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European Journal of Cell Biology 90 (2011) 1000– 1015

Contents lists available at ScienceDirect

European Journal of Cell Biology

jou rn al homepage: www.elsev ier .de /e jcb

onditioned medium from hypoxia-treated adipocytes renders muscle cellsnsulin resistant

unna Yua, Lihuan Shia, Hui Wanga, Philip J. Bilanb, Zhi Yaoa,. Constantine Samaanb, Qing Hea, Amira Klipb, Wenyan Niua,∗

Department of Immunology, Key Laboratory of Immuno Microenvironment and Disease of the Educational Ministry of China, Tianjin Medical University, Tianjin 300070, ChinaProgram in Cell Biology, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada

r t i c l e i n f o

rticle history:eceived 18 January 2011eceived in revised form 24 June 2011ccepted 24 June 2011

eywords:ypoxiadipocyte2C12 muscle cellslucose transporter 4onditioned medium

nsulin signalingL-6

CP-1diponectin

a b s t r a c t

Adipose tissue hypoxia is an early phenotype in obesity, associated with macrophage infiltration and localinflammation. Here we test the hypothesis that adipocytes in culture respond to a hypoxic environmentwith the release of pro-inflammatory factors that stimulate macrophage migration and cause muscleinsulin resistance. 3T3-L1 adipocytes cultured in a 1% O2 atmosphere responded with a classic hypoxiaresponse by elevating protein expression of HIF-1�. This was associated with elevated mRNA expressionand peptide release of cytokines TNF�, IL-6 and the chemokine monocyte chemoattractant protein-1(MCP-1). The mRNA and protein expression of the anti-inflammatory adipokine adiponectin was reduced.Conditioned medium from hypoxia-treated adipocytes (CM-H), inhibited insulin-stimulated and raisedbasal cell surface levels of GLUT4myc stably expressed in C2C12 myotubes. Insulin stimulation of Aktand AS160 phosphorylation, key regulators of GLUT4myc exocytosis, was markedly impaired. CM-H alsocaused activation of JNK and S6K, and elevated serine phosphorylation of IRS1 in the C2C12 myotubes.These effects were implicated in reducing propagation of insulin signaling to Akt and AS160. Heatinactivation of CM-H reversed its dual effects on GLUT4myc traffic in muscle cells. Interestingly, antibody-mediated neutralization of IL-6 in CM-H lowered its effect on both the basal and insulin-stimulatedcell surface GLUT4myc compared to unmodified CM-H. IL-6 may have regulated GLUT4myc trafficthrough its action on AMPK. Additionally, antibody-mediated neutralization of MCP-1 partly reversedthe inhibition of insulin-stimulated GLUT4myc exocytosis caused by unmodified CM-H. In Transwellco-culture, hypoxia-challenged adipocytes attracted RAW 264.7 macrophages, consistent with elevated

release of MCP-1 from adipocytes during hypoxia. Neutralization of MCP-1 in adipocyte CM-H preventedmacrophage migration towards it and partly reversed the effect of CM-H on insulin response in musclecells. We conclude that adipose tissue hypoxia may be an important trigger of its inflammatory responseobserved in obesity, and the elevated chemokine MCP-1 may contribute to increased macrophage migra-tion towards adipose tissue and subsequent decreased insulin responsiveness of glucose uptake in muscle.

ntroduction

Obesity is associated with chronic inflammation in adipose tis-ue (Sell and Eckel, 2010). However, how the inflammation isriggered is not well-defined. Recently adipose tissue has beenharacterized with low oxygen tension (hypoxia) in genetic andiet-induced obesity in mice and human obesity (Hosogai et al.,

007; Ye et al., 2007). In vivo, adipose tissue hypoxia is associ-ted with elevated expression of a hypoxia gene profile, includinglevated expression of the hypoxia inducible factor (HIF)1-�

∗ Corresponding author. Tel.: +86 13820512649; fax: +86 2227833287.E-mail addresses: wniu@tijmu.edu.cn, wyniu@hotmail.com (W. Niu).

171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.ejcb.2011.06.004

© 2011 Elsevier GmbH. All rights reserved.

transcription factor and the GLUT1 glucose transporter, elevatedexpression of pro-inflammatory genes such as TNF� and IL-6,infiltration by macrophages, and reduction in adiponectin geneexpression (Chen et al., 2006; Lolmede et al., 2003; Wang et al.,2007a). Indeed, cultured primary adipocytes and 3T3-L1 adipocytesincrease expression of hypoxia-inducible genes when cultured inlow oxygen conditions for several hours (Ye et al., 2007). Inter-estingly, cultured adipocytes also increase their pro-inflammatorygene expression when exposed to hypoxic conditions, suggestingthat adipocytes contribute to the inflammatory response of adi-

pose tissue in vivo. Yet, how hypoxia contributes to adipose tissueinflammation during obesity requires clarification.

Dysfunction of adipose tissue contributes to the pathophysiol-ogy of obesity-related metabolic diseases such as insulin resistance

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J. Yu et al. / European Journal o

nd type 2 diabetes, by promoting insulin resistance in liver andkeletal muscle (Guilherme et al., 2008). Skeletal muscle plays anmportant role in glucose homeostasis, since it is the major site fornsulin-stimulated glucose disposal after a meal. Insulin acts on itsurface receptors to elicit a cascade of intracellular signals throughRS1, PI3K, Akt and AS160/TBC1D4 to stimulate GLUT4 exocytosiso the muscle membrane and glucose removal from the circula-ion (Thong et al., 2005). Therefore, insulin resistance of glucoseptake in skeletal muscle can have a deleterious effect on whole-ody glucose homeostasis. At the molecular level, insulin resistance

n skeletal muscle is associated with blunted responsiveness of thensulin signaling pathway (Boura-Halfon and Zick, 2009; Kellerert al., 1998) that is often manifest by elevated serine phosphoryla-ion of IRS1 via activation of the stress kinase c-Jun NH2-terminalinase (JNK) and/or the negative feed-back of p70S6-kinase (S6K)Herschkovitz et al., 2007; Hotamisligil, 2006).

Given that adipose tissue is an endocrine organ it is postu-ated that, in obese animals, release of pro-inflammatory cytokineso the circulation with reduced production of insulin-sensitizingdipokines such as adiponectin and elevated release of free fattycids, conveys insulin resistance to skeletal muscle (Plomgaardt al., 2005; Schenk et al., 2008; Tsuchiya et al., 2010). Macrophagesre the main producers of cytokines in obese adipose tissueWeisberg et al., 2003; Xu et al., 2003). Prolonged exposure ofonditioned medium from adipocytes and macrophages rendersuscle cells insulin resistant (Samokhvalov et al., 2009; Sell et al.,

008), but the effect of hypoxia on this communication has noteen explored. Importantly, hypoxia in adipose tissue of geneti-ally obese ob/ob mice is observed as early as 6 weeks of age (Yint al., 2009), suggesting it could play a role in the pathophysiologyf obesity.

