Clinical Science (2014) 126, 739–752 (Printed in Great Britain) doi: 10.1042/CS20130678

Clinical Science

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Elevated levels of extracellular heat-shock protein 72 (eHSP72) are positively correlated with insulin resistance in vivo and cause pancreatic β-cell dysfunction and death in vitro Mauricio KRAUSE∗ †‡§, Kevin KEANE, Josianne RODRIGUES-KRAUSE§, Domenico CROGNALE∗ ‡, Brendan EGAN∗ ‡, Giuseppe DE VITO∗ ‡, Colin MURPHY§ and Philip NEWSHOLME∗  ∗ Food for Health Ireland (FHI), Department of Agriculture, Food Science & Veterinary Medicine, Institute of Food Health, University College Dublin, Dublin, Ireland †Laboratory of Cellular Physiology, Department of Physiology, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil ‡Institute for Sport and Health, University College Dublin, Dublin, Ireland §Biomedical Research Group, Department of Science, ITT Dublin, Ireland School of Biomedical Sciences, CHIRI - Biosciences, Curtin University, Perth, Western Australia 6845

Abstract eHSP72 (extracellular heat-shock protein 72) is increased in the plasma of both types of diabetes and is positively correlated with inflammatory markers. Since aging is associated with a low-grade inflammation and IR (insulin resistance), we aimed to: (i) analyse the concentration of eHSP72 in elderly people and determine correlation with insulin resistance, and (ii) determine the effects of eHSP72 on β-cell function and viability in human and rodent pancreatic β-cells. Fasting blood samples were collected from 50 older people [27 females and 23 males; 2 63.4 + − 4.4 years of age; BMI (body mass index) = 25.5 + − 2.7 kg/m ]. Plasma samples were analysed for eHSP72, insulin, TNF (tumour necrosis factor)-α, leptin, adiponectin and cortisol, and glycaemic and lipid profile. In vitro studies were conducted using rodent islets and clonal rat and human pancreatic β-cell lines (BRIN-BD11 and 1.1B4 respectively). Cells/islets were incubated for 24 h with eHSP72 (0, 0.2, 4, 8 and 40 ng/ml). Cell viability was measured using three different methods. The impact of HSP72 on β-cell metabolic status was determined using Seahorse Bioscience XFe96 technology. To assess whether the effects of eHSP72 were mediated by Toll-like receptors (TLR2/TLR4), we co-incubated rodent islets with eHSP72 and the TLR2/TLR4 inhibitor OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; 30 μg/ml). We found a positive correlation between plasma eHSP72 and HOMA-IR (homoeostasis model assessment of IR) (r = 0.528, P < 0.001), TNF-α (r = 0.389, P < 0.014), cortisol (r = 0.348, P < 0.03) and leptin/adiponectin (r = 0.334, P < 0.03). In the in vitro studies, insulin secretion was decreased in an eHSP72 dose-dependent manner in BRIN-BD11 cells (from 257.7 + − 33 to 84.1 + 10.2 μg/mg of protein per 24 h with 40 ng/ml eHSP72), and in islets in the presence of 40 ng/ml eHSP72 − + (from 0.48 + 0.07 to 0.33 0.009 μg/20 islets per 24 h). Similarly, eHSP72 reduced β-cell viability (at least 30 % − − for BRIN-BD11 and 10 % for 1.1B4 cells). Bioenergetic studies revealed that eHSP72 altered pancreatic β-cell metabolism. OxPAPC restored insulin secretion in islets incubated with 40 ng/ml eHSP72. In conclusion, we have demonstrated a positive correlation between eHSP72 and IR. In addition, we suggest that chronic eHSP72 exposure may mediate β-cell failure. Key words: aging, extracellular heat-shock protein 72 (eHSP72), insulin resistance, pancreatic β-cell, Type 1 diabetes, Type 2 diabetes

Abbreviations: BMI, body mass index; BP, blood pressure; CV, coefficient of variation; DMEM, Dulbecco’s modified Eagle’s medium; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GSIS, glucose-stimulated insulin secretion; HOMA-IR, homoeostasis model assessment of IR; HSP, heat-shock protein; eHSP72, extracellular HSP72; iHSP72, intracellular HSP72; IR, insulin resistance; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; OCR, O2 consumption rate; OxPAPC, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; PPR, proton production rate; ROS, reactive oxygen species; T1DM, Type 1 diabetes; T2DM, Type 2 diabetes; TLR, Toll-like receptor; TNF, tumour necrosis factor; UCP, uncoupling protein. Correspondence: Dr Mauricio Krause (email [email protected] or [email protected]) or Professor Philip Newsholme (email [email protected]).

