, which consisted of 25 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% NP-40, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 mmol/L Na3VO4, and 1X protease inhibitor cocktailP2714 (Sigma Chemical Company, St Louis, Missouri). The protein concentrations were determined by using a BCA Protein Assay kit (Thermo Scientific, Waltham, Massachusetts). Aliquots of 50 mg protein were boiled for 5 min in sample buffer (65 mmol/L Tris HCl, 10% glycerol, 2.3% sodium dodecyl sulfate [SDS], 0.01% bromophenol blue, and 1% dithiothreitol) before loading onto 10% SDS polyacrylamide gels. The electrophoresis was carried out for 50 minutes using a BioRad Mini Protein II system (Bio-Rad, Hercules, California), and the separated proteins were transferred to Immobilon PVDF membranes (EMD Millipore, Temecula, California ). The membranes were then blocked with 3% nonfat dried milk for 1 hour and incubated overnight at 4 C with rabbit antirat LHR polyclonal antibody (1:1000). Peroxidase-conjugated antirabbit IgG (Vector Laboratories, Burlingame, California ) was used as the secondary antibody. The membranes were incubated with the secondary antibody (1:2000) for 1 hour at room temperature. Immunoblotting signals were detected by Amersham enhanced chemiluminescence (ECL) plus Western blotting detection system and exposure to x-ray films (GE healthcare Biosciences, Pittsburgh, PA) according to the procedure recommended by the manufacturer.
Immunofluorescence Labeling of Cells and Human Pancreatic Islets The cells and islets were washed 3 times and then fixed in Bouin solution (Sigma Chemical Company, St Louis, Missouri) for 30 minutes at room temperature. They were then washed 3 times with phosphate-buffered saline (PBS), followed by dehydration using 50%, 70%, 95%, and 100% ethanol (EtOH) and rehydration using 100%, 95%, 70%, 50%, and 0% EtOH. The hydrated cells and islets were permeabilized for 10 minutes at room temperature with 0.2% Triton X-100 (Sigma Chemical Company, St Louis, Missouri) and washed 3 times with PBS. To block nonspecific antibodies binding sites, cells, and islets were incubated 3 successive times for 5 minutes each with PBS containing 2% BSA, followed by incubation for 30 minutes with PBS containing 5% normal goat serum. The cells and islets were
Figure 2. Quantitative polymerase chain reaction (qPCR) for hCG/ LH receptors in rat insulinoma cells. Lane 1, DNA ladder; lane 2, b-actin; lane 3, receptors in insulinoma cells; lane 4, receptors in mouse ovary; and lane 5, water as a procedural control.
then treated for 1 hour at room temperature with a 1:100 dilution of rat LHR antibody in blocking buffer or affinity purified 1:100 dilution of guinea pig antibody against insulin (gift of Professor William B. Rhoten, formerly of Marshall University, West Virginia). Then, the cells were washed 3 times with blocking buffer and treated for 1 hour at room temperature with 5 mg/mL of Alexa Fluor 488 goat antirabbit IgG and Alexa Fluor 546 goat antiguinea pig IgG fluorescent secondary antibodies (Invitrogen, Molecular Probes, Eugene, OR). The cells and islets were then washed 3 times with blocking buffer and 3 times with PBS and then coverslips were mounted onto the glass slide using water-soluble Fluoromont-G (SouthernBiotech, Birmingham, AL).
Confocal Fluorescence Microscopy The confocal fluorescence images were scanned on a Nikon TE2000U inverted fluorescence microscope equipped with a Nikon D-Eclipse C1 laser scanning confocal microscope system (Nikon Corp). The z-series scanning were done at every 1 mm up to a z-depth of 10 mm by using a Nikon 40 x 1.30 NA DIC H/N2 Plan Fluor oil immersion objective lens. The 488 nm excitation beam was provided by using a Spectra-Physics (Mountain View, California) Argon laser and 543 nm excitation beam was provided by using a Melles Griot (Carlsbad, California ) Helium-Neon laser. The
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Figure 3. Immunocytochemistry (A and B) and indirect immunofluorescence (C-F) for hCG/LH receptors in rat insulinoma cells. B and F, procedural controls; (C and E) green fluorescence for receptors; (C and D) blue color is nuclear DAPI staining. Mag 40 (A and B) and Mag. 20 (C-F). (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
Figure 4. Western blotting for hCG/LH receptors in rat insulinoma cells (lanes 1-4) and in rat testis as a positive control.
built-in Nikon EZ-C1 software and Metamorph 6 software (Universal Imaging Corp) were used for confocal image acquisition and analyses. 4
The histoscores were calculated on receptor positive RIN cells that contain insulin, using NIH imageJ software (freely available at the NIH website, http://imageJ.nih.gov/ij). Briefly, the cells were traced to calculate total area. The immunofluorescence intensities were calculated by the software.
