ERK1 is dispensable for mouse pancreatic beta cell function but is necessary for glucose-induced full activation of MSK1 and CREB
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Insufficient insulin secretion from pancreatic beta cells, which is associated with a decrease in beta cell mass, is a characteristic of type 2 diabetes. Extracellular signal-related kinase 1 and 2 (ERK1/2) inhibition in beta cells has been reported to affect insulin secretion, gene transcription and survival, although whether ERK1 and ERK2 play distinct roles is unknown. The aim of this study was to assess the individual roles of ERK1 and ERK2 in beta cells using ERK1 (also known as Mapk3)-knockout mice (Erk1 −/− mice) and pharmacological approaches.
NAD(P)H, free cytosolic Ca2+ concentration and insulin secretion were determined in islets. ERK1 and ERK2 subplasmalemmal translocation and activity was monitored using total internal reflection fluorescence microscopy. ERK1/2, mitogen and stress-activated kinase1 (MSK1) and cAMP-responsive element-binding protein (CREB) activation were evaluated by western blot and/or immunocytochemistry. The islet mass was determined from pancreatic sections.
Glucose induced rapid subplasmalemmal recruitment of ERK1 and ERK2. When both ERK1 and ERK2 were inhibited simultaneously, the rapid transient peak of the first phase of glucose-induced insulin secretion was reduced by 40% (p < 0.01), although ERK1 did not appear to be involved in this process. By contrast, ERK1 was required for glucose-induced full activation of several targets involved in beta cell survival; MSK1 and CREB were less active in Erk1 −/− mouse beta cells (p < 0.01) compared with Erk1 +/+ mouse beta cells, and their phosphorylation could only be restored when ERK1 was re-expressed and not when ERK2 was overexpressed. Finally, the islet mass of Erk1 −/− mice was slightly increased in young animals (4-month-old mice) vs Erk1 +/+ mice (section occupied by islets [mean ± SEM]: 0.74% ± 0.03% vs 0.62% ± 0.04%; p < 0.05), while older mice (10 months old) were less prone to age-associated pancreatic peri-insulitis (infiltrated islets [mean ± SEM]: 7.51% ± 1.34% vs 2.03% ± 0.51%; p < 0.001).
ERK1 and ERK2 play specific roles in beta cells. ERK2 cannot always compensate for the lack of ERK1 but the absence of a clear-cut phenotype in Erk1 −/− mice shows that ERK1 is dispensable in normal conditions.
KeywordsAgeing CREB ERK1/2 Insulin secretion MSK1 p90RSK Pancreatic beta cell mass Pancreatic islet biology Peri-insulitis
Free cytosolic Ca2+ concentration
Cyan fluorescent protein
Cyclic AMP-responsive element-binding protein
Extracellular signal-related kinase activity reporter
Extracellular signal-related kinase 1 and 2
Glucose-stimulated insulin secretion
Mitogen-activated protein kinase kinase
Mitogen and stress-activated kinase 1
Protein kinase A
p90 ribosomal S6 kinase
Total internal reflection fluorescence
Yellow fluorescent protein
Insufficient insulin secretion from pancreatic beta cells, often associated with a decrease in beta cell mass, is a characteristic of type 2 diabetes . Glucose-stimulated insulin secretion (GSIS) from beta cells is triggered by an increase in the free cytosolic Ca2+ concentration ([Ca2+]c) [2, 3, 4]. The serine/threonine kinases, extracellular signal-related kinase 1 and 2 (ERK1/2), are activated by glucose in a Ca2+-dependent manner and regulate key transcription factors, insulin gene expression and beta cell survival [5, 6]. Whether they also play a role in GSIS [7, 8, 9, 10] is still under debate [11, 12], mainly because clonal beta cell lines and/or PD98059 (a mitogen-activated protein kinase kinase [MEK]1/2 inhibitor), which display off-target effects on Ca2+ influx , have been used.
