Aberrant Expression of Fbxo2 Disrupts Glucose Homeostasis ...

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Aberrant expression of FBXO2 disrupts glucose homeostasis through

ubiquitin-mediated degradation of insulin receptor in obese mice

Bin Liu1 *

, Han Lu2 *

, Duanzhuo Li1, Xuelian Xiong

3,

Lu Gao4, 5

, Zhixiang Wu6, and Yan Lu

1, 3

1 Hubei Key Laboratory for Kidney Disease Pathogenesis and Intervention,

Huangshi Cental Hospital of Edong Healthcare Group, Hubei Polytechnic

University School of Medicine, Huangshi, Hubei 435003, PR China.

2 Department of Anesthesiology, Ruijin Hospital, Shanghai Jiao-Tong

University School of Medicine (SJTU-SM), Shanghai 200025, PR China.

3 Department of Endocrinology and Metabolism, Zhongshan Hospital,

Fudan University, Shanghai 200032, PR China.

4 College of Life Sciences, Northeast Agricultural University, No.59 Mucai

street, Harbin 150030, Heilongjiang, PR China.

5 Department of Pathology, University of Maryland School of Medicine,

655W. Baltimore Street, Baltimore, MD, 21202-1192,USA.

6 Department of Pediatric Surgery, Xinhua Hospital, Shanghai Jiao-Tong

University School of Medicine (SJTU-SM), Shanghai 200092, PR China.

* Co-first author

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Corresponding authors

Dr. Lu Gao, College of Life Sciences, Northeast Agricultural University,

No.59 Mucai street, Harbin 150030, Heilongjiang, PR China.

Tel: +86-045155191257

Fax: +86-045155191257

E-mail: [email protected]

Dr. Zhixiang Wu, Department of Pediatric Surgery, Xinhua Hospital,

Shanghai Jiao-Tong University School of Medicine (SJTU-SM), 1665

Kongjiang Road, Shanghai 200092, PR China.

Tel: +86-21-25078413,

Fax: +86-21-65791316,


Dr. Yan Lu, Department of Endocrinology and Metabolism, Zhongshan

Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, PR China.

Tel: +86-21-64041990

Fax: +86-21-64041990


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Abstract

Insulin resistance is a critical factor in the development of metabolic

disorders, including type 2 diabetes (T2DM). However, its molecular

mechanisms remain incompletely understood. In the present study, we

found that F-box only protein 2 (FBXO2), a substrate recognition

component of SKP1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase

complex, were up-regulated in livers of obese mice. Furthermore, using a

protein purification approach combined with high performance liquid

chromatography/tandem mass spectrometry (HPLC/MS/MS), we carried

out a system-wide screening of FBXO2 substrates, in which insulin

receptor (IR) was identified as a substrate for FBXO2. SCFFBXO2

acts as an

E3 ligase targeting the IR for ubiquitin-dependent degradation to

regulate insulin signaling integrity. As a result, adenovirus-mediated

overexpression of FBXO2 in healthy mice led to hyperglycemia, glucose

intolerance and insulin resistance, while ablation of FBXO2 alleviated

diabetic phenotypes in obese mice. Therefore, our results identify

SCFFBXO2

as an E3 ligase for IR in the liver, which might provide a novel

therapeutic target for treating T2DM and related metabolic disorders.

Key Words: Type 2 diabetes, Insulin resistance, Insulin signaling,

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Ubiquitination, Protein degradation,

Type 2 diabetes mellitus (T2DM), characterized by high blood glucose

levels, has become a pandemic problem worldwide. Usually,

hyperglycemia is caused by deficiency of insulin secretion and/or

reduced insulin sensitivity. In peripheral tissues, including liver, skeletal

muscle and adipose tissue, insulin binds to its receptor (IR), which then

phosphorylates and recruits insulin receptor substrates (IRS) to further

activate downstream signaling pathways (1). In the liver, the major node

of insulin signaling is activation of phosphoinositide-3-kinase (PI3K)/AKT,

which in turn inhibits the expression of phosphoenolpyruvate

carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), two key

gluconeogenic enzymes (2). As a result, hepatic insulin resistance is

characterized by excessive hepatic glucose production, contributing to

fasting hyperglycemia in T2DM (3). Therefore, identification of novel

molecules involved in regulating the hepatic insulin signaling pathway

will advance our understanding of the pathogenesis that leads to T2DM.