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INTRODUCTION Low-grade inflammation is a common feature of obesity, diabetes and aging [1,2]. The term ‘inflamm-aging’ has been used to highlight the increased levels of pro-inflammatory molecules that occur in older people [3]. Some pro-inflammatory cytokines are known to induce the inactivation of the insulin receptor and associated molecules [4], and the consequence of chronic receptor inactivation in aging is IR (insulin resistance) [5] and skeletal muscle atrophy [2]. It has been shown previously that HSPs (heat-shock proteins), which normally function as protein chaperones, are associated with inflammation and IR [4]. HSPs are considered part of a family of proteins known as ‘stress proteins’ since their expression is induced by a wide range of stressors, such as oxidative stress [6], thermal stress [7], ischaemia [8], exercise [6], metabolic stress [9] and many others. HSP72 (or HSPA1A) is inducible during cell stress and represents the most abundant of all HSPs, accounting for 1–2 % of cellular protein [10], including in skeletal muscle [11]. As a molecular chaperone, iHSP72 (intracellular HSP72) can interact with other proteins (unfolded, in non-native state and/or stress-denatured conformations) avoiding inappropriate interactions, formation of protein aggregates and degradation of damaged proteins, as well as helping the correct refolding of proteins [11]. Other functions include protein translocation [12], anti-apoptosis [13] and antiinflammatory responses [14]. More recently, the roles of HSPs have been expanded to include the control of cell signalling [15] and modulation of the immune response [16] in chronic diseases such as diabetes, obesity and IR [4,17]. HSPs were long thought to be cytoplasmic proteins with functions restricted to subcellular compartments. However, studies have reported that they may be released into the extracellular space, for example eHSP72 (extracellular HSP72), which induces effects in other cells [18], including activation of the immune system [19]. eHSP72 has been reported to stimulate neutrophil microbicidal capacity [20] and chemotaxis [21], and recruitment of NK (natural killer) cells [22], as well as cytokine release from various immune cells [16,23]. During inflammatory and oxidative stress states, the levels of eHSP72 in the extracellular medium (plasma and serum) may be elevated. Indeed, patients with T1DM (Type 1 diabetes) [24] and T2DM (Type 2 diabetes) exhibit higher levels of eHSP72 that appear to be dependent on the duration of the disease [25]. In addition, the serum eHSP72 concentration is positively correlated with markers of inflammation in humans, such as CRPs (C-reactive proteins), monocyte count and TNF (tumour necrosis factor)-α [26,27]. Recently, eHSP72 was suggested to be a potential biomarker/predictor of sarcopenia in elderly people [28], and this may reflect the release of HSP72 which normally would function in the maintenance of the muscle mass and signalling. Lastly, eHSP72 may be related to IR in aging; however, no study has investigated the relationship between insulin sensitivity and eHSP72 in aging or in conditions of compromised metabolism. In the present study, we have investigated the co-relationship between eHSP72, inflammation and IR in elderly people. In addition, since the level of eHSP72 is increased in low-grade inflammation in diabetes and in aging, we also investigated the effects

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of this protein on rodent and human pancreatic β-cell function using appropriate cell lines and rodent islets.

MATERIALS AND METHODS Participant characteristics and ethics A total of 50 sedentary non-smoking participants (63.4 + − 4.4 years of age) volunteered for the present study [27 females and 23 males; BMI (body mass in2 dex) = 25.5 + − 2.7 kg/m ]. Informed consent was obtained from all participants before beginning the study. Research assessments and protocols were approved by the local UCD Dublin Human Ethics Committee. All subjects were reported as non-diabetic after interview with an experienced physician, who considered their fasting glycaemia as the main criterion for diabetes exclusion.

Anthropometric measurements and body composition Standing height was measured using a stainless steel Harpenden Stadiometer (Holtain), with the participants’ shoes off and head at the Frankfort horizontal plane. Body mass was measured using a Seca 888 weighing scale). Body composition was assessed using dual energy X-ray absorptiometry (DEXA) (Lunar iDXA; GE Healthcare).

BP (blood pressure) and biochemistry Systemic arterial BP was measured in the brachial artery using an Omron M5-1 fully automated BP monitor (HEM 907). The mean of two measurements taken at 2 min intervals of supine rest was recorded. Venous blood samples were taken after fasting from an antecubital vein in heparin-coated and gel-clot VacutainerTM tubes using standard aseptic techniques. Samples were immediately centrifuged (at 4 ◦ C and 1000 g for 15 min), after which plasma and serum were removed and stored at − 80 ◦ C for further analysis.

Quantification of plasma HSP72 (eHSP72), leptin, adiponectin, cortisol and TNF-α A highly sensitive ELISA method (EKS-715; Stressgen) was used to determine the expression of HSP72 protein in serum, as described previously [29]. Absorbance was measured at 450 nm, and a standard curve was constructed from known dilutions of HSP72 protein to allow quantitative assessment of HSP72 concentration. Quantification was made using a microplate reader (Molecular Devices SpectraMax Plus 384). The intra- and interassay CV (coefficient of variation) ranged between 4.5 and 7 %. TNF-α was quantified using a human TNF-α Quantikine ELISA kit (catalogue number, DTA00C; R&D Systems). The intra- and inter-assay CV ranged between 3.5 and 8 %. Leptin (catalogue number, 10-1199-01; Mercodia) and adiponectin (catalogue number, 10-1193-01; Mercodia) were also measured in plasma using a highly sensitive ELISA method, according to the manufacturer’s instructions. The intra- and inter-assay CV ranged between 4 and 7 % for leptin and 4.5 to 8 % for adiponectin. We chose to analyse leptin and adiponectin since they are connected with insulin

eHSP72 correlates with insulin resistance and promotes pancreatic β-cell death

sensitivity and their ratio has been used as a marker for cardiovascular diseases [30].

The use of 16.7 mmol/l glucose + 10 mmol/l alanine is an accepted positive control for these cell lines [32,34].

Culture of BRIN-BD11 and 1.1B4 pancreatic β-cell lines, and measurement of insulin secretion at different HSP72 concentrations

Cell viability measurements

The clonal insulin-secreting β-cell lines BRIN-BD11 and 1.1B4 were chosen because their metabolic, signalling and secretory responses to glucose and amino acids, as well as other stimuli, have been extensively characterized [31–34]. Cells were maintained in culture overnight as described previously [35]. After washing with PBS, cells were incubated in fresh RPMI 1640 medium, supplemented with 11.1 mmol/l D-glucose and 2 mmol/l L-glutamine, in the absence or presence of rat or human recombinant HSP72 (catalogue numbers, ADI-SPP-758-F and ADI-NSP555-F respectively; Enzo Life Sciences) for 24 h. The HSP72 concentration range tested was chosen to include absence (0 ng/ml), the reported concentration found in obese patients with T2DM (0.2 ng/ml) [36], the range in human blood in response to acute exercise bouts (4 and 8 ng/ml) [37,38] and the concentration reported in the blood of subjects with T1DM (40 ng/ml) [24]. After 24 h, an aliquot of the medium was taken for analysis of insulin using the Mercodia ultrasensitive rat insulin ELISA kit.