In other tissues, ERK1 and ERK2 represent a major signalling pathway for proliferation, differentiation and cell death through the phosphorylation of many substrates [13, 14]. ERK1 and ERK2 share ~85% identity and are the two major members of the ERK family . Whether ERK1 and ERK2 are redundant or play distinct cellular roles is still disputed [15, 16]. Although large functional overlaps between ERK1 and ERK2 exist , discrete phenotypes are observed when they are targeted individually, suggesting that each ERK isoform cannot completely substitute for the other [18, 19, 20], partly because their relative tissue distributions differ . For example, although ERK1 and ERK2 are equally expressed in adipose tissues, ERK1 appears to be specifically involved in adipocyte differentiation  while ERK2 is much more abundant than ERK1 in the liver . ERK1 and ERK2 seem to be equally expressed in pancreatic islets [8, 22] but their individual roles have never been investigated. Nonetheless, Erk1 (also known as Mapk3)-knockout mice (Erk1 −/− mice) are resistant to high-fat-diet-induced obesity and are protected from insulin resistance [20, 23], and ERK1 expression is upregulated in islets isolated from db/+ mice , indicating that ERK1 and ERK2 could be modulated differently during the progression of type 2 diabetes.
Erk2 (also known as Mapk1)-knockout mice (Erk2 −/− mice) are not viable , and we observed that Erk2 depletion in MIN6 cells induced massive beta cell death [8, 22], suggesting that specific deletion of Erk2  in beta cells might not be a suitable model. Therefore, to discriminate between the roles of ERK1 and ERK2, we used Erk1 −/− mice and pharmacological inhibitors of MEK1/2. We revisited the roles of ERK1 and ERK2 in the regulation of GSIS from mouse islets and, for the first time in living beta cells, we monitored the recruitment and activity of ERK1 and ERK2 underneath the plasma membrane, using total internal reflection fluorescence (TIRF) microscopy. We also assessed the contributions of ERK1 and ERK2 to signalling cascades known to be involved in beta cell survival.
All reagents were from Sigma-Aldrich (St Louis, MO, USA), except for U0126 (Calbiochem, La Jolla, CA, USA) and trametinib (Selleckchem, Houston, TX, USA).
C57BL/6J mice were from Charles River (L’Arbresle, France). Erk1 +/− mice were bred to generate Erk1 +/+ vs Erk1 −/− mice . Four-month-old or 10-month-old male mice were used. Studies complied with the animal welfare guidelines of the European Community and complied with the authorisation of the Ministry of Agriculture, France (D34-172-13). Mice were housed in a pathogen-free animal house, with a 12 h light/dark cycle and a temperature of 20°C. Mice had free access to food (4% [kJ] fat diet) and water.
MIN6 cells were originally obtained from H. Ishihara (Tokyo, Japan) and were grown in Dulbecco’s modified Eagle’s medium containing 25 mmol/l glucose and 15% (vol./vol.) FBS and 75 nmol/l mercaptoethanol. See ESM Methods for further details.
Preparations and solutions
Isolated islets from collagenase digestion of the pancreas were used immediately or after overnight culture in RPMI-1640 medium containing 10 mmol/l glucose and 10% (vol./vol.) FBS. Clusters of cells were prepared by dispersion of isolated islets with trypsin and plated on glass coverslips (refractive index n = 1.53) in RPMI-1640 medium. The medium was a bicarbonate-buffered solution containing (in mmol/l): NaCl 120, KCl 4.8, CaCl2 2.5, MgCl2 1.2 and NaHCO3 24. Medium was gassed with O2:CO2 (95:5) to maintain pH 7.4 and was supplemented with 1 mg/ml BSA. The islets/cells were preincubated for 1 h with MEK inhibitors U0126 (Calbiochem,), trametinib (Selleckchem) or PD0325901 (Sigma-Aldrich) prior to the experiments.
