The International Journal of Biochemistry & Cell Biology 40 (2008) 2296–2302
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Neurotensin protects pancreatic beta cells from apoptosis ´ ´ Thierry Coppola, Sophie Beraud-Dufour, Aurelie Antoine, Jean-Pierre Vincent, Jean Mazella ∗ From Institut de Pharmacologie Mol´eculaire et Cellulaire, CNRS, UMR 6097, Universit´e de Nice Sophia Antipolis, 660 route des Lucioles, 06560 Valbonne, France
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Article history: Received 7 January 2008 Received in revised form 21 March 2008 Accepted 26 March 2008 Available online 1 April 2008 Keywords: Neurotensin Receptor Beta cell Apoptosis
a b s t r a c t The survival of pancreatic beta cells depends on the balance between external cytotoxic and protective molecular systems. The neuropeptide neurotensin (NT) has been shown to regulate certain functions of the endocrine pancreas including insulin and glucagon release. However, the mechanism of action of NT as well as the identification of receptors involved in the pancreatic functions of the peptide remained to be studied. We demonstrate here that NT is an efficient protective agent of pancreatic beta cells against cytotoxic agents. Both betaTC3 and INS-1E cell lines and the mouse pancreatic islet cells express the three known NT receptors. The incubation of beta cells with NT protects cells from apoptosis induced either by staurosporine or by IL-1beta. In beta-TC3 cells, NT activates both MAP and PI-3 kinases pathways and strongly reduces the staurosporine or the Il-1beta-induced caspase-3 activity by a mechanism involving Akt activation. The NTSR2 agonist levocabastine displays the same protective effect than NT whereas the NTSR1 antagonist is unable to block the effect of NT suggesting the predominant involvement of the NTSR2 in the action of NT on beta cells. These results clearly indicate for the first time that NT is able to protect endocrine beta cells from external cytotoxic agents, a role well correlated with its release in the circulation after a meal. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction Neuropeptides are known to display important roles in the central nervous system and to exhibit diverse biological actions in the periphery including regulation of metabolism and growth and cell survival in the gastroenteropancreatic-brain axis. At the level of endocrine pancreas, apart from the role recently attributed to melanin concentrating hormone in the function and growth of islet (Pissios et al., 2007), the regulatory functions of neuropeptides are poorly documented. However the neuropeptide neurotensin (NT) has been originally shown to behave as a regulator of endocrine pancreatic fate. Indeed, at low glucose concentration, NT stimulates insulin and glucagon
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release whereas at high glucose or arginine levels, NT inhibits insulin and glucagon release (Dolais-Kitabgi et al., 1979). Other important observations were that NT is released in the human circulation following a meal and that lipid absorption stimulated peptide release from the rat small intestine (Leeman and Carraway, 1982). Interestingly, NT has been also shown to be secreted from pancreas in streptozotocin-diabetic rats (Berelowitz and Frohman, 1982) and more recently to be colocalized with glucagon in the endocrine human fetal pancreas (Portela-Gomes et al., 1999). It should also be noted that NT administration increased pancreatic weight, DNA, RNA and protein contents, indicating a prominent proliferative effect of the peptide (Feurle et al., 1987; Wood et al., 1988). Although these data reveal strong evidences on the involvement of the neurotensinergic system in the regulation of endocrine pancreas, nothing is known about the cellular expression and localization of both NT and
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its receptors as well as about the intracellular pathways involved in these biological effects. NT exerts its actions through three known receptors. The first two, NTSR1 and NTSR2, belong to the family of G protein-coupled receptors (Vincent et al., 1999), whereas the third one, NTSR3, also called sortilin (Petersen et al., 1997), is a type I receptor belonging to a new class of receptors similar to the Vps10p sorting receptor (Mazella, 2001; Mazella and Vincent, 2006). Biochemical and functional studies have indicated that NTSR1 is implicated in several central actions of NT including hypothermia and neuroleptic-like behaviors whereas NTSR2 is rather responsible for the analgesic effect of the peptide (for review see Vincent et al., 1999). The third NT receptor behaves either as a co-receptor or a receptor depending on the cell system studied (Martin et al., 2002; Martin et al., 2003; Nykjaer et al., 2004). In this work, we provide for the first time evidence that the three NT receptors are expressed in murine beta cell lines and islets and that NT exerts a potent protective effect on these cells from both staurosporine and Il-1-induced apoptosis.
