Role of lnk in insulin resistance

aaaaaaaaaa Overexpression of Lnk in the Ovaries Is Involved inInsulin Resistance in Women With PolycysticOvary Syndrome

Meihua Hao,* Feng Yuan,* Chenchen Jin, Zehong Zhou, Qi Cao, Ling Xu,Guanlei Wang, Hui Huang, Dongzi Yang, Meiqing Xie, and Xiaomiao Zhao

Department of Obstetrics and Gynecology (M.H., H.H., D.Y., M.X., X.Z.), Sun Yat-Sen MemorialHospital, Sun Yat-Sen University, Guangzhou, 510120 China; Department of Pharmacology (F.Y., C.J.,G.W.), Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-SenUniversity, Guangzhou, 510120 China; Department of Obstetrics and Gynecology (Z.Z.), ReproductiveMedical Center, Peking University Third Hospital, Haidian District, Beijing, China; Division of Hematology/Oncology (Q.C.), Cedar-Sinai Medical Center, University of California, Los Angeles School of Medicine,Los Angeles, California; and Cedar-Sinai Medical Center (L.X.), University of California, Los AngelesSchool of Medicine, Los Angeles, California

Polycystic ovary syndrome (PCOS) progression involves abnormal insulin signaling. SH2 domain-containing adaptor protein (Lnk) may be an important regulator of the insulin signaling path-way. We investigated whether Lnk was involved in insulin resistance (IR). Thirty-seven womendue to receive laparoscopic surgery from June 2011 to February 2012 were included from thegynecologic department of the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. Sam-ples of polycystic and normal ovary tissues were examined by immunohistochemistry. Ovariancell lines underwent insulin stimulation and Lnk overexpression. Expressed Lnk underwentcoimmunoprecipitation tests with green fluorescent protein-labeled insulin receptor and His-tagged insulin receptor substrate 1 (IRS1), and their colocalization in HEK293T cells was ex-amined. Ovarian tissues from PCOS patients with IR exhibited higher expression of Lnk thanovaries from normal control subjects and PCOS patients without IR; mainly in follicular gran-ulosa cells, the follicular fluid and plasma of oocytes in secondary follicles, and atretic follicles.Lnk was coimmunoprecipitated with insulin receptor and IRS1. Lnk and insulin receptor/IRS1locations overlapped around the nucleus. IR, protein kinase B (Akt), and ERK1/2 activities wereinhibited by Lnk overexpression and inhibited further after insulin stimulation, whereas IRS1serine activity was increased. Insulin receptor (Tyr1150/1151), Akt (Thr308), and ERK1/2(Thr202/Tyr204) phosphorylation was decreased, whereas IRS1 (Ser307) phosphorylation wasincreased with Lnk overexpression. In conclusion, Lnk inhibits the phosphatidylinositol 3kinase-AKT and MAPK-ERK signaling response to insulin. Higher expression of Lnk in PCOSsuggests that Lnk probably plays a role in the development of IR. (Endocrinology 157:3709 –3718, 2016)

Polycystic ovary syndrome (PCOS) is a common endo-crine disorder that typically develops in reproductive-

age women characterized by hyperandrogenism andchronic anovulation (1). Its etiology remains unknown,

but PCOS is likely to result from a complex number ofgenetic and environmental factors including androgen ex-posure, obesity, environmental toxins, and socioeconomicstatus (2). The impact of PCOS includes major metabolic

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in USACopyright © 2016 by the Endocrine SocietyReceived April 15, 2016. Accepted July 22, 2016.First Published Online July 26, 2016

* M.H. and F.Y. contributed as cofirst authors.Abbreviations: Akt, protein kinase B; BMI, body mass index; BP, blood pressure; FBS, fetalbovine serum; GFP, green fluorescent protein; His, histidine; HOMA-IR, homeostasis modelassessment for IR; IIS, insulin-like growth signaling; IR, insulin resistance; IRS, insulin re-ceptor substrate; Lnk, SH2B adapter protein 3; OGTT, oral glucose tolerance test; PCOS,polycystic ovary syndrome; PI3K, phosphatidylinositol 3 kinase; SH2, Src homology 2; WHR,waist to hip ratio.

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doi: 10.1210/en.2016-1234 Endocrinology, October 2016, 157(10):3709–3718 press.endocrine.org/journal/endo 3709

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syndrome as well as reproductive morbidities that cancause female infertility (3). PCOS is strongly associatedwith obesity and insulin resistance (IR) (1). Lifestylemodification is the first line treatment for PCOS, andthis can improve ovulation and the success of fertilitytreatment (3). Improvement in PCOS symptoms can oc-cur with only moderate weight loss and is strongly re-lated to IR (4).

Although in some cases the development of PCOSmay be mediated through worsening IR (5), the mech-anism remains unclear. Therefore, it is important tostudy IR and the insulin signaling pathway in PCOS. IRin classic or nonclassic target tissues has been shown tobe a major contributor in PCOS pathophysiology (6, 7).Recent data also suggest that insulin action may be af-fected in granulosa cells (8), and PCOS-derived granu-losa cells are affected by impaired insulin action (9), sothere may be a direct relationship between IR andfolliculogenesis.

