Acute nutrient regulation of the unfolded protein response and integrated stress response in cultured rat pancreatic islets
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Inadequate chaperone function relative to client protein load in the endoplasmic reticulum triggers an adaptive unfolded protein response (UPR), including the integrated stress response (ISR), the latter being also activated by other types of stresses. It is well established that pancreatic beta cells, which synthesise and secrete insulin upon nutrient stimulation, are markedly affected by pathological disruption or excessive activation of the UPR. However, whether and how physiological nutrient stimulation affects the beta cell UPR has been little investigated.
Materials and methods
We compared the effects of increasing glucose concentrations and of endoplasmic reticulum Ca2+ emptying with thapsigargin on the UPR (X-box binding protein [Xbp1] mRNA splicing and XBP1/activating transcription factor [ATF] 6-target gene expression) and ISR (eukaryotic translation initiation factor 2A phosphorylation, ATF4 protein levels and target gene expression) in isolated rat islets.
Thapsigargin strongly increased both UPR and ISR. In comparison, glucose moderately increased the UPR between 5 and 30 mmol/l, but exerted complex effects on the ISR as follows: (1) marked reduction between 2 and 10 mmol/l; (2) moderate increase parallel to the UPR between 10 and 30 mmol/l. These glucose effects occurred within 2 h, were mimicked by other metabolic substrates, but were independent of changes in Ca2+ influx or insulin secretion. Remarkably, attenuating the glucose stimulation of protein synthesis with a low concentration of cycloheximide prevented UPR activation but not ISR reduction by high glucose.
Nutrient stimulation acutely activates rat islet UPR in a manner dependent on protein synthesis, while exerting complex effects on the ISR. These effects may contribute to nutrient-induced maintenance of the beta cell phenotype.
KeywordsBeta cell Endoplasmic reticulum stress Gene expression Integrated stress response α-Ketoisocaproate Nutrient stimulation Pancreatic islet Protein synthesis Succinic acid Unfolded protein response
activating transcription factor
immunoglobulin heavy chain binding protein
ER-degradation enhancer mannosidase alpha-like 1
eukaryotic translation initiation factor 2A
10 mmol/l glucose (G2, G5, G30 for 2, 5, 30 mmol/l glucose)
growth arrest and DNA damage
glucose regulated protein
integrated stress response
succinic acid monomethyl ester
pancreatic ER kinase
unfolded protein response
X-box binding protein
Folding of proteins destined for the secretory pathway in the endoplasmic reticulum (ER) critically depends on adequate production and function of ER chaperones such as immunoglobulin heavy chain binding protein (BiP; now known as heat shock 70 kDa protein 5 [glucose-regulated protein] [HSPA5]), glucose-regulated protein (GRP) 94 (now known as heat shock protein 90 kDa beta, member 1 [HSP90B1]), calnexin, calreticulin and protein disulphide isomerase . When ER chaperone function decreases or ER client protein load increases (following ER Ca2+ emptying, ATP depletion, increased protein synthesis, etc.), the unfolded protein response (UPR) is activated. This transiently reduces protein synthesis, improves the cell capacity to fold proteins and degrade unfolded proteins and increases cellular defences against various types of stresses [2, 3, 4]. This response, which is controlled by the ER stress sensors inositol requiring (IRE) 1, activating transcription factor (ATF) 6 and pancreatic ER kinase (PERK; now known as EIF2A kinase 3 [EIF2AK3]), consists of two branches. The first branch results from the unconventional splicing of X-box binding protein 1 (Xbp1) pre-mRNA by IRE1, with a subsequent increase in active XBP1, and from the proteolytic activation of ATF6. Both transcription factors contribute to increase the production of ER chaperones such as BiP and GRP94, as well as of components of the ER-associated degradation pathway like ER degradation-enhancer mannosidase alpha-like 1 (EDEM) . The second branch results from phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α; now known as EIF2A) on Ser51 by PERK, followed by a general translational pause with selective increase in Atf4 mRNA translation and subsequent stimulation of expression of ATF4-target genes like Atf3, growth arrest and DNA damage (Gadd)153 (now known as DNA-damage-inducible transcript 3 [Ddit3], also known as Chop), Gadd34 (now known as protein phosphatase 1, regulatory [inhibitor] subunit 15A [Ppp1r15a]) and BiP . Because eIF2α-Ser51 can also be phosphorylated by other kinases (EIF2AK1, 2 and 4) independently of UPR activation, events downstream from eIF2α-Ser51 phosphorylation (the shared branch of the UPR as defined by Ma et al. ) will be referred to here as the integrated stress response (ISR) regardless of the kinase involved [7, 8, 9].
