Superantigen activates the gp130 receptor on adipocytes resulting in altered adipocyte metabolism

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Available online at www.sciencedirect.com

Metabolism www.metabolismjournal.com

Superantigen activates the gp130 receptor on adipocytes resulting in altered adipocyte metabolism Elin Banke a,⁎, Karin Rödström a , Mikael Ekelund b , Jonathan Dalla-Riva a , Jens O. Lagerstedt a , Staffan Nilsson c , Eva Degerman a , Karin Lindkvist-Petersson a , Bo Nilson d, e a

Department of Experimental Medical Science, Lund University, BMC, 221 84 Lund, Sweden Department of Surgery, Skåne University Hospital & Lund University, 221 85 Lund, Sweden c Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, 221 00 Lund, Sweden d Department of Laboratory Medicine, Division of Medicinal Microbiology, Lund University, 223 62 Lund, Sweden e Department of Clinical Microbiology, University and Regional Laboratories in Region Skåne, 221 85 Lund, Sweden b

A R T I C LE I N FO Article history:

AB S T R A C T Objective. The bacteria Staphylococcus aureus is part of the normal bacterial flora and

Received 18 September 2013

produces a repertoire of enterotoxins which can cause food poisoning and toxic shock and

Accepted 4 March 2014

might contribute to the pathogenesis of inflammatory diseases. These enterotoxins directly cross-link the T cell receptor with MHC class II, activating large amounts of T cells and are

Keywords:

therefore called superantigens. It was recently discovered that the superantigen SEA binds

SEA

to the cytokine receptor gp130. As obesity and type 2 diabetes are highly associated with

Glucose- and lipid metabolism

inflammation of the adipose tissue and gp130 has been shown to play an important role in

Insulin sensitivity

adipocytes, we wanted to investigate the effect of SEA on adipocyte signaling and function. Materials/methods. Binding of SEA to gp130 was examined using surface plasmon resonance in a cell free system. Effects of SEA on adipocyte signaling, insulin sensitivity and function were studied using western blotting and biological assays for lipolysis, lipogenesis and glucose uptake. Results. We demonstrate that SEA binds to gp130 with a medium affinity. Furthermore, SEA induces phosphorylation of a key downstream target, STAT3, in adipocytes. SEA also inhibits insulin-induced activation of PKB and PKB downstream signaling which was associated with reduced basal and insulin induced glucose uptake, reduced lipogenesis as well as reduced ability of insulin to inhibit lipolysis. Conclusions. SEA inhibits insulin signaling as well as insulin biological responses in adipocytes supporting that bacterial infection might contribute to the development of insulin resistance and type 2 diabetes. © 2014 Elsevier Inc. All rights reserved.

Abbreviations: ERK, Extracellular signal-related kinase; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; HSL, Hormone sensitive lipase; IL-6R, Interleukin-6 receptor; Iso, Isoprenaline; JAK, Janus activated kinase; PKB, Protein kinase B; RU, Resonance units; SEA, Staphylococcal enterotoxin A; SEA-D227A, Staphylococcal enterotoxin A mutant D227A; SOCS3, Suppressor of cytokine signaling 3; STAT, Signal transducer and activator of transcription; SPR, surface plasmon resonance. ⁎ Corresponding author at: BMC C11, Sölvegatan 19, 221 84 Lund, Sweden. Tel.: +46 462228587; fax: +46 462224022. E-mail address: [email protected] (E. Banke). http://dx.doi.org/10.1016/j.metabol.2014.03.004 0026-0495/© 2014 Elsevier Inc. All rights reserved.

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1.