Polyubiquitination is the formation of an ubiquitin chain on a single

lysine residue on the substrate protein, leading to protein degradation

(4). It is carried out by a three-step cascade of ubiquitin-transfer

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reactions: activation, conjugation, and ligation, performed by

ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s),

and ubiquitin ligases (E3s), respectively (5). The largest subfamily of E3s

in mammalian is the Skp1-Cul1-F box protein ubiquitin ligases (SCFs),

which consist of Skp1, Cul1, Rbx1, and one of F box proteins (FBPs) (6).

Recent studies have shown that FBPs play a crucial role in many

biological events, such as inflammation, cell-cycle progression and

tumorigenesis, through ubiquitin-mediated degradation of cellular

regulatory proteins (7; 8). Besides, their dysregulation has been

implicated in several pathologies (6-8), suggesting that insights into

SCF-mediated biology may provide potential strategies to treat human

diseases. However, until now, whether FBPs play a role in the metabolic

diseases, especially insulin resistance and T2DM, remains poorly

understood.

Research design and Methods

Animal experiments

Male C57BL/6 and db/db mice aged 8-10 weeks were purchased from

the Shanghai Laboratory Animal Company (SLAC) and Nanjing Biomedical

Research Institute of Nanjing University, respectively. JNK1 knockout

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mice were obtained from Jackson Laboratories and backcrossed to

C57BL/6 background for 6 generations. All mice were housed at 21°C ±

1°C with humidity of 55% ± 10% and a 12-hour light/12-hour dark cycle.

HFD-induced obese mice were maintained with free access to high-fat

chow (D12492; Research Diets) containing 60% kcal from fat, 20% kcal

from carbohydrate, and 20% kcal from protein. For the depletion of

Kupffer cells, C57BL/6 mice were fed with a high-fat-diet for 12 weeks

and then injected with gadolinium chloride (GdCl3, 10 mg/kg, twice each

week) or sodium chloride (NaCl) by tail vein for another 2 weeks. All

study protocols comply with guidelines and institutional policies

prepared by the Animal Care Committee of Shanghai Jiao Tong University

School of Medicine.

Immuoprecipitation (IP) and in-solution digestion

The standard IP purification procedure has been previously described (9).

In brief, HEK293T cells stably expressing Flag-tagged wild-type or mutant

FBXO2 were lysed in 5 mL lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM

NaCl, 0.5% Nonidet P40, and 100mM PMSF) for 20 min with gentle

rocking at 4 °C. Lysates were cleared and subjected to IP with 50 μL of

anti-FLAG M2 beads overnight at 4 °C. Beads containing immune

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complexes were washed with 1 mL ice cold lysis buffer. Proteins were

eluted with 100 μL 3 × Flag-peptide (Sigma-Aldrich, St. Louis, MO, USA)

in TBS for 30 min and precipitated with cold acetone. The precipitated

proteins were in-solution digested with trypsin, and the tryptic peptides

were vacuum centrifuged to dryness for further analysis.

HPLC/MS/MS analysis

Nanoflow LC-MS/MS was performed by coupling an Easy nLC 1000

(Thermo Fisher Scientific, Waltham, MA) to an Orbitrap Fusion mass

spectrometer (Thermo Fisher Scientific, Waltham, MA). Tryptic peptides

were dissolved in 20 µL of 0.1% formic acid, and 10 µL were injected for

each analysis. Peptides were delivered to a trap column (2 cm length

with 100 µm inner diameter, packed with 5 µm C18 resin) at a flow rate

of 5 µL/min in 100% buffer A (0.1% FA in HPLC grade water). After 10 min

of loading and washing, the peptides were transferred to an analytical

column (17 cm× 79 μm, 3-μm particle size, Dikma, China) coupled to Easy

nLC 1000 (Thermo Fisher Scientific, Waltham, MA). The separated

peptides were ionized using NSI source, then analyzed in an Orbitrap

Fusion mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with

a top speed 3s data-dependent mode. For MS/MS scan, ions with

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intensity above 5,000 and charge state 2-6 in each full MS spectrum

were sequentially fragmented by Higher Collision Dissociation with

normalized collision energy of 32%. The dynamic exclusion duration was

set to be 60 s, and the precursor ions were isolated by quadrupole with

isolation window 1 Da. The fragment ions were analyzed in ion trap with

AGC 7,000 at rapid scan mode. The raw spectra data was processed by

protein discover and MS/MS spectra data was searched against the

Uniprot human database (88,817 sequences) by Mascot (v.2.4, Matrix

Science, London, UK)

Bioinformatics analysis

The molecular function, cellular component analysis of the glycoproteins

was performed using Database for Annotation, Visualization and

Integrated Discovery Bioinformatics Database (DAVID 6.7) (10; 11).

