Blockade of sphingosine 1-phosphate receptor 2 signaling attenuates streptozotocin-induced apoptosis of pancreatic \xce\xb2-cells
Sphingosine 1-phosphate (S1P) is a potent sphingolipid mediator that acts through five cognate G protein-coupled receptors (S1P1\xe2\x80\x93S1P5) and regulates many critical biological processes. Recent studies indicated that S1P at nanomolar concentrations significantly reduces cytokine-induced apoptosis of pancreatic \xce\xb2-cells in which genes for S1P1\xe2\x80\x93S1P4 are co-expressed. However, the S1P receptor subtype(s) involved in this effect remains to be clarified. In this study, we investigated the potential role of S1P2 in streptozotocin (STZ)-induced apoptosis of pancreatic \xce\xb2-cells and progression of diabetes. S1P2-deficient (S1P2\xe2\x88\x92/\xe2\x88\x92) mice displayed a greater survive ability, lower blood glucose levels, and smaller numbers of TUNEL-positive apoptotic \xce\xb2-cells to administration of a high dose of STZ than wild-type (WT) mice. S1P2\xe2\x88\x92/\xe2\x88\x92 mice showed higher insulin/glucose ratios (an index of relative insulin deficiency) and larger insulin-positive islet areas to administration of a low dose of STZ than WT mice. Moreover, administration of JTE-013, a S1P2-specific antagonist, to WT mice ameliorated STZ-induced blood glucose elevation and reduced the incidence of diabetes. Our findings indicate that blockade of S1P2 signaling attenuates STZ-induced apoptosis of pancreatic \xce\xb2-cells and decreases the incidence of diabetes.
Introduction<!>Animals<!>Induction of diabetes by STZ injection<!>Immunohistochemistry<!>Statistical analysis<!>S1P2\xe2\x88\x92/\xe2\x88\x92 mice were more resistant to administration of a high dose of STZ<!>More \xce\xb2-cells were preserved after STZ injection in S1P2\xe2\x88\x92/\xe2\x88\x92 mice<!>More islet cells were protected against STZ-induced apoptosis in S1P2\xe2\x88\x92/\xe2\x88\x92 mice<!>JTE-013 decreased the incidence of diabetes<!>Discussion<!>Conclusion
<p>Type 1 diabetes is an autoimmune disease that results in the destruction of pancreatic β-cells, whereas type 2 diabetes is a much more common disorder caused by insulin resistance and relative insulin deficiency. Insulin resistance in type 2 diabetes is initially managed by enhanced secretion of insulin from β-cells; however, this may gradually lead to a decrease in β-cell mass and deterioration of key β-cell functions such as glucose-stimulated insulin secretion. Therefore, β-cell failure is a key pathogenic process in both type 1 and 2 diabetes [1,2].</p><p>Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator that exhibits diverse biological functions in most cell types and regulates many pathological processes, acting through five cognate high-affinity receptors (S1P1–S1P5) [3,4]. S1P was found to ameliorate cytokine-induced apoptosis of β-cells [5,6] in which genes for S1P1–S1P4 are co-expressed [7]. Previous studies demonstrated that S1P2 signaling induces intracellular calcium mobilization leading to increased glucose uptake in myoblasts [8], and RNA-based screening for genes that confer insulin resistance to 3T3-L1 adipocytes identified S1pr2 as a candidate [9], suggesting that S1P2 plays an important role in the pathogenesis of diabetes. In the present study, we examined the role of S1P2 in streptozotocin (STZ)-induced apoptosis of β-cells and progression of diabetes using S1P2-deficient (S1P2−/−) mice as well as the S1P2-specific antagonist JTE-013.</p><!><p>S1P2−/− mice were generated and genotyped as described previously [10]. S1P2−/− mice were backcrossed with C57BL/6N (Clea Japan, Tokyo, Japan) for seven generations, and thus, littermate wild-type (WT) mice or age-matched (8-week-old) C57BL/6N were used as controls. All mice were fed ad libitum with standard chow/water and kept under a 12-hour light-dark cycle in an air-conditioned room. All animal protocols were approved by the animal care and use committee of Chiba-East National Hospital.</p><!