Stromal cell-derived factor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis activation induces intra-islet glucagon-like peptide-1 (GLP-1) production and enhances beta cell survival
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The endogenous production of stromal cell-derived factor-1 (SDF-1) in beta cells in transgenic mice attenuates the development of diabetes in response to streptozotocin. Here we propose that beta cell injury induces SDF-1 production, and the SDF-1/chemokine (C-X-C motif) receptor 4 (CXCR4) interaction auto-activates Sdf1 expression, resulting in the autocrine production of SDF-1 by beta cells and the paracrine activation of glucagon-like peptide-1 (GLP-1) production by alpha cells.
SDF-1 production in adult mouse and human islets and rat INS-1 cells was measured in models of beta cell injury. The paracrine actions of SDF-1 on GLP-1 production in alpha cells were explored. The potential synergism between the growth-promoting actions of GLP-1 and the pro-survival actions of SDF-1 on the preservation of cell mass was evaluated by cell viability assays.
In adult islets and INS-1 cells, Sdf1 expression was re-induced in response to injury. The interaction of SDF-1 with its receptor on alphaTC1 cells activated protein kinase Akt, stimulated cell proliferation and induced the expression of prohormone convertase 1/3 and the consequent production of GLP-1 in alpha cells. The combination of GLP-1 and SDF-1 additively enhanced both the growth and longevity of INS-1 beta cells.
The results of these studies suggest that in response to beta cell injury and the ensuing induction of SDF-1, the biological function of alpha cells switches from the production of glucagon to the provision of the local growth factor GLP-1 which, in combination with SDF-1, promotes the growth, survival and viability of the beta cells.
KeywordsAlpha cells Chemokine (C-X-C motif) receptor 4 Diabetes GLP-1 Islets Prohormone convertase SDF-1 Stromal cell-derived factor-1
Chemokine (C-X-C motif) receptor 4
Stromal cell-derived factor-1
Signal transducer and activator of transcription
Wingless-related MMTV integration
The prevalence of diabetes mellitus is increasing throughout the world . A fundamental underlying cause of diabetes is an inadequate mass of insulin-producing beta cells in the islets of the pancreas, resulting in insufficient insulin to meet the body’s needs. In type 1 diabetes beta cells are nearly completely destroyed by autoimmunity. In type 2 diabetes, most commonly associated with obesity, beta cell mass is modestly reduced (∼50%) and the remaining beta cells are functionally impaired by the acquired insulin resistance and resulting hyperlipidaemia and hyperglycaemia, so called gluco-lipotoxic stress. Gluco-lipotoxicity causes increased oxidative stress and shortens the lifespan of beta cells [2, 3]. A challenge in the treatment of diabetes is to find a means to preserve or enhance beta cell mass by stimulating the growth of new beta cells, prolonging the lifespan of beta cells, or both.
The architecture of the islets is remarkable in that the mass of insulin-producing beta cells is surrounded by non-beta endocrine cells: alpha, delta and pancreatic polypeptide-producing cells. In particular, in human islets the alpha cells are mixed in with the beta cells so that >70% of beta cells are in heterotypic contact with non-beta endocrine cells, most of which are the alpha cells that express the proglucagon gene (GCG) and produce the hormone glucagon in the islets . This close proximity of endocrine cells to one another in the islets is highly suggestive of intra-islet autocrine and paracrine regulatory interactions among the different endocrine cell types .
