Role of lnk in insulin resistance

aaaaaaaaaa Overexpression of Lnk in the Ovaries Is Involved in
Insulin Resistance in Women With Polycystic
Ovary 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 Memorial
Hospital, 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-Sen
University, Guangzhou, 510120 China; Department of Obstetrics and Gynecology (Z.Z.), Reproductive
Medical 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 Angeles
School 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 women
due to receive laparoscopic surgery from June 2011 to February 2012 were included from the
gynecologic department of the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. Sam-
ples of polycystic and normal ovary tissues were examined by immunohistochemistry. Ovarian
cell lines underwent insulin stimulation and Lnk overexpression. Expressed Lnk underwent
coimmunoprecipitation 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 than
ovaries 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/IRS1
locations overlapped around the nucleus. IR, protein kinase B (Akt), and ERK1/2 activities were
inhibited by Lnk overexpression and inhibited further after insulin stimulation, whereas IRS1
serine activity was increased. Insulin receptor (Tyr1150/1151), Akt (Thr308), and ERK1/2
(Thr202/Tyr204) phosphorylation was decreased, whereas IRS1 (Ser307) phosphorylation was
increased with Lnk overexpression. In conclusion, Lnk inhibits the phosphatidylinositol 3
kinase-AKT and MAPK-ERK signaling response to insulin. Higher expression of Lnk in PCOS
suggests 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 and
chronic anovulation (1). Its etiology remains unknown,
but PCOS is likely to result from a complex number of
genetic and environmental factors including androgen ex-
posure,obesity,environmentaltoxins,andsocioeconomic
status (2). The impact of PCOS includes major metabolic
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in USA
Copyright © 2016 by the Endocrine Society
Received 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, fetal
bovine serum; GFP, green fluorescent protein; His, histidine; HOMA-IR, homeostasis model
assessment 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,
polycysticovarysyndrome;PI3K,phosphatidylinositol3kinase;SH2,Srchomology2;WHR,
waist to hip ratio.
O R I G I N A L R E S E A R C H
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doi: 10.1210/en.2016-1234 Endocrinology, October 2016, 157(10):3709–3718 press.endocrine.org/journal/endo 3709
The Endocrine Society. Downloaded from press.endocrine.org by [Anirban Sinha] on 04 October 2016. at 22:08 For personal use only. No other uses without permission. . All rights reserved.

syndrome as well as reproductive morbidities that can
cause female infertility (3). PCOS is strongly associated
with obesity and insulin resistance (IR) (1). Lifestyle
modification is the first line treatment for PCOS, and
this can improve ovulation and the success of fertility
treatment (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 PCOS
may be mediated through worsening IR (5), the mech-
anism remains unclear. Therefore, it is important to
study IR and the insulin signaling pathway in PCOS. IR
in classic or nonclassic target tissues has been shown to
be 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), so
there may be a direct relationship between IR and
folliculogenesis.
As an important regulator of insulin and insulin sig-
naling in Drosophila (10), Lnk may play a role in the
development of IR and PCOS. The Lnk (Src homology
2 [SH2]B3) protein shares a central pleckstrin homol-
ogy (PH) domains and a SH2 domains and potential
tyrosine phosphorylation sites with SH2B (11, 12).
These domains function as protein-protein interaction
motifs and transduce signals downstream of a number
of 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 energy
metabolism (10). Moreover, mutations in SH2B in hu-
mans are associated with metabolic dysregulation and
obesity (10, 14). Recent study suggests that in ovarian
cancer Lnk may have a positive action in signal trans-
duction increasing cell proliferation and tumor size
(15). Our previous study demonstrated that Lnk almost
exclusively bound to the phosphorylated juxtamem-
brane domain of c-Kit directly without requiring other
interacting partners (16). Genetic study has shown that
Lnk is an important regulator of the insulin/insulin-like
growth factor pathway during growth, development de-
lay and female sterility (10, 17, 18). Insulin/insulin-like
growth signaling (IIS) is a major pathway involved in
growth 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 but
did not suppress overgrowth promoted by high activity
of phosphatidylinositol 3 kinase (PI3K) (21). This sug-
gests that Lnk acts between insulin receptor and PI3K in
IIS. Insulin receptor is a ligand-activated tyrosine kinase
receptor which mediates insulin signaling (22). The ac-
tivated insulin receptor then tyrosine phosphorylates
intracellular 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 cell
growth 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 synthase
kinase-3 through PI3K and protein kinase B (Akt) also
results 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 could
affect IR in granulosa cells during PCOS.
