Endocrine and exocrine pancreas pathologies crosstalk: Insulin regulates the unfolded protein response in pancreatic exocrine acinar cells

Experimental Cell Research 375 (2019) 28–35

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Endocrine and exocrine pancreas pathologies crosstalk: Insulin regulates the unfolded protein response in pancreatic exocrine acinar cells☆ Yekaterina Yatchenko, Avital Horwitz, Ruth Birk

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Department of Nutrition, Faculty of Health Sciences, Ariel University, 40700, Israel

A R T I C LE I N FO

A B S T R A C T

Keywords: ER stress Unfolded protein response (UPR) Diabetic mellitus (DM) Acute pancreatitis (AP) Insulin Glucose

Exocrine pancreas insufficiency is common in diabetic mellitus (DM) patients. Cellular stress is a prerequisite in the development of pancreatic pathologies such as acute pancreatitis (AP). The molecular mechanisms underlying exocrine pancreatic ER-stress in DM are largely unknown. We studied the effects of insulin and glucose (related to DM) alone and in combination with cerulein (CER)-induced stress (mimicking AP) on ER-stress unfolded protein response (UPR) in pancreatic acinar cells. Exocrine pancreas cells (AR42J) were exposed to high glucose (Glu, 25 mM) and insulin (Ins, 100 nM) levels with or without CER (10 nM). ER-stress UPR activation was analyzed at the transcript, protein, immunocytochemistry, western blotting, quantitative RT-PCR and XBP1 splicing, including; XBP1, sXBP1, ATF6, cleaved ATF6, IRE1-p, CHOP, Caspase-12 and Bax. Exocrine acinar cells exposed to high Ins or Ins+Glu concentrations (but not Glu alone) exhibited ER-stress UPR, demonstrated by significant increase of transcript and protein levels of downstream markers in the ATF6 and IRE1 transduction arms, including: sXBP1, cleaved ATF6, XBP1, CHOP, IRE1-p and caspase-12. UPR activation resulted in IRE1-p aggregation and nuclear trans-localization of cleaved activated ATF6 and sXBP1. Ins further aggravated UPR when cells were co-challenged with CER-induced stress, exacerbating the effects of CER alone. High Ins levels, typical to type-2-DM, activate the ER-stress UPR in pancreatic acinar cells, through the ATF6 and IRE1 pathways. This effect of Ins in naïve acinar cells further augments CER-induced UPR. Our data highlight molecular pathways through which DM enhances exocrine pancreas pathologies.

1. Introduction The molecular crosstalk between the exocrine and endocrine pancreas in health and disease is complex and not fully understood. Recent studies show clear link between pathological conditions of the exocrine and endocrine pancreas. For instance, chronic pancreatitis (CP) is estimated to constitute 25–75% of the risk of DM, depending on the etiology of disease. In fact, the most commonly identified causes of type 3c diabetes are exocrine pancreas pathologies [1]. Moreover, postpancreatitis DM is a novel disease entity being increasingly recognize [2]. Not only does the exocrine pancreas affect endocrine pancreas pathology; vice versa states have also been described: DM increases the severity and outcome of acute pancreatitis. Recent meta-analysis found that DM is associated with AP severe attacks and organ failure: AP patients with DM had higher morbidity of cardiovascular and renal failure than those without DM, with impact depending on the cause of

hyperglycemia, severity and duration of DM and its treatment [3]. The interaction between the exocrine and endocrine pancreas exists largely, apart from the closed proximity, due to direct blood flow. Thus, insulin secreted by the islets of Langerhans has direct contact with exocrine cells and can affect their function through their exposure to very high concentrations of insulin [4]. In fact, several initial studies demonstrated complex action of insulin on exocrine cells: promoting uptake of amino acids and controlling responsiveness to hyperglycemia [5]. Moreover, acinar atrophy due to lack of local trophic insulin could partly explain initial signals of pancreatitis expressed in altered pancreas morphology and function [6]. Furthermore, diabetic dogs exhibit exocrine pancreatic tissue damage with twenty-fold reduction in pancreatic amylase [7,8]. The exocrine pancreas exhibits the highest rates of protein synthesis in the body and consequently possesses abundant endoplasmic reticulum (ER) [9]. ER is a multifunctional organelle necessary for

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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Corresponding author. E-mail address: [email protected] (R. Birk).

https://doi.org/10.1016/j.yexcr.2019.01.004 Received 25 October 2018; Received in revised form 29 December 2018; Accepted 5 January 2019 Available online 06 January 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.