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2.3. RT-PCR analysis
2. Materials and methods
Total RNA was extracted from murine beta cells and islets by the method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987) using the RNAble kit (Promega). Briefly, 2 g of total RNAs were reverse transcribed using 1 g of oligo (dT)15 primer and 30 U of AMV reverse transcriptase as previously described (Dal Farra et al., 2001). PCR amplification was carried out with a first cycle at 94 ◦ C for 3 min, followed by 35 cycles 94 ◦ C for 1 min, 58 ◦ C for NTSR1-2, INS-2 and G6PC2 or 52 ◦ for NTSR3 C for 1 min, 72 ◦ C for 1 min and a final extension step at 72 ◦ C for 7 min. PCR products were analyzed on a 1.5% agarose gel. Primers used for PCR amplification were for NTSR1 F: 5 -GTTAACACCTTCATGTCCTTCCTG-3 , R: 5 -TACGTAAGACGAGGACTCCATGGCG-3 (200 bp); NTSR2 F: 5 -ATCAGGCCACCTCGAGACAGAGATG-3 , R: 5 -ATCAGGCCACCTCGAGACAGAGATG-3 (350 bp); NTSR3 F: 5 -ACGGACTTTACCAACGTGAC-3 , R: 5 -CAAGGATGTCTTTGAAATCGA3 (620 bp); mouse proinsuline-2 F: 5 -CCGCTACAATCAAAAACC-3 , R: 5 -GGTAGGCTGGGTAGTGGT-3 (390 bp) and mouse glucose-6-phosphate catalytic subunit 2 F: 5 TCTGGGTAGCGGTCATAG-3 , R-CGTGCTGTCAATGTGGAT-3 (590 bp).
2.1. Materials
2.4. Western blot analysis of NTSRs
Neurotensin (NT) was purchased from Peninsula Laboratories. RPMI 1640 was from Life Technologies Inc. and fetal calf serum from BioWest. Gentamycin, Bovine Serum Albumin (BSA), mowiol, paraformaldehyde, PD98059, mammalian protease and phosphatase inhibitor cocktails were from Sigma France. Wortmannin and LY294002 were from Calbiochem. Levocabastine was from Jenssen Pharmaceutica and SR48692 was a gift from Dr Gully (Sanofi-Avantis). Taq polymerase and Reverse Transcription system kit were from Promega. Antibodies against phosphorylated or total forms of Erk1/2 were from Santa Cruz Laboratory Inc. The rabbit polyclonal antiphospho-Akt antibodies were from Cell Signalling. The antiserum against the luminal domain of NTSR3/sortilin was a generous gift from Dr. C.M. Petersen (University of Aarhus, Aarhus, Denmark). Antibodies against NTSR1 and NTSR2 were from Santa Cruz Laboratories and from Neuromics, respectively. HRP conjugated goat anti-rabbit or anti-mouse was from Cell Signalling. The mouse TC3 and the rat INS-1E cell lines were gifts from Dr Abderahmani (Lausanne) and Dr Maechler (Geneva), respectively.
Proteins (20 g) from beta cells or mouse islets homogenates were denatured by boiling at 95 ◦ C for 3 min using 2× Laemmli sample buffer, resolved using 10 or 12% acrylamide gels and subsequently electroblotted onto nitrocellulose membranes. Membranes were blocked and incubated with primary antibodies either overnight at 4 ◦ C or for 2–4 h at room temperature. The bound antibody was visualized using an HRP-conjugated goat anti-rabbit followed by chemiluminescence reagents.
2.2. Cell culture and islets isolation The mouse -TC3 and the rat INS-1E cells were maintained in RPMI supplemented with 5% FCS and 50 mg/ml gentamycin in the presence of 0.1 mM sodium pyruvate and 0.001% -mercaptoethanol at 37 ◦ C under 5% CO2. Mouse pancreatic islets were isolated by the collagenase digestion method as described (Matsumoto et al., 2004). Isolated cells from pancreatic islets were cultured in the medium without sodium pyruvate and -mercaptoethanol, they were immediately used for the experiments.