As an important regulator of insulin and insulin sig-naling in Drosophila (10), Lnk may play a role in thedevelopment of IR and PCOS. The Lnk (Src homology2 [SH2]B3) protein shares a central pleckstrin homol-ogy (PH) domains and a SH2 domains and potentialtyrosine phosphorylation sites with SH2B (11, 12).These domains function as protein-protein interactionmotifs and transduce signals downstream of a numberof receptor tyrosine kinases (13). Lnk belongs to a fam-ily of intracellular adaptor proteins implicated in inte-gration and regulation of multiple signaling events in-cluding glucose homeostasis, reproduction and energymetabolism (10). Moreover, mutations in SH2B in hu-mans are associated with metabolic dysregulation andobesity (10, 14). Recent study suggests that in ovariancancer Lnk may have a positive action in signal trans-duction increasing cell proliferation and tumor size(15). Our previous study demonstrated that Lnk almostexclusively bound to the phosphorylated juxtamem-brane domain of c-Kit directly without requiring otherinteracting partners (16). Genetic study has shown thatLnk is an important regulator of the insulin/insulin-likegrowth factor pathway during growth, development de-lay and female sterility (10, 17, 18). Insulin/insulin-likegrowth signaling (IIS) is a major pathway involved ingrowth control and homeostasis in different cellular or-ganisms (19), and Lnk/SH2B3 has been described in IIS(20). Mutations in Lnk facilitated the overgrowth phe-notype caused by overexpression of insulin receptor butdid not suppress overgrowth promoted by high activityof phosphatidylinositol 3 kinase (PI3K) (21). This sug-gests that Lnk acts between insulin receptor and PI3K inIIS. Insulin receptor is a ligand-activated tyrosine kinase

receptor which mediates insulin signaling (22). The ac-tivated insulin receptor then tyrosine phosphorylatesintracellular substrates to initiate signal transduction(23). Tyrosine autophosphorylation increases the re-ceptor’s tyrosine kinase activity whereas serine phos-phorylation inhibits it (24). Insulin stimulates cellgrowth and differentiation through MAPK-Erk path-way and this pathway is activated by insulin receptor-mediated phosphorylation of insulin receptor substrate(IRS). Insulin induced inhibition of glycogen synthasekinase-3 through PI3K and protein kinase B (Akt) alsoresults in eukaryotic initiation factor 2B increasing pro-tein synthesis. Based on these previous studies, we hy-pothesized that Lnk may induce IR in PCOS patients.Therefore, in this study, we focused on how Lnk couldaffect IR in granulosa cells during PCOS.

Overall, we showed that Lnk expression level wassignificantly different in ovary tissue isolated fromwomen with both PCOS and IR, women with PCOSwithout IR and normal controls. We provided in vivoand in vitro evidence that Lnk is involved in controllingthe insulin signaling pathway in human granulosa cellsduring PCOS pathophysiology.

Materials and Methods

Study subjectsThirty-one women aged between 22 and 39 years with PCOS,

as defined by the Rotterdam criteria (25), and infertility whowere planning to receive laparoscopic surgery for tubal factor(18 cases), endometriosis (4 cases), hysteromyoma (2 cases), orothers (7 cases) were recruited at the gynecologic department ofthe Sun Yat-Sen Memorial Hospital, Sun Yat-Sen Universityfrom June 2011 to February 2012. The exclusion criteria weremenopause (FSH � 40 U/L) or gestation; hyperprolactinemia(prolactin � 25 mg/L), abnormal thyroid function, congenitaladrenal hyperplasia, androgen secreting tumors such as the-coma, Cushing syndrome or other diseases, or use of medicationsthat could potentially interfere with the evaluations carried outin the study. In particular, patients should not have received oralcontraceptives, insulin-sensitizing agents, antiandrogens, or glu-cocorticoids in the past 3 months.

The PCOS screening examined menstrual regularity, hir-sutism, acne, androgenic alopecia, gynecologic and obstetrichistory, medications, family history of related factors, and aphysical examination including blood pressure (BP), bodymass index (BMI) (weight in kilograms divided by height inmeters squared), and calculation of waist to hip ratio (WHR).The modified Ferriman Gallwey classification system (26) wasused to evaluate terminal hair growth. Levels of pituitary hor-mone, ovarian and adrenal steroids, TSH, lipids, fasting glu-cose, and insulin, or administration of a 75-g oral glucosetolerance test (OGTT) with blood obtained at 0, 1, and 2hours, were measured during the first 5 days of spontaneousmenstrual cycles or progestin-withdrawal bleeding. System-

3710 Hao et al Role of Lnk in IR Endocrinology, October 2016, 157(10):3709–3718


atic transvaginal ultrasound for the evaluation of polycysticovary morphology was carried out in the early follicular phaseif menses were regular, or randomly if menses were irregularwith a Toshiba Sonolayer SSA-220A (Toshiba) real-timesonograph fitted with a 6-MHz transvaginal transducer.These people were defined as having impaired fasting glucose(fasting plasma glucose levels 100 mg/dL [5.6 mmol/L] to 125mg/dL [6.9 mmol/L]), impaired glucose tolerance (2-h valuesin the 75-g OGTT of 140 mg/dL [7.8 mmol/L] to 199 mg/dL[11.0 mmol/L]), or type 2 diabetes mellitus (fasting plasmaglucose value of �126 mg/dL [7.0 mmol/L] or 2-h PG value of�200 mg/dL [11.1 mmol/L]0 according to the diagnosis andclassification of diabetes mellitus proposed by the AmericanDiabetes Association (27). Ovarian biopsies were performedduring the laparoscopic surgery.