><p>Streptozotocin (STZ, Sigma) was freshly dissolved in 20 mM citrate buffer (pH4.5) and intraperitoneally administered under various conditions in each experiment: 50 mg/kg body weight for 5 consecutive days (50 mg/kg for 5 days), 100 mg/kg for 1 day, or 100 mg/kg for 2 days. Control mice received injections of the citrate buffer. JTE-013 (Calbiochem), a specific S1P2 antagonist [11], was freshly dissolved in saline and intraperitoneally administered at 4 mg/kg for 6 days (one shot prior to STZ and five shots with STZ). Control mice received injections of saline. Blood was collected from the retro-orbital sinus of anesthetized mice and blood glucose levels were measured using the Accu-Chek Aviva system (Roche). Mice were diagnosed with diabetes mellitus (DM) when their blood glucose levels were ≥ 300 mg/dl on two consecutive days [12]. Serum insulin levels were measured using an insulin RIA kit (Millipore) in accordance with the manufacturer's instructions.</p><!><p>Pancreata were quickly removed from anesthetized mice, fixed with 3% formalin in phosphate-buffered saline, and embedded in paraffin. To count islet cells, deparaffinized pancreatic sections were immunostained with guinea pig polyclonal anti-insulin antibody (Cell Marque, Rocklin, CA) using a NexES IHC system (Ventana Medical Systems, Tucson, AZ). Full area sizes (mm2) of pancreatic sections (single section per mouse) were measured and the numbers of insulin-positive islets in each section were counted. Apoptotic cells were detected using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (Apotag Kit; Chemicon) in accordance with the manufacturer's recommendations. Apoptotic cells per nm2 of islet area were counted in ≥ 10 islets per section.</p><!><p>Results are expressed as mean ± SD. All statistical analyses were performed using Dr. SPSS II for Windows (SPSS Inc., Chicago, IL). The existence of significant differences between two groups (with an accuracy of at least 95%) was analyzed using a two-tailed unpaired t-test. Kaplan-Meier analysis was used to examine diabetes-free rates and survival rates, and the differences were determined by log-rank tests. A value of P < 0.05 was considered significant.</p><!><p>WT and S1P2−/− males were intraperitoneally injected with a high dose of STZ (100 mg/kg for 2 days), and their health status was monitored every week until the 15th week after the final injection. Forty percent (10/25) of WT mice died at the 2nd week, increasing to 64.0% (16/25) by the 11th week. In contrast, S1P2−/− mice show lower death rates of 11.1% (2/18) and 27.7% (5/18), respectively. Kaplan–Meier analysis indicates that S1P2−/− mice were significantly (P = 0.0334) more resistant to STZ toxicity than WT mice (Fig. 1A). At the 15th week after the final injection, serum glucose levels in surviving S1P2−/− mice were significantly lower than those in surviving WT mice (Fig. 1B).</p><!><p>WT and S1P2−/− males were injected with a low dose of STZ (50 mg/kg for 5 days) so that all mice survived until at least the 30th day after the final injection, and blood glucose levels were measured twice a week. There was no significant difference in glucose levels between WT and S1P2−/− mice (Fig. 2A). Eight of eleven (72.7%) WT mice were diagnosed with diabetes compared with only three of eight (37.5%) S1P2−/− mice; however, Kaplan-Meier analysis revealed no significant difference in diabetes-free rates between the two groups (Fig. 2B). Although blood insulin levels in S1P2−/− mice were comparable to those in WT mice at the 30th day after the final STZ injection (Fig. 2C), the insulin/glucose ratios (an index of relative insulin deficiency [13]) were significantly higher in S1P2−/− mice than in WT mice (Fig. 2D). This indicates that insulin was more efficiently secreted in response to blood glucose elevation in S1P2−/− mice than in WT mice. We counted the numbers of insulin-positive islets per pancreatic area at the 30th day after STZ injection. Insulin-positive islets were much more abundant in pancreatic sections from S1P2−/− mice (Fig. 2E). The numbers of insulin-positive islets per pancreatic area in S1P2−/− mice were comparable to those in WT mice without STZ, because more islet cells (per pancreatic area) withstood STZ-induced cytotoxicity in S1P2−/− mice (Fig. 2F). Taken together, these findings indicate that more islet β-cells were preserved after STZ injection in S1P2−/− mice than in WT mice.