IKKβ inhibition prevents fat-induced beta cell dysfunction in vitro and in vivo in rodents
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We have previously shown that oxidative stress plays a causal role in beta cell dysfunction induced by fat. Here, we address whether the proinflammatory kinase inhibitor of (nuclear factor) κB kinase β (IKKβ), which is activated by oxidative stress, is also implicated.
Fat (oleate or olive oil) was infused intravenously in Wistar rats for 48 h with or without the IKKβ inhibitor salicylate. Thereafter, beta cell function was evaluated in vivo using hyperglycaemic clamps or ex vivo in islets isolated from fat-treated rats. We also exposed rat islets to oleate in culture, with or without salicylate and 4(2′-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; BMS-345541 (BMS, another inhibitor of IKKβ) and evaluated beta cell function in vitro. Furthermore, oleate was infused in mice treated with BMS and in beta cell-specific Ikkb-null mice.
48 h infusion of fat impaired beta-cell function in vivo, assessed using the disposition index (DI), in rats (saline: 1.41 ± 0.13; oleate: 0.95 ± 0.11; olive oil [OLO]: 0.87 ± 0.15; p < 0.01 for both fats vs saline) and in mice (saline: 2.51 ± 0.39; oleate: 1.20 ± 0.19; p < 0.01 vs saline) and ex vivo (i.e., insulin secretion, units are pmol insulin islet−1 h−1) in rat islets (saline: 1.51 ± 0.13; oleate: 1.03 ± 0.10; OLO: 0.91 ± 0.13; p < 0.001 for both fats vs saline) and the dysfunction was prevented by co-infusion of salicylate in rats (oleate + salicylate: 1.30 ± 0.09; OLO + salicylate: 1.33 ± 0.23) or BMS in mice (oleate + BMS: 2.25 ± 0.42) in vivo and by salicylate in rat islets ex vivo (oleate + salicylate: 1.74 ± 0.31; OLO + salicylate: 1.54 ± 0.29). In cultured islets, 48 h exposure to oleate impaired beta-cell function ([in pmol insulin islet−1 h−1] control: 0.66 ± 0.12; oleate: 0.23 ± 0.03; p < 0.01 vs saline), an effect prevented by both inhibitors (oleate + salicylate: 0.98 ± 0.08; oleate + BMS: 0.50 ± 0.02). Genetic inhibition of IKKβ also prevented fat-induced beta-cell dysfunction ex vivo ([in pmol insulin islet−1 h−1] control saline: 0.16 ± 0.02; control oleate: 0.10 ± 0.02; knockout oleate: 0.17 ± 0.04; p < 0.05 control saline vs. control oleate) and in vivo (DI: control saline: 3.86 ± 0.40; control oleate: 1.95 ± 0.29; knockout oleate: 2.96 ± 0.24; p < 0.01 control saline vs control oleate).
Our results demonstrate a causal role for IKKβ in fat-induced beta cell dysfunction in vitro, ex vivo and in vivo.
KeywordsBeta cell dysfunction IKKβ In vivo Inflammation Lipotoxicity Oleate Olive oil Oxidative stress
5′ Adenosine monophosphate-activated protein kinase
Glucose infusion rate
Inhibitor of (nuclear factor) κB α
Inhibitor of (nuclear factor) κB kinase β
- IkkbΔbeta cell
Beta cell-specific IKK beta-deficient (mouse model)
Inducible nitric oxide synthase
IL-1 receptor antagonist
Monocyte chemoattractant protein 1
Insulin sensitivity index (glucose metabolism [M]/plasma insulin [I])
Rat insulin 2 promoter
Reactive oxygen species
NEFA have been shown to both stimulate and impair insulin secretion depending on the duration of beta cell exposure . Prolonged (>24 h) exposure is inhibitory and involves oxidative stress, endoplasmic reticulum stress and inflammation .