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Introduction

The incidence of type 2 diabetes (T2D) is increasing rapidly worldwide and there is a clear link between obesity and the development of T2D [1]. Obesity is associated with dysregulated lipid metabolism resulting in increased ectopic fat deposition as well as altered adipokine release and lowgrade inflammation, which can lead to local and peripheral insulin resistance and the development of T2D [2]. Besides obesity, acute infection can cause adipose tissue inflammation and subsequent insulin resistance affecting normal adipocyte biological responses such as lipolysis [3–5]. For example, Staphylococcus aureus infections are associated with T2D [6], however, the molecular links between bacterial infections and metabolic disorders are unclear. The bacteria S. aureus is part of the normal bacterial flora on skin and mucous membranes, as in nasal passages and in the gut [7]. S. aureus produces a repertoire of Staphylococcal enterotoxins abbreviated SE-A, -B, -C etc. These toxins can cause food poisoning and toxic shock and are associated with several acute and chronic inflammatory diseases and they can be produced in high concentrations (1 to 100 μg/ml) in vitro[8,9] and in vivo (in tampons)[10]. Collectively these are called “superantigens” due to their inherent property to activate large amounts of T cells, consequently causing the release of cytokines, such as IL-2 [11]. The Staphylococcal enterotoxins are known to interfere with the immune system by directly cross-linking the T cell receptor (TCR) on T cells with the MHC class II on antigen presenting cells, as native proteins [12]. The SEs have been shown to bind either the alpha chain or the beta chain of MHC class II, using their N- or C-terminal domain, respectively [13,14]. The interface between the alpha chain of MHC and the N-terminal domain of the SEs is classified as being the low affinity site (micromoles per liter range), while the interface between the beta chain of MHC and the Cterminal domain of the SEs is zinc dependent and of high affinity (nanomoles per liter range) [12]. Most superantigens use either the low or the high affinity site when binding to MHC class II, however, the SEA superantigen uses both sites to bind MHC [15]. It has been shown that a single substitution in the high affinity site of SEA, aspartate 227 to alanine (D227A), significantly reduces the binding to MHC class II, suggesting that the high affinity site is essential for MHC binding [16]. Nilson and co-workers discovered that in addition to binding to MHC class II and to the TCR, SEA and other superantigens can bind to the IL-6 signal transducer, a receptor also known as gp130 (Nilson et al., unpublished data 2014). MHC class II was shown to compete with gp130 for binding to SEA and SEA with the single substitution D227A also had considerably reduced binding for gp130. Gp130 is a cytokine receptor known to be involved in numerous biological processes, including inflammation, hematopoiesis, immune regulation, acute phase response and neuronal modulation [17]. The structure of the gp130 is well studied and consists of six domains (D1 to D6) with an immunoglobulin-like activation domain (D1) and a cytokine-binding homology domain (D2-D3) [18]. Gp130 has also been shown to play an important role in adipocytes [19], in particular as the signaling domain for the IL-6 cytokine receptor (IL-6R) [20]. The gp130 is known to activate the janus

activated kinase (JAK) and the signal transducer and activator of transcription (STAT) signaling pathways that both crossreact with the insulin signaling network [21]. More recently, it has also been shown that signaling via gp130 can affect the PKB (protein kinase B, also known as Akt) pathway in adipocytes and hence influence insulin signaling and its downstream events such as glucose uptake [20]. The aim of this study was to further examine the binding of SEA to the gp130 receptor using surface plasmon resonance (SPR) as well as to investigate whether SEA activates gp130 receptor signaling in adipocytes. Furthermore, the effect of a SEA-induced activation of gp130 on adipocyte insulin sensitivity was investigated. Our study shows for the first time that SEA can activate gp130 signaling in adipocytes and affect normal insulin signaling and adipocyte function, making them less insulin sensitive. Thus, bacterial infection might contribute to the development of insulin resistance and T2D.

2.

Materials and methods

2.1.

Ethical statement

Animal experiments were approved by the Animal Ethics Committee in Lund, Sweden (ethical permit number M212-09) and were carried out in accordance with EU directives for animal experiments. Animals were kept under standardized conditions in the animal house facilities and all efforts were made to minimize suffering. Human omental and subcutaneous adipose tissue was obtained from patients undergoing gastric bypass operations. Written consent was given and ethical approval was obtained from the Human Ethics Committee in Lund, Sweden. All work was carried out in accordance with the Declaration of Helsinki.

2.2.

Materials

For adipocyte stimulation, insulin (Novo Nordisk, Målöv, Denmark), isoprenaline (iso) (SIGMA, St. Louis, USA), recombinant rat IL-6 (R&D Systems, Abingdon, UK), wild-type SEA (Active Biotech, Lund Sweden) were used. The SEAD227A mutant used in both adipocyte stimulation and surface plasmon resonance was provided by Active Biotech. The gp130-Fc dimer used in surface plasmon resonance experiments was from R&D systems. The primary antibodies used in western blotting; anti-phospho-hormone sensitive lipase (HSL), anti-phospho-PKB-substrate, anti-phospho-PKB (pSer473) and anti-PKB were from Cell Signaling Technologies (Boston, USA). Anti-phospho-STAT3 (pTyr705) was from Santa Cruz (Santa Cruz, USA). Anti-GAPDH was from SIGMA. The secondary anti-mouse and anti-rabbit antibodies were from GE Healthcare (Buckinghamshire, UK) and Thermo Scientific (Rockford, USA) respectively.

