SDF-1 Is an Autocrine Insulin-Desensitizing Factor in Adipocytes · 2018. 5. 15. · a previously unnoticed chemokine, stromal derived factor-1 (SDF-1), also called CXCL12. We describe

SDF-1 Is an Autocrine Insulin-Desensitizing Factor inAdipocytesJihoon Shin,1,2,3 Atsunori Fukuhara,1,4 Toshiharu Onodera,1,3 Shunbun Kita,1,4 Chieko Yokoyama,1,5

Michio Otsuki,1 and Iichiro Shimomura1,2

Diabetes 2018;67:1068–1078 | https://doi.org/10.2337/db17-0706

Insulin desensitization occurs not only under the obesediabetic condition but also in the fasting state. However,little is known about the common secretory factor(s) thatare regulated under these two insulin-desensitized con-ditions. Here, using database analysis and in vitro andin vivo experiments, we identified stromal derived factor-1(SDF-1) as an insulin-desensitizing factor in adipocytes,overexpressed in both fasting and obese adipose tissues.Exogenously added SDF-1 induced extracellular signal–regulated kinase signal, which phosphorylated anddegraded IRS-1 protein in adipocytes, decreasing in-sulin-mediated signaling and glucose uptake. In contrast,knockdown of endogenous SDF-1 or inhibition of its re-ceptor in adipocytes markedly increased IRS-1 proteinlevels and enhanced insulin sensitivity, indicating the auto-crine action of SDF-1. In agreement with these findings,adipocyte-specific ablation of SDF-1 enhanced insulin sen-sitivity in adipose tissues and in the whole body. Theseresults point to a novel regulatory mechanism of insulinsensitivity mediated by adipose autocrine SDF-1 actionand provide a new insight into the process of insulin de-sensitization in adipocytes.

Adipose tissue has long been considered an energy-storageorgan. However, accumulating data indicate that adiposetissue also functions as a metabolic organ involved in theregulation of systemic insulin sensitivity and glucose ho-meostasis. Lipodystrophy (failure of normal adipose tissuedevelopment) is associated with severe insulin resistanceand hyperglycemia in humans andmice (1,2). Adipose-specificablation of the insulin receptor or GLUT4, an insulin-

responsive GLUT, impairs systemic insulin sensitivity andglucose homeostasis (3,4). Conversely, overexpression ofGLUT4 in adipocytes improves systemic glucose dis-posal (5). Furthermore, selective enhancement of adipocyte-insulin sensitivity is reported to improve systemic glucosehomeostasis (6).

Insulin resistance develops not only under obese condi-tions but also in the fasting state. Insulin resistance developsin obese individuals and is thought to be harmful in thecontext of being a major risk factor for type 2 diabetes anddyslipidemia (7). However, regulation of insulin action,especially insulin desensitization, is an important aspectof physiological metabolism. Insulin resistance also occursin fasting healthy subjects, represented by a decreased rate ofglucose disposal during hyperinsulinemic-euglycemic clamp(8). Fasting-induced insulin resistance suppresses glucoseutilization in peripheral tissues, including adipose tissue, tospare glucose for use by other tissues, such as the brain, thatrequire a large amount of glucose for cellular function andsurvival (8–10). Although several exogenous and endoge-nous factors are associated with insulin resistance in adiposetissues (7,11–13), little is known about the common factor(s)that are regulated under both obesity- and fasting-mediatedinsulin desensitized conditions.

Here, by integrating publicly available microarray datasets, we compiled gene lists abundantly present in theseinsulin-desensitized conditions, including fasting and obeseadipose tissues. This analysis led to the identification ofa previously unnoticed chemokine, stromal derived factor-1(SDF-1), also called CXCL12. We describe here the crucialrole of SDF-1 as an autocrine insulin-desensitizing factor in

1Department of Metabolic Medicine, Osaka University Graduate School of Med-icine, Suita, Osaka, Japan2Osaka University Graduate School of Frontier Biosciences, Suita, Osaka, Japan3Department of Diabetes Care Medicine, Osaka University Graduate School ofMedicine, Suita, Osaka, Japan4Department of Adipose Management, Osaka University Graduate School ofMedicine, Suita, Osaka, Japan5Department of Nutrition and Life Science, Kanagawa Institute of Technology,Atsugi, Kanagawa, Japan

Corresponding author: Atsunori Fukuhara, [email protected].

Received 19 June 2017 and accepted 14 March 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0706/-/DC1.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

1068 Diabetes Volume 67, June 2018

METABOLISM

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adipocytes, using in vitro adipocyte analysis and adipocyte-specific SDF-1 knockout (AdSDF-1 KO) mice.

RESEARCH DESIGN AND METHODS

Animal StudiesMice were housed in groups of one to three mice per cage,maintained in a room under controlled temperature (23 61.5°C) and humidity (45 6 15%) on a 12-h dark/12-h lightcycle, and had free access to water and chow (MF; OrientalYeast, Tokyo, Japan). SDF-1 flox mice (Stock No. 021773)(14) were purchased from The Jackson Laboratory, andother mice were purchased from Charles River Japan(Yokohama, Japan). Adiponectin-Cre mice were provided byE. Rosen (Beth Israel Deaconess Medical Center) (15). MaleAdSDF-1 KO mice and their littermate control (SDF-1flox/flox) mice were analyzed between 13 and 17 weeksof age after 8–12 weeks of a high-fat diet (HFD). Epididymalwhite adipose tissue (WAT), brown adipose tissue (BAT),liver, and gastrocnemius muscle were harvested and used forthe study. A diet-induced obesity mouse model was estab-lished by feeding an HFD containing 60% of calories fromfat for 8 to 12 weeks, starting at 5 weeks of age (HFD-60;Oriental Yeast).