Lipotoxic effects on the beta cell, i.e. the decrease in beta cell function and mass induced by chronically elevated NEFA, play a role in the pathogenesis of type 2 diabetes, at least in predisposed individuals . Previously, we demonstrated that oxidative stress mediates fat-induced beta cell dysfunction in vivo in rats  and in humans . Oxidative stress activates inhibitor of (nuclear factor) κB kinase β (IKKβ), which, by phosphorylating the inhibitor of (nuclear factor) κB α (IκBα), activates the transcription factor NFκB. The effect of IKKβ/NFκB on beta cell function is controversial. NFκB is important for cell survival and there are reports that NFκB is beneficial for glucose-stimulated insulin secretion , unless activated by cytokines . However, IKKβ which induces serine phosphorylation of IRSs  in addition to activating NFκB, decreases beta cell function . It is also controversial whether fat activates IKKβ/NFκB in beta cells: although fatty acids did not activate NFκB in INS-1 or primary rat beta cells in one study , lipotoxicity was associated with NFκB activation, and palmitate-induced apoptosis was inhibited by an IKKβ inhibitor in INS-1 beta cells in another study . The in vivo effect of fat on beta cell IKKβ/NFκB has not been investigated previously.
To address the role of IKKβ in fat-induced beta cell dysfunction in vivo, rats were infused i.v. with fat to elevate plasma NEFA 50–100% (elevation seen in obesity ) with or without the IKKβ inhibitor  salicylate for 48 h. Monounsaturated fat (oleate or olive oil) was infused, as in our previous study showing a role of oxidative stress in beta cell dysfunction . Although oleate has been found to protect beta cells against palmitate-induced toxicity in in vitro studies , the prolonged effect of oleate by itself on beta cell function is mainly inhibitory [2, 13]. After 48 h infusion, beta cell function was evaluated in vivo using hyperglycaemic clamps, or ex vivo in isolated islets. We also used the specific IKKβ inhibitor 4(2′-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; BMS-345541 (BMS)  in hyperglycaemic clamps performed after 48 h oleate infusion in mice. In addition, we exposed rat islets for 48 h to oleate with or without salicylate or BMS in vitro. Last, we performed oleate infusion in beta cell-specific IKKβ-deficient (Ikkb Δbeta cell) mice, followed by evaluation of beta cell function in vivo using hyperglycaemic clamps or ex vivo in isolated islets. In all these models, IKKβ inhibition prevented fat-induced beta cell dysfunction.
All procedures were approved by the Animal Care Committee of the University of Toronto and conducted according to the Canadian Council on Animal Care Guidelines and the appropriate sample sizes were determined prior to the studies (see also Electronic supplementary material [ESM]). Female Wistar rats (250–300 g, corresponding to 9–11 weeks of age, Charles River, Senneville, QC, Canada) were used, as in our previous studies [2, 15]. Female C57BL/6 mice (22–25 g, corresponding to 14–16 weeks of age, Jackson Laboratory, Bar Harbor, ME, USA) and male RIP2 (rat insulin 2 promoter)-Cre recombinase (Cre) positive or negative Ikkb floxed mice were also used . Female wild-type C57BL/6 mice underwent the BMS infusion studies. For studies in beta cell-specific Ikkb-null mice, Ikkb F/F mice on a C57BL/6 background were crossed with RIP2-Cre mice (also on a C57BL/6 background) obtained from Jackson Laboratory to generate RIP2-Cre positive or RIP2-negative Ikkb F/+ mice, which were interbred to generate RIP2-Cre−:Ikkb F/F (floxed controls) and RIP2-Cre+:Ikkb F/F (Ikkb Δbeta cell) mice. These mice were used for experiments at 11–13 weeks of age. Cre-mediated recombination was confirmed by PCR .
Surgery and i.v. infusion
Surgery was performed under general anaesthesia (isoflurane, to effect). Carotid artery and jugular vein cannulation of rats has been described previously [2, 15]. After 3 days’ post-surgical recovery, the rats were randomised to the following 48 h infusions: (1) NaCl (154 mmol/l; 5 μl/min) as control; (2) fat, either oleate (1.3 μmol/min) prepared as in our previous studies [2, 15] or 20% (vol./vol.) olive oil (OLO; 5.5 μl/min) prepared as in Dobbins et al ; (3) oleate or OLO + salicylate (0.7 μmol kg−1 min−1, the dose that reversed insulin resistance in Park et al ); or (4) salicylate only. Olive oil is a triacylglycerol mixture containing 75% oleate, and 16% saturated fat. Heparin was added to olive oil to a final concentration of 50 U/ml to activate lipoprotein lipase, which releases NEFA and glycerol from the triacylglycerol mixture of olive oil. We have shown that BSA, the vehicle for oleate, has no effect on insulin secretion [15, 20]; heparin  and glycerol  also have no effect. A two-step hyperglycaemic clamp or islet isolation was performed after the infusion period.