Pancreatic beta cells, which synthesise and secrete insulin in a manner dependent on glucose concentration , produce high levels of several UPR and ISR components. Surprisingly, both genetic disruption and excessive activation of the UPR and ISR induce beta cell apoptosis and type 1 diabetes [11, 12, 13, 14] (recently reviewed in ), suggesting that survival and function of beta cells critically depend on a continuous physiological activation of the UPR to cope with their high rate of proinsulin synthesis. More recently, it was also demonstrated that prolonged exposure to palmitate or high glucose concentrations triggers an ER stress response in insulin-secreting INS1 cells [15, 16, 17], suggesting that ER stress may play a role in beta cell gluco-lipotoxicity in type 2 diabetes.
Glucose is the main physiological regulator of protein synthesis in beta cells. As such, it can be expected to activate the UPR and ISR to compensate for an increase in ER client protein load. In support of that hypothesis, it was recently demonstrated that glucose acutely induces IRE1α phosphorylation and UPR activation in isolated mouse islets . However, these effects were not accompanied by Xbp1 mRNA splicing, a hallmark of UPR activation . In contrast, it has recently been shown that glucose rapidly decreases eIF2α phosphorylation and the ISR in MIN6 cells [20, 21]. To investigate whether and to what extent physiological nutrient stimulation affects the UPR in normal beta cells, we tested the effects of increasing glucose concentrations and of other metabolic substrates on the UPR and ISR in cultured rat islets, and compared these effects to those of the specific inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases, thapsigargin, which is a potent inducer of ER stress in many cell types .
Materials and methods
Thapsigargin, cycloheximide, succinic acid monomethyl ester (SAM), α-ketoisocaproate (KIC), l-glutamine and diazoxide were purchased from Sigma (St Louis, MO, USA). l-Leucine was from Calbiochem (San Diego, CA, USA).
Rabbit polyclonal anti-GADD153 (sc-575), anti-ATF4 (sc-200) and anti-XBP1 (sc-7160) antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Mouse anti-BiP antibody (clone 40) was from BD Biosciences (San Diego, CA, USA). Rabbit polyclonal anti-ACTIN antibody was from Sigma. Mouse monoclonal anti-total-eIF2α antibody was provided by D. Scheuner (Howard Hughes Medical Institute, University of Michigan, MI, USA). Rabbit anti-phospho-eIF2α antibodies were generated as described previously .
Islet isolation and culture
Islets were isolated from the pancreas of ∼200 g male Wistar rats (local animal facility), as described previously . They were precultured for 1 week at 37°C, in the presence of 5% CO2, in serum-free RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10 mmol/l glucose (G10), 5 g/l BSA fraction V (Roche, Basel, Switzerland), 100 IU/ml penicillin and 100 μg/ml streptomycin. Large islets (diameter >150 μm) were discarded. After preculture, islets were further cultured in medium containing 2–30 mmol/l glucose (G2, G5, G10 or G30) and various test substances, then collected for further analysis. All experiments were conducted in accord with accepted standards of humane animal care and were approved by the local Institutional Committee on Animal Experimentation.
RNA extraction and cDNA synthesis
Islet total RNA was extracted and reverse-transcribed in cDNA as described .