</p><!><p>The administration of STZ is known to induce apoptosis of islet β-cells [13,14]. WT and S1P2−/− males were intraperitoneally injected with a high dose of STZ (100 mg/kg for 1 day), and apoptosis of islet cells was evaluated the following day. Histological analysis revealed that TUNEL-positive apoptotic islet cells were less abundant in S1P2−/− mice than in WT mice (Fig. 3A). The number of apoptotic cells per nm2 of islet area in S1P2−/− mice was 27% of that in WT mice (Fig. 3B), indicating that the lack of S1P2 protects islet cells from STZ-induced apoptosis.</p><!><p>We examined the effect of JTE-013, a specific S1P2 antagonist, on the incidence of STZ-induced diabetes. JTE-013 (4 mg/kg) was intraperitoneally injected for 6 days (one shot prior to STZ and five shots with STZ (50 mg/kg)), and blood glucose elevation after the final STZ injection was examined every week. Glucose levels at the 4th week after the final STZ injection were significantly lower in JTE-013-treated mice than JTE-013-untreated mice (Fig. 4A). Glucose levels were not indistinguishable between JTE-013-treated and JTE-013-untreated mice in the absence of STZ. Among STZ-injected mice, 71.4% (10/14) of JTE-013-untreated mice were found to be diabetic compared with only 14.3% (2/14) of JTE-013-treated mice by the 6th week after the final STZ injection. Kaplan-Meier analysis indicates that JTE-013 treatment significantly (P = 0.0024) decreased the incidence of diabetes (Fig. 4B).</p><!><p>We found that the blockade of S1P2 signaling prevents the onset of diabetes by protecting islet β-cells from STZ-induced injury (Fig. 2 and 3). S1P2 is known to couple with Gq, activate phospholipase C that leads to Ca2+ mobilization, and induce the activation of extracellular-regulated kinase, stress-activated protein kinase/c-jun N-terminal kinase, and mitogen-activated protein kinase (MAPK) p38 [15,16]. The activation of MAPK p38 is a critical event leading to β-cell apoptosis and promoting peripheral insulin resistance [17,18,19], and thus mice lacking MAPK p38 were found to be protected against pancreatic β-cell failure and insulin resistance [14]. Therefore, blockade of S1P2 signaling may lead to deactivation of MAPK p38 and attenuation of STZ-induced β-cell failure/progression of diabetes. The present study bears analogy to our previous one, in which accelerated regeneration of hepatocytes was observed after liver injury in S1P2−/− mice [20], and may suggest generalization of cell protective effect by S1P2 inactivation. Recently, it has been suggested that S1P2 may protect against diabetes by preventing insulin resistance; S1P2−/− mice showed insulin resistance [9] and S1P2 signaling increased glucose uptake in myoblasts [8]. Although further studies are necessary to solve this inconsistency with our findings, the protective effect on pancreatic β-cells resulting from S1P2 inactivation may have surpassed the undesired insulin resistance in our system.</p><p>We further explored the possibility that the S1P2-specific antagonists have potential for use in the treatment of diabetes. JTE-013 decreased blood glucose levels (Fig. 4A) and reduced the incidence of diabetes in STZ-injected mice (Fig. 4B). It has been shown that FTY720, a pro-drug against all the S1P receptors except S1P2 (i.e., S1P1 and S1P3–5), reduces the incidence of diabetes in mice [21]. These results suggest that the protective effects of S1P against diabetes (including blockade of immune cell migration) may be mediated by non-S1P2-type S1P receptors. In this context, selective inactivation of pro-diabetic S1P2 and preservation of anti-diabetic non-S1P2-type S1P receptors by S1P2-specific antagonists may have strong potential as a future anti-diabetic strategy; the ligand S1P is abundant in the blood stream [22].</p><!><p>We report here that selective blockade of S1P2 signaling attenuates STZ-induced apoptosis of pancreatic β-cells and decreases the incidence of diabetes in mice. Modulation of S1P signaling may provide a new avenue for the treatment of diabetes.</p>