Overall, we showed that Lnk expression level was
significantly different in ovary tissue isolated from
women with both PCOS and IR, women with PCOS
without IR and normal controls. We provided in vivo
and in vitro evidence that Lnk is involved in controlling
the insulin signaling pathway in human granulosa cells
during PCOS pathophysiology.
Materials and Methods
Study subjects
Thirty-one women aged between 22 and 39 years with PCOS,
as defined by the Rotterdam criteria (25), and infertility who
were planning to receive laparoscopic surgery for tubal factor
(18 cases), endometriosis (4 cases), hysteromyoma (2 cases), or
others (7 cases) were recruited at the gynecologic department of
the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University
from June 2011 to February 2012. The exclusion criteria were
menopause (FSH Ͼ 40 U/L) or gestation; hyperprolactinemia
(prolactin Ն 25 mg/L), abnormal thyroid function, congenital
adrenal hyperplasia, androgen secreting tumors such as the-
coma,Cushingsyndromeorotherdiseases,oruseofmedications
that could potentially interfere with the evaluations carried out
in the study. In particular, patients should not have received oral
contraceptives, 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 obstetric
history, medications, family history of related factors, and a
physical examination including blood pressure (BP), body
mass index (BMI) (weight in kilograms divided by height in
meters squared), and calculation of waist to hip ratio (WHR).
The modified Ferriman Gallwey classification system (26) was
used 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 glucose
tolerance test (OGTT) with blood obtained at 0, 1, and 2
hours, were measured during the first 5 days of spontaneous
menstrual 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 polycystic
ovary morphology was carried out in the early follicular phase
if menses were regular, or randomly if menses were irregular
with a Toshiba Sonolayer SSA-220A (Toshiba) real-time
sonograph 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 125
mg/dL [6.9 mmol/L]), impaired glucose tolerance (2-h values
in 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 plasma
glucose value of Ն126 mg/dL [7.0 mmol/L] or 2-h PG value of
Ն200 mg/dL [11.1 mmol/L]0 according to the diagnosis and
classification of diabetes mellitus proposed by the American
Diabetes Association (27). Ovarian biopsies were performed
during the laparoscopic surgery.
The women with PCOS were also grouped according to
whether they displayed IR or not into PCOS with IR or PCOS
without IR. The homeostasis model assessment for IR (HOMA-
IR) is the most widely used index to evaluate the IR by using
measures based on fasting parameters (28, 29); 2.14 is consid-
ered as the cut-off value (22); HOMA-IR was calculated using
the formula: HOMA-IR ϭ (fasting insulin [␮IU/mL] ϫ fasting
glucose [mmol/L])/22.5. Therefore, diagnosis for IR was if either
of the following criteria were found: 1) delayed peak of insulin
release (defined as 2-h plasma insulin value higher than 1-h
plasma insulin value after 75-g OGTT), or impaired fasting
blood glucose (Ն 5.6 mmol/L), impaired glucose tolerance (2-h
glucose Ն 7.8 mmol/L) or HOMA-IR Ն 2.14; or 2) acanthosis
nigricans. Among the study population, 13 women were diag-
nosed as having PCOS without IR. In addition, 6 control subjects
were enrolled in this study. The age ranged from 22 to 39 years.
The control subjects without any PCOS or diabetes received
laparoscopic surgery. The baseline characteristics of the entire
study population are shown in Table 1. The study was approved
by the Institutional Review Board of the Sun Yat-Sen Memorial
Hospital, Sun Yat-Sen University, and written informed consent
was obtained from each woman. Our ethical standards were in
line with Helsinki Ethics.
The levels of free androgen and sex hormone-binding glob-
ulin (Diagnostic Systems Laboratories), androstenedione, de-
hydroepiandrosterone, and 17-hydroxyprogesterone (DRG
Instruments GmbH) in the blood were measured using ELISAs
as described by the manufacturers. Total testosterone, LH,
FSH, prolactin, and plasma insulin levels were measured by
chemiluminescence using the ACS180 SE autoanalyzer (Bayer
Diagnostics), glucose level by a glucose oxidase assay (Tosoh
Corp), and TSH level by chemiluminescence (Diagnostic
Products Corp).