Here we explore the hypothesis that hypoxic adipocytes releasenflammatory factors that can attract macrophages and confernsulin resistance to skeletal muscle. To test the fundamentals ofhis hypothesis, we explored the effect of conditioned mediumrom adipocytes grown under hypoxic conditions, on macrophage

igration and insulin-stimulated signaling and GLUT4 exocytosisn C2C12 myotubes.

aterials and methods

aterials

The protease inhibitor cocktail, o-phenylenediamine dihy-rochloride, anti-c-myc (epitope) polyclonal IgG, anti-�-Actinin-1,nti-�-actin, dexamethasone (Dex), 3-isobutyl-1-methylxanthineIBMX), porcine insulin and all other chemicals unless otherwiseoted were from Sigma Chemical (St. Louis, MO). Human insulinHumulin R) was from Eli Lilly Canada (Toronto, ON, Canada).ulbecco’s modified Eagle’s medium (DMEM), Horse serum (HS),enicillin/streptomycin and trypsin-EDTA were from InvitrogenCarlsbad, CA). Fetal bovine serum (FBS) was from BioInd (Israel).ompound C and blasticidin-hydrochloride were from CalbiochemSan Diego, CA). The RNApure High-purity Total RNA Rapid Extrac-ion Kit (Spin-column) was from BioTeke Corporation (Beijing,hina). The cDNA synthesis kit was from TaKaRa Biotechnol-gy (Dalian, China). FastStart Universal SYBY Green Master wasrom Roche (Switzerland). ELISA kits for TNF�, MCP-1 and IL-

were from RayBiotech (USA). The ELISA kit for adiponectin,ecombinant mouse MCP-1 and anti-mouse IL-6 antibody wererom R&D Systems, Inc. (USA). The anti-mouse MCP-1 anti-ody was from BioLegend (USA). The kit for measurement of

on-esterified fatty acids was from Wako Diagnostics (Japan).embrane inserts for 24-well culture dishes with a pore size

f 8 �m and insert companion plates were from Corning (Nework, NY). The Pierce BCA protein detection kit was from Thermo

Biology 90 (2011) 1000– 1015 1001

Fisher Scientific (Rockford, IL). Anti-Akt, anti-IRS1, anti-JNK, anti-S6K, anti-phospho-Akt (S473), anti-phospho-IRS1 (Ser636/639),anti-phospho-JNK (Thr183/Tyr185), anti-phospho-S6K (Thr389),anti-phospho-AS160 (T642), anti-phospho-AMPK (Thr172), anti-phospho-Acetyl-CoA carboxylase (ACC, Ser79) antibodies werefrom Cell Signaling Technology (Danvers, MA). Anti-phospho-IRS1(Ser307) was from Upstate (New York, USA). The anti-HIF-1� anti-body was from Abcam (Cambridge, UK). The antibody to GLUT1was prepared by rabbit immunization with keyhole limpet hemo-cyanin conjugated to peptides representing the last 12 amino acidsof rat GLUT1 in Complete Freund’s adjuvant (Sargeant and Paquet,1993). Horseradish peroxidase (HRP)-bound goat anti-mouse, goatanti-rabbit IgG antibodies were from Jackson ImmunoResearchLaboratories (West Grove, PA). The Immobilon Western Chemilu-minescent HRP Substrate was from Millipore (Billerica, MA). Filmwas from Fuji (Tokyo, Japan).

Cell culture

C2C12-GLUT4myc myoblasts were generated and differentiatedinto myotubes as described (Niu et al., 2010). C2C12-GLUT4mycmyoblasts were maintained in a humidified atmosphere of air and5% CO2 at 37 ◦C with DMEM containing 4.5 g/l glucose supple-mented with 10% FBS (v/v), antibiotics and 5 �g/ml blasticidin-HCl.Upon reaching confluency, the serum was lowered to 5% HS (v/v) toallow myotube formation, and cultures were used for experimen-tation within 7–8 days after seeding.

3T3-L1 fibroblasts were grown in DMEM containing 100 U/mlpenicillin, 100 �g/ml streptomycin and 10% FBS at 37 ◦C in 5%CO2. Cells were differentiated into adipocytes with a cocktailcontaining 0.5 mM isobutylmethylxanthine, 1 �M dexametha-sone, and 10 �g/ml insulin (differentiation cocktail) suspendedin DMEM + 10% FBS. Confluent cells were allowed to grow ingrowth medium for 2 days, exposed to a differentiation cocktailmedium for 2 days, treated with growth medium containing insulin(10 �g/ml) for an additional 2 days, then finally incubated withgrowth medium until full differentiation (defined as >80% surfacearea coverage by fat droplets) was achieved. Mouse macrophages(RAW 264.7) maintained in DMEM with 10% FBS were passagedby incubation in calcium-free PBS for 10 min at 37 ◦C, then agi-tated with a pipettor followed by centrifugation and suspensioninto DMEM with 10% FBS.

Conditioned media preparation

All conditioned media (CM) from adipocytes were collectedat day 8 after initial exposure to a differentiation cocktail. Fornormoxia or hypoxia treatment, the medium of 3T3-L1 cells wasreplaced with DMEM containing 5% HS (i.e. differentiation mediumof C2C12 cells) and the adipocytes were incubated at 37 ◦C under21% O2 and 5% CO2 (normoxic condition) or were transferred toan incubator (Model 3131, Thermo Fisher Scientific, USA) with ahumidified atmosphere of 1% O2, 94% N2 and 5% CO2 (hypoxiccondition). Compressed gases were from Kaiheng Gas Corpora-tion (Tianjin, China). CM from normoxic or hypoxic adipocytes wascollected after 4 h and centrifuged at 1000 rpm for 5 min and thesupernatant was used freshly when applied to muscle cultures orstored at −80 ◦C until required for cytokine analysis. Muscle cellswere incubated with adipocyte CM for 16 h as indicated in the figurelegends.

Quantitative real-time PCR

Adipocyte total RNA was isolated with the RNApure High-purity Total RNA Rapid Extraction Kit (Spin-column) following themanufacturer’s instructions. SYBR Green quantitative real-time

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002 J. Yu et al. / European Journal o

CR analysis was performed with an ABI PRISM 7500 Sequenceetection System (Applied Biosystems) and pre-validated primer

ets. All samples were run in triplicate. Threshold cycles werelaced in the logarithmic portion of the amplification curve, andhe results were normalized to �-actin. Actin mRNA abundance isot affected by hypoxia (Ye et al., 2007) and is therefore a validontrol for normalizing changes in cytokine mRNA. The fold differ-nce between two samples was determined using the delta-deltaethod [S1/S2 = 2−(T1−T2)], where S1 and S2 represent samples 1

nd 2 and T1 and T2 represent the threshold cycles of samples 1nd 2. The primers used were as follows: �-actin (NM 007393),′-GGCTGTATTCCCCTCCATCG-3′, 3′-CCAGTTGGTAACAATGCCATGT5′, TNF� (NM 013693), 5′-CGTCGTAGCAAACCACCAA-3′,′-GAGAACCTGGGAGTAGACAAGG-5′. IL-6 (NM 031168), 5′-CGGAGAGGAGACTTCACAG-3′, 3′-CAGAATTGCCATTGCACAAC-5′.CP-1 (NM 011333), 5′-GCAGTTAACGCCCCACTCA-3′, 3′-

CCAGCCTACTCATTGGGATCA-5′. Adiponectin (NM 009605),′-GCTCAGGATGCTACTGTTG-3′, 3′-TCTCACCCTTAGGACCAAG-5′.

ytokine analysis

3T3-L1 adipocytes were cultured in 6-well plates and treatednder normoxia or hypoxia conditions. The secreted levels of TNF�,CP-1, IL-6 and adiponectin in the medium in 4 h were determined

sing ELISA kits (see ‘Materials’ section).

acrophage migration

The macrophage migration assay was performed using co-ulture with adipocytes in 24-well Transwell plates (8-�m poreize). Three million RAW 264.7 macrophages were passaged andeeded into the upper chambers. After the macrophages settledown (30 min), the upper chambers were placed in the Tran-well plates with 3T3-L1 adipocytes grown in the lower chambers.wo groups of adipocytes (set 1 and set 2) were co-cultured withacrophages for 4 h under normoxia or hypoxia conditions. Each

roup of adipocytes received fresh media for the same length ofime (8 h). At the end of the co-culture incubation, macrophagesttached to the upper surfaces of the insert were wiped off andacrophages attached to the lower surfaces of the inserts were

xed with 3% PFA for 30 min and stained with crystal violet. Imagesf the inserts were taken after washing using a Motic BA400 lighticroscope with Motic Advanced 3.2 software. The number ofigrated macrophages was counted at 400 times magnification in

separate fields in each experiment and averaged as the numberf cells per field of view. In another set of experiments, additionf adipocyte growth medium alone (i.e. without cells) or the sameedium supplemented with 1 �g/ml MCP-1 to the lower chamber

f the Transwell co-culture system, served as negative and pos-tive macrophage migration controls. To neutralize MCP-1, equalortions of the different adipocyte CM were pre-incubated with