Measurements of Glucose-Induced Insulin Release The hCG effects on glucose-induced insulin release were measured at low (5.5 mmol/L) and high (20 mmol/L) glucose levels.
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Figure 5. Confocal fluorescence microscopy for hCG/LH receptors and insulin in rat insulinoma cells. Green fluorescence for receptors and red fluorescence for insulin. Mag 40. (The color version of this figure is available in the online version at http://rs.sagepub.com/.) Table 1. Histoscores on hCG/LH Receptor Positive RIN Cells that Contains Insulin.
Cell area (Arbitrary Cell Number Units) 1 2 3 4 5 6 7 8 9 10
1898 1063 1298 1461 1412 1669 950 1090 1093 1371
Histoscore (Integrated Fluorescence Intensity for Total Cell Area) 57 31 47 40 49 55 33 33 41 41
156 810 384 333 202 192 201 922 464 711
Histoscore (Integrated Immunofluorescence Intensity per Unit Cell Area) 30 30 37 28 35 33 35 31 38 30
Abbreviations: hCG, human chorionic gonadotropin; LH, luteinizing hormone.
Figure 6. The hCG/LH receptor positive cells that contain insulin were traced to calculate total cell area as well as to quantify immunofluorescence intensities by NIH image software.
Briefly, cells were cultured for 5 days at 37 C and then varying doses of hCG (1, 10, 25, 50, and 100 ng/mL) were added and culturing continued for another 24 hours. Culture media were then removed and the cells were washed twice with glucose free D-PBS buffer containing calcium and magnesium (GIBCO). The cells were incubated again for 1 hour with DPBS containing 5.5 mmol/L glucose and the supernatants were collected. The cells were then further incubated for 30 minutes with D-PBS containing 20 mmol/L glucose and the supernatants were again collected. Both sets of supernatants were assayed for the insulin as described subsequently. The cells were lysed with NP40 Cell Lysis RIPA buffer and the total protein concentrations were determined by BCA Protein Assay kit (Thermo Scientific, 23227).
Ultrasensitive rat insulin EIA kits (ALPCO Diagnostics, Salem, New Hampshire) were used for the measurement of insulin levels. A standard curve was constructed from the insulin standards provided in the kit. The absorbance of the 96-well ELISA culture plates was read at 450 nm at a reference wavelength of 640 nm in a Bio-Tek Synergy HT 96-well plate reader using KC4 junior software.
Results CG/LH Receptors in Pancreas of CG/LHR Knockout Animals The knockout animals have previously been developed and extensively characterized.8-13 Briefly, the wild-type animals contained receptors in gonadal as well as in nongonadal tissues. On the other hand, these tissues from homozygous animals did not contain them and as a result, they showed expected phenotypes.8-13
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Reproductive Sciences (Figure 3C-F) were performed. Both the techniques demonstrated the receptor protein in cells. The corresponding procedural controls showed no immunostaining (Figure 3B and F). The presence of receptor protein was further investigated by Western blotting. As shown in Figure 4, four different batches of rat insulinoma cells contained 80-kDa protein as does rat testis used as a positive control. This protein was not detected when preabsorbed receptor antibody was used (data not shown). These data led us to question whether CG/LHR-positive RIN cells are indeed b-cells, which of course will contain insulin. To check this possibility, we used confocal fluorescence microscopy after a single and dual labeling for CG/LHR and insulin (Figure 5). The merged images show that the insulin positive cells do indeed contain CG/LHR. The hCG/LH receptor histoscores were calculated on 10 RIN cells (Figure 6). The immunofluorescence intensities for total cell area as well as per unit cell area were calculated and presented in Table 1. As shown in this table, while total integrated immunofluorescence intensities varied, because of cell size differences, the intensities per unit cell area were less variable.
Response of Rat Insulinoma Cells
Figure 7. Effects of hCG on low (5.50 mmol/L; A) and high (20 mmol/L; B) glucose-induced release of insulin from rat insulinoma cells. *P < .05 compared with the control.
As a first step toward determining the possible presence of CG/LHR in pancreas, we harvested the tissues for immunostaining. As shown in Figure 1, islets from wild-type animals showed receptor immunostaining, which decreased in heterozygous animals and completely disappeared in homozygous animals. In parallel, ovaries from animals of 3 genotypes were also immunostained. As previously published,8 while ovaries of homozygous animals failed, those from wild-type immunostained for CG/LHR.