Immunohistochemistry
Formalin-fixed sections of tissue specimens from patients
with PCOS, PCOS with IR, and normal ovarian tissues were
obtained from the pathology department of University of Sun
Yat-Sen University. Eighteen samples were investigated. Six
samples from each group were randomly chosen and paraffin-
embedded. Four-micrometer sections were deparaffinized
with xylene and rehydrated with graded ethanol. After being
Table 1. Clinical Characteristics of PCOS Patients With or Without IR
PCOS With
IR (n ‫؍‬ 18)
PCOS Without
IR (n ‫؍‬ 13) Control (n ‫؍‬ 6)
P
Valuea
P
Valueb
P
Valuec
Age, y 30.17 Ϯ 4.97 30.36 Ϯ 3.23 30.00 Ϯ 4.54 .908 .727 .877
BMI, kg/m2
21.83 Ϯ 4.49 21.18 Ϯ 3.89 19.86 Ϯ 2.43 .694 .541 .175
WHR 0.84 Ϯ 0.07 0.81 Ϯ 0.05 0.79 Ϯ 0.05 .202 .466 .066
SBP 111.00 Ϯ 11.33 109.89 Ϯ 12.78 101.75 Ϯ 8.21 .62 .068 .100
DBP 73.14 Ϯ 6.44 71.67 Ϯ 6.86 68.00 Ϯ 5.83 .668 .097 .197
mFG score 3.14 Ϯ 3.49 1.88 Ϯ 3.36 1.17 Ϯ 0.75 .486 .702 .232
LH/FSH, IU/mL 1.05 Ϯ 0.0.83 1.08 Ϯ 0.0.75 0.75 Ϯ 0.33 .171 .592 .439
Total testosterone, nmol/mL 2.47 Ϯ 4.05 1.18 Ϯ 0.75 1.45 Ϯ 0.39 .309 .801 .472
Free testosterone, pg/mL 1.50 Ϯ 1.16 1.09 Ϯ 0.70 1.09 Ϯ 0.52 .305 .80 .467
DHEAS, ng/mL 1900.50 Ϯ 829.42 1547.09 Ϯ 575.68 1531.50 Ϯ 370.11 .233 .759 .226
SHBG, nmol/L 67.38 Ϯ 19.48 97.82 Ϯ 35.55 89.68 Ϯ 38.22 .008 .319 .796
FAI 2.38 Ϯ 1.60 1.50 Ϯ 0.55 1.83Ϯ.80 .226 .595 .340
Fasting insulin, mU/L 10.00 Ϯ 6.26 5.45 Ϯ 3.30 3.39 Ϯ 3.68 .037 .451 .005
1-h Ins, mU/L 62.19 Ϯ 36.18 70.90 Ϯ 32.83 75.80 Ϯ 39.67 .621 .947 .443
2-h Ins, mU/L 69.13 Ϯ 29.94 38.30 Ϯ 26.57 26.67 Ϯ 10.91 .015 .882 .008
Fasting glucose, mmol/L 4.78 Ϯ 0.81 4.55Ϯ.52 4.44 Ϯ 0.27 .404 .843 .440
1-h Glu, mmol/L 9.50 Ϯ 1.29 8.00 Ϯ 1.85 8.03 Ϯ 2.69 .181 .561 .787
2-h Glu, mmol/L 7.93 Ϯ 1.53 5.70 Ϯ 1.25 5.26 Ϯ 1.49 .001 .927 .002
HOMA 2.17 Ϯ 1.43 1.09 Ϯ 0.70 0.82 Ϯ 0.57 .012 .803 .002
CHOL, mmol/L 5.17Ϯ.75 4.86 Ϯ 0.69 4.22 Ϯ 0.52 .783 .119 .120
TG, mmol/L 1.17 Ϯ 0.98 0.71 Ϯ 0.49 0.95 Ϯ 0.36 .078 .122 .191
HDL, mmol/L 1.27 Ϯ 0.46 1.56Ϯ.52 2.60 Ϯ 0.78 .408 .003 Ͻ.0001
LDL, mmol/L 3.07 Ϯ 0.70 2.78 Ϯ 0.97 1.09 Ϯ 0.159 .925 Ͻ.0001 Ͻ.0001
Results are mean Ϯ 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-density
lipoprotein; Ins, insulin; Glu, glucose.
a
Comparison between PCOS without IR and PCOS with IR groups, P Ͻ .05 was considered statistically significant.
b
Comparison between PCOS without IR and normal control groups, P Ͻ .05 was considered statistically significant.
c
Comparison between PCOS with IR and normal control groups, P Ͻ .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 for
antigen retrieval, cooled for 30 minutes, and washed 3 times
in 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 sections
were washed twice in distilled water and then 3 times in PBS.