Islet isolation, culture and insulin secretion Pancreatic islets were isolated from wild-type Black mice (C57BL/6J, 8–12 weeks of age) [39]. Each pancreas was excised and inflated with a collagenase solution (0.5 mg/ml; C9891; Sigma) and chopped into small pieces. Digestion was initiated during sample incubation at 37 ◦ C for 3 min with constant shaking. The digest was washed with 0.1 % BSA/Krebs solution (5.6 mmol/l glucose) and the islets were sedimented by gentle centrifugation (500 g for 10 min at 4 ◦ C) with Histopaque 1077 (Sigma). Islets were resuspended in Krebs buffer containing 0.1 % BSA, individually picked and cultured in RPMI 1640 culture medium, in the presence of 0, 0.2 and 40 ng/ml recombinant HSP72 for 24 h. To test whether the action of eHSP72 is mediated by the activation of TLR2/TLR4 (Toll-like receptors 2 and 4), we co-incubated the islets with eHSP72 (in the same indicated concentrations) and the TLR2/TLR4 inhibitor OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero3-phosphocholine; 30 μg/ml; catalogue number, tlrl-oxp1; Invivogen) [40]. Insulin release was determined using the Mercodia ultrasensitive mouse insulin ELISA kit. To certify that all islets were healthy and functional, we performed incubations with low and high glucose to check the insulin secretion response. Indeed, mouse islets increased their insulin secretion from 0.3 + − 0.03 μg of insulin/20 islets per h (in the presence of 3 mmol/l glucose) to 1.57 + − 0.2 μg of insulin/20 islets per h (in the presence of 15 mmol/l glucose), whereas BRIN BD11 and 1.1B4 β-cells increased their insulin secretion from 0.58 + − 0.13 μg of insulin/mg of protein per 20 min and 0.096 + 0.007 ng of insulin/mg of pro− tein per 20 min (in the presence of 1.1 mmol/l glucose) respectively to 1.5 + − 0.09 μg of insulin/mg of protein per 20 min and 0.144 + 0.013 ng of insulin/mg of protein per 20 min (in the pres− ence of 16.7 mmol/l glucose + 10 mmol/l alanine) respectively.

Three different techniques were used to assess cell viability: Neutral Red assay, LDH (lactate dehydrogenase) release assay and WST-1 assay. The Neutral Red uptake assay provides a quantitative estimation of the number of viable cells based on their ability to incorporate and bind the dye Neutral Red in lysosomes. Cell lines were exposed (for 24 h) to different concentrations of recombinant HSP72 as described previously. After 2 h incubation in presence of Neutral Red (100 μg/ml), cells were washed with PBS, followed by disruption with acid ethanol [alcohol/glacial acetic acid, 50:1 (v/v)]. Aliquots of the resulting solution were transferred to 96-well plates and the absorbance at 540 nm was recorded using a microplate spectrophotometer (Molecular Devices SpectraMax Plus 384). In order to determine cell-membrane integrity (an indirect measurement of cell viability), an LDH release assay (catalogue number, K313-500; Biovision) was used, and the results expressed as percentage of cell LDH released. We also tested viability using a mitochondrial activity based test (WST-1; catalogue number, 05015944001; Roche). The cell chemical reagent WST-1 (10 μl) was added to each well and the cells were incubated at 37 ◦ C for a period of 1 h. Absorbance at 450 nm was measured using a microplate spectrophotometer and is an indirect measurement of cellular dehydrogenase activities.

Glucose consumption and nitrite production measurements The standard glucose concentration in RPMI 1640 medium at the beginning of incubation (11.1 mmol/l) and 24 h after culture was determined using a commercially available kit (catalogue number 10260; Liquicolor Human). The glucose utilized over the 24-h period was calculated by subtracting the concentration at 24 h from that at 0 h. The production of NO by the cell lines and rodent islets was assessed by measuring the formation of nitrites in the culture medium using the Griess Reagent System (catalogue number, G2930; Promega).

Seahorse XFe 96 measurements The Seahorse Bioscience XFe 96 Flux analyser and the Mito Stress Test kit were used according to the manufacturer’s instructions. In brief, cells were seeded into 96-well plates at a density of 5000 cells/well and were allowed to adhere overnight [the cell density was previously optimized so that the OCR (O2 consumption rate) and ECAR (extracellular acidification rate) measurements met the manufacturer’s criteria]. For 24 h treatments, the culture medium was then replaced with standard RPMI 1640 medium containing various concentrations of eHSP72 and incubated for a further 24 h at 37 ◦ C in 5 % CO2 . In the case of the 20 min exposure, the culture medium was replaced with standard RPMI 1640 without eHSP72 and cultured for the same 24 h period. On the day of the Seahorse analysis and stress test, the spent culture medium was changed using the Seahorse Prep Station, to serum-free DMEM (Dulbecco’s modified Eagle’s medium) (pH 7.4) containing 1 mmol/l sodium pyruvate and 2.5 mmo/l glucose without bicarbonate. Plates were then

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incubated for 60 min at 37 ◦ C in a non-CO2 incubator to allow the cells to become equilibrated with the new medium. The XFe 96 sensor cartridge, which was pre-hydrated for 24 h before the assay with calibrant solution at 37 ◦ C in a non-CO2 incubator, was inserted into the instrument and the calibration process initiated. Following successful calibration, the culture plate with cells was placed in the instrument. After recording the basal measurements, the injection strategy for cells that had undergone 24 h treatment consisted of DMEM (2.5 mmol/l glucose), followed by oligomycin (2 μmol/l), FCCP (carbonyl cyanide ptrifluoromethoxyphenylhydrazone; 0.4 μmol/l) and finally rotenone/antimycin A in combination (1 μmol/l). For cells that had undergone the 20 min exposure, the injection strategy consisted of DMEM without or with eHSP72 and containing 180 mmol/l glucose (final concentration of 25 mmol/l glucose in the plate), followed by oligomycin (2 μmol/l), FCCP (0.4 μmol/l) and finally rotenone/antimycin A in combination (1 μmol/l). OCR and ECAR were measured using three 3.5 min assay cycles of mix and measurement following each injection. Normalization of BRINBD11 and 1.1B4 β-cell line basal respiration was performed by determining the cell density using the Neutral Red uptake assay after the stress test, as described above. These measurements were compared to ensure there were no significant changes in relative cell numbers among the treatment groups. The concentrations of all of the inhibitors used were optimized to ensure the lowest concentration was used to produce the maximum effect. In addition, since these inhibitors were prepared in DMSO, we also measured the OCR/ECAR of cells receiving equal concentrations of DMSO in DMEM at every injection. No significant changes were observed (results not shown).