2.5. Determination of cell viability with Hoechst 33342 and propidium iodure Cells were cultured on polylysine-coated glass coverslips 24 h before experiments to reach 70% confluency. Then, the percent of viable, apoptotic, and necrotic cells was determined following 24 h exposure to interleukine1 (10 ng/ml) in the absence or in the presence of 0.1 M NT or various drugs in serum free medium. Cells were washed once in PBS then incubated for 15 min with the DNA binding dyes propidium iodure (10 g/ml, Immunotech SA, France) and Hoechst 33342 (100 g/ml, Molecular Probes) (Dive et al., 1992). The fluorescence of both Hoechst and propidium iodure was visualized under an inverted fluorescence microscope at 10× magnification. A minimum of 2000 cells was counted in each experimental condition. Percent of living or dead cells were calculated using Image J analysis program and expressed as means ± S.E.M. 2.6. Measurement of caspase-3 activity Cells were cultured in 12 wells culture dish up to 70% confluency and incubated for various times with 1 M stau-
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Fig. 1. Expression of NT receptors in beta cells. (A) RNAs from cultured ß-TC3, INS-1E and islet cells were reverse transcribed, and cDNAs were amplified by PCR using sense and antisense NTSR-specific primers. G6PR and insulin primers were used as positive controls for islets RT-PCR. Data shown here are representative from at least three independent determinations. (B) immunoblotting of membrane homogenates from -TC3, INS-1E and islet cells with anti-NTSR1, NTSR2 and NTSR3 reveals a protein of 65 kDa for NTSR1, between 52 and 56 kDa for NTSR2 and 105 kDa for NTSR3, respectively.
rosporine (Sigma–Aldrich) in the absence or in the presence of 0.1 M NT or levocabastine (0.1 M) or SR48692 (1 M) or NTSR3 propeptide (1 M). In some cases, cells were treated with 1 M wortmannin (Wt) or 50 M LY 294002 (LY) or 24 M PD98059 (PD). Samples were processed for caspase-3 activity as already described from cell extracts using the Ac-DEVD-al (Sigma–Aldrich) as a substrate (da Costa et al., 2000). 2.7. Detection of phosphorylated Akt and MAP Kinase Erk1/2 Cells at 70% confluence were incubated overnight in serum free medium. Cells were then incubated for various periods of time at 37 ◦ C with 10−7 or 10−8 M NT for stimulation of Erk1/2 and Akt signaling pathway, respectively. Cells were then lysed with a buffer containing 50 mM Tris/HCl pH7.5, 100 mM NaCl, 5 mM EDTA, 0.5% Na-Deoxycholate, 1% NP40, 0.1% SDS in the presence of phosphatase and protease inhibitor cocktails (1/100). Identical amounts of solubilized protein (about 40 g) were analyzed on SDSPAGE, electroblotted onto nitrocellulose and subjected to immunoblotting using an antibody directed against the active phosphorylated forms of Erk1/2 or Akt. Results were standardized within the same blot using the total antiErk1/2 and total anti-Akt antibodies. The amount of protein was assessed on digital images using the Image J software. The stimulation was calculated from the ratio between the density of each band and the density of the band obtained in the control condition, corrected by the loading variations assessed by the total non-phosphorylated protein. 2.8. Statistical analysis Statistical analysis between each experimental condition was made from the number of different independent assays by using the Student’s t test.
3. Results 3.1. Neurotensin receptors expression in pancreatic beta cells To determine which NT receptor type is expressed by pancreatic -cells, we isolated RNA from -TC3, INS-1E cells and from mouse islets. Fig. 1A shows the RT-PCR products obtained from cell extracts and plasmids expressing each receptor as positive controls, using specific oligonucleotides for each receptor. Pro-Insulin-2 and Glucose-6 Phosphate catalytic subunit-2 genes were used as positive controls of mouse islets preparations. These experiments detected the three NTSR both in cell and islet extracts. To confirm the specificity of the NTSR1, 2 and 3 RT-PCR products, we cloned the fragments into pTarget vector. Sequencing of the recombinant plasmids definitively identified the PCR products and demonstrated the expression of the murine NTSR1, NTSR2 and NTSR3 in all the beta cells studied. In order to follow up these data, we performed Western blotting analysis of cell extracts from -TC3 and INS-1E cells and from mouse islets. As shown in Fig. 1B, all cell homogenates expressed the three NT receptors. The NTSR1 was detected at 65 kDa in all samples as well as the NTSR3 with a labeling of a single protein at 105 kDa. Some differences have been observed for the NTSR2 whose apparent molecular weight was higher in -TC3 (54 kDa) than in islets and in CHO-cells stably transfected with the NTSR2 (52 kDa). In INS-1E cells, two proteins of 48 and 56 kDa were detected. 3.2. NT protects ß-TC3 and INS-1E cells from apoptosis To study the role of NT in beta cells, we first tested its effects on -TC3 and INS-1E cells proliferation by measuring 3 H-thymidine incorporation. We observed that incubation of both cells with increasing concentrations of NT did not increase 3 H-thymidine incorporation after 24 h,
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Fig. 2. Biological effects of NT on beta cells. Determination of -TC3 (A) and INS-1E (B) cell viability with Hoechst 33342 and propidium iodure. The percent of viable, apoptotic, and necrotic cells was determined following 24 h exposure to IL-1 (10 ng/ml) in the absence or in the presence of 0.1 M NT or various drugs in serum free medium. Cells were washed once in PBS and incubated with the DNA binding dyes propidium iodure (10 g/ml) and Hoechst 33342 (100 g/ml) as described in Materials and Methods. Values are expressed as percent of dead cells. Means ± S.E.M. are from four independent experiments. One star: p < 0.05, two stars: p < 0.02.