The sections were incubated with normal goat serum blocking
solution for 1 hour at room temperature, and then incubated
overnight 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 antimouse
immunoglobulins/biotinylated at 1:400 dilution in PBS was
incubated for 30 minutes at room temperature. The slides
were washed twice in PBS and then developed with 3,3Ј-di-
aminobenzidine tetra-chloride (DAB kit, K4100; Vector Lab-
oratories Ltd) for 3 minutes. After washing in distilled water
for 10 minutes, sections were counterstained with hematox-
ylin for 1 minute, and then coverslips were added. Sections for
negative controls were prepared using nonimmune rabbit IgG
antibody instead of the primary antibodies. Images were
captured randomly, and all sections were analyzed by using
inverted microscope (Nikon TS100) connected to Olympus
digital camera. Immunohistochemistry-positive area and
staining intensity were analyzed by Image-Pro Plus 5.0. For
each group, 6 ovarian sections were taken for the experiment,
respectively.
Cell culture
A steroidogenic human granulosa cell-like tumor cell line,
KGN, was kindly provided by Professor Huang Hefeng (Zhe
Jiang University, Hang Zhou, China). KGN cells were undif-
ferentiated and maintained the physiological characteristics
of human ovarian granulosa cells. KGN cells were cultured in
DMEM/F12 medium (Gibco) with 10% fetal bovine serum
(FBS) (Hyclone Laboratories) and 100-U/mL penicillin-strep-
tomycin (Invitrogen). HEK293T cells were purchased from
American Type Culture Collection. HEK293T cells were
maintained in DMEM containing 10% FBS with 100-U/mL
penicillin-streptomycin. Cells were grown at 37°C in a hu-
midified atmosphere of 5% CO2.
Real-time RT-PCR analysis
Total RNA was extracted using TRIzol reagent (Invitrogen)
and processed to cDNA with Superscript III (Invitrogen) accord-
ing to the manufacturer’s protocols. Lnk and glyceraldehyde
3-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) 5Ј-GCTCAACACCAAACTGGACAGTAGA-3Ј and
5Ј-CCGGGAGCTGTCAAGCTGTA-3Ј; glyceraldehyde 3-
phosphate dehydrogenase (138 bp) 5Ј-GCACCGTCAAGGCTG
AGAAC-3Ј and 5Ј-TGGTGAAGACGCCAGTGGA-3Ј. PCR was
performed 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 iQ
system (Bio-Rad).
Expression vectors and transfection
Human 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 LNK
pcDNA3.1, insulin receptor, IRS1, or empty pcDNA3.1 vector
by Lipofectamine 2000 reagent according to the manufacturer
(Invitrogen, Life Technologies, Inc).
Treatment with insulin
To 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 vector
for 48 hours were serum starved for 12 hours, and then
100nM insulin (Sigma) was added to the medium for 5 or 30
minutes.
Western blotting
Cells 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). Proteins
were resolved on 4%–15% gradient SDS-PAGE and transferred
to nitrocellulose membranes (Sigma-Aldrich). Immunoblots
were incubated with primary antibodies for Lnk (Santa Cruz
Biotechnology, Inc) or V5 (Santa Cruz Biotechnology, Inc) to
confirm the overexpression of Lnk in the cell lines, total IR (Cell
Signaling) and phosphorylated IR (p-IRTyr1150/1151
; Cell Signal-
ing), IRS1 and phosphorylated IRS1Ser307
Akt (Cell Signaling)
and phosphorylated Akt (pAKTThr308
; Cell Signaling), ERK1/2,
pERK1/2Thr202/Tyr204
(Cell Signaling) to detect the activity of
insulin signaling, anti-␤-actin (Cell Signaling) as the standard
control; followed by incubation with appropriate secondary IgG
antibody conjugated with horseradish peroxidase (Amersham
Pharmacia Biotech).
