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Am J Physiol Endocrinol Metab 293: E347-E354, 2007. First published April 10, 2007; doi:10.1152/ajpendo.00055.2007
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New functional aspects of the neuroendocrine marker secretagogin based on the characterization of its rat homolog

W. Gartner,1 G. Vila,1 T. Daneva,1,2 A. Nabokikh,1 F. Koc-Saral,1 A. Ilhan,1 O. Majdic,3 A. Luger,4 and L. Wagner1

1Department of Medicine III, 3Department of Immunology, and 4Division of Endocrinology and Metabolism, Medical University Vienna, Vienna, Austria; and 2Bulgarian Academy of Science, Sofia, Bulgaria

Submitted 23 January 2007 ; accepted in final form 4 April 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Secretagogin is a recently cloned human beta-cell-expressed EF-hand Ca2+-binding protein. Converging evidence indicates that it exerts Ca2+ sensor activity and is involved in regulation of insulin synthesis and secretion. To obtain a potent tool for the extension of its functional analysis in rat in vitro systems, we cloned the rat homolog of human secretagogin. Using comparative sequence analysis, immunostaining, and immunoblotting, we demonstrated a high degree of sequence homology and similar tissue expression patterns of human and rat secretagogin. Highest rat secretagogin expression levels were found in pancreatic beta-cells. On the basis of newly generated anti-rat secretagogin antibodies, we established a rat secretagogin-specific sandwich capture ELISA and demonstrated release of secretagogin from viable Rin-5F cells. Dexamethasone treatment of Rin-5F cells resulted in an increased secretagogin release rate, which was inversely correlated with insulin secretion. In contrast, the secretagogin transcription rate was markedly reduced. This resulted in a decreased intracellular secretagogin content under the influence of dexamethasone. Sucrose gradient cell fractionation analysis of Rin-5F cells confirmed the predominant cytosolic localization of secretagogin, with only limited association of secretagogin with insulin granules. The loss of intracellular secretagogin after dexamethasone treatment affected predominantly the insulin granule-associated secretagogin fractions. The sequence homology and the comparable tissue expression patterns of human and rat secretagogin indicate conserved intracellular functions. The effects of dexamethasone on the total secretagogin content in Rin-5F cells and on its intracellular distribution might result in an impaired Ca2+ sensitivity of dexamethasone-treated insulin-secreting cells.

EF-hand protein; insulin granules; dexamethasone; calcium sensor

IN PANCREATIC beta-CELLS, intracellular Ca2+ is the primary downstream second messenger (3, 20). Most prominently, oscillations of intracellular Ca2+ concentration are critically involved in nutrient-regulated insulin secretion (7). Upon glucose metabolism, the ATP-to-ADP ratio increases, with subsequent closure of ATP-dependent K+ channels, resulting in an increase of the membrane potential (11). This induces the opening of L-type Ca2+ channels and leads to an increase in intracellular Ca2+ concentration, which triggers the assembly of the exocytotic machinery and subsequent insulin secretion (5, 32). In this respect, the additional importance of Ca2+ in vesicle transport to the docking sites, which provides the substrate for the delayed phase of the biphasic insulin secretion, must be mentioned (5). In the field of Ca2+-mediated insulin secretion, the importance of spatial regulation of Ca2+ concentration, with specific impact of accumulation of voltage-gated L-type Ca2+ channels at the site of vesicle docking, seems of further interest (32). Additionally, intracellular Ca2+ is involved in the regulation of insulin gene transcription, and it has been demonstrated to mediate the activity of various beta cell-expressed kinases (22, 25).

Interestingly, steroid hormones have been demonstrated to exert profound effects on insulin transcription and secretion, which are at least partly attributable to altered Ca2+ signaling. For example, dehydroepiandrosterone decreased induced intracellular Ca2+ release, with subsequent impairment of insulin secretion (27). Glucocorticoids influence expression of the insulin gene transcription factors HES-1, BETA2/NeuroD, and PDX-1, which resulted in impaired insulin synthesis and suppression of beta-cell development (17, 41, 42). Most importantly, dexamethasone exerted direct inhibitory effects on the insulin-secreting capacity of mouse pancreatic islets (24). In light of adequate intracellular Ca2+ signaling, a genomic action of dexamethasone in beta-cells, leading to a decrease in the efficacy of cytoplasmic Ca2+ signaling, has been hypothesized as the underlying mechanism (24).