indicating no effect of NT on beta cells proliferation (not shown). Therefore, to determine whether NT triggered a biological effect on beta cells, we incubated cells with the cytotoxic agent IL-1 and measured the effect of NT, NT agonists or antagonists on cell viability. Incubation of -TC3 and INS1E with IL-1 strongly enhanced the number of dead cells after 24 h from 2–5% in control conditions up to 13–14% in the presence of the cytokine (Fig. 2A and B), this effect being observed in all the tested cells suggesting that IL-1 indeed induces the death of treated cells. Interestingly, the incubation of IL-1 in the presence of 100 nM NT reduced the amount of cell death by 60% in -TC3 cells (p < 0.05) (Fig. 2A) and by 35% in INS-1E cells (p < 0.1) (Fig. 2B). The NTSR1 antagonist SR48692 was unable to reverse the effect of NT whereas the NTSR2 agonist levocabastine displayed the same protective action than that of NT itself (Fig. 2A and B). These results suggest the predominant involvement of the NTSR2 in the protection of beta cells. We next investigated whether the protective effect of NT was mediated by a modulation of caspase activation by measuring Ac-DEVD-pNA hydrolysis from cells incubated in the same conditions than above. As shown in Fig. 3A and B, the treatment of -TC3 or INS-1E cells with IL-1 (10 ng/ml) for 24 h increased caspase-3 activity by a factor of 2.5 to 3, the presence of 100 nM NT with the cytokine efficiently blocked this increase to reach a value close to that obtained in control conditions. In both cells, the NTSR1 antagonist SR48692 was unable to block the effect of the peptide, the NTSR2 agonist levocabastine displayed the same activity than that of NT (Fig. 3A and B) confirming the predominant involvement of the NTSR2. We also tested the propeptide released from the precursor form of NTSR3 described as a NT antagonist in human microglial
cells (Martin et al., 2003). This propeptide did not modify the response of NT suggesting that in cells bearing several NT receptors, it was ineffective to antagonize the peptide action. The next step was to find conditions to be able to measure a reversible effect of NT on caspase-3 activity in a shorter period of time in order to study the signaling pathways that could be involved in the presence of selective drugs that are toxic during long time incubations (>8 h). Then, we tested the ability of staurosporine to induce activation of caspase-3 as a function of time both on -TC3 and INS-1E cells and measured the effect of NT on this activation. Fig. 3C and D clearly indicated that a significant protective effect of NT on staurosporine-induced caspase-3 activity was observable after 8 h. We verified on -TC3 cells that levocabastine displayed the same protective effect on cells treated with staurosporine (Fig. 3E) than on cells treated with IL-1 (Fig. 3A). The effects of the two substances (NT and levocabastine) were not additive. Finally, we examined the protective action of NT in isolated mouse islets. Fig. 3F showed that, as observed in beta cell lines, NT efficiently reverses the staurosporine-induced caspase3 activity in isolated islets. 3.3. Signaling pathways of NT in beta cells Since we observed identical effects of NT on both survival and caspase-3 activity on the different beta cells studied, the following experiments have been performed only on -TC3 cells. NT (10 nM) rapidly and transiently stimulated the phosphorylation of MAP kinases Erk1/2 in ß-TC3 cells. The effect was maximal after 5 min and returned to the basal level after 15 min (Fig. 4A). Standardization of the same blot using the anti Erk antibodies indicated a maxi-