Measurement of Xbp1 mRNA splicing
cDNAs corresponding to the unspliced and spliced forms of Xbp1 mRNA (NM_001004210) were amplified simultaneously with TATA-box binding protein (Tbp) cDNA by duplex radioactive PCR [19, 22] (see Electronic supplementary material [ESM] Table 1). The spliced:total Xbp1 mRNA ratio was calculated as an indicator of Xbp1 mRNA splicing.
Real-time fluorescent PCR
Real-time PCR was performed as described elsewhere  (ESM Table 2). Within each PCR, sample threshold cycles were compared with a standard curve prepared by serial fourfold dilutions of an appropriate control cDNA. Relative changes in gene mRNA levels were expressed relative to the mRNA levels in control islets and normalised for relative changes in Tbp mRNA levels.
After rinsing with ice-cold PBS, islets were precipitated with trichloracetic acid and their proteins were solubilised in Laemmli buffer. For eIF2α phosphorylation measurements, islet protein extracts were prepared as described previously . Proteins were separated by 8–12% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. The membrane was blocked (5% non-fat dry milk in Tris-buffered saline [pH 7.4] containing 0.05% Tween-20), incubated overnight with the primary antibody, washed, incubated for 1 h with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody and washed before detection of bound antibodies by enhanced chemiluminescence. Band intensities were quantified by scanning densitometry and normalised to Rouge Ponceau staining intensity.
Islets were washed, fixed in 1% formaldehyde and embedded in paraffin. Then, GADD153 was detected on 5 μm thick sections using anti-GADD153 antibody diluted 1:800 and the procedure described previously for haem oxygenase 1 immunodetection .
Measurement of protein biosynthesis
Islet protein synthesis was estimated from the incorporation of l-[3,5-3H]tyrosine (Amersham Biosciences, Bucks, UK) in total islet protein extracts during culture, as previously described .
Insulin secretion measurements
Insulin concentrations in the culture media were determined by RIA using rat insulin as a standard . The amount of insulin secreted during the whole culture period was expressed in pmol islet−1 h−1.
The results are presented as means ± SEM for the indicated number of experiments. In all cases, gene mRNA and protein levels were expressed relative to the level measured in the group of islets that had been maintained under the same condition as in the last 24 h of preculture (most frequently G10).
Most experiments were carried out in islets precultured for 1 week in serum-free RPMI medium containing G10 and 5 g/l BSA. These islets, which have recovered from the stress of isolation , have a well-preserved morphology and beta cell glucose responsiveness with low level of beta cell apoptosis , in marked contrast with islets cultured for several days in G5 . They express total Xbp1, BiP and Grp94 mRNA at much higher levels than Tbp, and Edem, Atf3, Gadd153 and Gadd34 mRNA at similar or lower levels (ESM Table 2).
Effects of thapsigargin and increasing glucose concentrations on the UPR and the ISR in isolated islets
We next tested the effects of a decrease or increase in glucose concentration. As shown in Fig. 1, 18 h culture in G2–G5 vs G10 resulted in a significant decrease in insulin secretion and Xbp1 mRNA splicing, the latter being always significantly higher than zero (p < 0.0001 by one sample t test). There were no changes in total Xbp1, Grp94, Edem and Tbp, and a strong increase in Atf3, Gadd153 and Gadd34 mRNA levels. The latter effect only partly resulted from a ∼2.3-fold increase in Atf3 and Gadd153 mRNA half-life in G2 vs G10 (ESM Fig. 1). Under these conditions, XBP1 protein levels decreased in G5 vs G10 while BiP and GADD153 protein levels increased in both G5 and G2 (Fig. 2). In G2, GADD153 increased in most islet cell nuclei (Fig. 2d). Clear increases in BiP mRNA and ATF4 protein levels were only observed in G2 vs G10 (Figs. 1h and 2a,c). Thus, lowering glucose from G10 to G5 and G2 attenuates the UPR while increasing mRNA of genes involved in ISR and corresponding protein levels.