Since insulin-containing cells were CG/LHR positive, we next examined whether hCG can regulate insulin release. As shown in Figure 7A, hCG had no effect at any of the doses on low glucose (basal)-induced release of insulin. When the same cells were subsequently challenged with 20 mmol/L glucose, which should induce the release of all the insulin, the cells responded (Figure 7B). While 10, 25, and 50 ng/mL hCG have further augmented the insulin release, lower and higher concentrations had no effect.
CG/LHR and Insulin Coexpression in Human Islets To provide human relevance of the findings on CG/LHR knockout animals and rat insulinoma cells, we used pancreatic islets obtained from human cadavers for confocal microscopy after a single and double immunostaining for CG/LHR and insulin. Merged images showed an overlap of CG/LHR with insulin, reaffirming the insulin producing cells do indeed contain CG/LHR (Figure 8).
Discussion CG/LHR in Rat Insulinoma Cells To further characterize b-cell CG/LHR, we used immortalized rat insulinoma cells, which have been extensively used for studying the control mechanisms of insulin secretion.14 Quantitative PCR data in Figure 2 show that rat insulinoma cells contained an expected size amplified CG/LHR fragment, as does rat ovaries. However, the band in b-cells is fainter than in rat ovaries. To determine the presence of receptor protein, immunocytochemistry (Figure 3A and B) and indirect immunofluorescence 6
The concept that hCG and LH can also act on multiple nongonadal tissues has now been around for more than 25 years.4,15,16 These findings opened new possibilities on how elevated or decreased levels of these hormones could explain several disease manifestations and how they might be treated with hCG.4,15,16 Maternal circulatory insulin levels increase during normal pregnancy.17-19 Whether this increase during first trimester could possibly due to hCG has never been questioned.18 However, the increases during mid to late pregnancy has generally
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Figure 8. Confocal microscopy for hCG/LH receptors and insulin in human pancreatic islets. Green fluorescence for receptors and red fluorescence for insulin. Mag. 40. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
been considered due to progressive increase in human placental lactogenic levels.17-21 In polycystic ovarian disease, plasma insulin levels are positively correlated with LH levels.6 These findings made us wonder whether hCG and LH could regulate insulin release from b-cells of pancreas. If this were to be the case, b-cells must contain hCG/LH receptors and their activation should result in changes in insulin levels. The possibility of b-cells containing hCG/LH receptors is not too farfetched, considering the evidence for the presence of these receptors in a number of nongonadal tissues.4,15,16 With this possibility in mind, we first began investigating well-characterized hCG/ LH receptor knockout mice.8-13 The null mice failed to show the receptors not only in gonadal tissues but also in nongonadal tissues examined.8-13 In the present studies, while null animals failed to show the receptor immunostaining in b-cells, wild-type mice did. Since further detailed studies could not be carried out on these mice, we turned to immortalized rat insulinoma cells, which have previously been used to investigate the control mechanisms of insulin release.14 The reverse transcription-PCR analysis showed that insulinoma cells contained an expected size hCG/LH receptor fragment. Immunocytochemistry as well as Western blotting demonstrated the presence of receptor protein. Its molecular size was about the same as in other nongonadal cells.4,22 In order to ascertain whether receptor positive insulinoma cells are indeed insulin secreting b-cells, confocal fluorescence microscopy was used after dual labeling for receptors and insulin. The results showed that insulin containing cells are indeed receptors positive. Measurement of media insulin levels showed that hCG had no effect on low glucose-induced release of insulin. However, when the same cells were challenged with high glucose levels, hCG further augmented the release of insulin at 10, 25, and 50 ng/mL doses. The failure of cells to respond to 1 and 100 ng/ mL hCG could be due to not enough or too much hCG to activate or down-regulate its receptors, respectively. The insulin release from pancreatic b-cells is tied to an increase in intracellular Ca2þ levels.23,24 Although it was not tested in RIN cells, hCG is known to increase intracellular Ca2þ levels in other target tissues,25 and perhaps this is one of the mechanisms that hCG uses in RIN cells.
Finally, confocal fluorescence microscopy was repeated on human islets obtained from cadavers of nondiabetic human subjects, after dual labeling for receptors and insulin, to determine the human relevance of findings on mice and rats. The results showed that insulin containing cells are receptor positive. In summary, evidences presented here demonstrate that b-cells of pancreas contain hCG/LH receptors and they may play a role in the release of insulin induced by high levels of glucose. These findings could have huge implications for causal relationships between insulin level changes seen during the periods of altered hCG and LH secretion, such as pregnancy and polycystic ovarian disease, respectively. In addition, a connection may be made between increasing or elevated LH levels and the development of metabolic syndrome in pre to postmenopausal women.26 Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
References 1. Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem. 1981;50:465-495. 2. Loosfelt H, Misrahi M, Atger M, et al. Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science. 1989;245(4917):525-528. 3. McFarland KC, Sprengel R, Phillips HS, et al. Lutropinchoriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science. 1989;245(4917): 494-499. 4. Rao CV. Nongonadal actions of LH and hCG in reproductive biology and medicine. Semin Reprod Med. 2001;19(1):1-119. 5. Dumesic DA, Abbott DH, Padmanabhan V. Polycystic ovary syndrome and its developmental origins. Rev Endocr Metab Disord. 2007;8(2):127-141.