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Fig. 3. Effect of NT on beta cells caspase-3 activity. (A and B) -TC3 and INS-1E cells were cultured in 12 wells culture dish up to 70% confluency and incubated with IL-1 (10 ng/ml) for 24 h in the absence or in the presence of NT (0.1 M), levocabastine (0.1 M), SR48692 (1 M) or NTSR3 propeptide (0.1 M). Samples were processed for caspase-3 activity as described in Section 2 from cell extracts using the Ac-DEVD-al as a substrate. Data are means ± S.E.M. expressed in arbitrary units from 5 different experiments. One star: p < 0.05, two stars: p < 0.02. (C and D) -TC3 and INS-1E cells incubated for various times with 1 M staurosporine in the absence or in the presence of 0.1 M NT. Samples were processed as above and data are means ± S.E.M. expressed in arbitrary units from 4 different experiments. Two stars: p < 0.02. (E) -TC3 cells were incubated for 8 h with 1 M staurosporine in the absence or in the presence of 0.1 M NT or 0.1 M levocabastine or with both agonists. Data are means ± S.E.M. expressed in percent of staurosporine effect from 3 different experiments. One star: p < 0.02. (F) Mouse islets were incubated for 8 h with 1 M staurosporine in the absence or in the presence of 0.1 M NT. Data are means ± S.E.M. expressed in percent of staurosporine effect from 3 different experiments. One star: p < 0.1.
mal 3–4-fold stimulation of phospho-Erk at 10 min (Fig. 4B). We next investigated the influence of NT on the PI 3-kinase pathway by measuring the phosphorylation of Akt. The effect of NT was more pronounced, with a 5–7-fold stimulation of Akt phosphorylation at 30 min (Fig. 4C and D). Finally, in order to identify the NT-induced signaling pathway involved in the protective effect, we performed the caspase-3 experiments on cells treated with staurosporine for 8 h in the presence of inhibitors of the Erk1/2 MAP
kinase and PI 3 kinase pathways. Although all inhibitors tested increased the level of caspase-3 activity by a factor of 2–3, only the inhibition of the PI-3 kinase by wortmannin or LY294002 was able to prevent the NT protective effect (Fig. 5A). PD98059, a blocker of the Erk1/2 MAP kinase pathway was unable to inhibit the action of NT on caspase-3 activity. Longer treatments (24 h) with either wortmannin, LY294002 or PD98059 led to the death of almost all beta cells, rending impossible the treatment with IL1-.
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Fig. 4. Signaling of NT in -TC3 cells. -TC3 cells were stimulated with 100 nM NT for various times. The phosphorylation of Erk1/2 (p-Erk1/2) and Akt (p-Akt) was determined by immunoblotting using antibodies directed against the phosphorylated form of Erk1/2 (A and B) and Akt (C and D). Immunoblots shown in A and C are from a typical experiment. (B and D) data were standardized from 3 different experiments using the labeling obtained on the same blot with total Erk or total Akt antibodies and expressed as means ± S.E.M.
Fig. 5. Intracellular pathways involved in the protective effect of NT on beta cells. (A) Cells were treated with staurosporine for 8 h in the absence of drugs or in the presence of 1 M wortmannin (Wt) or 50 M LY 294002 (LY) or 24 M PD98059 (PD) and the protective effect of NT was estimated by caspase-3 activity measurement Two stars: p < 0.001 (n = 7). Results are expressed as percent of values obtained in the presence of staurosporine plus the indicated drug. These values are 2–3 times higher than in the presence of saurosporine alone.