Immunoprecipitation
HEK293T 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 protease
inhibitor cocktail after being cotransfected with LNK pcDNA
3.1 and IR-green fluorescent protein (GFP) pcDNA 3.1 (IR,
kindly provided by Dr Lu-hai Wang, PhD, Mount Sinai School
of Medicine, New York, NY) or pENTER IRS1-HIS (ViGene
Biosciences) for 48 hours. Insoluble materials were removed af-
ter centrifugation at 12 000g for 12 minutes at 4°C, and the
supernatantwaspreparedastotalcelllysate.One-milligrampro-
tein of total cell lysates was incubated with 20-␮L anti-Lnk
(Santa Cruz Biotechnology, Inc), 20-␮L anti-GFP (Santa Cruz
Biotechnology, 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 a
control. The mixture was precipitated with 20-␮L Protein G
Plus/Protein An agarose suspension (Merck) for 4 hours on the
second day. Then, the immunocomplexes were washed 3 times
with ice-cold PBS and then boiled for 10 minutes. Proteins were
subjected to immunoblot analysis, probed by GFP, anti-HIS, or
anti-Lnk antibodies.
Colocalization
HEK293T cells were fixed with 200-␮L 4% paraformalde-
hyde for 30 minutes after being cotransfected with LNK pcDNA
3.1 and IR-GFP pcDNA 3.1 or pENTER IRS1-HIS (ViGene Bio-

sciences) for 48 hours. Cells were washed twice with PBS and
permeabilizedinPBScontaining0.1%TritonX-100for2.5min-
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) were
incubated overnight at 4°C. Washed twice with PBS, the cells
were then incubated for 1.5 hours with the secondary antibody
(Cy3-conjugated antimouse antibody was used to detect Lnk
protein, red, and fluorescein isothiocyanate (FITC)-conjugated
antirabbit antibody to detect IRS1 protein, green), at 1:200 in
5% BSA-PBS at 37°C. Washed 3 times with PBS, the cells were
then counterstained with Hoechst 33258 to stain the nuclei.
HEK293T cells were subsequently examined with confocal mi-
croscope (FV500; Olympus), and images were analyzed with
Metamorph version 6.3 software (Molecular Devices).
Statistical analysis
Data are expressed as the mean Ϯ SEM of at least 3 indepen-
dent experiments. The data were analyzed by 2-tailed Student’s
t test or one-way ANOVA followed by the Bonferroni multiple
comparison post hoc test with a 95% confidence interval. Sta-
tistical analyses were performed using PRISM version 6.0
(GraphPad Software). The correlation analyses were determined
by the Pearson correlation test. Statistical significance was con-
sidered to be P Ͻ .05.
Figure 1. Ovarian tissues from PCOS patients with IR exhibited higher expression of Lnk than the ovaries from control or PCOS patients
without IR. A, The mRNA expression level of Lnk in ovaries was detected in control (n ϭ 4) and PCOS with IR (n ϭ 4) or without IR (n ϭ 4)
patients according to the HOMA-IR (HOMA-IR defined as HOMA Ն 2.14). B–H, Quantitative real-time polymerase chain reaction validation
of the positive correlation between the level of Lnk mRNA and IR. B, Pearson correlation analysis showed a significant positive correlation of
Lnk vs HOMA-IR (Pearson r ϭ 0.672; P ϭ .0167, n ϭ 12). Line represents linear regression of data (y ϭ 0.01728xϩ0.01504; r2
ϭ 0.4515).