2.3.

Protein expression and purification

Staphylococcal enterotoxin A was expressed and purified as described elsewhere [14], apart from minor changes. Briefly, a vector harboring the SEA gene downstream of a constitutive

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promotor, as well as a kanamycin resistance gene was used for expression in Escherichia coli K12 strain UL635, cultured in 1 l 2xYT media (16 g/l tryptone, 10 g/l bacto yeast extract, 5 g/l NaCl) supplemented with 50 mg/l kanamycin, at 210 rpm and 29 °C. Cells were harvested by centrifugation at 8000 g for 30 min, and the pellet was resuspended in 300 ml sucrose buffer (20% w/v sucrose, 300 mmol/L Tris–HCl pH 8.0, 1 mmol/L EDTA). The suspension was centrifuged (10,000 g, 30 min, 30 min, 4 °C) and the pellet was resuspended in 150 ml MQ water containing 0.5 mmol/L MgCl2 and 0.1 mmol/L ZnCl2. After centrifugation (10,000 g, 30 min, 4 °C) sodium acetate was added to a final concentration of 20 mmol/L and the pH was adjusted to 4. The supernatant was centrifuged (25,000 g, 40 min, 4 °C) and filtered through a 0.2 μm filter. Purification was carried out using cation exchange chromatography on a 6 ml Resource S column (GE Healthcare), with a linear gradient of 20–250 mmol/L sodium acetate pH 5.5 and 0.025% v/v Tween-20, followed by size exclusion chromatography on a HiLoad Superdex 200 16/60 prep grade column (GE Healthcare) in TBS buffer (50 mmol/L Tris–HCl pH 7.4, 150 mmol/L NaCl). The gp130 receptor domains D1 to D3 with a hexa-histidine tag were cloned into the pAcgp67A secretion vector and produced in Hi5 insect cells according to previously published methods and purified by affinity and size exclusion chromatography [18]. MHC class II HLA-DR1 (α-chain DR*0101 and β-chain DRB1*0101), with bound Influenza HA peptide (residues 306– 318) with sequence PKYVKQNTLKLAT was produced as described [22], with minor modifications. Expression of the separate chains was carried out in E. coli BL21 (DE3) Star cells (Invitrogen), grown at 33 °C for 18 h in 1 l 2xYT media supplemented with 100 mg/l ampicillin. Prior to induction with 0.5 mmol/L isopropylthiogalactoside, cells were centrifuged (5000 g, 20 min) and resuspended in 1 l fresh 2xYT media. Cells were lysed and inclusion bodies were isolated, washed and solubilized. Subsequently, the solubilized chains were purified by anion exchange chromatography using a 6 ml Resource Q column (GE Healthcare). Refolding was carried out by dilution of α- and β-chains to a final concentration of 0.24 μmol/L each in 2 l refolding buffer (20 mmol/L Tris–HCl pH 8.5, 40% w/v glycerol, 1 mmol/L EDTA, 0.3 mmol/L oxidized glutathione, and 3 mmol/L reduced glutathione) in presence of 1 μmol/L Influenza HA peptide (Peptide Synthetics, Peptide Protein Research). Final purification was carried out using a 6 ml Resource Q column (GE Healthcare) followed by size exclusion chromatography on a Superdex 200 10/300 GL column (GE Healthcare) into TBS buffer pH 7.4.

2.4.

Surface plasmon resonance

For initial binding analysis, recombinant gp130 fragment D1D3 and gp130-Fc dimer (R&D Systems) were immobilized on separate channels on a CM5 chip (GE Healthcare), to 450 RU and 1100 RU respectively. Concentration series of wild-type SEA or the SEA D227A mutant, concentrations ranging from 40 nmol/L to 40 μmol/L, in HBS-P running buffer (10 mmol/L HEPES pH 7.4, 150 mmol/L NaCl, 0.005% v/v Tween-20, supplemented with 100 μmol/L ZnCl2) were injected and the

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response differences were recorded and the bulk refraction from a blank channel was subtracted. For calculation of the dissociation constant for the SEA-gp130 complex, gp130-Fc dimer was immobilized to 670 RU and a concentration series of SEA ranging from 7 nmol/L to 41 μmol/L was injected. The response was recorded and plotted against the respective concentration, and the equilibrium dissociation constant was obtained by fitting Eq. (1) to the data using nonlinear regression. Req ¼