For the intraperitoneal insulin tolerance test (ITT),mice were fasted for 5 h and injected with 0.7 units/kgbody weight (BW) (those fed a normal diet) or 2.0 units/kgBW (those fed HFD) insulin. For the intraperitonealglucose tolerance test (GTT), mice were fasted for 5 h beforeinjection of glucose at 1 g/kg BW. Glucose values weremeasured by tail vein sampling at the indicated times usinga portable glucose meter (Glutest Neo alpha; Sanwa KagakuKenkyusho, Nagoya, Japan). For measurement of insulin-mediated Akt phosphorylation, mice were fasted for 5 h,anesthetized, and injected intraperitoneallywith10units/kgBW insulin. The relevant tissues were quickly harvestedafter 15 min and frozen immediately in liquid nitrogen. Allmouse studies were approved by the Ethics Review Com-mittee for Animal Experimentation of Osaka University,Graduate School of Medicine, and performed in accordancewith the Osaka University Institutional Animal Care and UseCommittee Guidelines.

Adipose Tissue FractionationEpididymal WAT was excised and minced in DMEMsupplemented with 10% FBS and 1% AA (antibioticsand antimycotics). Collagenase (4,000 units/mL) andDNase (0.1 mg/mL) were added, and the tissue sampleswere incubated at 37°C for 30 min under constant shaking.The cell suspension was filtered through a 110-mm cellstrainer and then centrifuged at 500g for 5 min toseparate the stromal vascular fraction (SVF) pellet fromthe floating mature adipocytes fraction. Separated matureadipocytes and SVF cells were resuspended in different tubesand centrifuged at 500g for 5 min. The washing-centrifu-gation process was repeated twice.

Preparation of Mouse Primary Differentiated AdipocytesSubcutaneous WAT was excised and minced in DMEMsupplemented with 10% FBS and 1% AA. Collagenase(4,000 units/mL) and DNase (0.1 mg/mL) were added, andthe tissue samples were incubated at 37°C for 30 min underconstant shaking. The cell suspension was filtered througha 70-mm cell strainer and then centrifuged at 500g for 5 minto obtain the SVF pellet. The pellet was resuspended inDMEM containing 10% FBS and 1% AA and plated into anappropriate culture dish. At 4–6 h after seeding, the cellswere washed with culture medium to remove superfluouscells and debris. At 2 days after 100% confluence, the cellswere differentiated to adipocytes using differentiation me-dium containing 3-isobutyl-1-methylxanthine (0.5 mmol/L),dexamethasone (1 mmol/L), insulin (1 mmol/L), andpioglitazone (10 mmol/L). The cells were used in the exper-iment 5–7 days after differentiation.

Cell Culture3T3-L1 cells were differentiated into adipocytes using dif-ferentiation medium containing 3-isobutyl-1-methylxanthine(0.5 mmol/L), dexamethasone (1 mmol/L), and insulin(1 mmol/L). The cells were used in experiments 7 days afterdifferentiation. In all experiments, adipocytes were culturedin a serum-free DMEM to avoid unknown serum effects.PlatE cells were used to produce retrovirus, followed bytransfection in 3T3-L1 cells. Stable 3T3-L1 cells expressingtetracycline (tet)-inducible SDF-1 (3T3-L1–tet–SDF-1 cell),IRS-1 (3T3-L1–tet–IRS-1 cell), or control cells (3T3-L1–tet–empty cell) were produced using the Retro-X Tet-On Ad-vanced system according to the protocol supplied by themanufacturer (Clontech, Mountain View, CA). The codingregion of mouse SDF-1 or mouse IRS-1 (16) was subclonedinto the expression vector, pRetroX-Tight-Pur or pRetroX-Tight-hygro, respectively. Retroviral particles were generatedusing pRetroX-Tight-Pur-mSDF1, pRetroX-Tight-hygro-mIRS-1, or pRetroX-Tight-hygro-empty and pRetroX-tet-onadvanced vectors. Infected 3T3-L1 cells were selected in400 mg/mL G418 and 5 mg/mL puromycin or 200 mg/mLhygromycin. Recombinant proteins and othermaterials wereas described; murine SDF-1 (R&D Systems, Minneapolis,MN,), murine tumor necrosis factor-a (TNF-a) (PeproTech,Rocky Hill, NJ), U0126 (Sigma-Aldrich, St. Louis, MO),pertussis toxin (Sigma-Aldrich), and TC14012 (R&D Systemsand Cayman Chemical Company, Ann Arbor, MI).

Small Interfering RNAThe differentiated 3T3-L1 adipocytes or mouse primarydifferentiated adipocytes (day 5–7) in 10-cm dish weretreated with trypsin-EDTA and incubated at 37°C for 2 min.The cells were washed in a 50mL conical centrifuge tube andcentrifuged at 500g for 5 min. In the meantime, the smallinterfering (si)RNA mixture of Opti-MEM, siRNA solution(Qiagen, Valencia, CA; AllStars Negative Control siRNA andFlex tube siRNA), and RNAiMAX (Invitrogen, Carlsbad, CA)was prepared according to the instructions provided by themanufacturer. The cell pellet was gently resuspended in the

diabetes.diabetesjournals.org Shin and Associates 1069

culturemedium (106 cells/mL), plated onto 12-well dish withthe siRNA mixture, and incubated for 2 days. At 2 days aftersiRNA, the cells weremaintained in a serum-freemedium for12–36 h to allow SDF-1 accumulation in the medium.

Western Blot AnalysisCultured cells or tissue samples were lysed in lysis buffer(20 mmol/L Tris/HCl [pH 7.4], 1.0% Triton 3100,150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA)containing 1 mmol/L phenylmethylsulfonyl fluoride,1.6 g/mL aprotinin, 10 g/mL leupeptin, and protease inhibitorcocktail (Nacalai Tesque, Kyoto, Japan). Protein concen-tration was determined by the bicinchoninic acid method(Pierce, Rockford, IL). The samples were used for Westernblot analysis after concentrated sample buffer was addedand the samples were heated for 5 min at 95°C.