Seahorse data analysis Basal respiration was calculated by subtracting the minimum OCR following the addition of rotenone/antimycin A (nonmitochondrial respiration) from the last OCR measurement recorded before the addition of oligomycin. Proton leak was calculated by subtracting the minimum OCR following addition of rotenone/antimycin A (non-mitochondrial respiration) from the minimum OCR measurement recorded after addition of oligomycin. OCR related to ATP production (turnover) was calculated by subtracting the proton leak from the basal respiration above. The coupling efficiency percentage was calculated by dividing the ATP-turnover-dependent OCR by the basal respiration and multiplying by 100. Glycolytic response to 25 mmol/l (acute 20 min) or 2.5 mmol/l (chronic 24 h) glucose was measured by first correcting the ECAR experimental values to the PPR (proton production rate) by including the buffer capacity of the medium in the Seahorse software. Buffer capacity of the medium was determined by monitoring the pH change of DMEM following five additions of a known quantity of protons from 0.1 mol/l hydrochloric acid. Next, the glycolytic response was determined by subtracting the maximum PPR following the addition of glucose (25 or 2.5 mmol/l) and before oligomycin addition from the last PPR measurement before the addition of glucose. Each treatment was measured in at least duplicate wells and on at least four independent occasions (n = 4).

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Statistical analysis The Ryan–Joiner normality test was applied before all analyses. For samples that were not normally distributed, a logarithmic transformation (log10 ) was applied. Values are presented as medians and interquartile range or means + − S.D, depending on the measured level. Pearson correlations (two tails) were performed for human eHSP72, blood lipids, fasting glucose, insulin, TNF-α and HOMA-IR (homoeostasis model assessment of IR). After having checked for co-linearity, a stepwise multiple regression analysis (entry/removal threshold at P < 0.15) was conducted to investigate the strength of the association of HOMA-IR with the following parameters: percentage body fat, adipokines and eHSP72. An ANOVA general linear model (GLM) was performed to compare the subgroups (eHSP72 doses). A post-hoc Tukey test was further applied when appropriate. The α level was set at P < 0.05. Data were analysed using Minitab 16 software.

RESULTS Human blood analysis and eHSP72 correlations We detected a positive correlation between fasting plasma eHSP72, HOMA-IR (r = 0.528, P = 0.001) and TNFα (r = 0.389, P = 0.014) in elderly subjects (Table 1). In addition, the levels of triacylglycerols (triglycerides) (r = 0.339, P = 0.035), cortisol (r = 0.348, P = 0.03) and the leptin/adiponectin ratio (r = 0.334, P = 0.038) were also positively correlated with eHSP72. In addition, the stepwise multiple regression analysis revealed that, among the selected parameters (adiponectin, percentage body fat, TNF-α and HSP70), only HSP70 contributed significantly to the variability of HOMA-IR (R2 = 27.91 %, P = 0.001).

Chronic effects of eHSP72 on rat and human pancreatic β-cell lines

Clonal pancreatic BRIN-BD11 β-cell line insulin secretion was significantly reduced in a concentration-dependent manner by HSP72 (Figure 1A). Glucose consumption was also reduced by eHSP72 (Figure 1B). However, no significant changes in insulin secretion or glucose consumption was observed for the human β-cell line 1.1B4 (Figures 1C and 1D), indicating a cell-specific effect. In order to assess whether cell viability was affected, we tested three different assays that could provide information on eHSP72 toxicity. All assays indicated a significant decrease in cell viability that was most pronounced in the WST-1 assay, demonstrating dose-dependent effects in both cell lines (Figure 2B and 2E). Chronic exposure to eHSP72 induced a loss of BRIN-BD11 β-cell viability of at least 30 % in all assays (Figure 2A–2C), whereas human 1.1B4 β-cell viability was reduced by a maximum of 10 % (Figure 2D–2F).

Effect of HSP72 on BRIN BD11 and 1.1B4 bioenergetics Interestingly, acute (20 min) addition of eHSP72 to BRIN-BD11 and 1.1B4 β-cell lines resulted in significant changes in their bioenergetic profiles. Although no significant effect was observed

eHSP72 correlates with insulin resistance and promotes pancreatic β-cell death

Table 1

Median and interquartile ranges of body composition, inflammatory and metabolic variables in relation to eHSP72 in elderly people ∗ P  0.05 and ∗∗ P  0.001. IQR, interquartile range. AU, arbitrary units; LDL, low-density lipoprotein; HDL, high-density lipoprotein. Value Parameter

Median

Pearson correlation IQR

r

P value

eHSP72 (ng/ml)

1.06

0.13–13.7

–

–

Glucose (mmol/l)

5.4

4.7–6.4

0.130

0.429

Insulin (milli-units/l)

9.01

1.8–36.4

0.537

0.001∗∗

HOMA-IR (AU)

2.4

0.4–9.9

0.528

0.001∗∗

HOMA-β (AU)

97.5

18.9–280

0.521

0.001∗∗

TNF-α (pg/ml)

18.15

16.1–29.6

0.389

0.014∗

Leptin (ng/ml)

40.4

1.5–182.4

0.313

0.052

Adiponectin (ng/ml)

7.15

2.55–18.6

− 0.072

0.662

Leptin/adiponectin ratio (AU)

