Free Fatty Acid Induces Endoplasmic Reticulum Stress And Apoptosis Of β-cells By Ca²⁺/calpain-2Pathways

Dysfunction of β-cells is a major characteristic in the pathogenesis of type2diabetesmellitus （T2DM）. The combination of obesity and T2DM is associated with elevatedplasma free fatty acids （FFAs）. However, molecular mechanisms linking FFAs to β-celldysfunction remain poorly understood. In the present study, we identified that the majorendoplasmic reticulum stress （ERS） marker, Grp78and ERS-induced apoptotic factor,CHOP, were time-dependently increased by exposure of β-TC3cells to FFA. Theexpression of ATF6and the phosphorylation levels of PERK and IRE1, which triggerERS signaling, markedly increased after FFA treatments. FFA treatments increased cellapoptosis by inducing ERS in β-TC3cells. We also found that FFA-induced ERS wasmediated by the store-operated Ca²⁺entry through promoting the association of STIM1and Orai1. Moreover, calpain-2was required for FFA-induced expression of CHOP and activation of caspase-12and caspase-3, thus promoting cell apoptosis in β-TC3cells.Together, these results reveal pivotal roles for Ca²⁺/calpain-2pathways in modulatingFFA-induced β-TC3cell ERS and apoptosis. The study is composed of four parts.Part1. FFAtreatments induce ERS in β-TC3cellsand increase cell apoptosis.To examine whether FFA triggers ERS in β-TC3cells, we examined the expressionpatterns of several molecular indicators of ERS. The ER chaperon glucose regulated78-kDa protein （Grp78）, also named Bip, and the transcription factor CHOP are centralregulators of ERS. Mouse pancreatic β-TC3cells were treated with FFA （0.5mM） fordifferent times （0,8,16, or24h）. We used Bovine serum albumin （BSA） as a negativecontrol. The results showed that Grp78and CHOP protein levels were time-dependentlyincreased in cells treated with FFA compared with corresponding BSA treated cells（P<0.05）. The mRNA levels of Grp78and CHOP were also increased in a time-dependentlymannerin cells treated with FFA compared with corresponding BSA treated cells （P<0.05）.In order tofurther assess whether FFA treatments could induce ERS, we examined theexpression and phosphorylation levels of ATF6, PERK, and IRE1, the three key factorsthat trigger ERS signaling in β-TC3cells. We observed that the expression of ATF6andthe phosphorylation levels of PERK and IRE1markedly increased after FFA treatments for16h （P<0.05）.All these data demonstrated that ERS can be activated by FFA treatments in β-TC3cells.ERS might be a mediator of cell death, thus β-TC3cell death was examined byannexin V/PI double staining after FFA treatments. The results showed that FFAtreatments caused more cell apoptosis than BSA control treatments （P<0.05）.It is known that activation of murine caspase-12is associated with ERS-induced cellapoptosis. Thus, we detected the activation of caspase-12. Compared with the activation in BSA-treated β-TC3cells, caspase-12activation in FFA-treated β-TC3cells wassignificantly increased （P<0.05）.Caspase-3（also named CPP32, apopain, and YAMA） has been identified as a keymediator of apoptosis in mammalian cells. We further assessed the activation of caspase-3（cleaved caspase-3）, and the results showed that FFA treatments increased caspase-3activation （P<0.05）.From the above results, we concluded that FFA treatments could induce ERS andincrease apoptosis in β-TC3cells.Part2. FFA treatments promote store-operated Ca²⁺entry to induce ERS in β-TC3cells.It is well known that the disruption of intracellular Ca²⁺homeostasis can disturb ERfunction and induce ERS. In nonexcitable cells, such as pancreatic β-cells, Ca²⁺influx isthe predominant mechanism to maintain intracellular Ca²⁺homeostasis. To assess whetherFFA treatments-induced ERS is mediated by Ca²⁺influx changes in β-TC3cells, NiCl2, aCa²⁺channel blocker, was used. The results showed that FFA treatments were not able toincrease Grp78and CHOP levelsin the condition that Ca²⁺influx was blocked with NiCl2treatments （P>0.05）.The results suggested that FFA treatments induced-ERS is mediatedby the increase of Ca²⁺influx.One of the major routes for Ca²⁺influx into cells is through store-operated Ca²⁺entrychannels. To further assess whether FFA treatments may inducestore-operated Ca²⁺entryinβ-TC3cells, store-operated Ca²⁺entry was examined after FFA treatments. The resultsdemonstrated that FFA treatments resulted in more than a2-fold increase of store-operatedCa²⁺entry in β-TC3cells compared with BSA treatments （P<0.05）.Recent studies have identified that STIM1and Orai1are responsible forstore-operated Ca²⁺entry. We found that the expression levels of STIM1and Orai1incells treated with FFA was similar to cells treated with BSA （P>0.05）. However,immunoprecipitation with anti-STIM1antibody followed by western blot with anti-Orai1antibody revealed that the association of STIM1with Orai1in samples from FFA-treated cells, was much more than the association observed in BSA-treated cells （P<0.05）. Theresults were further strengthened with Orai1as the bait （P<0.05）. The results suggestedthat FFA enhanced the association of STIM1with Orai1.Therefore, these results indicated that FFA treatments induced-ERS is mediated bythe store-operated Ca²⁺entry which is regulated by the association of STIM1with Orai1.Part3. By increasing calpain-2activity, FFA treatments promote CHOP induction inβ-TC3cells.Calpain-2, a neutral Ca²⁺-dependent protease, mediates a variety of physiologicalfunctions such as cytoskeleton remodeling, vesicle trafficking, and membrane fusion. Toassess whether FFA treatments may influence calpain-2activity in β-TC3cells, calpain-2activity was examined after FFA treatments. The results demonstrated that after FFAtreatments, calpain-2activity was dramatically enhanced in β-TC3cells（P<0.05,）.To examine whether FFA-induced ERS is dependent on calpain-2, we used smallinterfering RNA （siRNA） to knock down calpain-2in β-TC3cells. Calpain-2siRNA ledto a marked reduction of calpain-2protein and mRNA levels （P<0.05）.When the cells were treated with calpain-2siRNA, FFA treatments did not increasecalpain-2activity compared with corresponding BSA treatments in β-TC3cells （P>0.05）.To further validate whether calpain-2is involve in FFA-triggered ERS in β-TC3cells,we examined the protein and mRNA levels of Grp78and CHOP in the condition thatcalpain-2activity was inhibited with calpain-2siRNA. The results showed that FFAtreatments increased Grp78protein and mRNA levels following calpain-2siRNAtreatments （P<0.05）.However, FFA treatments were not able to increase CHOP proteinand mRNA levels following calpain-2siRNA treatments （P>0.05）.These resultssuggested that calpain-2is involved in FFA-induced CHOP expression, but not involvedin FFA-induced Grp78expression. Part4. Calpain-2is required for FFA-induced β-TC3cell apoptosis.The above results demonstrated that FFA treatments can increase apoptosis in β-TC3cells. To further confirm that calpain-2is involved in this process, calpain-2siRNA wasused. Annexin V/PI double staining demonstrated that FFA treatments were not able toincrease the percentage of apoptotic cells compared with corresponding BSA treatmentsfollowing calpain-2siRNA treatments （P>0.05）.To corroborate these results, we detected the activation of caspase-12and caspase-3.The results indicated that FFA treatments were not able to increase caspase-12andcaspase-3activation compared with corresponding BSA treatments following calpain-2depletion （P>0.05）.Therefore, we concluded that calpain-2is required for FFA-induced β-TC3cellapoptosis.