C, Pearson correlation analysis showed a significant positive correlation of Lnk vs BMI (Pearson r ϭ 0.702; P ϭ .0108, n ϭ 12). Line
represents linear regression of data (y ϭ 0.004983xϪ0.05860; r2
ϭ 0.4935). D, Pearson correlation analysis showed a significant positive
correlation of Lnk vs WHR (Pearson r ϭ 0.735; P ϭ .0065, n ϭ 12). Line represents linear regression of data (y ϭ 0.3296xϪ0.2252; r2
ϭ
0.5400). E, Pearson correlation analysis showed a significant positive correlation of Lnk vs 0-hour glucose (Glu) (Pearson r ϭ 0.791; P ϭ
.0022, n ϭ 12). Line represents linear regression of data (y ϭ 0.02953xϪ0.09443; r2
ϭ 0.6253). F, Pearson correlation analysis showed a
significant positive correlation of Lnk vs 2-hour Glu (Pearson r ϭ 0.731; P ϭ .0069, n ϭ 12). Line represents linear regression of data (y ϭ
0.009978xϪ0.02884; r2
ϭ 0.5340). G, Pearson correlation analysis showed a significant positive correlation of Lnk vs 0-hour insulin (INS)
(Pearson r ϭ 0.769; P ϭ .0035, n ϭ 12). Line represents linear regression of data (y ϭ 0.005166xϩ0.004672; r2
ϭ 0.5914). H, Pearson
correlation analysis showed a significant positive correlation of Lnk vs 2-hour Ins (Pearson r ϭ 0.5822; P ϭ .05, n ϭ 12). Line represents
linear regression of data (y ϭ 0.0003434xϩ0.01669; r2
ϭ 0.3309).

Results
The level of Lnk mRNA increases in the ovaries of
PCOS women with IR
Quantitative real-time polymerase chain reaction was
usedtoidentifythelevelofLnkmRNAinovaries.Ovarian
tissues from PCOS patients with IR exhibited higher ex-
pression of Lnk thanovaries from normal control and
PCOS patients without IR (Figure 1A). The difference be-
tween the PCOS/IR group and control group is significant
(P Ͻ .05), whereas the level of Lnk between PCOS group
and control group had no significance. Pearson correla-
tion analysis showed a significant positive correlation of
the level of Lnk mRNA vs HOMA-IR, BMI, WHR, 0-hour
glucose, 2-hour glucose, 0-hour insulin, and 2-hour insu-
lin (Figure 1, B–H).
The Lnk protein levels were significantly higher in
the group with PCOS/IR compared with the
normal control and PCOS without IR group
Immunohistochemistryofovariesfrompatientswithor
without PCOS/IR showed the expression of Lnk in the
ovaries from patients. There were more primary and sec-
ondary follicles, thickened follicles, and small vascular
structures in polycystic ovaries than normal ovaries. Lnk
was significantly increased in the ovaries in the group with
PCOS/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 the
secondary follicles (Figure 2A). The positive rates of Lnk
were significantly higher in the ovaries in the group with
PCOS/IR and PCOS without IR compared with that in the
normal control group (P Ͻ .05) (Figure 2B).
Lnk binds and colocalizes with insulin receptor
and IRS1
Insulin receptor and IRS1 binds with Lnk
Cell lysates from HEK293T cells cotransfected with
Lnk pcDNA 3.1 and GFP-tagged insulin receptor pcDNA
3.1 or pENTER IRS1-HIS were immunoprecipitated with
anti-Lnk, anti-GFP, or anti-HIS antibody. The pellet was
separatedbySDS-PAGEandsubjectedtoimmunoblotting
with anti-GFP or anti-Lnk antibody to assess the binding
of insulin receptor and IRS1 to Lnk. We found that Lnk
bound to insulin receptor (Figure 3, A and B) and IRS1
(Figure 3, C and D).
Colocalization of Lnk and insulin receptor/IRS1 were
tracked in cells with intracellular immunofluorescence
HEK293T cells were cotransfected with LNK pcDNA
3.1 and insulin receptor-GFP pcDNA 3.1 or pENTER
IRS1-HIS. Immunofluorescent staining of HEK293T cells
with FITC-conjugated antirabbit antibody (to detect
IRS1, green) and Cy3-conjugated antimouse antibody (to
detect Lnk, red) showed Lnk and insulin receptor-GFP
(Figure 3E) or IRS1-HIS (Figure 3F) were coexpressed in
HEK293 cells. The expression and position of Lnk, insulin
receptor, and IRS1 were observed with intracellular flu-
orescence under a confocal microscope. Lnk was mainly
expressed in plasma, around the nucleus, whereas insulin
receptor and IRS1 were both in plasma and nuclear loca-
tions. The intracellular localizations of Lnk and insulin
receptor/IRS1 overlapped around the nucleus.
Figure 2. Immunohistochemistry of ovaries from patients with or
without PCOS/IR. A, The expression of Lnk in ovaries from patients.