The transduction and efficacy of intracellular Ca2+ signals mainly depend on Ca2+-binding proteins (33). In the pancreatic beta-cell, expression of multiple Ca2+-binding proteins has been described. We recently cloned the Ca2+-binding protein secretagogin from a human beta-cell library (46, 47). Secretagogin belongs to the EF-hand family Ca2+-binding proteins, which exert their Ca2+-binding capacity via the evolutionary highly conserved helix-loop-helix EF-hand motifs (9, 18, 23, 31). In addition to its strong expression in beta-cells, secretagogin is also found in other endocrine tissues and in distinct neurons of the central nervous system (4, 8, 16, 37). Although the exact function of secretagogin is still a matter of research, its strong expression in insulin-secreting cells (10, 48), its influence on insulin expression (46), and recent data indicating glucose-induced upregulation of secretagogin expression in beta-cells (1, 43) highlight the functional importance of this Ca2+-binding protein in pancreatic beta-cells.

Because of the lack of human in vitro beta cell systems, studies on beta-cell function mostly rely on rodent systems (21). In this light, the focus of this study was the characterization of the rat homolog of human secretagogin. Using the rat secretagogin-expressing insulinoma cell line Rin-5F, we demonstrated dexamethasone-inducible reduction of intracellular secretagogin, which might contribute to the effects of dexamethasone on insulin homeostasis.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Rin-5F cells were cultured in RPMI 1640 supplemented with 10% (vol/vol) FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO2. After 5–6 days, cell passage was performed using trypsin (0.25%) and EDTA (0.03%) in Hanks' buffered saline solution.

Cloning of rat secretagogin. For RNA isolation from Rin-5F cells, Trizol (Roche Diagnostics, Mannheim, Germany) was used according to the manufacturer's instructions. First-strand cDNA was synthesized using the Advantage RT-for-PCR Kit (Clontech, Palo Alto, CA). PCR thermal cycling parameters (30 cycles) were set to 94°C (30 s) for denaturation, 57°C (30 s) for annealing, and 68°C (60 s) for synthesis. For generation of the full-length coding sequence, PfuUltra High-Fidelity DNA polymerase (Stratagene, La Jolla, CA) was used, along with the following PCR primers: 5'-caccatggacaacgcacacag-3' (forward) and 5'-tcaggggtttatttttagccc-3' (reverse). The initial denaturation step was performed at 94°C for 4 min. An A-overhang was attached to the PCR products using Taq DNA polymerase (GenScript, Piscataway, NJ), and the PCR products were cloned into the pCR 2.1 vector (Invitrogen, Carlsbad, CA). The ligated vector was amplified in DH5{alpha} Escherichia coli. The insert was cut out using EcoR I (Roche Diagnostics) and analyzed by electrophoretic separation. Plasmids containing the right-sized insert were subjected to sequence analysis.

Stimulation assays. For stimulation assays, Rin-5F cells were seeded into 12-well tissue culture plates. At a confluence of ~75% (~72 h of culture), the tissue culture medium was removed, and the cells were subsequently incubated in native tissue culture medium (see above) supplemented with one of the following substrates: 100 nM dexamethasone, 10–5 M LY-294002, 10–5 M PD-098059, or 500 µM cAMP (all from Sigma-Aldrich, Vienna, Austria). After an incubation period of 1–24 h, tissue culture medium was harvested and frozen at –30°C until further analysis using the newly established rat secretagogin-specific sandwich capture ELISA (see below) and a rat insulin-specific RIA (Linco Research, St. Louis, MO), respectively. Adherent cells were washed three times with PBS and lysed using Trizol reagent (Roche Diagnostics).

Generation of recombinant rat secretagogin variants. To obtain a COOH terminally truncated rat secretagogin exopeptide, we chose a plasmid containing a stop codon following amino acid 113. It was cloned into the pGEX1{lambda}T vector (Amersham Pharmacia Biotech, Uppsala, Sweden) by means of a PCR-based modification [primers: 5'-tcggatccatggacaacgcacacag-3' (forward) and 5'-tcaggggtttatttttagccc-3' (reverse)]. The peptide was subsequently expressed in E. coli BL21 as a glutathione S-transferase (GST)-fusion protein. After 4 h of induction with 100 µM isopropyl-1-thio-beta-D-galactopyranoside (Sigma-Aldrich), the bacteria were pelleted, suspended in ice-cold PBS containing 0.1 mM PMSF, and sonicated until the cell suspension became translucent. Subsequently, Triton X-100 (Sigma-Aldrich) was added to a final concentration of 0.1%, and insoluble material was removed by centrifugation. The resultant supernatant was incubated with glutathione-Sepharose 4B slurry (Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 h at 4°C under constant rotation. After four washing steps with ice-cold PBS, the 113-aa rat secretagogin exopeptide was liberated by thrombin cutting and subsequent pelleting of the slurry. The purified peptide was frozen at –80°C or lyophilized for further use. Engineering of recombinant full-length rat secretagogin was similar using the pGEX1{lambda}T vector and BL21 as the host E. coli strain.