In contrast with culture in low glucose, 18 h culture in G30 vs G10 induced a significant increase in insulin secretion, Xbp1 mRNA splicing and XBP1 protein levels without changes in total Xbp1 and Tbp mRNA levels (Fig. 1b,d,f,h,j, Fig. 2a,b). High glucose also induced a small similar increase in BiP, Grp94, Edem, Atf3, Gadd153 and Gadd34 mRNA levels (Fig. 1h,j). Under these conditions, culture in G30 vs G10 significantly increased BiP, but neither ATF4 nor GADD153 protein levels (Fig. 2b,c). When the islets were cultured for 1 week in G10 or G30 immediately after isolation, the spliced : total Xbp1 mRNA ratio was also significantly increased (from 0.18 ± 0.03 in G10 to 0.24 ± 0.02 in G30; n = 7; p < 0.05 by unpaired t test). Thus, increasing glucose from G10 to G30 for 18 h up to 1 week moderately activates the UPR and increases mRNA levels of genes involved in ISR in cultured rat islets.
Altogether, these results suggest that, in contrast to thapsigargin, which strongly increases both UPR and ISR, glucose moderately increases the UPR between G5 and G30 but exerts complex effects on the ISR, namely a marked reduction from G2 to G10, and a moderate increase parallel to the UPR from G10 to G30. The effects of glucose on Xbp1 mRNA splicing and Gadd153 mRNA levels were similar in medium containing 10% FCS instead of BSA (data not shown).
Time-dependence of the effects of glucose on the UPR
Time-dependence of the effects of glucose on the ISR
Role of mitochondrial metabolism, Ca2+ influx and insulin secretion
Effects of mitochondrial metabolic substrates and diazoxide on insulin secretion during culture, Xbp1 mRNA splicing and Gadd153 mRNA expression in cultured rat islets
Insulin secretion (pmol islet−1 h−1)
Spliced:total Xbp1 mRNA ratio
Gadd153:Tbp mRNA ratio (relative to G10)
0.045 ± 0.007
0.10 ± 0.01
8.46 ± 1.16
G5 + Leu-Gln
0.356 ± 0.046*
0.15 ± 0.01*
2.72 ± 0.19*
G5 + SAM
0.401 ± 0.107*
0.21 ± 0.03*
1.20 ± 0.17*
G5 + KIC
0.455 ± 0.084*
0.19 ± 0.01*
1.51 ± 0.13*
0.350 ± 0.069*
0.20 ± 0.01*
1.00 ± 0.09*
G10 + DZ
0.067 ± 0.007**
0.21 ± 0.01
0.62 ± 0.04
0.582 ± 0.059*
0.30 ± 0.01*
1.86 ± 0.22*
G30 + DZ
0.250 ± 0.034**
0.33 ± 0.03*
1.47 ± 0.23
To evaluate the role of plasma membrane depolarisation, stimulation of Ca2+ influx and insulin secretion, we tested the effects of diazoxide, a drug that reduces Ca2+ influx and insulin secretion by opening ATP-dependent K+ channels . As expected, addition of diazoxide to G10 and G30 significantly reduced insulin secretion. However, it did not alter Xbp1 mRNA splicing or Gadd153 mRNA levels (Table 1). Similar results were obtained when insulin secretion was inhibited by clonidine, an α2-adrenergic agonist that inhibits cAMP production  and uncouples exocytosis from the rise in cytosolic Ca2+ concentration  (data not shown). Thus, glucose activates the UPR and reduces the ISR independently of its effects on beta cell membrane potential, Ca2+ influx and insulin secretion.
Role of protein synthesis
Our results slightly contrast with a recent study showing that glucose increases IRE1α phosphorylation and expression of genes involved in UPR without increasing Xbp1 mRNA splicing in isolated mouse islets . This discrepancy is unlikely to be due to species differences, because glucose also increases Xbp1 mRNA splicing in mouse islets under our culture conditions (unpublished observations, M. Bensellam). It may rather result from the use of freshly isolated vs precultured islets, since Xbp1 mRNA splicing was significantly higher in fresh vs cultured rat islets (spliced : total Xbp1 mRNA ratio 0.55 ± 0.02 in fresh islets vs 0.20 ± 0.01 in islets precultured for 1 week; n = 21–28; p < 0.0001 by unpaired t test). This high level of Xbp1 mRNA splicing, which may result from the stress of isolation, was observed regardless of the pre-isolation condition, thereby preventing us from studying the impact of in vivo hyperglycaemia on UPR activation.
