Rfamide Peptides 43Rfa and 26Rfa Both Promote Survival of Pancreatic B-Cells and Human Pancreatic Islets but Exert Opposite Effects on Insulin Secretion

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2380 Diabetes Volume 63, July 2014

Riccarda Granata,1 Fabio Settanni,1 Letizia Trovato,1 Davide Gallo,1 Iacopo Gesmundo,1 Rita Nano,2 Maria Pia Gallo,3 Loredana Bergandi,4 Marco Volante,4 Giuseppe Alloatti,3 Lorenzo Piemonti,2 Jérôme Leprince,5 Mauro Papotti,4 Hubert Vaudry,5 Huy Ong,6 and Ezio Ghigo1

RFamide Peptides 43RFa and 26RFa Both Promote Survival of Pancreatic b-Cells and Human Pancreatic Islets but Exert Opposite Effects on Insulin Secretion

Diabetes 2014;63:2380–2393 | DOI: 10.2337/db13-1522

RFamide peptides 43RFa and 26RFa have been shown because of their survival effects along with the effects to promote food intake and to exert different peripheral on insulin secretion, these ﬁndings suggest potential for actions through G-protein–coupled receptor 103 (GPR103) 43RFa and 26RFa as therapeutic targets in the treatment binding. Moreover, 26RFa was found to inhibit pancreatic of diabetes. insulin secretion, whereas the role of 43RFa on b-cell function is unknown, as well as the effects of both peptides on b-cell survival. Herein, we investigated the Pancreatic b-cell mass plays an essential role in glucose ISLET STUDIES effects of 43RFa and 26RFa on survival and apoptosis homeostasis. The reduced capacity of the endocrine pan- of pancreatic b-cells and human pancreatic islets. In creas to maintain an adequate insulin secretion, due to addition, we explored the role of these peptides on decreased b-cell mass and function, underlies both type insulin secretion and the underlying signaling mecha- 1 and type 2 diabetes (1). In type 1 diabetes, immune- nisms. Our results show that in INS-1E b-cells and hu- mediated release of inﬂammatory cytokines such as tu- man pancreatic islets both 43RFa and 26RFa prevented mor necrosis factor-a (TNF-a), interferon-g (IFN-g), and cell death and apoptosis induced by serum starva- interleukin-1b (IL-1b) has been implicated in b-cell apo- tion, cytokine synergism, and glucolipotoxicity, through ptosis (2). In type 2 diabetes, b-cell apoptosis results from phosphatidylinositol 3-kinase/Akt- and extracellular the combined action of increased plasma glucose/free signal–related kinase 1/2-mediated signaling. More- fatty acid levels (glucolipotoxicity) (3) and cytokines (4). over, 43RFa promoted, whereas 26RFa inhibited, Therefore, identifying molecules capable of increasing glucose- and exendin-4–induced insulin secretion, pancreatic b-cell survival may be crucial for the treatment

through Gas and Gai/o proteins, respectively. Inhibition and prevention of diabetes. of GPR103 expression by small interfering RNA blocked RFamide-related peptides constitute a family of biologically 43RFa insulinotropic effect, but not the insulinostatic active peptides terminating in arginine-phenylalanine-amide action of 26RFa. Finally, 43RFa, but not 26RFa, induced (Arg-Phe-NH2) at their C-terminus. They include a 26– cAMP increase and glucose uptake. In conclusion, amino acid RFamide peptide (26RFa), which was isolated

