Restricted Expression of Somatostatin Receptor 3 to Primary Cilia in the Pan- Creatic Islets and Adenohypophysis of Mice

Biomedical Research 32 (1) 73-81, 2011

Restricted expression of somatostatin receptor 3 to primary cilia in the pancreatic islets and adenohypophysis of mice

1 2 1 Toshihiko Iwanaga , Takashi Miki , and Hiromi Takahashi-Iwanaga 1 Laboratory of Histology and Cytology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638 and 2 Department of Medical Physiology (C3), Chiba University Graduate School of Medicine, Chiba 260-8670, Japan (Received 2 December 2010; and accepted 25 December 2010)

ABSTRACT The primary cilium is now considered to function as a fundamental, not rudimentary, structure for mechanical and chemical sensing by individual cells. Primary cilia in neurons express type III adenylyl cyclase (ACIII) and GPCRs for somatostatin (somatostatin receptor 3, SSTR3), serotonin, and melanin-concentrating hormone. The present immunohistochemical and electron microscopic study revealed an abundant occurrence of SSTR3-expressing solitary cilia in insulin- and growth hormone-secreting cells of the mouse. The SSTR3 immunoreactivity was restricted to the plasma membrane of cilia in both cell types, differing from previously reported immunohistochemical localization of SSTRs to cell bodies. The primary cilia in the islet cells were longer than those in the pituitary cells and extended for a long distance in the intercellular canalicules endowed with microvilli. No other endocrine organs were provided with the SSTR3-expressing primary cilia, while the primary cilia in these organs were frequently immunolabeled with ACIII antibody. Since the somatostatin inhibition of both insulin and GH release is regulated mainly by SSTR1 and SSTR5, the primary cilia expressing SSTR3 may be involved in a signaling which differs from that via other SSTR subtypes expressing in cell bodies.

Morphologists have long observed a single cilium, spond to fluid flow in the lumen (21, 26, 27). Bend- termed the primary cilium, in a variety of cells un- ing of the ciliary axoneme by fluid movement has der light and electron microscopes. The primary cil- been shown to induce a Ca2+-response in the cells of ium is an immotile cilium that is extended from the the collecting tubules (26). Neuronal primary cilia centriole—usually surrounded by the Golgi appara- in the brain express G protein-coupled receptors tus—in almost all cell types (38). The primary cilia (GPCRs), including somatostatin receptor 3 (SSTR3) in sensory cells serve specialized sensing functions, (11), 5-HT6 serotonin receptor (10), and melanin- as represented by photoreceptor cells, auditory hair concentrating hormone receptor 1 (MCHR1) (3), cells, and olfactory receptor cells. However, evi- suggesting an important role for the primary cilia in dence is accumulating that raises the idea that the the extra-synaptic signaling of neurons. primary cilium in non-sensory cells functions as a The five SSTRs identified and characterized to sensory device at a cellular level. Long solitary cilia date predominate in having inhibitory actions (6, on epithelial cells in the collecting tubules of the 23). Neurons in the central nervous system express kidney act as mechanoreceptors to detect and re- all SSTR subtypes on the plasma membrane of so- mata and proximal dendrites and on beaded fibers— Address correspondence to: Dr. Toshihiko Iwanaga, Lab- including axons (17), while the immunoreactive oratory of Histology and Cytology, Hokkaido University SSTR3 gathers in the primary cilia of neurons in Graduate School of Medicine, Sapporo 060-8638, Japan several regions of the brain (11, 31). In addition to Tel: +81-11-706-5033, Fax: +81-11-706-7151 SSTR3, the primary cilia of neurons can be easily E-mail: [email protected] labeled by immunostaining for type III adenylyl cy- 74 T. Iwanaga et al. clase (ACIII) in vitro and in vivo (3, 4). Immuno The fixed tissues were dipped in 30% sucrose staining for ACIII and tubulin, though not SSTR3, solution overnight at 4°C, embedded in OCT com- has been applied to detect the primary cilia in vis- pound, and quickly frozen in liquid nitrogen. Frozen ceral organs such as the pancreas