What could be the physiological role of nutrient-activation of the UPR in islet beta cells? Its rapid onset upon glucose stimulation and fast reduction upon lowering of glucose concentration suggest that meal-related fluctuations in plasma nutrient concentration may induce synchronous fluctuations of Xbp1 mRNA splicing and other early steps of the UPR in beta cells. By adapting the ER chaperone to client protein ratio and the ER-associated degradation capacity to physiological variations in protein synthesis, temporal integration of these UPR fluctuations could contribute to the plasticity of the beta cell phenotype . Our hypothesis may seem at variance with the observation, by electron microscopy, of normal beta cells in Xbp1 knockout mice with a liver-specific rescue of Xbp1 expression . However, the argument is not sufficient to exclude a role for XBP1 in beta cell plasticity for several reasons. First, because apolipoprotein E mRNA is expressed in rat islets (unpublished observations, M. Bensellam), expression of Xbp1 under the apolipoprotein E promoter may have corrected XBP1 deficiency not only in the liver but also in beta cells. Second, beta cell function was not evaluated in that study, leaving open the possibility that beta cells were defective in those mice despite their normal morphology. Third, it is possible that XBP1 is not important in the early post-natal period, but becomes so later in life, when beta cells are more responsive to nutrient stimulation.
Although we had expected the ISR to be activated by nutrients in parallel with Xbp1 mRNA splicing, as after thapsigargin treatment, we unexpectedly observed a partial dissociation between both responses. Thus, the UPR was low in G2–G5 and concentration-dependently activated by G10 and G30, whereas the ISR was high in G2, minimal (though not zero) in G10 and slightly increased in G30, as shown by changes in eIF2α phosphorylation, ATF4 protein levels and ATF4-target gene mRNA levels. It has also been recently shown in MIN6 cells that low glucose strongly induces the ISR in beta cells . As there were no detectable increases in eIF2α phosphorylation and ATF4 production in G5 vs G10, the rise in ATF4-target gene mRNA levels in G5 may have resulted from their transcriptional activation by a mechanism different from the ISR and from their concomitant stabilisation. However, our interpretation that the ISR is activated in G5 is supported by the observations that eIF2α phosphorylation rapidly decreases upon stimulation from G5 to G30 in rat islets (see Fig. 4d) and within the whole physiological range of glucose concentrations in MIN6 cells [20, 37].
It was not our primary aim to investigate the mechanism of ISR activation in low glucose. Nevertheless, our results clearly demonstrate that ISR activation can be prevented or acutely reduced by stimulation with glucose (and SAM, KIC and leucine + glutamine) independently of the stimulation of Ca2+ influx, insulin secretion and protein synthesis. It could result either from the activation of any eIF2α kinase, among which PERK and EIF2AK4 are the most likely candidates , or by the inactivation of protein phosphatase 1, mediated by inhibitor-1, which is highly expressed in beta cells . In other cell types, complete glucose deprivation is known to induce the UPR, an effect generally attributed to ER Ca2+ emptying and reduced ER chaperone function at very low ATP concentrations [1, 3]. However, the situation was clearly different in G2–G5, where the islet ATP:ADP ratio is only moderately reduced as compared with G10 [27, 38] and Xbp1 mRNA splicing was not increased.