The women with PCOS were also grouped according towhether they displayed IR or not into PCOS with IR or PCOSwithout IR. The homeostasis model assessment for IR (HOMA-IR) is the most widely used index to evaluate the IR by usingmeasures based on fasting parameters (28, 29); 2.14 is consid-ered as the cut-off value (22); HOMA-IR was calculated usingthe formula: HOMA-IR � (fasting insulin [�IU/mL] � fastingglucose [mmol/L])/22.5. Therefore, diagnosis for IR was if eitherof the following criteria were found: 1) delayed peak of insulinrelease (defined as 2-h plasma insulin value higher than 1-hplasma insulin value after 75-g OGTT), or impaired fastingblood glucose (� 5.6 mmol/L), impaired glucose tolerance (2-hglucose � 7.8 mmol/L) or HOMA-IR � 2.14; or 2) acanthosisnigricans. Among the study population, 13 women were diag-

nosed as having PCOS without IR. In addition, 6 control subjectswere enrolled in this study. The age ranged from 22 to 39 years.The control subjects without any PCOS or diabetes receivedlaparoscopic surgery. The baseline characteristics of the entirestudy population are shown in Table 1. The study was approvedby the Institutional Review Board of the Sun Yat-Sen MemorialHospital, Sun Yat-Sen University, and written informed consentwas obtained from each woman. Our ethical standards were inline with Helsinki Ethics.

The levels of free androgen and sex hormone-binding glob-ulin (Diagnostic Systems Laboratories), androstenedione, de-hydroepiandrosterone, and 17-hydroxyprogesterone (DRGInstruments GmbH) in the blood were measured using ELISAsas described by the manufacturers. Total testosterone, LH,FSH, prolactin, and plasma insulin levels were measured bychemiluminescence using the ACS180 SE autoanalyzer (BayerDiagnostics), glucose level by a glucose oxidase assay (TosohCorp), and TSH level by chemiluminescence (DiagnosticProducts Corp).

ImmunohistochemistryFormalin-fixed sections of tissue specimens from patients

with PCOS, PCOS with IR, and normal ovarian tissues wereobtained from the pathology department of University of SunYat-Sen University. Eighteen samples were investigated. Sixsamples from each group were randomly chosen and paraffin-embedded. Four-micrometer sections were deparaffinizedwith xylene and rehydrated with graded ethanol. After being

Table 1. Clinical Characteristics of PCOS Patients With or Without IR

PCOS WithIR (n � 18)

PCOS WithoutIR (n � 13) Control (n � 6)

PValuea

PValueb

PValuec

Age, y 30.17 � 4.97 30.36 � 3.23 30.00 � 4.54 .908 .727 .877BMI, kg/m2 21.83 � 4.49 21.18 � 3.89 19.86 � 2.43 .694 .541 .175WHR 0.84 � 0.07 0.81 � 0.05 0.79 � 0.05 .202 .466 .066SBP 111.00 � 11.33 109.89 � 12.78 101.75 � 8.21 .62 .068 .100DBP 73.14 � 6.44 71.67 � 6.86 68.00 � 5.83 .668 .097 .197mFG score 3.14 � 3.49 1.88 � 3.36 1.17 � 0.75 .486 .702 .232LH/FSH, IU/mL 1.05 � 0.0.83 1.08 � 0.0.75 0.75 � 0.33 .171 .592 .439Total testosterone, nmol/mL 2.47 � 4.05 1.18 � 0.75 1.45 � 0.39 .309 .801 .472Free testosterone, pg/mL 1.50 � 1.16 1.09 � 0.70 1.09 � 0.52 .305 .80 .467DHEAS, ng/mL 1900.50 � 829.42 1547.09 � 575.68 1531.50 � 370.11 .233 .759 .226SHBG, nmol/L 67.38 � 19.48 97.82 � 35.55 89.68 � 38.22 .008 .319 .796FAI 2.38 � 1.60 1.50 � 0.55 1.83�.80 .226 .595 .340Fasting insulin, mU/L 10.00 � 6.26 5.45 � 3.30 3.39 � 3.68 .037 .451 .0051-h Ins, mU/L 62.19 � 36.18 70.90 � 32.83 75.80 � 39.67 .621 .947 .4432-h Ins, mU/L 69.13 � 29.94 38.30 � 26.57 26.67 � 10.91 .015 .882 .008Fasting glucose, mmol/L 4.78 � 0.81 4.55�.52 4.44 � 0.27 .404 .843 .4401-h Glu, mmol/L 9.50 � 1.29 8.00 � 1.85 8.03 � 2.69 .181 .561 .7872-h Glu, mmol/L 7.93 � 1.53 5.70 � 1.25 5.26 � 1.49 .001 .927 .002HOMA 2.17 � 1.43 1.09 � 0.70 0.82 � 0.57 .012 .803 .002CHOL, mmol/L 5.17�.75 4.86 � 0.69 4.22 � 0.52 .783 .119 .120TG, mmol/L 1.17 � 0.98 0.71 � 0.49 0.95 � 0.36 .078 .122 .191HDL, mmol/L 1.27 � 0.46 1.56�.52 2.60 � 0.78 .408 .003 �.0001LDL, mmol/L 3.07 � 0.70 2.78 � 0.97 1.09 � 0.159 .925 �.0001 �.0001