CRmax KD þ Cn

ð1Þ

where Req is the response, C is the analyte concentration, KD is the equilibrium dissociation constant, Rmax the maximum response and n the steric interference factor. In order to further investigate the gp130 binding properties to SEA, recombinant gp130 fragment D1-D3 was immobilized to approximately 470 RU. The analytes injected were 1 μmol/L wild type SEA, followed by a concentration series of 40 nmol/L to 22 μmol/L MHC class II, all with a constant concentration of 1 μmol/L SEA. All Surface Plasmon Resonance (SPR) measurements were made on a BIACore2000 (GE Healthcare).

2.5.

Isolation of primary rat and human adipocytes

Primary adipocytes were isolated from the epididymal fat pads of male Sprague–Dawley rats (35–39 days old, B&K Universal, Stockholm, Sweden) as well as from omental and subcutaneous fat from patients undergoing gastric bypass operation, by collagenase digestion [23] and subsequently diluted to 1.5%–10% cell suspensions (depending on following method) in Krebs Ringer buffer with 25 mmol/L HEPES (KRH), 1% BSA, 2 mmol/L glucose and 200 nmol/L adenosine, pH 7.5 (exceptions, see glucose uptake and lipogenesis).

2.6.

SDS-PAGE and western blotting

Cell suspensions (10%) were incubated at 37 °C for 10 min with stimuli as indicated. Subsequently, the cells were washed with BSA free KRH buffer and cells were lysed in lysis buffer containing 50 mmol/L Tris–HCl, 1 mmol/L EGTA, 1 mmol/L EDTA, 0.05 mmol/L Na-orthovanadate, 50 mmol/L NaF, 5 mmol/L Na-pyrophosphate, 0.27 mol/L sucrose, 1 mmol/L dithioerythriol, complete protease inhibitor (1 tablet/50 ml, Roche, Bromma, Sweden) and 1% NP40, pH 7.4. Cell lysates were centrifuged at 13,000 × g at 4 °C for 10 min, supernatant saved and protein concentration determined by the Bradford method [24]. Samples (20 μg protein) were mixed with LDS sample buffer 4 × (Invitrogen, Carlsbad, USA) containing 46.5 mg/ml dithiothreitol (DTT) and subjected to electrophoresis on 7.5% bisacrylamide gels. Protein bands were transferred to Hybond-C Extra membranes (Amersham Biosciences, Uppsala, Sweden), blocked with 10% milk in tris-buffered saline Tween-20 (TBST: 50 mmol/L Tris, 150 mmol/L NaCl and 0.1% Tween-20, pH 7.6) for 1 h and subsequently incubated at 4 °C overnight with primary antibodies as indicated. Membranes were incubated with secondary antibodies conjugated with horse radish peroxidase for 1 h in room temperature and subsequently incubated with SuperSignal West Pico ECL (enzymatic chemoluminescence) reagent (Thermo Scientific, Rockford, USA) for 10 min

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followed by imaging (Molecular Imager ChemiDoc XRS+, Biorad Laboratories, Solna, Sweden) and quantification (Image lab software, version 3.0, Bio-Rad Laboratories).

2.7.

Glucose uptake

After collagenase digestion of rat adipose tissue [23] (in a glucose-free buffer), uptake of 14C-glucose was measured as previously described [25] in triplicates of 5% cell suspensions in Krebs–Ringer Bicarbonate–HEPES buffer (120 nmol/L NaCl, 4 mmol/L KH2PO4, 1 mmol/L MgSO4, 0.75 mmol/L CaCl2, 10 mmol/L NaHCO3, 30 mmol/L HEPES, pH 7.4) with 200 nmol/L adenosine, 1% BSA and stimuli as indicated for 1 h.

2.8.

Lipogenesis

Cells were washed in KRH buffer with low glucose (3.5% BSA, 0.55 mmol/L glucose and 200 nmol/L adenosine, pH 7.5) and diluted into a 1.5% cell suspension. The incorporation of 6-3HGlucose into cellular lipids was measured as previously described [26] in triplicates with stimuli as indicated for 30 min.

2.9.

Lipolysis

Glycerol release, as a measure of lipolysis, was measured after incubating 5% cell suspensions at 37 °C for 30 min in duplicates with stimuli as indicated. Stimulations were stopped on ice, medium separated from the cells and transferred to new tubes for enzymatic measurement of glycerol release as previously described [27].

