Mitogen-activated protein kinases and protein phosphatase 5 mediate glucocorticoid-induced cytotoxicity in pancreatic islets and β-cells

Molecular and Cellular Endocrinology 383 (2014) 126–136

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Mitogen-activated protein kinases and protein phosphatase 5 mediate glucocorticoid-induced cytotoxicity in pancreatic islets and b-cells Liselotte Fransson a, Victoria Rosengren a, Titu Kumar Saha a, Nina Grankvist a,1, Tohidul Islam a, Richard E. Honkanen a,b, Åke Sjöholm b,c, Henrik Ortsäter a,d,⇑ a

Department of Clinical Science and Education, Södersjukhuset, Karolinska Institutet, Stockholm, Sweden Department of Internal Medicine, Södertälje Hospital, SE 152 86 Södertälje, Sweden Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, Mobile, AL, USA d Research Unit, Södertälje Hospital, SE-152 86 Södertälje, Sweden b c

a r t i c l e

i n f o

Article history: Received 18 June 2013 Received in revised form 15 December 2013 Accepted 16 December 2013 Available online 20 December 2013 Keywords: Glucocorticoids Apoptosis Pancreatic islet JNK p38 MAPK Protein phosphatase 5

a b s t r a c t Glucocorticoid excess is associated with glucose intolerance and diabetes. In addition to inducing insulin resistance, glucocorticoids impair b-cell function and cause b-cell apoptosis. In this study we show that dexamethasone activates mitogen-activated protein kinases (MAPKs) signaling in MIN6 b-cells, as evident by enhanced phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK). In contrast, the integrated stress response pathway was inhibited by dexamethasone. A p38 MAPK inhibitor attenuated dexamethasone-induced apoptosis in b-cells and isolated islets and decreased glucocorticoid receptor phosphorylation at S220. In contrast, a JNK inhibitor augmented DNA fragmentation and dexamethasone-induced formation of cleaved caspase 3. We also show that inhibition of protein phosphatase 5 (PP5) augments apoptosis in dexamethasone-exposed islets and b-cells, with a concomitant activation of p38 MAPK. In conclusion, our data provide evidence that in islets and b-cells, p38 MAPK and JNK phosphorylation work in concert with PP5 to regulate the cytotoxic effects exerted by glucocorticoids. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Hyperglycemia and Diabetes mellitus are important causes of mortality and morbidity worldwide. The number of people with impaired glucose tolerance or manifest type 2 Diabetes mellitus (T2DM) is rising in all regions of the world. A systemic analysis of health examination surveys and epidemiological studies show that between 1980 and 2008 there were nearly 194 million new cases of diabetes (Danaei et al., 2011). Of these, 70% can be attributed to population growth and aging, but the cause for the remaining 30% are likely due to environmental changes. Abbreviations: ASK-1, apoptosis signal-regulating kinase 1; dexa, dexamethasone; eIF2a, eukaryotic translation initiation factor 2a; GC, glucocorticoid; GR, glucocorticoid receptor; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PP5, serine/threonine protein phosphatase 5; ROS, reactive oxygen species; RU, RU486; SB, SB203580; SP, SP600125; T2DM, type 2 diabetes mellitus; UPR, unfolded protein response. ⇑ Corresponding author. Address: Department of Clinical Science and Education, Södersjukhuset, Karolinska Institutet, Sjukhusbacken 10, SE-118 83 Stockholm, Sweden. Tel.: +46 (0) 8 616 3952; fax: +46 86162933. E-mail address: [email protected] (H. Ortsäter). 1 Present address: Degenerative Disease Program, Del E. Webb Neuroscience, Aging & Stem Cell Research Center, Sanford-Burnham Medical Research Institute, Building 7, Room 7124, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. 0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.12.010

Glucocorticoids (GCs) are steroid hormones produced by the adrenal cortex. These hormones promote processes that raise blood glucose levels (Wajchenberg et al., 1984) and due to these effects, patients exposed to elevated levels of GCs for longer periods of time suffer a heightened risk of glucose intolerance or even overt diabetes (Raul Ariza-Andraca et al., 1998; van Raalte et al., 2009; Vegiopoulos and Herzig, 2007). Endogenous overproduction of GCs by the adrenal cortex, as observed in patients with Cushing’s syndrome, is associated with an increased 30–40% risk of developing diabetes (Biering et al., 2000). T2DM is also over-represented in patients with elevated GCs at levels that are subclinical for the diagnosis of Cushing’s syndrome (Di Dalmazi et al., 2012). Furthermore, in a retrospective study of 1258 human volunteers, a positive correlation was found between long term cortisol levels and the prevalence of metabolic syndrome, which is a risk factor for T2DM (Stalder et al., 2013). Supraphysiological concentrations of GCs are also evident during treatment with GC-based drugs, which have become a mainstay therapy for conditions of inflammation, autoimmune disease and cancer (McDonough et al., 2008; Schacke et al., 2002). Current knowledge indicates that new-onset diabetes after starting low-dose GC treatment seems rare, but progression of existing impaired glucose tolerance to overt diabetes is more

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common (van der Goes et al., 2010). At higher dosages, especially during long-term treatment, increased risk for diabetes and inadequate glucose control among patients on GC treatment is a clinically well-known side effect of GC therapy, that has been found both in patients with asthma (Dendukuri et al., 2002; Suissa et al., 2010) and with rheumatic diseases (Origuchi et al., 2011). The systemic metabolic actions of GCs include a general decrease in the uptake and utilization of glucose alongside with an increase in hepatic gluconeogenesis (McMahon et al., 1988). Importantly, GCs antagonize the anabolic effects of insulin and thus causes insulin resistance. In skeletal muscle, GCs interfere with insulin signaling causing an inhibition of GLUT-4 mediated glucose uptake as well as inhibition of glycogen synthesis (Ruzzin et al., 2005; Weinstein et al., 1998). GC-induced hepatic insulin resistance results in impaired suppression of hepatic glucose production by insulin (Andrews and Walker, 1999). In addition, GCs directly increase endogenous glucose production by induction of several genes involved in hepatic carbohydrate metabolism (Vegiopoulos and Herzig, 2007). These GC-mediated effects on muscle and hepatic tissue are augmented by GC-induced release of nonesterified fatty acids (NEFA) and adipokines from adipose tissue (Galic et al., 2010). Taken together, increased circulating levels of GCs heighten the need for insulin in order to maintain glucose homeostasis. In response to alterations in insulin sensitivity pancreatic bcells can adjust their insulin secretory capacity to meet the need for glucose homeostasis. Thus, experimental studies performed both in healthy human subjects and in rodent models show that steroid treatment results in hyperinsulinemia but maintained normal fasting blood glucose levels and glucose tolerance (Ahrén, 2008; Binnert et al., 2004; Hollingdal et al., 2002; Nicod et al.,

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2003; Schneiter and Tappy, 1998; Willi et al., 2002). Compensatory b-cell hypertrophy is the most plausible explanation for the increased secretory capacity (Rafacho et al., 2011). However, when people with any degree of susceptibility towards glucose intolerance are given GCs, b-cells fail to adapt, and in such individuals GC treatment may disrupt glucose homeostasis (Besse et al., 2005; Grill et al., 1990; Henriksen et al., 1997; Larsson and Ahren, 1999; Wajngot et al., 1992). Similarly, in diabetes prone Zucker rats (Ogawa et al., 1992; Ohneda et al., 1993) or in normal rats and mice treated with high dosages of GCs, glucose homeostasis is disrupted (Fransson et al., 2013; Rafacho et al., 2009). The establishment of any direct GC effects on pancreatic b-cells under in vivo conditions is difficult since systemic metabolic consequences of GC treatment (e.g. glucose, NEFA) interfere with direct GC-mediated effects. Although direct evidence for bcell apoptosis during development of steroid-induced diabetes is lacking, GC-mediated cytotoxicity conditions is a striking feature upon GC exposure to isolated islets and insulinoma cell lines (Avram et al., 2008; Ranta et al., 2006; Reich et al., 2012). While these direct negative effects of GCs on b-cells are well established, the molecular mechanisms regulating GC action in b-cells remain largely unknown. To elucidate pathways that can regulate GC action in b-cells, we have in the present study investigated the role of mitogen-activated protein kinases (MAPKs) in the regulation of GC-induced cytotoxicity. Our results show that SB203580, a p38 MAPK inhibitor, protected against dexamethasone-induced cell death in islet cells and in clonal insulin-producing cells, whereas SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, augmented this death. Furthermore, we show that inhibition of the enzyme serine/threonine protein phosphatase 5 (PP5) also enhances death of dexamethasone-exposed b-cells.