Equal amounts of protein were separated by SDS-PAGEand transferred electrophoretically to polyvinylidene difluoridemembranes. The membranes were blocked for 1 h at roomtemperature using Tris-buffered saline (137 mmol/LNaCl, 20 mmol/L Tris–HCl, pH 7.6) containing 0.05%Tween-20 (TBS-T) and 5% skim milk. After triple washingwith TBS-T, each for 10min, the membranes were incubatedovernight at 4°C with primary antibodies against phosphor-ylated (phospho)–extracellular signal–regulated kinase (Erk;Thr202/Tyr204; Cell Signaling Technology, Danvers, MA),total Erk (Cell Signaling), phospho-Akt (Ser473; Cell Signal-ing), total Akt (Cell Signaling), total IRS-1 (Upstate,Charlottesville, VA), total IRS-2 (Cell Signaling), phospho–IRS-1 (Ser636/639; Cell Signaling), adiponectin (R&D Sys-tems), b-actin (Sigma-Aldrich), GAPDH (Cell Signaling), anda-tubulin (Cell Signaling) in TBS-T and 5% skim milk. Aftertriple washing with TBS-T, each for 10 min, the membraneswere incubated for 1 h at room temperature with enhancedchemiluminescence horseradish peroxidase–linked second-ary antibodies (GE Healthcare, Piscataway, NJ) in TBS-T and5% skim milk. After extensive triple washing in TBS-T, theimmunoreactive bands were visualized by Pierce WesternBlotting Substrate Plus. Quantification was conducted bydensitometry using ImageJ software (National Institutes ofHealth).

ELISAELISA assay kits for SDF-1 (R&D Systems), MCP-1 (R&DSystems), and insulin (Morinaga, Yokohama, Japan) werepurchased, and analysis was performed according to theinstructions provided by the manufacturer.

RNA Isolation and Quantitative PCRTotal RNA was isolated from cells or tissues using TRIreagent (Sigma-Aldrich). RT-PCR was performed using theTranscriptor First Strand cDNA Synthesis Kit (Roche,Indianapolis, IN). Quantitative PCR was performed withLightCycler-DNA Master SYBR Green I mix (Roche). Allprocedures were performed using the instructions pro-vided by the manufacturer. The specific primers were

purchased from Sigma-Aldrich, and the sequences areavailable upon request.

DNA Isolation and PCRTotal DNA was isolated from tissues with TRI reagent(Sigma-Aldrich) using the procedure recommended by themanufacturer.

ImmunohistochemistryEpididymal WAT and liver were excised and fixed in 10%formalin. After paraffin embedding and sectioning, the sec-tions were stained using anti-Mac2 antibodies (Abcam).

Flow CytometryFACS analysis was performed as described previously.Briefly, cells in the SVF from epididymal WAT weresuspended in FACS buffer and incubated with anti-mouseCD16/CD32 (93; BioLegend, San Diego, CA) for 15 min.Then, the cells were rinsed and resuspended in FACS bufferand stained for 25 min with anti-CD45 (30F-11; BioLegend),anti-CD11b (M1/70; BioLegend), anti-MHC class II (M5/114.15.2; eBioscience, San Diego, CA), anti-F4/80 (BM8;BioLegend), CD11c (N418; BioLegend), and anti–siglec-F(E50-2440; BD Pharmingen, San Diego, CA) for macro-phages, dendritic cells, and eosinophils. For B cells, CD4+

T cells, CD8+ T cells, natural killer (NK) cells, and NKT cells, SVF was incubated with anti-B220 (RA3-6B2;BioLegend), anti-CD19 (6D5; BioLegend), anti-NK1.1(PK136; BioLegend), anti-CD8 (53-6.7; BioLegend), anti-CD4 (RM4-5; BioLegend), and anti-CD3 (17A2; BioLegend).For preadipocytes, endothelial cells, and hematopoieticcells, SVF was incubated with anti-CD45, anti-CD31(390; BioLegend), anti-CD34 (RAM34; eBioscience), andanti–platelet-derived growth factor receptor-a (APA5; BioL-egend). The SVF was washed twice and resuspended with400 mL FACS buffer, and 20 mL precision count beads(BioLegend) was added as internal control and analyzed withFACSVerse (BD Biosciences, San Diego, CA). The absolutecell count was determined according to instructions providedby the manufacturer.

Glucose Uptake Assay2-Deoxyglucose (2-DG) uptake kit (Cosmo Bio, Tokyo, Japan)was purchased and analysis was performed according to theinstructions provided by the manufacturer.

Ex Vivo Glucose Uptake AssayEpididymalWATwas dissected frommouse, and cut into 50-to 100-mg pieces, washed, and incubated for 1 h with KrebsRinger Phosphate HEPES buffer at 37°C. Insulin (1 nmol/L)was added and incubated for 20 min, followed by treatmentwith 1mmol/L 2-DG, further incubated for 20min, and thenwashed four times with PBS. Tissue samples were lysed bysonication in 10 mmol/L Tris-HCl pH 8.0 buffer and in-tracellular 2-DG-6-phosphate levels weremeasured accordingto the 2-DG uptake kit instructions provided by the manu-facturer (Cosmo Bio).

1070 SDF-1 and Insulin Desensitization Diabetes Volume 67, June 2018

Statistical AnalysisAll data are presented as mean6 SEM. Differences betweentwo groups were examined for statistical significance by theStudent t test. A P value ,0.05 denoted the presence ofa statistically significant difference.