Although we did not fully investigate the mechanisms behind the asymmetric V-shaped glucose-concentration response curve for changes in expression of genes involved in islet ISR, our results allow us to propose the following model. On the one hand, the ISR is activated in parallel with nutrient activation of the classical UPR (pathway 1, Fig. 6b), as shown by the parallel increases in Xbp1 mRNA splicing and in mRNA levels of genes involved in UPR and ISR between G10 and G30. This ISR activation is very low, as neither eIF2α phosphorylation nor an increase in ATF4 or GADD153 protein expression could be detected in G30 vs G10. The ∼1.6-fold increase in ATF4-target gene mRNA levels in G30 vs G10 is, however, compatible with our estimation (based on changes in Xbp1 mRNA splicing) that the maximal glucose activation of the UPR is less than that produced by 10−7 mol/l thapsigargin. On the other hand, low glucose concentrations (G2 and G5 vs G10) rapidly increase mRNA levels of genes involved in ISR by inducing their transcription and, to some extent, increasing their mRNA stability (pathway 2, Fig. 6b). The reduction in mRNA of genes involved in ISR and in corresponding protein levels between G5 and G10, despite concurrent activation of the UPR, therefore indicates that, within that physiological range of glucose concentrations, pathway 2 predominates over pathway 1 for regulation of the ISR (Fig. 6b).
What could be the role of the asymmetric V-shaped glucose concentration-dependent changes in ISR in islet beta cells? In the short term, changes in eIF2α phosphorylation may play a role in the nutrient regulation of protein synthesis [12, 14, 20, 37]. This hypothesis is compatible with our finding of an inverse correlation between glucose-induced changes in mRNA levels of genes involved in islet ISR and total protein synthesis after 18 h culture. It is strongly supported by the observation that inhibiting glucose-induced eIF2α de-phosphorylation markedly reduced the acute glucose stimulation of protein synthesis in MIN6 cells . In the long term, beta cell apoptosis after 2–7 days of culture also displayed a V-shaped glucose concentration-dependent curve [27, 28, 39, 40]. We, in fact, confirmed, by measuring caspase activation, that islet cell apoptosis is minimal after 1 week of culture in G10 and significantly increased by G2–G5 and, to a lesser extent, by G30 (ESM Fig. 3). The rather good correlation between rapid ISR activation and slower induction of apoptosis reinforces the hypothesis of a causal link between both events under various conditions [13, 41]. Thus, although the activation of islet ISR and apoptosis by G30 vs G10 was rather small in comparison to that induced by low glucose, the sustained activation of the islet UPR by G30 is compatible with recent studies indicating that prolonged exposure to high glucose or high NEFA concentrations induces an ER stress response that may contribute to the slow deterioration of beta cell function and survival in type 2 diabetes [15, 16, 17, 42, 43]. In this context, it will be crucial to clarify whether ISR activation contributes to beta cell apoptosis [13, 41, 44], protects against it [45, 46] or is just a marker of beta cell exposure to pro-apoptotic stimuli.
In conclusion, our results demonstrate that nutrient stimulation of rat islets moderately activates the classical UPR in a manner dependent on protein synthesis and inhibits the strongly increased expression, induced by low glucose concentrations, of genes involved in ISR. Both effects lead to an asymmetric V-shaped glucose-concentration response curve for changes in islet ISR. The kinetics of these effects suggests their possible role in nutrient-induced changes of the beta cell phenotype under both physiological and pathological conditions.
The authors thank A. Dannau and F. Knockaert for expert technical help and D. Scheuner (University of Michigan, Ann Arbor, MI, USA) for supply of the eIF2α antiserum. This work was made possible by a grant from the European Foundation for the Study of Diabetes/MSD Research Partnership for European Studies on beta cell function and survival. It was supported by Grant 1.5.182.04 from the Fonds de la Recherche Scientifique (Scientific Research Foundation; FRS-FNRS, Belgium), Grants 3.4616.05 and 3.4506.05 from the Fonds de la Recherche Scientifique Médicale (Medical Research Foundation, Belgium), the Interuniversity Poles of Attraction Program (P5/17)-Belgian Science Policy, Grant ARC 05/10-328 from the General Direction of Scientific Research of the French Community of Belgium and Grant (GOA/2004/11) from the Catholic University of Leuven (to F. C. Schuit). M. Bensellam is Research Fellow and J. C. Jonas is Senior Research Associate of the FRS-FNRS (Belgium).
Duality of interest
The authors have no duality of interest to disclose.