Fig. 1. GCs reduce cell viability in MIN6 cells and isolated islets of Langerhans. (A) MIN6 cells were cultured in the absence or presence of 100 nM of dexamethasone (Dexa), prednisolone (Pred), triamcinolone (Tri), or betamethasone (Beta) with or without 1 lM of RU486 (RU) for 24 h after which cells were lysed and lactate dehydrogenase activity was measured as indicator of cell viability (n = 8). (B) MIN6 cells were cultured at various concentrations of dexamethasone in the absence (closed circles) or presence (open circles) of 1 lM of RU486 for 24 h, after which cell viability was measured as above (n = 8). (C) MIN6 cells were cultured with 100 nM dexamethasone 0–48 h. Apoptosis was evaluated at different time points after exposure by measuring cytoplasmic levels of oligonucleosomes as an indicator of DNA fragmentation (n = 6). (D) MIN6 cells were cultured with 100 nM dexamethasone with or without 1 lM of RU486 for 24 h and apoptosis was evaluated as above (n = 4–15). Dexamethasone-induced apoptosis was also evaluated in islets of Langerhans isolated from obese ob/ob mice (E) or lean C57Bl/6J mice (F). Islets were exposed to dexamethasone and RU486 for 48 h after which apoptosis was detected as above (n = 5). Bars and circles represent mean ± SEM. A  denotes a significant (p < 0.05) difference from untreated cells and a # denotes a significant (p < 0.05) effect of RU486.

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Fig. 2. Cellular stress responses activated by dexamethasone in MIN6 cells. MIN6 cells were cultured in the absence or presence of 100 nM of dexamethasone (Dexa) with or without 1 lM of RU486 (RU) for 24 h. After culture, phosphorylation of eIF2a (A), p38 MAPK (C) and JNK (D) was evaluated by Western blot analysis (images show representative immunoblots). (B) mRNA levels of Ddit3 and Ppp1r15a were evaluated by qPCR in untreated cells (white bars) and cells treated with 100 nM of dexamethasone (black bars) for 24 h. Bars represent mean ± SEM, n = 5. A * denotes a significant (p < 0.05) difference from untreated cells and a # denotes a significant (p < 0.05) effect of RU486.

2. Materials and methods

national law and approved by the local animal ethics committee (Stockholm South Animal Ethics Committee, permit # S201-10).

2.1. Reagents 2.3. MIN6 cell culture and in vitro treatment Reagents of analytical grade and deionized water were used. Dexamethasone, prednisolone, triamcinolone, betamethasone and RU486 came from Sigma–Aldrich (St. Louis, MO, USA). SB203580 and SP600125 were obtained from Calbiochem (La Jolla, CA, USA) and collagenase was from Roche (Roche Diagnostics GmbH, Mannheim, Germany).

2.2. Animals and isolation of islets of Langerhans For this study, islets isolated from either C57Bl/6J mice (Scanbur, Stockholm, Sweden), ob/ob mice (local breed, C57Bl/6J background) or PP5 knockout (Ppp5c/, local breed, C57Bl/6J background) mice and their wild-type littermates (Amable et al., 2011) were used. Experiments were performed on 3–6-monthold male mice, which were sacrificed by exposure to CO2 followed by decapitation. The pancreatic gland was excised and islets were isolated by collagenase digestion and placed in RPMI-1640 culture medium (SVA, Uppsala, Sweden) containing 11 mM glucose and supplemented with 10% (w/v) FBS (Sigma–Aldrich), 2 mM L-glutamine (SVA), 6 mg/ml penicillin G and 5 mg/ml streptomycin sulfate (Invitrogen, Carlsbad, CA, USA) for an overnight recovery at 37 °C in 5% CO2. Islets were then transferred to medium (described above) supplemented with additions as indicated in figures and legends. The use of laboratory animals was performed according to the guidelines of Karolinska Institutet, Sweden, in accordance with

MIN6 cells (Miyazaki et al., 1990), derived from mouse pancreatic b-cells, were maintained in DMEM containing 1 mM sodium pyruvate and 25 mM glucose (Invitrogen), and supplemented with 15% (w/v) FBS, 6 mg/ml penicillin G, 5 mg/ml streptomycin sulfate, 2 mM L-glutamine and 50 lM b-mercaptoethanol (Sigma–Aldrich) at 37 °C and 5% CO2. 2.4. Suppression of PP5 in cultured cells using siRNA Short interfering RNAs (siRNAs), targeting three different regions of mouse PP5 (NM_011155), or the corresponding scrambled (negative control) siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), were used to suppress PP5 expression in MIN6 cells. Cell transfection was aided by electroporation, using a NucleofectorÒ II (Lonza, Cologne, Germany) and 100 pM of the indicated siRNA. 2.5. Assessment of cell viability and apoptosis Cell viability was assessed by a Cytotoxicity Detection KitPlus (Roche Diagnostics). After exposure, cells were washed twice with PBS and then lysed in PBS supplemented with 1% Triton X100. In this setting, the amount of lactate dehydrogenase released after cell lysis correlates with the amount of living cells after treatment. Apoptosis was detected by measuring cytoplasmic DNA-histone

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protein concentrations were determined as described by Lowry et al. (1951). Immunoblot analyses were performed using antibodies that recognize phosphorylated glucocorticoid receptor (GR) at position serine 220 (cat# ab55189), total GR (cat# ab52190) (Abcam Cambridge, UK), PP5 (cat# sc-32588), phosphorylated apoptosis signal-regulating kinase 1 (ASK-1) (cat# sc-109911), total ASK-1 (cat# sc-7931) (Santa Cruz Biotechnology), phosphorylated eukaryotic translation initiation factor 2a (eIF2a) (cat# 9721), total eIF2a (cat# 9722), phosphorylated p38 MAPK (cat# 9211), total p38 MAPK (cat# 9212), phosphorylated JNK (cat# 9251), total JNK (cat# 9252) and the cleaved form of caspase 3 (cat# 9661) (Cell Signaling Technology, Danvers, MA). Immunoreactive bands were detected using ECL (GE Healthcare, Uppsala, Sweden), imaged with a GelDoc system and quantified with Quantity One software (Bio-Rad Laboratories, Hercules, CA). To verify equal protein loading after imaging, the poly(vinylidene difluoride) membranes were stained with Coomassie Blue (BioRad Laboratories). 2.7. Quantitative PCR Total mRNA was isolated from cells and islets as previously described (Rosengren et al., 2012). Complementary DNA (cDNA) was produced using reverse transcriptase (iScript™ cDNA Synthesis Kit; Bio-Rad Laboratories). The expression levels of mRNAs were measured by SYBR green-based quantitative RT-PCR (SYBR Green Master mix; Thermo Scientific, Waltham, MA, USA). Actin (Actb) mRNA was used as an internal standard. The following primer sequences were used: Actb forward: 50 -GACGTTGACATCCGTAAAGA-30 Actb reverse: 50 -GCCAGAGCAGTAATCTCCTT-30 , DNA damage-inducible transcript 3 protein (Ddit3) mRNA forward: 50 -TCTGTCTCTCCGGAAGTGTA-30 Ddit3 reverse: 50 -CTGGTCTACCCTCAGTCCTC-30 , Protein phosphatase 1 regulatory subunit 15A (Ppp1r15a) mRNA forward: 50 -GGTGGTCCAGCTGAGAATGA-30 Ppp1r15a reverse: 50 -TCTTCCGTGGCTTGATGTTC-30 , Cyclin-dependent kinase inhibitor 1 (Cdkn1a) mRNA forward: 50 -ATATACGCT GCCTGCCCTCT-30 Cdkn1a reverse: 50 -AAGGGCCCTACCGTCCTACT30 , Serum/glucocorticoid-regulated kinase 1 (Sgk1) mRNA forward: 50 -AGCCATCCTGAAGAAGAAAG-30 Sgk1 reverse: 50 -CCCTCTGGAGATGGTAGAAC-30 . 2.8. Statistical analysis Data are presented as mean ± SEM. Student’s t test was used to compare the difference between two groups. For multiple comparisons, differences were determined by one- or two-way ANOVA followed by the appropriate post hoc test (GraphPad Prism version 5 software). A value of p < 0.05 was considered significant. 3. Results Fig. 3. Dexamethasone increase phosphorylation of ASK-1, p38 MAPK and JNK in an exposure-time dependent manner in MIN6 cells. MIN6 cells were cultured in the presence of 100 nM dexamethasone (Dexa) between 0 and 24 h. Phosphorylation of ASK-1 (A), p38 MAPK (B) and JNK (C) was evaluated by Western blot analysis (images show representative immunoblots). Bars represent mean ± SEM, n = 3. A  denotes a significant (p < 0.05) difference from untreated (0 h) cells.