�g/ml of neutralizing antibody MCP-1 (+) or IgG control anti-ody (−) for 1 h at 37 ◦C. These CM samples were then added tohe lower chambers of Transwell systems and macrophages in thepper chambers were inserted for 4 h incubation under normoxiand processed for migration as above.

ell surface GLUT4myc density

GLUT4myc levels were detected at the cell surface of intactyotubes using the protocol described in Niu et al. (2011). Essen-

ially, cells grown in 24-well plates and treated as indicated were

ashed twice with ice-cold PBS, fixed with 3% (v/v) paraformalde-yde for 10 min at 4 ◦C then 20 min at room temperature. Allubsequent steps were at room temperature. Cells were rinsednd incubated for 10 min with 0.1 M glycine in PBS. Following

Biology 90 (2011) 1000– 1015

blocking with 5% non-fat milk (w/v) in PBS for 10 min, cells werereacted with anti-myc polyclonal antibody (1:250) in 5% milk for1 h. After washes with PBS, cells were incubated with horseradishperoxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000) for 1 h,extensively washed with PBS, and incubated with 1 ml/well of0.4 mg/ml o-phenylenediamine dihydrochloride reagent. The reac-tion was stopped by addition of 0.25 ml of 3 N HCl. The supernatantwas collected to read optical absorbance at 492 nm. The backgroundabsorbance obtained from wild type C2C12 myotubes was sub-tracted from all values.

Cell lysates and immunoblotting

C2C12-GLUT4myc myotubes grown in 6-well plates were lysedwith 300 �l RIPA buffer (100 mM NaCl, 0.25% w/v sodium deoxy-cholate, 1.0% w/v NP40, 0.1% w/v SDS, 2 mM EDTA, 50 mMNaF, 10 nM okadaic acid, 1 mM sodium orthovanadate, proteaseinhibitor cocktail and 50 mM Tris-HCl, pH 7.2) on ice. Lysates werecentrifuged (13,000 rpm) for 10 min at 4 ◦C, and the protein concen-tration was determined using BCA protein assay reagent. Lysates ofequal protein were mixed with 5 × Laemmli sample buffer (finalconcentration: 1% (w/v) SDS, 7.5% (v/v) �-mercaptoethanol, 10%glycerol, 0.05% (w/v) bromophenol blue, 50 mM Tris-HCl, pH 6.8),heated for 15 min at 65 ◦C, resolved by SDS-PAGE, transferredto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA).After blocking with 3% BSA in Tris-buffered saline (TBS) containing0.05% (w/v) each of Tween-20 and NP40, the blots were incubatedwith antibodies to: GLUT1 (1:1000), HIF-1� (1:600), Akt (1:1000),IRS1 (1:1000), JNK (1:1000), S6K (1:1000), phospho-S473-Akt(1:1000), phospho-S636/639-IRS1 (1:1000), phospho-S307-IRS1(1:1000), phospho-T183/Y185-JNK (1:1000), phospho-T389-S6K(1:1000), phospho-T642-AS160 (1:1000), phospho-T172-AMPK(1:1000), phospho-S79-ACC (1:1000), �-actin (1:5000), �-Actinin-1 (1:20,000) in TBS-Tween/NP40 containing 1% BSA and sodiumazide. Immunoblots were incubated with appropriate HRP-conjugated secondary antibodies in TBS-Tween/NP40 + 1% BSA andfollowing washes they were developed with chemiluminescentreagent and autoradiographic film. Densitometric quantification ofprotein bands was performed using National Institutes of Health(NIH) Image J software.

Statistical analysis

Statistical analysis was performed using Prism 3.0 software(San Diego, CA). Two groups were compared using the Student’st-test and data sets of more than two groups were compared usingANOVA with Tukey’s post hoc analysis.

Results

Hypoxia increases HIF-1 ̨ and GLUT1 expression in adipocytes

Increased HIF-1� and GLUT1 expression is a prototypicalresponse of cells to a hypoxic environment (Bashan et al., 1993;Trayhurn and Wood, 2004; Wood et al., 2007; Yin et al., 2009). 3T3-L1 adipocytes were incubated in a low oxygen atmosphere (1% O2)for 4 h while 3T3-L1 control adipocytes were incubated in parallelunder normal oxygen conditions (normoxia, 21% O2). Expression ofthe hypoxia-responsive protein, HIF-1� and GLUT1 were examinedby immunoblotting of cell lysates. HIF-1� and GLUT1 protein lev-

els increased by 3.1 ± 0.6- and 5.0 ± 0.4-fold, respectively, underconditions of hypoxia compared to control, whereas the proteinlevels of the cytoskeletal protein, �-Actinin-1 remained unalteredby hypoxia (Fig. 1A). These observations confirm that a bona fide

J. Yu et al. / European Journal of Cell Biology 90 (2011) 1000– 1015 1003

Fig. 1. Hypoxia regulates the expression of HIF-1�, GLUT1 and adipokine secretion in adipocytes. 3T3-L1 adipocytes were incubated for 4 h in 1% O2 atmosphere (hypoxia) orn and Ga ± S.E.c

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ormal atmosphere (normoxia) prior to (A) cell lysis and immunoblotting for HIF-1�nd adiponectin concentrations measured by ELISA, respectively. Results are meansells.

ypoxic response has been achieved by culturing adipocytes in lowxygen for 4 h.

ypoxia regulates expression and release of inflammatoryytokines and adiponectin from adipocytes

Ye et al. have shown that 2 h of hypoxia induces rapid expressionf inflammatory cytokine genes in 3T3-L1 adipocytes, typified by

1.5–2-fold induction of TNF� and IL-6 mRNA (Ye et al., 2007).uilding on these findings, we searched for inflammation-relatedolypeptides secreted into the medium by adipocytes challenged

ith hypoxia for 4 h. In the conditioned medium (CM) derived fromormoxic 3T3-L1 adipocytes (CM-N), inflammatory cytokines and

actors were present, however, the amount of TNF�, IL-6 and MCP- polypeptides was comparatively elevated in CM from hypoxic

LUT1 protein or (B) collection of the CM followed by detection of TNF�, IL-6, MCP-1 of 6 independent experiments. *p < 0.05, **p < 0.01, #p < 0.001 vs. normoxia-treated

adipocytes (CM-H). The fold increases were 2.6 ± 0.3, 3.7 ± 0.5 and3.5 ± 0.7, respectively (Fig. 1B). In contrast, the level of adiponectin(an anti-inflammatory adipokine with beneficial effects on insulin-regulated glucose homeostasis (Yamauchi et al., 2002)) was lowerin CM-H by 42 ± 12% compared to CM-N (p < 0.001, Fig. 1B). Thesechanges are consistent with the mRNA changes of these factors attimes as early as 2 h of hypoxia treatment in adipocytes (Ye et al.,2007). Others have also reported stimulation of IL-6 polypeptiderelease from 3T3-L1 after hypoxia (Regazzetti et al., 2009; Wanget al., 2007b).

Hypoxia treated-adipocytes promote macrophage recruitment

The observation that MCP-1 polypeptide is elevated in CM-H(Fig. 1B) is reminiscent of the augmented MCP-1 levels observed

1004 J. Yu et al. / European Journal of Cell Biology 90 (2011) 1000– 1015

Fig. 2. Hypoxia-treated adipocytes recruit more macrophages. (A) Two sets of 3T3-L1 adipocytes were cultured for 4 h in normoxia prior to the addition of an upper chamberof RAW 264.7 macrophages (3 million per well) as illustrated by the time-line. The co-cultures were then incubated under normoxia or hypoxia conditions for another 4 h.Afterwards, macrophages on the under-side of the Transwell were fixed and stained with crystal violet and imaged using light microscopy, as described in Materials andmethods. Macrophage numbers were calculated as the mean number of cells per field of view (seven fields per condition). (B) Shown are representative fields of view fornegative and positive macrophage migration controls in which macrophages were co-cultured with culture medium alone (no adipocytes) or the same media supplementedwith MCP-1 (1 �g/ml) in the lower chamber of the Transwell culture system, respectively. Conditioned medium collected from adipocytes treated under hypoxia or normoxiawere pre-treated without (−) or with (+) anti-MCP-1 antibody (1 �g/ml) before adding them to the lower chamber followed by addition of the macrophage to the upperchambers.