6.39

0.2–26.67

0.334

0.038∗

Cortisol (pg/ml)

24.3

10.2–53

0.348

0.030∗

LDL (mmol/l)

2.89

1–4.8

0.036

0.834

HDL (mmol/l)

1.42

0.85–2.35

0.053

0.747

Total cholesterol (mmol/l)

5.08

3–7

0.072

0.664

Triacyglycerols (mmol/l)

1.11

0.4–4.47

0.339

0.035∗

Body fat (%)

33.7

13.1–48.6

0.099

0.552

Lean mass (g)

46.8

31.1–70.2

0.085

0.611

in the BRIN-BD11 cell OCR (an indirect measure of oxidative phosphorylation), the glycolytic response of these cells to high glucose was weakened in the presence of eHSP72, as reflected by a decrease in the PPR of 56.3 pmol of H + /min (Figure 3A). However, this effect did not appear to be concentration-dependent (Figure 3A). Conversely, the OCR for 1.1B4 β-cells decreased by approximately 2 pmol of O2 /min in response to glucose, and there was a slight enhancement of the PPR by 16 pmol of H + /min in the presence of eHSP72 (40 ng/ml), further indicating a celltype-dependent effect of HSP72 (Figure 3A). Moreover, chronic exposure of both cell lines to eHSP72 pre-treatment significantly reduced the OCR by approximately 2 pmol of O2 /min in a concentration-dependent manner, and this was observed for both cell lines (Figure 3B). However, no significant modulation of the PPR was detected following eHSP72 pre-treatment for 24 h (Figure 3B). eHSP72 also had an impact on other bioenergetic parameters in a time-, dose- and cell-type-specific manner. Acute exposure of the BRIN-BD11 β-cell line to eHSP72 caused a significant concentration-dependent and transient increase in β-cell proton leak that was not observed under chronic 24 h conditions (Figure 4A). Here, the level of O2 consumption attributed to proton leak increased from 18 pmol of H + /min at 0 ng/ml eHSP72, to 24 pmol of H + /min at 40 ng/ml eHSP72 following 24 h pretreatment. Interestingly, this effect was not evident in the human cell line at either treatment time (Figure 4A). However, chronic eHSP72 pre-treatment enhanced the level of O2 consumption that was directly required for ATP production in both cell lines and by approximately the same proportion (Figure 4B). eHSP72 at 0.2 ng/ml increased BRIN-BD11 β-cell ATP turnover by 6 pmol of O2 /min and by 4 pmol of O2 /min in the 1.1B4 β-cell line (Figure 4B). At 40 ng/ml eHSP72, ATP turnover was increased in BRIN-BD11 cells by 14 pmol of O2 /min, whereas in 1.1B4 cells it

was increased by 12 pmol of O2 /min (Figure 4B). Acute exposure (20 min) to eHSP72 increased the OCR related to ATP turnover in BRIN-BD11 cells by approximately 10 pmol of O2 /min, but no significant effect was detected in the human cell line following these short time frames, indicating cell-line-specific effects. Coupling efficiency was also determined in both cell lines and under both treatment methods, but no significant modulation was observed (Figure 4C). However, it was clear from these data that the human cells exhibited a greater coupling efficiency (75 %) than the rat cells (60 %) (Figure 4C). This may be explained directly by the greater proton leak observed in rat control cells (approximately 20 % compared with 10 % for human cells;, Figure 4A).

Chronic effects of eHSP72 on rodent pancreatic islets and TLR inhibition Insulin secretion from rodent islets was decreased significantly when incubated in the presence of 40 ng/ml eHSP72 compared with control islets (Figure 5). Interestingly, the presence of the TLR2/TLR4 inhibitor OxPAPC was able to restore insulin secretion in the groups incubated with 40 ng/ml eHSP72 (Figure 5). Curiously, OxPAPC alone increased insulin secretion in all of the groups, suggesting that the isolation procedure may lead to activation of TLRs in isolated islets that may limit insulin secretion under normal culture conditions. No difference was found in nitrite production in any of the cultures or conditions tested.

DISCUSSION A major finding of the present study was that elevated eHSP72 was positively correlated with IR in elderly people. We also

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Figure 1 Chronic insulin secretion after 24 h incubation with eHSP72 at different concentrations (A and C) Chronic insulin secretion after 24 h incubation with eHSP72 in different concentrations. (B and D) Chronic glucose consumption in the presence of eHSP72. Values are means + − S.D. of three separate preparations. ∗ P < 0.05 compared with control; †P < 0.05 compared with 0.2 ng/ml eHSP72; ‡P < 0.05 compared with 4 ng/ml eHSP72; θ P < 0.05 compared with 8 ng/ml eHSP72; and ε P < 0.05 compared with 40 ng/ml eHSP72.