Generation of mouse anti-rat secretagogin monoclonal antibodies. The NH2-terminal 113-aa exopeptide was emulsified in Freund's adjuvant and used for immunization of 6-wk-old BALB/c mice by subcutaneous injection. Incomplete Freund's adjuvant was used for additional immunizations on days 14 and 28. Monoclonal antibody generation was performed as described previously (30). Briefly, the spleens of the immunized mice were removed 4 days after the last immunization, and spleen cell suspensions were fused with the prepared P3 cell line under standard conditions. Outgrowing clones were screened after 14 days of selection by a direct ELISA technique with use of rat secretagogin-coated plates. Positive clones were further selected by the fluorescence-activated cell sorting technique using fixed and permeabilized Rin-5F cells. Clones generating a significant shift in the median fluorescence intensity were subjected to further subcloning and expansion.

Immunostaining. Rin-5F cytoslide preparations or cryosections of rat pancreas were fixed with acetone (3 min). Subsequently, the slides were wetted in PBS. Pancreatic tissue sections and Rin-5F cytoslides were coincubated (2 h) with a rabbit polyclonal anti-secretagogin antibody (16) and a murine monoclonal anti-rat insulin antibody (1:200 dilution, MAb D3E7, Research Diagnostics, Flanders, NJ). The primary antibodies were followed by a parallel incubation (30 min) with a goat anti-mouse tetramethylrhodamine isothiocyanate-labeled affinity-purified F(ab')2 fragment (for insulin detection) and a goat anti-rabbit Alexa Fluor 488-labeled affinity-purified F(ab')2 fragment (for secretagogin detection). The secondary antibodies were obtained from Accurate Chemical and Scientific (Westbury, NY). Nuclear staining was performed with 4,6-diamidino-2-phenylindole. The slides were subsequently mounted in Fluoprep (BioMerieux, Marcy L'Etoile, France). Immunofluorescence signals were visualized by confocal microscopy using the Axiovert 200M confocal fluorescence microscope (Zeiss, Jena, Germany), and the images were processed using LSM 5 software (Zeiss). In each experiment, negative controls with BSA-containing PBS without the primary antibody were included. All incubation steps were performed in a moist chamber protected from light. Each incubation step was followed by three 5-min PBS washes.

Cell fractionation. Rin-5F cells were cultured in 75-cm2 tissue culture flasks. At 75% confluence, Rin-5F cells were washed twice with ice-cold PBS and then scraped into 2.5 ml of ice-cold homogenization buffer (250 mM sucrose and 3 mM imidazole, pH 7.4) supplemented with protease inhibitors (10 µg/ml aprotinin, 1 µg/ml pepstatin, 10 µg/ml leupeptin, and 0.8 mM Pefabloc). The cell suspension was then passed four times through a 22-gauge needle. After centrifugation (2,500 rpm, 10 min, 4°C), the postnuclear supernatant was overlaid on a discontinuous sucrose gradient (50% and 20%, respectively) in 14 x 95-mm polyallomer centrifuge tubes (Beckman Instruments, Palo Alto, CA) and centrifuged at 40,000 rpm for 1 h at 4°C in the SW40 TI rotor of an ultracentrifuge (model L-80, Beckman). The resultant density equilibrium was then fractionated using a peristaltic pump. Starting at the bottom, we collected 21 aliquots. The fractions were subjected to immunoblot analysis for rat secretagogin, 25-kDa synaptosomal-associated protein (SNAP-25), and rat insulin.