Glucose and insulin tolerance tests

Glucose tolerance tests were performed by intraperitoneal injection of

D-glucose (Sigma-Aldrich, USA) at a dose of 2.0 mg/g body weight after a

16-hour fast. For insulin tolerance tests, mice were injected with regular

human insulin (Eli Lily, Indianapolis, Indiana, USA) at a dose of 0.75 U/kg

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body weight after a 6-hour fast. Blood glucose was measured by a

portable blood glucose meter (Lifescan, Johnson & Johnson, New

Brunswick, New Jersey, USA).

Western blots

Hepatic tissues or cells were lysed in radioimmunoprecipitation buffer

containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1

mM NaF, 1% NP-40, and 0.1% sodium dodecyl sulfate. Western blots

were performed using antibodies against FBXO2 (ab133717; Abcam), IRβ

(ab131238; Abcam), AKT (13038, 4821; Cell Signaling) and GAPDH (5174;

Cell Signaling). Analysis of tyrosine phosphorylation of IRS1 was

performed by immunoprecipitation of IRS1 with anti-IRS1 from total

lysate, followed by western blot with anti-pTyr antibody (PY100).

Luciferase reporter and Chromatin immunoprecipitation assays

All the transient transfections were conducted using Lipofectamine 2000

(Invitrogen, Shanghai, China). The FBXO2 promoter was amplified from

the mouse genomic DNA templates and inserted into pGL4.15 empty

vector (Promega). Luciferase activity was measured using the

Dual-Luciferase Reporter Assay System (Promega). For chromatin

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immunoprecipitation (ChIP) assays, a commercial kit was employed

(Upstate, Billerica, Massachusetts, USA). In short, MPHs were fixed with

formaldehyde and chromatin was incubated and precipitated with

antibodies against p65 (ab16502, Abcam), or control IgG (ab172730,

Abcam). DNA fragments were subjected to real-time PCR using primers

flanking NF-κB binding site in the FBXO2 promoter. The primer

sequences are listed below: Forward (5’-ACCAGCGCGACGCGG

TATGGGA-3’), Reverse (5’-TGGGGCAGCCGGACTAAAAGCT-3’).

Statistical analysis

Values were shown as mean ± SEM. Statistical differences were

determined by a Student t test. Statistical significance is displayed as

*P<0.05, ** P<0.01 or *** P<0.001.

Results

Up-regulation of FBXO2 in livers of obese mice

To identify genes that are differentially expressed in obesity, we

previously performed a clustering analysis of Affymetrix arrays, which

showed that a large number of mRNAs were markedly dys-regulated in

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the liver of mice fed a high-fat-diet (HFD) compared with mice fed a

normal chow diet (ND) (12; 13). Here, we describe work on the FBPs.

There are more than 70 FBPs in mammals (6). Our data showed that 11

FBPs were significantly changed (P < 0.05), of which 8 were increased

and 3 were decreased (Supplementary Table 1). Here, FBXO2 was chosen

for further experiments, since its expression was enriched in the liver

and hepatocytes (Supplementary Fig. 1A-B). In contrast, its expression in

other tissues, including skeletal muscle, white adipose tissue, heart and

kidney, was relatively low (Supplementary Fig. 1A). Increased mRNA and

protein expression of FBXO2 in HFD-fed mice was further confirmed by

quantitative real-time PCR (qPCR) and western blots (WB), respectively

(Fig. 1A-B). The upregulation of FBXO2 was also detected in the livers of

db/db mice (Fig. 1C-D), a well-established genetic model of T2DM,

suggesting that abnormal expression of FBXO2 represents a typical

feature of insulin resistance in obese animals.

Identification of insulin receptor as a novel substrate for FBXO2

FBXO2 was shown to preferentially target N-linked high mannose

oligosaccharides in glycoproteins for ubiquitination and degradation (14).

The F-box associated (FBA) domain of FBOX2 is essential for its activity of

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recognizing glycoprotein, which is completely abolished by mutations of

two residues (15; 16). In order to systematically identify the

FBXO2-interacting proteins, HEK293T cells were transfected with

retroviruses expressing Flag-tagged wild-type (WT) FBXO2 or a FBA

domain mutant (MUT), which could not recognize glycoprotein as

previously described (15; 16). Immunoprecipitation (IP) against Flag was

subsequently performed with the lysates of cells carrying WT or MUT

FBXO2 proteins, respectively. As depicted in Supplementary Fig. 2, the

whole purification procedures were monitored by Coomassie Brilliant

Blue staining as well as western blots with anti-Flag antibody, showing

that both WT and MUT FBXO2 proteins were highly enriched in the final

elution fraction. Consistent with previous results (16), the concanavalin

(ConA)-positively signals were dramatically accumulated in WT final

elution fraction, but not MUT. The final immunoprecipitates from WT

and MUT cells were further subjected to mass spectrometry analysis.