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6. Baptiste C, Battista MC, Trottier A, Baillargeon JP. Insulin and hyperandrogenism in women with polycystic ovary syndrome. J Steroid Biochem Mol Biol. 2010;122(1-3):42-52. 7. Sliwowska JH, Fergani C, Gawalek M, Skowronska B, Fichna P, Lehman MN. Insulin: its role in the central control of reproduction. Phys Behav. 2014;133:197-206. 8. Lei ZM, Mishra S, Zou W, Xu B, Foltz M, Li X, Rao CV. Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene. Mol Endocrinol. 2001;15:184-200. 9. Rao CV, Lei ZM. Consequences of targeted inactivation of LH receptors. Mol Cell Endocrinol. 2002;187(1-2):57-67. 10. Lei ZM, Rao CV. Knockout of gonadotropins and their receptor genes. In: Henry HL, Norman AW (eds), Encyclopedia of Hormones, Simpson E, section ed Female Reproduction, Academic Press, San Diego, California. Article #EOH129-MRW581, 2003; pp. 554-562. 11. Lin DX, Lei ZM, Li X, Rao CV. Targeted disruption of LH receptor gene revealed the importance of uterine LH signaling. Mol Cell Endocrinol. 2005;234(1-2):105-116. 12. Lin DX, Lei ZM, Rao CV. Dependence of uterine cyclooxygenase-2 expression on luteinizing hormone signaling. Biol Reprod. 2005;73(2):256-260. 13. Lin J, Li X, Yuan FP, et al. Genetic ablation of luteinizing hormone receptor improves the amyloid pathology in a mouse model of Alzheimer disease. J Neuropathol Exp Neurol. 2010;69(3):253-261. 14. Parkash J, Chaudhry MA, Amer AS, Christakos S, Rhoten WB. Intracellular calcium ion response to glucose in b-cells of calbindin-D28k nullmutant mice and in bHC13 cells overexpressing calbindin-D28k. Endocrine. 2002;18(3):221-229. 15. Rao CV, Lei ZM. The past, present and future of nongonadal LH/ hCG actions in reproductive biology and medicine. Mol Cell Endocrinol. 2007;269(1-2):2-8. 16. Rahman N, Rao CV. Recent progress in luteinizing hormone/ human chorionic gonadotrophin hormone research. Mol Human Reprod. 2009;15(11):703-711.
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17. Spellacy WN, Goetz FC. Plasma insulin in normal late pregnancy. N Engl J Med. 1962;268:988-991. 18. Spellacy WN, Goetz FC, Greenberg BZ, Ells J. Plasma insulin in normal ‘‘early’’ pregnancy. Obstet Gynecol. 1965;25: 862-865. 19. Spellacy WN, Goetz FC, Greenberg BZ, Ells J. Plasma insulin in normal midpregnancy. Am J Obstet Gynecol. 1965;92: 11-15. 20. Sorenson RL, Brelje TC. Adapatation of islets of Langerhans to pregnancy: ß-cell growth, enhanced insulin secretion and the role of lactogenic hormones. Horm Metab Res. 1997; 29(6):301-307. 21. Green IC, Taylor KW. Effects of pregnancy in the rat on the size and insulin secretory response of the islets of Langerhans. J Endocrinol. 1972;54(2):317-325. 22. Lojun S, Bao S, Lin J, Lei ZM, Rao CV. The presence of functional luteinizing hormone/chorionic gonadotropin (hCG) receptors in human breast cell lines: Implications supporting the premise that hCG protects women against breast cancer. Biol Reprod. 1997;57(5):1202-1210. 23. Reddy D, Pollock AS, Clark SA, et al. Transfection and overexpression of the calcium binding protein calbindin-D28k results in a stimulatory effect on insulin synthesis in a rat b-cell line (RIN 1046-38). Proc Natl Acad Sci USA. 1997; 94(5):1961-1966. 24. Yang SN, Berggren PO. b-Cell CaV channel regulation in physiology and pathophysiology. Am J Physiol Endocrinol Metab. 2005;288(1):E16-E28. 25. Eta E, Ambrus G, Rao CV. Direct regulation of human myometrial contractions by human chorionic gonadotropin. J Clin Endocrinol Metab. 1994;79(6):1582-1586. 26. Janssen I, Powell LH, Crawford S, Lasley BL, Sutton-Tyrrell K. Menopause and metabolic syndrome: the study of women’s health across the nation. Arch Intern Med. 2008;168(14): 1568-1575.