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protein samples (as indicated below).

synthesis, folding and processing of secretory and transmembrane proteins. In some pathological conditions, such as pancreatitis, obesity and different types of cancer, ER function is disrupted, resulting in accumulation of misfolded and unfolded proteins in the ER lumen, leading to ER stress. In fact, rodent studies show rapid activation of UPR within minutes of pancreatitis initiation [10–12]. Additionally, UPR activation in pancreatic exocrine cells following induction of ER stress by different fatty acids was demonstrated in our previous studies [13,14]. Moreover, chronic ER stress in pancreatic acinar cells might contribute to AP recovery [15]. However, the molecular mechanisms linking AP and ER stress remain not fully understood. UPR activation is targeted to alleviate ER stress, restore ER homeostasis and promote cell survival and adaptation. However, under irresolvable ER stress conditions, UPR promotes apoptosis [16]. UPR includes three different ER stress transducers localized to the ER membrane: protein kinase RNA (PKR)-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6). Upon activation, each sensor elicits unique downstream responses [10]. UPR activation in different stress conditions can occur through all three parallel signaling pathways; however, in many situations, particular pathways are more dominant [17,18]. ER stress conditions balance between factors that lead to adaptation and survival, vs factors that lead to death. In the context of DM, pancreatic β-cells depend on efficient UPR signaling to meet the demands for constantly varying levels of insulin synthesis [19]. This large biosynthetic load can overwhelm the ER folding capacity, resulting in ER stress. PERK deficient mice are prone to DM and progressive hyperglycemia. Furthermore, PERK mutation preferentially affects insulin biosynthesis over total protein biosynthesis [20]. Induction of ER stress or reduction in the compensatory capacity through down-regulation of XBP1 leads to suppression of insulin receptor signaling in intact cells via IRE-1 dependent activation of c-Jun N-terminal protein kinase (JNK). Deletion of XBP1 allele in mice leads to enhanced ER stress, hyperactivation of JNK, reduced insulin receptor signaling, systemic insulin resistance, and type 2 DM [21]. The pathophysiological interactions between DM and AP are mostly unknown and only recently are re-evaluated. The cellular and molecular processes causing DM and AP are possibly intertwined, as both initiate and develop due to deleterious effects on cellular components of the pancreas. Recent evidence points to ER stress as an important process involved both in DM and in exocrine pancreas pathologies, such as AP and CP; however, possible interaction between DM and ER stress of exocrine pancreas cells has scarcely been studied to date. We set out to explore the dynamics of the UPR in DM and in DM in the presence of AP, studying ER stress and UPR in AR42J exocrine pancreas acinar cells. To simulate DM state, cells were treated acutely with different concentrations of glucose (Glu), insulin (Ins) and their combination (Glu + Ins) for 24 h. To simulate DM in the presence of AP state and investigate the influence of this combination on UPR markers, Cerulein (CER) was added to the above treatments.