Mouse jugular vein cannulation is described in Koulajian et al . In mice, oleate (0.4 μmol/min)/equivolume saline was infused for 48 h , starting 4–5 days after surgery with or without BMS (0.12 mmol kg−1 day−1) . After 48 h infusion, mice received hyperglycaemic clamps. Sampling was through the tail vein.
Insulin secretion was determined by measuring plasma insulin and C-peptide during a two-step (13 and 22 mmol/l) hyperglycaemic clamp in rats  and a one-step (22 mmol/l) hyperglycaemic clamp in mice . Insulin sensitivity (M/I, where M is glucose metabolism and I is plasma insulin) and beta cell function (disposition index [DI]; units are μmol kg−1 min−1 glucose/pmol insulin × nmol C-peptide) were assessed as described in the ESM.
Insulin sensitivity was determined using the gold-standard technique, i.e. hyperinsulinaemic–euglycaemic clamp .
Ex vivo studies
Islets of the in vivo infused rats and Ikkb Δbeta cell mice were isolated and insulin secretion studies were performed as described previously . ELISA for phosphorylated IκBα and active nuclear NFκB (Active Motif, Carlsbad, CA, USA), reactive oxygen species (ROS) measurements (see ESM) and RT-PCR were also performed in rat islets. Total rat islet RNA was prepared as described previously [23, 24] according to the manufacturer’s instructions (Qiagen, Hombrechtikon, Switzerland), and was reverse transcribed using random hexamers. Mouse primers for Il1b, Tnfa (also known as Tnf), monocyte chemoattractant protein 1 (Mcp1; also known as Ccl2), IL-1 receptor antagonist (Il1ra; also known as Il1rn), Tgfb (also known as Tgfb1), the macrophage marker Cd68, and cyclooxygenase (Cox)2 (also known as Ptgs2) (see ESM Table 1) from Applied Biosystems (Foster City, CA, USA) were used and quantitative PCR was done with a fluorescein amidite (FAM)-based reference dye using commercial TaqMan gene expression assays and the 7500 Fast Real-Time PCR System according to the manufacturer’s protocol (Applied Biosystems). Changes in mRNA expression were calculated using difference in Ct values, and normalised to the housekeeping gene 18S [23, 24].
Studies in cultured islets
Rat islets were cultured for 48 h in RPMI 1640 without antioxidants, containing 0.4 mmol/l oleate in 0.5% NEFA-free BSA with or without 0.25 mmol/l salicylate  or 3 μmol/l BMS, a dose based on pilot dose–response studies. Islets were also cultured in control/oleate media with or without the COX-2 inhibitor SC-236 (10 μmol/l, based on Castaño et al ) or the COX-1 inhibitor SC-560 (100 μmol/l, based on Smith et al ). Thereafter, glucose-stimulated insulin secretion was assessed. Prostaglandin E2 (PGE2) in the medium was measured via ELISA (Enzo Life Sciences, Farmingdale, NY, USA).
Western blots were performed as previously described  using antibody against phospho-Ser307-IRS-1 (Millipore, Billerica, MA, USA, 07-247, raised in rabbit, RRID:AB_310463, 1:500), total IKKβ (Cell Signaling Technology, Danvers, MA, USA, 2370, raised in rabbit, RRID:AB_2122154, 1:100), phospho-Thr172-5′ adenosine monophosphate-activated protein kinase (AMPK)α (Cell Signaling Technology, 2535, raised in rabbit, RRID:AB_331250, 1:1000), total AMPKα (Cell Signaling Technology, 2793, raised in mouse, RRID:AB_915794, 1:1000), total IκBα (Santa Cruz Biotechnology, Dallas, TX, USA, sc-371, raised in rabbit, RRID:AB_2235952, 1:250), α-actinin (Cell Signaling Technology, 3134S, raised in rabbit, RRID:AB_2223798, 1:1000), β-actin (Abcam, Cambridge, UK, ab6276, RRID:AB_2223210, raised in mouse, 1:10,000) or γ-tubulin (Sigma-Aldrich, St Louis, MO, USA, T6557, RRID:AB_477584, raised in mouse, 1:1000) (see also ESM). The antibodies had been validated in previous studies carried out in the laboratory (see ESM).