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The Presence of Human Chorionic Gonadotropin/Luteinizing Hormone Receptors in Pancreatic b-Cells
Reproductive Sciences 1-8 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719115570910 rs.sagepub.com
Jai Parkash, PhD1,2, Zhenmin Lei, MD, PhD3, and C. V. Rao, PhD4
Abstract We investigated the possible presence of functional human chorionic gonadotropin (hCG)/luteinizing hormone (LH) receptors in b-cells of pancreas, using a combination of techniques on hCG/LH receptor knockout mice, immortalized rat insulinoma cells, and human pancreatic islets. The results showed the presence of receptors and their activation resulted in a dose-dependent increase in glucose-induced release of insulin. These findings place hCG and LH among the regulators of insulin release with potential implications for insulin-level changes during the periods of altered hCG and LH secretion. Keywords hCG/LH receptors, b-cells of pancreas, colocalization, insulin release, glucose
Introduction Human placenta and anterior pituitary gland secrete human chorionic gonadotropin (hCG) and luteinizing hormone (LH), respectively.1 They are structural and functional homologous that bind to the same G-protein-coupled cell surface receptors in target cells.2,3 For a long time, gonadal cells were thought to be the only targets of hCG/LH actions. However, this has been changed by the data published from around the world during the last 25 years to include many nongonadal tissues.4 The activation of nongonadal receptors results in tissue and cell-specific effects.4 In view of these advances, we considered it possible due to seemingly close relationship between hCG/LH and insulin levels and the role of insulin in reproduction5-7 that b-cells of pancreas could contain hCG/LH receptors and their activation may influence glucoseinduced release of insulin. The present studies investigated these possibilities by using pancreas of hCG/LH receptor knockout mice, immortalized rat b-cells and human pancreatic islets. The results showed that b-cells of pancreas do contain hCG/LH receptors and their activation results in a dose-dependent increase in glucose-induced release of insulin. These findings should place hCG and LH among the regulators of insulin release, which would require the reevaluation of insulin level changes during the periods of altered hCG and LH secretion.
Materials and Methods hCG/LH Receptor Knockout Mice These animals have previously been generated by homologous DNA recombination technology and extensively characterized.8 Briefly, the homozygous animals exhibited expected phenotypes
from the lack of hCG/LH receptors in gonadal as well as in nongonadal tissues.8-13 Pancreas and ovaries were harvested for the present studies from adult females of all 3 genotypes and frozen until used.
Immortalized Pancreatic b-Cells and Human Pancreatic Islets Rat insulinoma cells (RINr 1046-38, abbreviated as RIN), which are insulin-producing b-cells, were kindly provided by Dr Bruce Chertow (previously at the Veterans Administration Medical Center, Huntington, West Virginia). Human islets, isolated from cadavers of nondiabetic human subjects, were obtained from the National Disease Research Interchange, Philadelphia, Pennsylvania. Exemption 4 approval was obtained from the Institutional Review Board of Florida International University for their use. 1 Department of Environmental and Occupational Health, Robert Stempel College of Public Health and Social Work, Florida International University, Miami, FL, USA 2 Keiser University, Fort Myers, FL, USA 3 Department of Obstetrics and Gynecology, University of Louisville Medical Center, Louisville, KY, USA 4 Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, USA
Corresponding Author: C. V. Rao, Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL 33199, USA. Email: [email protected]
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Figure 1. Immunocytochemistry for hCG/LH receptors in pancreas of hCG/LH receptor knockout mice. White arrows point out islets. Mag 40. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
Cell and Islets Culture RIN cells were grown in 5% CO2 and 95% air incubator at 37 C in 6-well culture plates or in T 75 culture dishes in RPMI-1640 supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, and 100 mg/mL of streptomycin (Invitrogen Corporation, Grand Island, New York). The human islets were maintained under culture conditions in 6-well plates in a 5% CO2 incubator at 37 C in CMRL culture medium containing 15% fetal calf serum and 100 units/mL of penicillin and 100 mg/mL of streptomycin.