Western blot analysis. Protein aliquots were loaded on 12% SDS-polyacrylamide gels, which were run at 200 V using Tris-glycine or Tris-tricine (<20-kDa protein) running buffer. A semidry blotting device was used to electrophoretically transfer the protein onto nitrocellulose. The blotted membrane was blocked with 10% skim milk for 1 h. Antigen detection was performed by incubations (2 h, constant shaking at room temperature) with the polyclonal rabbit anti-secretagogin antibody (16), an anti-beta-actin monoclonal antibody (MAb AC-15, Novus Biologicals, Littleton, CO), a mouse monoclonal anti-rat insulin antibody (MAb D3E7, Research Diagnostics, Flanders, NJ), and the anti-SNAP-25 antibody (MAb SP12, Sigma-Aldrich). Secondary antibody was a peroxidase-conjugated goat anti-mouse antibody or a peroxidase-conjugated goat anti-rabbit antibody (both from Dako, Glostrup, Denmark), respectively (incubation time 30 min). Each incubation step was followed by two washes with PBS containing 0.1% Tween 20 for 10 min. Sites of specific antibody binding were visualized by BM chemiluminescent reagents (Roche Molecular Biochemicals), and electronic images were recorded using Lumi-Imager F1 (Roche Molecular Biochemicals).

Rat secretagogin-specific sandwich capture ELISA. The ELISA was performed in flat-bottomed, 96-well Reacti-Bind goat anti-rabbit plates (catalog no. 15135, Pierce, Rockford, IL), which were coated overnight at 4°C with rabbit anti-secretagogin antiserum (1:200 dilution) (16). After a brief wash with PBS, 100-µl aliquots of biological fluids and tissue culture supernatant were loaded and incubated for 2 h. Bound antigen was subsequently detected by sequential incubations with a rat secretagogin-specific mouse antibody (1:1,000 dilution, 90 min) and a peroxidase-labeled goat anti-mouse antibody (1:10,000 dilution, 90 min; Dako). The incubations were performed in a moist chamber under constant shaking. Each incubation was followed by three washes with PBS containing 0.1% Tween 20 in the ELX Autostrip Washer (Biotex Instruments, Vinooski, VT). The ELISA was developed using the tetramethylbenzidine dihydrochloride two-component peroxidase substrate solution (Kirkegaard & Perry, Gaithersburg, MD). The reaction was stopped by addition of 1 M H3PO4 and quantified by absorbance at 450 nm using a Powerwave ELISA reader (Biotek Instruments), including a standard curve with rat recombinant secretagogin.

Quantitative real-time PCR. Total RNA was isolated using the Trizol reagent (Roche Diagnostics) according to the manufacturer's instructions. Isolated RNA was reverse-transcribed after treatment with DNase I using Superscript II and random hexamer primers according to the test manual (Invitrogen). Briefly, 1 µg of RNA diluted in 9 µl of DNase reaction buffer was incubated with 1 µl of DNase I at room temperature for 15 min. The reaction was stopped using 1 µl of 25 mM EDTA followed by incubation at 65°C for 15 min. For cDNA synthesis, 4 µl of 5x first-strand buffer together with 2 µl of DTT, 1 µl of dNTP mix (10 mM), 1 µl of random primers, and RNase inhibitor with 1 µl of SuperScript II were incubated for 10 min at 25°C and then for 50 min at 42°C. The reaction was stopped by incubation at 70°C for 15 min, and 80 µl of H2O were added. Quantitative real-time PCR amplifications were performed using gene-specific FAM-TAMRA-labeled Assay-on-Demand (Applied Biosystems, Foster City, CA) normalized to 18S FAM-TAMRA-labeled endogenous control (Applied Biosystems). Gene-specific mRNA was quantified in duplicates in a PRISM 7000 cycler (Applied Biosystems).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of rat secretagogin. To accomplish our goal of functional analysis of the human beta cell-expressed Ca2+-binding protein secretagogin using the rat insulinoma cell line Rin-5F, our initial attempts focused on the characterization of its rat homolog. For this purpose, we performed reverse-transcription PCR of cell extracts derived from Rin-5F cells. Using 5'- and 3'-terminal exon-specific primers according to the rat secretagogin gene sequence (Scgn, gene identification no. 306942, location 17p11), we obtained a ~950-bp cDNA PCR product, which was cloned and subjected to sequencing. The predicted amino acid sequences of rat secretagogin and its human and mouse homologs exhibit a high degree of homology (Fig. 1). The greatest extent of sequence variation is found at the NH2-terminal protein part of rat secretagogin, which results in loss of two EF-hand motifs. The predicted size of the rat secretagogin full-length protein was 30 kDa. The full-length cDNA sequence of rat secretagogin is available under National Center for Biotechnology Information accession no. NM_201561.