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confirmed that eHSP72 was correlated with inflammatory status as indicated by the plasma TNF-α level. The consequences of chronically elevated eHSP72 on β-cell function were explored in vitro. We report, for the first time, that exposure to eHSP72 for chronic periods (24 h) can have an impact on β-cell line (BRIN-BD11 and 1.1B4) metabolism and functional integrity. Additionally, pharmacological-dependent inhibition of TLR2 and TLR4 ligation in the presence of eHSP72 may benefit islet function, including insulin secretion, indicating that TLRs may regulate insulin secretion in vivo. The fact that eHSP72 is increased in T1DM and T2DM may indicate that this protein is involved in β-cell failure and dysfunction in both types of diabetes. Adipose tissue expansion is a hallmark of IR in diabetes and aging. Aging is associated with fat redistribution, which is characterized by the loss of peripheral subcutaneous fat and the accumulation of visceral fat [41]. Visceral adipose tissue is more important for development of metabolic diseases than subcutaneous adipose tissue [41]. Visceral fat accumulation occurs in aging even in people with a normal BMI, increasing the release of adipokines such as leptin, diminishing adiponectin levels and increasing macrophage infiltration, which leads to low-grade inflammation in obese and older people [42]. Leptin is associated with IR, whereas higher adiponectin levels are correlated with improved insulin sensitivity [43]. The ratio between the two adipokines may be used as an index for IR and cardiovascular diseases [43]. Interestingly, eHSP72 was correlated with the leptin/adiponectin ratio, indicating that the higher levels of eHSP72 may be associated with adiposity. Indeed, divergence between the content of eHSP72 and iHSP72 were found to be dependent on the body fat composition in obese people without and with T2DM [36]. Lean mass is associated with hormonal status, thus changes in anabolic [growth hormone, testosterone and IGFs (insulin-like growth factors)] and catabolic (cortisol) hormones may be provoked by aging [44]. Although eHSP72 was correlated with the level of hormones that reflect body composition, no direct correlation was found with respect to lean or fat mass. Thus the level of adipokines may be as important as the adiposity level as determinants of eHSP72 release. The positive correlation with HOMA-β (HOMA for β-cell function) may be a consequence of elevated IR. However, our results for insulin secretion, function and viability in clonal pancreatic β-cells and pancreatic islets demonstrated that eHSP72 had negative effects on these parameters and exclude any possible positive effect on insulin secretion. Thus we believe that increased IR leads to a compensatory increase in insulin secretion by the β-cell that is maintained while these cells are still healthy and before a dysfunctional effect becomes dominant. In contrast with its intracellular anti-inflammatory effect, eHSP70 plays a role as an activator of inflammation [17]. At the extracellular compartment, this protein can bind to cellsurface receptors known as the TLRs (TLR2/TLR4) [46]. This interaction can lead to the activation of pro-inflammatory signalling proteins such as MyD88 (myeloid differentiation factor 88) and TIRAP (Toll/interleukin-1 receptor domain-containing adaptor protein), that activate IKK (inhibitor of nuclear factor κB kinase), p38, JNK (c-Jun N-terminal kinase) and ultimately

eHSP72 correlates with insulin resistance and promotes pancreatic β-cell death

Figure 2

Effects of 24 h culture in the presence of various concentrations of eHSP72 on cell viability in BRIN-BD11 (A–C) and 1.1B4 (D–F) cells (A and D) Cell viability after 24 h incubation with eHSP72 at different concentrations using the Neutral Red assay, (B and E) using the cell proliferation WST-1 reagent assay and (C and F) using the LDH release assay. Values are means + − S.D. of three separate preparations. ∗ P < 0.05 compared with control; †P < 0.05 compared with 0.2 ng/ml eHSP72; ‡P < 0.05 θ ε compared with 4 ng/ml eHSP72; P < 0.05 compared with 8 ng/ml eHSP72 and P < 0.05 compared with 40 ng/ml eHSP72.

NF-κB (nuclear factor κB), and induce changes in gene expression [47]. The underlying mechanisms which may lead to IR could involve eHSP70-mediated stimulation of TLR2/TLR4. Accordingly, TLR2/TLR4-dependent activation of JNKs promotes phosphorylation of IRS-1 (insulin receptor substrate-1) at Ser307 in rodents (equivalent to Ser312 in humans), leading to inhibition of Akt activation [48], to a reduced glucose uptake by sensitive tissues and to a state of resistance to insulin action. Primary β-cells and some cell lines, such as HP62 and RINm5f cells, have been reported to express TLR2 and TLR4 [49], and our group has detected mRNA expression of the vari-

ous TLRs in the rat β-cell line BRIN-BD11, possibly contributing to the deleterious effects of LPS (lipopolysaccharide) on β-cell insulin secretion [50]. Although TLR2 is involved in the responses to a range of constituents of the cell walls of pathogens, TLR4 is a subclass that can be ligated by LPS and by non-bacterial agonists, such as saturated fatty acids [47]. Activation of TLR4 signalling at the cell surface induces activation of specific intracellular inflammatory pathways, which in sensitive cells and tissues are related to the induction of IR due to suppression of insulin signalling pathways. Interestingly, TLR2 maps to the same chromosome region as the NOD (non-obese

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Figure 3

Effects of acute and chronic eHSP72 exposure on basal mitochondrial respiration (OCR) and glycolysis (PPR) in BRIN-BD11 and 1.1B4 cells (A) Effect of acute (20 min) eHSP72 exposure on the OCR and PPR in response to 25 mmol/l glucose. (B) Effect of chronic (24 h) eHSP72 pre-treatment on the OCR and PPR following addition of medium containing 2.5 mmol/l glucose. ∗ P < 0.05 compared with the OCR for BRIN-BD11 control; †P < 0.05 compared with the OCR for BRIN-BD11 0.2 ng/ml; ‡P < 0.05 compared with the OCR for BRIN-BD11 4 ng/ml; θ P < 0.05 compared with the PPR for BRIN-BD11 control; P < 0.05 compared with the OCR for 1.1B4 control; ¥ P < 0.05 compared with the OCR for 1.1B4 4 ng/ml; P < 0.05 compared with the PPR for 1.1B4 control.

diabetic) mouse diabetes-susceptibility gene Idd17 [51], and the gene encoding TLR4 maps to chromosome 9q33 [52], which has also been mapped to an unnamed T1DM locus [53]. Interestingly, HSP72 has been found to serve as a ligand for innate TLRs that regulate the inflammatory functions of macrophages and other leucocytes [54]. Thus such molecules may have a broad role in