Results are mean � SD. SBP, systolic BP; DBP, diastolic BP; mFG, modified Ferriman Gallwey classification; DHEAS, dehydroepiandrosterone; SHBG,sex hormone-binding globulin; FAI, free androgen index; CHOL, cholesterol; TG, triglyceride; HDL, high-density lipoprotein; LDL, low-densitylipoprotein; Ins, insulin; Glu, glucose.a Comparison between PCOS without IR and PCOS with IR groups, P � .05 was considered statistically significant.b Comparison between PCOS without IR and normal control groups, P � .05 was considered statistically significant.c Comparison between PCOS with IR and normal control groups, P � .05 was considered statistically significant.

doi: 10.1210/en.2016-1234 press.endocrine.org/journal/endo 3711


washed in distilled water twice (5 min), the slides were pre-treated with citrate buffer (pH 6) in a pressure cooker forantigen retrieval, cooled for 30 minutes, and washed 3 timesin distilled water. Endogenous peroxidase activity was inhib-ited by 3% hydrogen peroxide (Biocare Medical) for 10 min-utes in a dark chamber at room temperature. Tissue sectionswere washed twice in distilled water and then 3 times in PBS.The sections were incubated with normal goat serum blockingsolution for 1 hour at room temperature, and then incubatedovernight at 4°C with an anti-Lnk mouse monoclonal anti-body (Santa Cruz Biotechnology, Inc) at 1:40 dilution. Sec-tions were then washed 3 times with PBS. Goat antimouseimmunoglobulins/biotinylated at 1:400 dilution in PBS wasincubated for 30 minutes at room temperature. The slideswere washed twice in PBS and then developed with 3,3�-di-aminobenzidine tetra-chloride (DAB kit, K4100; Vector Lab-oratories Ltd) for 3 minutes. After washing in distilled waterfor 10 minutes, sections were counterstained with hematox-ylin for 1 minute, and then coverslips were added. Sections fornegative controls were prepared using nonimmune rabbit IgGantibody instead of the primary antibodies. Images werecaptured randomly, and all sections were analyzed by usinginverted microscope (Nikon TS100) connected to Olympusdigital camera. Immunohistochemistry-positive area andstaining intensity were analyzed by Image-Pro Plus 5.0. Foreach group, 6 ovarian sections were taken for the experiment,respectively.

Cell cultureA steroidogenic human granulosa cell-like tumor cell line,

KGN, was kindly provided by Professor Huang Hefeng (ZheJiang University, Hang Zhou, China). KGN cells were undif-ferentiated and maintained the physiological characteristicsof human ovarian granulosa cells. KGN cells were cultured inDMEM/F12 medium (Gibco) with 10% fetal bovine serum(FBS) (Hyclone Laboratories) and 100-U/mL penicillin-strep-tomycin (Invitrogen). HEK293T cells were purchased fromAmerican Type Culture Collection. HEK293T cells weremaintained in DMEM containing 10% FBS with 100-U/mLpenicillin-streptomycin. Cells were grown at 37°C in a hu-midified atmosphere of 5% CO2.

Real-time RT-PCR analysisTotal RNA was extracted using TRIzol reagent (Invitrogen)

and processed to cDNA with Superscript III (Invitrogen) accord-ing to the manufacturer’s protocols. Lnk and glyceraldehyde3-phosphate dehydrogenase primers and probes were synthe-sized by Invitrogen and Biosearch Technologies, respectively.The sequences of the sense and antisense primers used were Lnk(88 bp) 5�-GCTCAACACCAAACTGGACAGTAGA-3� and5�-CCGGGAGCTGTCAAGCTGTA-3�; glyceraldehyde 3-phosphate dehydrogenase (138 bp) 5�-GCACCGTCAAGGCTGAGAAC-3� and 5�-TGGTGAAGACGCCAGTGGA-3�. PCR wasperformed for 30 cycles, with each cycle at 95°C for 1 minute,56°C for 1 minute, and 72°C for 1 minute, in an iCycler iQsystem (Bio-Rad).

Expression vectors and transfectionHuman Lnk cDNA was cloned into the pcDNA3.1 vector.

Cells were plated in tissue culture plates. When grown to

50% confluence, cells were transfected with 1.0-�g/mL LNKpcDNA3.1, insulin receptor, IRS1, or empty pcDNA3.1 vectorby Lipofectamine 2000 reagent according to the manufacturer(Invitrogen, Life Technologies, Inc).