Figure 1
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  		Fig. 1. Amino acid sequences of rat, mouse, and human secretagogin (sec). Rat secretagogin primary structure was deduced from cloned rat secretagogin cDNA sequence. Amino acid sequences of human, mouse, and rat secretagogin exhibit a high degree of homology (shaded). Highest degree of sequence variability is localized within the NH2-terminal protein. This results in loss of the 2 NH2-terminal EF-hand motifs of rat secretagogin. Rat secretagogin specific antibody was generated using a 113-aa NH2-terminal exopeptide of rat secretagogin as immunogen.

 
To test whether the high degree of sequence homology between human and rat secretagogin is reflected by comparable tissue expression patterns, we performed rat secretagogin expression analysis. Immunoblotting using a rabbit polyclonal anti-secretagogin antibody revealed the highest secretagogin expression levels in rat pancreas. Additionally, considerable rat secretagogin expression was found in the prostate and adrenal glands; lower rat secretagogin expression levels were found in other tissues known to contain neuroendocrine cells (Fig. 2). The immunoblot analysis confirmed the predicted size of rat secretagogin of ~30 kDa but did not detect protein variants. Confocal immunofluorescence analysis using the rabbit anti-secretagogin antibody revealed strong expression of secretagogin in Rin-5F cells (Fig. 3A). Secretagogin was mainly found at cytoplasmic sites, but it was also detectable within the nuclei (Fig. 3). Rat insulin-specific coimmunostaining (Fig. 3B) revealed only a limited extent of intracellular secretagogin-insulin colocalization (Fig. 3C). In analogy to its human homolog, confocal immunofluorescence analysis of secretagogin-insulin double-stained rat pancreatic tissue sections showed a nearly exclusive localization of rat secretagogin within the pancreatic islets (Fig. 3D). Additionally, singular cells located within the ductal epithelium were characterized by secretagogin immunostaining (data not shown). Within the islets, rat secretagogin was expressed by beta-cells (Fig. 3E), but it was also present in insulin-negative cells at the periphery, most probably representing A and D cells. Our findings in Rin-5F cells were confirmed by only a limited colocalization of rat secretagogin and insulin (Fig. 3F).


Figure 2
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  		Fig. 2. Multiple-tissue immunoblot of rat secretagogin. Multiple-tissue immunoblot analysis was performed using a rabbit anti-secretagogin antibody. Secretagogin expression of individual tissues was quantified by parallel beta-actin immunoblotting and subsequent densitometric scanning of immunosignals. Results are expressed as secretagogin-to-beta-actin ratio of individual tissues. Highest expression of rat secretagogin was found in pancreas-derived tissue extracts. Secretagogin was additionally expressed to a considerable extent in prostate and adrenal glands and to a lesser extent in gastrointestinal-derived tissues and thyroid. Only full-length ~30-kDa secretagogin was detectable. Results represent values from 1 of 4 experiments with similar results.

 

Figure 3
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  		Fig. 3. Rat secretagogin expression in Rin-5F cells and pancreatic islets. Rin-5F cell cytoslides and pancreatic crysections were coimmunostained with a rabbit anti-secretagogin antibody and a murine monoclonal anti-rat insulin antibody and then subjected to confocal microscopy. 4,6-Diamidino-2-phenylindole was used for nuclear staining. A–C: expression of rat secretagogin in Rin-5F cells. Secretagogin (green) was found predominantly within the cytoplasm; secretagogin-specific immunofluorescence was only marginal at nuclear sites (blue). Parallel insulin immunostaining (red) revealed limited secretagogin-insulin colocalization (yellow). D–F: confocal immunofluorescence analysis of pancreatic tissue sections. Note strong secretagogin-specific immunofluorescence (green) of pancreatic islets. Beta cells were identified by parallel secretagogin (green) and insulin (red) immunostaining (yellow). Additionally, secretagogin was found in non-insulin-producing islet cells.