Stromal cell-derived factor-1 (SDF-1; also known as chemokine [C-X-C motif] ligand 12 [CXCL12]) is a small peptide chemokine that regulates many essential biological processes, including stem cell motility, cardiac and neuronal development, neovascularisation and tissue repair and regeneration . In particular, SDF-1 is produced in reactive stromal tissue in response to injuries of the liver  and heart , where it is believed to recruit bone-marrow-derived somatic stem cells involved in tissue repair . Injury to the mouse pancreas in response to the transgenic overexpression of IFNγ activates production of SDF-1 in duct and stromal tissues . In earlier studies we demonstrated that the SDF-1 receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), is produced in the adult mouse pancreas. However, in adult islets the production of SDF-1 is restricted to vascular endothelial and stromal cells and is not detected in beta cells . The forced endogenous production of SDF-1 in beta cells in transgenic mice (RIP-SDF-1) attenuates the development of diabetes in response to the ablation of beta cells by the administration of streptozotocin. An important and surprising finding was that 6 h after the administration of a high dose of streptozotocin to cause injury in beta cells, phospho-Akt, a pro-proliferative signal, appeared only in the alpha cells and not in the beta cells . When the islets were examined 2 weeks after the single dose of streptozotocin the alpha cells had nearly completely replaced the beta cells in the islets in the normal mice, but many beta cells had persisted or regenerated in the SDF-1 transgenic mice that produce high levels of SDF-1 in beta cells . These observations suggested the existence of paracrine cross-talk between beta and alpha cells as streptozotocin is known to be a beta cell-specific toxin that does not affect alpha cells [12, 13]. These findings led to our current hypothesis that injured beta cells might be communicating with alpha cells in the islets via SDF-1/CXCR4 signalling. Further, we showed that SDF-1 exerts cytoprotective actions on islet beta cells, and promotes their survival by the activation of the pro-survival kinase Akt and β-catenin/transcription factor 7-like 2 (T cell specific, HMG-box) (TCF7L2)-mediated wingless-related MMTV integration (WNT) signalling . Likewise, the glucoincretin hormone glucagon-like peptide-1 (GLP-1) stimulates WNT signalling in beta cells resulting in an increase in their proliferation .
Here we report that in adult islets and in rat insulinoma INS-1 cells Sdf1 expression is re-induced in beta cells in response to injury invoked by cytokines, streptozotocin and thapsigargin. The SDF-1 receptor, CXCR4, is expressed on both alpha and beta cells. The paracrine actions of SDF-1 on its receptor on alpha cells activate protein kinase Akt, stimulate cell proliferation and induce the production of prohormone convertase (PC)1/3 and the consequent production of GLP-1 in alpha cells. GLP-1 predominantly promotes proliferation whereas SDF-1 exerts anti-apoptotic actions and promotes survival. We found the combination of GLP-1 and SDF-1 additively enhances both growth and longevity of cultured beta cells and thereby preserves beta cell mass against injury, glucotoxicity and nutrient deprivation.
We propose that injury to beta cells activates the SDF-1/CXCR4 axis in islets, resulting in the autocrine production of SDF-1 by beta cells and paracrine activation of GLP-1 production by alpha cells. The combination of the local intra-islet actions of GLP-1 and SDF-1 enhances the growth and survival of beta cells. Our findings suggest that SDF-1 agonists may be a means to stimulate the growth of alpha cells and their production of GLP-1 in the local intra-islet environment and thereby stimulate regeneration of injured beta cells in diabetes.
Thapsigargin was obtained from Biomol (Plymouth Meeting, PA, USA). Cytokines, SDF-1 IFNγ, IL-1β and TNFα were obtained from R&D Systems (Minneapolis, MN, USA). Exendin-4 (EXD4) and streptozotocin were from Sigma-Aldrich (St Louis, MO, USA)
Culture of INS-1, MIN6 cells and alphaTC1 cells
Rat INS-1 cells (obtained from C. Wollheim, Geneva, Switzerland) were maintained in RPMI supplemented with 10% FBS, 1.0 mmol/l sodium pyruvate, 10 mmol/l HEPES, penicillin and streptomycin at 37°C under 5% CO2 and at 95% humidity. Mouse MIN6 cells (obtained from J. Miyazaki, University of Osaka, Japan) were cultured in DMEM containing 15% (vol./vol.) FBS. Mouse alphaTC1 cells (ATCC #2350, American Type Culture Collection, Gaithersberg, MD, USA) were maintained in RPMI supplemented with 10% (vol./vol.) FBS and 15 mmol/l HEPES.