2.2. Protein extraction Protein extraction was done according to standard techniques, using RIPA lysis buffer with protease inhibitors cocktail (Sigma-Aldrich, Israel). Protein was quantified by Bradford assay (Bio-Rad, Israel). 2.3. Western blot analysis Sodium dodecyl sulfate/polyacrylamide gel electrophoresis, protein transfer, and western blotting were performed using standard techniques. Antibodies were obtained from commercial sources and protocols were per manufacturer's instructions. The following primary antibodies were used: Primary rabbit antibodies against XBP1 (1:1000, Abcam, Israel), IREα-p (1:1000, Abcam, Israel) or CHOP/GADD 153(R-20) (1:1000, Santa Cruz, Israel) and primary mouse antibodies against ATF6 (1:500, Santa Cruz, Israel) or a-tubulin (1:500, SIGMA, Israel). Primary antibodies were detected using goat anti rabbit-IgG (1:3000, Abcam, Israel) or goat Anti-Mouse (1:3000, Abcam, Israel) antibodies. EZ-ECL solutions (Biological Industries, Israel) were used to develop blots. Specific bands were subjected to densitometry analysis using image J software 1.46j version. Proteins were quantified relative to housekeeping gene. 2.4. RNA isolation and cDNA synthesis RNA was isolated using standard laboratory techniques. Briefly, RNA was isolated using 1 ml Trizol (Rhenium, Israel) per 10 cm2 of culture dish area. Next, chloroform (Ornat, Israel) was added and centrifuged for phase separation. The aqueous phase, containing the RNA, was removed and isopropanol (Ornat, Israel) was added. After centrifugation samples were washed with 75% ethanol, air dried and dissolved in ultra pure water (Biological Industreis, Israel). RNA was quantified by UV absorption at 260 nm (Thermo Scientific NanoDrop 2000, UV–Vis Spectrophotometer, USA). Samples were stored at − 80 °C. Total RNA was reverse transcribed into cDNA using Tetro RT Enzyme, Random Hexamer Primer Mix and dNTP Mix (Tetro, BIOLINE, Israel) according to the manufacturer's protocol. 2.5. Quantitative RT-PCR (qPCR) Transcript levels were determined by qPCR using SYBRs Green PCR Master Mix (Life Technologies, Rhenium, Israel). Gene specific primers were designed using the Primer Express Software (Life Technologies, Rhenium, Israel). Primer and cDNA concentrations were optimized (including melt curve analyses). Each 20 µl reaction contained 2 µl (1–2 µg first strand) cDNA, 10 µl Power SYBR Green PCR master mix (Life Technologies, Rhenium, Israel), 300–700 nM of each forward and reverse primer (according to the optimization of the primers). All reactions were performed under the following conditions: pre-incubation at 50 °C (2 min), denaturation at 95 °C (10 min) and 40 cycles of 95 °C (15 s) followed by annealing and elongation at 60 °C (1 min). Primers' sequences are available upon request. Software (Applied Biosystems, Carlsbad, CA, USA) was used to calculate relative expression values for all genes studied, normalized to control.

2. Material and methods 2.1. AR42J cell culture Rat pancreatoma AR42J cells (American Type Culture Collection, Rockville, MD, USA) were maintained as a sub-confluent monolayer culture in Dulbecco's Modified Eagle Medium (Rhenium, Israel) containing 10% Fetal Bovine Serum (FBS; Biological industries, Israel) and 1% Penicillin-Streptomycin (Biological industries, Israel) under humidified condition of 5% CO2 at 37 °C. Dexamethasone (100 nM; SigmaAldrich, Israel) was added 48 h before the experiments to induce cell differentiation. Cell survival following the different treatments was studied using trypan blue staining (Sigma-Aldrich, Israel). Mortality rates were low (4–15%) for treatment groups (data not shown). All further analyses were equilibrated ensuring equal amounts of RNA and

2.6. Detection of sXBP1 Amplification of XBP1 transcripts was done using PCR kit MyTaq DNA Polymerase (Origolab, Israel). PCR products were subjected to Pst1 restriction enzyme (Thermo Scientific, Israel) according to the manufacturer's protocol. Products were run on 4% agar-gel. The unspliced (non-stressed) variant of Xbp1 (289 bp) results in two smaller fragments of 173 bp and 116 bp, whereas the spliced (stressed) form size is 266 bp. 29

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the nucleus. In untreated Con AR42J cells, both XBP and ATF6 were located in a typical ER cellular location. However, both XBP1 and ATF6 were extensively translocated to the nucleus following Ins treatment (ICC, white head arrows). Furthermore, also Glu + Ins treatment resulted in XBP1 and ATF6 significant nuclear translocation compared to Glu-only treatment [white arrows] (Fig. 1D). Taken together, these results indicate that in DM-simulated state the UPR ATF6 arm activation in pancreatic exocrine acinar cells is mainly due to high Ins levels.

2.7. Immunocytochemistry (ICC) staining AR42J cells were grown and differentiated on cover slips and were subject to treatments as indicated. After fixation with 4% paraformaldehyde (Sigma- Aldrich, Israel) cells were permeabilized with 10% TritonX100 and incubated in blocking solution (1% TritonX100, 3% iNGS), followed by over-night incubation with primary rabbit antibodies against XBP1 (Abcam, Israel) or IREα-p (Abcam, Israel) or primary mouse antibodies against ATF6 (Santa cruz, Israel). Primary antibodies were detected using goat anti rabbit-IgG coupled to alexa Fluor 488 (Abcam, Israel) or goat Anti-Mouse IgG coupled to Alexa Fluor 555 (Abcam, Israel). Nuclear labeling was performed with DAPI (Sigma, Israel). The samples were analyzed using fluorescence confocal microscope (LSM 700 Zeiss). For quantification of positive cells, clusters were randomly selected from triplicates of two independent experiments and the average value ± SEM was determined.