 
Rat secretagogin is released from Rin-5F cells. To test whether rat secretagogin is released from Rin-5F cells, we used a newly established rat secretagogin specific sandwich capture ELISA to measure the rat secretagogin content in Rin-5F tissue culture supernatant. The species specificity of this ELISA is based on a newly generated mouse anti-rat secretagogin antibody directed against the NH2 terminus of rat secretagogin. Using this antibody for antigen detection and a rabbit anti-secretagogin antibody for antibody capture, we obtained reproducible linear standard curves within a range of 0.15–16 ng/ml. The inter- and intra-assay coefficients of variation were 5% and 7%, respectively. Analysis of Rin-5F supernatant after 24 h of culture revealed considerable detectability of rat secretagogin, with a release rate of 349 ± 58 pg·ml–1·24 h–1.

Consecutively, we screened various agents (100 nM dexamethasone, 10–5 M LY-294002, 10–5 M PD-098059, and 500 µM cAMP) known to potentially affect beta cell physiology for their influence on the secretagogin release rates (Fig. 4). After a 24-h incubation period, Rin-5F cell culture supernatants were harvested and subjected to ELISA. Interestingly, dexamethasone exhibited the strongest stimulatory effect on rat secretagogin release, which increased to 717 ± 136 pg·ml–1·24 h–1 (205 ± 39% of control, P < 0.005 by paired t-test). Whereas addition of the phosphatidylinositol 3-kinase inhibitor LY-294002 also exerted stimulatory effects on rat secretagogin release (541 ± 45 pg·ml–1·24 h–1, 155 ± 10% of control, P < 0.05 by paired t-test), exogenous cAMP (475 ± 43 pg·ml–1·24 h–1, 136 ± 21% of control) and the MAPK inhibitor PD-098059 (331 ± 46 pg·ml–1·24 h–1, 95 ± 13% of control) did not significantly affect the release of rat secretagogin. In parallel with the determination of rat secretagogin content, we measured the insulin concentration in the supernatant of Rin-5F cells. Interestingly, dexamethasone induced an inverse release pattern of insulin and rat secretagogin, with a marked suppression of the insulin release (646 ± 46 µU/ml, 33 ± 2% of control, P < 0.0001 by paired t-test). In contrast, LY-294002 and PD-098059 had no significant effects on insulin secretion [1,809 ± 108 µU/ml (93 ± 6% of control) and 2,158 ± 140 µU/ml (110 ± 7% of control), respectively]. Expectedly, cAMP induced an upregulation of insulin secretion (3,161 ± 173 µU/ml, 162 ± 9% of control, P < 0.005 by paired t-test).


Figure 4
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  		Fig. 4. Influence of dexamethasone (Dex), cAMP, the MAPK inhibitor PD-098059 (PD-09), and the phosphatidylinositol 3-kinase inhibitor LY-294002 (LY-29) on rat secretagogin (sec) and insulin (ins) release from Rin-5F cells. A newly established rat secretagogin-specific sandwich capture ELISA was used to confirm release of rat secretagogin from viable Rin-5F cells. In parallel, a rat insulin-specific RIA was used to determine insulin secretion rates of Rin-5F cells. Note antiparallel effects of dexamethasone (100 nM) on secretagogin and insulin release rates, with induction of rat secretagogin release from Rin-5F cells. Exogenous cAMP (500 µM) exerted minor stimulatory effects on insulin and rat secretagogin, whereas LY-294002 (10–5 M) stimulated release of rat secretagogin but had no effect on insulin secretion capacity. PD-098059 (10–5 M) influenced neither insulin nor secretagogin release rate. Values are means ± SD; all experiments were performed in quadruplicates. *P < 0.05; **P < 0.005; ***P < 0.0001 (paired t-test).

 
Effect of dexamethasone on rat secretagogin expression. To evaluate whether the increased release of rat secretagogin from dexamethasone-treated Rin-5F cells relies on induction of rat secretagogin expression, Rin-5F cells were incubated with dexamethasone for 1, 6, 12, and 24 h and secretagogin-specific quantitative RT-PCR of Rin-5F cells was performed. Surprisingly, we observed a profound and significant suppression of secretagogin expression in dexamethasone-treated Rin-5F cells compared with untreated Rin-5F cells (Fig. 5, top). The suppressive effect became overt after 6 h (relative secretagogin expression 33 ± 2% of control, P < 0.0005), reached a maximum after 12 h (relative secretagogin expression 27 ± 20%, P < 0.05), and slightly decreased after 24 h (relative secretagogin expression 30 ± 14%, P < 0.05). To confirm the reliability of the chosen in vitro system, Pdx-1, which is known to be downregulated under dexamethasone treatment, was included as control (Fig. 5, bottom). Expectedly, dexamethasone decreased Pdx-1 expression, with a peak suppressive effect after 12 h (relative Pdx-1 expression 17 ± 6%, P < 0.005).