Plasma glucose in rats was measured on a Beckman Analyser II (Beckman, Fullerton, CA, USA) and in mice was measured on a HemoCue Glucose 201 Analyser (HemoCue, Brea, CA, USA). Plasma NEFA were measured with an enzymatic colorimetric kit (Wako Industries, Neuss, Germany). Radioimmunoassays specific for rat/mouse insulin and C-peptide (Linco, St Charles, MO, USA) were used to determine their plasma concentrations. Plasma triacylglycerol levels were measured by a colorimetric kit (Boehringer Mannheim, Laval, QC, Canada).
Data are means ± SEM. One-way non-parametric ANOVA for repeated measurements followed by Tukey’s test was used to compare treatments. Calculations were performed using SAS version 8.0 (Cary, NC, USA).
Hyperglycaemic clamp in rats
Plasma NEFA, triacylglycerol, glucose and insulin levels after 48 h infusions
Saline (n = 12)
0.693 ± 0.026
0.17 ± 0.019
5.8 ± 0.2
79 ± 12
Oleate (n = 10)
1.050 ± 0.118**
0.21 ± 0.014
5.9 ± 0.1
72 ± 13
OLO (n = 7)
0.925 ± 0.104*
0.60 ± 0.063***
5.5 ± 0.2
110 ± 80
Oleate + SLY (n = 8)
1.123 ± 0.168**
0.25 ± 0.020
5.2 ± 0.1
68 ± 14
OLO + SLY (n = 11)
1.027 ± 0.095*
0.54 ± 0.057***
5.0 ± 0.1
82 ± 15
SLY (n = 9)
0.580 ± 0.095
0.15 ± 0.024
5.4 ± 0.1
57 ± 90
Clamp plasma insulin was lower with oleate infusion and was restored with oleate + salicylate, whereas insulin was not different from control with OLO and OLO + salicylate (Fig. 1e, f). Clamp C-peptide showed the same pattern as insulin (Fig. 1g, h), indicating unchanged absolute insulin secretion in OLO-treated groups.
When evaluated by hyperinsulinaemic–euglycaemic clamp, insulin sensitivity tended to be decreased by oleate (n = 4), and was significantly decreased by OLO (n = 3) compared with saline (n = 4) (ESM Fig. 1).
Ex vivo studies in rat islets
mRNA levels of inflammatory markers in freshly isolated islets of infused rats
Oleate + SLY
0.82 ± 0.17 (n = 8)
2.02 ± 0.42††,‡‡‡,§§§ (n = 8)
0.30 ± 0.12 (n = 5)
0.10 ± 0.04† (n = 3)
0.58 ± 0.22 (n = 6)
1.97 ± 0.28†,§§ (n = 7)
0.81 ± 0.46 (n = 4)
0.08 ± 0.04 (n = 3)
0.76 ± 0.28 (n = 6)
5.64 ± 1.89††,‡‡‡,§§§ (n = 10)
0.52 ± 0.24 (n = 5)
0.01 ± 0.00† (n = 3)
1.00 ± 0.31 (n = 5)
15.52 ± 4.67†† (n = 10)
2.79 ± 1.34 (n = 4)
4.70 ± 2.79 (n = 3)
0.85 ± 0.26 (n = 7)
2.47 ± 0.51†,‡,§§ (n = 7)
0.81 ± 0.37 (n = 4)
0.54 ± 0.33 (n = 4)
0.62 ± 0.14 (n = 8)
4.21 ± 1.07†††,‡‡,§§ (n = 7)
0.54 ± 0.12 (n = 4)
0.75 ± 0.37 (n = 3)
1.00 ± 0.10 (n = 5)
2.66 ± 0.53††,‡‡‡,§§§ (n = 9)
0.36 ± 0.10† (n = 3)
0.33 ± 0.09† (n = 4)
Hyperglycaemic clamp in mice
Studies in beta cell-specific IKKβ-deficient mice
In vitro studies in islets
Oleate decreased total IκBα (a marker of IKKβ activity as phosphorylated IκBα is degraded) and the decrease was prevented with salicylate (Fig. 7b). Salicylate is known to activate AMPK by preventing its dephosphorylation  and AMPK activation can result in IKKβ inhibition , though salicylate has also been reported to directly inhibit IKKβ . There was a tendency for oleate to decrease phosphorylation of AMPK, which appeared to be prevented with salicylate, but there was no significant effect (Fig. 7c). BMS, which inhibits IKKβ in AMPKα-null cells , prevented oleate-induced beta cell dysfunction similar to salicylate (Fig. 7d).