Reverse Transcription-Polymerase Chain Reaction RNA preparation. The total RNA was isolated from RIN cells using TRIzol reagent (Invitrogen, Carlsbad, California) and its concentrations determined by absorbance at 260 nm, using a Biotek Synergy HT 96-well plate reader. The purity was 2
checked by electrophoresis in 1.5% agarose gels. The reverse transcription was carried out using the kits and instructions from Applied Biosystems (Foster City, California ), BioRad CFX96 Real-TimeSystem (Hercules, California ), and SsoFast Probes Supermix (Bio-Rad). A total of 100 ng of complementary DNA (cDNA) was used per reaction. The polymerase chain reaction (PCR) sample consisted of 10 mL containing cDNA, 5 mL SsoFast EvaGreen Supermix (BioRad, cat #1725200), and 1 mL 200 nmol/L probes (LHR and b-actin) from IDT Technologies (Coralville, Iowa). PCR amplification protocol was set for 1 min at 95 C, followed by 44 cycles at 95 C for 5 seconds, 60 C for 25 seconds. The data was analyzed by using the BioRad CFX Manager Software. The Primers were Synthesized by IDT Technologies (Coralville, Iowa) and their Sequences were: Rat b actin (forward): 50 -AGGGTGTAGAGTGTTTGCAGTCGT-30 ;
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(reverse): 50 -AACGCCTGACTCCCAATACTGTGT-30 Rat LHR (forward): 50 -TGTATGCGATCTTCACGAAGGCGT-30 (reverse): 50 -TGCACTGTGGACAACTTCAGGGTA-30 No cDNA, only primer set, cDNA but no primer set and water were used for controls. The amplified products were analyzed by electrophoresis in 1.5% agarose gels.
Western Blotting The cell lysates were prepared using RIPA buffer, which consisted of 25 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% NP-40, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 mmol/L Na3VO4, and 1X protease inhibitor cocktailP2714 (Sigma Chemical Company, St Louis, Missouri). The protein concentrations were determined by using a BCA Protein Assay kit (Thermo Scientific, Waltham, Massachusetts). Aliquots of 50 mg protein were boiled for 5 min in sample buffer (65 mmol/L Tris HCl, 10% glycerol, 2.3% sodium dodecyl sulfate [SDS], 0.01% bromophenol blue, and 1% dithiothreitol) before loading onto 10% SDS polyacrylamide gels. The electrophoresis was carried out for 50 minutes using a BioRad Mini Protein II system (Bio-Rad, Hercules, California), and the separated proteins were transferred to Immobilon PVDF membranes (EMD Millipore, Temecula, California ). The membranes were then blocked with 3% nonfat dried milk for 1 hour and incubated overnight at 4 C with rabbit antirat LHR polyclonal antibody (1:1000). Peroxidase-conjugated antirabbit IgG (Vector Laboratories, Burlingame, California ) was used as the secondary antibody. The membranes were incubated with the secondary antibody (1:2000) for 1 hour at room temperature. Immunoblotting signals were detected by Amersham enhanced chemiluminescence (ECL) plus Western blotting detection system and exposure to x-ray films (GE healthcare Biosciences, Pittsburgh, PA) according to the procedure recommended by the manufacturer.
Immunofluorescence Labeling of Cells and Human Pancreatic Islets The cells and islets were washed 3 times and then fixed in Bouin solution (Sigma Chemical Company, St Louis, Missouri) for 30 minutes at room temperature. They were then washed 3 times with phosphate-buffered saline (PBS), followed by dehydration using 50%, 70%, 95%, and 100% ethanol (EtOH) and rehydration using 100%, 95%, 70%, 50%, and 0% EtOH. The hydrated cells and islets were permeabilized for 10 minutes at room temperature with 0.2% Triton X-100 (Sigma Chemical Company, St Louis, Missouri) and washed 3 times with PBS. To block nonspecific antibodies binding sites, cells, and islets were incubated 3 successive times for 5 minutes each with PBS containing 2% BSA, followed by incubation for 30 minutes with PBS containing 5% normal goat serum. The cells and islets were
Figure 2. Quantitative polymerase chain reaction (qPCR) for hCG/ LH receptors in rat insulinoma cells. Lane 1, DNA ladder; lane 2, b-actin; lane 3, receptors in insulinoma cells; lane 4, receptors in mouse ovary; and lane 5, water as a procedural control.
then treated for 1 hour at room temperature with a 1:100 dilution of rat LHR antibody in blocking buffer or affinity purified 1:100 dilution of guinea pig antibody against insulin (gift of Professor William B. Rhoten, formerly of Marshall University, West Virginia). Then, the cells were washed 3 times with blocking buffer and treated for 1 hour at room temperature with 5 mg/mL of Alexa Fluor 488 goat antirabbit IgG and Alexa Fluor 546 goat antiguinea pig IgG fluorescent secondary antibodies (Invitrogen, Molecular Probes, Eugene, OR). The cells and islets were then washed 3 times with blocking buffer and 3 times with PBS and then coverslips were mounted onto the glass slide using water-soluble Fluoromont-G (SouthernBiotech, Birmingham, AL).