Isolation of mouse pancreas and immunohistochemistry
Pancreases were removed from mice, fixed in formaldehyde or frozen, and immunostaining was performed on tissue sections as described by Yano et al. .
Isolation of mouse islets
Mouse islets were isolated from the pancreases of mice according to the standard protocol (see legend to Fig. 2). From each 12–18 week old male C57BL/6J mouse, 200–300 islets were obtained. Dispersed cells were prepared from islets isolated from 20–22 week old male mice for immunochemical studies by digestion of the islets in 0.05% (wt/vol.) trypsin at 37°C for 15 min with gentle shaking. The dispersed cells were plated on poly-d-lysine-coated slides (BD, Franklin Lakes, NJ, USA) and cultured overnight before treatment and fixation for immunostaining.
Human islets from donors
Human islet tissue was obtained from the Integrated Islet Distribution Program (Madison, WI, USA). Use of human tissues was approved by the MGH Human Studies Committee. Dispersed cells were prepared from human islets by incubation in 0.01% trypsin (wt/vol.)-EDTA for 20 min at 37°C.
Treatment of islets and cells with cytokines and thapsigargin
Dispersed islet cells or INS-1 cells were cultured in 96 well plates or 10 cm dishes in the presence of 2 nmol/l EXD4 and/or 10 nmol/l SDF-1 on a background of nutrient deprivation by serum withdrawal or cell-stress-inducing reagent including thapsigargin 50 nmol/l or a cytokine cocktail of IL-1β 50 ng/ml, TNFα 10 ng/ml and IFNγ 50 ng/ml. Cell viability was measured by ATPlite assay (PerkinElmer, Waltham, MA, USA) or cell mass measurement.
Cell mass assay
INS-1 cells were cultured for 6 days in media with and without combinations of thapsigargin or cytokines with and without SDF-1 and EXD4. At the end of the incubation the cells were harvested by scrape loading and weighed.
RNA isolation and real-time
RT-PCR RNA isolation and real-time RT-PCR were performed as previously reported by Yano et al. and Liu et al. [11, 15]. For each treatment, all samples were performed in triplicate and the data are presented as ratio of Sdf1 (also known as Cxcl12)/Gapdh compared with the normalised control.
AlphaTC1 cells (5 × 105 cells/ml) were treated with 10 nmol/l SDF-1 or vehicle controls for the times indicated. Streptozotocin was dissolved in 50 mmol/l sodium citrate buffer (pH 4.7). Cell lysates were prepared and proteins were analysed on western immunoblots as described earlier by Liu et al. [14, 15]. Protein density on immunoblots was quantified by densitometric analysis using a Kodak Image Station 440 CF (Eastman Kodak, Rochester, NY, USA). The primary antibodies used were phospho-Akt (Ser473) (587F11) monoclonal antibody (catalogue no. 4051; Cell Signaling Technologies, Beverly, MA, USA) and Akt antibody, which recognises Akt1, 2 and 3 (catalogue no. 9272; Cell Signaling Technologies).
GLP-1 and insulin assays
GLP-1 content in the culture medium and cell lysates was measured using an ELISA kit from BioVender LLC (Candfer, NC, USA) according to the instruction manual. Insulin content in the culture medium was measured by ELISA kit from Millipore (Billerica, MA, USA) according to the instruction manual.
Cell viability assay
Cell viability was measured using the ATPlite one-step assay according to manufacturer’s manual (PerkinElmer).
Cell proliferation assay
Proliferation of alphaTC1 cells was determined by incorporation of BrdU into newly synthesised DNA of proliferating cells. Cells in 96 well plates were treated with SDF-1 (10 nmol/l) or PBS overnight, then pulse-labelled with BrdU for 4 h. BrdU staining was measured using the DELFIA cell proliferation kit (PerkinElmer).
