Physiological concentrations of interleukin-6 directly promote insulin secretion, signal transduction, nitric oxide release, and redox status in a clonal pancreatic β-cell line and mouse islets

in Journal of Endocrinology
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  • 1 Biomedical Research Group (BMRG), UCD School of Biomolecular and Biomedical Science, School of Public Health, Department of Physiology, Federal University of Rio Grande do Sul School of Physical Education, School of Biomedical Sciences, School of Biomedical Sciences, Department of Science, ITT Dublin, Tallaght, Dublin 24, Ireland

Interleukin-6 (IL6) has recently been reported to promote insulin secretion in a glucagon-like peptide-1-dependent manner. Herein, the direct effects of IL6 (at various concentrations from 0 to 1000 pg/ml) on pancreatic β-cell metabolism, AMP-activated protein kinase (AMPK) signaling, insulin secretion, nitrite release, and redox status in a rat clonal β-cell line and mouse islets are reported. Chronic insulin secretion (in μg/mg protein per 24 h) was increased from 128.7±7.3 (no IL6) to 178.4±7.7 (at 100 pg/ml IL6) in clonal β-cells and increased significantly in islets incubated in the presence of 5.5 mM glucose for 2 h, from 0.148 to 0.167±0.003 ng/islet. Pretreatment with IL6 also induced a twofold increase in basal and nutrient-stimulated insulin secretion in subsequent 20 min static incubations. IL6 enhanced both glutathione (GSH) and glutathione disulphide (GSSG) by nearly 20% without changing intracellular redox status (GSSG/GSH). IL6 dramatically increased iNOS expression (by ca. 100-fold) with an accompanying tenfold rise in nitrite release in clonal β-cells. Phosphorylated AMPK levels were elevated approximately twofold in clonal β-cells and mouse islet cells. Calmodulin-dependent protein kinase kinase levels (CaMKK), an upstream kinase activator of AMPK, were also increased by 50% after IL6 exposure (in β-cells and islets). Our data have demonstrated that IL6 can stimulate β-cell-dependent insulin secretion via direct cell-based mechanisms. AMPK, CaMKK (an upstream kinase activator of AMPK), and the synthesis of nitric oxide appear to alter cell metabolism to benefit insulin secretion. In summary, IL6 exerts positive effects on β-cell signaling, metabolism, antioxidant status, and insulin secretion.

Abstract

Interleukin-6 (IL6) has recently been reported to promote insulin secretion in a glucagon-like peptide-1-dependent manner. Herein, the direct effects of IL6 (at various concentrations from 0 to 1000 pg/ml) on pancreatic β-cell metabolism, AMP-activated protein kinase (AMPK) signaling, insulin secretion, nitrite release, and redox status in a rat clonal β-cell line and mouse islets are reported. Chronic insulin secretion (in μg/mg protein per 24 h) was increased from 128.7±7.3 (no IL6) to 178.4±7.7 (at 100 pg/ml IL6) in clonal β-cells and increased significantly in islets incubated in the presence of 5.5 mM glucose for 2 h, from 0.148 to 0.167±0.003 ng/islet. Pretreatment with IL6 also induced a twofold increase in basal and nutrient-stimulated insulin secretion in subsequent 20 min static incubations. IL6 enhanced both glutathione (GSH) and glutathione disulphide (GSSG) by nearly 20% without changing intracellular redox status (GSSG/GSH). IL6 dramatically increased iNOS expression (by ca. 100-fold) with an accompanying tenfold rise in nitrite release in clonal β-cells. Phosphorylated AMPK levels were elevated approximately twofold in clonal β-cells and mouse islet cells. Calmodulin-dependent protein kinase kinase levels (CaMKK), an upstream kinase activator of AMPK, were also increased by 50% after IL6 exposure (in β-cells and islets). Our data have demonstrated that IL6 can stimulate β-cell-dependent insulin secretion via direct cell-based mechanisms. AMPK, CaMKK (an upstream kinase activator of AMPK), and the synthesis of nitric oxide appear to alter cell metabolism to benefit insulin secretion. In summary, IL6 exerts positive effects on β-cell signaling, metabolism, antioxidant status, and insulin secretion.

Introduction

Interleukin-6 (IL6) is a pleiotropic cytokine that influences metabolism in health and disease (Kamimura et al. 2003). IL6 plasma levels are acutely elevated following muscle contraction and chronically during obesity and type 2 diabetes (T2DM; Ostrowski et al. 1998, Spranger et al. 2003, Herder et al. 2005). It is accepted that pro-inflammatory cytokine levels (such as TNFα, IL1β, and/or IFNγ) are correlated with disease progression and severity of both type 1 and T2DM due to promotion of β-cell dysfunction. However, given that contracting skeletal muscle produces and releases substantial amounts of IL6, we hypothesized that this cytokine actually has positive effects on pancreatic β-cell function, so as to enhance insulin secretion.

The mechanism(s) by which IL6 exerts its diverse effects within target cells (such as skeletal muscle, hepatocytes, and adipocytes) has been suggested to include activation of AMP-activated protein kinase (AMPK). There is now accumulating evidence for a direct correlation between IL6 concentration and AMPK activity in metabolically sensitive tissues (Ruderman et al. 2006, Steinberg & Jorgensen 2007). AMPK activation stimulates fatty acid oxidation and increases glucose uptake (Alquier et al. 2007). Indeed, IL6 enhanced AMPK activity in both skeletal muscle and adipose tissue (Kelly et al. 2004), while enhancing glucose uptake in skeletal muscle (Carey et al. 2006b). Furthermore, IL6 provoked an increase in suppressor of cytokine signaling-3 (SOCS3) expression, as reported in the liver and skeletal muscle cells, which resulted in significant changes in glucose metabolism (Senn et al. 2003, Carey et al. 2006a).

It has recently been demonstrated that IL6 promotes insulin secretion in pancreatic islets via a mechanism that is dependent on the release of glucagon-like peptide-1 (GLP1; Ellingsgaard et al. 2011). Ellingsgaard et al. (2011) have shown that administration of IL6 or elevated IL6 concentrations in response to exercise stimulated GLP1 secretion from intestinal L-cells and pancreatic α-cells, so improving insulin secretion and glycemia.