Treatment with insulinTo study the effect of Lnk on the phosphorylation of insulin

receptor, IRS1, protein kinase B and ERK1/2, ovarian gran-ulosa cells transfected with Lnk PCDNA3.1 or empty vectorfor 48 hours were serum starved for 12 hours, and then100nM insulin (Sigma) was added to the medium for 5 or 30minutes.

Western blottingCells were washed twice with PBS and lysed on ice with lysis

buffer (50mM Tris-HCl [pH 7.4], 150mM NaCl, and 0.5%Nonidet P-40) containing complete protease inhibitor cocktail(Roche) and phosphatase inhibitors (1mM phenylmethanesul-fonyl fluoride, 100mM NaF, and 10mM Na3VO4). Proteinswere resolved on 4%–15% gradient SDS-PAGE and transferredto nitrocellulose membranes (Sigma-Aldrich). Immunoblotswere incubated with primary antibodies for Lnk (Santa CruzBiotechnology, Inc) or V5 (Santa Cruz Biotechnology, Inc) toconfirm the overexpression of Lnk in the cell lines, total IR (CellSignaling) and phosphorylated IR (p-IRTyr1150/1151; Cell Signal-ing), IRS1 and phosphorylated IRS1Ser307Akt (Cell Signaling)and phosphorylated Akt (pAKTThr308; Cell Signaling), ERK1/2,pERK1/2Thr202/Tyr204 (Cell Signaling) to detect the activity ofinsulin signaling, anti-�-actin (Cell Signaling) as the standardcontrol; followed by incubation with appropriate secondary IgGantibody conjugated with horseradish peroxidase (AmershamPharmacia Biotech).

ImmunoprecipitationHEK293T cells were lysed on ice with a mild ice-cold lysis

buffer (50mM Tris-HCl [pH 8.0], 150mM NaCl, 10% glycerol,1% Triton X-100, 0.1% Igepal) containing complete proteaseinhibitor cocktail after being cotransfected with LNK pcDNA3.1 and IR-green fluorescent protein (GFP) pcDNA 3.1 (IR,kindly provided by Dr Lu-hai Wang, PhD, Mount Sinai Schoolof Medicine, New York, NY) or pENTER IRS1-HIS (ViGeneBiosciences) for 48 hours. Insoluble materials were removed af-ter centrifugation at 12 000g for 12 minutes at 4°C, and thesupernatant was prepared as total cell lysate. One-milligram pro-tein of total cell lysates was incubated with 20-�L anti-Lnk(Santa Cruz Biotechnology, Inc), 20-�L anti-GFP (Santa CruzBiotechnology, Inc), or 5-�L antihistidine (Cell Signaling) anti-body overnight at 4°C with constant shaking. Nonspecific im-munoprecipitation was carried out by using normal IgG as acontrol. The mixture was precipitated with 20-�L Protein GPlus/Protein An agarose suspension (Merck) for 4 hours on thesecond day. Then, the immunocomplexes were washed 3 timeswith ice-cold PBS and then boiled for 10 minutes. Proteins weresubjected to immunoblot analysis, probed by GFP, anti-HIS, oranti-Lnk antibodies.

ColocalizationHEK293T cells were fixed with 200-�L 4% paraformalde-

hyde for 30 minutes after being cotransfected with LNK pcDNA3.1 and IR-GFP pcDNA 3.1 or pENTER IRS1-HIS (ViGene Bio-



sciences) for 48 hours. Cells were washed twice with PBS andpermeabilized in PBS containing 0.1% Triton X-100 for 2.5 min-utes and then blocked by 5% BSA for 1 hour. Primary antibodies(anti-Lnk and anti-IRS1, diluted at 1:100 in 5% BSA-PBS) wereincubated overnight at 4°C. Washed twice with PBS, the cellswere then incubated for 1.5 hours with the secondary antibody(Cy3-conjugated antimouse antibody was used to detect Lnkprotein, red, and fluorescein isothiocyanate (FITC)-conjugatedantirabbit antibody to detect IRS1 protein, green), at 1:200 in5% BSA-PBS at 37°C. Washed 3 times with PBS, the cells werethen counterstained with Hoechst 33258 to stain the nuclei.HEK293T cells were subsequently examined with confocal mi-

croscope (FV500; Olympus), and images were analyzed withMetamorph version 6.3 software (Molecular Devices).

Statistical analysisData are expressed as the mean � SEM of at least 3 indepen-

dent experiments. The data were analyzed by 2-tailed Student’st test or one-way ANOVA followed by the Bonferroni multiplecomparison post hoc test with a 95% confidence interval. Sta-tistical analyses were performed using PRISM version 6.0(GraphPad Software). The correlation analyses were determinedby the Pearson correlation test. Statistical significance was con-sidered to be P � .05.