and kidney. sections of 10 μm in thickness were mounted on Somatostatin is widely distributed outside of the poly-l-lysine-coated glass slides. After immersion in brain and exerts various inhibitory actions for endo- 0.01 M phosphate buffered saline containing 0.3% crine and exocrine secretions (28). Among the endo- Triton-X 100, the sections were preincubated with a crine cells and organs, somatostatin is produced and normal donkey serum. For double immunofluores- released from the gastro-entero-pancreatic (GEP) en- cence, sections were stained with one of following docrine system, thyroid gland (parafollicular cells), antibodies: rabbit anti-SSTR3 (1 : 5,000 in dilution; adrenal medulla, and dispersed endocrine cells of Gramsch Laboratories, Schwabhausen, Germany), the respiratory and urogenital tracts. In most regions, rabbit anti-ACIII (0.2 μg/mL; Santa Cruz Biotechnol- somatostatin functions as a local hormone which is ogy, Inc., Santa Cruz, CA), or mouse anti-acetylated conveyed by local circulation or diffusion through tubulin (Clone 6-11B-1, 1 μg/mL; Sigma-Aldrich, St. an intercellular space to regulate neighboring cells. Louis, MO). The sections were then incubated with Many electron microscopic observations have dem- one of the following antibodies: guinea pig anti- onstrated the solitary cilium in endocrine cells such insulin (1 : 100; Invitrogen, Camarillo, CA), guinea as pancreatic islet cells (39), pituitary endocrine pig anti-glucagon (1 : 600, Progen, Heidelberg, Ger- cells (2), and adrenal endocrine cells (37, 38). How- many), goat anti-somatostatin (1 : 750, Santa Cruz ever, it remains unknown whether the primary cilia Biotechnology), goat anti-GH (1 : 250; R&D Sys- in these endocrine cells express SSTR3 and other tem, Inc., Minneapolis, MN), or rabbit anti-GH signal molecules for sensing. antisera (1 : 100; Zymed Laboratories, South San The present immunohistochemical study exam- Francisco, CA). The sites of antigen-antibody reac- ined the existence of primary cilia expressing SSTR3 tions were detected by incubation with a combina- and ACIII in a variety of endocrine cells. Insulin- tion of Cys3-labeled anti-rabbit or mouse IgG and secreting cells in the pancreas and growth hormone FITC-labeled anti-guinea pig or goat IgG (Jackson (GH)-secreting cells in the adenohypophysis of mice ImmunoResearch, West Grove, PA). Stained sections were found to develop the primary cilia expressing were mounted with glycerin-PBS and observed un- SSTR3, suggesting the involvement of primary cilia der a confocal laser scanning microscope (Fluoview; in a special sensing of somatostatin in the mouse. Olympus, Tokyo, Japan). Some of immunostained sections were counterstained with SyTO 13 (Invitro- gen) before observation. SSTR3 and ACIII antibod- MATERIALS AND METHODS ies used specifically labeled the primary cilia on Immunohistochemistry. Adult male BALB/c mice, neurons in several regions of the mouse and rat weighing about 25 g each, were used. For immuno- brain, in agreement with previous studies (4, 11). histochemistry, five mice were perfused with a phys- The specificity of immunoreactions with the anti- iological saline through the heart, followed by 4% SSTR3 antibody was confirmed by disappearance of paraformaldehyde plus 0.2% picric acid in 0.1 M the immunoreactivity when the antibody was prein- phosphate buffer, pH 7.4. The hypophysis, pancreas, cubated with a synthetic peptide antigen (Yanaihara other endocrine organs, and sexual glands were dis- Institute, Shizuoka, Japan). sected out and immersed in the same fixative for an additional 8 h. The stomach, duodenum, lung, and Silver-intensified immunogold method for electron urethra containing dispersed paraneuronal cells were microscopy. Frozen sections of 15 μm in thickness processed according to the same protocol. In addi- were mounted on poly-l-lysine-coated glass slides, tion, the pancreas was collected from male rats incubated with the rabbit anti-SSTR3 (1 : 2,000 in (Wistar strain, 250 g in body weight), hamsters (Syr- dilution; Gramsch Laboratories), and subsequently ian strain, 300 g), and guinea pigs (Hartley strain, reacted with goat anti-rabbit IgG covalently linked 350 g) and processed as