nucleosomes generated during apoptotic DNA fragmentation using the Cell Death Detection Kit ELISAPLUS (Roche Diagnostics), according to the manufacturer’s instructions. 2.6. Western blot analyses Protein samples from islets or cells were prepared for Western blot analysis as previously described (Sargsyan et al., 2008). Total

3.1. Cytotoxic effects of GCs on pancreatic islets and MIN6 cells The effect of synthetic GCs on cell viability was investigated in insulin-producing clonal MIN6 cells. After 24 h of continuous exposure to 100 nM dexamethasone, prednisolone, triamincon or betamethasone, cell viability was reduced by 20–30% (Fig. 1A). The different GC variants produced no discernible differences, and for all GC species used, the GR antagonist RU486 reversed their effects. A dose response experiment revealed that the maximal reduction in cell viability was obtained at 200 nM of dexamethasone (Fig. 1B). Based on this observation, dexamethasone at 100 nM was used in further experiments with MIN6 cells since this concentration allowed detecting both attenuation and augmentation of the degree of dexamethasone-induced cytotoxicity. To verify these

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Fig. 4. Inhibition of p38 MAPK attenuates dexamethasone-induced cell death in MIN6 cells and isolated islets of Langerhans and decreases dexamethasone-induced GR phosphorylation at S220. MIN6 cells (A, B and D) or isolated islets of Langerhans from C57Bl/6J mice (C) were cultured in the absence or presence of 100 nM, 24 h (A and B), 100 nM, 2 h (D) or 200 nM, 48 h (C) of dexamethasone (Dexa) with or without either 1 lM of RU486 (RU) or the p38 MAPK inhibitor SB203580 (SB; 20 lM in C and D). After culture, DNA fragmentation (A), cell viability (B), formation of cleaved caspase 3 (C) and GR phosphorylation at S220 (D), were evaluated. Images show representative immunoblots. Bars represent mean ± SEM, n = 5–8. A  denotes a significant (p < 0.05) difference from untreated cells and a # denotes a significant (p < 0.05) effect of RU486 or SB203580.

data, we performed a time series analysis of DNA fragmentation as an indicator of apoptosis in dexamethasone-exposed MIN6 cells. In alignment with the cell viability results, the steroid time-dependently induced DNA fragmentation (Fig. 1C). Also, the DNA fragmentation induced by dexamethasone was reversed by the GR antagonist RU486 (Fig. 1D). The cytotoxic effect of dexamethasone was also investigated in islets isolated from either obese leptin-deficient ob/ob mice or lean C57Bl/6J mice. Both obese and lean mice were investigated since earlier reports have indicated a difference in GC sensitivity between these mouse strains (Ortsäter et al., 2005). However, based on the analysis of DNA fragmentation, the ability of dexamethasone to induce apoptosis in islet cells was similar for both strains of mice (Fig. 1E and F), and the GR antagonist reversed the cytotoxic effect of dexamethasone in islets isolated from ob/ob mice (Fig. 1E). These data indicate that GCs induce apoptotic death both in islet cells and in MIN6 cells. 3.2. Activation of cellular stress responses after dexamethasone exposure in MIN6 cells To gain insights into the mechanisms behind GC-induced cytotoxicity, the activity of cellular stress responses was investigated in MIN6 cells following dexamethasone exposure. We first analyzed if dexamethasone exposure could stimulate the integrated stress response, which is known to be activated both by high and low glucose (Elouil et al., 2007; Vander Mierde et al., 2007) and by saturated fatty acids (Cunha et al., 2008; Sargsyan et al., 2008). The stress response results in phosphorylation of eIF2a (Prostko

et al., 1993) and induction of pro-apoptotic genes, such as Ddit3 and Ppp1r15a (Ortsäter and Sjöholm, 2007). In contrast to the apoptosis-inducing properties of dexamethasone, eIF2a phosphorylation was reduced after dexamethasone exposure in MIN6 cells (Fig. 2A), as were mRNA levels of both Ddit3 and Ppp1r15a (Fig. 2B) indicating that GCs rather reduce, not increase, activity in this pathway. For further investigation relating to the apoptotic effects of GCs, the MAPK signaling pathway was studied following dexamethasone exposure. The two MAPK proteins known to regulate cell differentiation and apoptosis, p38 MAPK and JNK, are activated by various forms of environmental stress and by inflammation (Ichijo et al., 1997). Following dexamethasone exposure, the phosphorylation of both p38 MAPK and JNK was induced, an effect that was reversed by the GR antagonist RU486 (Fig. 2C and D). Also ASK-1, an upstream kinase activating both p38 MAPK and JNK pathways (Ichijo et al., 1997), was phosphorylated by dexamethasone, in a time dependent manner (Fig. 3A). In the same manner, the phosphorylation of p38 MAPK and JNK also increased with increasing dexamethasone exposure-time (Fig. 3B and C). Thus, our data show that dexamethasone exposure activates ASK-1 and its downstream targets p38 MAPK and JNK in these cells. To further investigate the involvement of p38 MAPK and JNK in GC-induced b-cell death, pharmacological inhibition of both MAPKs was used. Co-culturing MIN6 cells with dexamethasone and either an inhibitor of p38 MAPK (SB203580) or an inhibitor of JNK (SP600125) yielded opposing results. SB203580 dose-dependently attenuated dexamethasone-induced DNA fragmentation (Fig. 4A) and improved cell viability (Fig. 4B). These effects were

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Fig. 5. Inhibition of JNK augments dexamethasone-induced death of MIN6 cells. MIN6 cells were cultured in the absence or presence of 100 nM of dexamethasone (Dexa) with or without 1 lM of RU486 (RU) or the JNK inhibitor SP600125 (SP, 25 lM in B, C and D) for 24 h. After culture, DNA fragmentation (A and B), formation of cleaved caspase 3 (C) and phosphorylation of p38 MAPK (D) were evaluated. Images show representative immunoblots. Bars represent mean ± SEM, n = 4–8. A  denotes a significant (p < 0.05) difference from untreated cells and a # denotes a significant (p < 0.05) effect of RU486 or SP600125.