RESULTS

SDF-1 Gene Expression Correlates With Insulin-Desensitized Conditions in AdipocytesTo search for factors involved in “insulin desensitization”common to fasting and obesity, we compiled and inter-crossed gene lists from four independent microarray datasets related to insulin-desensitized or -sensitized condi-tions: 1) fasting-induced genes in mouse adipose tissue(GSE46495); 2) obesity-induced genes in human adiposetissue (GDS3602); 3) TNF-a–induced genes in 3T3-L1adipocytes (GSE62635) as upregulated genes in an insulin-resistance model in vitro (17); and 4) peroxisome prolifer-ator–activated receptor-g (PPARg) agonist–reduced genes inrat adipose tissue (GDS3850) as downregulated genes in aninsulin-sensitive model in vivo (18). Comprehensive analysisof the data sets yielded two overlapping genes, Cyp1b1 and

SDF-1 (Fig. 1A). The Cyp1b1 gene has been well describedpreviously, and its deficiency ameliorates glucose intoler-ance induced by an HFD (19). SDF-1 is a secretary proteinclassified as a CXC chemokine (20). Its gene expression isexceptionally high compared with other chemokines in RNAsequencing data sets of 3T3-L1 adipocytes (GSM2322563and GSE50612) (Fig. 1B and Supplementary Fig. 1A). Quan-titative PCR and ELISA data showed high expression ofSDF-1 in the culture media of 3T3-L1 adipocytes andmouse primary differentiated adipocytes (SupplementaryFig. 1B–E).

We validated the original microarray data sets by quan-titative PCR. Fasting increased SDF-1 gene expression inepididymal WAT (Fig. 1C). Treatment with culture mediummimicking fasting conditions, such as insulin depletion orglucose starvation, increased SDF-1 expression in 3T3-L1adipocytes (Supplementary Fig. 2A and B). We also con-firmed that SDF-1 expression was augmented in epididymalWAT of various obese mouse models compared with thecontrol mice, including ob/ob mice (Fig. 1D), KKAy mice(Supplementary Fig. 2C), and HFD-fed mice (SupplementaryFig. 2D), as reported previously (21,22). In the human

Figure 1—SDF-1 gene expression correlates with insulin-desensitized conditions in adipocytes. A: Schematic diagram of microarray analysis toidentify insulin-resistance factors expressed in adipose tissue and adipocytes. The following Gene Expression Omnibus DataSets were usedfor the analysis: fasting-induced genes in mouse adipose tissue (GSE56248, fold-change .1.5, P , 0.05; 781 genes), obesity-induced genesin human adipose tissue (GDS3602, fold-change .3, P , 0.05; 86 genes), TNF-a–induced genes in 3T3-L1 adipocytes (GSE62635, fold-change.3,P, 0.05; 375 genes), and PPARg agonist–reduced genes in rat adipose tissue (GDS3850, fold-change,0.6,P, 0.05; 477 genes).B:Chemokine gene expressions in 3T3-L1 adipocytes (GSM2322563). RPKM, reads per kilobase million. C: SDF-1 gene expression in epididymalWAT after 24 h feeding or fasting (n= 6).D: SDF-1 gene expression in epididymalWATof control C57BL/6Jmice (WT,wild-type) orob/obmice (n=4–6). E: SDF-1 gene expression with or without TNF-a (10 ng/mL) for 12 h in 3T3-L1 adipocytes (n = 3). F: SDF-1 gene expression with or withoutpioglitazone (Pio; 10 mmol/L) in 3T3-L1 adipocytes (n = 3). Data are mean 6 SEM. **P , 0.01; ***P , 0.001.


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microarray data set, SDF-1 expression was higher in epidid-ymal WAT of obese subjects than in nonobese subjects(GDS3602) (Supplementary Fig. 2E). Furthermore, SDF-1expression tended to be higher in adipose tissues of obeseinsulin-resistant subjects with diabetes compared withage- and BMI-matched normal glucose-tolerant subjects(GDS3665) (Supplementary Fig. 2F), suggesting the involve-ment of adipose–SDF-1 in human obesity and diabetes aswell. Furthermore, the addition of TNF-a to cultured 3T3-L1adipocytes markedly increased SDF-1 gene expression (Fig.1E), whereas pioglitazone, a PPAR-g agonist, decreased SDF-1gene expression in 3T3-L1 adipocytes (Fig. 1F). Thesedata suggest the upregulation of SDF-1 gene under insulin-desensitized conditions and its downregulation in insulin-sensitive states in adipose tissues and adipocytes.

SDF-1 Directly Induces Insulin Desensitization inAdipocytes With Reduced IRS-1 Protein LevelsTo assess the direct effects of SDF-1 on adipocyte insulinsensitivity, we conducted in vitro experiments using culturedadipocytes and evaluated SDF-1–related factors. IRS-1 is animportant signaling molecule that regulates insulin actionin adipocytes (23,24). IRS-1 deficiency induces insulin re-sistance in adipocytes with decreased insulin-induced Akt

phosphorylation and glucose uptake (25,26). SDF-1 markedlyreduced IRS-1 protein levels in 3T3-L1 adipocytes (Fig. 2A)in a manner similar to TNF-a, as reported previously (27).On one hand, IRS-1 protein levels were significantly reducedafter 9-h treatment with SDF-1 (Supplementary Fig. 3A andB). On the other hand, SDF-1 did not alter IRS-2 proteinlevels significantly (Supplementary Fig. 3A and C).

The reduction in IRS-1 protein was associated withattenuated insulin-mediated Akt phosphorylation (Fig. 2Band Supplementary Fig. 3D). SDF-1 also inhibited insulin-mediated glucose uptake in 3T3-L1 adipocytes (Fig. 2C).Similarly, doxycycline-induced overexpression of SDF-1in 3T3-L1 adipocytes (Fig. 2D and Supplementary Fig.3E) decreased IRS-1 protein levels, insulin-mediated Aktphosphorylation, and glucose uptake (Fig. 2E and F). Wegenerated 3T3-L1 adipocytes that overexpressed IRS-1 (Sup-plementary Fig. 3F) to examine the importance of IRS-1expression level on SDF-1–induced insulin desensitization.SDF-1 inhibited insulin-mediated glucose uptake in 3T3-L1adipocytes, which was partially reversed by overexpressionof IRS-1 (Supplementary Fig. 3G). These results clearly showthat SDF-1 directly induces insulin desensitization in adi-pocytes with reduced IRS-1 protein level and its downstreameffects.