Confocal Fluorescence Microscopy The confocal fluorescence images were scanned on a Nikon TE2000U inverted fluorescence microscope equipped with a Nikon D-Eclipse C1 laser scanning confocal microscope system (Nikon Corp). The z-series scanning were done at every 1 mm up to a z-depth of 10 mm by using a Nikon 40 x 1.30 NA DIC H/N2 Plan Fluor oil immersion objective lens. The 488 nm excitation beam was provided by using a Spectra-Physics (Mountain View, California) Argon laser and 543 nm excitation beam was provided by using a Melles Griot (Carlsbad, California ) Helium-Neon laser. The
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Figure 3. Immunocytochemistry (A and B) and indirect immunofluorescence (C-F) for hCG/LH receptors in rat insulinoma cells. B and F, procedural controls; (C and E) green fluorescence for receptors; (C and D) blue color is nuclear DAPI staining. Mag 40 (A and B) and Mag. 20 (C-F). (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
Figure 4. Western blotting for hCG/LH receptors in rat insulinoma cells (lanes 1-4) and in rat testis as a positive control.
built-in Nikon EZ-C1 software and Metamorph 6 software (Universal Imaging Corp) were used for confocal image acquisition and analyses. 4
The histoscores were calculated on receptor positive RIN cells that contain insulin, using NIH imageJ software (freely available at the NIH website, http://imageJ.nih.gov/ij). Briefly, the cells were traced to calculate total area. The immunofluorescence intensities were calculated by the software.
Measurements of Glucose-Induced Insulin Release The hCG effects on glucose-induced insulin release were measured at low (5.5 mmol/L) and high (20 mmol/L) glucose levels.
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Figure 5. Confocal fluorescence microscopy for hCG/LH receptors and insulin in rat insulinoma cells. Green fluorescence for receptors and red fluorescence for insulin. Mag 40. (The color version of this figure is available in the online version at http://rs.sagepub.com/.) Table 1. Histoscores on hCG/LH Receptor Positive RIN Cells that Contains Insulin.
Cell area (Arbitrary Cell Number Units) 1 2 3 4 5 6 7 8 9 10
1898 1063 1298 1461 1412 1669 950 1090 1093 1371
Histoscore (Integrated Fluorescence Intensity for Total Cell Area) 57 31 47 40 49 55 33 33 41 41
156 810 384 333 202 192 201 922 464 711
Histoscore (Integrated Immunofluorescence Intensity per Unit Cell Area) 30 30 37 28 35 33 35 31 38 30
Abbreviations: hCG, human chorionic gonadotropin; LH, luteinizing hormone.
Figure 6. The hCG/LH receptor positive cells that contain insulin were traced to calculate total cell area as well as to quantify immunofluorescence intensities by NIH image software.
Briefly, cells were cultured for 5 days at 37 C and then varying doses of hCG (1, 10, 25, 50, and 100 ng/mL) were added and culturing continued for another 24 hours. Culture media were then removed and the cells were washed twice with glucose free D-PBS buffer containing calcium and magnesium (GIBCO). The cells were incubated again for 1 hour with DPBS containing 5.5 mmol/L glucose and the supernatants were collected. The cells were then further incubated for 30 minutes with D-PBS containing 20 mmol/L glucose and the supernatants were again collected. Both sets of supernatants were assayed for the insulin as described subsequently. The cells were lysed with NP40 Cell Lysis RIPA buffer and the total protein concentrations were determined by BCA Protein Assay kit (Thermo Scientific, 23227).
Ultrasensitive rat insulin EIA kits (ALPCO Diagnostics, Salem, New Hampshire) were used for the measurement of insulin levels. A standard curve was constructed from the insulin standards provided in the kit. The absorbance of the 96-well ELISA culture plates was read at 450 nm at a reference wavelength of 640 nm in a Bio-Tek Synergy HT 96-well plate reader using KC4 junior software.
Results CG/LH Receptors in Pancreas of CG/LHR Knockout Animals The knockout animals have previously been developed and extensively characterized.8-13 Briefly, the wild-type animals contained receptors in gonadal as well as in nongonadal tissues. On the other hand, these tissues from homozygous animals did not contain them and as a result, they showed expected phenotypes.8-13
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Response of Rat Insulinoma Cells
Figure 7. Effects of hCG on low (5.50 mmol/L; A) and high (20 mmol/L; B) glucose-induced release of insulin from rat insulinoma cells. *P < .05 compared with the control.
As a first step toward determining the possible presence of CG/LHR in pancreas, we harvested the tissues for immunostaining. As shown in Figure 1, islets from wild-type animals showed receptor immunostaining, which decreased in heterozygous animals and completely disappeared in homozygous animals. In parallel, ovaries from animals of 3 genotypes were also immunostained. As previously published,8 while ovaries of homozygous animals failed, those from wild-type immunostained for CG/LHR.