Figure 1. Ovarian tissues from PCOS patients with IR exhibited higher expression of Lnk than the ovaries from control or PCOS patientswithout IR. A, The mRNA expression level of Lnk in ovaries was detected in control (n � 4) and PCOS with IR (n � 4) or without IR (n � 4)patients according to the HOMA-IR (HOMA-IR defined as HOMA � 2.14). B–H, Quantitative real-time polymerase chain reaction validationof the positive correlation between the level of Lnk mRNA and IR. B, Pearson correlation analysis showed a significant positive correlation ofLnk vs HOMA-IR (Pearson r � 0.672; P � .0167, n � 12). Line represents linear regression of data (y � 0.01728x�0.01504; r2 � 0.4515).C, Pearson correlation analysis showed a significant positive correlation of Lnk vs BMI (Pearson r � 0.702; P � .0108, n � 12). Linerepresents linear regression of data (y � 0.004983x0.05860; r2 � 0.4935). D, Pearson correlation analysis showed a significant positivecorrelation of Lnk vs WHR (Pearson r � 0.735; P � .0065, n � 12). Line represents linear regression of data (y � 0.3296x0.2252; r2 �0.5400). E, Pearson correlation analysis showed a significant positive correlation of Lnk vs 0-hour glucose (Glu) (Pearson r � 0.791; P �.0022, n � 12). Line represents linear regression of data (y � 0.02953x0.09443; r2 � 0.6253). F, Pearson correlation analysis showed asignificant positive correlation of Lnk vs 2-hour Glu (Pearson r � 0.731; P � .0069, n � 12). Line represents linear regression of data (y �0.009978x0.02884; r2 � 0.5340). G, Pearson correlation analysis showed a significant positive correlation of Lnk vs 0-hour insulin (INS)(Pearson r � 0.769; P � .0035, n � 12). Line represents linear regression of data (y � 0.005166x�0.004672; r2 � 0.5914). H, Pearsoncorrelation analysis showed a significant positive correlation of Lnk vs 2-hour Ins (Pearson r � 0.5822; P � .05, n � 12). Line representslinear regression of data (y � 0.0003434x�0.01669; r2 � 0.3309).



Results

The level of Lnk mRNA increases in the ovaries ofPCOS women with IR

Quantitative real-time polymerase chain reaction wasused to identify the level of Lnk mRNA in ovaries. Ovariantissues from PCOS patients with IR exhibited higher ex-pression of Lnk thanovaries from normal control andPCOS patients without IR (Figure 1A). The difference be-tween the PCOS/IR group and control group is significant(P � .05), whereas the level of Lnk between PCOS groupand control group had no significance. Pearson correla-tion analysis showed a significant positive correlation ofthe level of Lnk mRNA vs HOMA-IR, BMI, WHR, 0-hourglucose, 2-hour glucose, 0-hour insulin, and 2-hour insu-lin (Figure 1, B–H).

The Lnk protein levels were significantly higher inthe group with PCOS/IR compared with thenormal control and PCOS without IR group

Immunohistochemistryofovaries frompatientswithorwithout PCOS/IR showed the expression of Lnk in theovaries from patients. There were more primary and sec-ondary follicles, thickened follicles, and small vascularstructures in polycystic ovaries than normal ovaries. Lnkwas significantly increased in the ovaries in the group withPCOS/IR and PCOS compared with the normal controls,mainly in the granulosa cells of the follicles at all stages,and in the follicular fluid and plasma of oocytes among thesecondary follicles (Figure 2A). The positive rates of Lnkwere significantly higher in the ovaries in the group withPCOS/IR and PCOS without IR compared with that in thenormal control group (P � .05) (Figure 2B).

Lnk binds and colocalizes with insulin receptorand IRS1

Insulin receptor and IRS1 binds with LnkCell lysates from HEK293T cells cotransfected with

Lnk pcDNA 3.1 and GFP-tagged insulin receptor pcDNA3.1 or pENTER IRS1-HIS were immunoprecipitated withanti-Lnk, anti-GFP, or anti-HIS antibody. The pellet wasseparated by SDS-PAGE and subjected to immunoblottingwith anti-GFP or anti-Lnk antibody to assess the bindingof insulin receptor and IRS1 to Lnk. We found that Lnkbound to insulin receptor (Figure 3, A and B) and IRS1(Figure 3, C and D).

Colocalization of Lnk and insulin receptor/IRS1 weretracked in cells with intracellular immunofluorescence

HEK293T cells were cotransfected with LNK pcDNA3.1 and insulin receptor-GFP pcDNA 3.1 or pENTERIRS1-HIS. Immunofluorescent staining of HEK293T cells

with FITC-conjugated antirabbit antibody (to detectIRS1, green) and Cy3-conjugated antimouse antibody (todetect Lnk, red) showed Lnk and insulin receptor-GFP(Figure 3E) or IRS1-HIS (Figure 3F) were coexpressed inHEK293 cells. The expression and position of Lnk, insulinreceptor, and IRS1 were observed with intracellular flu-orescence under a confocal microscope. Lnk was mainlyexpressed in plasma, around the nucleus, whereas insulinreceptor and IRS1 were both in plasma and nuclear loca-tions. The intracellular localizations of Lnk and insulinreceptor/IRS1 overlapped around the nucleus.