However, the possibility of direct actions of IL6 at physiological concentrations in the pancreatic islet is still open to question. Given that i) elevated IL6 levels are an independent predictor of T2DM, ii) high-fat diet feeding increases systemic IL6 levels in vivo, which are necessary for expansion of pancreatic α-cell mass and maintenance of fasting circulating glucagon levels, and iii) evidence exists that high concentrations of IL6 directly regulate α- and β-cell function in the pancreatic islet (Ellingsgaard et al. 2008, 2011) while IL6 can promote insulin secretion via GLP1 release and subsequent β-cell action, we decided to investigate the possibility that this cytokine may directly regulate pancreatic β-cell/islet metabolism and insulin secretion at physiological concentrations. We hypothesized that pancreatic β-cell exposure to various physiological concentrations of IL6 (0–100 pg/ml) and one pathological concentration (1000 pg/ml) would provoke changes in metabolism that would favor both cell defense and insulin secretion. We suggest that IL6, through the activation of key metabolic enzymes such as AMPK and also by its anti-inflammatory action, is able to induce beneficial metabolic changes in β-cells. In addition, as nitric oxide (NO) has been shown to stimulate glucose-elicited insulin secretion at low intracellular concentrations (Krause et al. 2011), we investigated the effects of IL6 on l-arginine/NO metabolism, by studying the expressions of inducible NO synthase (iNOS) and arginase, as well as the metabolism of glutamate, which may be diverted to glutathione (GSH)-based defense of β-cells against NO excesses.

Materials and Methods

Culture of BRIN-BD11 β-cells, measurement of insulin secretion, and cell viability

Clonal rat insulin-secreting BRIN-BD11 cells were maintained in RPMI 1640 tissue culture medium supplemented with 10% (v/v) FCS, 0.1% (v/v) antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin), and 11.1 mmol/l d-glucose, pH 7.4. The clonal β-cell line BRIN-BD11 was chosen for this work as its metabolic, signaling, insulin secretory, and cell-viability responses to glucose, amino acids, as well as other stimuli have been well characterized (McClenaghan et al. 1996, McClenaghan & Flatt 1999, Brennan et al. 2003). The origin and the characteristics of BRIN-BD11 cells are described elsewhere (McClenaghan et al. 1996). The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air using a Forma Scientific incubator (Marietta, OH, USA). The cells were cultured in 50–70 ml RPMI 1640 tissue culture medium in T175 sterile tissue culture flasks. Cells were subsequently seeded into 24-well plates (0.75×105 cells/well) and allowed to adhere overnight. Cells were then washed with PBS after which they were incubated in fresh media, containing 11.1 mM d-glucose and 2 mM l-glutamine, in the absence or presence of IL6 (50 pg/ml). After 24 h incubation, an aliquot of the media was removed and centrifuged at 200 g for 5 min and used for quantitation of insulin and at 16 000 g for 10 min for the determination of metabolites (d-glucose consumption, urea, and nitrites). After the 24 h incubation, the cells were washed and ‘rested’ for 40 min in the presence of 1.1 mmol/l glucose in Krebs–Ringer bicarbonate buffer (KRB), pH 7.4 (and absence of IL6), followed by an acute stimulation period of 20 min in the presence of either 16.7 mM glucose and 10 mM alanine (a widely validated stimulus that results in a robust and reproducible secretory response in clonal BRIN-BD11 cells (Brennan et al. 2002)) or basal 1.1 mM glucose in KRB, pH 7.4, when an aliquot of the incubation medium was removed and centrifuged at 200 g for 5 min for analysis of insulin secretion using the Mercodia Ultrasensitive Rat Insulin ELISA kit (Mercodia, Uppsala, Sweeden). Cell viability was determined using the cell proliferation reagent WST-1, a tetrazolium salt that is cleaved by mitochondrial dehydrogenases in viable cells. Briefly, 100 μl of cell suspension (containing 2×104 cells/well) were plated in 96-well plates. After a 24 h reattachment period in culture, the cells were treated with IL6 (50 pg/ml) for a further 24 h. At the end of each experiment, the cell proliferation reagent WST-1 (10 μl) was added to each well and the cells were incubated at 37 °C for a period of between 0.5 and 1.5 h. Absorbance at 450 nm was measured at 0.5, 1.0, and 1.5 h using a kinetic plate reader (Spectramax Plus; Molecular Devices, Sunnyvalle, CA, USA).

Mouse islet isolation, culture, viability, and insulin secretion

Pancreatic islets were isolated from WT C57BL/6J mice. Pancreata were dissected and inflated with a Liberase TL grade solution (Roche 1815032). Digestion was initiated by shaking and mixing in a 37 °C water bath. The homogenate was washed with 0.1% (w/v) BSA KHB solution (5.5 mM glucose) and the islets were purified and isolated by centrifugation (800 g for 10 min at 4 °C) using Histopaque 1077 (Sigma–Aldrich). Islets were resuspended in fresh 0.1% (w/v) BSA Krebs buffer, manually picked and cultured for 24 h in the presence of RPMI 1640 culture medium supplemented with 10% (v/v) FCS, antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin), and 11.1 mmol/l d-glucose, pH 7.4, containing 11.1 mM d-glucose and 2 mM l-glutamine, in the presence or absence of a IL6 and kept at 37 °C in a humidified atmosphere of 5% CO2 in air.

In order to assess the early influence of IL6 on insulin secretion, ten islet equivalents (IEQs) per well (50 μl total volume) were manually picked (five replicates per test concentration) and prepared for culture as described earlier. The islets were then immediately plated in RPMI 1640 medium containing 5.5 mM glucose supplemented with FCS (10% v/v) and antibiotics. Cell viability was assessed using a MTT-based TOX-1 viability assay kit (Sigma) by cultivating ten IEQs per well (50 μl total volume) in phenol red-free RPMI 1640 culture medium supplemented with FCS (10% v/v) and antibiotics in the absence or presence of increasing concentrations of IL6 for 2 h. Glucose was added to incubation media at 5.5 mM. Cell viability in all the groups did not significantly change during the 2 h incubation. Insulin secretion was measured using Mercodia Ultrasensitive Mouse Insulin ELISA kit.