3.3. High insulin levels activate the IRE1 arm of ER stress UPR in exocrine pancreas cells Upon ER stress and UPR activation, IRE1 is activated through autophosphorylation (IRE1-p). Following Ins treatment, IRE1-p protein levels increased significantly by 2-fold compared to Glu treatment and Con groups. Additionally, following Glu + Ins treatment, IRE1-p protein level increased significantly by 3 fold compared to Glu treatment and untreated Con groups (Fig. 2A). One of the major IRE-p functions is cleavage of XBP1 mRNA to sXBP1. Percent of sXBP1 increased significantly by 1.6, 4 and 7-fold following Glu, Ins and Glu + Ins treatments, respectively, compared to the Con group; and by 2 and 4-fold following Ins and Glu + Ins treatments, respectively, compared to Glu treatment alone (Fig. 2B), indicating a partly synergistic effect mostly due to Ins. Similarly and in accordance to our finding concerning IRE1-p protein levels and sXBP1 levels (Fig. 2A–B), IRE1-p ICC studies demonstrated that IRE-p foci were significantly expressed following challenge with high Ins levels (prime effect) and combined Glu + Ins treatments compared to Glu only treatment and untreated Con cells [white arrows] (Fig. 2C). Although IRE is reported only in ER, we verified our results using a confocal microscope (data not shown). The ICC results reinforce our findings that high levels of Ins and Glu, simulating those found in DM, induce ER stress UPR in pancreatic acinar cells, and this effect is mostly due to the high Ins levels.

2.8. Statistical analysis Results were collected from 3 independent experiments, each performed in triplicate. Data are expressed as mean ± standard error (SD). Statistical analysis was performed using GraphPad Prism 7.0; comparisons using one-way analysis of variance (ANOVA). Statistical significance (p < 0.05) of differences between treatment groups presented by (*). 3. Results 3.1. Insulin activates ER stress UPR in AR42J exocrine pancreas cells To test possible activation of ER stress UPR in acinar cells in the presence of high Ins (100 nM) and Glu (25 mM) levels similar to those found in DM, AR42J cells were exposed to high levels of Ins, Glu or their combination, and the different pathways of ER stress UPR were assayed.

3.4. High insulin levels augment CER-induced ER stress UPR in exocrine pancreas cells

3.2. High insulin levels activate the ATF6 arm of ER stress UPR in exocrine pancreas cells

3.4.1. ATF6 arm activation CER (10 nM) treatment, simulating AP state in exocrine acinar cells in-vitro, is known to induce ER stress in those cells. We next set out to test whether high Ins and Glu levels seen in DM further affect the CERmediated ER stress in acinar AR42J cells. Total ATF6 protein level increased significantly by 1.2 and 2-fold following Glu + CER, Ins + CER and Glu + Ins + CER treatments, respectively, compared to cells treated with CER alone (Fig. 3A). Parallel to total ATF6 levels, cleaved ATF6 protein level showed significant increase of 3.5, 8 and 6-fold following Glu+CER, Ins+CER and Glu + Ins + CER compared to the cells treated with CER alone. Additionally, cleaved ATF6 protein levels increased significantly by 2 and 1.8-fold following Ins+CER and Glu + Ins + CER treatments compared to Glu+CER treatment (Fig. 3A). No significant changes were found in uncleaved ATF6 protein expression levels. XBP1 protein levels increased significantly by 3-fold following Glu + CER, Ins + CER and combined Glu + Ins + CER treatments compared to CER-only treatment (Fig. 3A). Similar results were demonstrated regarding XBP1 transcript levels: significant increase of 3fold following Glu + CER, Ins + CER and combined Glu + Ins + CER treatments compared to CER only treatment (Fig. 3B). CHOP protein levels increased significantly by 2.5-fold following Ins + CER treatment compared to CER only and Glu + CER treatments. Additionally, following combined Glu + Ins + CER treatment, CHOP protein levels increased significantly by 3-fold compared to CER only and Glu + CER treatments (Fig. 3A). Parallel to CHOP protein levels, CHOP transcript levels significantly increased by 3-fold following Ins + CER treatment compared to CER and Glu + CER treatments, and