Insulin, NAD(P)H and [Ca2+]c measurements
Freshly isolated islets from 10-month-old mice or cultured islets from 4-month-old mice were used. The insulin content of islets or pancreases were determined after extraction in acid–ethanol (see electronic supplementary materials (ESM) Methods for further details). For [Ca2+]c, islets were loaded with 2 μmol/l Fura2-Leak-Resistant-AM (Tef Labs, Austin, TX, USA) for 2 h, placed into a temperature-controlled perifusion chamber and excited every 3 s at 340 nm and 380 nm; the emitted light was recorded at 510 nm. For NAD(P)H, islets were preincubated for 30 min in 1.1 mmol/l glucose and were then transferred to the same experimental setup that was used for [Ca2+]c measurements. The reduced forms of NAD and NADP, (referred as NAD(P)H) were excited at 360 nm every 36 s and the emitted light was recorded at 470 nm.
Vectors and adenovirus generation
Dispersed islet cells were infected for 4 h with adenoviruses. Briefly, ERK activity reporter (EKAR)-CeruleanVenus plasmid (www.addgene.com) was digested and the released fragment was ligated into EcoRV sites of pShuttle-CMV. For ERK1–yellow fluorescent protein (YFP) and/or ERK2–YFP (ATCC, Molsheim France), plasmids were digested and the released fragments were ligated into pShuttle-CMV (www.coloncancer.org/adeasy.htm). Recombination with pAdEasy-1, transfection into HEK 293 cells (ATCC) and viral amplification were performed as described (www.coloncancer.org/adeasy.htm). Probes were preferentially expressed in beta cells compared with non-beta-cells. See ESM Methods for further details.
Histological analysis, immunostaining of pancreas, TUNEL and immunocytochemistry of dispersed cells
Pancreases were fixed with 4% (vol./vol.) formaldehyde, paraffin embedded and longitudinally sectioned through the pancreatic head-to-tail axis (4 μm thickness). Pancreas sections were stained with H&E or were incubated overnight at 4°C with primary antibodies (ESM Table 1), followed by incubation for 1 h with secondary antibodies (ESM Table 1) and DAPI. The percentage of apoptotic islet beta cells (TUNEL- and insulin-positive cells) was determined using the In Situ Cell Death Detection Kit-TMR red (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s instructions. Overnight-cultured dispersed mouse islet cells were preincubated for 2 h in 1.1 mmol/l glucose and then stimulated with 16.7 mmol/l glucose for 10 or 20 min in the presence or absence of MEK inhibitors. Dispersed islet cells were fixed for 20 min (4% formaldehyde; 80 mmol/l NaF for phosphorylated proteins), incubated overnight with primary antibodies and then incubated for 1 h with secondary antibodies and DAPI (see ESM Table 1). For p-ERK1/2, a methanol treatment after paraformaldehyde fixation was required. See ESM Methods for further details. The antibodies used for immunocytochemistry were against insulin, glucagon, Ki-67, p-ERK1/2, phosphorylated mitogen and stress-activated kinase 1 (MSK1), phosphorylated cyclic AMP-responsive element-binding protein (CREB), CD3 and CD8. All antibodies are commercially available (see ESM Table 1) and were validated by companies for immunofluorescence. Results were reproducible over time, using different antibody lots. Experimenters were blinded to group assignment.
A ×100 lens with 1.49 numerical aperture was used. EKAR, a genetically encoded FRET-based sensor that reports ERK1/2 activation, was excited with a 405 nm laser (Roper Scientific, Evry, France) and the emitted light was filtered at 475 nm for cyan fluorescent protein (CFP) and 540 nm for venus, a variant of YFP. YFP was excited with a 491 nm laser (Roper Scientific) and the emitted light was filtered at 540 nm. Images were collected every 10 s with 30 ms exposure using a charge-coupled device camera (Quantem; Roper Scientific) and MetaMorph software (Molecular Devices, Downingtown, PA, USA). Experiments were performed in a 37°C temperature-controlled perifusion chamber. See ESM Methods for further details.