corroborated in experiments using isolated islets from C57Bl/6J mice where dexamethasone-induced induction of cleaved caspase 3 was prevented by SB203580 almost to the same extent as with RU486 (Fig. 4C). Since p38 MAPK has been shown to regulate GR phosphorylation at serine 220 to promote GR activity (Miller et al., 2005), we hypothesized that SB203580 could influence the phosphorylation of this residue. Indeed, treatment of MIN6 cells with dexamethasone induced a protein band immunoreactive towards both an antibody targeting total GR and another antibody specifically targeting GR phosphorylated at position S220 (Fig. 4D). These data show that dexamethasone promotes cellular accumulation of GR in an S220-phosphorylated state. These effects of dexamethasone were clearly prevented albeit not completely by SB203580 (Fig. 4D), supporting the notion that p38 MAPK regulates GR signaling by modulating GR phosphorylation. In contrast to the cytoprotective effect of SB203580 and p38 MAPK inhibition, treatment with SP600125, the JNK inhibitor, dose-dependently augmented dexamethasone-induced DNA fragmentation (Fig. 5A and B), as well as formation of cleaved caspase 3 (Fig. 5C), in MIN6 cells. This effect of SP600125 was clearly dependent on a functional GR since blocking of the GR with RU486 abolished the augmenting effect of SP600125 on dexamethasone-induced DNA fragmentation (Fig. 5B). Furthermore, there seems to be a cross-talk between JNK and p38 MAPK signaling, since the inhibition of JNK with SP600125 promoted p38 MAPK phosphorylation, both in the absence and presence of dexamethasone (Fig. 5D). Taken together, these data suggest that dexamethasone decreases activity in the integrated stress response pathway but enhances MAPK signaling activity, via the GR. Furthermore, our data indicate that p38 MAPK signaling augments, while JNK signaling dampens, dexamethasone-induced cytotoxicity.

3.3. Dexamethasone cytotoxicity is augmented in MIN6 cells and islets with reduced levels of serine/threonine protein phosphatase 5 The actions of protein kinases are countered by protein phosphatases. Endogenous phosphatase activity of the GR is catalyzed by PP5 that binds to GR via interaction with heat-shock protein 90 (Chen et al., 1994; Silverstein et al., 1997; Zuo et al., 1999). We have recently shown that PP5 can regulate MAPK signaling in MIN6 cells and pancreatic islets (Grankvist et al., 2012). Consequently, we investigated if the suppression of PP5 could alter dexamethasone effects on cell death in b-cells and islets. Since there are no specific inhibitors for PP5, we used siRNA to silence PP5 expression in MIN6 cells. We also used islets isolated from male Ppp5c/ mice, with islets from Ppp5c+/+ littermates serving as controls (Amable et al., 2011). Dexamethasone did not change PP5 protein expression levels (Fig. 6A) but MIN6 cells transfected with PP5 siRNA displayed a 60% reduction of PP5 protein levels 48 h post transfection (Fig. 6B and C). Cells with reduced levels of PP5 displayed increased susceptibility towards dexamethasone-induced apoptosis, as evident by increased formation of cleaved caspase 3 (Fig. 6B and D) and augmented DNA fragmentation (Fig. 6E). When PP5 levels were reduced, dexamethasone-induced p38 MAPK phosphorylation was enhanced (Fig. 6B and F), lending further credence to p38 MAPK as a mediator of the cytotoxic effect of GCs. To investigate the role of PP5 in regulating GC action in pancreatic islets, we isolated islets from Ppp5c+/+ and Ppp5c/ mice and exposed them to dexamethasone for 48 h. Surprisingly, islets isolated from Ppp5c+/+ mice were not as sensitive to dexamethasone as islets isolated from standard C57Bl/6J mice. Hence, 200 nM dexamethasone failed to induce any significant elevation of p38 MAPK phosphorylation and caspase 3 cleavage in wild-type islets (Fig. 7). However, entirely consistent

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Fig. 6. Silencing of protein phosphatase 5 augments dexamethasone-induced death of MIN6 cells. (A) MIN6 cells were cultured in the presence of 100 nM of dexamethasone (Dexa) between 0 and 24 h. Protein expression of PP5 was evaluated by Western blot analysis (images show representative immunoblots). Bars represent mean ± SEM, n = 3. (B–F) MIN6 cells were transfected with siRNA targeting PP5 (black bars) or a scrambled negative control siRNA (white bars). Cells were thereafter cultured in the absence or presence of 100 nM of dexamethasone (Dexa) for 24 h. Protein levels of PP5 (C), cleaved caspase 3 (D), and phosphorylated p38 MAPK (F) were evaluated by Western blot analysis. Images in B show representative immunoblots. Apoptosis was determined by measuring cytoplasmic levels of oligonucleosomes as an indicator of DNA fragmentation (E). Bars represent mean ± SEM, n = 6. A * denotes a significant (p < 0.05) difference from untreated cells and a # denotes a significant (p < 0.05) effect of PP5 siRNA.

with the results obtained in MIN6 cells, islets lacking PP5 showed increased p38 MAPK phosphorylation (Fig. 7A and B) and enhanced formation of cleaved caspase 3 (Fig. 7C and D) following dexamethasone exposure, as compared to wild-type islets. These data indicate that, by down-regulating or silencing PP5, MIN6 cells and islets become sensitized to the cytotoxic actions of GCs. To further scrutinize this hypothesis, we analyzed mRNA levels of Sgk1 and Cdkn1a, whose expressions are known to be positively regulated by GCs (Cha et al., 1998; Ullrich et al., 2005). Indeed, dexamethasone-induced Sgk1 and Cdkn1a mRNA expression was enhanced in MIN6 cells with reduced levels of

PP5 (Fig. 8A and B), thus further corroborating the increased susceptibility to GCs in cells and islets with decreased levels or lack of PP5.

4. Discussion GCs exert direct cytotoxic effects on pancreatic b-cells in culture. This observation is reproduced in the current study and corroborates previous findings (Avram et al., 2008; Ranta et al., 2006, 2008). While GC administration for a short period in healthy

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Fig. 7. Genetic disruption of protein phosphatase 5 augments dexamethasone-induced p38 MAPK phosphorylation and apoptosis in isolated islets of Langerhans. Pancreatic islets were isolated from Ppp5c+/+ (white bars) or Ppp5c/ (black bars) mice and then treated with 200 nM dexamethasone (Dexa) for 48 h in culture. Protein levels of phosphorylated p38 MAPK (A, B) and cleaved caspase 3 (C, D) were then evaluated by Western blot analysis. Images show representative immunoblots. Bars represent mean ± SEM, n = 5. A # denotes a significant (p < 0.05) difference vs. islets isolated from Ppp5c/ mice.

Fig. 8. Silencing of protein phosphatase 5 augments dexamethasone-induced gene transcription in MIN6 cells. MIN6 cells were transfected with siRNA targeting PP5 (black bars) or a scrambled control (white bars). Cells were then cultured in the absence or presence of 100 nM of dexamethasone (Dexa) for 30 h. mRNA levels of Sgk1 (A) and Cdkn1a (B) were measured by qPCR. Bars represent mean ± SEM, n = 6. A  denotes a significant (p < 0.05) difference from untreated cells and a # denotes a significant (p < 0.05) effect of PP5 siRNA.

human volunteers neither raises fasting blood glucose levels (Ahrén, 2008; Binnert et al., 2004; Nicod et al., 2003) nor produce glucose intolerance (Hollingdal et al., 2002; Schneiter and Tappy, 1998; Willi et al., 2002), it is well known that long term GC treatment can induce glucose intolerance or even precipitate overt diabetes in susceptible individuals (Besse et al., 2005; Henriksen et al., 1997; Larsson and Ahren, 1999). In such cases, a GC-induced decrease in functional b-cell mass may be relevant. Although evidence of b-cell apoptosis after in vivo exposure to GCs is lacking, impaired b-cell function was observed following prednisolone treatment in humans (van Raalte et al., 2011). b-cell susceptibility to GCs may also be relevant in the natural progression towards diabetes, since mice overexpressing the GR restricted to the b-cells develop b-cell failure and diabetes (Delaunay et al., 1997; Ling et al., 1998) and humans with impaired b-cell function (low insulin responders) are predisposed to becoming overtly diabetic during GC therapy (Wajngot et al., 1992).