We examined the effects of prolonged NEFA exposure with or without IKKβ inhibitors on beta cell function in vivo, ex vivo and in vitro. We used our in vivo models of lipotoxicity in rats [2, 15] and mice . These are models of beta cell dysfunction, as beta cell mass is not decreased by 48 h fat infusion [20, 24] and apoptosis is not increased . During hyperglycaemic clamps in vivo, both insulin and C-peptide levels were lower in rats treated with oleate, indicating reduced insulin secretion; reduced glucose-stimulated insulin secretion was also found in isolated islets ex vivo. With olive oil, glucose-stimulated insulin secretion ex vivo was reduced but insulin and C-peptide levels (indices of absolute insulin secretion) during hyperglycaemic clamps were unaffected. The different absolute insulin secretion between oleate and olive oil is explained by the effect of olive oil to induce a greater degree of insulin resistance. This was demonstrated using hyperinsulinaemic–euglycaemic clamps, which are the gold-standard assessment for insulin sensitivity in vivo. In vivo, the beta cell compensates for insulin resistance by increasing secretion. In the absence of insulin resistance, absolute insulin secretion corresponds to DI. However, in the presence of insulin resistance, DI rather than absolute insulin secretion should be taken as a measure of beta cell function, which includes the ability of the beta cell to compensate for insulin resistance. DI was impaired by both oleate and olive oil, showing a decrease in beta cell function with both types of fat, consistent with the ex vivo results with islets. The different effects of oleate and olive oil on insulin sensitivity may reflect the amount of saturated fat in olive oil (16%) and/or the plasma triacylglycerol elevation induced by olive oil .
Our results showing that fat-induced beta cell dysfunction was prevented by salicylate in vivo and ex vivo in isolated islets suggest a role for inflammatory pathways involving IKKβ in lipid-induced beta cell dysfunction.
Inflammatory pathways are known to be activated in beta cell glucotoxicity  and may enhance lipotoxicity . Our previous results show that oxidative stress plays a causal role in beta cell dysfunction induced by monounsaturated fat . ROS are known activators of IKKβ which, in addition to phosphorylating IκBα and thereby activating NFκB, phosphorylates IRS, thus inhibiting insulin signalling. In our ex vivo study, salicylate prevented the increase in phosphorylated IκBα and nuclear active NFκB induced by oleate or olive oil, although, interestingly, salicylate alone had no effect at this dose, as previously seen in the liver . An increase in ROS was induced by oleate, but salicylate did not prevent this effect. This suggests IKKβ is a downstream effector required for the previously demonstrated effect of oxidative stress to induce beta cell dysfunction . IKKβ activation could also be unrelated to oxidative stress in the case of olive oil, which contains saturated fat. Saturated fatty acids can activate IKKβ via toll-like receptors (TLR) 2 and 4. Tlr4-null mice are protected from beta cell dysfunction induced by palmitate  and Tlr2-null mice are protected from beta cell dysfunction induced by high-fat diet . Although saturated fat is believed to exert a more deleterious effect on beta cells than unsaturated fat, this is mostly based on in vitro data because, until recently, palmitate infusion has been a challenge .
Salicylate has previously been found to restore glucose-stimulated insulin secretion in an in vitro glucotoxicity model . Although not all studies are concordant , the majority report that salicylate improves beta cell function in humans [40, 41, 42]. This effect was initially attributed to inhibition of COX-2 , the gene for which is controlled by NFκB, and the consequently decreased synthesis of PGE2, a prostaglandin which inhibits insulin secretion. In addition to inhibiting Cox2 transcription via NFκB, salicylate is a direct inhibitor of both COX-1 and COX-2 , and an activator of AMPK . Salicylate did not likely protect against beta cell dysfunction through COX-1 inhibition as a COX-1 inhibitor did not prevent oleate-induced beta cell dysfunction. AMPK phosphorylation, however, did tend to decrease with oleate and this decrease was prevented by salicylate, which raises the question as to whether the protective effect of salicylate was mediated in part by AMPK activation. The effect of AMPK on insulin secretion is generally considered to be inhibitory ; however, AMPK may also deplete islet fat  and inhibit IKKβ . To further implicate IKKβ in the effect of oleate to decrease beta cell function, we also used BMS, an inhibitor that, to our knowledge, has not been reported to activate AMPK; we obtained the same results for beta cell function as with salicylate both in vitro and in vivo. Importantly, the effect of IKKβ to mediate fat-induced beta cell dysfunction is also supported by our ex vivo and in vivo data using a genetic inhibition model, the Ikkb Δbeta cell mouse.