2.10.

Statistics

Paired parametrical Student´s t-test was used to calculate statistical significance and data are presented as means ± SEM. p-values ≤ 0.05 were considered significant.

2.11.

Figures

The figures depicting three-dimensional structures were made and rendered using PyMOL [28].

3.

Results

3.1. SEA binds the gp130 receptor using the C-terminal domain In SPR experiments, wild-type SEA and the SEA-D227A mutant were injected over immobilized gp130 receptor, both a gp130D1-D3 and a disulphide-linked gp130-Fc dimer. SEA clearly binds both variants of the gp130 protein (Fig. 1A and C) and it confirms previous analysis showing that the SEA binding site is located to the D1 to D3 region of gp130 (Nilson et al., unpublished data 2014). Binding was not detectable for the SEA-D227A variant to any of the gp130 proteins, which suggests that the gp130 binding site on SEA is located in the C-terminal β-grasp domain of the superantigen (Fig. 1A).

Fig. 1 – SEA binds the gp130 receptor. Wild-type SEA and mutant D227A SEA (A) were injected over immobilized gp130-Fc dimer. The concentrations shown are 13 nmol/L, 50 nmol/L, 0.2 μmol/L, 0.8 μmol/L, 3.1 μmol/L, and 11.2 μmol/L for wt SEA and 5.4 μmol/L for the D227A mutant. The response at t = 199 s is shown against concentration of wt SEA (B). (C) SEA (1 μmol/L) was injected over immobilized gp130 fragment D1-D3 (top curve), followed by a concentration series of MHC class II with a constant SEA concentration of 1 μmol/L. The concentrations of MHC class II ranged from 0 μmol/L to 5.5 μmol/L and are indicated in the figure. SEA: Staphylococcal enterotoxin A, SEA-D227A: Staphylococcal enterotoxin A mutant D227A, RU: Resonance units.

Furthermore, the equilibrium dissociation constant (KD) for the SEA-gp130-Fc complex was calculated to be approximately 0.9 μmol/L (Fig. 1B). To further investigate the location of the gp130 binding site on SEA, 1 μmol/L SEA was injected over

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immobilized gp130-D1-D3, followed by a step-wise increase of MHC concentration accompanied by a constant concentration of SEA (1 μmol/L). Upon increasing concentration of MHC, the response for SEA decreased, suggesting a competitive binding between MHC and gp130 on the superantigen (Fig. 1C). This is consistent with the lack of binding to gp130 for the SEAD227A variant.

3.2.

SEA activates STAT3 of the gp130 signaling pathway

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[21] at the activity-controlling site Tyr705 [29]. To assess whether SEA can activate the gp130 receptor on adipocytes, both primary rat- (Fig. 2A, n = 11) and human (Fig. 2B, n = 4, exception IL-6 n = 3) adipocytes were stimulated with IL-6 (100 ng/ml) and SEA (5–50 μg/ml). Stimulation of rat adipocytes with SEA resulted in phosphorylation and activation of STAT3 to about 50% of the IL-6-induced STAT3 activation (Fig. 2A). The inactive variant of SEA, SEA-D227A, was used as a negative control and showed no phosphorylation of STAT3 (Fig. 2A, 5 μg/ml, n = 4). Stimulation with SEA (5 μg/ml) of human adipocytes

The gp130 receptor complex activates the JAK/STAT-signaling pathway resulting in phosphorylation and activation of the transcription factor STAT3 [21]. The inflammatory cytokine IL-6 binds to the IL-6R, which subsequently forms a complex with and activates gp130. IL-6 was therefore used as a positive control of gp130 activation and subsequent phosphorylation of STAT3

Fig. 2 – SEA activates STAT3 of the gp130 signaling pathway. Primary adipocytes from rat (A) and human (B) were stimulated for 10 min as indicated (IL-6: 100 ng/ml, SEA: 5–50 μg/ml (A) 5 μg/ml (B), SEA-D227A: 5 μg/ml) and western blot analysis was performed using an antibody specific against phospho-STAT3 (Tyr705). Immunoblots from 11 (A) and 4 (B) separate experiments were quantified (exception SEAD227A: n = 4) and representative blots are shown. * p < 0.05, *** p < 0.001 Ctrl: Control, GAPDH: Glyceraldehyde 3-phosphate dehydrogenase, IL-6: Interleukin-6, SEA: Staphylococcal enterotoxin A, SEA-D227A: Staphylococcal enterotoxin A mutant D227A, pSTAT3: phosphorylated Signal transducer and activator of transcription 3.