J. Yu et al. / European Journal of Cell Biology 90 (2011) 1000– 1015 1005

Cell Surface GLUT4myc Density (fold above each basal)

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Fig. 3. The insulin-induced gain of cell surface GLUT4myc in myotubes is reducedby conditioned medium (CM) from adipocytes. C2C12-GLUT4myc myotubes wereincubated with CM from normoxia- (CM-N) or hypoxia-treated (CM-H) adipocytesfor 16 h followed by insulin treatment (100 nM, 20 min), then cell surface GLUT4mycdensity was measured as indicated in Materials and methods. Illustrated are themeans ± S.E. of at least 6 independent experiments. #p < 0.001, †p < 0.001 vs. basal(

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Fig. 4. Conditioned medium (CM) from hypoxia-treated adipocytes reduces Akt andAS160 phosphorylation by insulin in myotubes. C2C12-GLUT4myc myotubes weretreated with CM from normoxia- (CM-N) or hypoxia-treated (CM-H) adipocytesfor 16 h, followed by insulin treatment (100 nM, 10 min) prior to cell lysis andimmunoblotting for phospho-Akt S473 (A) and phospho-AS160 T642 (B). Shownare immunoblots representative of 6. The densitometry mean ± S.E. from 6 inde-pendent experiments is plotted as the fold change relative to the result from the

temic influence on glucose metabolism in skeletal muscle. To

untreated) in the regular medium (RM) group or as indicated.

n visceral adipose tissue during obesity (Ye et al., 2007). There-ore, we hypothesized that 3T3-L1-derived CM-H may stimulate

acrophage migration. To test this prediction, we cultured RAW64.7 macrophages in the upper well of a Transwell co-cultureystem along with 3T3-L1 adipocytes in the lower chamber for

h under different conditions (see the time-line, Fig. 2A). In set and set 2, adipocytes were pre-incubated under normoxia for h. Set 1 adipocytes continued under normoxia upon additionf macrophages to the Transwell system. Following the additionf macrophages, set 2 adipocytes were switched to hypoxia for

h. It is important to stress that the timing of the last mediahange prior to experimentation in the two sets of adipocytesas the same – 4 h prior to addition of the Transwell containingacrophages. The light microscopy images (400 x magnification)

n Fig. 2A show the underside of the Transwell chambers. Fixednd crystal violet-stained macrophages are indicated by arrows,nd the darkened or clear circles point to pores in the basef the Transwell chamber. Few macrophages migrated throughhe Transwell chamber towards set 1 adipocytes pre-incubatednder normoxia (Fig. 2A). During hypoxia co-culture (set 2), webserved an increased number of macrophages had moved towardsdipocytes (Fig. 2A). Adding MCP-1 alone to growth medium (with-ut cells) in the lower chamber attracted macrophages (Fig. 2B).nterestingly, adding CM-N or CM-H pre-treated with MCP-1 block-ng antibody to the lower chamber of the Transwell inhibited the

acrophages migration towards both types of CM compared toigration towards untreated CM-N and CM-H (Fig. 2B). These

esults suggested that products from hypoxia-treated adipocytes

ave enhanced macrophage chemoattracting properties and MCP-

plays a major role in this process.

basal (untreated) in the regular medium (RM) group. **p < 0.01 vs. basal in RM or asindicated.

Conditioned medium from hypoxia-treated adipocytes evokesinsulin resistance in muscle cells

Secretion of increased inflammatory cytokines and decreasedadiponectin release from hypoxic adipose tissue may have sys-

investigate this possibility in a cell culture system, CM-N and CM-H were applied to C2C12-GLUT4myc myotubes for 16 h before

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hallenging the latter with 100 nM insulin for 20 min. GLUT4mycxocytosis was measured with an enzyme-linked absorbent assays described before (Niu et al., 2011). As shown in Fig. 3, insulinnhanced GLUT4myc exocytosis by 1.6 ± 0.1-fold (p < 0.001 vs.asal, untreated) in myotubes incubated with regular mediumRM). Incubation with CM-N slightly increased the basal cell sur-ace GLUT4myc level by 1.1 ± 0.04-fold (p < 0.001 vs. basal in RM),hile the maximal response to insulin reached the same level as

n cells treated with RM (1.6 ± 0.1-fold, p < 0.001, Fig. 3). There-ore, the fold increase of GLUT4myc exocytosis above basal waslightly, yet significantly reduced compared to the RM controlondition (1.4 ± 0.1-fold, p < 0.001 vs. RM, Fig. 3). This suggestshat CM-N induces modest insulin resistance of GLUT4myc exo-ytosis. Interestingly, incubation of myotubes with CM-H furtherncreased basal surface GLUT4myc by 1.2 ± 0.1-fold (p < 0.001 vs.asal in RM) and also significantly reduced the insulin-stimulatedesponse compared to CM-N (p < 0.001 vs. insulin in CM-N, Fig. 3)uch that the fold increase of insulin was reduced to 1.1 ± 0.1-foldhen calculated relative to its own basal response (p < 0.001 vs.M or CM-N, Fig. 3). These results demonstrate that adipocyteM-H provoked marked insulin resistance in myotube culturesompared to CM-N.

Akt and AS160 are important signals for insulin-induced GLUT4xocytosis (Zaid et al., 2008), therefore we assessed these responsesn muscle cultures treated with CM from adipocytes. Relative tokt Ser473 phosphorylation in control C2C12-GLUT4myc myotubes,M-N did not affect basal or insulin-stimulated Akt Ser473 (Fig. 4A).owever, CM-N did raise basal AS160 Thr642 phosphorylation,ithout affecting its insulin-stimulated levels in myotubes (p < 0.05

s. basal in RM, Fig. 4B). Consistent with the marked effect of CM-Hn GLUT4myc exocytosis in myotube cultures, CM-H significantlympaired insulin-stimulated Akt Ser473 phosphorylation (p < 0.01s. insulin in CM-N, Fig. 4A) and AS160 Thr642 phosphorylation,n addition to raising the basal AS160 Thr642 phosphorylationp < 0.001 vs. insulin in CM-N; p < 0.01 vs. basal in RM, Fig. 4B).hese results link the effect of CM-H on GLUT4myc exocytosis with

large reduction in the phosphorylation of the Rab-GAP AS160,he down-stream effector of Akt responsible for regulating insulin-timulated GLUT4 traffic in insulin-responsive tissues (Ng et al.,010; Sun et al., 2010).

In C2C12 myotubes, phosphorylation of IRS1 at serine residuess detectable by immunoblotting with phospho-specific antibod-es (Lim et al., 2006; Müssig et al., 2005; Wang et al., 2007a) andRS-1 serine phosphorylation reduces its insulin-dependent tyro-ine phosphorylation and downstream signaling, implicating thisechanism in insulin resistance (Boura-Halfon and Zick, 2009;otamisligil and Spiegelman, 1994). We therefore examined phos-horylation of IRS1 Ser307 and Ser636/639 in myotube culturesreated with adipocyte-derived CM. As shown in Fig. 5A, CM-N hado effect on IRS1 Ser307, whereas CM-H increased both basal and

nsulin-stimulated IRS1 Ser307 phosphorylation. In addition, onlyM-H increased the basal and insulin-stimulated levels of IRS1er636/639 phosphorylation (Fig. 5B). These results are consistentith the reduced insulin-stimulated signaling of Akt and AS160

n CM-H treated myotubes (Fig. 4A and B) and the reduction ofnsulin-stimulated GLUT4myc exocytosis (Fig. 3).

