Palmitate induces a pro-inflammatory response in human pancreatic islets that mimics CCL2 expression by beta cells in type 2 diabetes
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Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate.
Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes.
Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1β, TNF-α, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1β signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor κB (NF-κB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-κB activation similar to that occurring with palmitate.
Saturated-fatty-acid-induced NF-κB activation and endoplasmic reticulum stress may contribute to IL-1β production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.
KeywordsChemokine Cytokine Endoplasmic reticulum Fatty acid Inflammation Interleukin-1β Islet Palmitate Pancreatic beta cell Type 2 diabetes
Activating transcription factor 6
Chemokine (C-C motif) ligand
Chemokine (C-X-C motif) ligand 1
Eukaryotic translation initiation factor 2, subunit 1 α
Nuclear factor κB inhibitor, α
IL-1 receptor antagonist
Laser capture microdissection
Nuclear factor κB
PRKR-like endoplasmic reticulum kinase
Insulin deficiency and insulin resistance contribute to the development of type 2 diabetes. Pancreatic beta cell dysfunction is present early in the pathogenesis of type 2 diabetes and worsens over time , at least in part as a result of loss of functional beta cell mass . By increasing insulin requirements, insulin resistance can precipitate the onset of hyperglycaemia. Both beta cell failure and insulin resistance are determined by genetic and environmental factors, the latter causing the rapidly increasing prevalence of type 2 diabetes worldwide. Sedentary lifestyle and energy-dense diets rich in refined sugars and saturated fats are known risk factors for type 2 diabetes, but the mechanisms involved are only partially understood.
Glucotoxicity and lipotoxicity are terms coined for the deleterious effects of high circulating concentrations of glucose and lipids, both at the level of insulin sensitivity and in beta cells. Chronically elevated glucose concentrations affect beta cell function and survival through increased generation of reactive oxygen species and mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and JNK signalling [3, 4, 5]. NEFA, and in particular saturated NEFA, impair beta cell function and cause apoptosis through ceramide synthesis, JNK activation and oxidative and ER stress [5, 6, 7, 8, 9]. Saturated NEFA cause ER stress by depleting ER calcium, which secondarily affects protein folding in the organelle . Human islets exposed in vitro to palmitate activate the three branches of the ER stress response under the control of PRKR-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring 1 (IRE1) . Markers of oxidative  and ER stress [8, 12, 13] are present in islets of type 2 diabetic patients, suggesting these stress responses may indeed play a role in the development and progression of the disease.
Mild inflammation by activation of the innate immune system has been suggested to play a role in the pathogenesis of type 2 diabetes, and it is present years before disease onset . In the adipose tissue of obese individuals, infiltrating macrophages produce cytokines, modulate adipocyte adipokine secretion and contribute to the development of insulin resistance . The spill over of inflammatory cytokines into the circulation has been associated with the development of type 2 diabetes. While the single increase in circulating IL-6 does not enhance disease risk, the combined elevation of IL-6 and IL-1β is associated with a threefold increased risk for type 2 diabetes . A new role for inflammation in type 2 diabetes has been recently proposed, namely as a trigger of beta cell dysfunction and death . Macrophage infiltration was observed in islets from type 2 diabetic individuals compared with non-diabetic (ND) individuals, with 20% of the type 2 diabetic islets containing >3 CD68+ cells per islet compared with 5% of the ND islets [18, 19]; the role of these macrophages is unknown. It has been proposed that beta cells contribute to the inflammatory process in type 2 diabetes by their own secretion of IL-1β in response to high glucose. In a series of studies, it was suggested that glucotoxicity in human beta cells is mediated by Fas (TNF receptor superfamily, member 6) receptor upregulation and nuclear factor κB (NF-κB) activation, culminating in beta cell dysfunction and apoptosis [20, 21], though this was not confirmed by others [22, 23]. Altogether, the cause and role of islet inflammation in type 2 diabetes remains controversial and poorly understood.
We have used human pancreatic beta cells and islets from ND and type 2 diabetic individuals to answer the following questions: (1) is an inflammatory process present in type 2 diabetic beta cells, as evaluated by array analysis of laser-capture microdissected beta cells from type 2 diabetic and normoglycaemic individuals; (2) do NEFA and/or high glucose contribute to this process; (3) by which signalling events and cellular response mechanisms do saturated NEFA induce an inflammatory response in human islets; and (4) does NEFA-induced inflammation contribute to lipotoxicity? The results demonstrate the presence of mild inflammation in islets from type 2 diabetic individuals, a process that is mimicked by in vitro exposure of ND human islets to palmitate. The saturated-NEFA-induced inflammation could be a consequence of NEFA-induced ER stress and does not directly contribute to human islet lipotoxicity.
