In this present study we showed that activation of GPR40 by a small molecule, CNX-011-67, rescued inflammation-mediated apoptosis in pancreatic β-cells. We demonstrated that chronic inflammation, which has been shown to severely impact β-cells’ function and mass , caused an augmentation in cellular oxidative and ER stress, expression of pro-inflammatory cytokines genes and apoptosis. We found that all these impacts of chronic inflammation were reversed by GPR40 activation. Also we have observed no significant change in the expression of GPR40 under inflammation condition with or without the receptor activation (data not shown).
Chronic inflammation has been a mechanism implicated for β-cell apoptosis in both type-1 and type-2 diabetes [4, 40–42]. Hence, it is important to counteract inflammation-mediated changes in β-cells in order to protect them from apoptosis. Therefore, we evaluated the potential of GPR40 activation as a mechanism to neutralize inflammation mediated consequences. We have activated GPR40 using a small molecule agonist (CNX-011-67) which showed an increase in calcium flux. CNX-011-67 is a specific GPR40 agonist as it enhanced calcium flux only in cells expressing GPR40. Moreover, inhibition of PLC reversed CNX-011-67 induced calcium flux as GPR40 activation mediates its effect via PLC-Ca2+ pathway .
Since, inflammatory cytokines exert their effects through activation of JNK and NFκB, we have showed that treatment with GPR40 agonist was able to reduce their activation. These findings were also supported by a decrease in pro-inflammatory cytokines gene expression by GPR40 agonist. Activation of JNK and NFκB has been shown to up-regulate these pro-inflammatory cytokines , hence their reduced activation by GPR40 agonist led to a decreased expression of these genes (TNFα, IL1β, Nos2a). Thus, GPR40 activation not only blocked impact of those inflammatory cytokines added to culture medium but also inhibited their production from β-cells thereby plunging chronic low grade inflammation maintenance.
Elevated cellular oxidative and ER stress under nutrient overload condition can cause β-cells apoptosis [7–11]. In fact, we also observed increased oxidative and ER stress after pro-inflammatory cytokines treatment which was abolished by GPR40 agonist. Under inflammatory conditions, an increased level of β-cell death was observed as measured by cytochrome-c release, TUNEL positivity, Caspase-3 activation and nuclear fragmentation. In consistent with above mentioned findings with GPR40 activation, we observed diminished apoptosis in pancreatic β-cells upon GPR40 agonist treatment.
In order to dissect the mechanism by which GPR40 activation reduced β-cells apoptosis, we used various pharmacological agents to inhibit or activate key intracellular signaling pathways. As expected, inhibition of PLC signaling by U73122 abolished GPR40 agonist impact on apoptosis because GPR40 exerts its effect by PLC activation. Interestingly, PLC inhibition caused a higher increase in Caspase-3 activity compared to inflammation alone, indicating its importance for normal physiological functions of β-cells. In fact, elevated glucose level can cause activation of PLC signaling in islets . Activation of PLC leads to ER calcium release; hence we next tested the role of calcium signaling for GPR40 induced reduction in apoptosis. Increase in cytoplasmic calcium can lead to activation of calcium-calmodulin dependent protein kinase (CaMK) and calcineurin-NFAT pathways. In order to specify which of these pathways was involved in the ability of GPR40 to counteract inflammation induced apoptosis, we inhibited each of these and observed their effect. Inhibition of CaMKII, the major isoform present in β-cells, showed a pronounced increase in caspase-3 activity. Similar results were obtained after calcineurin inhibition. Interestingly, inhibition of either CaMKII or calcineurin caused increased caspase-3 activity higher than that achieved by inflammation itself, signifying their importance in normal physiology of β-cells.
Increase in cytoplasmic calcium level is coupled with an increase in cAMP level due to presence of calcium activated ADCY in β-cells. We tested the possibility that increase in cytoplasmic calcium level by GPR40 activation would lead to a corresponding increase in cAMP level. We found that cAMP levels were decreased under inflammation condition and were restored by GPR40 agonist. To demonstrate the importance of GPR40-induced cAMP level for caspase-3 activity, we inhibited a soluble isoform of ADCY present in β-cells which is known to mediate cAMP oscillation [38, 45]. Similar to data obtained with PLC, CaMKII and calcineurin inhibitor; inhibition of sADCY caused a drastic increase in caspase-3 activity at levels higher than that achieved with inflammation alone. Since these inhibitors are known to suppress insulin secretion, their exposure might have reduced autocrine survival ‘insulin-PI3K-Akt’ signaling. In contrast, increase in cAMP level by forskolin led to a significant reduction in caspase-3 activity indicating an independent role of cAMP for reduction in β-cell death. Treatment with GLP1, which can also increase cAMP levels, has been shown to reduce apoptosis in pancreatic β-cells [28–30]. Thus, our data collectively demonstrate that GPR40 activation relayed its signal to multiple arms of intracellular signaling pathways to reduce inflammation mediated apoptosis.
GPR40 activation not only inhibited β-cell apoptosis but also activated cell survival pathways. Its activation led to an increase in ATP level which together with elevated Ca2+ and cAMP level caused a significant increase in glucose-stimulated insulin secretion. This secreted insulin can lead to Akt/PKB phosphorylation in an autocrine manner. The level of phosphorylated Akt, a well known marker for cell survival and proliferation, was reduced under chronic inflammation conditions, together with a reduction in insulin secretion. GPR40 agonist reversed this decrease in pAkt level. Similarly, BCL2 and PDX1 expression were reduced under inflammation and were restored by GPR40 agonist. PDX1 maintains β-cell phenotype and also regulates insulin synthesis. In consistent with PDX1 data, inflammation reduced insulin gene transcription and intracellular insulin content and their levels were restored by GPR40 agonist. Under this condition we did not observe any change in the glucagon expression in rat islets (data not shown). This is consistence with the fact that GPR40 expression in predominant only in β-cells.
We have earlier showed that activation of GPR40 by oral administration of CNX-011-67 in male ZDF rats reduces β-cells apoptosis, increases insulin and PDX1 positive cell number and insulin secretion . It should be noted that ZDF rat is an animal model of diabetes having high chronic systemic inflammation [47, 48]. Hence, our in vitro data are in agreement with in vivo findings. Taken together, our data demonstrate that GPR40 activation by CNX-011-67 reduces inflammation induced apoptosis, enhances β-cell survival and improves β-cell function as measured by insulin synthesis and secretion.