Fig. 3 – SEA reduces insulin signaling. Western blot analysis was performed using antibodies specific against phospho-PKB (Ser473) (A) as well as PKB phosphorylated PKB substrates (B) on lysates from primary rat adipocytes stimulated for 10 min as indicated (Ins: 1 nmol/L, SEA: 5 μg/ml (A), 5–50 μg/ml (B)). Immunoblots from 3 different experiments were quantified and representative blots are shown. * p < 0.05, ** p < 0.01, Ctrl: Control, Ins: Insulin, PKB: Protein kinase B, SEA: Staphylococcal enterotoxin A.

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resulted in a phosphorylation and activation of STAT3 greater than that induced by IL-6 (Fig. 2B).

3.3.

SEA reduces insulin signaling

Activation of the gp130 receptor has previously been observed to induce insulin resistance in adipocytes [20]. Phosphorylation of PKB at the activity-controlling site Ser473 can be used as an indicator for insulin sensitivity in adipocytes [30]. To examine whether SEA affects insulin sensitivity in adipocytes, primary rat adipocytes were stimulated with insulin (1 nmol/L) with or without SEA (Fig. 3A: 5–50 μg/ml, 3B: 5 μg/ml). SEA caused a reduction in insulin-induced phosphorylation of PKB (Fig. 3A, n = 3) as well as a reduced insulin-induced phosphorylation of downstream PKB substrates (Fig. 3B, n = 3).

3.4.

SEA modulates adipocyte glucose uptake

Insulin stimulates glucose uptake in adipocytes via the translocation of glucose transporter GLUT4 to the plasma membrane after activation of PKB [31]. Since SEA was shown to reduce the insulin-induced PKB phosphorylation, whether SEA could affect glucose uptake in adipocytes was investigated. Stimulation with SEA (5 μg/ml) resulted in a small inhibition of basal adipocyte glucose uptake (Fig. 4A, n = 4), and a significant reduction of insulin-induced glucose uptake (Fig. 4B, n = 4).

Fig. 5 – SEA reduces insulin-induced lipogenesis. Lipogenesis was measured after stimulating primary rat adipocytes for 30 min as indicated (SEA: 5 μg/ml, Ins: 10 nmol/L, n = 7). ** p < 0.01, *** p < 0.001, Ins: Insulin, SEA: Staphylococcal enterotoxin A.

3.5.

An important role of the adipocyte is to store energy in the form of triglycerides, a process stimulated by insulin [32]. The effect of SEA on insulin-induced lipogenesis was therefore analyzed. As expected, stimulation with SEA (5 μg/ml) resulted in reduced insulin-induced lipogenesis in primary rat adipocytes (Fig. 5, n = 7). When energy substrates are needed, the breakdown of triglycerides is increased, a process called lipolysis. Stimulation of adipocytes with the beta adrenergic agonist isoprenaline results in elevated intracellular cAMP levels, PKA-dependent phosphorylation of the lipase HSL and an increase in lipolysis [33]. Insulin antagonizes lipolysis by PKB-dependent activation the cAMP-hydrolyzing enzyme PDE3B [34]. Therefore, the effect of SEA on lipolysis was investigated. Stimulation with SEA (5 μg/ml) resulted in a reduction of the basal lipolysis in primary rat adipocytes (Fig. 6A, n = 5). In the presence of isoprenaline (30 nmol/L), SEA significantly potentiated isoinduced lipolysis (Fig. 6B, n = 5) accompanied by an increase in HSL phosphorylation at Ser563 (Fig. 6C, n = 4). SEA also reduced the ability of insulin (1 nmol/L) to antagonize lipolysis (Fig. 6B, n =5), which is in agreement with the reduced PKB phosphorylation.

4.

Fig. 4 – SEA modulates adipocyte glucose uptake. Glucose uptake was measured after stimulating primary rat adipocytes for 30 min as indicated (SEA: 5 μg/ml, Ins: 1 nmol/L, n = 4). A: Basal glucose uptake, B: Insulin-stimulated glucose uptake. * p < 0.05, ** p < 0.01, Ctrl: Control, Ins: Insulin, SEA: Staphylococcal enterotoxin A.