mentioned above. A mouse with 1-nm gold particles (1 : 200; Nanoprobes, cell line secreting insulin, MIN6, was fixed for 3 h Yaphank, NY). Following silver enhancement using with 4% paraformaldehyde. All experiments using a kit (HQ silver; Nanoprobes), the sections were animals were performed under protocols following osmicated, dehydrated, and directly embedded in the Guidelines for Animal Experimentation, Hokkaido Epon (Nisshin EM, Tokyo, Japan). Ultrathin sections University Graduate School of Medicine. were prepared and stained with both uranyl acetate Primary cilia in pancreatic islets 75 and lead citrate for observation under an electron active cilia (Fig. 1a). A population of glucagon cells microscope (H-7100; Hitachi, Tokyo, Japan). possessed the primary cilia with SSTR3 immunoreactivity, whereas other populations of glucagon cells Scanning electron microscopy (SEM). Under pento- lacked SSTR3-expressing cilia (Fig. 1c). No soma- barbital anesthesia (40 mg/kg body weight), another tostatin cells appeared to possess the immunoreac- five mice were perfused transcardially with Locke’s tive primary cilia. In the adenohypophysis, cells with solution and subsequently with a mixture containing the SSTR3-expressing primary cilia corresponded to 2.5% glutaraldehyde and 1.0% paraformaldehyde in GH-secreting cells (Fig. 1b), though it was difficult 0.1 M phosphate buffer, pH 7.4. The hypophysis and to deny the existence of SSTR3-expressing cilia in pancreas were removed and immersed in the same other hormone-secreting cells. fixative overnight. The anterior lobe of the hypoph- In the silver-intensified immunogold method, ysis and the islets of the pancreas were cut out of SSTR3-immunolabeled primary cilia were easily the fixed tissues under a dissecting microscope, and found under the electron microscope (Fig. 1e, f). rinsed in 0.1 M phosphate buffer (pH 7.4). The Many pancreatic B cells possessed a very long pri- specimens were macerated with 6N NaOH at 60°C mary cilium extending from the deeper region of the for 18 min (34). After maceration, the tissue pieces cytoplasm into intercellular spaces among islet cells. were rinsed in 0.1 M phosphate buffer and postfixed The primary cilium possessed two centrioles, the with 1% tannic acid in the phosphate buffer for 1 h, proximal centriole and the distal centriole, the latter followed by 1% OsO4 buffered with phosphate being connected with the axial fibrils of primary cil- (0.1 M, pH 7.2) for 1 h. The osmicated specimens ium (an arrow in Fig. 1e). In the adenohypophysis, were dehydrated through a graded series of ethanol, immunolabeled primary cilia were found in endo- transferred to isoamyl acetate, and critical-point- crine cells filled with large secretory granules- char dried with liquid CO2. The dried specimens were acteristic of GH cells (Fig. 1f). Unlike the B cells, coated with osmium in a plasma osmium coater the primary cilia of GH cells tended to project from (Nippon Laser and Electronics Laboratory, Nagoya, the centrioles close to the cell surface, thus being Japan), and examined in a Hitachi H-4500 scanning shorter than those of B cells. The plasma membrane electron microscope (Hitachi, Tokyo, Japan) at an of cell bodies in both B cells and GH cells com- acceleration voltage of 10 kV. pletely avoided any immunolabeling by the anti- SSTR3 antibody. RESULTS Scanning electron microscopy Immunohistochemistry of SSTR3 in mice Scanning electron microscopy (SEM) confirmed We first immunostained using the anti-SSTR3 anti- primary cilia extending from pancreatic islet cells body major endocrine organs (the hypophysis, pine- (Fig. 2a–d) and pituitary endocrine cells of mice al body, thyroid/parathyroid glands, adrenal gland, (Fig. 2e, f). Our specimens for SEM revealed the and pancreas), gonads (ovary and testis), and some whole image of cilia after removing the extracellu- visceral organs containing dispersed endocrine cells. lar matrix and basement membrane by an alkaline The primary cilia immunoreactive for SSTR3 were maceration. B cells occupying the central region of found only in the pancreatic islets and the anterior pancreatic islets displayed large angular shapes with lobe of adenohypophysis (Fig. 1a-c). This