Enzymatic determination of metabolites

Urea production

Urea, a metabolite of l-arginine produced by the action of arginase, which competes with iNOS for l-arginine use, was measured in 50 μl samples using the 96-well QuantiChrom Urea Assay kit (DIUR- 500; Biovision, CA, USA) according to the manufacturer's instructions.

Nitrite production

The production of NO by BRIN-BD11 cells was assessed by measuring, in the culture medium, the formation of nitrites that were determined using the Griess Reagent System (Promega Medical Supply Co.).

Glucose consumption and production of glutamate

Glucose and glutamate concentrations were measured in the incubation medium using an YSI 7100 amino acid analyzer (YSI Inc., Yellow Springs, OH, USA). Briefly, an enzyme specific for the substrate of interest is immobilized between two membrane layers, polycarbonate and cellulose acetate. The substrate is oxidized as it enters the enzyme layer, producing hydrogen peroxide, which passes through cellulose acetate, thus reaching a platinum electrode where the hydrogen peroxide is oxidized. The resulting current is proportional to the concentration of the substrate.

Protein determination

Cellular protein was determined using a BCA protein assay kit, according to the manufacturer's instructions (Pierce, Rockford, IL, USA kit no. 23225). The assay uses a modification of the biuret reaction.

Western blot analysis of specific cell protein content

BRIN-BD11 cells and islets were seeded into six-well plates (1.5×106 cells/well), allowed to adhere overnight, and then washed with PBS after which they were incubated in fresh media, containing 11.1 mM d-glucose and 2 mM l-glutamine, in the presence or absence of IL6 for a further 24 h. After culture and cell lysis (using lysis buffer containing 0.1% SDS containing protease inhibitors (100 μM phenylmethanesulphonyl fluoride (PMSF), 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 20 μM TLCK (Nα-Tosyl-L-Lysine Chloromethyl Ketone)) and phosphatase inhibitors (1 mM sodium orthovanadate, 1 mM sodium molybdate, and 1 mM β-glycerophosphate)), equal amounts of cell protein extracts (30 μg) were prepared and subsequently subjected to 10% SDS–PAGE and electrophoretically transferred onto nitrocellulose sheets. The sheets were blocked in 5% milk protein or BSA and probed with polyclonal antibodies anti-NOS2, calmodulin-dependent kinase kinase (CaMKKα), or LKB1 (Sigma–Aldrich); anti-AMPK; and phosphorylated AMPK (AMPK-P) (Cell Signalling Technologies, Danvers, MA, USA). The blots were washed and visualized with a HRP-based Supersignal West Pico chemiluminescent substrate (Pierce).

In order to investigate early effects of IL6 on signaling protein expression in mouse pancreatic islets, 50 IEQs per well (50 μl total volume) were incubated in RPMI 1640 medium supplemented with FCS (10% v/v), antibiotics, and 5.5 mM glucose for just 2 h in the absence or presence of 100 pg/ml IL6. Afterward, islets were either immediately harvested or cultured for an additional 6 h in the absence of IL6. After that, islets were homogenized in 0.1% SDS containing 100 μM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 20 μM TLCK for protein quantitation. Then, samples were dissolved in Laemmli sample buffer and equal amounts of protein were electrophoresed, electrotransferred to nitrocellulose membranes, and immunoblotted for iNOS, heat-shock proteins (HSP70) (both 73-kDa constitutive and 72-kDa inducible forms), AMPK catalytic subunit, phospho (Ser487)-AMPK, or tubulin using monoclonal primary antibodies (Sigma) and biotin-labeled secondary antibodies (Sigma) to be revealed by enhanced chemiluminescence (ECL-Plus, GE HealthCare (Little Chalfont, UK)). A 70-kDa family of HSP70 was included in the study because HSP70 is known to block NF-κB-dependent iNOS expression in 6 h cultures (Gabai et al. 1997, Rossi et al. 2000).

Measurement of GSH and glutathione disulphide content

As GSH is the most important antioxidant systems that β-cells use against redox-threatening situations (for instance, in immunoinflammatory situations triggered by inflammatory cytokines (Krause et al. 2007)), BRIN-BD11 cells were seeded into six-well plates (2×106 cells/well), allowed to adhere overnight, and then washed with PBS after which they were incubated in fresh media, containing 11.1 mM d-glucose and 2 mM l-glutamine, in the presence or absence of sublethal pro-inflammatory cytokine cocktail with different concentrations of l-arginine (above). Afterward, cells were rinsed twice with PBS and disrupted in 200 μl of 5% (w/v) metaphosphoric acid on ice. After centrifugation (14 000 g, 5 min at room temperature), cell lysates were spectrophotometrically (415 nm) assayed on a temperature-controlled microplate reader (Molecular Devices SpectraMax Plus 384) by a modification of the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)/glutathione disulphide (GSSG) reductase recycling method, using the N-ethylmaleimide conjugating technique for GSSG sample preparation (Krause et al. 2007). Samples (10 μl) were assayed in 105 μl final volume in 96-well polystyrene plates at 37 °C in the presence of 10 mM DTNB, 0.17 mM β-NADPH (dissolved in 0.5% (w/v) NaHCO3 as a stabilizing agent), and 0.5 U/ml GSSG reductase (EC 1.6.4.2).

Statistical analysis

The results are presented as mean±s.d. Results of digitalized images were expressed as the mean±s.d. using GAPDH or tubulin (where appropriate) as an expression control. Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software (La Jolla, CA, USA). Differences were considered significant at a P value of <0.05.

Results

The effect of various concentrations of IL6 on i) insulin secretion and glucose consumption in 24 h cultured BRIN-BD11 cells, ii) insulin secretion from a subsequent 20 min acute stimulation assay using clonal β-cells, and iii) insulin secretion from 2 h incubated mouse islets

Insulin secretion from clonal BRIN-BD11 β-cells incubated for 24 h with various IL6 concentrations (0–100 pg/ml) was increased in a dose-dependent manner (Fig. 1A). Glucose consumption was also increased from 20.3±1.2 (μmol/mg protein) to 25.13±0.93 at 50 pg/ml IL6 (ca. 14% compared with control groups), indicating enhanced metabolic stimulus-secretion coupling. IL6 treatment also increased insulin secretion over 24 h in a time-dependent manner (Fig. 1C) and also positively influenced nutrient-induced insulin secretion in an acute (20 min) stimulation assay immediately following 24 h incubation in the presence of IL6 (Fig. 1B), while IL6 promoted a dose-dependent increase in basal insulin secretion (at 1.1 mM glucose) in equivalent experimental conditions, by 82% compared with cells cultured in the absence of IL6 for the prior 24 h period (Fig. 1B). Moreover, when BRIN-BD11 β-cells were submitted to the robust nutrient challenge (incubation in the presence of 16.7 mM glucose plus 10 mM l-alanine for 20 min), following the 24 h treatment with IL6, an additional dose-dependent increase in insulin release was observed, over and above the level produced from cells cultured in the absence of IL6 for the previous 24 h period (Fig. 1B).