The positive rates of Lnk were significantly higher in the group with
PCOS and IR compared with the control group. Results show: con,
weak positive; PCOS, moderate positive; PCOSϩIR, strong positive.
Scale bar, 50 ␮m; n ϭ 6 per group. B, The expression of Lnk was
significantly higher in the PCOS/IR and PCOS group compared with the
control group; **, P Ͻ .05 vs control; ##, P Ͻ .05 vs PCOS.

Overexpression of Lnk inhibits the insulin
response PI3K-AKT and MAPK-ERK1/2 signaling
pathways in ovarian granulosa cells
Lnk inhibits phosphorylation of insulin receptor
(Tyr1150/1151) and IRS1 (Ser307)
We overexpressed Lnk in ovarian granulosa cells by
Lnk pcDNA 3.1 transfection. Five minutes after insulin
addition, there was a significant increase in insulin recep-
tor tyrosine and IRS1 serine phosphorylation levels. Lnk
overexpression affected insulin receptor autophosphory-
lation induced by insulin. Insulin-dependent tyrosine
phosphorylation 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 by
insulin (100nM) were time dependent (Figure 4E). The
basal activations of Akt and ERK1/2 were inhibited by
Lnk in granulosa cells with 100nM insulin for 30 minutes
(Figure 4F). Thr308 phosphorylation level of Akt and
Thr202/Tyr204 phosphorylation of ERK1/2 were all fur-
ther inhibited by Lnk overexpression (P Ͻ .01). The total
levels of Akt and ERK Akt were not affected by these
treatments.
Discussion
In this study, we demonstrated that Lnk may play an im-
portant role in inhibiting insulin signaling in the ovary
during PCOS. The results showed that ovarian Lnk ex-
pression was increased in patients with IR-PCOS. We also
found that women with IR-PCOS had higher levels of
HOMA-IR, BMI, WHR, glucose, and insulin expression.
Lnk expression was positively correlated with HOMA-IR,
BMI, and WHR. Moreover, we found that overexpression
of 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 of
noninsulin-dependent diabetes mellitus (32). A study in
PCOS women found a significantly increased the preva-
lence of noninsulin-dependent diabetes mellitus and hy-
pertension especially in obese women (6). Approximately
50%–70% of women with PCOS have some degree of IR
(1, 33). IR also has been found in PCOS women of many
racialgroupsincludingJapanese,Caribbean,Chinese,and
AfricanAmericas(34).Ourclinicalstudiesshowthatmost
PCOS 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 tissues
such as liver, adipose tissue and muscle (35). It also has
nonclassic target tissues such as stromal and follicular
compartment (36). In the nonclassic target tissues, insulin
could stimulate steroidogenesis in ovarian cells because of
the prevalence of insulin receptor. Hyperinsulinemia in IR
alters the enzymes and proteins that are important for
steroidogenesis then affects follicle development and fer-
tility (37). This study mainly focused on investigating the
effectofIRongranulosacellsthatultimatelyaffectfertility
through folliculogenesis.
Figure 3. Lnk interacts with insulin receptor and IRS1. A, Coimmunoprecipitation of Lnk with insulin receptor. B, Coimmunoprecipitation of Lnk
with IRS1. HEK293T cells were cotransfected with LNK pcDNA 3.1 and IR-GFP pcDNA 3.1 or pENTER IRS1-HIS. C and D, Immunofluorescent
staining 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 association
studies have linked Lnk to hyperten-
sion, renal disease, and diabetes (21,
38). Lnk is an intracellular adaptor
protein that functions as negative
regulator in many signaling path-
ways (39). Mutations in Drosophila
Lnk produce a phenotype reminis-
cent of reduced insulin signaling dur-
ing development of female fertility
(8, 10). In this study, we provided the
first evidence that Lnk is an impor-
tant factor in PCOS patients with IR
and is associated with the insulin sig-
naling pathway in ovary tissue. In
addition, Lnk expression was signif-
icantly up-regulated in IR-PCOS and
also in granulosa cells treated with
insulin. Up-regulation of Lnk was
correlated with the severity of IR-
PCOS. These findings suggest that
up-regulation of Lnk contributes to
the development of PCOS and asso-
ciated IR.