restricted many dark spots disseminated on the cell surface. staining dose not imply a lack of primary cilia, These spots indicate the presence of insulin granules since the antibody against ACIII—another marker of that only poorly emit secondary electrons (Fig. 2c, the primary cilia—detected many primary cilia in d). The B cells issued single long primary cilia mea- the intermediate lobe of adenohypophysis, thyroid suring 7–14 μm in arbitrary directions (Fig. 2a, b). parafollicular cells, adrenal medulla (Fig. 1d), and The cilia of the B cells occurred on the cell surfaces corpus luteum in the ovary. Interestingly, the prima- apposed to islet cells and not on those surrounding ry cilia in the adenohypophysis were also immuno- vessels. The cilia were projected from invaginations labeled by the ACIII antibody with the same staining of the plasma membrane, subsequently pursued pattern as SSTR3 while those in pancreatic islets tortuous courses in narrow intercellular gaps, and were immunonegative for ACIII. ultimately entered a canalicular space bordered by In the pancreas, cells with SSTR3-immunoreactive microvilli of the endocrine cells (Fig. 2b). Such a cilia were restricted to the islets of Langerhans, in microvillous channel coursed along every cell edge which all insulin cells possessed SSTR3-immunore- where three or four endocrine cells met, to consti- 76 T. Iwanaga et al.

Fig. 1 Immunohistochemistry of primary cilia in pancreatic islets (a, c, e), adenohypophysis (b, f), and the adrenal gland of mice (d). The pancreas and hypophysis were double-stained for somatostatin receptor 3 (SSTR3, red) together with insulin, glucagon or GH. An antibody against SSTR3 labels primary cilia in insulin cells (a) and pituitary GH cells (b). Some, though not all, glucagon cells appear to possess SSTR3-immunoreactive cilia (c). Adrenal chromaffin cells possess short primary cilia immunoreactive for type III adenylyl cyclase (but not for SSTR3) (d). Electron microscopically, gold particles showing the existence of SSTR3 gather selectively in the plasma membrane of primary cilia of a B cell (e) and a GH cell (f). An arrow indicates centrioles located at the base of a primary cilium in the B cell (e). N: nucleus Bars 20 μm (a–d), 1 μm (e, f) tute a reticular system of intercellular canalicule cluding large round cells which corresponded closely that communicated with perivascular spaces in the to GH-secreting cells in shape—were seen to extend islets. Some cilia in the intercellular passages were a short primary cilium ranging in length from 2.8 to directed toward the blood vessels, while others were 7.5 μm (Fig. 2e, f). The round endocrine cells dis- extended in the opposite direction (Fig. 2c). Occa- played many bright spots on the cell surfaces, sug- sionally, double primary cilia were found to protrude gesting the presence of secretory granules that emit from a single invagination of the plasma membrane excess amounts of secondary electrons (Fig. 2e). (Fig. 2d). Primary cilia always originated from the contact In the hypophysis, all the parenchymal cells—in- surfaces between endocrine cells. The cilia occurred Primary cilia in pancreatic islets 77

Fig. 2 Scanning electron micrographs of primary cilia in pancreatic islet cells (a–d) and pituitary endocrine cells of mice (e, f). The extracellular matrix has been thoroughly removed by alkaline maceration to expose the surfaces of cells. Arrows indicate exposed primary cilia. One long primary cilium projects from each islet cell, pursues a tortuous course, and frequently enters an intercellular canalicule endowed with microvilli (a–c). Occasional islet cells possess double primary cilia projecting from a deep invagination of the cell membrane (d). Primary cilia of pituitary endocrine cells are shorter and less flexuous than those of islet cells e( , f). Bars 5 μm (a, e), 1 μm (b–d, f) solitarily, or in small groups. In the latter case, two Primary cilia in pancreatic islets of other animal to four endocrine cells projected their own cilium species and MIM6 cells into a narrow space that was surrounded by their To examine the existence of SSTR3-expressing cilia small facets sparsely covered with microvilli and in