1Division of Endocrinology, Diabetes, and Metabolism, Department of Medical 6Faculty of Pharmacy, University of Montréal, Montréal, Québec, Canada Sciences, University of Torino, Torino, Italy Corresponding author: Riccarda Granata, [email protected]. 2Diabetes Research Institute, Division of Immunology, Transplantation, and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy Received 3 October 2013 and accepted 5 March 2014. 3Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy © 2014 by the American Diabetes Association. See http://creativecommons.org 4Department of Oncology, University of Torino, Torino, Italy /licenses/by-nc-nd/3.0/ for details. 5Laboratory of Neuronal and Neuroendocrine Differentiation and Communication, In- ternational Associated Laboratory Samuel de Champlain, Institute for Research and Innovation in Biomedicine, INSERM U-982, University of Rouen, Rouen, France diabetes.diabetesjournals.org Granata and Associates 2381 from the frog brain (5), and a longer form of 26RFa, of Geneva, Geneva, Switzerland) and cultured as previously a43–amino acid peptide with a pyroglutamylated described (24). RFamide (termed QRFP-43 or 43RFa). Both peptides Human Islet Isolation were identified as the cognate ligands of the orphan Human islets were obtained from pancreases of multi- G-protein–coupled receptor 103 (GPR103) (6,7). In hum- organ donors as previously described (24). Islet prepara- ans and rodents, 43RFa, 26RFa, and GPR103 are mainly tions with purity .70%, not suitable for transplantation, expressed in brain, particularly in the hypothalamus (8), were used after approval by the local ethical committee. as well as in peripheral tissues, including the eye, testis, – Islets (10,000) were cultured in CMRL (Invitrogen) with thyroid, adipose tissue, and macrophages (6,9 11). 10% FBS. 43RFa and 26RFa have been implicated in many physio- logical functions, including stimulation of food intake RT-PCR and Real-Time PCR (5,12–17), regulation of gonadotropic axis (18,19), aldoste- Total RNA extraction and reversed transcription to cDNA rone secretion (7,20), bone formation (21), adipogenesis from 3 mg RNA was performed as previously described and inflammation (10,11), blood pressure (12), and pros- (24). The following primer sequences were used: rat/ tate cancer differentiation and migration (22). mouse/human GPR103, forward 59-TAGGATCACCCATG Recently, 26RFa has been shown to regulate peripheral TGGCACGT-39, reverse 59-AAGAGAGCCACCACTGTCACC glucose metabolism through inhibition of glucose-induced ATC-39 (AB109629.1) (21); human QRFP, forward 59-ATG insulin secretion in the perfused rat pancreas, without GTAAGGCCTTACCCCCTGATCTAC-39, reverse 59-CCTG affecting glucagon secretion. This effect involved inhibi- TGGCTGTGAAGCTCT-39 (BC101127.2); rat QRFP, for- tion of the adenylyl cyclase/cAMP system via a pertussin ward 59-AGCACACTGGCTTCCGTCTAG-39, reverse 59-CG 9 toxin (PTX)-sensitive Gi protein (23). On the other hand, CTGGCCTTCTCTGAGTCA-3 (NM198200.1) (7); 18S rRNA, to our knowledge, the role of 43RFa on insulin secretion is forward 59-GTGGAGCGATTTGTCTGGTT-39,reverse59-CG unknown, as well as the effect of both peptides on pan- CTGAGCCAGTTCAGTGTA-39 (X_01117). cDNA (9 mL) was creatic b-cell survival. Therefore, in the current study, we amplified (GeneAmp PCR System; PerkinElmer, Milan, sought to determine the role of both 43RFa and 26RFa on Italy) in 50 mL under the following conditions: 94°C for survival and apoptosis of pancreatic b-cells and human 30 s, 60°C for 30 s annealing; 72°C for 60 s, 72°C for pancreatic islets, particularly in stress conditions such as 7 min. The final PCR products (318 bp for GPR103, serum starvation, cytokine synergism, or glucolipotoxic- 196 bp for human QRFP, 300 bp for rat QRFP, and ity. Furthermore, we investigated the effects of the pep- 199 bp for 18s rRNA) were separated by 1.5% agarose tides on insulin secretion and the underlying signaling gel electrophoresis and visualized by ethidium bromide mechanisms. staining. GPR103 Immunofluorescence RESEARCH DESIGN AND METHODS INS-1E b-cells and human pancreatic islets were seeded Reagents on glass coverslips in complete medium for 2 days and Rat QRFP-43 (43RFa), QRFP-26 (26RFa), and exendin-4 then washed twice with PBS. INS-1E cells were allowed to (Ex-4) were from Phoenix Pharmaceuticals (Karlsruhe, Ger- grow up to 50–60% confluence. The cells were then fixed many). PD-98059, wortmannin, PTX, NF449, Hoechst-33258, with 4% paraformaldehyde and incubated with goat anti- 3-isobutyl-1-methylxanthine (IBMX), 3-(4,5-dimethylthiazol-2- GPR103 antibody overnight at 4°C. After washing three yl)-2,5-diphenyltetrazolium bromide (MTT), forskolin, glucose, times with PBS, cells were incubated with anti-goat sec- palmitate, and mammalian cell lysis kit were from Sigma- ondary TRITC-conjugated antibody (Life Technologies, Aldrich (Milano, Italy). Cytokines (TNF-a,INF-g,andIL-1b) Milan, Italy) for 1 h at room temperature. Nuclei were 3 were from Life Technologies (Milan, Italy). [ H]-2-deoxy-D- stained with DAPI. Red and blue channels were assigned glucose was from PerkinElmer Life Sciences, Inc. (Boston, to GPR103 and nuclei, respectively. Images were taken MA). Cell culture and RT-PCR reagents were from Life using a Leica DM2000 fluorescent microscope and a Leica – Technologies. P-Akt (Ser473), P-extracellular signal related DFC340 FX camera. Adult rat hippocampal cells were used kinase 1/2 (P-ERK1/2), ERK1/2, and Akt antibodies were as positive control. from Cell Signaling Technology (Euroclone, Milan, Italy). b-Actin antibody was from Santa Cruz Biotechnology (Italia, Immunohistochemistry Milan, Italy). GPR103 antibody was from Abcam (Prodotti Human pancreatic tissue (n = 3 samples) was obtained Gianni, Milano, Italy) and QRFP from Phoenix Pharmaceu- from surgical samples of nonpancreatic diseases; pituitary ticals (Burlingame, CA) for either immunohistochemistry, im- samples (n = 3 samples) were obtained from autopsies. munofluorescence, or Western blot experiments. Primers for Tissues were fixed in buffered formalin and embedded in RT-PCR were from IDT (Tema Ricerca, Bologna, Italy). paraffin. Sections (5-mm thick) were deparaffinized and rehydrated in graded alcohols and phosphate buffer, pH Cell Culture 7.5, and endogenous peroxidase activity was blocked INS-1E rat b-cells were provided by Claes B. Wollheim by absolute methanol and 0.3% hydrogen peroxide for (Department of Cell Physiology and Metabolism, University 15 min. To assess the presence of GPR103 and QRFP 2382 43RFa and 26RFa in Pancreatic b-Cells Diabetes Volume 63, July 2014 proteins, the following primary antibodies and conditions radioimmunoassay kit (Linco Research, Labodia, Yens, were used: rabbit polyclonal antibody to GPR103 (diluted Switzerland) that recognizes human insulin and cross- 1:150; Abcam, Cambridge, U.K.) and rabbit polyclonal an- reacts with rat insulin. tibody to QRFP-43 (diluted 1:150; Phoenix Pharmaceut- Small Interfering RNA icals, Burlingame, CA). Immunoreactions were revealed by Rat Qrfpr Silencer Select Predesigned siRNA (s160302) a dextran chain (biotin-free) detection system (EnVision; (GPR103 siRNA), Silencer Negative Control siRNA #1 Dako) using 3,3-diaminobenzidine (DAB; Dako) as a (catalog number AM4611), and Lipofectamine RNAiMAX chromogen. were purchased from Life Technologies (Monza, Italy). Cell Proliferation and Survival INS-1E cells (2 days postseeding) were transfected in Cell proliferation was assessed by BrdU incorporation RPMI-1640 serum-free medium (Sigma-Aldrich) without ELISA (Roche), as previously described (25). Cells were antibiotics, with 50 nmol/L control or GPR103 small in- seeded on 96-well plates at 5 3 103 cells/well in serum- terfering RNAs (siRNAs) using Lipofectamine RNAiMAX, containing medium until 60–70% confluence and serum according to the manufacturer’s instructions. After 24 h, starved for 24 h before treatments. Cell survival was the cells were used for insulin measurement experiments. assessed by MTT as previously described (25). Efficiency of transfection was assessed by RT-PCR. 3 Hoechst Staining [ H]-2-deoxyglucose Uptake 3 Morphological changes in the nuclear chromatin of apo- [ H]-2-deoxyglucose uptake was performed in both INS- ptotic cells were detected by Hoechst 33258 staining 1E b-cells and human pancreatic islets. Pancreatic islets as previously described (24). Stained nuclei (500) were were dissociated with StemPro Accutase (Gibco, Invitrogen, double counted under a fluorescence microscope (DAPI San Diego, CA) (25). b-Cells were incubated in HEPES filter). buffer without glucose, with either Ex-4, 43RFa, or 26RFa for 20 min, and then with 6 mmol/L 2-deoxyglucose and Caspase 3 Activity 1 Ci/mL [3H]-2-deoxyglucose for 5 or 10 min, without Caspase 3 activity was assessed by Caspase-3 Colorimetric HEPES buffer removal. Human islets were incubated in Kit (Assay Designs, Florence, Italy) in INS-1E b-cells serum-free medium with the peptides for 20 min, and and human islet cell lysates, according to the manu- then the media was aspirated and HEPES buffer con- ’ facturer sinstructions. taining 20 mmol/L 2-deoxyglucose and 1 Ci/mL [3H]-2- cAMP Assay deoxyglucose was added for 10 min. The reaction was Starved 8 3 105 INS-1E b-cells and 1 3 103 human islets stopped by washing three times with ice-cold PBS. Cells were seeded in 100-mm dishes. After incubations, in the were solubilized on ice with lysis buffer (NaOH 50 mmol/L). presence of IBMX (100 mmol/L), cAMP was measured Equal amounts of lysates were mixed with scintillation from lysates, using the Direct Cyclic AMP EIA Kit (Assay fluid, and specific activity was counted in a liquid scintil- Designs) according to the manufacturer’s instructions. lation counter (Beckman Instruments, Fullerton, CA) in du- 3 Forskolin was used as positive control. plicate. [ H]-2-deoxyglucose uptake, expressed as pmol/mg, was normalized by using a bicinchoninic acid protein quan- Western Blotting tification of each sample, as previously described (26). P-ERK and P-Akt (40 mg protein) and GPR103 and QRFP (60 mg) were resolved in 12% SDS-PAGE. Proteins were Statistical Analysis 6 treated as previously described (24) and incubated with the Results are presented as means SE. Results were ana- t specific antibody (dilution 1:1,000). Blots were reprobed lyzed using two-tailed Student test or two-way ANOVA fi with the respective total antibodies or with b-actin for followed by Tukey honestly signi cant difference for post- normalization. Immunoreactive proteins were visualized ANOVA comparisons (GraphPad Prism 5.0 software, San fi P , with ChemiDoc XRS (Bio-Rad, Milan, Italy), and densito- Diego, CA). Signi cance was established when 0.05. metric analysis was performed with Quantity One software RESULTS (Bio-Rad). GPR103 and QRFP Are Expressed in INS-1E b-Cells Insulin Secretion and Human Pancreatic Islets Insulin secretion in INS-1E b-cells was performed as pre- RT-PCR analysis showed mRNA expression of both viously described (24). Human pancreatic islets (n =3) GPR103 and QRFP, the precursor of 43RFa and 26RFa, were incubated for 1 h at 37°C in HEPES-buffered in pancreatic INS-1E b-cells and human pancreatic islets Krebs-Ringer bicarbonate buffer containing 0.5% BSA (Fig. 1A and B). At the protein level, Western blot analysis with 2 mmol/L glucose. The medium was changed, and showed presence of both GPR103 and QRFP, the precur- the cells were incubated again for 1 h in Krebs-Ringer sor peptide of 43RFa and 26RFa, in INS-1E b-cells and bicarbonate buffer/0.5% BSA containing 1.25, 7.5, 15, human pancreatic islets (Fig. 1C and D). Furthermore, or 25 mmol/L glucose, with or without the different immunofluorescence analysis revealed GPR103 protein peptides or inhibitors. After acid ethanol extraction expression in both b-cells (Fig. 1E) and human islets, of the hormone, secreted insulin was quantified by a where GPR103 showed partial colocalization with insulin diabetes.diabetesjournals.org Granata and Associates 2383