Figure 2—SDF-1 directly desensitizes adipocytes to insulin action with decreased IRS-1 protein levels. A: IRS-1 protein levels in 3T3-L1adipocytes treated with the vehicle (Control), TNF-a (10 ng/mL), or SDF-1 (500 ng/mL) for the indicated duration (1, 3, or 6 h).B: IRS-1 protein andAkt phosphorylation levels in 3T3-L1 adipocytes pretreatedwith SDF-1 (500 ng/mL) for the indicated duration (0, 1, 3, or 9 h), followed by treatmentwith insulin (1 nmol/L) for the indicated intervals (0, 5, or 15min).C: Relative glucose uptake in 3T3-L1 adipocytes pretreatedwith or without SDF-1(1,000 ng/mL) in serum-free medium for 18 h, followed by treatment with insulin (0, 0.1, or 1 nmol/L) for 20 min. 2-DG uptake for 10 min wasmeasured (n = 3–4).D: SDF-1 gene expression in 3T3-L1–tet–SDF-1 adipocytes treatedwith (+) or without (2) 30mg/mL doxycycline (Dox) for 48 h(n = 5, 4). E: IRS-1 protein and Akt phosphorylation levels in 3T3-L1–tet–SDF-1 adipocytes pretreated with or without 30 mg/mL of Dox for 48 h,followed by treatment with insulin (1 nmol/L) for the indicated intervals (0, 10, 30, or 60 min). F: Relative glucose uptake in 3T3-L1–tet–SDF-1adipocytes pretreated with or without 30 mg/mL Dox for 48 h, incubated with serum-free medium for 18 h, followed by treatment with insulin(0.1 nmol/L) for 20 min. 2-DG uptake for 10 min was measured (n = 6). Data are mean 6 SEM. *P , 0.05; ***P , 0.001.


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SDF-1 Induces Insulin Desensitization in Adipocytes viaCXCR4/Erk/IRS-1 AxisWe next investigated the molecular mechanism of SDF-1–induced reduction of IRS-1 protein level in adipocytes. Withregard to gene expression, SDF-1 did not change the IRS-1mRNA level (Supplementary Fig. 3H). Previous studiesshowed that IRS-1 is degraded by proteasome activity inadipocytes (28). Pretreatment with lactacystin, a proteasomeinhibitor, significantly restored SDF-1–induced reduction ofIRS-1 protein in 3T3-L1 adipocytes (Fig. 3A and Supplemen-tary Fig. 4A and B), suggesting the involvement of SDF-1 inactivation of the IRS-1 proteasome degradation pathway.SDF-1 activates Erk signals (29), although this has not beenexamined in adipocytes. Furthermore, Erk participates inIRS-1 degradation via serine phosphorylation (30). In ourexperiments, 3T3-L1 adipocytes and mouse primary differ-entiated adipocytes cultured in the presence of SDF-1showed marked activation of Erk (Supplementary Fig. 4C),and the effect was dose-dependent (Supplementary Fig. 4D).SDF-1–induced Erk activation reached a peak level at 5 min,followed by a gradual decrease (Supplementary Fig. 4E), andwas concomitantly associated with phosphorylation of IRS-1

at Ser636 (Fig. 3B). Inhibition of Erk signal by U0126, anupstreamMEK inhibitor, completely blocked SDF-1–inducedIRS-1 phosphorylation (Fig. 3C and Supplementary Fig. 4F)and IRS-1 degradation (Fig. 3D) in 3T3-L1 adipocytes andmouse primary differentiated adipocytes. Furthermore,U0126 markedly reversed SDF-1 action on impaired glucoseuptake in the adipocytes (Supplementary Fig. 4G). Thesedata show that SDF-1–induced IRS-1 degradation in adipo-cytes is Erk signal dependent.

SDF-1–induced Erk activation and its biological functionsaremediatedmainly by CXCR4, a G protein-coupled receptor(GPCR) that uses the Gai subunit for its signal transduction(20,31,32). Blockade of the Gai subunit by pertussis toxinmarkedly reduced SDF-1–induced Erk activation in adipo-cytes (Supplementary Fig. 4H). TC14012, a peptidomimeticCXCR4 antagonist (33), inhibited SDF-1–induced Erk acti-vation, but not the epidermal growth factor-induced one, inC2C12 myocytes (Supplementary Fig. 4I), as reported pre-viously (34). Under similar conditions, TC14012 significantlyinhibited SDF-1–induced phosphorylation of Erk1/2 andIRS-1 at Ser636 and downregulation of IRS-1 in mouseprimary differentiated adipocytes, without changing IRS-1