4. Discussion In the present study, we investigated the role of NT and its receptors on the regulation of pancreatic beta cells fate. We demonstrated an efficient protective effect of the peptide on two beta cell lines submitted to apoptotic conditions like IL-1 or staurosporine treatment. Previous physiological studies performed during the first decade following the discovery of NT in 1973 revealed its potential involvement in the regulation of pancreatic functions such as insulin and glucagon release and pancreatic growth (Dolais-Kitabgi et al., 1979; Leeman and Carraway, 1982). However the molecular mechanisms by which NT exerts these effects were
not established. In this work, we demonstrate for the first time the expression of the three known NT receptors in the same beta cells and in islets. The NTSR1 is expressed as a 65 kDa protein and the NTSR3 as a 105 kDa protein in all beta cells. By contrast, the NTSR2 protein varies from 52 kDa in islet cells and in CHO cells stably transfected with the mouse NTSR2 to about 54 kDa in -TC-3 cells and to 56 kDa in INS-1E cell line. These discrepancies observed for the NTSR2 relative mobility are certainly not due to differences in glycosylation levels since murine NTSR2 are devoid of N-glycosylation site (Vincent et al., 1999). Rather, they are the consequences of distinct phospholipids environments leading to slightly different solubilities in detergent. The coexpression of NTSR2 and NTSR3 has already been observed in rat brain astrocytes (Nouel et al., 1999) and in rat cerebellar neurones (Sarret et al., 2002) whereas the co-expression of NTSR1 and NTSR3 was demonstrated in the HT29 cell line (Martin et al., 2002) but nothing was known about the coexistence of the three proteins. Pharmacological approaches indicated that only the NTSR2 could be implicated in the action of NT since the selective NTSR2 ligand levocabastine displays identical response than NT in both assays and that neither the NTSR1 antagonist SR48692 nor the propeptide antagonist of NTSR3 counteracts NT activity. Moreover, the fact that no additive effect was observed when cells were incubated with both NT and levocabastine is not in favour of the involvement of another receptor than NTSR2. In -TC3 cells, NT stimulates the phosphorylation of Erk1/2 and Akt in a time- and concentration-dependent manner (not shown). However, by measuring the influence of the MAP kinases (PD98059) and PI-3 kinase (wortmannin, LY294002) inhibitors on the final protective effect of NT, we noticed that only PI-3 kinase inhibitors were able to counteract the action of NT in the caspase-3 assay (Fig. 5). This suggests that the NT protective effect on ß-TC3 cells
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involves only the PI-3 kinase pathway. The MAP kinase pathway also stimulated by the peptide could mediate another NT effect which remains to be characterized. These results highlight the pivotal role of protein kinase B in controlling beta cell survival as initially suggested by Elghazi et al. (2006). However, the downstream targets of Akt upon NT activation remain to be investigated. The cell protection of NT against apoptosis has already been observed in some cancer cells in which NT also behaves as a proliferative agent, e.g. in breast cancer cells (Somai et al., 2002; Souaze et al., 2006). However, in mammary gland, the MAP kinase pathway was shown to be involved in the anti-apoptotic effect of NT but not in the present study. This discrepancy could be explained by the fact that the activation of this pathway in breast cancer cells was essentially mediated by NTSR1 whereas in beta cells (present study), the NT effect is rather mediated by NTSR2 leading to the involvement of the PI-3 kinase pathway. In another biological system where the NTSR3/sortilin is associated with the neurotrophin receptor p75NTR to trigger neuronal cell death induced by the precursor of the nerve growth factor (proNGF), NT was also shown to counteract this effect (Nykjaer et al., 2004). Thus, it seems that one of the major functions of NT, both in the central nervous system and in peripheral tissues, is to activate cell survival pathways. Although NT has no effect on beta cell growth, its protective role against external cytotoxicity could be of importance and physiologically relevant. Indeed, the fact that NT is released in the human circulation following a meal and that lipid absorption stimulates peptide release from rat small intestine (Leeman and Carraway, 1982) could be closely related with a physiological role for NT in the protection of endocrine pancreas towards fatty acids and glucose cytotoxic effects. In conclusion, the present work demonstrates that NT exerts a potent protective action on beta cells and that this effect appears to be mediated by NTSR2. Further in vivo studies carried out on lesionned or streptozotocin-treated animals or on NTSR2-KO mice should help to confirm the role of this receptor in the protective effect of NT and to evaluate the potency of the peptide or of bioavailable NT analogues (Hadden et al., 2005) as protective or regenerating agents of the endocrine pancreas. Acknowledgments This work was supported by the Centre National de la Recherche Scientifique. We thank Claus Petersen (Aarhus, Denmark) for the gift of NTSR3 antibodies, Pierre Maehler (Geneva, Switzerland) for the INS-1E cell line, and Amar Abderahmani (Lausannne, Switzerland) for the -TC3 cell line. References Berelowitz M, Frohman LA. The role of neurotensin in the regulation of carbohydrate metabolism and in diabetes. Ann NY Acad Sci 1982;400:150–9.