Figure 1—Gene and protein expression of GPR103 and QRFP (the 43RFa and 26RFa precursor) in INS-1E b-cells and human pancreatic islets (H. islets). GPR103 (A)andQRFP(B) mRNA assessed by RT-PCR. Rat brain (A and B) and adult hippocampal progenitor cells (AHPs) (A)were used as positive control; 18s rRNA was used as internal control. GPR103 (C)andQRFP(D) protein expression assessed by Western blot on whole lysates from INS-1E b-cells or human pancreatic islets. Equal protein loading was determined by reprobing with antibodies to b-actin. The blots are representative of three independent experiments. E: Immunofluorescent staining of GPR103 in INS-1E b-cells. GPR103 is shown in red and nuclei in blue (DAPI); AHPs were used as positive control. Each image is representative of three independent experiments (original magnification 340). F: GPR103 expression in human pancreatic islets assessed by immunofluorescent staining. Insulin is shown in green and GPR103 in red (original magnification 340) (n = 3). Merge shows colocalization of GPR103 and insulin (yellow), as indicated by arrows. G:Immunohis- tochemical analysis of GPR103 and QRFP expression in INS-1E b-cells. Cells incubated without the primary antibody were used as negative control (2). H: Immunohistochemical analysis of GPR103 and QRFP expression in human pancreas; pituitary was used as positive control (n =3). 2384 43RFa and 26RFa in Pancreatic b-Cells Diabetes Volume 63, July 2014 in b-cells (Fig. 1F). Immunohistochemical studies also cause of b-cell dysfunction and death (3). Based on the confirmed expression of both GPR103 and QRFP in results obtained in cytokine-treated cells, 43RFa and INS-1E b-cells and human pancreatic islets (Fig. 1G and 26RFa were tested at 100 nmol/L, the concentration H). In human pancreas, GPR103 positivity was found in that was chosen for the following experiments. The pep- endocrine pancreatic cells, being negative in acinar cells. tides equally increased INS-1E b-cell survival and prolif- Similarly, QRFP was positive in pancreatic islets and neg- eration, which were both reduced in cells incubated with ative in acinar cells (Fig. 1H). Pituitary was used as pos- high glucose (30 mmol/L) and palmitate (0.4 mmol/L), itive control, showing strong staining for GPR103 in the compared with serum-free medium alone (Fig. 2E and posterior lobe (neurohypophysis) but almost negative F). These effects were similar to those of Ex-4. staining in the anterior lobe (adenohypophysis) (Fig. As assessed by Hoechst staining of apoptotic nuclei 1H). QRFP was negative in the neurohypophysis and (Fig. 2G) and caspase 3 activation (Fig. 2H), apoptosis was weakly positive in isolated cell clusters of the adenohy- increased in cells cultured in serum-free medium, com- pophysis (corresponding to acydophilic cells) (Fig. 1H). pared with those in serum, and even more with cytokines or high glucose/palmitate. Both 43RFa and 26RFa, at 100 43RFa and 26RFa Both Promote Survival and nmol/L and similarly to Ex-4 (100 nmol/L), strongly re- Proliferation and Inhibit Apoptosis in INS-1E b-Cells duced apoptosis in all experimental conditions (Fig. 2G One of the major pathological conditions, responsible for and H). the progression of diabetes, is b-cell loss by apoptosis and Overall, these results indicate that 43RFa and 26RFa decreased b-cell mass. Therefore, we tested the hypothe- protect pancreatic b-cells against different diabetogenic sis as to whether 43RFa and 26RFa may protect b-cells stimuli. and human pancreatic islets from the harmful effects of stimuli classically involved in diabetes, such as cytokines 43RFa and 26RFa Both Promote Survival and Inhibit Apoptosis in Human Pancreatic Islets or glucolipotoxicity. Similarly to INS-1E b-cells, 43RFa and 26RFa increased 43RFa and 26RFa effects on survival and proliferation survival and inhibited apoptosis in human pancreatic were assessed in INS-1E b-cells cultured in serum-free medium, in either absence or presence of the inflamma- islets cultured for 72 h under either serum starvation, cytokine synergism, or high glucose/palmitate. The pep- tory cytokines IFN-g, TNF-a, and IL-1b. Serum starvation tides and Ex-4, which was used as positive control, were per se is known to reduce survival in many cell types, and tested at 1, 10, 25, 50, and 100 nmol/L in islets cultured cytokine synergism has been implicated in b-cell destruc- in either serum-free medium alone or with IFN-g/TNF-a/ tion and apoptosis in both type 1 and type 2 diabetes IL-1b. Under serum starvation, 43RFa and 26RFa in- (4,25). 43RFa and 26RFa were initially tested at different creased survival at 100 nmol/L, having no significant ef- concentrations (1, 10, 25, 50, and 100 nmol/L). Both fect at lower concentrations (Fig. 3A). In the presence of peptides increased cell survival under serum starvation from 10 to 100 nmol/L, as assessed by MTT assay cytokines, they both increased cell survival from 25 to 100 nmol/L, which was the best concentration tested (Fig. (Fig. 2A). In cytokine-treated cells, they both promoted 3B). Conversely, Ex-4 was effective at all concentrations survival at all the concentrations tested, showing the in both experimental conditions and in a dose-dependent greatest effect at 100 nmol/L (Fig. 2B). Similar results manner (Fig. 3A and B). At 100 nmol/L, 43RFa- and were obtained by performing the Trypan blue assay 26RFa-induced survival was similar to that of Ex-4, and (data not shown). 43RFa and 26RFa also increased cell a dose response was observed under cytokine treatment proliferation under serum starvation at all the concentra- (Fig. 3B). In islets cultured in high glucose/palmitate, 100 tions tested (Fig. 2C), whereas with cytokines, they were effective from 25 to 100 nmol/L (Fig. 2D). As for cell nmol/L was also used, where the peptides equally increased islet cell survival (Fig. 3C). Apoptosis, assessed survival, the best proliferative effect was observed at by Hoechst staining and caspase 3 activity, was inhibited 100 nmol/L, for both peptides, with a slight dose re- by both 43RFa and 26RFa in all experimental settings and sponse under cytokine treatment. Notably, under either similarly to Ex-4 (Fig. 3D and E). serum starvation or cytokines, at 100 nmol/L, the pep- These results indicate that, as for b-cells, in human tides increased survival and proliferation up to levels islets, 43RFa and 26RFa exert protective effects against comparable to those of serum and serum-free medium, different detrimental diabetogenic stimuli. respectively. The glucagon-like peptide 1 receptor (GLP-1R) agonist 43RFa Survival Effects Involve Activation of Ex-4, which exerts antiapoptotic, survival, and prolifera- Phosphoinositide 3-Kinase/Akt and ERK1/2, Whereas tive effects in b-cells (27), was used as control peptide. Only ERK1/2 Is Required for 26RFa-Induced Cell The survival and proliferative effects of Ex-4 were com- Survival parable to those of 43RFa and 26RFa, particularly at 100 We next investigated the signaling pathways involved in the nmol/L (Fig. 2A–D). survival effects of the peptides, and specifically, phosphati- Glucolipotoxicity, caused by excessive glucose and/or dylinositol 3-kinase (PI3K)/Akt and ERK1/2, whose activa- lipids, is a consequence of type 2 diabetes and is a major tion plays a key role in b-cell growth and survival (28–30). diabetes.diabetesjournals.org Granata and Associates 2385