The mechanisms conveying GC-induced cytotoxic effects on bcells are not well understood. A role for reactive oxygen species (ROS) has been proposed, based on observations showing that (1) dexamethasone induces ROS production in RINm5F insulinoma cells, (2) the overexpression of catalase in these cells prevents the cytotoxic effects of dexamethasone (Roma et al., 2009), and (3) dexamethasone enhances expression of the thioredoxin-interacting protein TXNIP, which is a negative regulator of the antioxidant protein thioredoxin (Reich et al., 2012) and provokes b-cell death (Chen et al., 2008a). GC-induced expression of TXNIP in bcells is dependent on p38 MAPK activation (Reich et al., 2012). These observations are in complete alignment with our current findings, which show activation of MAPK signaling as evidenced by enhanced phosphorylation of ASK-1 and its downstream targets p38 MAPK and JNK following dexamethasone exposure. Thus, the effect of GCs on MAPK activity is complex. Our data clearly show that by treating MIN6 cells and isolated C57Bl/6J islets with

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dexamethasone, the MAPK signaling pathway is activated, lending support to the observations by Reich et al. (2012) and Furst et al. (2008). At the same time it is known from studies in other tissues that dexamethasone can attenuate p38 MAPK and JNK phosphorylation induced by cytokines (Burke et al., 2012), glucose starvation (Galliher-Beckley et al., 2011) or UV light exposure (Lasa et al., 2001). In contrast to dexamethasone-induced activation of MAPK signaling, the integrated stress response was not induced by dexamethasone in our experiments. In fact, we observed lowered phosphorylation of eIF2a, as previously noted (Linssen et al., 2011) and decreased mRNA levels of Ddit3 and Ppp1r15a. Phosphorylation of eIF2a results in inhibition of protein synthesis and constitutes a part of the cytoprotective unfolded protein response (UPR) (Wu and Kaufman, 2006). Thus, our results indicate that UPR activity is compromised in dexamethasone-exposed b-cells. Failure to induce UPR has been linked to abnormalities in b-cell gene expression and progression towards diabetes in db/db mice (Chan et al., 2013). The results presented herein also provide insights into the regulation of GC action in pancreatic b-cells. GCs exert their effects by binding to an intracellular GR that, upon ligand binding, is phosphorylated and translocated to the nucleus (Mangelsdorf et al., 1995). In the nucleus, activated GR affects gene expression by binding to GC-responsive elements in the promoter region of GCregulated genes. GR transcriptional activity is regulated via phosphorylation (Chen et al., 2008b). Up to eight different phosphorylation sites have been identified on the GR (Wang et al., 2002). Phosphorylation of serines 212, 220 and 234 (mouse form, corresponding to S203, S211 and S226 in the human form) is particularly associated with GR transcriptional activity (Ismaili and Garabedian, 2004). Serine to alanine mutations of S203 and S211, individually or in combination, impede transcriptional receptor activation in mammalian cells, suggesting that phosphorylation of these residues is required for full GR activity (Almlof et al., 1995; Miller et al., 2005; Webster et al., 1997). In contrast, an alanine substitution for S234 increases GR transcriptional activity relative to the wild-type receptor, suggesting that phosphorylation of S234 is inhibitory to GR function (Itoh et al., 2002; Rogatsky et al., 1998). Thus, phosphorylation appears to provide both positive and negative regulatory inputs with respect to GR transcriptional activation. Of relevance to our results, serine residue 220 in the murine GR is a substrate for p38 MAPK activity. Changing, via site directed mutagenesis, serine 220 into alanine 220 greatly diminishes GRdriven gene transcription and apoptosis in lymphoid cells and hepatocytes as does pharmacological inhibition of p38 MAPK (Miller et al., 2005; Nader et al., 2010). Thus, our finding that a p38 MAPK inhibitor reduces dexamethasone-induced phosphorylation of GR at S220 and attenuates dexamethasone-induced cytotoxicity aligns well with reduced GR activity. In our experiments, the p38 MAPK inhibitor reduced dexamethasone-induced apoptosis in MIN6 cells (Fig. 4A) and dexamethasone-induced formation of cleaved caspase 3 in isolated pancreatic islets (Fig. 4C). Although these levels are in agreement with previous data (Reich et al., 2012), it is clear that activation of p38 MAPK is not the sole mechanism behind GC-induced apoptosis in insulin-producing cells. We also investigated the effect of a JNK inhibitor on dexamethasone-induced cytotoxicity. JNK is the kinase primarily responsible for phosphorylation of S234 of the GR (Rogatsky et al., 1998). Unfortunately, we were not able to produce reliable results with a commercially available antibody directed towards GR phosphorylated at position S234. However, our results showing that inhibition of JNK resulted in augmented apoptosis are entirely consistent with the concept that JNK action negatively influences GR activity. Indeed, blockade of GR action by RU486 completely blocked the

augmenting effect of SP600125 on GC-induced apoptosis in MIN6 cells. GR activity is not only regulated by kinases. Perturbations in protein phosphatase activity have also been shown to affect GR function in other cell types. Treatment of cells with okadaic acid, a general serine/threonine protein phosphatase inhibitor, results in GR hyper-phosphorylation, retaining of the receptor in the nucleus and, hence, transcriptional activation in mammalian cells (DeFranco et al., 1991; Somers and DeFranco, 1992). In vitro experiments with A549 cells showed that suppression of PP5 expression through antisense oligonucleotides increased GR transcriptional activity both in the absence and presence of GCs (Zuo et al., 1999). In airway smooth muscle cells, PP5 was recently shown to suppress GR phosphorylation at S220/S211 and in so doing the enzyme reduced cell sensitivity to GCs (Bouazza et al., 2012). The data presented herein show that PP5 is suppressing GC cytotoxicity in b-cells, supporting the notion that endogenous phosphatase activity of the GR is catalyzed by PP5 (Wang et al., 2007). Cells and islets with reduced levels of PP5 display increased susceptibility towards dexamethasone-induced apoptosis, and GC-induced p38 MAPK phosphorylation is augmented in these cells. Of note, the protective effect of the p38 MAPK inhibitor is in the same range as the augmenting effects of PP5 inhibition, which do not prove but supports the conclusion that p38 MAPK and PP5 is operating via the same mechanism. These observations corroborate our previous results where we have shown that mouse embryonic fibroblasts isolated from Ppp5c/ mice show increased sensitivity to various DNA-damaging agents such as UV light, hydroxyurea, and camptothecin (Amable et al., 2011) and that islets from Ppp5c/ mice and MIN6 cells with suppressed PP5 expression display increased sensitivity towards palmitate- and H2O2-induced cytotoxicity (Grankvist et al., 2012). Taken together, these results point to PP5 as a mediator of b-cell cytoprotection. In conclusion, our results provide evidence that GC exposure of b-cells induces p38 MAPK and JNK phosphorylation that work in opposite to regulate the cytotoxic effects exerted by GCs. Our data are in line with previous results, showing the activating effect of p38 MAPK-induced phosphorylation at S220, and the inhibiting effect of JNK-induced phosphorylation at S234 of the GR, after stimulation with GCs. In this context, inhibition of JNK augments GC cytotoxicity in b-cells, whereas inhibition of p38 MAPK protects. We also show that PP5 plays a protective role in b-cell defense against GC toxicity. Thus, PP5 is a novel player in the GC response and might serve as an important and potentially target in therapeutic attempts to protect against b-cell demise and, hence, glucose intolerance during GC therapy and perhaps also the natural unfolding of polygenic T2DM. Acknowledgements We thank the personnel at the animal facility of Södersjukhuset for excellent animal care and the research center at Södersjukhuset for providing laboratory facilities. This study was supported by grants from the Diabetes Research Wellness Foundation, Folksam Research Foundation and the Tore Nilsson Foundation. Henrik Ortsäter is funded by the Swedish Society for Medical Research. Liselotte Fransson is partly funded by KID (Karolinska Institutet, Faculty funds for partial funding of doctoral students). References Ahrén, B., 2008. Evidence that autonomic mechanisms contribute to the adaptive increase in insulin secretion during dexamethasone-induced insulin resistance in humans. Diabetologia 51 (6), 1018–1024. Almlof, T., Wright, A.P., Gustafsson, J.A., 1995. Role of acidic and phosphorylated residues in gene activation by the glucocorticoid receptor. J. Biol. Chem. 270 (29), 17535–17540.