Figure 2. Immunohistochemistry of ovaries from patients with orwithout PCOS/IR. A, The expression of Lnk in ovaries from patients.The positive rates of Lnk were significantly higher in the group withPCOS and IR compared with the control group. Results show: con,weak positive; PCOS, moderate positive; PCOS�IR, strong positive.Scale bar, 50 �m; n � 6 per group. B, The expression of Lnk wassignificantly higher in the PCOS/IR and PCOS group compared with thecontrol group; **, P � .05 vs control; ##, P � .05 vs PCOS.



Overexpression of Lnk inhibits the insulinresponse PI3K-AKT and MAPK-ERK1/2 signalingpathways in ovarian granulosa cells

Lnk inhibits phosphorylation of insulin receptor(Tyr1150/1151) and IRS1 (Ser307)

We overexpressed Lnk in ovarian granulosa cells byLnk pcDNA 3.1 transfection. Five minutes after insulinaddition, there was a significant increase in insulin recep-tor tyrosine and IRS1 serine phosphorylation levels. Lnkoverexpression affected insulin receptor autophosphory-lation induced by insulin. Insulin-dependent tyrosinephosphorylation of insulin receptor (Tyr1150/1151) de-creased, whereas serine phosphorylation of IRS1 (Ser307)(Figure 4, A–D) increased significantly. However, expres-sion of total insulin receptor and IRS1 were not altered.

The phosphorylation of Akt and ERK1/2 increased byinsulin (100nM) were time dependent (Figure 4E). Thebasal activations of Akt and ERK1/2 were inhibited byLnk in granulosa cells with 100nM insulin for 30 minutes(Figure 4F). Thr308 phosphorylation level of Akt andThr202/Tyr204 phosphorylation of ERK1/2 were all fur-ther inhibited by Lnk overexpression (P � .01). The totallevels of Akt and ERK Akt were not affected by thesetreatments.

Discussion

In this study, we demonstrated that Lnk may play an im-portant role in inhibiting insulin signaling in the ovaryduring PCOS. The results showed that ovarian Lnk ex-

pression was increased in patients with IR-PCOS. We alsofound that women with IR-PCOS had higher levels ofHOMA-IR, BMI, WHR, glucose, and insulin expression.Lnk expression was positively correlated with HOMA-IR,BMI, and WHR. Moreover, we found that overexpressionof Lnk inhibited insulin receptor (Tyr1150/1151) phos-phorylation and the Akt/Erk signaling pathway.

Most women with PCOS have endocrine abnormalities(30, 31). IR is an important defect in the development ofnoninsulin-dependent diabetes mellitus (32). A study inPCOS women found a significantly increased the preva-lence of noninsulin-dependent diabetes mellitus and hy-pertension especially in obese women (6). Approximately50%–70% of women with PCOS have some degree of IR(1, 33). IR also has been found in PCOS women of manyracial groups including Japanese, Caribbean, Chinese, andAfrican Americas (34). Our clinical studies show that mostPCOS patients have a high level of insulin, HOMA-IR,BMI, and WHR. These are in line with previous research.In PCOS pathophysiology, IR has classic target tissuessuch as liver, adipose tissue and muscle (35). It also hasnonclassic target tissues such as stromal and follicularcompartment (36). In the nonclassic target tissues, insulincould stimulate steroidogenesis in ovarian cells because ofthe prevalence of insulin receptor. Hyperinsulinemia in IRalters the enzymes and proteins that are important forsteroidogenesis then affects follicle development and fer-tility (37). This study mainly focused on investigating theeffect of IR on granulosa cells that ultimately affect fertilitythrough folliculogenesis.

Figure 3. Lnk interacts with insulin receptor and IRS1. A, Coimmunoprecipitation of Lnk with insulin receptor. B, Coimmunoprecipitation of Lnkwith IRS1. HEK293T cells were cotransfected with LNK pcDNA 3.1 and IR-GFP pcDNA 3.1 or pENTER IRS1-HIS. C and D, Immunofluorescentstaining of HEK293T cells with FITC-conjugated antirabbit antibody (to detect IRS1, green) and Cy3-conjugated antimouse antibody (to detect Lnk,red). The expression and position of Lnk, insulin receptor, and IRS1 were observed with intracellular fluorescence under a confocal microscope.Nuclei were identified by DAPI staining. Scale bar, 20 �m.



Human genome-wide associationstudies have linked Lnk to hyperten-sion, renal disease, and diabetes (21,38). Lnk is an intracellular adaptorprotein that functions as negativeregulator in many signaling path-ways (39). Mutations in DrosophilaLnk produce a phenotype reminis-cent of reduced insulin signaling dur-ing development of female fertility(8, 10). In this study, we provided thefirst evidence that Lnk is an impor-tant factor in PCOS patients with IRand is associated with the insulin sig-naling pathway in ovary tissue. Inaddition, Lnk expression was signif-icantly up-regulated in IR-PCOS andalso in granulosa cells treated withinsulin. Up-regulation of Lnk wascorrelated with the severity of IR-PCOS. These findings suggest thatup-regulation of Lnk contributes tothe development of PCOS and asso-ciated IR.