Figure 2—43RFa and 26RFa effects on survival, proliferation, and apoptosis of INS-1E b-cells. The cells were cultured in serum-free medium (SF) for 12 h, and then incubated for a further 24 h without or with the cytokines (CK) TNF-a/IFN-g/IL-1b (100, 50, and 5 ng/mL, respectively), or with high glucose (30 mmol/L) and palmitate (0.4 mmol/L) (P/G), and with either 43RFa, 26RFa, or Ex-4. The peptides were added 40 min prior to CK or P/G. Cell survival and proliferation (assessed by MTT and BrdU, respectively) in the absence (A and C)or presence (B and D) of CK and the peptides, at the indicated concentrations. Data are expressed as percent of control (SF) and are the mean 6 SE of ﬁve replicates (n =3)(*P < 0.05 and **P < 0.01 vs. SF; ns, not signiﬁcant). Cell survival (E) and cell proliferation (F) in cells cultured in SF medium, alone or with P/G and the indicated peptides (100 nmol/L each). Results are expressed as percent of control (SF); n = 3. (**P < 0.01 vs. SF; #P < 0.05 and ##P < 0.01 vs. P/G.) G: Apoptosis assessed by counting condensed/fragmented Hoechst-stained 2386 43RFa and 26RFa in Pancreatic b-Cells Diabetes Volume 63, July 2014