Immunofluorescent staining of dispersed mouse islet cells
Trypsin-treated cells were plated on slides and cultured overnight in RPMI media (Gibco, Grand Island, NY, USA) containing 10% (vol./vol.) FBS. Cells were treated, washed and fixed in 10% formalin in PBS for 15 min at room temperature for fluorescence immunostaining using a rabbit antiserum to mouse PC1/3 (a gift from D. F. Steiner and M. Hara, Chicago, IL, USA) and antisera to insulin (Millipore) and glucagon (Sigma-Aldrich). A mouse anti-glucagon monoclonal antibody was used and the antiserum to insulin was from guinea pig. Secondary fluorophore antibodies used were Cy2 (green)-labelled donkey anti-rabbit IgG and Cy3 (red)-labelled donkey anti-mouse IgG (glucagon) and donkey anti-guinea pig IgG (insulin). Fluorescent images were captured with a Nikon Optiphot 2 microscope using Photometric Cool Snap HQ camera (Photometrics, Huntington Beach, CA, USA) and IP Lab 3.6.5 software (Scanalytics, Falls Church, VA, USA).
Data are presented as the mean ± SD. Statistical analysis was performed using paired t test. Values of p < 0.05 were considered statistically significant.
Neonatal expression of SDF-1 in beta cells is extinguished in the adult pancreas
Injuries of islets and beta cells induces the production of SDF-1
SDF-1 stimulates the proliferation of alphaTC1 cells and induces the production of PC1/3 and GLP-1
To investigate production of PC1/3 in primary mouse alpha cells, dispersed mouse islet cells were prepared and cultured overnight before immunostaining with antisera to PC1/3, glucagon and insulin. Immunocytochemical analyses of alpha and beta cells in dispersed mouse islet cells showed weak, but definite, specific production of PC1/3 in alpha cells compared with stronger production in beta cells (electronic supplementary material [ESM] Fig. 1a, b). Dispersed mouse islet cells were treated with SDF-1 (100 nmol/l) for 6 h. Examination of several hundred cells by immunocytofluorography with antisera to PC1/3, glucagon and insulin suggested that the intensity of PC1/3 fluorescence increased in alpha cells with the addition of SDF-1 (ESM Fig. 1c). However, the paucity of cells precluded attempts at quantificative assessment of PC1/3 production in alpha cells. It remains possible, however, that the production of PC1/3 in alpha cells is perturbed by the stresses induced in the cells by islet isolation and the enzymatic dispersal of the islet cells. The effect of SDF-1 on GLP-1 production is an important finding because GLP-1 is known to stimulate the growth [14, 26] and promote the survival [27, 28] of beta cells. The production of GLP-1 locally within the islet could exert short-range tropic actions on adjacent beta cells. The addition of SDF-1 to alphaTC1 cells appears to stimulate their proliferation as determined by BrdU incorporation into the cells in response to SDF-1 (Fig. 5d). This finding suggests that the production of SDF-1 by injured beta cells might be a mechanism for the development of alpha cell hyperplasia seen in mice after the ablation of beta cells by the administration of streptozotocin [11, 12].
SDF-1 and GLP-1 additively enhance INS-1 cell mass
We demonstrate that when islet beta cells are injured by cytokines, streptozotocin, thapsigargin and glucotoxicity, SDF-1 production resembling neonatal production is re-induced. The SDF-1 receptor CXCR4 is expressed on alpha cells and is capable of activating the Akt protein kinase. Importantly, the apparent paracrine actions of SDF-1 on its receptor in alpha cells stimulate the production of GLP-1 in islets. We further explored the potential synergism between the growth-promoting actions of GLP-1 and the pro-survival actions of SDF-1 on the preservation of INS-1 cell mass, and found that the combination of GLP-1 and SDF-1 additively enhances both growth and longevity of beta cells.