Real-time quantitative RT-PCR
Freshly isolated mouse islets were distributed in batches of 120–150 islets. Total RNAs were extracted and DNase-treated using the RNAeasy microkit (Qiagen, Courtaboeuf, France) and then reverse-transcribed with MoMuLVRT (Life Technology, Paisley, UK), according to the manufacturers’ instructions. To quantify the expression of genes, we designed and validated primer pairs (Qiagen-Operon; see ESM Table 2 for primer sequences) for real-time quantitative PCR, which was performed with Roche LC480 and the Light Cycler 480 SybrGreen Master Mix (Roche Diagnostics). The level of expression of each gene was normalised to the geometric mean of the levels of expression of Hprt and Tubb2a. The genes analysed were Ins2, Ins1, Erk2, Irs2. See ESM Methods for further details.
Western blotting of mouse islet samples
Freshly isolated mouse islets were distributed in batches of 120–150, preincubated for 2 h in bicarbonate buffer with 1.1 mmol/l glucose and then stimulated with 16.7 mmol/l glucose for 20 min, as detailed above. A 2 h preincubation in 1.1 mmol/l glucose did not trigger apoptosis (ESM Fig. 1). At the end of the incubation with glucose, MIN6 cells, mouse islets or dispersed islets cells were washed with ice-cold PBS and lysed in NP40 lysis buffer. The antibodies used for western blotting were against β-actin, p-ERK1/2, phosphorylated p90 ribosomal S6 kinase (p90RSK), p-MSK1, p-CREB, total ERK1/2, total CREB, cleaved caspase 3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). See ESM Methods for further details and ESM Table 1 for antibodies. All antibodies were commercially available (see ESM Table 1) and were validated by companies for western blotting. All antibodies produced bands that were at the expected molecular weights for the targeted proteins. Results were reproducible over time with different antibody lots.
Blood glucose, glucose and insulin tolerance tests, and plasma insulin or glucagon measurements
Blood glucose levels were measured using a OneTouch Verio glucometer (LifeScan Issy les Moulineaux, France). Glucose tolerance tests were performed on overnight-fasted mice. Blood glucose levels were measured before (0 min) and 15, 30, 60, 90, 120 and 150 min after i.p. glucose injection (1.5 g/kg body weight). Insulin tolerance tests were performed on 4 h-fasted mice. Blood glucose levels were measured before (0 min) and 15, 30, 60, 90, 120 and 150 min after i.p. insulin injection (0.75 U/kg body weight) (Umuline; Lilly, Giessen, Germany). Plasma insulin was determined using the Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem, Zaandam, Netherlands). Plasma glucagon was determined using a glucagon radioimmunoassay kit (Millipore, St Charles, MO, USA).
Data are shown as means ± SEM for the indicated number of observations. For kinetic experiments, SEM are indicated only at selected time points. GraphPad Prism 7.2 software (GraphPad, La Jolla, CA, USA) was used. Samples were not randomised. All data obtained during the experiments were analysed. Statistically significant differences between groups were assessed by Student’s t test or by ANOVA. For ANOVA, when the global factor effect was shown to be significant, post hoc tests for multiple comparisons (indicated in figure legends) were performed to obtain adjusted p values. One-way ANOVA was followed by Sidak’s post hoc test when selected pairs of columns were compared, or by Dunnett’s test when columns were compared with a control column. Two-way ANOVA was followed by Tukey’s post hoc testing for multiple comparisons.
ERK1 and ERK2 are recruited underneath the plasma membrane by glucose and ERK activity contributes to the first phase of insulin secretion in mouse islets
To determine whether ERK1/2 are actively recruited underneath the plasma membrane, the fluorescence of EKAR, a genetically encoded FRET-based sensor that reports ERK1/2 activation , was imaged using TIRF microscopy  in living mouse beta cells. EKAR fluorescence intensity was increased by 16.7 mmol/l glucose (p < 0.01), reaching a maximum intensity 9 min after stimulation (Fig. 1g); this response was suppressed by U0126 (Fig. 1g). This indicates that native ERK1/2 phosphorylated EKAR beneath the plasma membrane. Moreover, 16.7 mmol/l glucose induced recruitment of both ERK1 (ERK1–YFP, Fig. 1h) and ERK2 (ERK2–YFP, Fig. 1i) underneath the plasma membrane, reaching maximum recruitment after 9 min of stimulation; this action was blocked by U0126 (Fig. 1h; data not shown for ERK2–YFP) and was absent when YFP was expressed alone (Fig. 1i). Thus, our data suggest that ERK1/2 are rapidly recruited beneath the plasma membrane by glucose and contribute to the first phase of GSIS.