Figure 3—43RFa and 26RFa effects on survival and apoptosis of human pancreatic islets. Islet cells were incubated for 72 h in the presence of serum or in serum-free medium (SF), either alone or with IFN-g/TNF-a/IL-1b (CK) (5 ng/mL each) and 43RFa or 26RFa. Cell viability assessed by MTT in islets cultured in SF, alone (A) or with cytokines (CK) (B), and the peptides at the indicated concentrations. Results are expressed as percent of control (SF, n =3)(*P < 0.05 and **P < 0.01 vs. SF for A or vs. CK for B; ns, not signiﬁcant). C: Cell survival (MTT) in human islets treated in SF medium alone or with high glucose (30 mmol/L)/palmitate (P/G) (0.4 mmol/L) and the indicated peptides (100 nmol/L each). Results are expressed as percent of control (SF); n =3(*P < 0.05 and **P < 0.01 vs. SF; ##P < 0.01 vs. P/G). D: Apoptosis assessed by Hoechst staining. Values are expressed as percent of apoptotic cells from duplicate determinations (500 cells each, n = 3). E: Apoptosis assessed by caspase 3 activation. Results are expressed as percent of control (SF). For both D and E,*P < 0.05 and **P < 0.01; ##P < 0.01 vs. SF; n = 3. Each peptide was used at 100 nmol/L.

In INS-1E cells, 43RFa increased the phosphorylation action of 43RFa, whereas that of 26RFa was reduced only of both Akt (at 15 min) and ERK1/2 (5–30 min, compared by PD98059 and not by wortmannin. These results sug- with basal time point) (Fig. 4A and B). 26RFa displayed no gest that both PI3K/Akt and ERK1/2 are involved in the effect on Akt, at any time point (Fig. 4C), whereas it prosurvival effects of 43RFa, whereas for 26RFa, only ERK1/2 moted ERK1/2 increase, from 5 to 60 min compared with is engaged. D basal (Fig. 4 ). Similar effects were observed in human 43RFa Promotes Glucose-Stimulated Insulin Secretion pancreatic islets, where 43RFa induced Akt and ERK1/2 and Potentiates Ex-4 Insulinotropic Action in b-Cells phosphorylation (both at 5–30 min) (Fig. 4G and H), and Human Islets, Whereas 26RFa Inhibits Insulin whereas 26RFa promoted ERK1/2 (5 and 15 min) (Fig. Release and Reduces Ex-4 Effects 4J), but not Akt activation (Fig. 4I). Accordingly, in both We next sought to determine 43RFa and 26RFa effects INS-1E and human islets cultured in serum-free medium on glucose-stimulated insulin secretion (GSIS) in both (Fig. 4E and K) or in the presence of cytokines (Fig. 4F INS-1E and human pancreatic islets. The peptides were and L), preincubation with the inhibitors for either Akt tested at different glucose concentrations either alone or (wortmannin) or ERK1/2 (PD98059) blocked the survival in combination with Ex-4, which was used as positive apoptotic nuclei. Values are expressed as percent of apoptotic cells and are the mean 6 SE of duplicate determinations (500 cells each) from three independent experiments. H: Apoptosis assessed as caspase 3 activation and expressed as percent of control (SF). For both G and H,**P < 0.01; ##P < 0.01 vs. SF; n = 3. Each peptide was used at 100 nmol/L. diabetes.diabetesjournals.org Granata and Associates 2387