SEA affects adipocyte lipid metabolism

Discussion

It was recently discovered that superantigens can bind to the cytokine receptor gp130. Here we further stress the importance of this finding by showing significant effects of SEA in mammalian fat cells. The binding of SEA to the gp130 receptor on both rat and human adipocytes leads to phosphorylation and activation of STAT3, a crucial step in the activation of the gp130 signaling pathway [21]. We also revealed that SEA affects adipocyte insulin signaling and several biological processes essential for the regulation of normal adipocyte function such as lipolysis, glucose uptake and lipogenesis. Using SPR, we confirm the previous finding that the binding site on gp130 resides in the outermost D1-D3 domains (Fig. 7B), which is in the same region that forms complex with IL-6/IL-6R [35]. The superantigen likely uses its C-terminal domain where the aspartate 227 residue is located (Fig. 7A),

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Fig. 6 – SEA affects adipocyte lipolysis. Lipolysis was measured in primary rat adipocytes after stimulation for 30 min as indicated (SEA 5 μg/ml, Iso: 30 nmol/L, Ins: 1 nmol/L, n = 5). A: Basal lipolysis, B: Iso-induced lipolysis. Western blot analysis was performed using an antibody specific against phosphor-HSL (Ser563) (C) on lysates from primary rat adipocytes stimulated for 10 min as indicated (IL-6 100 ng/ml, SEA in μg/ml, SEA D227A 5 μg/ml) * p < 0.05, ** p < 0.01, *** p < 0.001, Ctrl: Control, IL-6: Interleukin 6, Ins: Insulin, Iso: Isoprenaline, SEA: Staphylococcal enterotoxin A, SEA D227A: Staphylococcal enterotoxin A mutant D227A.

since a substitution to an alanine results in abolished gp130 binding. The loss of binding is not a result of incorrect folding, since the D227A mutant has been shown with X-ray crystallography to retain its three-dimensional structure [14]. In addition, the finding that MHC class II is able to inhibit gp130

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Fig. 7 – Three-dimensional structures of SEA and gp130. Three-dimensional structures of SEA (A) and gp130 (B), with aspartate 227 marked in SEA and the binding site for herpes virus IL-6 marked in purple on the gp130 receptor. Structure 1ESF [55] was used for the SEA figure and structures 1I1R [18] and 3L5H (RCSB Protein Data Bank, DOI:10.2210/pdb3l5h/pdb) for the gp130 figure. SEA: Saphylococcal enterotoxin A, IL-6: interleukin 6. binding to SEA further supports this. However, this inhibition is most likely not of relevance for the biological role of SEA in adipocyte metabolism, due to the fact that MHC class II is only present on more specialized antigen presenting cells, such as dendritic cells [36]. The prominent role of D227 in gp130 binding is of interest due to its conservation in a number of other superantigens, such as SE-D, E and J [13], which also bind to gp130 (Nilson et al., unpublished data 2014). Phosphorylation of STAT3 is an indicator of gp130 activation [21]. The degree of STAT3 activation in response to SEA is much higher in human than in rat and it has previously been shown that the response to SEs can be species specific and it has been attributed to differences in affinity to its receptors [37,38]. We found that SEA reduces basal as well as insulininduced glucose uptake. Although the decrease was only 10%, a small decrease in glucose uptake can be of great