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156–9. da Costa CA, Ancolio K, Checler F. Wild-type but not Parkinson’s diseaserelated ala-53 → Thr mutant alpha-synuclein protects neuronal cells from apoptotic stimuli. J Biol Chem 2000;275:24065–9. Dal Farra C, Sarret P, Navarro V, Botto JM, Mazella J, Vincent JP. Involvement of the neurotensin receptor subtype NTR3 in the growth effect of neurotensin on cancer cell lines. Int J Cancer 2001;92: 503–9. Dive C, Gregory CD, Phipps DJ, Evans DL, Milner AE, Wyllie AH. Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochim Biophys Acta 1992;1133:275–85. Dolais-Kitabgi J, Kitabgi P, Brazeau P, Freychet P. Effect of neurotensin on insulin, glucagon, and somatostatin release from isolated pancreatic islets. Endocrinology 1979;105:256–60. Elghazi L, Balcazar N, Bernal-Mizrachi E. Emerging role of protein kinase B/Akt signaling in pancreatic beta-cell mass and function. Int J Biochem Cell Biol 2006;38:157–63. Feurle GE, Muller B, Rix E. Neurotensin induces hyperplasia of the pancreas and growth of the gastric antrum in rats. Gut 1987;28(Suppl.): 19–23. Hadden MK, Orwig KS, Kokko KP, Mazella J, Dix TA. Design, synthesis, and evaluation of the antipsychotic potential of orally bioavailable neurotensin (8–13) analogues containing non-natural arginine and lysine residues. Neuropharmacology 2005;49:1149–59. Leeman SE, Carraway RE. Neurotensin: discovery, isolation, characterization, synthesis and possible physiological roles. Ann NY Acad Sci 1982;400:1–16. Martin S, Navarro V, Vincent JP, Mazella J. Neurotensin receptor-1 and -3 complex modulates the cellular signaling of neurotensin in the HT29 cell line. Gastroenterology 2002;123:1135–43. Martin S, Vincent JP, Mazella J. Involvement of the neurotensin receptor-3 in the neurotensin-induced migration of human microglia. J Neurosci 2003;23:1198–205. Matsumoto M, Miki T, Shibasaki T, Kawaguchi M, Shinozaki H, Nio J, et al. Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells. Proc Natl Acad Sci USA 2004;101:8313–8. Mazella J. Sortilin/neurotensin receptor-3: a new tool to investigate neurotensin signaling and cellular trafficking? Cell Signal 2001;13: 1–6. Mazella J, Vincent JP. Functional roles of the NTS2 and NTS3 receptors. Peptides 2006;27:2469–75. Nouel D, Sarret P, Vincent JP, Mazella J, Beaudet A. Pharmacological, molecular and functional characterization of glial neurotensin receptors. Neuroscience 1999;94:1189–97. Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen MS, et al. Sortilin is essential for proNGF-induced neuronal cell death. Nature 2004;427:843–8. Petersen CM, Nielsen MS, Nykjaer A, Jacobsen L, Tommerup N, Rasmussen HH, et al. Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography. J Biol Chem 1997;272:3599–605. Pissios P, Ozcan U, Kokkotou E, Okada T, Liew CW, Liu S, et al. Melanin concentrating hormone is a novel regulator of islet function and growth. Diabetes 2007;56:311–9. Portela-Gomes GM, Johansson H, Olding L, Grimelius L. Co-localization of neuroendocrine hormones in the human fetal pancreas. Eur J Endocrinol 1999;141:526–33. Sarret P, Gendron L, Kilian P, Nguyen HM, Gallo-Payet N, Payet MD, et al. Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells. J Biol Chem 2002;277:36233–43. Somai S, Gompel A, Rostene W, Forgez P. Neurotensin counteracts apoptosis in breast cancer cells. Biochem Biophys Res Commun 2002;295:482–8. Souaze F, Dupouy S, Viardot-Foucault V, Bruyneel E, Attoub S, Gespach C, et al. Expression of neurotensin and NT1 receptor in human breast cancer: a potential role in tumor progression. Cancer Res 2006;66: 6243–9. Vincent JP, Mazella J, Kitabgi P. Neurotensin and neurotensin receptors. Trends Pharmacol Sci 1999;20:302–9. Wood JG, Hoang HD, Bussjaeger LJ, Solomon TE. Effect of neurotensin on pancreatic and gastric secretion and growth in rats. Pancreas 1988;3:332–9.