Figure 4—43RFa and 26RFa effects on activation of PI3K/Akt and ERK1/2. Akt and ERK1/2 phosphorylation was assessed by Western blot on whole lysates from INS-1E cells or human pancreatic islets stimulated with either 100 nmol/L 43RFa (A, B, G,andH) or 100 nmol/L 26RFa (C, D, I,andJ) for the indicated times (top panels). Equal protein loading was determined by reprobing with antibodies to the respective total proteins (bottom panels). Blots are representative of three independent experiments. Graphs show the densitometric analysis of phosphor- ylated proteins normalized to total proteins and reported as percent of basal (*P < 0.05 and **P < 0.01; ns, not significant). Cell survival assessed by MTT in INS-1E cells that were starved for 12 h and then incubated for a further 24 h in the absence or presence of either 43RFa (43) or 26RFa (26) (100 nmol/L each), and either without serum-free medium (SF) (E)orwithTNF-a/IFN-g/IL-1b (cytokines, CK) (100, 50, and 5ng/mL,respectively)(F), and wortmannin (WM) (100 nmol/L) or PD98059 (40 mmol/L). Inhibitors were added 30 and 60 min before the peptides and cytokines, respectively. **P < 0.01 vs. SF (E)orvs.CK(F); ##P < 0.01 vs. 43; §P < 0.05 vs. 26; ns, not significant; n = 3. Cell survival assessed by MTT in human pancreatic islets incubated for 72 h in the presence of serum or in serum-free medium (SF), either alone (K) or with IFN-g/TNF-a/IL-1b (CK) (5 ng/mL each) (L), and 43RFa or 26RFa (100 nmol/L each). Inhibitors were added as for E and F.**P < 0.01 vs. SF (K) or vs. CK (L); #P < 0.05 and ##P < 0.01 vs. 43; §P < 0.05 and §§P < 0.01 vs. 26; ns, not significant; n =3. 2388 43RFa and 26RFa in Pancreatic b-Cells Diabetes Volume 63, July 2014 control. As expected, Ex-4 increased GSIS at all glucose Glucose uptake by pancreatic b-cells, through the concentrations, in both b-cells and islets. Similarly, 43RFa glucose transporter GLUT2 and glucokinase activity, is increased insulin release at all glucose concentrations in essential for the normal insulin secretory response to b-cells and islets, to an extent comparable to that of Ex-4. hyperglycemia (35,36). To further ascertain the stimula- 43RFa even increased basal insulin levels, without glucose tory and inhibitory roles of 43RFa and 26RFa, respectively, stimulation. Moreover, Ex-4– and 43RFa-induced insulin on GSIS, glucose uptake was determined in INS-1E b-cells secretion was further potentiated when Ex-4 and 43RFa and human pancreatic islets. In INS-1E b-cells, at 10 min were given together, at 15 and 25 mmol/L glucose in INS- incubation, 43RFa increased glucose 2-[3H]-deoxyglucose 1E cells, and at 7.5, 15, and 25 mmol/L glucose in human uptake at 10 nmol/L and showed a remarkable effect at islets (Fig. 5A and B). On the other hand, 26RFa strongly 100 nmol/L, similarly to Ex-4 and to the cAMP analog inhibited both basal and GSIS and dramatically reduced 8-Br-cAMP. At 5 min, 43RFa had no effect at 10 nmol/L Ex-4 insulinotropic effects at all glucose concentrations, but stimulated glucose uptake at 100 nmol/L. Conversely, in either INS-1E b-cells or human islets (Fig. 5A and B). 26RFa had no effect at any concentration and incuba- To investigate the signaling pathways involved in the tion time tested (Fig. 6E). Similarly, in human pancreatic insulinotropic and insulinostatic effects of 43RFa and islets that were tested for 10 min with 100 nmol/L of each 26RFa, respectively, INS-1E b-cells and human islet cells peptide, 43RFa, but not 26RFa, increased 2-deoxyglucose were preincubated with either PTX, an inhibitor of Gai/o- uptake (Fig. 6F). – coupled receptor (31), or NF449, a selective Gas protein coupled receptor antagonist (32). 43RFa-induced increase DISCUSSION of insulin secretion was unaffected by pretreatment with The current study shows that the RFamide peptides 43RFa PTX, whereas it was blocked by NF449, in both b-cells and and 26RFa both display survival and antiapoptotic effects islets. Conversely, PTX restored up to control levels GSIS, in pancreatic b-cells and human pancreatic islets. Further- which was inhibited by 26RFa, whereas NF449 had no more, 43RFa stimulated, whereas 26RFa inhibited, the in- effect on the insulinostatic action of the peptide (Fig. sulin response to glucose and Ex-4 in both b-cells and 5C–F). These results suggest that the opposite effects of human pancreatic islets. These effects involved activation 43RFa and 26RFa on insulin secretion involve signaling of distinct G proteins and different regulation of cAMP through Gas and Gai/o, respectively. Furthermore, the levels and glucose uptake. role of GPR103 in 43RFa and 26RFa effects on GSIS In mammals, the mRNAs encoding for the 43RFa and was determined by transfecting INS-1E b-cells with siRNA 26RFa precursor (QRFP) and for its receptor GPR103 are targeted to GPR103. As assessed by RT-PCR, at 24 after mainly expressed in the brain, particularly in the hypo- transfection, GPR103 mRNA expression was strongly re- thalamus, and in different peripheral organs (6,9). QRFP duced by siGPR103, compared with scrambled (control) mRNA has been found expressed in mouse and rat pan- siRNA (Fig. 5G). The insulinotropic effect of 43RFa, creas, but not in human pancreas (37). GPR103 was assessed at both 0 and 7.5 mmol/L glucose, was com- reported to be absent in human pancreas and expressed pletely blocked in siGPR103 cells, as compared with at a very low level in mouse pancreas (6,7,37). Here we siControl, whereas 26RFa-induced inhibition of GSIS show QRFP and GPR103 expression in both rat INS-1E was unchanged in siGPR103-transfected cells (Fig. 5H). b-cells and human pancreatic islets, either at mRNA or These findings suggest that 43RFa promotes insulin protein level. Interestingly, in human islets, GPR103 pro- secretion through binding to GPR103, whereas 26RFa tein colocalized with insulin, suggesting direct interaction inhibits insulin release likely through a different receptor. of 43RFa and 26RFa with their receptor in b-cells. Fur- thermore, QRFP expression in b-cells and human islets 43RFa, but Not 26RFa, Promotes cAMP Increase and may suggest autocrine/paracrine effects of the peptides. Glucose Uptake in b-Cells and Human Pancreatic Accordingly, 26RFa autocrine/paracrine effects have been Islets recently proposed in prostate cancer cells, where the pep- cAMP elevation is linked to increased insulin secretion in tide induced cell migration and neuroendocrine differen- pancreatic b-cells and protection from apoptosis (33,34). tiation, and in macrophages and adipocytes, where both Therefore, we investigated the effects of 43RFa and 43RFa and 26RFa promoted adipogenesis (10,11,22). Fu- 26RFa on cAMP levels in both INS-1E b-cells and human ture studies will help elucidate whether autocrine/para- pancreatic islets. In INS-1E b-cells, 43RFa elevated intra- crine mechanisms may also occur in pancreatic islets. cellular cAMP at 5–30 min with respect to basal (time 0), We firstly report here the survival and antiapoptotic but not at 60 min, where cAMP returned to basal levels effects of 43RFa and 26RFa. Pancreatic b-cells and human (Fig. 6A). Conversely, 26RFa reduced cAMP levels, at the pancreatic islets were chosen as in vitro models because, same time points (Fig. 6B). Similarly, in human pancreatic besides the survival actions, we were interested in the islets, cAMP was increased by 43RFa at 15 and 30 min metabolic effects of these peptides. Indeed, a role in adi- and inhibited by 26RFa, at 5–30 min (Fig. 6C and D). The pogenesis, lipid metabolism, and inflammation, as well as cAMP-increasing agent forskolin was used as positive an association between GPR103 and QRFP expression and control. obesity, has been recently described (10,11). Moreover, as diabetes.diabetesjournals.org Granata and Associates 2389