Lnk has 20 known binding part-
ners that interact predominantly
through the pleckstrin homology and
SH2 domains to activate major sig-
naling pathways such as Janus kinase-
signal transducer and activator of
transcription and ERK1/2 (39). Lnk/
SH2B has potential tyrosine phos-
phorylation sites and functions as
protein-protein interaction motifs and
transduces signals downstream of a
number 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 insulin
induced insulin receptor tyrosine
phosphorylation and facilitated insu-
lin receptor serine phosphorylation.
We demonstrated that overexpression
of Lnk suppressed insulin induced
MAPK and Akt phosphorylation.
This study has some limitations. It
isworthnotingthat,inthisstudy,the
women with PCOS were diagnosed
according to the Rotterdam criteria
developed in 2003 because these are
the criteria used by the physicians in
Figure 4. Overexpression of Lnk impairs insulin signaling in ovarian granulosa cells. A, Time
course of insulin stimulated insulin receptor and IRS1 phosphorylation in ovarian granulosa cells.
Representative blots of phosphorylation of insulin receptor and IRS1 by 100nM insulin. Insulin
increased the phosphorylation of insulin receptor and IRS1 but did not alter the level of total
insulin receptor and IRS1. B, Representative blot of phosphorylation of insulin receptor and IRS1
in Lnk overexpression granulosa cells. Ovarian granulosa were transfected with Lnk pcDNA 3.1
for 48 hours and then starved for 12 hours with 1% FBS culture medium. After that, 100nM
insulin was added to the culture medium for 5 minutes. C and D, Densitometry analysis of
phosphorylation of insulin receptor and IRS1 in granulosa cells. The Tyr1150/1151
phosphorylation level of insulin receptor was inhibited by Lnk in ovarian granulosa cells; n ϭ 4;
*, P Ͻ .05 vs control; #, P Ͻ .05 vs insulin-treated control group by one-way ANOVA. E, Time
course 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 increases
the 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 granulosa
cells. Ovarian granulosa cells were transfected with Lnk pcDNA 3.1 for 48 hours and then starved
for 12 hours with 1% FBS culture medium. After that, 100nM insulin was added to the culture
medium for 30 minutes. G and H, Densitometry analysis of phosphorylation of Akt and ERK1/2 in
granulosa cells. The basal Thr308 phosphorylation level of Akt and Thr202/Tyr204
phosphorylation of ERK1/2 were all inhibited by Lnk in ovarian granulosa cells. After insulin
treatment, the phosphorylation level of Akt and ERK1/2 were all further inhibited; P Ͻ .01; n ϭ
4, *, P Ͻ .05 vs control; #, P Ͻ .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 now
define PCOS by updated criteria (40). The PCOS women
included in this study without IR might be classified as
having ovulatory dysfunction with polycystic ovarian
morphology but not hyperandrogenism, that is phenotype
D(2,40).TheBMIscoresofthestudypopulationwerenot
within the obese range; however, the mean WHRs of the
PCOS patients with or without IR was both more than or
equal to 0.8, which is a marker of Asian central obesity
(41). So further study is needed to understand whether
theseresultswouldtranslatetoWesternobesepopulations
becauseoftheinteractionbetweenandrogensBMIandIR.
In summary, our findings show that Lnk expression is
increased 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 Lnk
could 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 for
the treatment of IR-PCOS.
Acknowledgments
We thank Dr Philip H. Koeffler and Sigal Gery (Cedars-Sinai
Medical Center, the University of California, Los Angeles) for
their ideas and guidance of performing the experiments and their
helps 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: zhaoxiaomiao2015@sina.com; or
Dongzi Yang, MD, PhD, Department of Obstetrics and Gyne-
cology, Memorial Hospital of Sun Yat-Sen University, Guang-
zhou, 510120 China. E-mail: yangdz@mail.sysu.edu.cn; or
Meiqing Xie, MD, PhD, Department of Obstetrics and Gyne-
cology, Memorial Hospital of Sun Yat-Sen University, Guang-
zhou, 510120 China. E-mail: mqxiegz@163.com.
This work was supported by National Natural Science Foun-
dation General Program Grants 81471425 and 81100402; the
Science Technology Research Project of Guangdong Province Grants
2013B022000016, 2014A020213006, and 2015A030313091; and
the Sun Yat-Sen scholarship for young scientist.
Disclosure Summary: The authors have nothing to disclose.
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