L. Fransson et al. / Molecular and Cellular Endocrinology 383 (2014) 126–136 Amable, L., Grankvist, N., Largen, J.W., Ortsäter, H., Sjöholm, A., Honkanen, R.E., 2011. Disruption of serine/threonine protein phosphatase 5 (PP5:PPP5c) in mice reveals a novel role for pp5 in the regulation of ultraviolet light-induced phosphorylation of serine/threonine protein Kinase Chk1 (CHEK1). J. Biol. Chem. 286 (47), 40413–40422. Andrews, R.C., Walker, B.R., 1999. Glucocorticoids and insulin resistance: old hormones, new targets. Clin. Sci. (Lond.) 96 (5), 513–523. Avram, D., Ranta, F., Hennige, A.M., Berchtold, S., Hopp, S., Haring, H.U., Lang, F., Ullrich, S., 2008. IGF-1 protects against dexamethasone-induced cell death in insulin secreting INS-1 cells independent of AKT/PKB phosphorylation. Cell. Physiol. Biochem. 21 (5–6), 455–462. Besse, C., Nicod, N., Tappy, L., 2005. Changes in insulin secretion and glucose metabolism induced by dexamethasone in lean and obese females. Obes. Res. 13 (2), 306–311. Biering, H., Knappe, G., Gerl, H., Lochs, H., 2000. Prevalence of diabetes in acromegaly and Cushing syndrome. Acta Med. Austriaca. 27 (1), 27–31. Binnert, C., Ruchat, S., Nicod, N., Tappy, L., 2004. Dexamethasone-induced insulin resistance shows no gender difference in healthy humans. Diabetes Metab. 30 (4), 321–326. Bouazza, B., Krytska, K., Debba-Pavard, M., Amrani, Y., Honkanen, R.E., Tran, J., Tliba, O., 2012. Cytokines alter glucocorticoid receptor phosphorylation in airway cells: role of phosphatases. Am. J. Respir. Cell Mol. Biol. 47 (4), 464–473. Burke, S.J., Goff, M.R., Updegraff, B.L., Lu, D., Brown, P.L., Minkin Jr., S.C., Biggerstaff, J.P., Zhao, L., Karlstad, M.D., Collier, J.J., 2012. Regulation of the CCL2 gene in pancreatic beta-cells by IL-1beta and glucocorticoids: role of MKP-1. PLoS ONE 7 (10), e46986. Cha, H.H., Cram, E.J., Wang, E.C., Huang, A.J., Kasler, H.G., Firestone, G.L., 1998. Glucocorticoids stimulate p21 gene expression by targeting multiple transcriptional elements within a steroid responsive region of the p21waf1/ cip1 promoter in rat hepatoma cells. J. Biol. Chem. 273 (4), 1998–2007. Chan, J., Luzuriaga, J., Bensellam, M., Biden, T., Laybutt, D., 2013. Failure of the adaptive unfolded protein response in islets of obese mice is linked with abnormalities in b-cell gene expression and progression to diabetes. Diabetes 62 (5), 1557–1568. Chen, M.X., McPartlin, A.E., Brown, L., Chen, Y.H., Barker, H.M., Cohen, P.T., 1994. A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus. Embo J. 13 (18), 4278–4290. Chen, J., Saxena, G., Mungrue, I.N., Lusis, A.J., Shalev, A., 2008a. Thioredoxininteracting protein: a critical link between glucose toxicity and beta-cell apoptosis. Diabetes 57 (4), 938–944. Chen, W., Dang, T., Blind, R.D., Wang, Z., Cavasotto, C.N., Hittelman, A.B., Rogatsky, I., Logan, S.K., Garabedian, M.J., 2008b. Glucocorticoid receptor phosphorylation differentially affects target gene expression. Mol. Endocrinol. 22 (8), 1754– 1766. Cunha, D.A., Hekerman, P., Ladriere, L., Bazarra-Castro, A., Ortis, F., Wakeham, M.C., Moore, F., Rasschaert, J., Cardozo, A.K., Bellomo, E., Overbergh, L., Mathieu, C., Lupi, R., Hai, T., Herchuelz, A., Marchetti, P., Rutter, G.A., Eizirik, D.L., Cnop, M., 2008. Initiation and execution of lipotoxic ER stress in pancreatic b-cells. J. Cell. Sci. 121 (Pt 14), 2308–2318. Danaei, G., Finucane, M.M., Lu, Y., Singh, G.M., Cowan, M.J., Paciorek, C.J., Lin, J.K., Farzadfar, F., Khang, Y.H., Stevens, G.A., Rao, M., Ali, M.K., Riley, L.M., Robinson, C.A., Ezzati, M., 2011. National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2.7 million participants. Lancet 378 (9785), 31–40. DeFranco, D.B., Qi, M., Borror, K.C., Garabedian, M.J., Brautigan, D.L., 1991. Protein phosphatase types 1 and/or 2A regulate nucleocytoplasmic shuttling of glucocorticoid receptors. Mol. Endocrinol. 5 (9), 1215–1228. Delaunay, F., Khan, A., Cintra, A., Davani, B., Ling, Z.C., Andersson, A., Ostenson, C.G., Gustafsson, J., Efendic, S., Okret, S., 1997. Pancreatic b cells are important targets for the diabetogenic effects of glucocorticoids. J. Clin. Invest. 100 (8), 2094–2098. Dendukuri, N., Blais, L., LeLorier, J., 2002. Inhaled corticosteroids and the risk of diabetes among the elderly. Br. J. Clin. Pharmacol. 54 (1), 59–64. Di Dalmazi, G., Vicennati, V., Rinaldi, E., Morselli-Labate, A.M., Giampalma, E., Mosconi, C., Pagotto, U., Pasquali, R., 2012. Progressively increased patterns of subclinical cortisol hypersecretion in adrenal incidentalomas differently predict major metabolic and cardiovascular outcomes: a large cross-sectional study. Eur. J. Endocrinol.. Elouil, H., Bensellam, M., Guiot, Y., Vander Mierde, D., Pascal, S.M., Schuit, F.C., Jonas, J.C., 2007. Acute nutrient regulation of the unfolded protein response and integrated stress response in cultured rat pancreatic islets. Diabetologia 50 (7), 1442–1452. Fransson, L., Franzen, S., Rosengren, V., Wolbert, P., Sjöholm, Å., Ortsäter, H., 2013. Bcell adaptation in a mouse model of glucocorticoid-induced metabolic syndrome. J. Endocrinol. 219 (3), 231–241. Furst, R., Zahler, S., Vollmar, A.M., 2008. Dexamethasone-induced expression of endothelial mitogen-activated protein kinase phosphatase-1 involves activation of the transcription factors activator protein-1 and 30 ,50 -cyclic adenosine 50 -monophosphate response element-binding protein and the generation of reactive oxygen species. Endocrinology 149 (7), 3635–3642. Galic, S., Oakhill, J.S., Steinberg, G.R., 2010. Adipose tissue as an endocrine organ. Mol. Cell. Endocrinol. 316 (2), 129–139. Galliher-Beckley, A.J., Williams, J.G., Cidlowski, J.A., 2011. Ligand-independent phosphorylation of the glucocorticoid receptor integrates cellular stress pathways with nuclear receptor signaling. Mol. Cell. Biol. 31 (23), 4663–4675.