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autoimmunity and other types of inflammation through their multiple roles in signalling both innate receptors on leucocytes and antigen-specific receptors on lymphocytes. Furthermore, activation of TLRs with various TLR agonists, including LPS, was able to boost ATP synthesis and secretion in macrophages and platelets [55,56]. These features may correlate with the results

eHSP72 correlates with insulin resistance and promotes pancreatic β-cell death

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Figure 5 Effects eHSP72 on insulin secretion in rodent islets Pancreatic islets were isolated from wild-type Black mice (C57BL/6J, 8–12 weeks of age). Each pancreas was excised and inflated with a collagenase solution (0.5 mg/ml) and chopped into small pieces. Digestion was initiated during sample incubation at 37 ◦ C for 3 min with constant shaking. The digest was washed with 0.1 % BSA/Krebs solution (5.6 mmol/l glucose) and the islets were sedimented by gentle centrifugation (500 g for 10min at 4 ◦ C) with Histopaque 1077. Islets were resuspended in Krebs buffer containing 0.1 % BSA, individually picked and cultured in RPMI 1640 culture medium in the presence of 0, 0.2 and 40 ng/ml of recombinant HSP72 for 24 h. To test whether the action of eHSP72 is mediated by the activation of TLR2/TLR4, the islets were co-incubated with eHSP72 (at the same indicated concentrations) and the TLR2/TLR4 inhibitor OxPAPC. ∗ P < 0.05 compared with same eHSP72 concentration but no OxPAPC; †P < 0.05 compared with no eHSP72 plus OxPAPC; ‡P < 0.05 compared with 0.2 ng/ml eHSP72 plus OxPAPC; ε P < 0.05 compared with 40 ng/ml eHSP72 plus OxPAPC.

described in the present study, and the increase in ATP turnover detected may be a function of cellular stress and may also explain some of the observations discussed below. We have clearly demonstrated that eHSP72 induced bioenergetic and metabolic changes in BRIN-BD11 cells which had an impact on insulin secretion. The fact that we did not observe differences in insulin secretion in primary islets in the presence of 0.2 ng/ml eHSP72 does not exclude the possibility that eHSP72 may induce β-cell dysfunction in T2DM. Since the time of incubation was only 24 h, perhaps a longer exposure is needed. Furthermore, as no significant modulation of insulin secretion or glucose consumption was observed in human 1.1B4 cells, this may indicate that either the physiological concentrations of eHSP72 chosen were not high enough to promote this effect in vitro or that this relatively new clonal β-cell line has an altered sensitivity to HSPs. Indeed, eHSP72 promoted a small but significant reduction in 1.1B4 cell viability (10 %) and also altered the kinetics of respiration in this cell line, which may indicate that these cells are responsive to eHSP72, but at the concentrations used had no effect on the intracellular regulation of insulin secretion in this model. The variability of the results between the cell types may be explained by the sensitivity and expression of TLRs among the cells. Although BRIN-BD11 β-cells express all

isoforms of TLRs [50], the expression in 1.1B4 cells remains to be determined. The fact that HSPs have an impact on cellular bioenergetics is a novel and important finding. eHSP72 induced effects that were dependent on cell line and time of exposure (summarized in Table 2). In summary, chronic exposure (24 h) to eHSP72 increased ATP production and turnover (as determined by the ATP-production-dependent OCR, see Figure 4B), while decreasing basal oxygen consumption. Keeping in mind that eHSP72 did not have an impact on human insulin secretion, it is interesting that it also had no significant effect on proton leak in this cell line, although enhanced proton leak and decreased insulin secretion were observed in BRIN-BD11 β-cells following chronic exposure to eHSP72. Proton leak refers to the process by which protons from the intramembrane space leak back across the inner mitochondrial membrane to the matrix without generating ATP via ATP synthase, and this process may be facilitated by membrane proteins such as UCP (uncoupling protein)-2 or adenine nucleotide translocase [57,58]. UCP-2, in particular, has been implicated in regulating GSIS (glucose-stimulated insulin secretion), and deletion of UCP-2 via interfering RNA in vitro or by genetic knockout in mice has been shown to improve GSIS [59,60]. Intriguingly, no significant change in coupling efficiency was detected in either cell line or under either treatment condition (Figure 4C). However, others have recently reported that, in glutaredoxin-2 whole-body knockout mice, muscle tissue showed enhanced expression of UCP-3, along with increased proton leak, but no significant change in coupling efficiency was evident [61]. The underlying mechanisms remain unclear, but it is believed that UCP-3 activity is regulated by glutathionylation and redox interactions with thioredoxin-2 and glutaredoxin-2, and that these interactions may limit ROS (reactive oxygen species) generation that potentially influences ATP turnover [61]. This concept further indicates that ROS and cell stress may play a role in our observations. Intriguingly, ATP turnover was found to be elevated in all eHSP72 treatments with the exception of acute exposure in the human cell line (Figure 4B). These results may partially explain the fact that, although proton leak was increased in BRIN-BD11 cells, there was also a parallel increase in ATP turnover (reflecting demand) that occurred as early as 20 min after eHSP72 exposure, continuing to 24 h, and consequently no change in coupling efficiency was observed. Furthermore, the glycolytic flux of BRINBD11 cells in response to elevated glucose (25 mmol/l) was significantly blunted in the presence of eHSP72 (Figure 3A), while eHSP72 increased glycolysis in 1.1B4 cells (Figure 3A). These findings suggested that, even though glycolysis was dampened in BRIN-BD11 cells, ATP turnover was still elevated, potentially through oxidative phosphorylation, although no significant acute changes in the OCR were evident (Figure 3A). It may also suggest that the response observed in the rat cell line was a response

Figure 4 Effects of acute and chronic eHSP72 exposure on proton leak, ATP turnover and coupling efficiency in BRIN-BD11 and 1.1B4 cells Effect of acute (20 min) and chronic (24 h) eHSP72 exposure on the OCR related to proton leak (A), the OCR related to ATP turnover (B) and coupling efficiency (C) in BRIN-BD11 and 1.1B4 cells. ∗ P < 0.05 compared with BRIN-BD11 control; †P < 0.05 compared with BRIN-BD11 0.2 ng/ml; P < 0.05 compared with 1.1B4 control; and P < 0.05 compared with 1.1B4 0.2 ng/ml.