Figure 5
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  		Fig. 5. Effect of dexamethasone on rat secretagogin transcription. Top: quantitative RT-PCR of rat secretagogin revealed a strong, suppressive effect of 100 nM dexamethasone on secretagogin expression in Rin-5F cells. Peak inhibitory effect of dexamethasone was observed after 12–24 h of incubation. Bottom: relative expression of Pdx-1, which is known to be suppressed by dexamethasone. Co, control. Values are means ± SD; all experiments were performed in triplicates. *P < 0.05; **P < 0.005 (paired t-test).

 
Effect of dexamethasone on intracellular rat secretagogin. To characterize the intracellular distribution pattern of rat secretagogin and to evaluate effects of dexamethasone on the compartmentalization and overall content of secretagogin, dexamethasone-treated Rin-5F cells and controls were subjected to sucrose gradient fractionation. The individual cell fractions were analyzed by parallel immunoblotting against rat secretagogin and SNAP-25, as well as by immunoblotting against rat insulin (Fig. 6A, top). The rat secretagogin quantity of the individual fractions was determined by subsequent densitometric immunosignal scanning (Fig. 6A, bottom).


Figure 6
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  		Fig. 6. Effect of dexamethasone on intracellular localization of rat secretagogin in Rin-5F cells. A: analysis of intracellular distribution of rat secretagogin in Rin-5F cells by sucrose density gradient cell fractionation. Individual fractions were subjected to rat secretagogin immunoblotting and subsequent densitometric scanning of individual immunosignals. Granular cell fractions were identified by additional insulin and 25-kDa synaptosomal-associated protein (SNAP-25) immunoblotting. Under unstimulated conditions, rat secretagogin was mainly found in fractions representing the cytosol, but was also found in SNAP-25- and insulin-containing cell fractions. After 24 h of stimulation with 100 nM dexamethasone, especially the secretagogin content of the insulin and SNAP-25-associated fractions decreased. Results are representative of values from 1 of 3 experiments with similar results. B: cumulative secretagogin signal intensities of individual fractions revealed a significant reduction of total Rin-5F secretagogin content after 24 h of treatment with dexamethasone (relative secretagogin content of dexamethasone-treated cells 76 ± 6.4% of control, P < 0.05, n = 3). Results are representative of values from 1 of 3 experiments with similar results.

 
Rat secretagogin was found in a majority of the cytosolic fractions. Only a minor portion of rat secretagogin was associated with the secretory granules and with the membrane fractions, which were specified by insulin and SNAP-25 immunostaining, respectively. Interestingly, dexamethasone decreased the total Rin-5F secretagogin content (Fig. 6B; relative secretagogin content of dexamethasone-treated cells 76 ± 6.4% of control, P < 0.05, n = 3). Notably, the decrease of intracellular secretagogin affected predominantly the granular cell compartments (Fig. 6A). The cell fractionation analysis is consistent with the immunfluorescence data and with earlier findings indicating that only a subfraction of intracellular secretagogin colocalizes with the insulin-containing granules.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we characterized the rat homolog of the beta-cell-expressed Ca2+-binding protein secretagogin and demonstrated its release from Rin-5F, which was inducible by dexamethasone.

Because of the limited number of human in vitro systems to study insulin-secreting cells (6, 15, 28, 29), much of our knowledge on the intracellular signaling pathways in beta-cells relies on rodent cell systems (12, 21). Therefore, the determination of homologous protein characteristics between humans and rodents is essential. Because of its specific expression pattern, human secretagogin was established as a marker for neuroendocrine differentiation in humans (8). The restriction of rat secretagogin expression to the rat pancreatic islets, its strong expression in endocrine cell-containing tissues, and the similarity of its intracellular distribution to that of human secretagogin underline conserved intracellular functions of the protein in humans and rodents. We detected exclusively a full-length variant of rat secretagogin, in contrast to our recent findings in humans, which revealed additional expression of truncated secretagogin variants due to posttranscriptional processing (49).