The ATF6 arm of ER stress was determined through assays of expression levels of downstream transduction factors, including; ATF6, cleaved ATF6, XBP1 and CHOP (Fig. 1A–D). Total ATF6 protein levels increased significantly (p < 0.05) by 1.6-fold following high Glu and by 2-fold following high Ins and Glu + Ins treatments compared to the untreated control (Con) group. Compared to the Con group, activated cleaved ATF6 protein levels increased significantly by 1.4 and 2-fold following Glu, Ins and Glu + Ins treatments, respectively. No significant changes were found in uncleaved ATF6 protein expression levels following all DM stressors (high Glu, Ins and combination) compared to the Con group (Fig. 1A). Activated cleaved ATF6 translocates to the nucleus and acts as a transcription factor for XBP1. XBP1 protein levels increased significantly following Glu, Ins and Glu + Ins treatments by 1.5, 1.9 and 2.3 fold, respectively, compared to the untreated Con group. XBP1 transcript levels increased significantly following Ins and Glu + Ins treatments by 2 and 3-fold, respectively, compared to the Glu treated group (Fig. 1A; 1B). Activated cleaved ATF6 regulates the UPR pro-apoptotic protein CHOP. CHOP protein levels increased significantly following Ins and Glu + Ins treatments by 3.5 and 5-fold, respectively compared to Glu and Con groups. In parallel to the protein levels, CHOP transcript levels increased significantly by 8-fold following Ins treatment compared to Glu and Con treatments. Following Glu + Ins treatment, CHOP transcript levels increased significantly by 4-fold compared to Glu treatment (Fig. 1A; 1C). Following ER stress and UPR activation, sXBP1 and the cleaved form of ATF6 function as transcript factors for UPR genes, translocating to 30

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Fig. 1. UPR ATF6 arm activation following exposure of AR42J cells to high levels of insulin and glucose. AR42J cells were exposed to high concentrations of Glucose (25 mM), Insulin (100 nM) and their combination for 24 h. (A) Total protein was isolated and ATF6, XBP1 and CHOP were analyzed (western blot). Protein levels were normalized to house-keeping gene β-Tubulin. (B–C) Total RNA was isolated. cDNA was synthesized and subjected to qRT-PCR using specific XBP1 and CHOP primers as described in Section 2. Results were normalized to house-keeping gene S18. (D) Immunocytochemistry (ICC) staining of XBP1 and ATF6 protein. Nuclear labeling was performed with DAPI. For quantification of positive cells (n = 100–150), clusters were randomly selected from triplicates of two independent experiments. Photos were taken using Olympus fluorescent microscope at ×100 magnification (10 µm scale). Results are expressed as mean ± SD (n = 3). * represent significant difference at p ≤ 0.05.

CER-induced UPR activation in acinar cells following exposure to high Ins and Glu levels is mainly due to the high Ins levels.

by 2-fold following Glu + Ins + CER treatment compared to CER only and Glu+CER treatments (Fig. 3C). These results suggest that in CERinduced stress state of acinar cells, Ins levels are a major regulator of CHOP (protein and mRNA) during UPR activation. Our ICC studies (Fig. 3D) demonstrated that both XBP1 and ATF6 were primarily localized in the nucleus following Ins+CER treatment (white arrows). XBP1 was primarily localized in the cytosol and ER surroundings and less in the nucleus following CER, Glu + CER and combined Glu + Ins + CER (green head arrows) treatments. ATF6 cellular localization was primarily in the ER following all treatments (more so following the CER alone and Glu + Ins + CER treatments) except Ins + CER treatment (white head arrows) (Fig. 3D). Ins + CER treatment induced significant translocation of XBP1 and ATF6 to the nucleus. Taken together, these results indicate that the enhancement of

3.5. IRE1 arm activation Upon ER stress induction and activation of the UPR by CER, IRE1 became active (IRE1-p). No further changes were found in IRE1-p protein expression levels following addition of DM stressors (high Glu and Ins levels) (Fig. 4A) As we demonstrated, IRE1-phosphorylation resulted in XBP1 splicing in acinar cells exposed to DM-simulating high Ins and Glu concentrations, mostly due to the Ins levels (Fig. 2B). We therefore set out to test whether this effect of Ins is still evident also in acinar cells already stressed by CER (in-vitro simulating AP): %sXBP1 was 31