Laser-capture microdissection and microarray analysis
Pancreatic samples from ten ND multiorgan donors (age 60 ± 2 years, four women/six men, BMI 30.6 ± 1.6 kg/m2) and ten type 2 diabetic donors (age 67 ± 7 years, three women/seven men, BMI 30.9 ± 6.2 kg/m2, known duration of diabetes 5.3 ± 2.3 years, insulin/oral therapy 33%/66%) were obtained with approval of the Ethics Committee in Pisa, Italy. The causes of death were similar. The available clinical characteristics of the donors are provided in Electronic supplementary material (ESM) Table 1; no data were available on levels of circulating lipids or inflammatory markers. Laser-capture microdissection (LCM) was performed as previously detailed [24, 25]. Briefly, frozen pancreatic sections were fixed, dehydrated and air-dried. LCM was performed using PixCell II Laser Capture Microdissection System (Arcturus Engineering, Mountain View, CA, USA) by melting thermoplastic films mounted on transparent LCM caps (Arcturus) on beta cells, identified by their intrinsic autofluorescence, in islets with no signs of amyloid deposits. The microdissected beta cells were incubated in guanidine isothiocyanate extraction buffer and RNA was extracted by modification of the RNA micro-isolation protocol . The total RNA underwent two amplification rounds using the RiboAmp HS RNA Amplification Kit (Arcturus) and was biotinylated using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY, USA). RNA products were fragmented and hybridised to GeneChip Human X3P Array (Affymetrix, Santa Clara, CA, USA). Array data were normalised and analysed using DNA-Chip Analyzer (dChip) software (available from http://biosun1.harvard.edu/complab/dchip/) that assesses the standard errors for the expression indices and calculates confidence intervals for fold changes. Lower confidence bound (used at a cut-off value of 1.2), a conservative estimate of the fold change, and the p value were used to assess differentially expressed genes. The complete array data will be reported elsewhere (L. Marselli, D. C. Sgroi, J. Thorne, S. Dahiya, A. Sharma, S. Bonner-Weir, P. Marchetti and G.C. Weir, unpublished results).
Pancreatic islet preparation and culture
With the approval of the Ethics Committee in Pisa, Italy, human islets were isolated from 12 ND donors (age 58 ± 6 years, seven women/five men, BMI 24.1 ± 0.7 kg/m2) by collagenase digestion and density gradient purification , cultured in M199 medium (5.5 mmol/l glucose) and shipped to Brussels, Belgium, within 1–5 days of isolation. After overnight recovery in Ham’s F-10 containing 6.1 mmol/l glucose, 10% (vol./vol.) FCS, 2 mmol/l GlutaMAX (Gibco, Invitrogen, Merelbeke, Belgium), 50 μmol/l 3-isobutyl-1-methylxanthine, 1% (wt/vol.) charcoal-absorbed BSA (Boehringer, Indianapolis, IN, USA), 50 U/ml penicillin and 50 μg/ml streptomycin, islets were exposed to NEFA in the same medium without FCS and a glucose concentration of 6.1 or 28 mmol/l . The percentage of beta cells, examined by insulin immunofluorescence (see below), in the islet preparations was 50 ± 3% (range 35% to 70%, n = 12).
Oleate and palmitate (sodium salt, Sigma, Bornem, Belgium) were dissolved in 90% (vol./vol.) ethanol and diluted 1:100 to a final concentration of 0.5 mmol/l, corresponding to a NEFA/BSA ratio of 3.4 [10, 26]. The control condition contained 1% charcoal-absorbed BSA and a similar ethanol dilution. IL-1 receptor antagonist (IL-1ra; R&D Systems, Abingdon, UK) was used alone, or in combination with palmitate, at a concentration of 30 ng/ml. In combination with recombinant human IL-1β (50 U/ml) and IFN-γ (1,000 U/ml; PeproTech, Paris, France), 300 ng/ml IL-1ra was used (>100-fold concentration compared with the added IL-1β) and added 30 min prior to cytokines . Recombinant murine TNF-α (Innogenetics, Gent, Belgium) was used at a concentration of 1000 U/ml. Cyclopiazonic acid (CPA; Sigma) was used at 50 μmol/l  and salubrinal (ChemBridge, San Diego, CA, USA) at 75 μmol/l .