ERK1 is required for glucose-induced full activation of p90RSK, MSK1 and, consequently, CREB, but not for insulin gene expression
Finally, it has been reported that glucose-induced ERK1/2 activation plays an important role in insulin gene transcription through the direct regulation of transcription factors that bind to the insulin gene promoter . Ins1 and Ins2 mRNA levels (Fig. 4d) and the insulin content in size-matched islets (Fig. 4e) were not affected in Erk1 −/− mouse islets. Therefore, ERK1 does not appear to play a major role in the regulation of insulin gene expression.
ERK1 is not required for GSIS
ERK1 contributes to the regulation of the islet mass and to the pancreatic peri-insulitis caused by ageing
In this study, we aimed to determine whether ERK1 and ERK2 have a specific role in mouse beta cells. We report that ERK1 and ERK2 are equally expressed and that the lack of ERK1 was not compensated for by an upregulation (expression and activation) or relocation of ERK2. Moreover, we reveal a crucial role for ERK1 in glucose-induced MSK1, p90RSK and CREB activation in mouse pancreatic islets since ERK2 was unable to fully compensate for the lack of ERK1. Furthermore, because MSK1 and CREB activity was restored when ERK1 was reintroduced in Erk1 −/− mouse beta cells, and not when ERK2 was overexpressed, this indicates a specific role for ERK1.
The expression of Irs2 was reported to be regulated by CREB . Nevertheless, despite a 65% reduction in activation of CREB by glucose, Irs2 gene expression was not affected in Erk1 −/− mouse islets and the islet mass was even slightly increased in 4-month-old mice. CREB has been primarily found to be phosphorylated at S133 by protein kinase A (PKA) and, more recently, by MSK1 and p90RSK [14, 31]. It is important to bear in mind that CREB expression was not altered in Erk1 −/− mouse islets. Therefore, in vivo, CREB can still be activated by stimuli other than glucose that directly activate PKA, such as glucagon-like peptide-1 . Nevertheless, it has been reported that a beta cell-specific CREB ablation in adult mice did not produce a major phenotype in vivo and did not affect Irs2 mRNA level or beta cell mass unless the mice/islets were challenged with high-fat diet or with a cocktail of cytokines . ERK1/2 have been shown to be involved in insulin gene expression , but ERK1 is not specifically required for insulin gene expression, or at least ERK2 activation seems to be sufficient to compensate for the lack of ERK1.
We have reinvestigated the role of ERK1 and ERK2 in GSIS using three different MEK inhibitors and Erk1 −/− mice. Although we cannot rule out the possibility that another protein kinase was also targeted by MEK inhibitors, our study and those of others [12, 26] point to the necessity of carefully investigating whether MEK inhibitors affect changes in metabolism and [Ca2+]c before drawing any conclusions about ERK1/2 signalling pathways. We showed that ERK1 activation and recruitment underneath the plasma membrane only accounts for 25% of the ERK1/2 activation. We have not investigated whether a knockdown of ERK2 expression was sufficient to decrease the first phase of insulin secretion but we report that ERK2 activity is likely sufficient for its full regulation. As possible targets for regulating insulin secretion, focal adhesion kinase (FAK) and paxillin  and Rac1  have been proposed in clonal beta cells. Interestingly, a specific activation of paxillin by ERK2 (and not ERK1) was shown in the lungs . Although ERK1 is actively recruited beneath the plasma membrane by glucose, ERK1 seems dispensable for the regulation of insulin secretion. Synapsin I, which is not involved in the regulation of insulin secretion [39, 40], has been proposed to be a target of ERK1/2 . Interestingly, Synapsin I seems to co-localise with small vesicles such as synaptic-like microvesicles , the release of which is regulated by glucose [41, 42]. The mechanisms leading to ERK1/2 translocation to the plasma membrane are unknown. Different scaffold proteins target ERK1/2 to specific sites , such as Sef1 for Golgi/endoplasmic reticulum or MEK partner 1 (MP-1) for endosomes. The specific anchors at the plasma membrane are kinase suppressor of Ras (KSR)1/2 (in cholesterol-rich domains) and paxillin (in focal adhesion), which are both expressed in beta cells .