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√

X √

X √

X

X √

Agreement between cell lines

to stress and that the elevation in ATP turnover was related to processes unconnected to the insulin secretory mechanism. Consequently, a decrease in secretion was observed. Taken together, the lower viability and function of the β-cells, along with the changes in ATP turnover, may indicate that the increased energy expenditure could be related to DNA repair and the synthesis of antioxidants, such as glutathione, that are crucial for the maintenance of the redox state [62].

− Proton leak

↓ Basal OCR (2.5 mmol/l glucose)

− Basal glycolysis (2.5 mmol/l glucose)

↓ Basal OCR (2.5 mmol/l glucose)

− Basal glycolysis (2.5 mmol/l glucose)

↑ ATP turnover ↑ ATP turnover

↑ Proton Leak

↑ Basal glycolysis (25 mmol/l glucose)

Chronic eHSP72 exposure (24 h)

↓ Basal OCR (25 mmol/l glucose)

− Proton leak − Proton leak

− Basal OCR (25 mmol/l glucose)

− ATP turnover

↓ Basal glycolysis (25 mmol/l glucose)

1.1B4

↑ ATP turnover Acute eHSP72 exposure (20 min)

Cell line

BRIN-BD11

Conclusions and perspectives

Treatment

Table 2 Summary of acute and chronic eHSP72 effects on β-cell bioenergetics using the Seahorse XFe 96 technology Comparison of the bioenergetic response observed between both types of pancreatic β-cell lines as a consequence of the two different treatment regimens with eHSP72. ↑, increase in the parameter response; ↓, decrease in the √ parameter response; − , no change in the parameter response; X, disagreement between the parameter response between the two cell lines and at the same eHSP72 treatment time point; and , agreement between the parameter response between the two cell lines and at the same eHSP72 treatment time point.

eHSP72 correlates with insulin resistance and promotes pancreatic β-cell death

In the present study, we have shown that eHSP72 is positively correlated with IR and inflammation in elderly people. This may indicate a role for eHSP72 in the impairment of insulin signalling in the skeletal muscle that occurs with advanced age and in T2DM. In addition, we have demonstrated, for the first time, that chronic exposure of clonal pancreatic β-cells (BRIN-BD11 and 1.1B4) and islets to increased eHSP72 levels induced β-cell death and altered cell bioenergetics. Since in T1DM there is a dramatic increase in eHSP72 and in T2DM and aging there is a slow chronic increase in the concentration of this protein, we suggest that the chronic exposure of pancreatic β-cells to eHSP72 may lead to β-cell failure and loss of functional integrity in vivo. TLRs may contribute to the mechanism of transduction of eHSP72 signalling in β-cells, indicating a potential target for drug development for diabetes. The function of HSP72 as a molecular chaperone may suggest that increases in HSP72 levels in the plasma are necessary to combat the oxidative damage induced by ROS and RNS (reactive nitrogen species) against proteins. In addition, as lymphocytes are able to release eHSP72, we suggest that this protein is a component of the inflammatory process. We suggest that eHSP72 could be used as an important marker of elevated oxidative stress and inflammation. Nevertheless, our results indicate that eHSP72 is more than a marker of disease: it appears to have its own role for the pathogenesis of IR and β-cell dysfunction.

CLINICAL PERSPECTIVES • eHSP72 is increased in the plasma of both types of diabetes and is positively correlated with inflammatory markers; however, no studies have investigated the relationship between eHSP72, insulin sensitivity and pancreatic β-cell function. • In the present study, we have shown that eHSP72 is positively correlated with IR and inflammation in elderly people. This may indicate a role for eHSP72 in the impairment of insulin signalling that occurs with advanced age and in T2DM. In addition, we demonstrated, for the first time, that chronic exposure of pancreatic β-cells and islets to increased eHSP72 induced β-cell death and altered cell bioenergetics. • Since in T1DM there is a dramatic increase in eHSP72 and in T2DM and aging there is a slow chronic increase in the concentration of this protein, we suggest that the chronic exposure of pancreatic β-cells to eHSP72 may lead to β-cell failure and loss of functional integrity.

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AUTHOR CONTRIBUTION

Mauricio Krause, Kevin Keane and Josianne Rodrigues-Krause completed all of the experiments described in the paper. Mauricio Krause, Domenico Crognale, Brendan Egan and Giuseppe De Vito were responsible for the human study. Mauricio Krause and Philip Newsholme were responsible for the study design and production of the first version of the paper. Philip Newsholme, Kevin Keane, Josianne Rodrigues-Krause and Giuseppe De Vito contributed with research advice and revision of the paper. Philip Newsholme, Colin Murphy and Giuseppe De Vito were responsible for grant support with respect to Technological Sector Research: Strand III - Core Research Strengths Enhancement Scheme (Ireland), and Giuseppe De Vito for the Enterprise Ireland grant.

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The work was supported by Enterprise Ireland [grant number CC20080001], the Institute of Technology Technological Sector Research (TSR): Strand III - Core Research Strengths Enhancement Scheme (Ireland), the School of Biomedical Sciences, Curtin University (Perth, Australia), and : Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq - Brazil).

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ACKNOWLEDGEMENTS

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We thank the Department of Science, Institute of Technology Tallaght, Dublin, Ireland, UCD School of Biomolecular and Biomedical Science, UCD Institute for Sport and Health and the TSR: Strand III - Core Research Strengths Enhancement Scheme (Ireland), UCD School of Public Health, Physiotherapy & Population Science and the UCD Institute for Sport and Health (Dublin, Republic of Ireland) and The Enterprise Ireland for supporting this work. We also thank Professor Paulo Ivo Homem de Bittencourt Jr (Laboratory of Cellular Physiology, Department of Physiology Federal University of Rio Grande do Sul, Porto Alegre, Brazil) for his scientific advice on the HSP studies and the School of Biomedical Sciences, Faculty of Health Sciences, Curtin University, Perth, WA, Australia (for provision of research support and equipment).

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Received 29 October 2013/3 December 2013; accepted 11 December 2013 Published as Immediate Publication 11 December 2013, doi: 10.1042/CS20130678

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