Lnk has 20 known binding part-ners that interact predominantlythrough the pleckstrin homology andSH2 domains to activate major sig-naling pathways such as Janus kinase-signal transducer and activator oftranscription and ERK1/2 (39). Lnk/SH2B has potential tyrosine phos-phorylation sites and functions asprotein-protein interaction motifs andtransduces signals downstream of anumber of receptor tyrosine kinases.Our data suggests that there is an in-teraction between Lnk and insulin re-ceptor and IRS. We also found over-expression of Lnk inhibited insulininduced insulin receptor tyrosinephosphorylation and facilitated insu-lin receptor serine phosphorylation.We demonstrated that overexpressionof Lnk suppressed insulin inducedMAPK and Akt phosphorylation.

This study has some limitations. Itis worth noting that, in this study, thewomen with PCOS were diagnosedaccording to the Rotterdam criteriadeveloped in 2003 because these arethe criteria used by the physicians in

Figure 4. Overexpression of Lnk impairs insulin signaling in ovarian granulosa cells. A, Timecourse of insulin stimulated insulin receptor and IRS1 phosphorylation in ovarian granulosa cells.Representative blots of phosphorylation of insulin receptor and IRS1 by 100nM insulin. Insulinincreased the phosphorylation of insulin receptor and IRS1 but did not alter the level of totalinsulin receptor and IRS1. B, Representative blot of phosphorylation of insulin receptor and IRS1in Lnk overexpression granulosa cells. Ovarian granulosa were transfected with Lnk pcDNA 3.1for 48 hours and then starved for 12 hours with 1% FBS culture medium. After that, 100nMinsulin was added to the culture medium for 5 minutes. C and D, Densitometry analysis ofphosphorylation of insulin receptor and IRS1 in granulosa cells. The Tyr1150/1151phosphorylation level of insulin receptor was inhibited by Lnk in ovarian granulosa cells; n � 4;*, P � .05 vs control; #, P � .05 vs insulin-treated control group by one-way ANOVA. E, Timecourse of insulin-stimulated Akt and ERK1/2 phosphorylation in ovarian granulosa cells.Representative blots of phosphorylation of Akt and ERK1/2 by 100nM insulin. Insulin increasesthe phosphorylation of Akt and ERK1/2 but have no effects on the level of total Akt and ERK1/2.F, Representative blot of phosphorylation of Akt and ERK1/2 in Lnk overexpression granulosacells. Ovarian granulosa cells were transfected with Lnk pcDNA 3.1 for 48 hours and then starvedfor 12 hours with 1% FBS culture medium. After that, 100nM insulin was added to the culturemedium for 30 minutes. G and H, Densitometry analysis of phosphorylation of Akt and ERK1/2 ingranulosa cells. The basal Thr308 phosphorylation level of Akt and Thr202/Tyr204phosphorylation of ERK1/2 were all inhibited by Lnk in ovarian granulosa cells. After insulintreatment, the phosphorylation level of Akt and ERK1/2 were all further inhibited; P � .01; n �4, *, P � .05 vs control; #, P � .05 vs insulin-treated control group by one-way ANOVA.



the hospital. More recently, the criteria for PCOS diag-nosis have become stricter; therefore, many studies nowdefine PCOS by updated criteria (40). The PCOS womenincluded in this study without IR might be classified ashaving ovulatory dysfunction with polycystic ovarianmorphology but not hyperandrogenism, that is phenotypeD (2, 40). The BMI scores of the study population were notwithin the obese range; however, the mean WHRs of thePCOS patients with or without IR was both more than orequal to 0.8, which is a marker of Asian central obesity(41). So further study is needed to understand whetherthese resultswould translate toWesternobesepopulationsbecause of the interaction between androgens BMI and IR.

In summary, our findings show that Lnk expression isincreased in IR-PCOS ovarian tissues compared with con-trol group. Lnk expression is closely related to insulin lev-els, HOMA-IR, BMI, and WHR. Overexpression of Lnkcould inhibit the insulin response PI3K-AKT and MAPK-ERK1/2 signaling pathways in ovarian granulosa cells.This study suggests that Lnk might be a new strategy forthe treatment of IR-PCOS.

Acknowledgments

We thank Dr Philip H. Koeffler and Sigal Gery (Cedars-SinaiMedical Center, the University of California, Los Angeles) fortheir ideas and guidance of performing the experiments and theirhelps of experimental materials.

Address all correspondence and requests for reprints to:Xiaomiao Zhao, MD, PhD, Department of Obstetrics and Gy-necology, Memorial Hospital of Sun Yat-Sen University, Guang-zhou, 510120 China. E-mail: [email protected]; orDongzi Yang, MD, PhD, Department of Obstetrics and Gyne-cology, Memorial Hospital of Sun Yat-Sen University, Guang-zhou, 510120 China. E-mail: [email protected]; orMeiqing Xie, MD, PhD, Department of Obstetrics and Gyne-cology, Memorial Hospital of Sun Yat-Sen University, Guang-zhou, 510120 China. E-mail: [email protected].

This work was supported by National Natural Science Foun-dation General Program Grants 81471425 and 81100402; theScience Technology Research Project of Guangdong Province Grants2013B022000016, 2014A020213006, and 2015A030313091; andthe Sun Yat-Sen scholarship for young scientist.

Disclosure Summary: The authors have nothing to disclose.

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Role of lnk in insulin resistance

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