135

Grankvist, N., Amable, L., Honkanen, R.E., Sjöholm, Å., Ortsäter, H., 2012. Serine/ threonine protein phosphatase 5 regulates glucose homeostasis in vivo and apoptosis signalling in mouse pancreatic islets and clonal MIN6 cells. Diabetologia 55 (7), 2005–2015. Grill, V., Pigon, J., Hartling, S.G., Binder, C., Efendic, S., 1990. Effects of dexamethasone on glucose-induced insulin and proinsulin release in low and high insulin responders. Metabolism 39 (3), 251–258. Henriksen, J.E., Alford, F., Ward, G.M., Beck-Nielsen, H., 1997. Risk and mechanism of dexamethasone-induced deterioration of glucose tolerance in non-diabetic first-degree relatives of NIDDM patients. Diabetologia 40 (12), 1439–1448. Hollingdal, M., Juhl, C.B., Dall, R., Sturis, J., Veldhuis, J.D., Schmitz, O., Porksen, N., 2002. Glucocorticoid induced insulin resistance impairs basal but not glucose entrained high-frequency insulin pulsatility in humans. Diabetologia 45 (1), 49–55. Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K., Gotoh, Y., 1997. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275 (5296), 90–94. Ismaili, N., Garabedian, M.J., 2004. Modulation of glucocorticoid receptor function via phosphorylation. Ann. N. Y. Acad. Sci. 1024, 86–101. Itoh, M., Adachi, M., Yasui, H., Takekawa, M., Tanaka, H., Imai, K., 2002. Nuclear export of glucocorticoid receptor is enhanced by c-Jun N-terminal kinasemediated phosphorylation. Mol. Endocrinol. 16 (10), 2382–2392. Larsson, H., Ahren, B., 1999. Insulin resistant subjects lack islet adaptation to shortterm dexamethasone-induced reduction in insulin sensitivity. Diabetologia 42 (8), 936–943. Lasa, M., Brook, M., Saklatvala, J., Clark, A.R., 2001. Dexamethasone destabilizes cyclooxygenase 2 mRNA by inhibiting mitogen-activated protein kinase p38. Mol. Cell. Biol. 21 (3), 771–780. Ling, Z.C., Khan, A., Delauny, F., Davani, B., Ostenson, C.G., Gustafsson, J.A., Okret, S., Landau, B.R., Efendic, S., 1998. Increased glucocorticoid sensitivity in islet betacells: effects on glucose 6-phosphatase, glucose cycling and insulin release. Diabetologia 41 (6), 634–639. Linssen, M.M., van Raalte, D.H., Toonen, E.J., Alkema, W., van der Zon, G.C., Dokter, W.H., Diamant, M., Guigas, B., Ouwens, D.M., 2011. Prednisolone-induced beta cell dysfunction is associated with impaired endoplasmic reticulum homeostasis in INS-1E cells. Cell. Signal. 23 (11), 1708–1715. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 (1), 265–275. Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., Evans, R.M., 1995. The nuclear receptor superfamily: the second decade. Cell 83 (6), 835–839. McDonough, A.K., Curtis, J.R., Saag, K.G., 2008. The epidemiology of glucocorticoidassociated adverse events. Curr. Opin. Rheumatol. 20 (2), 131–137. McMahon, M., Gerich, J., Rizza, R., 1988. Effects of glucocorticoids on carbohydrate metabolism. Diabetes Metab. Rev. 4 (1), 17–30. Miller, A.L., Webb, M.S., Copik, A.J., Wang, Y., Johnson, B.H., Kumar, R., Thompson, E.B., 2005. P38 Mitogen-activated protein kinase (MAPK) is a key mediator in glucocorticoid-induced apoptosis of lymphoid cells: correlation between p38 MAPK activation and site-specific phosphorylation of the human glucocorticoid receptor at serine 211. Mol. Endocrinol. 19 (6), 1569–1583. Miyazaki, J., Araki, K., Yamato, E., Ikegami, H., Asano, T., Shibasaki, Y., Oka, Y., Yamamura, K., 1990. Establishment of a pancreatic b cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127 (1), 126–132. Nader, N., Ng, S.S., Lambrou, G.I., Pervanidou, P., Wang, Y., Chrousos, G.P., Kino, T., 2010. AMPK regulates metabolic actions of glucocorticoids by phosphorylating the glucocorticoid receptor through p38 MAPK. Mol. Endocrinol. 24 (9), 1748– 1764. Nicod, N., Giusti, V., Besse, C., Tappy, L., 2003. Metabolic adaptations to dexamethasone-induced insulin resistance in healthy volunteers. Obes. Res. 11 (5), 625–631. Ogawa, A., Johnson, J.H., Ohneda, M., McAllister, C.T., Inman, L., Alam, T., Unger, R.H., 1992. Roles of insulin resistance and b-cell dysfunction in dexamethasoneinduced diabetes. J. Clin. Invest. 90 (2), 497–504. Ohneda, M., Johnson, J.H., Inman, L.R., Unger, R.H., 1993. GLUT-2 function in glucose-unresponsive b cells of dexamethasone-induced diabetes in rats. J. Clin. Invest. 92 (4), 1950–1956. Origuchi, T., Yamaguchi, S., Inoue, A., Kazaura, Y., Matsuo, N., Abiru, N., Kawakami, A., Eguchi, K., 2011. Increased incidence of pre-diabetes mellitus at a department of rheumatology: a retrospective study. Mod. Rheumatol. 21 (5), 495–499. Ortsäter, H., Sjöholm, A., 2007. A busy cell – endoplasmic reticulum stress in the pancreatic b-cell. Mol. Cell. Endocrinol. 277 (1–2), 1–5. Ortsäter, H., Alberts, P., Warpman, U., Engblom, L.O., Abrahmsén, L., Bergsten, P., 2005. Regulation of 11b-hydroxysteroid dehydrogenase type 1 and glucosestimulated insulin secretion in pancreatic islets of Langerhans. Diabetes Metab. Res. Rev. 21 (4), 359–366. Prostko, C.R., Brostrom, M.A., Brostrom, C.O., 1993. Reversible phosphorylation of eukaryotic initiation factor 2a in response to endoplasmic reticular signaling. Mol. Cell. Biochem. 127–128, 255–265. Rafacho, A., Cestari, T.M., Taboga, S.R., Boschero, A.C., Bosqueiro, J.R., 2009. High doses of dexamethasone induce increased b-cell proliferation in pancreatic rat islets. Am. J. Physiol. Endocrinol. Metab. 296 (4), E681–E689. Rafacho, A., Abrantes, J.L., Ribeiro, D.L., Paula, F.M., Pinto, M.E., Boschero, A.C., Bosqueiro, J.R., 2011. Morphofunctional alterations in endocrine pancreas of