In parallel to the comparable tissue expression patterns, our data revealed a high degree of sequence homology between human and rat secretagogin. This is consistent with other EF-hand family members, highlighting their importance in Ca2+-mediated intracellular processes (23, 31). The highest extent of sequence variation between the human and the rat homolog was found within the NH2-terminal part of the protein, which results in loss of two NH2-terminal EF-hand motifs in rat secretagogin. It was recently demonstrated that human secretagogin, despite its hexa-EF-hand structure, binds only four Ca2+ ions, with Ca2+-binding capacity absent in the two NH2-terminal EF-hand motifs (39). This underlines the minor functional relevance of the NH2-terminal sequence variation of rat secretagogin compared with its human counterpart.

Neither human nor rat secretagogin contains a signal peptide sequence, which is a characteristic of secreted proteins. Thus the detectability of secretagogin in the cell culture supernatant of viable Rin-5F cells stably expressing human secretagogin was a surprising finding (46). There are two possible explanations for this phenomenon: 1) a spillover effect due to the overexpression of secretagogin in transfected Rin-5F cells and 2) a release of human secretagogin from the rat insulinoma cell line because of species incompatibilities. However, in this study, we specifically detected considerable amounts of rat secretagogin in the supernatant of wild-type Rin-5F cells, implicating secretagogin secretion via signal sequence-independent pathways. At this point, the recently revealed association of secretagogin to the soluble NSF attachment protein receptor (SNARE) complex via binding of secretagogin to SNAP-25 seems of interest (39). The tight association of secretagogin with the secretory machinery might explain its release in the course of insulin granule fusion and membrane pore formation. However, the antiparallel regulation of insulin and secretagogin release observed in our studies argues against this hypothesis. Alternatively, recently deduced constitutive secretion processes in beta-cell might underlie the detectability of secretagogin in Rin-5F supernatant. These include transfer and secretion of immature granules as well as a trans-Golgi network-independent pathway involving the endosome-lysosome compartments (2, 44). Dexamethasone induced predominantly a decrease of the secretagogin content of the corpuscular cell compartments. This further supports the latter notion and makes a passive transmembrane diffusion of cytosolic secretagogin to the extracellular space less plausible. It has to be stressed that the extent of constitutive secretion seems to depend on the cellular differentiation state (45). Moreover, considerable differences between individual rodent cell lines (21) must be considered when our results are interpreted in relation to other cell lines or in vivo systems.

The diabetogenic effects of glucocorticoids are well known (26). The underlying mechanisms involve not only downregulation of GLUT2 expression with subsequent induction of insulin resistance but, also, include direct effects of glucocorticoids on insulin synthesis and secretion (13, 14, 19, 24, 35, 36, 40, 42). We observed an inverse effect of dexamethasone on insulin and secretagogin secretion of Rin-5F cells, with suppression of insulin secretion but induction of secretagogin release compared with untreated cells. At this point, the disproportionate secretion of insulin and islet amyloid polypeptide under the influence of dexamethasone, which was also attributed to constitutive islet amyloid polypeptide secretion, must be mentioned (34, 38, 45). In parallel, dexamethasone strongly inhibits secretagogin expression in Rin-5F cells, resulting in a reduction of the intracellular secretagogin content with dexamethasone treatment. On the basis of its Ca2+-binding capability, secretagogin exerts Ca2+ sensor, rather than Ca2+ buffer, functions (39). Thus, especially the loss of the secretagogin fraction associated with the insulin secretion machinery might result in a decreased efficacy of Ca2+ responsiveness under dexamethasone treatment. At this point, it seems of specific interest that attenuation of Ca2+ signaling has been hypothesized to underlie the impaired insulin secretion observed under the influence of dexamethasone (24).

In conclusion, our data demonstrate a high degree of homology between the sequences and tissue expression patterns of human and rat secretagogin, which implies comparable functions. Furthermore, we provide evidence for dexamethasone-induced loss of intracellular secretagogin. This might underlie the impaired Ca2+-sensing efficacy observed under the influence of dexamethasone.


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 ABSTRACT
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T. Daneva was supported by Bulgarian Ministry of Education and Science National Science Fund Grant B-1517/05.


   ACKNOWLEDGMENTS
 
We thank Rita Lang for technical support and Barbara Brunnmair for rat tissue dissection.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Wagner, Medical Univ. Vienna, Dept. of Medicine III, Waehringer Guertel 18-20, A-1090 Vienna, Austria (e-mail: ludwig.wagner{at}meduniwien.ac.at)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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