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Fig. 2. UPR IRE1 arm activation following exposure of AR42J cells to high levels of insulin and glucose. AR42J cells were exposed to high concentrations of Glucose (25 mM), Insulin (100 nM) and their combination for 24 h. (A) Total protein was isolated and IRE1-p were analyzed (western blot). Protein levels were normalized to house-keeping gene β-Tubulin. (B) %XBP1 splicing expression levels. Total RNA was isolated and subjected to XBP1 splicing analysis as described in Section 2. (C) Immunocytochemistry (ICC) staining of IRE1-p protein. Nuclear labeling was performed with DAPI. For quantification of positive cells (n = 100–150), clusters were randomly selected from triplicates of two independent experiments. Photos were taken using Olympus fluorescent microscope at ×100 magnification (10 µm scale). Results are expressed as mean ± SD (n = 3). * represent significant difference at p ≤ 0.05.

(activated in mitochondrial stress pathway) decreased significantly by 1.4-fold following Glu + CER (gray column) treatment compared to the Glu treated group (white column), and significantly increased by 3 and 2-fold following Ins + CER and Glu + Ins + CER treatments, respectively (gray column), compared to Ins and Glu + Ins treated groups (white column). Our results show no significant change in Bax transcript levels following all DM treatments, could suggests that in the presence of high Ins and Glu levels, the activation of apoptotic markers is caused by ER stress rather than mitochondrial stress. Moreover, the data show that in high Ins levels, UPR acts toward survival mode. Bax transcript levels increased significantly following Ins + CER treatments by 6.5 and 4.5-fold compared to CER only and Glu + CER treatments, respectively, and by 5 and by 3-fold following Glu + Ins + CER treatment compared to CER and Glu + CER treatments, respectively (Fig. 5C). Altogether, our results regarding the pro-apoptotic pathway activation indicate that in acinar cells CER-induced stress activates the UPR pro-apoptotic arm, which further aggravates significantly by the addition of Glu, Ins and their combination (synergistically). In DM in the presence of AP state, Ins is the major effector of the pro-apoptotic arm of the UPR activation.

significantly increased by 2-fold following treatment with CER and Glu +CER (gray column) compared to Con and Glu treatments without CER (white column). %sXBP1 was significant decrease by 1.2 and 1.4-fold following high Ins+CER and Glu + Ins + CER treatments (gray column) compared to Ins and Glu + Ins treatment groups without CER (white column). Additionally, there was significant increase in %sXBP1 levels by 1.5-fold following Glu + Ins + CER treatment compared to Ins + CER and Glu + CER treatment groups (gray column) (Fig. 4B). Thus, in CER-induced stress, high levels of Ins cause decreased expression of the pro-survival protein sXBP1. 3.6. The pro-apoptotic IRE1 arm is activated in acinar cells exposed to Ins and CER-induced stress CHOP is known to be involved in ER stress-mediated apoptosis. CHOP protein and transcript levels were significantly activated and elevated in acinar cells following exposure to Ins or to Ins + CER (Fig. 1A; 3A). Caspase-12, a member of the caspase family, is ER-localized and activated by ER stress. Caspase-12 transcript levels were significantly increased by 6, 2 and 4-fold following Glu + CER, Ins + CER and Glu + Ins + CER treatments, respectively (gray column) compared to these treatment groups without CER (white column). Additionally, there was significant increase of Caspase-12 levels by 2 and 4-fold following Ins and Glu + Ins treatments, respectively, compared to Glu treatment (white column) and by 2, 1.4 and 1.2-fold following Glu + Ins + CER treatment compared to CER only, Glu + CER and Ins + CER treatments, respectively (gray column) (Fig. 5A). Interestingly, mRNA levels of the pro-apoptotic factor Bax

4. Discussion Exocrine and endocrine pancreas crosstalk in normal and pathological states is an emerging and important clinical and basic research field, with scarce understanding to date. Both exocrine and endocrine cells are secretory cells, facing challenges of increasing protein 32

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Fig. 3. UPR ATF6 arm activation following exposure of AR42J cells to CER and high levels of insulin and glucose. AR42J cells were exposed to high concentrations of Glucose (25 mM), Insulin (100 nM) and their combination for 24 h. In the last 4 h, cerulein (10 nM) was added, in-vitro simulating AP. (A) Total protein was isolated and ATF6, XBP1 and CHOP were analyzed (western blot). Protein levels were normalized to house-keeping gene β-Tubulin. (B–C) Total RNA was isolated; cDNA was synthesized and subjected to qRT-PCR using specific XBP1 and CHOP primers as described in Section 2. Results were normalized to house-keeping gene S18. (D) Immunocytochemistry (ICC) staining of XBP1 and ATF6 protein. Nuclear labeling was performed with DAPI. For quantification of positive cells (n = 100–150), clusters were randomly selected from triplicates of two independent experiments. Photos were taken using Olympus fluorescent microscope at ×100 magnification (10 µm scale). Results are expressed as mean ± SD (n = 3). * represent significant difference at p ≤ 0.05.