Figure 3—SDF-1 degrades IRS-1 protein via CXCR4/Erk signaling in adipocytes. A: IRS-1 protein levels in 3T3-L1 adipocytes pretreated withlactacyctin (Lacta) (250 nmol/L) for 30min, followed by treatment with SDF-1 (1,000 ng/mL) for 18 h.B: Erk1/2 and IRS-1 (Ser636) phosphorylationin 3T3-L1 adipocytes treated with SDF-1 (500 ng/mL) for the indicated intervals (0, 5, 15, 30, or 60 min). C: Erk1/2 and IRS-1 (Ser636)phosphorylation in 3T3-L1 adipocytes pretreated with U0126 (10 mmol/L) for 30 min, followed by treatment with SDF-1 (500 ng/mL) for theindicated intervals (0, 5, or 15 min). D: IRS-1 protein levels in 3T3-L1 adipocytes pretreated with U0126 (250 nmol/L) for 30 min, followed bytreatment with SDF-1 (1,000 ng/mL) for 18 h. E: Erk1/2 and IRS-1 (S636) phosphorylation levels in mouse primary differentiated adipocytespretreated with the vehicle (Control) or TC14012 (200 mg/mL) for 30 min, followed by treatment with SDF-1 (500 ng/mL) for the indicated intervals(0, 10, or 20min). F: IRS-1 protein levels in mouse primary differentiated adipocytes pretreated with vehicle (Control) or TC14012 (TC) (200 mg/mL)for 30 min, followed by treatment with the vehicle or SDF-1 (500 ng/mL) for 12 h. G: Relative glucose uptake in mouse primary differentiatedadipocytes pretreated with vehicle (Control) or TC14012 (200 mg/mL) for 30min, treated with the vehicle or SDF-1 (1,000 ng/mL) for 12 h, followedby treatment with insulin (0.1 nmol/L) for 20 min (n = 3). Data are mean 6 SEM. **P , 0.01.


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mRNA levels (Fig. 3E and F and Supplementary Fig. 4J).Also, TC14012markedly reversed SDF-1 action on impairedglucose uptake in the adipocytes (Fig. 3G). CXCR7, anotherreceptor for SDF-1, does not use the Gai subunit for itssignal transduction, but rather b-arrestin (35). Knockdownof CXCR7 did not affect the SDF-1–induced Erk activation in3T3-L1 adipocytes and mouse primary differentiated adipo-cytes (Supplementary Fig. 4K–M). These results indicate thatCXCR4 mainly mediates the insulin-desensitizing action ofSDF-1 in adipocytes.

Autocrine SDF-1 Action Controls Insulin Sensitivity inAdipocytesBased on the above findings, we hypothesized that adipocyte-derived SDF-1 acts in an autocrine manner to controladipocyte insulin sensitivity. To confirm this hypothesis,we estimated the autocrine action of SDF-1 in adipocytesin vitro. Knockdown of SDF-1 resulted in a marked increase

in IRS-1 protein level, insulin-mediated Akt phosphoryla-tion, and glucose uptake in 3T3-L1 adipocytes (Fig. 4A–Cand Supplementary Fig. 4N) and mouse primary differenti-ated adipocytes (Fig. 4D and E), confirming the autocrineaction of SDF-1. Moreover, blockade of its receptor CXCR4with TC14012 alone, without exogenous SDF-1 treatment,augmented IRS-1 protein level and insulin-mediated Aktphosphorylation in mouse primary differentiated adipo-cytes (Fig. 4F). These results confirm that adipocyte-derivedSDF-1 controls adipocyte-insulin sensitivity in an auto-crine manner.

Adipocyte-Specific SDF-1 Ablation Enhances InsulinSensitivity in Adipose TissueTo access the effect of SDF-1 on insulin sensitivity in vivo,we generated AdSDF-1 KO mice by crossing SDF-1 flox/floxmice (14) with adiponectin-Cre mice (36). In contrast toembryonic lethality of whole-body KO of SDF-1, AdSDF-1

Figure 4—Autocrine SDF-1 action downregulates insulin sensitivity in adipocytes. A: SDF-1 concentration in conditioned medium of 3T3-L1adipocytes transfected with control siRNA or SDF-1 siRNA (n = 3). B: IRS-1 protein and Akt phosphorylation levels in 3T3-L1 adipocytestransfectedwith control or SDF-1 siRNA, followed by treatment with insulin (1 nmol/L) for the indicated intervals (0, 5, 15, 30, or 60min).C: Relativeglucose uptake in 3T3-L1 adipocytes transfected with control or SDF-1 siRNA, followed by treatment with insulin (0.1 nmol/L) for 20 min. 2-DGuptake for 10 min was measured (n = 6). D: SDF-1 concentration in conditioned medium of mouse primary differentiated adipocytes transfectedwith control siRNA or SDF-1 siRNA (n = 3). E: IRS-1 protein and Akt phosphorylation levels in mouse primary differentiated adipocytes transfectedwith control or SDF-1 siRNA, followed by treatment with insulin (1 nmol/L) for the indicated intervals (0, 10, 30, or 60min). F: IRS-1 protein and Aktphosphorylation levels in mouse primary differentiated adipocytes pretreated with the vehicle (Control) or TC14012 (200mg/mL) for 12 h, followedby treatment with insulin (1 nmol/L) for the indicated intervals (0, 10, or 30 min). Data are mean 6 SEM. *P , 0.05; ***P , 0.001.


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KO mice were born at the expected Mendelian ratio andappeared grossly normal, with no apparent differences inbody weight (Supplementary Fig. 5A), organ weight (Sup-plementary Fig. 5B), and food intake (Supplementary Fig.5C) compared with control flox/flox mice. In AdSDF-1 KOmice, Cre-mediated recombination occurred specifically inWAT and BAT, but not in other tissues such as the liver ormuscle (Supplementary Fig. 6A). Fractionation data showedspecific SDF-1 gene ablation in mature adipocytes of KOmice but not in the SVF (Supplementary Fig. 6B and C).

We also evaluated the effects of adipocyte-specific SDF-1ablation on whole-body insulin sensitivity and glucose me-tabolism. In mice fed the normal diet, fasting insulin levelswere significantly lower in AdSDF-1 KO mice than in thecontrol, with normal fasting glucose levels (Fig. 5A and B).Systemic insulin sensitivity (Fig. 5C) and glucose tolerance(Fig. 5D) were also better in AdSDF-1 KO mice than thecontrol mice. These data suggest that adipose SDF-1 con-tributes to physiological insulin desensitization in adipocytesas well as in the whole body.