GLP-1, an incretin hormone initially recognised for its glucose-dependent insulin-releasing actions, is now known to stimulate both the proliferation [15, 26] and the survival  of beta cells. GLP-1 agonists given systemically are currently used to stimulate insulin secretion in type 2 diabetic individuals. Alpha cells normally produce PC2, which cleaves proglucagon to release glucagon, a hormone that stimulates hepatic glucose production to maintain euglycaemia during fasting. GLPs are cleaved from proglucagon by PC1/3, another member of the PC protein family not normally produced in alpha cells. Activation of PC1/3 production and the ensuing GLP-1 production were recently found in alpha cells of prediabetic NOD mice, pregnant mice, ob/ob mice and db/db mice . Therefore the stimulation of GLP-1 production from alpha cells within islets, specifically by SDF-1 agonists, might provide high local paracrine concentrations of GLP-1 for adjacent beta cells at a critical time during their injury and thereby might promote beta cell regeneration. We propose that a mechanism for the induction of GLP-1 production in alpha cells of islets is via the induction of PC1/3 by the paracrine actions of SDF-1. PC1/3 production and resultant GLP-1 production convert the alpha cell from a hyperglycaemia-promoting cell to one that promotes beta cell growth and survival.
In pathophysiological conditions, such as exposure to sustained high glucose because of the development of insulin resistance in type 2 diabetes, the beta cells increase in number (hyperplasia) in an attempt to produce more insulin to counteract the hyperglycaemia. Likewise, sustained hypoglycaemia induced by the experimental disruption of glucagon signalling, e.g. knockout of the glucagon receptor gene [33, 34], or knockout of the gene encoding PC2, which is required for the post-translational cleavage of proglucagon to produce glucagon [35, 36, 37], resulting in impaired gluconeogenesis, leads to marked alpha cell hyperplasia.
Paradoxically, alpha cell hyperplasia also occurs in conditions of insulin deficiency resulting from injury of beta cells, for example in mice given the beta cell toxin streptozotocin [11, 12, 37, 38], or in the NOD mouse in which beta cells are injured by autoimmune attack . By all physiological arguments of feedback regulation of glucagon secretion, hyperglycaemia should suppress glucagon secretion and alpha cell functions, and not cause hyperplasia of the alpha cells. We propose that this paradox of alpha cell hyperplasia might be explained by the fact that in response to beta cell injury (resulting in hypoinsulinaemia and hyperglycaemia) the injured beta cells produce SDF-1 and the activation of the SDF-1/CXCR4 axis in alpha cells adjacent to beta cells in islets. The activation of CXCR4-mediated signalling stimulates alpha cell proliferation and switches the biological functions of the alpha cells from the regulation of glucose metabolism to that of providing local growth factors, such as GLP-1, involved in the regeneration of the injured beta cells. Whether or not SDF-1 stimulates the proliferation of primary alpha cells in vivo or in vitro remains uncertain. Although we have demonstrated increased proliferation of cultured alphaTC1 cells by SDF-1, attempts at demonstrating SDF-1-mediated stimulation of primary mouse and human islet cells in vitro were equivocal because of the sparsity of alpha cells in dispersed mouse islet cell preparations and the rapid (1 day in culture) de-differentiation of dispersed cells prepared from donor human islets. The validation of the hypothesis that SDF-1 might stimulate the growth of alpha cells awaits appropriate studies in vivo, such as using the transgenic RIP-SDF-1 mouse that constitutively expresses Sdf1 in islet beta cells . Our results, and the previous study in Pc2 (also known as Pcsk2)-null mice , demonstrate that the alpha cells are endowed with an unusual plasticity in their abilities to switch their hormone production from glucagon to GLP-1, which has functions within islets proposed to promote the growth and the survival of beta cells.
We thank K. McManus and L. Brindamour for expert experimental assistance. We thank M. Hara and D. F. Steiner for the generous gift of the PC1/3 antiserum. The studies were supported in part by research grants (J. F. Habener) and a postdoctoral fellowship (Z. Liu) from the Juvenile Diabetes Research Foundation and a grant from the Charles H. Hood Foundation of the Medical Research Foundation to Z. Liu.
Duality of interest
The authors declare there is no duality of interest associated with this manuscript.
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