IRS1 Ser307 and Ser636/639 can be phosphorylated by JNK and6K, respectively (Aguirre et al., 2000; Zhang et al., 2008). Indeed,e detected increased activating phosphorylation of JNK and S6K

n myotubes treated with CM-H (Fig. 5C and D) and these patternsf phospho-JNK Thr183/Tyr185 and phospho-S6K Thr389 are consis-ent with the heightened serine phosphorylation of IRS1 on Ser307

nd Ser636/639 in myotubes treated with CM-H (Fig. 5A and B). Theseesults suggest that factors in CM-H increase the activity of IRS1-argeting serine kinases to elevate serine phosphorylation of IRS1,ossibly reducing downstream signaling that lead to the observed

Biology 90 (2011) 1000– 1015

reduction in insulin-stimulated Akt and AS160 phosphorylationand GLUT4myc exocytosis.

The above results outline a likely chain of events that explainhow CM-H induced changes in insulin signaling and insulin-stimulated cell surface levels of GLUT4myc, yet they cannot explainhow basal levels of GLUT4myc and AS160 phosphorylation mightbe elevated in myotube cultures under these conditions. Factorspresent in the CM, such as adiponectin and IL-6, can stimulate AMP-activated protein kinase (AMPK) in muscle (Carey et al., 2006; Yoonet al., 2006) and AMPK activation is known to regulate GLUT4 traf-fic (Jessen and Goodyear, 2005). Therefore, we analyzed the effectof CM on AMPK activity in myotube cultures. Both CM-N and CM-H elevated phosphorylation of AMPK Thr172 and its downstreamsubstrate ACC Ser79 (Fig. 6A and B). These results are consistentwith the observed insulin-independent increase of GLUT4myc atthe cell surface. To explore this possible causal relationship, weco-incubated muscle cultures with the AMPK inhibitor, CompoundC and CM-N or CM-H. As shown in Fig. 6C, Compound C inhib-ited the phosphorylation of ACC in myotubes caused by CM. In thecontrol RM-treated muscle cells, Compound C had a tendency toslightly increase the basal cell surface levels of GLUT4myc (Fig. 6D).CM-N and CM-H each raised the basal levels of GLUT4myc andthese were reduced by Compound C to levels of GLUT4myc seen inRM-treated cells incubated with Compound C. Similarly, insulin-stimulated surface levels of GLUT4myc in CM-N and CM-H groupswere also reduced from 1.5 ± 0.1- and 1.3 ± 0.06-fold to 1.3 ± 0.08-and 1.2 ± 0.1-fold by Compound C, respectively (p < 0.05), whereasno effect of Compound C was observed on insulin response in theRM group. These findings suggest that AMPK mediated the eleva-tion of basal GLUT4myc at the cell surface in response to CM and thatthis contributes to an insulin-independent elevation of GLUT4mycunder insulin conditions (Fig. 6D).

To begin investigation of the components of CM that may stim-ulate AMPK, we tested the effect of IL-6 blocking antibody onphosphorylation of ACC and AMPK by CM. As shown in Fig. 7A,neutralization of IL-6 in RM had no effect on pACC and pAMPK.However, the CM-stimulated phosphorylation of ACC and AMPKwas reduced by neutralization of IL-6 (p < 0.05, p < 0.001 vs. eachcontrol). Consistent with the significant effect of the IL-6 block-ing antibody on pACC and pAMPK, neutralization of IL-6 decreasedboth basal- and insulin-stimulated surface GLUT4myc levels in CM-N and CM-H groups, without an effect on basal or insulin responsesin the RM group. The fold increase of basal cell surface GLUT4mycwas reduced by IL-6 neutralization from 1.2 ± 0.02- to 1.1 ± 0.06-fold in the CM-N group and from 1.3 ± 0.06- to 1.1 ± 0.07-fold inthe CM-H group. The insulin-enhanced surface GLUT4myc densi-ties were reduced from 1.5 ± 0.05- to 1.4 ± 0.09-fold in the CM-Ngroup and from 1.4 ± 0.1- to 1.3 ± 0.09-fold in the CM-H group(p < 0.05 vs. each control, Fig. 7B). In summary, IL-6 present in theCM is able to stimulate GLUT4myc levels at the cell surface in aninsulin-independent manner.

MCP-1 contributes to muscle cell insulin resistance promoted byconditioned medium from hypoxia-treated adipocytes

Both inflammatory cytokines and free fatty acids derived fromadipocytes can influence the insulin response of muscle cells. Wetherefore examined the levels of non-esterified fatty acids (NEFA)in CM under normoxia or hypoxia treatment. As shown in Fig. 8A,the concentration of NEFA was not significantly different betweenCM-N and CM-H. This indicated that NEFA may not contribute to theharsher effects of CM-H on muscle cell insulin responses compared

to CM-N. We further heat-treated CM-N and CM-H to determineif protein factors in CM were responsible for the dysregulation ofGLUT4myc traffic. Heat treatment did not affect the concentrationof NEFA in either CM (Fig. 8A). As shown in Fig. 8B, heat treatment

J. Yu et al. / European Journal of Cell Biology 90 (2011) 1000– 1015 1007

Fig. 5. Conditioned medium (CM) from hypoxic adipocytes affects IRS1, JNK and S6K phosphorylation in myotubes. C2C12-GLUT4myc myotubes were incubated with CM fromnormoxia- (CM-N) or hypoxia-treated (CM-H) adipocytes for 16 h, followed by insulin treatment (100 nM, 10 min) prior to cell lysis and immunoblotting for phospho-IRS1S307 (A), phospho-IRS1 S636/639 (B), phospho-JNK T183/Y185 (C), or phospho-S6K T389 (D). Shown are immunoblots representative of 6. The densitometry mean ± S.E. from6 the bv

hRihCM2tcGisnv

independent experiments is plotted as the fold change relative to the result froms. basal in RM or as indicated.

ad no effect on insulin-stimulated surface GLUT4myc density inM and CM-N groups. However, heat treatment of CM-H increased

nsulin-stimulated cell surface GLUT4myc levels and reduced theeightened basal levels of surface GLUT4myc caused by unmodifiedM-N and CM-H (p < 0.05 vs. control, Fig. 8B). There are reports thatCP-1 can mediate insulin resistance in muscle cells (Kamei et al.,

006), therefore we treated CM-N and CM-H with anti-MCP-1 neu-ralizing antibody prior to and during their incubation with muscleells. Neutralization of MCP-1 increased insulin-stimulated surfaceLUT4myc levels in the CM-H group, without an effect on basal or

nsulin responses in the RM and CM-N groups (Fig. 9). The insulin-timulated cell surface GLUT4myc level was restored by MCP-1eutralization from 1.4 ± 0.05-fold to 1.5 ± 0.07-fold (p < 0.05s. control, Fig. 9). Taken together, the heat inactivation and

asal (untreated) in the regular medium (RM) group. *p < 0.05, **p < 0.01, #p < 0.001

MCP-1 neutralization strategies suggest that MCP-1 contributes tothe inhibition of insulin-stimulated GLUT4myc exocytosis by CM-H. Overall, these results suggest cytokines and chemokines, ratherthan free fatty acids, are the active components in CM-H that conferinsulin resistance to muscle cells.