Figure 1
Figure 1

Chronic insulin secretion (24 h), acute response (20 min), and time course of insulin secretion in BRIN-BD11 cells under exposure to IL6. Clonal insulin-secreting BRIN-BD11 cells were seeded into 24-well plates (0.75×105 cells/well) and allowed to adhere overnight. Cells were then washed with PBS and cultured in fresh media, containing 11.1 mM d-glucose and 2 mM l-glutamine, in the absence or presence of IL6 (0–100 pg/ml). An aliquot of the media was removed for the measurement of chronic insulin secretion response (A). After a 24 h incubation period, cells were washed and ‘rested’ for 40 min in the presence of 1.1 mM glucose in KRB, pH 7.4 (and absence of IL6), followed by an acute stimulation period of 20 min in the presence of either 16.7 mM glucose or 10 mM alanine (a standardized stimulus that results in a robust and reproducible secretory response in normal conditions for BRIN-BD11 (Brennan et al. 2002)) or basal 1.1 mM glucose in KRB, pH 7.4, when an aliquot of the incubation medium was assessed for insulin secretion response as described in the Materials and Methods section (B). In addition, to observe the time course of insulin secretion in response to IL6, cells were incubated for 24 h (with 50 pg/ml of IL6) where an aliquot of the media was collected at different times (C). Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Data are the mean±s.d. of three independent determinations using eight wells per experimental condition. Significance: ΔP<0.05 vs control group; P<0.05 vs 25 pg/ml IL6; P<0.05 vs 50 pg/ml IL6; ψP<0.05 vs 100 pg/ml IL6; ¥P<0.05 vs respective basal treatment at the same IL6 concentration).

Citation: Journal of Endocrinology 214, 3; 10.1530/JOE-12-0223

With respect to primary mouse islet cells, insulin secretion at a basal glucose concentration of 5.5 mM over a standard islet incubation period of 2 h was increased in the presence of IL6 in a dose-dependent manner (Fig. 2). Insulin secretion increased from 0.148±0.003 to 0.167±0.003 ng/islet (13%) on increasing IL6 from zero to 100 pg/ml and to 0.173±0.005 ng/islet (at 1000 pg/ml, 17%). Insulin secretion was not significantly increased by IL6 when mouse islets were incubated in the presence of 16.7 mM glucose for 2 h (from 1.668±0.073 ng/ml without IL6 to 1.674±0.073 ng/ml in the presence of 10 000 pg/ml of IL6).

Figure 2
Figure 2

Pancreatic islet insulin secretion under exposure to IL6. Pancreatic islets were isolated from WT C57BL/6J mice. Islets were resuspended in fresh 0.1% (w/v) BSA containing Krebs buffer, picked, and cultured for 24 h in the presence of RPMI 1640 culture medium supplemented with 10% (v/v) FCS, antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin), and 11.1 mM d-glucose, pH 7.4. Subsequently, islets were cultured in KRB containing 5.5 mM d-glucose and 2 mM l-glutamine, in the absence or presence of IL6 and kept at 37 °C in a humidified atmosphere of 5% CO2 in air for 2 h. Insulin secretion was measured using Mercodia Ultrasensitive Mouse Insulin ELISA kit. Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Data are the mean±s.d. of three separate mouse extractions of primary islets. *P<0.05 vs basal treatment.

Citation: Journal of Endocrinology 214, 3; 10.1530/JOE-12-0223

The effect of IL6 on i) AMPK levels and extent of protein phosphorylation, ii) CaMKKα levels, and iii) LKB1 levels

AMPK is a serine/threonine protein kinase, which serves as an energy sensor in all eukaryotic cell types. It responds to a decreased ATP/AMP ratio by enhancing processes that generate cellular ATP and inhibiting others that consume ATP but are not acutely necessary for survival. In addition to allosteric control and to achieve full activation of AMPK, the enzyme must be phosphorylated. In our model, treatment with IL6 (50 pg/ml) for 24 h induced an increase in the level of AMPK-P protein by 89%, whereas the level of the total amount of AMPK protein (phosphorylated plus dephosphorylated forms) was reduced by 43% (Figs 3B and 4B). The level of the possible upstream regulator of AMPK, CaMKK, was increased by more than 50% by 24 h incubation in the presence of IL6 (Figs 3C and 4D). However, the levels of an alternative upstream regulator of AMPK, LKB1, were not significantly altered by IL6 (Figs 3D and 4C). We also investigated the early effects of IL6 on signaling protein levels in mouse pancreatic islets after 2 and 6 h in the absence or presence of 100 pg/ml IL6. We demonstrated that IL6 significantly increased the levels of AMPK-P without any significant affect on HSP72 or iNOS after 6 h (Fig. 5).

Figure 3
Figure 3

Determination of AMPK, AMPK-P, LKB1, and CaMKKα expression in clonal insulin-secreting cells incubated in the presence of IL6. BRIN-BD11 cells were seeded into six-well plates (1.5×106 cells/well) and allowed to adhere overnight and cultured for 24 h. Cells were then washed with PBS after which they were incubated in fresh media, containing 11.1 mM d-glucose and 2 mM l-glutamine, in the absence or presence of IL6 (50 pg/ml) for 24 h as described in the legend of Fig. 1. Afterward, equal amounts of BRIN-BD11 cell protein extracts were prepared, electrophoresed (10% SDS–PAGE), and transferred onto a nitrocellulose membrane. A representative image of the protein levels is shown (A). AMPK and its phosphorylated form (B), CaMKKα and LKB1 (C), were then probed with specific antibodies and visualized after HRP/chemiluminescent reaction. Results are presented in arbitrary units relative to GAPDH expression. Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Data are the mean±s.d. of at least three independent determinations using three wells per experimental condition. Significance: ΔP<0.05 vs control group.