We also assessed the insulin-desensitizing action of SDF-1in vivo adipose tissue. AdSDF-1 KO mice showed specificenhancement of insulin sensitivity in epididymal WAT andBAT, with a marked increase in IRS-1 protein level andinsulin-mediated Akt phosphorylation (Fig. 5E and Sup-plementary Fig. 7A, B, andD). Furthermore, insulin-mediatedglucose uptake was enhanced in epididymal WAT fromAdSDF-1 KOmice compared with control mice (Supplementary

Fig. 7C). These differences were not observed in other insulin-target organs, such as the liver and muscle (SupplementaryFig. 7E and F), again confirming the autocrine action ofSDF-1 in adipose tissue.

Because SDF-1 was also increased in obese adipose tissue,another insulin desensitizing tissue, we analyzed the effectof lack of adipocyte SDF-1 in the HFD-induced obese insulinresistance condition. AdSDF-1 KO mice maintained on theHFD exhibited slightly lower body and organ weights, in-cluding adipose tissue and liver weights, than the controlmice, without changes in food intake (Supplementary Fig.5D–F). In addition, the AdSDF-1 KO mice had lower fastingglucose and insulin levels and improved insulin sensitivityand glucose tolerance compared with the control mice (Fig.5F–I). AdSDF-1 KO mice showed specific enhancement ofinsulin sensitivity in adipose tissue, withmarked elevation ofIRS-1 protein level and insulin-mediated Akt phosphoryla-tion under the HFD condition (Fig. 5J). These data clearlydemonstrated that adipocyte SDF-1 attenuated systemicinsulin sensitivity in obese conditions as well.

Lastly, we assessed the function of SDF-1 as a chemo-attractant factor in AdSDF-1 KO mice fed the normal chowdiet. Immunostaining of macrophages from AdSDF-1 KOmice showed crown-like structures in epididymal WAT,similar to the control mice (Supplementary Fig. 8A andB). Also, quantitative PCR indicated that SDF-1 ablationdid not change immune cell marker genes in epididymalWAT (Supplementary Fig. 8C). Finally, we quantitatively

Figure 5—SDF-1 deletion in adipocytes enhances insulin sensitivity and glucose homeostasis. Metabolic analysis of AdSDF-1 KO micemaintainedon a normal diet (A–E) orHFD (F–J). Fastingglucose (n55) (A) and fasting insulin (n5 10) (B) levels of flox/flox andAdSDF-1KOmice at10 weeks of age. Results of ITT at 12 weeks of age (C) and GTT at 14 weeks of age (D) of flox/flox and AdSDF-1 KOmice (n = 6). E: IRS-1 proteinand insulin-mediated Akt phosphorylation levels in epididymal WAT. After a 5-h fast, control (flox/flox) and AdSDF-1 KO (SDF-1 KO) mice fed thenormal diet were injected intraperitoneally with the vehicle (Insulin2) or insulin (10 units/kg BW) (Insulin+). The samples were collected for analysisafter 15min. A diet-induced obesity mousemodel was established by feeding an HFD containing 60%calories from fat for 8–12 weeks starting at5 weeks of age. Fasting glucose (n5 5, 4) (F) and fasting insulin (n5 12) (G) levels of flox/flox and AdSDF-1 KOmice fed the HFD at 13 weeks ofage. ITT at 15 weeks of age (H) and GTT at 17 weeks of age (I) of flox/flox and AdSDF-1 KOmice (n = 8). J: IRS-1 protein and insulin-mediated Aktphosphorylation levels in epididymal WAT. After a 5-h fast, control (flox/flox) and AdSDF-1 KO (SDF-1 KO) fed the HFD were injectedintraperitoneally with the vehicle (Insulin2) or insulin (10 units/kg BW) (Insulin+).The samples were collected for analysis after 15 min. Dataare mean 6 SEM. *P , 0.05; **P , 0.01.


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profiled SVF components in epididymal WAT by FACSanalysis. SDF-1 knockout did not alter the proportion ornumber of preadipocytes (CD452CD312PDGFRa+) (37),vascular endothelial cells (CD452CD31+), or hematopoieticcells (CD45+) in epididymal WAT (Supplementary Fig. 8D–F). In addition, there were no differences in the proportionand number of adipose-immune cells between AdSDF-1 KOand control mice (Supplementary Fig. 8G and H). Theseresults clearly show that adipocyte-derived SDF-1 does notaffect immune cell profile in adipose tissue and rule out theinvolvement of adipose-immune cells in the observedenhanced insulin sensitivity in AdSDF-1 KO mice, at leastunder the chow diet condition.

DISCUSSION

In the current study, we identified SDF-1 as an autocrineinsulin-desensitizing factor in adipocytes. Fasting and obe-sity both induced SDF-1 expression in adipocytes. Its auto-crine action activated Erk signaling, which concomitantlyinduced serine phosphorylation of IRS-1 protein, degradedIRS-1 protein in adipocytes, and attenuated insulin-mediatedAkt phosphorylation and glucose uptake (Fig. 6).

SDF-1 is a ubiquitously expressed and highly conservedsecretary factor with 99% homology between human andmouse. It plays important roles in development, tissueregeneration, hematopoiesis, immunity, and carcinogen-esis (20). SDF-1 contributes to the pathogenesis, progres-sion, and diverse pathological effects of type 2 diabetes, suchas insulitis, nephropathy, and adipose tissue inflammation(22,38,39). Furthermore, plasma SDF-1 levels correlatedwith the type 2 diabetes disease state (40). However,

the lethal phenotype of global SDF-1 KO mice has madeit difficult to assess the metabolic functions of SDF-1 inadult mice (20,41). In the current study, we successfullygenerated viable AdSDF-1 KO mice with an insulin-sensitivephenotype.