other animal species, the pancreas of the rat, microplicae. Such intercellular spaces were separat- hamster, and guinea pig was stained using the same ed from one another instead of forming an intercon- antibody against the C-terminal amino acids of nected system of canalicule. SSTR3 which are common to the mouse and rat molecules. At first, we stained the brain of these animals for checking the crossreactivity of the anti- 78 T. Iwanaga et al. body. The SSTR3 antibody immunolabeled many DISCUSSION primary cilia in the brain of the rat and hamster, but did not any cilia in the guinea pig brain, implying a Hypothalamic somatostatin is released at the median sufficient crossreactivity to the rat and hamster. In eminence, conveyed by the hypophysial portal sys- the hamster pancreas, primary cilia of insulin cells tem, and regulates GH secretion in the adenohy- were immunoreactive for SSTR3 (Fig. 3a), whereas pophysis. In the pancreas, somatostatin is secreted no immunoreactive primary cilia were found in the from D cells and exerts inhibitory actions against is- islets of the rat as well as the guinea pig. The anti- let hormone secretion, possibly in a paracrine man- body against acetylated tubulin could detect many ner. It is worth noting that the localization of SSTR3 immunoreactive primary cilia in the islet cells— in the primary cilia of endocrine cells is restricted largely insulin cells—of the hamster and guinea pig to the two main target cells of somatostatin in the (Fig. 3b). In the rat (Fig. 3c), glucagon cells were body. Among other endocrine cells, somatostain is endowed with tubulin-immunoreactive cilia, but in- secreted by thyroid parafollicular cells (13), adrenal sulin cells lacked any cilia immunoreactive for acet- chromaffin cells (15), gut endocrine cells (12), and ylated tubulin. A cell line of murine insulin cells dispersed endocrine cells in the respiratory (8) and (MIN6) possessed the primary cilia immunoreactive urogenital tracts (29). However, no adjacent endo- for SSTR3 as well as tubulin (Fig. 3d). crine and exocrine cells were provided with the SSTR3-expressing primary cilia in endocrine sys- tems other than the adenohypophysis and pancreas, though the primary cilia immunoreactive for ACIII

Fig. 3 Primary cilia in the pancreatic islets of other animal species and a murine insulin cell line. Primary cilia and insulin cells are labeled red and green, respectively (a–c). SSTR3 antibody stains many but short primary cilia on insulin cells of the hamster (a). In Fig. 3b and 3c, sections from the guinea pig and rat pancreas are double-stained for tubulin (red) and insulin (green). The tubulin-immunoreactive cilia are largely localized on insulin cells in the guinea pig (b), while in the rat (c) they are restricted to the periphery of an islet, namely the glucagon cell region (asterisks). Arrowheads indicate nerve fibers immunoreactive for tubulin (c). A mouse insulin cell line (MIN6) possesses the primary cilia immunoreactive for tubulin (d). In Fig. 3d, nuclei are stained green with a nucleic acids-sensitive dye. Bars 20 μm Primary cilia in pancreatic islets 79 were found in a variety of cells, such as the adrenal lation or via intercellular spaces to act upon other chromaffin cells and thyroid parafollicular cells. islet cells in a paracrine manner. Some morpholo- There are five subtypes of somatostatin receptors gists have recognized the development of a special (SSTRs) that have been identified so far, each hav- intercellular canalicular system apart from the peri- ing different sequence homologies, distribution pat- vascular spaces within pancreatic islets (7, 39). As terns, and affinities to antagonists and agonists. shown by the present SEM observation, the primary Expressions of all SSTR subtypes can be recognized cilia were usually located in these intercellular cana- in both the pancreatic islets (33) and adenohypophy- licules, i.e., microvillous channels among islet cells, sis (40). Immunohistochemical studies for SSTRs— but did not face the perivascular spaces. Thus, the including SSTR3—localized the immunoreactivities primary cilia on insulin cells are well situated to de- in the whole cell bodies of endocrine cells in pan- tect