Human ductal cell lines
Pancreatic ductal adenocarcinoma, Capan-2, and epithelioid carcinoma, Panc-1, cell lines  were a kind gift of L. Bouwens (Vrije Universiteit Brussel, Brussels, Belgium). Capan-2 cells were cultured in Advanced-RPMI medium (Gibco, Paisley, UK) containing 10% (vol./vol.) FCS, 2 mmol/l GlutaMAX and antibiotics (as above). Panc-1 cells were cultured in DMEM (without pyruvate; Gibco) containing 10% (vol./vol.) serum, MEM non-essential amino acids (Sigma) and antibiotics. For the NEFA treatment, medium contained 1% (vol./vol.) FCS and 1% (wt/vol.) BSA.
Assessment of cell death
The percentage of islet cell death was determined by at least two observers (one unaware of sample identity) using inverted fluorescence microscopy following staining with propidium iodide (5 μg/ml; Sigma) and Hoechst 33342 (10 μg/ml; Sigma) as described [10, 30].
Poly(A)+ RNA was isolated from human islets and reverse transcribed. The real-time PCR amplification was done using iQ SyBR Green Supermix on iCycler MyiQ Single Color (BIO-RAD, Hercules, CA, USA) as described . The gene expression level was normalised to the housekeeping gene β-actin, expression of which is not modified by the treatments and comparable with that of ornithine decarboxylase antizyme 1 (OAZ1; ESM Fig. 1). Primer sequences are given in ESM Tables 2 and 3.
Dispersed human islet cells were stained with anti-p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-insulin antibodies (Sigma) as described in the ESM. To determine the islet cell type(s) producing chemokine (C-C motif) ligand (CCL)2 pancreatic specimens were stained by double immunofluorescence using anti-CCL2 (Abcam, Cambridge Sciences Park, Cambridge, UK), anti-insulin (Dako, Glostrup, Denmark), and anti-glucagon (R&D Systems) antibodies and analysed with a Leica TCS SP5 laser scanning confocal microscope (see ESM).
Insulin accumulated in the culture medium was measured as described  and normalised to β-actin mRNA expression.
Human chemokine (C-X-C motif) ligand 1 (CXCL1) and IL-6 protein were measured in the culture medium by ELISA following the manufacturer’s instructions (Quantikine, R&D Systems, Minneapolis, MN, USA) and normalised to β-actin mRNA expression.
Data are means±SEM. Real-time PCR data were log transformed to obtain normal distribution and compared by ANOVA followed by paired t test with the Bonferroni correction for multiple comparisons. Microarray data were analysed by Mann–Whitney test. Linear regression was done using the least squares method. Multiple regression models were built to determine whether the association between a dependent and an independent variable remained significant after adjusting for other independent variables. A p value < 0.05 was considered significant.
Microarray profiling of human beta cells reveals increased cytokine and chemokine expression in type 2 diabetes
Array data on IL1-β (also known as IL1B) and IL8 from our LCM samples has already been reported and confirmed by real-time PCR . These previously published data were obtained for nine ND and ten type 2 diabetic patients; one of the ten controls with no type 2 diabetes history was excluded because of some high glycaemia readings during the antemortem period. Values were obtained from an initial analysis in which filtering criteria were used and the signals and statistical results of 30,990 probe sets were retrieved. The published results for IL1-β were 28 ± 3.9 vs 58 ± 14.2 (p = 0.032) and for IL8 22.2 ± 3.8 vs 50.7 ± 12.9 (p = 0.034) for ND vs type 2 diabetic patients, respectively ; for all ten ND, IL1-β was 27 ± 3.7 (p = 0.057 vs type 2 diabetes) and IL8 22.2 ± 3.8 (p = 0.059).
Palmitate, but not oleate or high glucose, induces expression of chemokines and cytokines in human islets
Human islet chemokine and cytokine expression is positively correlated with IL-1β and TNF-α
IL-1β mediates the chemokine/cytokine induction by palmitate
Cell source of the chemokines and cytokines produced by human islets
Because the purification of human beta cells from islet preparations is technically challenging, we evaluated the cell type in which palmitate induces NF-κB activation by staining dispersed human islet cells for insulin and p65, the major NF-κB component in beta cells. Palmitate, and the combination of IL-1β plus IFN-γ plus TNF-α used as positive control, induced NF-κB activation in beta and non-beta cells, indicated by nuclear translocation of p65 after 2 h (ESM Fig. 2). Palmitate also activated NF-κB in the clonal human ductal cells Panc-1 and Capan-2 (ESM Fig. 3), and it induced chemokines and cytokines in ductal cells, while oleate did not (ESM Fig. 3). This suggests that both human beta cells and exocrine ductal cells exhibit pro-inflammatory responses to saturated NEFA.