Citation: Journal of Endocrinology 214, 3; 10.1530/JOE-12-0223

Figure 4
Figure 4

Determination of AMPK, AMPK-P, LKB1, and CaMKKα expression in pancreatic islets from WT mice incubated in the presence of IL6 for 24 h. Determination of AMPK, AMPK-P, LKB1, and CaMKKα expression in pancreatic islets from WT mice incubated in the presence of IL6 for 24 h. Isolated mouse islets were seeded into six-well plates (150 IEQs/well) and cultured for 24 h, as described in the legend of Fig. 2. Afterward, equal amounts of islet protein extracts were prepared, electrophoresed (10% SDS–PAGE), and transferred onto a nitrocellulose sheet. Protein forms were then probed with specific antibodies and visualized after HRP/chemiluminescent reaction. A representative image of the protein levels is shown (A). AMPK and its phosphorylated form (B), LKB1 (C) and CaMKKα (D). Results are presented in arbitrary units relative to GAPDH expression. Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Data are the mean±s.d. of three separate mouse extractions of primary islets using six wells per experimental condition. Significance: ΔP<0.05 vs control group.

Citation: Journal of Endocrinology 214, 3; 10.1530/JOE-12-0223

Figure 5
Figure 5

Determination of AMPK, AMPK-P, iNOS, and HSP72 expression in pancreatic islets from WT mice incubated in the presence of IL6 for 2 and 6 h. iNOS (A), HSP70 (B), P-AMPK (C), total AMPK (D), P-AMPK / Total AMPK (E). Results are presented in arbitrary units relative to Tubulin expression. A representative image of the protein levels is shown (F). In order to investigate the early effects of IL6 on signaling protein expression in mouse pancreatic islets, 50 IEQs per well (50 μl total volume) were incubated in RPMI 1640 medium supplemented with FCS (10% v/v), antibiotics, and 5.5 mM glucose for just 2 h in the absence or presence of 100 pg/ml IL6. Afterward, islets were either immediately harvested or cultured for additional 6 h in the absence of IL6. After that, islets were homogenized in 0.1% SDS containing 100 μM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 20 μM TLCK for protein quantitation. Then, samples were dissolved in Laemmli sample buffer and equal amounts of protein were electrophoresed, electrotransferred to nitrocellulose membranes, and immunoblotted for iNOS, HSP70 (both constitutive and inducible forms), AMPK catalytic subunit, phospho (Ser487)-AMPK or tubulin using monoclonal primary antibodies (Sigma), and biotin-labeled secondary antibodies (Sigma) to be revealed by enhanced chemiluminescence (ECL-Plus, GE HealthCare). Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. *P<0.05 vs basal treatment.

Citation: Journal of Endocrinology 214, 3; 10.1530/JOE-12-0223

The effect of IL6 on iNOS expression and the production of NO or urea production

The addition of IL6 to the BRIN-BD11 β-cell culture medium caused a dramatic increase in the expression of iNOS from barely detectable values to concentrations ∼100-fold greater than in control β-cells, but this was not observed in mouse islets (Fig. 6A and B). Indeed, IL6 incubation markedly enhanced NO production in BRIN-BD11 cells, as measured by nitrite release (from 0.34±0.12 to 3.59±0.86 μmol NO/mg protein per 24 h; Fig. 6C). IL6 decreased the level of urea production by about 20% (Fig. 6D), suggesting a diversion of the route of l-arginine metabolism in favor of NO production rather than urea production in response to IL6, so that at physiological concentrations, IL6 preferentially stimulated β-cell NO production in long culture periods (24 h) but did not to interfere in NO signalling via iNOS in shorter periods (2 h, above).

Figure 6
Figure 6

Determination of iNOS expression and NO metabolism in clonal insulin-secreting cells incubated in the presence of IL6. BRIN-BD11 cells (1.5×106 per well) were prepared, cultivated, and electrophoretically analyzed as described in the legend of Fig. 3 to be assessed for iNOS expression. Gel results are presented in arbitrary units relative to GAPDH expression. A representative gel in duplicate is given (A and B). NO production was inferred from the nitrite production to the 24 h incubation medium by the Griess reaction (C) whereas urea levels were measured using QuantiChrom Urea Assay kit (D). Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Data are the mean±s.d. of at least three separate determinations using three wells per experimental condition. Significance: ΔP<0.05 vs control group.

Citation: Journal of Endocrinology 214, 3; 10.1530/JOE-12-0223

The effects of IL6 on BRIN-BD11 cell and islet viability, GSH metabolism, and redox status

No significant viability changes occurred in BRIN-BD11 β-cells or in islets with respect to any of the treatments described in this study over 24 h of incubation. The level of GSH and the BRIN-BD11 β-cell redox status, assessed by the GSSG/GSH ratio (Krause et al. 2007), was assessed in relation to addition of IL6, which induced an increase in the content of GSH by ∼21% (Table 1) and an equivalent 21% increment in the oxidized form (GSSG) (Table 1), thus ensuring that redox status was unaltered (Table 1). Production of glutamate, a necessary amino acid for GSH synthesis (and a putative stimulus-secretion coupling factor for insulin secretion), was also augmented (ca. 36%) in the presence of IL6 (Table 1) in BRIN-BD11 cells. For GSH assays, an intra-assay mean coefficient of variance was identified as being <4.5%.

Table 1

Pancreatic β-cell metabolite consumption/production (μmol/mg protein) over 24 h incubation. Glutathione metabolism and redox state in clonal insulin-secreting cells incubated in the presence of IL6. BRIN-BD11 cells were seeded into six-well plates (2×106 per well) and treated as described in the legends of the previous figures. After 24 h incubation, cells were prepared for the spectrophotometric measurement of GSH and GSSG contents by the DTNB/GSSG reductase recycling method, using the N-ethylmaleimide (NEM) conjugating technique for GSSG sample preparation as described in the Materials and Methods section. Intracellular redox status, given by the GSSG/GSH ratio, was also calculated. Glutamate released into the culture medium was also assessed. Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Results are expressed in μmoles of each metabolite per milligram of cellular protein in terms of mean±s.d. of at least three separate determinations using three wells per experimental condition

Experimental conditionGlucose consumptionGSH contentGSSG contentGSSG/GSH ratio (redox state)Glutamate
IL6 addition+++++
20±0.825±0.8*0.759±0.020.913±0.02*0.221±0.010.267±0.06*0.291±0.070.293±0.072.937±0.193.997±0.30*

Significance: *P<0.05 vs control group.