Prolonged adipocyte hypoxia produces conditioned medium thatconfers further insulin resistance to muscle cells

Whereas a 4 h hypoxia treatment of adipocytes elevated mRNA

expression of TNF�, IL-6 and MCP-1, 24 h of hypoxia elevatedthese cytokines even further (Fig. 10A). Conversely, 4 h of hypoxiasignificantly reduced mRNA expression of adiponectin, and 24 hof hypoxia reduced these levels further (Fig. 10A). Thus, 4 h

1008 J. Yu et al. / European Journal of Cell Biology 90 (2011) 1000– 1015

Fig. 6. Conditioned medium (CM) from adipocytes promotes AMPK and ACC phosphorylation and GLUT4myc cell surface levels in myotubes. C2C12-GLUT4myc myotubeswere incubated with CM from normoxia- (CM-N) or hypoxia-treated (CM-H) adipocytes for 16 h. In one set of experiments this was followed by insulin treatment (100 nM,10 min) prior to cell lysis and immunoblotting for phospho-ACC S79 (A), phospho-AMPK T172 (B). Shown are immunoblots representative of 6. The densitometry mean ± S.E.from 6 independent experiments is plotted as the fold change relative to the result from the basal (untreated) in the regular medium (RM) group. **p < 0.01, #p < 0.001 vs.basal in RM or as indicated. In another set of experiments, cells were treated with CM for 16 h including incubation with or without Compound C (CC, 10 �M) in the last30 min, then phospho-ACC S79 (C) and surface GLUT4myc density (D) were measured as indicated in Materials and methods. Illustrated are the means ± S.E. of 6 independente r as in

hi

tMf2f2tctd2i

xperiments. *p < 0.05, **p < 0.01, #p < 0.001 vs. basal (untreated) in the RM group o

ypoxia induced a significant and robust inflammatory responsen adipocytes that was sustained for up to 24 h.

We next determined how CM collected from 24 h hypoxia-reated adipocytes affects GLUT4 traffic in muscle cells (Fig. 10B).

uscle cells grown in RM responded to insulin with a 1.65 ± 0.04-old gain in surface GLUT4. Exposing muscle cells to adipocyte4 h CM-N or 24 h CM-H increased the basal level of sur-ace GLUT4myc (Fig. 10B). In the insulin-stimulated condition,4 h CM-N, caused a somewhat blunted response comparedo that in RM-cultured cells (Fig. 10B). Moreover, 24 h CM-Haused further insulin resistance vs. 24 h normoxia. Although

hese responses to 24 h CM-N and CM-H were only slightlyifferent from the responses to 4 h CM-N and CM-H (Fig. 3),4 h CM-H nearly eliminated the response of GLUT4myc to

nsulin.

dicated.

Discussion

In obesity, the expanded adipose tissue develops localizedhypoxia that is associated with adipose tissue inflammation(Trayhurn and Wood, 2004). Elevated adipose tissue production ofpro-inflammatory cytokines and elevated numbers of macrophagesand other cell types in this tissue contribute to localized insulinresistance of adipocytes that impairs the anti-lipolytic actions ofthe hormone. These cytokines also enter the circulation along withelevated free fatty acids and, coupled to a reduction in adiponectinrelease, culminate in a combination of changes that impair insulin

action in other tissues, primarily muscle and liver (Zhou et al.,2007). Importantly, the role of adipose tissue hypoxia in the devel-opment of these events is unclear. Nonetheless, 6-week-old ob/obmice present adipose tissue hypoxia, raising the possibility that

J. Yu et al. / European Journal of Cell Biology 90 (2011) 1000– 1015 1009

Fig. 7. IL-6 is partly responsible for the effects of adipocyte-conditioned medium (CM) on basal-state GLUT4myc levels in myotubes. CM from normoxia- (CM-N) or hypoxia-treated (CM-H) adipocytes were incubated with 2.5 �g/ml IL-6 neutralization antibody (+) or IgG control antibody (−) for 1 h prior to application to C2C12-GLUT4mycmyotubes for 16 h. In one set of experiments this was followed by insulin treatment (100 nM, 10 min) prior to cell lysis and immunoblotting for phospho-ACC S79 andp metryt 05, #pt ateria

hricaettbt

a13

hospho-AMPK T172 (A). Shown are immunoblots representative of 6. The densitoo the result from the basal (untreated) in the regular medium (RM) group. *p < 0.reated as in (A) and surface GLUT4myc density (B) was measured as indicated in M

ypoxia is an early event or possible trigger of the inflammatoryesponse associated with obesity (Ye et al., 2007). Indeed, hypoxianduces cell-autonomous expression of several pro-inflammatoryytokines and reduces adiponectin expression in primary humandipocytes and 3T3-L1 adipocytes in vitro (Wang et al., 2007b; Yet al., 2007). Despite some specific differences in the profile ofhe inflammatory gene response between these systems, overallhey suggest the possibility that hypoxic adipocytes release a com-ination of factors that may confer insulin resistance to muscleissue.

In the present study, we show that hypoxic 3T3-L1 adipocyteslso attract macrophages by producing and secreting MCP-. Further, we demonstrate that CM derived from murineT3-L1 adipocytes cultured under hypoxic conditions reduces

mean ± S.E. from 6 independent experiments is plotted as the fold change relative < 0.001 vs. basal in RM or as indicated. In another set of experiments, cells werels and methods. *p < 0.05 vs. basal in RM or as indicated.

insulin-stimulated GLUT4myc exocytosis and Akt and AS160phosphorylation in murine C2C12-GLUT4myc muscle cells. Thisimpaired insulin response is not ascribed to fatty acids but insteaddepends in part upon the chemokine MCP-1 and other unidentified,heat-sensitive factors in CM-H.

Hypoxia regulates expression of inflammatory cytokines andadiponectin in adipocytes in culture

Adipocytes adapt to the anaerobic condition of hypoxia by up-

regulating genes encoding glycolytic enzymes and GLUT1 via theHIF-1� transcription factor (Semenza, 2003). Increased HIF-1� andGLUT1 gene expression has been observed in mice and humanadipocytes in response to low O2 tension (Wang et al., 2007a; Ye

1010 J. Yu et al. / European Journal of Cell Biology 90 (2011) 1000– 1015

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CM-N 16.02 ± 0.016Heated CM-N 18.28 ± 0.018CM-H 18.37 ± 0.018Heated CM-H 19.29 ± 0.019

Concentration of non-esterified fatty acids in CM mean ± SD (nM)

B

A

Fig. 8. Heat-treatment abolishes the effect of adipocyte-conditioned medium (CM) on GLUT4myc traffic in myotubes. CM from normoxia- (CM-N) or hypoxia-treated (CM-H)adipocytes were heated at 95 ◦C for 15 min, then supplemented with 5% HS and applied to C2C12-GLUT4myc myotubes for 16 h. Then insulin (100 nM, 20 min) was addedto myotubes followed by measurement of surface GLUT4myc density as indicated in Materials and methods. Illustrated are the means ± S.E. of 6 independent experiments.*p < 0.05, #p < 0.001 vs. basal (untreated) in the regular medium (RM) group or as indicated.

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MCP-1

Fig. 9. MCP-1 neutralization relieves the insulin resistance imparted by conditioned medium (CM) from hypoxic adipocytes to GLUT4myc traffic in myotubes. CM fromnormoxia- (CM-N) or hypoxia-treated (CM-H) adipocytes were incubated with 1 �g/ml MCP-1 neutralization antibody (+) or IgG control antibody (−) for 1 h prior toapplication to C2C12-GLUT4myc myotubes for 16 h. Then insulin (100 nM, 20 min) was added to myotubes followed by measurement of surface GLUT4myc density asindicated in Materials and methods. Illustrated are the means ± S.E. of 6 independent experiments. *p < 0.05, **p < 0.01, #p < 0.001 vs. basal (untreated) in the regular medium(RM) group or as indicated.