an IRE1-p aggregation phenotype. Interestingly and similarly, upon UPR activation in human T-REx293 cells, mammalian IRE1 oligomerizes to tightly packed foci on the ER membrane correlating with the onset of IRE1 phosphorylation [24]. The IRE1 UPR arm mediates mainly pro-survival mode; however, if homeostasis in the ER protein folding environment cannot be re-established, IRE1-p suggestively serves as the tipping point in the life–death cellular decision process, and can also activate two pro-apoptosis downstream pathways: caspase-12 and ASK1-p. We demonstrate significant activation of caspase-12 transcripts following exposure to high levels of Ins and Ins + Glu. Similarly, we demonstrate significant elevation in CHOP transcript and protein levels following Ins and Ins + Glu treatments, suggesting again that Ins is the main cause of CHOP activation. Similar to our results, Ins was previously shown to induce CHOP gene expression in adipocytes [25]. CHOP is a component

synthesis several-fold during acute and chronic stimulations, consequently having highly developed ER susceptible to severe stress upon harmful situations. Previous studies demonstrated that exocrine pancreas insufficiency is frequent in DM, and that DM increases the severity and outcome of acute pancreatitis. Exocrine pancreas stress is a prerequisite in the development of pancreatic pathologies [22,23]. The cellular and molecular mechanisms underlying exocrine pancreatic stress in diabetic patients are not understood. Our results demonstrate that in the presence of both high Glu and Ins levels, as occur in DM, ER stress is induced and UPR activation occurs in exocrine pancreas acinar cells. Ins-induced ER stress in acinar cells significantly activated all the studied downstream markers of the ATF6 and IRE1 UPR transduction arms: sXBP1, cleaved ATF6, XBP1, CHOP and IRE1-p were significantly up-regulated at the transcript and/ or protein levels. Furthermore, Ins-induced UPR activation resulted in 33

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genes during ER stress [26]. High Ins and/or Glu levels did not alter Bax transcript levels, suggesting that activation of apoptotic markers is caused by ER stress rather than by mitochondrial stress. Furthermore, these data indicate that despite the high activation of the apoptotic process (as seen by CHOP and Caspase 12) the pro-survival pathways were more active than the pro-apoptotic ones. In ER-stressed cells, following activation of the ATF6 and IRE1 arms, cleaved ATF6 and sXBP1 (respectively) re-localize to the nucleus, where they function as transcription factors for UPR target genes [27–30]. We show that cleaved ATF6 and sXBP1 translocated to the nucleus upon activation by Ins and Glu, with high Ins levels inducing the highest cellular translocation response, highlighting Ins as the main player in the UPR activation. Correspondingly, we previously showed that sXBP1 translocates to the nucleus in pancreatic exocrine cells following induction of ER stress by different fatty acids [13,14]. Additionally, ER stress-induced ATF6 translocation to the nucleus was shown in other tissues and cells (substantia nigra; hepatocellular carcinoma; pancreatic β cells) [31–35]. Taken together, our results indicate that in response to acute Ins challenge, pancreatic exocrine cells activate the ER stress UPR towards a pro-survival mode. A similar phenomenon was demonstrated in a different system, showing that Ins-like growth factor 1 (IGF1) was able to reduce pancreatic islet cell death and is involved in protection against the induction of apoptosis [36]. Moreover, Ins and IGF-I receptors signaling were reported to protect from apoptosis in several cells and organs [37–40]. Interestingly, Ins was previously reported as regulator of UPR [25,41,42]. In human adipose tissue, Ins-induced UPR is not due to increased Glu uptake/metabolism and oxidative stress, but rather, due to increased protein synthesis [41]. Having demonstrated the effects of high Ins levels on naïve exocrine cells, we went on to study whether exposure to high Ins and Glu levels further augments ER-stress UPR induced by CER (in-vitro simulating AP). Combined exposure to these two states induced further activation and higher UPR response compared to CER-induced state without high Ins and Glu. Addition of CER induced the activation of both the ATF6 and IRE1 UPR transduction arms, as demonstrated by significant increase in all downstream transcript and/or protein levels assayed, including: sXBP1, ATF6, cleaved ATF6, XBP1, CHOP and IRE1-p. Furthermore, cleaved ATF6 and sXBP1 translocated to the nucleus, mainly following acute Ins exposure. While without CER Ins induced the highest UPR response, in the presence of CER, both Glu, Ins and combined treatments significantly elevated XBP1 protein and transcript levels, suggesting that this combined state induced the highest activation of the ATF6 arm pro-survival mode. IRE1-p protein expression levels following CER-induced stress did not differ from those following combined treatments. However, because other IRE1 arm downstream markers were up-regulated, it is likely that