Discussion

Previous studies have reported that the exposure of pancreatic β-cells to a sublethal concentration of pro-inflammatory cytokines (IFNγ, TNFα, IL1β) appeared to shift β-cell metabolism away from a key role in stimulus-secretion coupling toward a catabolic state, which may be related to cell defense (Kiely et al. 2007). The results reported herein indicate that IL6, at exercise-related doses, robustly increased insulin secretion from BRIN-BD11 β-cells over 24 h in culture and in response to a subsequent acute (20 min) stimulation assay. These findings support the previously reported positive effects of IL6 on glucagon secretion (Ellingsgaard et al. 2008) and insulin secretion (Ellingsgaard et al. 2011). IL6 has also been found to protect MIN6 cells (an insulin-secreting cell line) and isolated primary islets against the cytotoxic and insulin-inhibiting effects of IL1β, TNFα, and IFNγ (Choi et al. 2004). In this work, we detected lower but significantly different changes in primary mouse islet insulin secretion induced by IL6 at concentrations in the range 100–1000 pg/ml (Fig. 2). Higher concentrations of IL6 (600–1200 pg/ml) were recently reported to significantly increase insulin secretion from mouse islets and also MIN6 β-cells (Suzuki et al. 2011), but the physiological significance of such a high-concentration finding remains to be established. The authors speculate that IL6 β-cell action is of physiological importance following termination of exercise in vivo, when insulin secretion and subsequent action at the level of skeletal muscle will aid in recovery mechanisms. While in vivo animal-based intervention (pharmacological) would perhaps appear to be a logical next step, due to the complexity of IL6 actions on GLP1 secretion and pancreatic α-cell effects (Ellingsgaard et al. 2011), the dissection of the results obtained with respect to obtaining information concerning direct IL6 action on β-cells would be impossible. In our studies, IL6 (at physiological concentrations, from 25 to 100 pg/ml) showed robust and reproducible effects on insulin secretion that were dose and time dependent. In long-term incubations of both islets and clonal β-cell lines, 50 pg/ml was sufficient to evoke a rise in insulin secretion, both chronically (24 h accumulation) and acutely (after 20 min of secretagogue stimulation). On the other hand, in short-term incubations (2 h in the presence of IL6 followed by withdrawal of the cytokine), the 50 pg/ml dose did work, but the maximum physiological dose was 100 pg/ml. The significance of higher doses (to which islets still continue to respond) is under investigation.

The enhancement of the ATP/ADP ratio due to increased glycolysis, glucose oxidation, and mitochondrial metabolism is directly linked to insulin secretion (Newsholme et al. 2007). Hence, activation of lipid and glucose oxidation will promote, in most circumstances, both acute and chronic insulin secretion. We observed an increase in glucose use promoted by IL6, suggesting enhanced glucose metabolism, oxidation, and generation of metabolic stimulus-secretion coupling factors. In order to explore the molecular signaling mechanisms underlying the action of IL6 on insulin secretion, we measured the expression of AMPK and AMPK-P, which regulates energy generation in β-cells. While a number of studies have reported an inhibitory effect of AMPK on insulin secretion, based on pharmacological activation or high levels of overexpression, other authors have described a stimulatory effect of AMPK on glucose-induced insulin secretion (Zhang et al. 2009, Dufer et al. 2010). IL6 enhanced the levels of the active form of the enzyme AMPK-P (phosphorylated state) in both clonal β-cells and primary islet cells, while that of the total (phosphorylated, active, plus dephosphorylated, inactive) form was reduced (perhaps due to an IL6-mediated ATP-sparing reduction in protein synthesis) in clonal β-cells, suggesting that IL6 may increase fatty acid oxidation and energy production. As we detected a substantial increase in CaMKKα expression in response to IL6, we speculate that CaMKK may be also an upstream kinase activator of AMPK, and thus an activator of β-cell and islet substrate metabolism and insulin secretion. Activation of AMPK can result in phosphorylation and inhibition of acetyl-CoA carboxylase (which produces malonyl-CoA, which subsequently enters the pathway of fatty acid synthesis). Thus, the increased level of activated AMPK-P in the face of an elevated ATP/AMP ratio will result in a decreased concentration of malonyl-CoA (which is an allosteric inhibitor of carnitine palmitoyltransferase 1, CPT1, the rate-limiting step for long-chain fatty acyl-CoA transfer into mitochondria), and thus will promote fatty acid oxidation while reducing fatty acid synthesis. Pancreatic β-cells have a large ‘fatty acid reserve’ in the form of triacylglycerol, which may undergo hydrolysis to provide necessary fatty acyl-CoA in the presence of IL6 (Newsholme et al. 2007). However, in the present work, we did not measure fatty acid oxidation or triacylglycerol hydrolysis, and for this reason, our hypothesis remains to be tested.

Recently, it has been reported that IL6 promotes insulin secretion though a phospholipase C-IP3-dependent pathway (Suzuki et al. 2011). IP3 promotes Ca2+ release from intracellular endoplasmic reticulum stores, hence elevating cytosolic Ca2+ concentration and activation of CaMKK, AMPK, and substrate oxidation. IL6 may additionally impact on insulin secretion through its action on GLP1 secretion, release, and subsequent β-cell signaling, as recently demonstrated (Ellingsgaard et al. 2011). However, the local effect of IL6 on islet function should also be proposed from the present results.