Insulin plays a key role in maintaining glucose homeo-stasis. Its action is regulated by endogenous signaling mol-ecules and exogenous counterregulatory factors in fastingand obese conditions. Fasting induces counterregulatoryhormones, such as catecholamines, cortisol, and growthhormone, and reduces insulin signaling in the target organs,including liver, skeletal muscle, and adipose tissue (11–13).In the development of obesity, infiltrating macrophages se-crete proinflammatory cytokines, such as TNF-a, and induceinflammation and insulin resistance in adipose tissues(3,7,42). In this study, we found SDF-1 as a common factorresponsible for fasting- and obesity-related insulin desensi-tization in adipose tissue. To our knowledge, this is the firstevidence that insulin target cells, in this case adipocytes,secrete an autocrine factor that desensitizes insulin action.

Liver and skeletal muscle are also important insulin targetorgans regulating glucose metabolism. IRS-1 is responsiblefor insulin-mediated suppression of gluconeogenesis in theliver (43) and insulin-mediated glucose uptake in the skeletalmuscle (44). In addition, the liver and skeletalmuscle expressSDF-1 and its receptor CXCR4 (45,46), and SDF-1 inducedErk phosphorylation in H4CII hepatocytes (data not shown)and C2C12 myocytes (Supplementary Fig. 4F), suggestingthe possible role of SDF-1 in insulin signaling in these tissuesas well. However, regulation of SDF-1 expression was dif-ferent among these tissues. SDF-1 expression was aug-mented in adipose tissues under both insulin-desensitizedconditions in fasting and obesity. In contrast, its expressionshowed no difference in the liver and was reduced in theskeletal muscle in the fasting condition (data not shown).Furthermore, it was decreased in liver and exhibited nodifference in skeletal muscle of ob/ob mice compared withcontrol mice (data not shown). Based on these results, wesuspect that SDF-1has a specific role in adipocytes as a commonfactor responsible for both obesity- and fasting-mediatedinsulin desensitization.

Previous studies suggested that SDF-1 acts as a patholog-ical chemoattractant factor in obese adipose tissues (21,22).Treatment with AMD3100, a potent antagonist of CXCR4,reduced macrophage infiltration and inflammation in adi-pose tissues under the HFD condition (22). The studyconcluded that systemic inhibition of SDF-1 signaling ame-liorated macrophage infiltration into adipose tissues, result-ing in improvement of whole-body insulin resistance (22). Incontrast, the current study showed that AdSDF-1 KO miceexhibited adipose tissue-specific enhancement of insulinsensitivity without changes in adipose-immune cell profiles,which clearly differed from the chemokine/inflammation/insulin resistance scenario in the adipose tissue. One possibleexplanation for this point is the distinct function of SDF-1according to the cell type. In addition to adipocytes, adi-pose tissue contains the SVF, including endothelial cells,

Figure 6—Schematic diagram of autocrine SDF-1 as an insulin-desensitizing factor in adipocytes. 1) Fasting or obesity inducesSDF-1 expression in adipocytes. 2) Its autocrine action activates Erksignaling. 3) SDF-1–induced Erk signal concomitantly induces serinephosphorylation of IRS-1 protein and degrades IRS-1 protein.This attenuates 4) insulin-mediated Akt phosphorylation and 5) glu-cose uptake.


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preadipocytes, and immune cells. Considering that SVF alsoexpresses SDF-1 (GDS2428) (47), SDF-1 from adipocytes actson adipocytes, and SDF-1 fromother cell types in SVF shouldbe related to the chemotactic property on macrophages.

SDF-1 has received little attention in the metabolic andphysiological research fields. Our findings, however, shednew light on its function as a crucial autocrine insulin-desensitizing factor in adipocytes.

Acknowledgments. The authors thank all members of the ShimomuraLaboratory, Interdisciplinary Program for Biomedical Sciences (IPBS), T. Nagasawaand T. Sugiyama (Laboratory of Stem Cell Biology and Developmental Immunology,Graduate School of Frontier Biosciences, Osaka University) for the helpful discussion,E. Rosen (Department of Genetics, Harvard Medical School) and J. Eguchi (Divisionof Nephrology, Diabetology and Endocrinology, Okayama University) for providingadiponectin-Cre, and J. Zimmermann and T. Hunter (Molecular and Cell BiologyLaboratory, Salk Institute for Biological Studies) for providing pcDNA3-mIRS-1 vectors.Funding. This work was partly supported by the Japan Society for the Promotionof Science Grant-in-aid for Scientific Research (C) (grant no. 17K09829), and theOsaka University Institute for Academic Initiatives. J.S. is supported by a JapaneseGovernment Ministry of Education, Culture, Sports, Science and Technology scholar-ship.Duality of Interest. This work was partially supported by Sanofi (grant),AstraZeneca (grant), and Merck Sharp & Dohme (grant). A.F. and S.K. belong toa department endowed by Takeda Pharmaceutical Company; Sanwa Kagaku Ken-kyusho Co., Ltd.; Rohto Pharmaceutical Co., Ltd.; Fuji Oil Holdings Inc.; and Roche DCJapan.

The funders had no role in the study design, data collection and analysis, decisionto publish, or preparation of the manuscript.Author Contributions. J.S. designed and performed the experiments andacquired data. J.S. and A.F. interpreted the data and wrote the manuscript.T.O. performed FACS analysis. S.K., C.Y., M.O., and I.S. supervised the project andwrote the manuscript. A.F. is the guarantor of this work and, as such, had fullaccess to all the data in the study and takes responsibility for the integrity of thedata and the accuracy of data analysis.

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SDF-1 Is an Autocrine Insulin-Desensitizing Factor in Adipocytes · 2018. 5. 15. · a previously unnoticed chemokine, stromal derived factor-1 (SDF-1), also called CXCL12. We describe