Figure 5—43RFa and 26RFa regulate insulin secretion in INS-1E b-cells and human pancreatic islets. Insulin release was assessed by radioimmunoassay in conditioned medium from INS-1E b-cells or islets, incubated alone or with 2 mmol/L glucose for 1 h and then for a further 1 h with the indicated concentrations of glucose, in the presence or absence of either 43RFa, 26RFa, Ex-4, or a combination of Ex- 4/43RFa or Ex-4/26RFa (100 nmol/L each). A: Insulin secretion in INS-1E b-cells. B: Insulin secretion in human pancreatic islets. For A and B, values are the means 6 SE of triplicate determinations from at least three independent experiments (*P < 0.05 and **P < 0.01 vs. control at each glucose concentration; #P < 0.05 and ##P < 0.01). Insulin secretion in INS-1E cells (C and D) and human pancreatic islets (E and F) in either absence or presence of PTX (50 ng/mL) or NF449 (10 mmol/L). *P < 0.05 and **P < 0.01 vs. control (c) at each glucose concentration; #P < 0.05 and ##P < 0.01; ns, not significant. G: GPR103 mRNA assessed by RT-PCR in INS-1E b-cells after 24 h transfection with either nonsilencing siRNA (siControl) or siRNA to GPR103 (siGPR103). As expected, the amplified products corresponded 2390 43RFa and 26RFa in Pancreatic b-Cells Diabetes Volume 63, July 2014 for QRFP, other orexigenic peptides such as ghrelin exert used in the study by Egido et al. (23), where the experi- antiapoptotic and metabolic effects in different cell types, ments were performed in perfused rat pancreas, at vari- including pancreatic b-cells and human islets (24,38,39). ance with our in vitro models. Here, both 43RFa and 26RFa increased survival and Interestingly, either 43RFa or Ex-4 alone showed inhibited apoptosis of INS-1E b-cells and human pancre- comparable insulinotropic action in both b-cells and hu- atic islets. They also increased proliferation of INS-1E man islets. Moreover, insulin secretion was markedly b-cells, which was not investigated in human islets due increased when 43RFa and Ex-4 were administered to- to the low proliferative rate of human b-cells in vitro gether, particularly at the highest concentrations of glu- (40,41). Besides serum starvation, which induces b-cell cose. On the other hand, besides exerting inhibitory death (24,42), b-cells and human islets were challenged action per se, 26RFa hampered Ex-4 insulinotropic effect. with diabetogenic stimuli, such as cytokine synergism and These results imply that 43RFa may either potentiate glucolipotoxicity, which are major causes of b-cell dys- Ex-4 action or act synergistically with Ex-4 to increase function and death in both type 1 and 2 diabetes (1–4). insulin secretion, whereas 26RFa behaves in an opposite Interestingly, 43RFa and 26RFa both displayed prolifera- manner. They also suggest clinical implications in diabe- tive, survival, and antiapoptotic actions, comparable to tes, as 43RFa may become a therapeutic target for en- those of the long-acting GLP-1 agonist Ex-4 (27). hancing b-cell function, alone or in combination with Phosphorylation of either PI3K/Akt or ERK1/2 has incretin hormones, whereas inhibition of 26RFa effect been shown to mediate b-cell proliferation and survival in may prevent loss of b-cell competence. response to different external signals, including incretin Activation of G-protein–coupled receptors by metabo- hormones (24,27,30,43). Here, 43RFa and 26RFa, al- lites and hormones can modulate glucose-induced insulin though displaying similar survival effect in b-cells and secretion through different pathways. Gas-coupled recep- human islets, differently regulated these signaling path- tors increase insulin secretion through stimulation of ways. In fact, whereas 43RFa increased the phosphoryla- adenylyl cyclase and activation of protein kinase A (PKA) tion of both PI3K/Akt and ERK1/2, 26RFa increased by cAMP. In turn, Gai/o-coupled receptors reduce insulin ERK1/2, but not PI3K/Akt. Accordingly, inhibition of release by inhibition of adenylyl cyclase and cAMP for- PI3K and ERK1/2 phosphorylation with wortmannin mation and also through inhibition of voltage-dependent 2+ and PD98059, respectively, blocked 43RFa-induced sur- Ca channels involving the bg subunits of Gai/o (27,44). vival, whereas only ERK1/2 inhibitor decreased 26RFa- We found here that the Gas antagonist NF449 blocked the induced survival. These results indicate that ERK1/2, insulinotropic effect of 43RFa, but not the insulinostatic and not PI3K/Akt, is required for 26RFa survival effect, action of 26RFa. Conversely, the Gai/o inhibitor PTX did whereas 43RFa uses both pathways. Notably, a cross-talk not affect 43RFa-induced insulin release but blocked the between ERK1/2 and cAMP in pancreatic b-cell survival inhibitory effect of 26RFa, in both b-cells and human has been previously described (29). However, here only islets. This in agreement with the findings by Egido et al. 43RFa elevated cAMP levels, suggesting that 26RFa- (23), except for the fact that here PTX was used in combi- induced activation of ERK1/2 is independent of the nation with glucose only, and not with Ex-4. cAMP pathway. Of note, PTX alone had no effect here on GSIS, To further understand the effects of 43RFa and 26RFa whereas with 26RFa, insulin levels were found increased. in b-cells, we next determined their role on insulin secre- This is likely because glucose alone, in the absence of tion. To our knowledge, 43RFa effect on insulin secretion G-protein–coupled receptor ligands, causes inhibition of has never been studied. Instead, 26RFa was recently ATP-regulated K+ channels, opening of voltage-dependent shown to inhibit glucose- as well as arginine- and Ex-4– Ca2+ channels, and increase in intracellular Ca2+ concen- induced insulin secretion in perfused rat pancreas (23). tration ([Ca2+]i), which triggers exocytosis of insulin- Here, we firstly show that 43RFa promoted insulin secre- containing vesicles (45). Conversely, in the presence of tion in INS-1E b-cells and human islets, both in basal 26RFa, PTX may restore cAMP levels, which are blunted conditions and in the presence of either different glucose by the peptide, as well as ([Ca2+]i), and may thus contrib- concentrations or Ex-4. Conversely, 26RFa showed oppo- ute to increased insulin release. site effects and inhibited both glucose- and Ex-4–induced Our results indicate that the opposite effects of 43RFa insulin secretion, in agreement with the previous findings and 26RFa on insulin secretion are due to activation (23). 26RFa was used here at 100 nmol/L, which was also of distinct pathways. In fact, 43RFa promotes insulin the best concentration to promote survival and prevent secretion, at least in part, through Gas and adenylyl apoptosis. Conversely, 10 nmol/L was the concentration cyclase/cAMP increase, whereas 26RFa inhibits insulin to 318 bp for rat GPR103 and 199 bp for 18s rRNA, which was used as internal control. H: Insulin secretion in siControl- or siGPR103- transfected INS-1E b-cells, incubated for 1 h with the indicated concentrations of glucose, and with 43RFa or 26RFa (100 nmol/L each). Values are the means 6 SE of triplicate determinations from at least three independent experiments (*P < 0.05 vs. control at each glucose concentration; #P < 0.05). diabetes.diabetesjournals.org Granata and Associates 2391