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physiological relevance [39]. Until now, nothing has been published about the effects of superantigens on adipocyte function, however, since SEA activates the same signaling receptor as IL-6 (gp130) it is anticipated that SEA has similar effects as IL-6. Several studies have shown a reduced adipocyte glucose uptake in response to IL-6 [20,40–42]. However, Carey et al. observed an increased glucose uptake by incubating 3 T3-L1 adipocytes with IL-6 [43] and Päth et al. observed no difference in glucose transport in human adipocytes [44]. Insulin increases adipocyte glucose uptake via phosphorylation and activation of PKB [31]. Our results demonstrate that SEA reduces both insulin-induced activation of PKB as well as insulin-induced phosphorylation of several PKB substrates, further verifying the observed decrease in adipocyte glucose uptake. In accordance with this observation, IL-6 [20,42], as well as other gp130 receptor ligands, [45] has previously been seen to inhibit insulin signaling via decreased phosphorylation of PKB. Additionally, chronic activation of the gp130 receptor increases the expression of suppressor of cytokine signaling (SOCS3), a protein known to inhibit insulin receptor signaling [46]. In this project the focus is on acute effects of SEA, although it would be very interesting to investigate whether chronic SEAdependent activation of gp130 also increases the expression of SOCS3, further inhibiting insulin signaling. One of the most important functions of the adipose tissue is to regulate lipid metabolism by storing energy in the form of triglycerides via lipogenesis [32] and to release free fatty acids to be used as energy substrates, via lipolysis [33], two processes that are regulated by insulin. We observed a reduced lipogenesis in the presence of insulin, which in part could be explained by the reduced uptake of glucose, a substrate for triglyceride synthesis, but also by the observed inhibition of PKB activation by SEA. In line with this, Lagathu et al. showed a reduced insulin-induced lipogenesis after preincubating 3 T3-F442A preadipocytes with IL-6 for 8 days [20]. Our experiments show similar results when stimulating primary rat adipocytes with SEA for only 30 min. Insulin regulates lipolysis via PKB-dependent activation of the cAMP-hydrolyzing enzyme PDE3B, having an antilipolytic effect [34]. In agreement with a reduction in insulininduced glucose uptake, PKB phosphorylation and reduced lipogenesis, SEA reduced the antagonistic effect of insulin on lipolysis and potentiated iso-induced lipolysis. Several studies have demonstrated an association between IL-6 and increased lipolysis both in vitro[44,47–49] and in vivo[50–52], supporting our results with SEA. In parallel with a reduction in PKB-phosphorylation, SEA induced phosphorylation of HSL, in line with the observed potentiation of lipolysis. Until now, nothing has been reported on a gp130-induced phosphorylation of HSL, however, more long term effects of IL-6 stimulation have been found to increase the transcription of HSL [47]. Although bacterial infections might not be entirely responsible for the development of obesity and T2D, it has been suggested that infections could be the last trigger causing disease onset [53]. Our results suggests that SEA from S. aureus infections could contribute to the development of adipose tissue insulin resistance, tissue inflammation and the development of T2D by binding to the gp130 receptor on adipocytes. Physiolog-

ically, the net effect of decreased lipogenesis, reduced inhibition of lipolysis as well as an increase in lipolysis would result in elevated levels of free fatty acids systemically, causing lipotoxicity and peripheral insulin resistance [54]. The strength of this study is that we have looked at the effects of SEA on a number of biological processes and cellular signaling events in a model highly relevant in the context of regulation of whole body energy homeostasis and insulin sensitivity. Indeed, all results demonstrate that SEA-stimulation leads to less insulin sensitive adipocytes. A weakness of this study is that it focuses on acute effects. It would be interesting to investigate more long-term effects of SEA on adipocyte metabolism including studies on SEAdependent activation of gp130 expression of SOCS3 in relation to insulin signaling and action. The translational potential for the findings relates to interface between bacterial infections and dysregulation of metabolism. Since infections have previously been associated with metabolic disorders, our results suggest that SEA from S. aureus infections could be a contributing factor to insulin resistance seen in type 2 diabetes. Also, since gp130 is expressed on most cell types in the body, SEA as well as other superantigens may target and regulate gp130 on cells not expressing MHC class II or TCR. This could further modulate local inflammation and immune responses seen during for example chronic skin and airway diseases, pneumonia, infective endocarditis and food poisoning caused by S. aureus infections. In conclusion, by examining the effect of SEA on primary adipocytes we have revealed that SEA binds to the gp130 receptor and activates STAT3-signaling and that SEA has a number of effects on adipocyte glucose and lipid metabolism and signaling events, all in agreement with an ability of SEA to lower insulin sensitivity.

Author contributions All authors contributed to the design of the study. EB, KR, ME, JD and BN contributed to the conduct of the study, data collection and data analysis. All authors contributed to data interpretation and manuscript writing.

Funding This work was supported by FLÄK (the research school in pharmaceutical science), The Swedish medical research council [VR, ED project 3362], Lund University Diabetes Center (LUDC), AFA Försäkringar, Olle Engkvist Fondation and Åke Wibergs Stiftelse.

Acknowledgment The authors would also like to thank Eva Ohlson and AnnKristin Holmén-Pålbrink for excellent technical assistance. We thank Active Biotech Research for providing purified SEA and SEA-D227A mutant, for the gp130 and SEA plasmids. For the gift of the MHC plasmids, we thank Prof. Lawrence Stern. The authors have no conflict of interest related to this manuscript.

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Conflict of interest The authors have no conflict of interest related to this manuscript.

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