Beta cell functions are altered in aged animals . We observed that the natural process of ageing was associated with the development of a moderate pancreatic inflammation, which was less severe than in NOD mice  since it concerned only ~7% of the islets and our mice never developed diabetes. Similar peri-insulitis has been reported by others in aged C57BL/6 J mice . Most importantly, CD3+ T cells and macrophages were also reported in peri-islet regions of aged control human pancreases . This peri-insulitis was significantly decreased in Erk1 −/− mice. Since we have used a global knockout, we think that this reduced peri-insulitis is not specific to beta cells. Indeed, obese leptin-deficient mice lacking ERK1 (ob/ob–Erk1 −/−) display reduced expression of mRNA encoding inflammatory cytokines and T lymphocyte markers in the adipose tissue . Additionally, ERK1 is involved in thymocyte maturation , and both ERK1 and ERK2 are involved in the process of positive selection and affect CD4+ and CD8+ T cell maturation  and macrophage development . The reason why aged Erk1 −/− mice showed better glucose tolerance is unclear but is certainly caused by a combination of several factors, such as a reduced pancreatic inflammation and the tendency of both plasma insulin concentration and insulin sensitivity to be increased.
In conclusion, ERK1 is necessary for glucose-induced full activation of key proteins involved in beta cell survival. Nevertheless, as shown by the absence of a clear-cut phenotype in Erk1 −/− mice, ERK1 is dispensable in normal conditions in living mice. ERK1 has been shown to be involved in the development of insulin resistance  and its role in beta cells indicates that dysregulation of ERK1 activity could play a role in the progression of type 2 diabetes. By contrast, ERK2 activity is likely sufficient for insulin gene expression and the first phase of GSIS. Future studies are required to identify the targets involved. The phenotypic differences observed between ERK1 and ERK2 could reside either in differential substrate specificity or differences in ERK intensity signal. The existence of a viable Erk2 −/− transgenic mouse that overexpresses ERK1 to compensate for the absence of ERK2  should help to determine whether ERK2 is specifically required for the first phase of GSIS and insulin gene expression, or whether it is the global ERK activity that is necessary.
The authors thank L. Ruiz (Institut de Génomique Fonctionnelle, France) and RHEM and MRI facilities (Montpellier, France) for technical assistance, Q. Durrix and E. Gavois (Institut de Génomique Fonctionnelle, France) for technical assistance with animals, C. Jopling (Institut de Génomique Fonctionnelle, France) for editorial assistance and C. Reynes (Faculté des sciences pharmaceutiques et biologiques, Montpellier, France) for statistical assistance.
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
This work was supported by the Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Fondation pour la Recherche Médicale (grant DRM20101220453) and research allocation from SFD-ALFEDIAM.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
ML, JR, SC, DM, VC, JM, AV and GB researched data, contributed to analysis and discussions of data and revised the manuscript. GP and JP provided the mice, contributed to analysis and discussion of data and revised the manuscript. J-FT and SD contributed to analysis and discussion of data and revised the manuscript. MAR contributed to the conception and design of the study, the acquisition, analysis and interpretation of data and to drafting/revising the manuscript, and is the guarantor of this work. All authors approved the final version to be published.
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