136

L. Fransson et al. / Molecular and Cellular Endocrinology 383 (2014) 126–136

short- and long-term dexamethasone-treated rats. Horm. Metab. Res. 43 (4), 275–281. Ranta, F., Avram, D., Berchtold, S., Dufer, M., Drews, G., Lang, F., Ullrich, S., 2006. Dexamethasone induces cell death in insulin-secreting cells, an effect reversed by exendin-4. Diabetes 55 (5), 1380–1390. Ranta, F., Dufer, M., Stork, B., Wesselborg, S., Drews, G., Haring, H.U., Lang, F., Ullrich, S., 2008. Regulation of calcineurin activity in insulin-secreting cells: stimulation by Hsp90 during glucocorticoid-induced apoptosis. Cell. Signal. 20 (10), 1780– 1786. Raul Ariza-Andraca, C., Barile-Fabris, L.A., Frati-Munari, A.C., Baltazar-Montufar, P., 1998. Risk factors for steroid diabetes in rheumatic patients. Arch. Med. Res. 29 (3), 259–262. Reich, E., Tamary, A., Sionov, R.V., Melloul, D., 2012. Involvement of thioredoxininteracting protein (TXNIP) in glucocorticoid-mediated beta cell death. Diabetologia 55 (4), 1048–1057. Rogatsky, I., Logan, S.K., Garabedian, M.J., 1998. Antagonism of glucocorticoid receptor transcriptional activation by the c-Jun N-terminal kinase. Proc. Natl. Acad. Sci. USA 95 (5), 2050–2055. Roma, L.P., Bosqueiro, J.R., Cunha, D.A., Carneiro, E.M., Gurgul-Convey, E., Lenzen, S., Boschero, A.C., Souza, K.L., 2009. Protection of insulin-producing cells against toxicity of dexamethasone by catalase overexpression. Free Radic. Biol. Med. 47 (10), 1386–1393. Rosengren, V., Johansson, H., Lehtio, J., Fransson, L., Sjoholm, A., Ortsater, H., 2012. Thapsigargin down-regulates protein levels of GRP78/BiP in INS-1E cells. J. Cell. Biochem. 113 (5), 1635–1644. Ruzzin, J., Wagman, A.S., Jensen, J., 2005. Glucocorticoid-induced insulin resistance in skeletal muscles: defects in insulin signalling and the effects of a selective glycogen synthase kinase-3 inhibitor. Diabetologia 48 (10), 2119–2130. Sargsyan, E., Ortsäter, H., Thörn, K., Bergsten, P., 2008. Diazoxide-induced b-cell rest reduces endoplasmic reticulum stress in lipotoxic b-cells. J. Endocrinol. 199 (1), 41–50. Schacke, H., Docke, W.D., Asadullah, K., 2002. Mechanisms involved in the side effects of glucocorticoids. Pharmacol. Ther. 96 (1), 23–43. Schneiter, P., Tappy, L., 1998. Kinetics of dexamethasone-induced alterations of glucose metabolism in healthy humans. Am. J. Physiol. 275 (5 Pt 1), E806–E813. Silverstein, A.M., Galigniana, M.D., Chen, M.S., Owens-Grillo, J.K., Chinkers, M., Pratt, W.B., 1997. Protein phosphatase 5 is a major component of glucocorticoid receptor.hsp90 complexes with properties of an FK506-binding immunophilin. J. Biol. Chem. 272 (26), 16224–16230. Somers, J.P., DeFranco, D.B., 1992. Effects of okadaic acid, a protein phosphatase inhibitor, on glucocorticoid receptor-mediated enhancement. Mol. Endocrinol. 6 (1), 26–34. Stalder, T., Kirschbaum, C., Alexander, N., Bornstein, S.R., Gao, W., Miller, R., Stark, S., Bosch, J.A., Fischer, J.E., 2013. Cortisol in hair and the metabolic syndrome. J. Clin. Endocrinol. Metab. 98 (6), 2573–2580. Suissa, S., Kezouh, A., Ernst, P., 2010. Inhaled corticosteroids and the risks of diabetes onset and progression. Am. J. Med. 123 (11), 1001–1006. Ullrich, S., Berchtold, S., Ranta, F., Seebohm, G., Henke, G., Lupescu, A., Mack, A.F., Chao, C.M., Su, J., Nitschke, R., Alexander, D., Friedrich, B., Wulff, P., Kuhl, D., Lang, F., 2005. Serum- and glucocorticoid-inducible kinase 1 (SGK1) mediates

glucocorticoid-induced inhibition of insulin secretion. Diabetes 54 (4), 1090– 1099. van der Goes, M.C., Jacobs, J.W., Boers, M., Andrews, T., Blom-Bakkers, M.A., Buttgereit, F., Caeyers, N., Cutolo, M., Da Silva, J.A., Guillevin, L., Kirwan, J.R., Rovensky, J., Severijns, G., Webber, S., Westhovens, R., Bijlsma, J.W., 2010. Monitoring adverse events of low-dose glucocorticoid therapy: EULAR recommendations for clinical trials and daily practice. Ann. Rheum. Dis. 69 (11), 1913–1919. van Raalte, D.H., Ouwens, D.M., Diamant, M., 2009. Novel insights into glucocorticoid-mediated diabetogenic effects: towards expansion of therapeutic options? Eur. J. Clin. Invest. 39 (2), 81–93. van Raalte, D.H., van Genugten, R.E., Linssen, M.M., Ouwens, D.M., Diamant, M., 2011. Glucagon-like peptide-1 receptor agonist treatment prevents glucocorticoid-induced glucose intolerance and islet-cell dysfunction in humans. Diabetes Care 34 (2), 412–417. Vander Mierde, D., Scheuner, D., Quintens, R., Patel, R., Song, B., Tsukamoto, K., Beullens, M., Kaufman, R.J., Bollen, M., Schuit, F.C., 2007. Glucose activates a protein phosphatase-1-mediated signaling pathway to enhance overall translation in pancreatic b-cells. Endocrinology 148 (2), 609–617. Vegiopoulos, A., Herzig, S., 2007. Glucocorticoids, metabolism and metabolic diseases. Mol. Cell. Endocrinol. 275 (1–2), 43–61. Wajchenberg, BL., Prestes Cesar, F., Okada, H., Torres de Toledo e Souza, I., Lerario, AC., Borghi, VC., Malerbi, DA., Giurna Filho, A., Liberman, B., Gianella, D., 1984. Glucocorticoids, glucose metabolism and hypothalamic-pituitary-adrenal axis. Adv. Exp. Med. Biol. 171, 25–44. Wajngot, A., Giacca, A., Grill, V., Vranic, M., Efendic, S., 1992. The diabetogenic effects of glucocorticoids are more pronounced in low- than in high-insulin responders. Proc. Natl. Acad. Sci. USA 89 (13), 6035–6039. Wang, Z., Frederick, J., Garabedian, M.J., 2002. Deciphering the phosphorylation ‘‘code’’ of the glucocorticoid receptor in vivo. J. Biol. Chem. 277 (29), 26573– 26580. Wang, Z., Chen, W., Kono, E., Dang, T., Garabedian, M.J., 2007. Modulation of glucocorticoid receptor phosphorylation and transcriptional activity by a Cterminal-associated protein phosphatase. Mol. Endocrinol. 21 (3), 625–634. Webster, J.C., Jewell, C.M., Bodwell, J.E., Munck, A., Sar, M., Cidlowski, J.A., 1997. Mouse glucocorticoid receptor phosphorylation status influences multiple functions of the receptor protein. J. Biol. Chem. 272 (14), 9287–9293. Weinstein, S.P., Wilson, C.M., Pritsker, A., Cushman, S.W., 1998. Dexamethasone inhibits insulin-stimulated recruitment of GLUT4 to the cell surface in rat skeletal muscle. Metabolism 47 (1), 3–6. Willi, S.M., Kennedy, A., Wallace, P., Ganaway, E., Rogers, N.L., Garvey, W.T., 2002. Troglitazone antagonizes metabolic effects of glucocorticoids in humans: effects on glucose tolerance, insulin sensitivity, suppression of free fatty acids, and leptin. Diabetes 51 (10), 2895–2902. Wu, J., Kaufman, R.J., 2006. From acute ER stress to physiological roles of the unfolded protein response. Cell Death Differ. 13 (3), 374–384. Zuo, Z., Urban, G., Scammell, J.G., Dean, N.M., McLean, T.K., Aragon, I., Honkanen, R.E., 1999. Ser/Thr protein phosphatase type 5 (PP5) is a negative regulator of glucocorticoid receptor-mediated growth arrest. Biochemistry 38 (28), 8849– 8857.