It has long been recognized that NO is a potent secretagogue for β-cell insulin release (Palmer et al. 1976), while its deficiency is associated with insulinopenia and failure to secrete insulin in response to glucose (Spinas 1999, Newsholme et al. 2009). Hence, although NO may be cytotoxic for β-cells at high concentrations (for review, see Krause Mda & de Bittencourt (2008)), at lower levels NO might be important for promotion of insulin secretion (Krause et al. 2011). The large increase in IL6-induced iNOS expression in clonal insulin-secreting β-cells reported in this work and subsequent NO production (Fig. 5C) may be related to an in vivo mechanism that uses the pathway of NO facilitation of insulin release, in response to post-stress (e.g. exercise). The observed reduction of BRIN-BD11 urea production but augmented NO release is similar to the competition observed between iNOS and l-arginase with respect to the common substrate, l-arginine, in stimulated macrophages (Murphy & Newsholme 1996). Moreover, NO may stimulate insulin secretion by directly acting on β-cell KATP channels (Sunouchi et al. 2008). Besides possible NO-dependent effects on β-cell electrical activity, this agent can also induce metabolic changes in β-cells by increasing the rates of glucose transport and oxidation, augmenting lipolysis and evoking mitochondrial biogenesis, effects that are mediated by multiple cGMP-dependent pathways (Jobgen et al. 2006). Therefore, the important stimulatory effects of IL6 on glucose use and insulin secretion reported herein support the concept of a regulatory role for IL6 in mitochondrial activity and stimulus-secretion coupling (Smukler et al. 2002). The fact that in our study IL6 did not change the levels of iNOS in short-term primary mouse islet incubations (2 h) does not exclude the possibility that NO is elevated in islets in response to high concentrations of IL6 under prolonged stimuli (e.g. 24 h).

In a wider context, it is known that IL6 also operates as a signal for the metabolic communication between l-arginine and energy metabolism, as NO (derived from l-arginine) regulates AMPK activity. Accordingly, it has recently been demonstrated that NO donors may activate AMPK-dependent responses via the generation of cGMP and activation of NO-dependent guanylate cyclase, whereas inhibition of either AMPK or NO synthase abolishes these effects (Lira et al. 2007). Peroxynitrite (ONOO) generated at nontoxic concentrations, from the spontaneous reaction of NO (NO) with superoxide anion (O2• −), activates AMPK through a c-Src-mediated and phosphatidylinositol 3-kinase (PI3K)-dependent pathway (Zou et al. 2003), which may account for some of the IL6-dependent β-cell effects. Conversely, there are a number of NF-κB-linked pro-inflammatory pathways that involve subsequent iNOS expression and NO production, which are dependent on upstream AMPK activation, while pharmacological ablation of AMPK activation blocked inflammatory responses (Huang et al. 2008, Jeong et al. 2009). The oral hypoglycemic agent metformin impairs NF-κB-dependent activation of TNFα production through an inhibition of PI3K-dependent AMPK phosphorylation in endothelial cells (Huang et al. 2008). It is therefore possible that physiological elevations in IL6 (e.g. during and after exercise) may connect muscle with pancreatic islet signal transduction cells via NO-AMPK coupling pathways.

NO is a strong redox reactant able to oxidize the sulfidryl moiety of GSH and cysteine-containing proteins and transcription factors. Hence, as described for other electrophiles (Gutierrez et al. 2008), IL6-induced NO production is capable of enhancing GSH production, at a level proportional to GSH consumption (GSSG formation), via the enhanced transcription of γ-glutamylcysteine synthetase (γ-GCS, also known as glutamate cysteine ligase), the rate-limiting enzyme of de novo GSH biosynthesis, through the activation of Nrf2 transcription factor. Cytosolic NRF2 inhibiting protein (KEAP1) has a critical cysteine moiety, which is redox sensitive (Gutierrez et al. 2008). The IL6-stimulated increase in the production of glutamate (a necessary amino acid for GSH synthesis) may be related to the maintenance of redox status in conditions that result in increased oxidative stress. The absence of IL6-dependent oxidative stress (i.e. maintenance of redox status) was confirmed by the lack of HSP72 responses (a widely used marker for cell stress (Rodrigues-Krause et al. 2012)) in pancreatic islets.

Finally, our data confirms that IL6 promotes insulin secretion from clonal β-cells and pancreatic islets (Ellingsgaard et al. 2011) confirming that this cytokine may exert GLP1-independent effects in the islet in vivo. It has been reported that the expression of IL6 receptor in human- and rodent-isolated islets is associated with α-cells and that the addition of IL6 (200 ng/ml) resulted in an increased glucagon secretion and α-cell mass expansion without influence on β-cell insulin release (Kiely et al. 2007). The IL6 concentration used in the study described herein was much lower (50 pg/ml). It is possible that insulin secretion by β-cells, which is extremely susceptible to oxidative or nitrosative stress, is sensitive to IL6 at low concentrations. Indeed, it was recently reported that IL6 promoted insulin release from a β-cell line and primary islets using high concentrations of IL6 (600 to 1200 pg/ml), an effect dependent on activation of the phospholipase C-IP3 pathway (Suzuki et al. 2011), which would be expected to elevate intracellular Ca2+. CaMKKα is activated by Ca2+, thus our data suggest that IL6-mediated effects in β-cells may occur via an initial elevation in Ca2+, subsequent activation of CaMKKα and AMPK, and stabilization of redox status. These possibilities are currently under further investigation in our laboratories.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Author contribution statement

M da S K and A B completed all experiments described in this manuscript. M da S K and P N co-wrote the manuscript. N H M, P R F, P I H de B Jr, and C M provided experimental advice and helped with manuscript revision. C M and P N were responsible for grant support with respect to TSR: Strand III – Core Research Strengths Enhancement Scheme (Ireland).

Acknowledgements

The authors thank UCD School of Biomolecular and Biomedical Science; the Department of Science, Institute of Technology Tallaght (Dublin, Ireland) and TSR: Strand III – Core Research Strengths Enhancement Scheme (Ireland); the School of Biomedical Sciences, Curtin University, Perth, Western Australia, and the Brazilian National Council for Scientific and Technological Development (CNPq, grants no 563870/2010-9 – ‘Amino acid metabolism in Diabetes’ and no 551097/2007-8 – ‘LipoCardium’) for their support of this work.