directly somatostatin released from D cells and creatic islets (16, 18, 25) and adenohypophysis (17, undiluted by blood. In contrast, pituitary GH cells 19), except for one study showing the SSTR2A im- must be regulated by somatostatin which is con- munoreactivity in the plasma membrane of islet veyed by the pituitary portal system. This fact may cells in the rat and human pancreas (14). In con- affect the length of primary cilia: short primary cilia trast, the present study showed the selective local- on GH cells is enough for sensing. ization of SSTR3 to the primary cilia of both insulin The use of SSTR subtype-selective drugs and cells and GH cells, with a heavy immunolabeling gene-knockout studies suggest that the inhibition of for SSTR3 along the plasma membrane of the pri- insulin secretion by somatostatin is mediated pre- mary cilia, while the plasma membrane of cell bod- dominantly by SSTR1 and SSTR5 in rodents (32, ies lacked any immunoreactivity for SSTR3. Recent 33, 36). An in vitro study of human pancreatic islets studies have identified several proteins, such as Bar- also reported that somatostatin inhibited both insulin det-Biedl syndrome (BBS) proteins, which mediate and glucagon release through SSTR1, SSTR2, and vesicular transport to the primary cilia (20). BBS SSTR5 (30). Thus, it is likely that SSTR3 on the proteins in neurons of the mouse brain are required primary cilia may regulate insulin secretion and oth- for functional development of the primary cilia; the er B cell functions via a unique mechanism which genetic deletion of BBS proteins failed to gather differs from the reception of somatostatin on the SSTR3 and MCHR1 into the primary cilia of neu- cell body of B cells. This idea accords with a find- rons, while another membrane-bound ciliary protein, ing that a genetic deletion of the primary cilia with ACIII, was normally localized to the primary cilia inactivated Kif3a in the whole pancreas induced se- in the knockout mice (3). BBS is characterized vere dilation of the duct system in the exocrine pan- by obesity as well as renal anomalies (35), and creas but did not affect basic islet formation MCHR1 is involved in the regulation of feeding and (arrangement of A and B cells) and functions (glu- energy balance (24). Therefore, an interesting inqui- cose tolerance tests) (5). Since a cell line of murine ry to be addressed in future studies is whether BBS insulin cells (MIN6) consistently possessed the pri- patients and BBS protein-knockout mice normally mary cilia expressing SSTR3, the precise function express SSTR3 on the primary cilia of insulin cells. of the primary cilia in B cells may be verified in fu- Cells of pancreatic ducts and islets possess the ture studies by cell-biological methods including a primary cilia, but acinar cells of the exocrine pan- controlled formation of cilia. creas apparently lack them (1, 5, 22, 39). An elec- There was a species difference in the existence of tron microscopic study by Yamamoto and Kataoka primary cilia that must be linked to species differ- (39) has reported in detail the ultrastructure of pri- ence in the function of somatostatin/SSTRs. The mary cilia on islet cells of some animal species. The lack of SSTR3-immunoreactive primary cilia in the primary cilia were projected from the Golgi region pancreatic islets of guinea pigs may be due to non- to intercellular spaces and their basal part was usu- crossreactivity of the antibody raised against the ally surrounded by deep invaginations of the plasma mouse/rat SSTR3. However, this dose not hold true membrane. In an immunostaining of mouse pancre- of the rat, because the antibody could detect SSTR3- atic islets with an antibody against acetylated tubu- expressing primary cilia in various regions of the rat lin, the primary cilia were mostly localized in the brain (11 and the present study). Instead, insulin lateral surface of B cells away from capillaries (9), cells in the rat pancreas did not possess the primary in agreement with the present SEM study. It is cilium itself, which should be detectable by immu- generally believed that somatostatin released from nostaining for tubulin. 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