J. Yu et al. / European Journal of Cell Biology 90 (2011) 1000– 1015 1011

Fig. 10. Short and prolonged hypoxia alters mRNA expression of cytokines in adipocytes, and conditioned medium (CM) from prolonged hypoxia treatment exacerbatesinsulin resistance in muscle cells. 3T3-L1 adipocytes were incubated for 4 h or 24 h in 1% O2 atmosphere (hypoxia) or normal atmosphere (normoxia) prior to (A) cell lysisfor total RNA and quantitative PCR analysis of mRNA expression for TNF�, IL-6, MCP-1 and adiponectin as detailed in Materials and methods. The relative abundance of eachcytokine mRNA was compared to normoxia-treated adipocytes. Results are means ± S.E. of 6 independent experiments. *p < 0.05, **p < 0.01, #p < 0.001 vs. normoxia-treatedcells or as indicated. Following the collection of adipocyte CM after 24 h, C2C12-GLUT4myc myotubes were incubated for 16 h with CM from normoxia- (CM-N) or hypoxia-treated (CM-H) adipocytes followed by insulin stimulation (100 nM, 20 min). Cell surface GLUT4myc density was measured as indicated in Materials and methods. Illustrateda in the

eatp

oTct

re the means ± S.E. of 6 independent experiments. #p < 0.001 vs. basal (untreated)

t al., 2007) and, in our model system, 4 h of culturing of 3T3-L1dipocytes in 1% O2 induces a hypoxic response compared to cul-ures kept in normoxia, evinced by elevated HIF-1� and GLUT1rotein levels within this time frame.

Adipose tissue secretes multiple cytokines and adipokines, some

f which (e.g. TNF� and IL-6) are known to modulate insulin action.NF� and IL-6 are well-known cytokines that are elevated in theirculation of obese animals and are released in high levels fromhe expanding adipose tissue (Odegaard and Chawla, 2008; Olefsky

regular medium (RM) group or as indicated.

and Glass, 2010; Sell and Eckel, 2010). Here we observed that 4 h ofhypoxia up-regulates adipocyte TNF� and IL-6 mRNA expressionwith concomitant release of the corresponding polypeptides to themedium. MCP-1 is a pro-inflammatory chemokine that is releasedfrom the expanded adipose tissue in obese animals (Xu et al., 2003;

Ye et al., 2007), and we similarly found it elevated in CM-H com-pared to CM-N. Okada et al. similarly observed induction of MCP-1mRNA in 3T3-L1 adipocytes exposed to hypoxia (Okada et al., 2008).In contrast to the elevated cytokines and chemokine, we found

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012 J. Yu et al. / European Journal o

hat hypoxia reduced the gene and protein expressions of the anti-nflammatory adipokine adiponectin by 3T3-L1 adipocytes. Similareductions in adiponectin release have been observed in adiposeissue from high-fat fed mice or streptozotocin-induced diabeticats (Palanivel et al., 2008; Ye et al., 2007). Therefore, our resultsupport a model whereby hypoxia associated with expanded adi-ose tissue (Ye et al., 2007) is reflective of ensuing changes ofytokines/chemokines/adipokines in the circulation of obese ani-als, in vivo.Both TNF� and IL-6 directly cause insulin resistance in adipose,

epatic and muscle cells in vitro (Austin et al., 2008; Kim et al.,004; Plomgaard et al., 2005) although the effects of IL-6 on mus-le depend on the time of exposure (Nieto-Vazquez et al., 2008),t short times primarily elevating surface GLUT4 levels in mus-le cells (Carey et al., 2006; Roher et al., 2008). Similar to TNF�nd IL-6, MCP-1 can impair insulin signaling in skeletal muscleells (Kamei et al., 2006). In contrast, a reduction in the releasef adiponectin has been associated with reduced insulin sensitiza-ion in L6 muscle cells by CM from rat adipocytes (Vu et al., 2007).ogether, the changes in these cytokines/chemokines/adipokinesbserved in vivo and their associated biological effects suggestedhat our findings of elevated TNF�, IL-6 and MCP-1 and reduceddiponectin in hypoxia-treated 3T3-L1 adipocytes may be respon-ible for the insulin resistance imparted onto muscle tissue (seeext).

ypoxic adipocytes attract macrophages

In the present study we observe augmented MCP-1 release fromnd increased macrophage migration towards hypoxia-cultureddipocytes or CM-H. Moreover, neutralization of MCP-1 in CM-Nr CM-H markedly reduced macrophage migration compared tontreated CM-N and CM-H. These observations highlight MCP-1s a major factor responsible for macrophage attraction towardsypoxic adipocytes. Thus, our findings suggest elevated MCP-1 inhe medium of hypoxia-treated adipocytes plays an important rolen attracting macrophages. In a similar vein, we recently observedndogenous MCP-1 release from palmitate-challenged L6 mus-le cells also contributes to macrophage chemoattraction (Samaant al., unpublished). Importantly, in adipose tissue from obeseice, macrophages are highly associated with areas of greater

ypoxia (Rausch et al., 2008), buttressing a model whereby hypoxicdipocytes in vivo may attract macrophages towards adipose tis-ue in the context of obesity. Indeed, in vivo studies show thatn increased production of inflammatory factors in adipose tis-ue is associated with a rise in the number of macrophages in thisissue of obese mice and humans (Harman-Boehm et al., 2007;

eisberg et al., 2003). MCP-1 is a potent inducer of macrophageigration (Guo et al., 2009; Sozzani et al., 1996) and substan-

ial evidence suggests that MCP-1 is a likely candidate to initiateacrophage infiltration of adipose tissue during obesity. In par-

icular, expression of endogenous MCP-1 (also known as, Ccl2)nd its receptor (CCR2) are elevated in adipose tissue of obesendividuals and this positively correlates with the levels of theD68 macrophage marker in adipose tissue (Huber et al., 2008).

n mice, a rise in MCP-1 in adipose tissue precedes the expressionf other macrophage markers during the development of geneticnd diet-induced obesity (Xu et al., 2003). Further, overexpres-ion of an adipocyte-specific MCP-1 transgene in mice results inacrophage recruitment into adipose tissue and adipose tissue

nflammation (Kamei et al., 2006). However, the prominence ofCP-1 as the major macrophage chemoattractant to adipose tis-

ue in vivo is not without controversy (Inouye et al., 2007; Kandat al., 2006). The complicated phenotypes of transgenic and knock-ut mouse models suggest multiple actions of MCP-1 or its receptornd the potential of various consequences and compensations in

Biology 90 (2011) 1000– 1015

their absence. Instead, simple co-culture experimental systems likethe one described herein may provide more specific information asto the role of MCP-1 in macrophage attraction towards adipocytes.

Conditioned media from hypoxia-treated adipocytes impairsinsulin action in muscle cells

CM-H diminished activation of muscle cell insulin-dependentsignals, lowering insulin-stimulated Akt Ser473 and AS160 Thr642

phosphorylation. Interestingly, we observe elevated serine phos-phorylation of IRS1 at Ser307 and Ser636/639 residues, which areknown to be associated with impaired propagation of insulinsignals from IRS-1 (Boura-Halfon and Zick, 2009; Kellerer et al.,1998) to Akt and AS160 (Bouzakri et al., 2006; Huang et al., 2005;Tamemoto et al., 1994).

JNK is responsible for phosphorylation of Ser307 in IRS1 (Aguirreet al., 2000) and consistent with this, CM-H induced activation ofJNK in C2C12-GLUT4myc myotubes. Similarly, CM-H also causedactivation of S6K, which can phosphorylate IRS1 on Ser636/639 anddiminish downstream signaling to Akt (Tremblay et al., 2005).Although, insulin normally activates JNK and S6K, low-level chronicactivation of these kinases has tonic negative feedback on IRS1activity through their IRS1 serine phosphorylation (Boura-Halfonand Zick, 2009; Kellerer et al., 1998). Indeed in muscle tissue, JNKand S6K are overactive during high fat-feeding (Tremblay et al.,2005; Tuncman et al., 2006) as well as upon exposure to TNF�(Bouzakri and Zierath, 2007; Plomgaard et al., 2005). It is notewor-thy that conditioned media from untreated primary adipocytes orco-cultures of adipocytes and skeletal muscle cells impaired insulinsignaling to Akt in the muscle cells (Dietze et al., 2002; Skurk et al.,2009). However, in those studies CM was collected from adipocytesor co-cultures were maintained for 24 h and 48 h, respectively andmay have accumulated greater levels of adipocyte-associated fac-tors. Since a goal of our study was to establish parameters in whichCM from hypoxia-treated adipocytes would cause insulin resis-tance compared to control CM, we collected CM at shorter timesthat mitigated the effects of CM-N on muscle cells.