Figure 6—43RFa and 26RFa effects on intracellular cAMP and glucose uptake. A–D: Serum-starved INS-1E cells and human islets were cultured for the indicated times with either 100 nmol/L 43RFa or 100 nmol/L 26RFa, in the presence of the phosphodiesterase inhibitor IBMX (100 mmol/L), which was added 30 min before stimulation. Forskolin (Forsk) (50 mmol/L for 1 min) was used as positive control. Results are the mean 6 SE of three independent experiments performed in triplicate (**P < 0.005 vs. basal time point; ns, not signiﬁcant). E: 2-[3H]-deoxyglucose uptake assessed in serum-starved INS-1E cells cultured for 5 or 10 min with either Ex-4, 43RFa, or 26RFa, at the indicated concentrations. F: 2-[3H]-deoxyglucose uptake in human pancreatic islets cultured for 10 min with the peptides that were used at 100 nmol/L each. 8-Br-cAMP was used at 1 mmol/L. For E and F, n =3;*P < 0.05, **P < 0.005, and ***P < 0.001; ns, not signiﬁcant vs. control. 2392 43RFa and 26RFa in Pancreatic b-Cells Diabetes Volume 63, July 2014

release through Gai/o and inhibition of cAMP formation. 2008EFHJ5H_02 to R.G., PRIN 2010B5B2NL to E.G.]); by Compagnia di San This assumption is further sustained by our results show- Paolo (2011 to R.G. and 2007 to E.G.); and by the Studio delle Malattie Endocrino ing that 43RFa increased, whereas 26RFa inhibited, in- Metaboliche Foundation (Turin, Italy). Human islets were provided through tracellular cAMP levels in both INS-1E b-cells and human Juvenile Diabetes Research Foundation Award 31-2008-416 (ECIT, Islet for Research Program). pancreatic islets. At variance with these findings, 43RFa has Duality of Interest. This study was supported by Sanofi S.p.A., Italy. No been recently found to decrease forskolin-induced cAMP in other potential conflicts of interest relevant to this article were reported. human adrenocortical cells, likely through Gq-mediated Author Contributions. R.G. contributed to study design and interpreta- mechanisms (20). 26RFa, instead, in line with our results, tion and wrote the manuscript. F.S., L.T., D.G., I.G., and L.B. researched data and was shown to decrease cAMP production in CHO- or hu- reviewed and edited the manuscript. R.N. and L.P. provided the human islets and man embryonic kidney (HEK293)–GPR103 transfected reviewed and edited the manuscript. M.P.G. and G.A. researched data on 2+ cells, suggesting GPR103 coupling to Gi/o and Gq (7). In Ca transients and reviewed and edited the manuscript. M.V. and M.P. addition, we found here that similarly to the cAMP analog contributed with the immunohistochemical studies and reviewed and edited 8-Br-cAMP and to Ex-4, 43RFa, but not 26RFa, increased the manuscript. J.L., H.V., H.O., and E.G. critically revised the manuscript glucose uptake, which is required in the initial step of and helped with the discussion. R.G. is the guarantor of this work and, as GSIS by pancreatic b-cells and has been shown to be such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. impaired in both type 1 and type 2 diabetes (35,46). Col- lectively, these findings further support the positive role References of 43RFa on b-cell function, which is opposed to that of 1. Muoio DM, Newgard CB. Mechanisms of disease: molecular and metabolic 26RFa. mechanisms of insulin resistance and beta-cell failure in type 2 diabetes. Nat Rev An open question during the study was why two Mol Cell Biol 2008;9:193–205 peptides that likely bind the same receptor exert opposite 2. Eizirik DL, Colli ML, Ortis F. The role of inflammation in insulitis and beta- effects on insulin secretion. To solve this issue, we cell loss in type 1 diabetes. Nat Rev Endocrinol 2009;5:219–226 knocked down GPR103 expression in INS-1E b-cells using 3. 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