In mice, however, two important differences from the rat studies were noted. First, 48 h exposure to oleate induced marked insulin resistance in mice in accordance with our previously published data . Besides species difference, the reason behind this finding may be the strain of mice, as C57BL/6 mice are very susceptible to fat-induced insulin resistance . Oleate infusion, which induced insulin resistance in mice, did not result in lower absolute insulin secretion during the clamp, but decreased beta cell function (DI), similar to our results with olive oil in rats. Second, BMS did restore DI but did not affect oleate-induced insulin resistance, as it likely effected a decrease in insulin sensitivity itself, which resulted in increased plasma insulin and C-peptide during clamps. Decreased insulin sensitivity with an IKKβ inhibitor may be dose related and due to the inhibition of COX-2-derived prostaglandins that increase insulin action .
The mechanisms whereby IKKβ inhibition prevents fat-induced beta cell dysfunction deserve further study. However, both upregulation of insulin signalling and COX-2 inhibition are plausible mechanisms, as suggested by two lines of evidence. First, salicylate prevented serine phosphorylation of IRS, which is known to decrease insulin-induced tyrosine phosphorylation (i.e. insulin signalling). Beta cell insulin signalling is known to be important for beta cell function . Second, salicylate prevented the oleate-induced PGE2 production and a COX-2 inhibitor mimicked the effect of salicylate. Previously, the effect of COX-2 on beta cell function has been studied mainly in the context of cytokine exposure, with contrasting results, presumably due to COX-1 vs COX-2 specificity of the inhibitors used [32, 49]. Also, the effects of exposure to COX-2 products yielded variable results among laboratories [49, 50] but a dose-dependent inhibitory effect [49, 50] suggests COX-2 may be implicated in decreasing beta cell function. As oleate increased Il1ra gene expression to a greater extent than that of Il1b, the increase in Cox2 mRNA expression is likely due to an oxidative stress-induced activation of IKKβ, independent of IL-1β. Nonetheless, salicylate did prevent upregulated gene expression of cytokines and chemokines, which may have contributed to beta cell dysfunction by further activating IKKβ and possibly other inflammatory pathways.
In summary, we demonstrated that prolonged exposure to fatty acids, which induces oxidative stress in islets, decreases beta cell function both in vitro and in vivo via activation of IKKβ. The novelty of our findings is the demonstration that IKKβ mediates beta cell dysfunction induced by NEFA selectively, and that the IKKβ/NFκB pathway is a therapeutic target to prevent NEFA-induced beta cell dysfunction in vivo.
The authors thank L. Lam, Department of Physiology, University of Toronto, for her excellent technical 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 Canadian Institutes of Health Research grants MOP-69018 (AG). AI was supported by Novo Nordisk Scholarships from the Banting and Best Diabetes Centre (University of Toronto) and Ontario Graduate Scholarships. AIO was supported by Novo Nordisk Scholarships from the Banting and Best Diabetes Centre (University of Toronto). KK was supported by scholarships from the Banting and Best Diabetes Centre (University of Toronto), Ontario Graduate Scholarship, and Ontario Graduate Scholarship for Science and Technology. YM was funded by a Showa University Research Grant for Young Researchers.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
AI, AIO and KK researched and analysed data and drafted the manuscript. YM, JAE, LZ, RNF and JE researched and analysed data and contributed to revising the manuscript. GFL, MYD, MK, MBW, JE, AV and CBC contributed to data analysis and interpretation and reviewed and edited the manuscript. AG designed the study, contributed to the discussion and reviewed and finalised the manuscript. All the authors gave final approval to the submission of the manuscript. AG is the guarantor of this work and is responsible for the integrity of the work as a whole.
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