Since insulin-containing cells were CG/LHR positive, we next examined whether hCG can regulate insulin release. As shown in Figure 7A, hCG had no effect at any of the doses on low glucose (basal)-induced release of insulin. When the same cells were subsequently challenged with 20 mmol/L glucose, which should induce the release of all the insulin, the cells responded (Figure 7B). While 10, 25, and 50 ng/mL hCG have further augmented the insulin release, lower and higher concentrations had no effect.
CG/LHR and Insulin Coexpression in Human Islets To provide human relevance of the findings on CG/LHR knockout animals and rat insulinoma cells, we used pancreatic islets obtained from human cadavers for confocal microscopy after a single and double immunostaining for CG/LHR and insulin. Merged images showed an overlap of CG/LHR with insulin, reaffirming the insulin producing cells do indeed contain CG/LHR (Figure 8).
Discussion CG/LHR in Rat Insulinoma Cells To further characterize b-cell CG/LHR, we used immortalized rat insulinoma cells, which have been extensively used for studying the control mechanisms of insulin secretion.14 Quantitative PCR data in Figure 2 show that rat insulinoma cells contained an expected size amplified CG/LHR fragment, as does rat ovaries. However, the band in b-cells is fainter than in rat ovaries. To determine the presence of receptor protein, immunocytochemistry (Figure 3A and B) and indirect immunofluorescence 6
The concept that hCG and LH can also act on multiple nongonadal tissues has now been around for more than 25 years.4,15,16 These findings opened new possibilities on how elevated or decreased levels of these hormones could explain several disease manifestations and how they might be treated with hCG.4,15,16 Maternal circulatory insulin levels increase during normal pregnancy.17-19 Whether this increase during first trimester could possibly due to hCG has never been questioned.18 However, the increases during mid to late pregnancy has generally
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Figure 8. Confocal microscopy for hCG/LH receptors and insulin in human pancreatic islets. Green fluorescence for receptors and red fluorescence for insulin. Mag. 40. (The color version of this figure is available in the online version at http://rs.sagepub.com/.)
been considered due to progressive increase in human placental lactogenic levels.17-21 In polycystic ovarian disease, plasma insulin levels are positively correlated with LH levels.6 These findings made us wonder whether hCG and LH could regulate insulin release from b-cells of pancreas. If this were to be the case, b-cells must contain hCG/LH receptors and their activation should result in changes in insulin levels. The possibility of b-cells containing hCG/LH receptors is not too farfetched, considering the evidence for the presence of these receptors in a number of nongonadal tissues.4,15,16 With this possibility in mind, we first began investigating well-characterized hCG/ LH receptor knockout mice.8-13 The null mice failed to show the receptors not only in gonadal tissues but also in nongonadal tissues examined.8-13 In the present studies, while null animals failed to show the receptor immunostaining in b-cells, wild-type mice did. Since further detailed studies could not be carried out on these mice, we turned to immortalized rat insulinoma cells, which have previously been used to investigate the control mechanisms of insulin release.14 The reverse transcription-PCR analysis showed that insulinoma cells contained an expected size hCG/LH receptor fragment. Immunocytochemistry as well as Western blotting demonstrated the presence of receptor protein. Its molecular size was about the same as in other nongonadal cells.4,22 In order to ascertain whether receptor positive insulinoma cells are indeed insulin secreting b-cells, confocal fluorescence microscopy was used after dual labeling for receptors and insulin. The results showed that insulin containing cells are indeed receptors positive. Measurement of media insulin levels showed that hCG had no effect on low glucose-induced release of insulin. However, when the same cells were challenged with high glucose levels, hCG further augmented the release of insulin at 10, 25, and 50 ng/mL doses. The failure of cells to respond to 1 and 100 ng/ mL hCG could be due to not enough or too much hCG to activate or down-regulate its receptors, respectively. The insulin release from pancreatic b-cells is tied to an increase in intracellular Ca2þ levels.23,24 Although it was not tested in RIN cells, hCG is known to increase intracellular Ca2þ levels in other target tissues,25 and perhaps this is one of the mechanisms that hCG uses in RIN cells.
Finally, confocal fluorescence microscopy was repeated on human islets obtained from cadavers of nondiabetic human subjects, after dual labeling for receptors and insulin, to determine the human relevance of findings on mice and rats. The results showed that insulin containing cells are receptor positive. In summary, evidences presented here demonstrate that b-cells of pancreas contain hCG/LH receptors and they may play a role in the release of insulin induced by high levels of glucose. These findings could have huge implications for causal relationships between insulin level changes seen during the periods of altered hCG and LH secretion, such as pregnancy and polycystic ovarian disease, respectively. In addition, a connection may be made between increasing or elevated LH levels and the development of metabolic syndrome in pre to postmenopausal women.26 Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
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