Fig. 4. UPR IRE1 arm activation following exposure of AR42J cells to CER and high levels of insulin and glucose. AR42J cells were exposed to high concentrations of Glucose (25 mM), Insulin (100 nM) and their combination for 24 h. At the last 4 h, cerulein (10 nM) was added, simulating AP in-vitro. (A) Total protein was isolated and IRE1-p were analyzed (western blot). Protein levels were normalized to house-keeping gene β-Tubulin. (B) %XBP1 splicing expression levels. Total RNA was isolated and subjected to XBP1 splicing analysis as described in Section 2. Results are expressed as mean ± SD (n = 3). Columns not sharing the same symbol(s) are statistically significantly different. (*)p < 0.05 for DM treatment group vs control. (¥)p < 0.05 for DM + AP treatment group vs CER. (#)p < 0.05 for DM + AP vs DM treatment groups respectively.

of the ER stress-mediated apoptosis pathway. Our results show that Insinduced CHOP elevation in acinar cells is mediated through either the ATF6 or IRE arms; however, all three arms of the UPR can induce elevation in CHOP transcription, as CHOP is one of highest inducible

Fig. 5. Pro-apoptotic IRE1 arm activation following exposure of AR42J cells to high levels of insulin and glucose in the absence and presence of CER. AR42J cells were exposed to high concentrations of Glucose (25 mM), Insulin (100 nM) and their combination for 24 h. In one of the experiments, at the last 4 h cerulein (10 nM) was added, in-vitro simulating AP (gray column). Total RNA was isolated, cDNA was synthesized and subjected to qPCR using specific (A) Caspase-12 and (B) Bax primers, as described in Section 2. Results were normalized to reference gene GAPDH. Results are expressed as mean ± SD (n = 3). Columns not sharing the same symbol (s) are statistically significantly different. (*)p < 0.05 for DM treatment group vs control. (¥)p < 0.05 for DM + AP treatment group vs CER. (#)p < 0.05 for DM + AP vs DM treatment groups respectively. 34

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IRE1-p reached the maximum activation level with CER-induced stress treatment alone. Downstream apoptotic markers CHOP protein levels and transcript levels of caspase-12 and Bax were significantly elevated mainly following addition of acute Ins exposure to the CER-induced stress state. In contrast to exposure to Ins alone, in co-exposure with CER there were significantly augmented levels of Bax apoptotic marker, indicating that in CER-induced stress, high levels of Ins cause a decrease in the pro-survival state. Furthermore, upon activation, CHOP downregulates the transcription of Bcl-2, a BAX inhibitor and increases the expression of the BAX activating protein, death receptor 5 (DR5) [43]. Bax induces mitochondria to release cytochrome c, caspase activation and cell death by activating progressive apoptotic processes [44]. Thus, we can suggest that in the response to acute challenge of combined high Ins and CER, the pancreatic exocrine cells commit to pro-apoptosis state. In conclusion, we demonstrated that in the presence of high insulin and glucose levels, simulating those seen in type 2 DM, ER stress UPR in AR42J pancreatic acinar cells is activated. This UPR activation, affecting both the ATF6 and the IRE1 arms, is mostly due to the high Ins levels (and not high Glu levels), and drives a UPR pro-survival mode of activation. In pancreatic acinar cells exposed to CER, the presence of high Ins causes further ER stress through the same two transduction arms (ATF6 and IRE1), enhancing apoptosis. Thus, high Ins levels seen in DM enhance ER stress UPR in both naive and CER-treated pancreatic acinar cells, likely enhancing pathological processes of the exocrine pancreas.

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