The negative effects of CM-H on insulin signaling in musclecells were characterized by impairing insulin-stimulated GLUT4translocation in C2C12-GLUT4myc myotubes. In skeletal muscletissue, GLUT4 is the major glucose transporter that mediatesinsulin-stimulated glucose uptake (Konrad et al., 2005). In con-trast, wild type C2C12 myotubes express high levels of GLUT1,which accounts for 83% and 58% of basal and insulin-stimulated glu-cose uptake, respectively (Niu et al., 2010). Therefore, we createdthe C2C12-GLUT4myc muscle cell line to facilitate the measure-ment of cell surface GLUT4 levels, rather than measuring themore integrated response of glucose uptake that is reflective of allglucose transporters in the cell/tissue (Niu et al., 2010). Accord-ingly, we found that CM-H markedly reduced the fold increase ininsulin-stimulated surface GLUT4myc levels in C2C12-GLUT4mycmyotubes, while CM-N induced a smaller, yet significant reduc-tion. In the case of CM-N, the reduction was not due to a changein the responsiveness to insulin, but to the aforementioned risein basal cell surface GLUT4myc levels. In contrast, CM-H produceda greater rise in the basal levels and a significant reduction in themaximal insulin response of cell surface GLUT4myc. CM-N and CM-H collected for 24 h from adipocytes, produced slightly exaggerateddecreases in the insulin-stimulated gain in cell surface levels ofGLUT4myc in muscle cells. Thus, the insulin resistance of musclecultures induced by factors in 4 or 24 h CM-H may be similar. Thisis consistent with the modest differences in the mRNA expression of

TNF�, IL-6 and MCP-1 in adipocytes treated for 4 vs. 24 h of hypoxia.Nevertheless, future experimentation with adipocyte CM producedby prolonged hypoxia may reveal novel findings with respect toadipocyte factor cross-talk with muscle cells and macrophages.

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The complex changes we outlined above, led us to search forhe responsible components in the CM and to begin to dissect theirpecific roles in regulating GLUT4 traffic and insulin resistance.

NEFA are released by lipolysis from adipocytes and impart directegative effects on insulin signaling in muscle within a short periodf time (Bilan et al., 2009). However, the levels of NEFA were notifferent between CM-N and CM-H. Moreover, heat inactivation ofM-N and CM-H, which would not be expected to damage NEFAhile eliminating polypeptide activities, completely prevented the

ffects of CM-N and CM-H on basal and CM-H on insulin-stimulatedLUT4myc cell surface levels. This led us to investigate polypep-

ide components of CM that might be responsible for the effects onuscle cells.

L-6 and MCP-1 released from adipocytes regulate basal andnsulin-stimulated GLUT4 traffic in muscle cells

TNF� directly administered to L6 muscle cells raises basal andnsulin-stimulated GLUT4myc levels in L6 muscle cells (Roher et al.,008), as is the case for IL-6 administration (Carey et al., 2006).owever, the biology of IL-6 on muscle is complex, since it canlso activate AMPK and thereby elevate both basal and insulin-timulated glucose uptake (Carey et al., 2006). Thus, the contextr environment in which IL-6 is released and the time of exposureay influence the effect it exerts on muscle cells.Given that AMPK can stimulate GLUT4 exocytosis in muscle cells

n an insulin independent manner and its activation is additive tonsulin-stimulated GLUT4 exocytosis (Jessen and Goodyear, 2005),

e explored whether AMPK may partake in the actions of CM onLUT4myc traffic. Indeed, CM-N and CM-H activated AMPK (phos-horylation of T172 of its alpha-subunit and phosphorylation of theMPK target, ACC). Interestingly, AMPK activation may account for

he increase in basal AS160 phosphorylation that we observe inesponse to CM-N and CM-H (Fig. 5), since AMPK can phosphory-ate AS160 at T642 (Kramer et al., 2006). Moreover, incubation ofM-treated C2C12-GLUT4myc myotubes with the AMPK inhibitor,ompound C revealed an AMPK-dependent component for both theise in basal and insulin-stimulated cell surface levels of GLUT4mycn CM-treated myotubes (Fig. 6D). Because Compound C did notffect basal nor insulin-stimulated GLUT4myc cell surface levelsn myotubes cultured in regular medium, the above effects refero specific actions of CM on the myotubes that manifest throughMPK.

These observations suggest CM-N and CM-H contain factors thatctivate AMPK, and affect insulin action in muscle cells. BecauseNF� actually inhibits AMPK in muscle (Steinberg et al., 2006),e focused on the possibility that IL-6 present in CM-H might be

esponsible for activating AMPK and conferring insulin resistanceo muscle cells. To this end we antagonized the action of IL-6 inM by adding a neutralizing antibody. Our results confirm thatM-N or CM-H contain IL-6 that when neutralized reduces AMPKctivation and basal and insulin-stimulated cell surface GLUT4mycevels induced by CM-N and CM-H (Fig. 7). Future experimentation

ill be needed to ascertain whether TNF� has additional input onLUT4myc traffic in this cellular model.

MCP-1 is a marker of pro-inflammatory macrophages and itsxpression is elevated upon feeding mice a high-fat diet (Fujisakat al., 2009). Interestingly, an acute administration of recombinantCP-1 to mice during a euglycemic-hyperinsulinemic clamp pro-

oked skeletal muscle insulin resistance within hours, independentf macrophage infiltration of adipose tissue (Tateya et al., 2010).urther, direct MCP-1 administration to cultured skeletal muscle

ells or isolated muscle ex vivo, induced insulin-resistant Akt phos-horylation and glucose uptake (Kamei et al., 2006). Our resultshow that blocking MCP-1 in CM-H with a neutralizing antibodyartly reversed the muscle cell insulin resistance of GLUT4myc

Biology 90 (2011) 1000– 1015 1013

traffic imparted by CM-H. Notably, this effect was not complete andMCP-1 neutralization did not prevent the rise in basal-state surfaceGLUT4myc. These results suggest that additional factors contributeto the insulin resistance induced by CM-H, and that MCP-1 doesnot regulate GLUT4myc traffic in an insulin-independent manner.It will be of interest to explore the mechanism of MCP-1-inducedinsulin resistance in muscle, but earlier studies indicate that ERK1/2inhibition reverses the muscle insulin resistance caused by MCP-1(Kamei et al., 2006).

In summary, C2C12-GLUT4myc myotubes exposed to CM fromadipocytes show modest reductions in insulin-induced gains in sur-face GLUT4. CM from hypoxic adipocytes markedly worsens thiseffect and reduces insulin signaling. Through the use of neutraliz-ing antibodies we show that MCP-1 is partly responsible for theinhibition of insulin-stimulated GLUT4 externalization provokedby CM-H. IL-6 is also present in CM-H and is responsible for theinsulin-independent elevation rise in surface GLUT4 levels, most-likely mediated through the activation of AMPK. Finally, hypoxicadipocytes show elevated ability to attract macrophages and thisis largely attributed to the release of MCP-1. These cell culturestudies therefore provide strong proof of concept that hypoxia pro-motes adipocyte release of chemokines and cytokines that attractmacrophages and provoke muscle cell insulin resistance. Com-prehension of the factors released from hypoxic adipocytes thatinitiate these two hallmarks of obesity-associated insulin resis-tance – macrophage migration to adipose tissue and muscle tissueinsulin resistance – will help in our understanding of the patho-physiology of obesity-related diseases.

Acknowledgements

This work was supported by the National Natural Science Foun-dation of China (grants #30611120532 and #30570912), by theTianjin Municipal Science and Technology Commission (grant #09ZCZDSF04500) to W. Niu, and by a grant to A. Klip from the Cana-dian Diabetes Association. M. Constantine Samaan was supportedby a grant from the Canadian Pediatric Endocrine Group (CPEG).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ejcb.2011.06.004.

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