Role of ER stress in the induction of chemokines and cytokines by palmitate
ER stress is present in islets from type 2 diabetic individuals , and can be elicited in human islets in vitro by saturated NEFA . To examine whether ER stress signalling contributes to the observed induction of chemokines and cytokines, we exposed human islets to the synthetic ER stressor CPA. This ER stressor induced cytokine expression and NF-κB activation to a similar extent as palmitate (ESM Fig. 4). To evaluate the role of the PERK/eukaryotic translation initiation factor 2, subunit 1 α (eIF2α) pathway on chemokine/cytokine expression we used salubrinal, a selective inhibitor of eIF2α dephosphorylation [28, 30]. Salubrinal did not lead to chemokine/cytokine or IκBα expression in human islets (ESM Fig. 5), suggesting that other branches of the ER stress response mediate the pro-inflammatory response.
Systemic low-grade inflammation precedes the onset of type 2 diabetes by years , but its role in the pathogenesis of the disease is debated. In genetic studies, a polymorphism in CCL2 has been associated with type 2 diabetes risk . In a meta-analysis of type 2 diabetes genome-wide association scans, however, gene variants influencing circulating levels of IL-1ra, IL-18, the IL-6 receptor, macrophage migration inhibitory factor and C-reactive protein did not predispose to type 2 diabetes , suggesting that inflammation might be secondary to rather than causal for diabetes. Human and animal studies point to a role for inflammation in enlarged adipose tissues, increased secretion of cytokines and chemokines by adipocytes and fat-infiltrating macrophages, and insulin resistance [14, 15]. Whether inflammation is also present in islets and whether this contributes to the development of type 2 diabetes are important questions because of the central role of beta cell dysfunction in the disease pathogenesis . Moreover, IL-1ra therapy is under evaluation for the treatment of type 2 diabetes [34, 35], based on studies suggesting that high-glucose-induced IL-1β secretion by beta cells causes beta cell dysfunction and death .
In the present study, we observed a gene expression signature of mild inflammation in human type 2 diabetic beta cells obtained by LCM. There was increased expression of the mRNA for: the β-chemokines CCL2 and CCL13, implicated in monocyte chemotaxis ; α-chemokines CXCL1 and IL-8, potent chemoattractants and activators of neutrophils ; and the pro-inflammatory cytokines IL-1β and IL-6. The chemokine and cytokine mRNA expression was heterogeneous in the type 2 diabetic group, suggesting that mild inflammation is present in some but not all type 2 diabetic individuals. Mild macrophage infiltration has been observed in islets from type 2 diabetic individuals in two recent studies, with 20% of the islets containing >3 CD68+ cells per islet compared with 5% of the ND islets [18, 19]. This islet inflammation could be mediated by CCL2 and CCL13 produced in human beta cells. Islet inflammation was also present in animal models for type 2 diabetes, namely the high-fat-diet-fed and db/db mice [18, 36], the Cohen diabetic rat  and the Goto–Kakizaki rat [37, 38]. The role of these infiltrating macrophages is unknown. Some propose them to be beneficial to islet function by promoting angiogenesis ; others suggest that activated CD68+ cells could cause beta cell dysfunction and death [36, 37]. The beta cell inflammatory response was presently reproduced at the RNA and protein level in ND human islets by their in vitro exposure to the saturated NEFA palmitate, and was dependent on the canonical mediator of early inflammation IL-1β. While confocal microscopy suggests that CCL2 protein production in ND and type 2 diabetic pancreas is largely restricted to beta cells, palmitate induced a pro-inflammatory response in both beta and islet non-beta and ductal cells.