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    Chronic insulin secretion (24 h), acute response (20 min), and time course of insulin secretion in BRIN-BD11 cells under exposure to IL6. Clonal insulin-secreting BRIN-BD11 cells were seeded into 24-well plates (0.75×105 cells/well) and allowed to adhere overnight. Cells were then washed with PBS and cultured in fresh media, containing 11.1 mM d-glucose and 2 mM l-glutamine, in the absence or presence of IL6 (0–100 pg/ml). An aliquot of the media was removed for the measurement of chronic insulin secretion response (A). After a 24 h incubation period, cells were washed and ‘rested’ for 40 min in the presence of 1.1 mM glucose in KRB, pH 7.4 (and absence of IL6), followed by an acute stimulation period of 20 min in the presence of either 16.7 mM glucose or 10 mM alanine (a standardized stimulus that results in a robust and reproducible secretory response in normal conditions for BRIN-BD11 (Brennan et al. 2002)) or basal 1.1 mM glucose in KRB, pH 7.4, when an aliquot of the incubation medium was assessed for insulin secretion response as described in the Materials and Methods section (B). In addition, to observe the time course of insulin secretion in response to IL6, cells were incubated for 24 h (with 50 pg/ml of IL6) where an aliquot of the media was collected at different times (C). Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Data are the mean±s.d. of three independent determinations using eight wells per experimental condition. Significance: ΔP<0.05 vs control group; P<0.05 vs 25 pg/ml IL6; P<0.05 vs 50 pg/ml IL6; ψP<0.05 vs 100 pg/ml IL6; ¥P<0.05 vs respective basal treatment at the same IL6 concentration).

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    Pancreatic islet insulin secretion under exposure to IL6. Pancreatic islets were isolated from WT C57BL/6J mice. Islets were resuspended in fresh 0.1% (w/v) BSA containing Krebs buffer, picked, and cultured for 24 h in the presence of RPMI 1640 culture medium supplemented with 10% (v/v) FCS, antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin), and 11.1 mM d-glucose, pH 7.4. Subsequently, islets were cultured in KRB containing 5.5 mM d-glucose and 2 mM l-glutamine, in the absence or presence of IL6 and kept at 37 °C in a humidified atmosphere of 5% CO2 in air for 2 h. Insulin secretion was measured using Mercodia Ultrasensitive Mouse Insulin ELISA kit. Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Data are the mean±s.d. of three separate mouse extractions of primary islets. *P<0.05 vs basal treatment.

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    Determination of AMPK, AMPK-P, LKB1, and CaMKKα expression in clonal insulin-secreting cells incubated in the presence of IL6. BRIN-BD11 cells were seeded into six-well plates (1.5×106 cells/well) and allowed to adhere overnight and cultured for 24 h. Cells were then washed with PBS after which they were incubated in fresh media, containing 11.1 mM d-glucose and 2 mM l-glutamine, in the absence or presence of IL6 (50 pg/ml) for 24 h as described in the legend of Fig. 1. Afterward, equal amounts of BRIN-BD11 cell protein extracts were prepared, electrophoresed (10% SDS–PAGE), and transferred onto a nitrocellulose membrane. A representative image of the protein levels is shown (A). AMPK and its phosphorylated form (B), CaMKKα and LKB1 (C), were then probed with specific antibodies and visualized after HRP/chemiluminescent reaction. Results are presented in arbitrary units relative to GAPDH expression. Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Data are the mean±s.d. of at least three independent determinations using three wells per experimental condition. Significance: ΔP<0.05 vs control group.

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    Determination of AMPK, AMPK-P, LKB1, and CaMKKα expression in pancreatic islets from WT mice incubated in the presence of IL6 for 24 h. Determination of AMPK, AMPK-P, LKB1, and CaMKKα expression in pancreatic islets from WT mice incubated in the presence of IL6 for 24 h. Isolated mouse islets were seeded into six-well plates (150 IEQs/well) and cultured for 24 h, as described in the legend of Fig. 2. Afterward, equal amounts of islet protein extracts were prepared, electrophoresed (10% SDS–PAGE), and transferred onto a nitrocellulose sheet. Protein forms were then probed with specific antibodies and visualized after HRP/chemiluminescent reaction. A representative image of the protein levels is shown (A). AMPK and its phosphorylated form (B), LKB1 (C) and CaMKKα (D). Results are presented in arbitrary units relative to GAPDH expression. Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Data are the mean±s.d. of three separate mouse extractions of primary islets using six wells per experimental condition. Significance: ΔP<0.05 vs control group.

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    Determination of AMPK, AMPK-P, iNOS, and HSP72 expression in pancreatic islets from WT mice incubated in the presence of IL6 for 2 and 6 h. iNOS (A), HSP70 (B), P-AMPK (C), total AMPK (D), P-AMPK / Total AMPK (E). Results are presented in arbitrary units relative to Tubulin expression. A representative image of the protein levels is shown (F). In order to investigate the early effects of IL6 on signaling protein expression in mouse pancreatic islets, 50 IEQs per well (50 μl total volume) were incubated in RPMI 1640 medium supplemented with FCS (10% v/v), antibiotics, and 5.5 mM glucose for just 2 h in the absence or presence of 100 pg/ml IL6. Afterward, islets were either immediately harvested or cultured for additional 6 h in the absence of IL6. After that, islets were homogenized in 0.1% SDS containing 100 μM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 20 μM TLCK for protein quantitation. Then, samples were dissolved in Laemmli sample buffer and equal amounts of protein were electrophoresed, electrotransferred to nitrocellulose membranes, and immunoblotted for iNOS, HSP70 (both constitutive and inducible forms), AMPK catalytic subunit, phospho (Ser487)-AMPK or tubulin using monoclonal primary antibodies (Sigma), and biotin-labeled secondary antibodies (Sigma) to be revealed by enhanced chemiluminescence (ECL-Plus, GE HealthCare). Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. *P<0.05 vs basal treatment.

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    Determination of iNOS expression and NO metabolism in clonal insulin-secreting cells incubated in the presence of IL6. BRIN-BD11 cells (1.5×106 per well) were prepared, cultivated, and electrophoretically analyzed as described in the legend of Fig. 3 to be assessed for iNOS expression. Gel results are presented in arbitrary units relative to GAPDH expression. A representative gel in duplicate is given (A and B). NO production was inferred from the nitrite production to the 24 h incubation medium by the Griess reaction (C) whereas urea levels were measured using QuantiChrom Urea Assay kit (D). Groups of data were compared using unpaired Student's t-test or ANOVA where appropriate using GraphPad Prism 5 software. Data are the mean±s.d. of at least three separate determinations using three wells per experimental condition. Significance: ΔP<0.05 vs control group.

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