At variance with previous reports that elevated glucose concentrations increase IL1-β expression in human islets cultured either in suspension or on an extracellular matrix [20, 21], glucose did not induce an inflammatory response in human islets in this or a previous study by our group . Rather, the chemokine/cytokine expression and NF-κB activation was induced by the saturated NEFA palmitate, but not by the unsaturated oleate. Saturated and unsaturated NEFA have different functional effects in beta cells [40, 41, 42]. Saturated NEFA are pro-inflammatory in several cell types [43, 44] and also cause ER stress . Because palmitate (but not oleate or high glucose) induces ER stress in human islets  and there is a crosstalk between ER stress and NF-κB pathways [45, 46, 47], we explored this mechanism further. We have previously shown that palmitate activates PERK, and also leads to ATF6 and IRE1 activation through depletion of ER Ca2+ stores . The crosstalk between ER stress and NF-κB activation can occur at different levels. Ca2+ efflux from the ER activates NF-κB through generation of reactive oxygen species  and release of a diffusible, still unknown, NF-κB activator . IRE1 and TNF receptor-associated factor 2 (TRAF-2) can activate NF-κB , and stalled protein translation caused by eIF2α phosphorylation decreases IκBα protein availability, consequently activating NF-κB . CPA, a canonical ER stressor, upregulates chemokines and cytokines and activates NF-κB in human islets with an effect comparable with that of palmitate (present data). The CPA-induced p65 nuclear translocation in beta and non-beta cells occurred after short (2 h) exposure, in line with previously reported kinetics showing maximal NF-κB activation by CPA and thapsigargin after 1 h due to ER Ca2+ depletion . To examine involvement of the PERK pathway in NF-κB activation, we used salubrinal as a selective inhibitor of eIF2α dephosphorylation [28, 30]. Salubrinal did not induce chemokines/cytokines or IκBα (ESM Fig. 5), suggesting that ER Ca2+ depletion or ER stress-induced IRE1 signalling but not inhibition of protein translation mediates palmitate-induced NF-κB activation in human islets. ER stress is not the only mechanism by which palmitate could induce mild inflammation in human islets. Toll-like receptors (TLRs) may also recognise saturated NEFA . These TLRs have been suggested to mediate some of the NEFA-induced islet inflammation in a recent study .
Blocking IL-1β signalling with IL-1ra prevented the palmitate-induced expression of chemokines and cytokines and NF-κB activation, showing that the observed inflammatory response is largely dependent on IL-1β production. The magnitude of IL-1β induction is modest, and probably insufficient to cause human islet cell damage. In fact, low concentrations of IL-1β have been shown to stimulate human islet function . IL-1ra did not prevent palmitate-induced human islet cell death in vitro, suggesting that the mild IL-1β induction is not directly deleterious to the human islets and does not mediate lipotoxicity. Administration of IL-1ra in vivo to high-fat-fed mice improved beta cell function but also fully protected against changes in insulin sensitivity . IL-1ra reduced adipose tissue inflammation, altered adipokine secretion and decreased circulating lipid levels, which may have contributed to the preservation of insulin secretion and glucose tolerance in the high-fat-fed mice. We propose, therefore, that the induction of IL-1β and downstream chemokines and cytokines by saturated NEFA results in mild islet inflammation in type 2 diabetes, but that this does not directly contribute to beta cell dysfunction and apoptosis. ER stress  and other signalling pathways  contribute to lipotoxicity in human islets in type 2 diabetes.
In conclusion, we demonstrate here that there is a mild inflammatory process in islets from some but not all type 2 diabetic patients. Palmitate, but not oleate or high glucose, induces in vitro chemokine and cytokine expression in ND human islets, a process largely dependent on IL-1β production. ER stress could be one of the mechanisms involved in saturated-fatty-acid-induced human islet inflammation. The induction of IL-1β and downstream chemokines and cytokines by saturated NEFA does not directly contribute to lipotoxicity.
The mild inflammation in islets from type 2 diabetic patients could be the result of in vivo exposure to saturated NEFA. Whether this islet inflammation causes loss of functional beta cell mass and thereby contributes to the development and progression of type 2 diabetes remains to be elucidated.
We thank G. Vandenbroeck, M. Urbain, J. Schoonheydt and R. Leeman (Laboratory of Experimental Medicine) for expert technical assistance. This work was supported by the European Union (Integrated Project EuroDia in the Framework Programme [FP] 6 and Collaborative Projects CEED3 and NAIMIT in FP7); the Belgian Program on Interuniversity Poles of Attraction (IUAP P6/40); the Fonds National de la Recherche Scientifique (FNRS), Fonds de la Recherche Scientifique Médicale (FRSM) and Actions de Recherche Concertées de la Communauté Française, Belgium (M. Cnop and D. L. Eizirik); the Italian Diabetes Research Foundation-FORISID and the Italian Ministry of Health (F. Dotta); the European Association for the Study of Diabetes–European Foundation for the Study of Diabetes GlaxoSmithKline research grant (L. Marselli).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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