Protein identification was carried out using Mascot software and

identified proteins filtered with overall false discovery rate < 0.01% were

considered as potential interacting candidates. Using these criteria, we

finally identified 2643 proteins from WT samples and 1138 proteins from

MUT samples (Supplementary Table 2). To exclude the unspecific binding,

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we then focused on the proteins that were exclusively identified in WT

cells, resulting in 1569 potential substrates. Importantly, by comparing

with the Uniprot database, we found that more than one third of these

proteins (528, 33.7%) were glycoproteins. In contrast, only 82 (7.6%)

proteins from MUT elutes were classified as glycoproteins in Uniprot

database (Fig. 2A). Together, our data found that a significant enrichment

of glycoproteins interacted with WT but not MUT FBXO2. Interestingly,

the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway showed

that part of these glycoproteins was involved in N-Glycan biosynthesis

and oxidative phosphorylation, suggesting a potential role for FBXO2 in

energy metabolism (Fig. 2B and Supplementary Table 3). Bioinformatics

analysis further showed that these glycoproteins were highly enriched in

membrane, endoplasmic reticulum and lysosome (Fig. 2C and

Supplementary Table 3). Given the relevance of FBXO2 in obese animals,

we questioned if any molecules involved in the insulin signaling pathway

are potential substrates of FBXO2. Intriguingly, we found that insulin

receptor (IR), a large transmembrane glycoprotein containing multiple

N-linked glycosylation sites (17; 18), was only co-eluted with WT but not

MUT FBXO2 in two replicates (Fig. 2D).

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FBXO2 negatively regulates the stability of insulin receptor

Next, we confirmed the specific interaction between FBXO2 and IR in

transiently transfected HEK293T cells using co-immunoprecipitations (Fig.

3A). The endogenous interaction of these two proteins was also detected

in mouse primary hepatocytes (MPHs) (Fig. 3B). As FBXO2 could interact

with IR, we tested if FBXO2 could regulate IR stability or accelerate its

protein degradation. Indeed, endogenous IR protein contents were

dramatically decreased in MPHs transfected with adenovirus expressing

FBXO2 (Fig. 3C), while its mRNA levels remained unchanged (Fig. 3D).

Besides, protein abundance of IRS-1, IRS-2, Glut1 and Glut4 were not

affected by FBXO2 overexpression (Fig. 3C). IGF1 receptor, which is

closely related to the insulin receptor and has overlapping functions, was

slightly reduced, suggesting the specificity of FBXO2-induced IR

degradation (Fig. 3C). The ubiquitination of IR was also increased by

ectopic expression of FBXO2 in MPHs treated with MG132, a proteasome

inhibitor (Fig. 3E). Furthermore, overexpression of FBXO2 reduced the

half-life of IR to less than 2 hour (Fig. 3F), supporting the notion that

FBXO2 could regulate IR stability and promote its degradation. In

agreement, post-transcriptional downregulation of hepatic IR was also

observed in obese mice (Supplementary Fig. 3A-D).

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Moreover, insulin inhibited dexamethasone/foskolin-induced

glucose production, which was largely attenuated by overexpression of

FBXO2 (Fig. 4A). In agreement, FBXO2 expression also blocked the

suppressive effects of insulin on dexamethasone/foskolin-induced

expression of gluconeogenic enzymes (PEPCK and G6Pase) (Fig. 4B). In

addition, FBXO2-induced downregulation of IR protein was attenuated

by MG132 (a proteasome inhibitor), but not Leupeptin (an inhibitor of

lysosomal protease) (Fig. 4C). MG132 treatment also restored

insulin-suppressed glucose production and gluconeogenic genes

expression (Fig. 4D-E), indicating the involvement of the proteasome

system in FBXO2-mediated inhibition of insulin signaling.

Liver-specific overexpression of FBXO2 promotes hyperglycemia and

insulin resistance

To investigate the role of FBXO2 in regulating insulin signaling in vivo,

FBXO2 or GFP adenovirus was delivered into C57BL/6 mice via tail-vein

injection. As shown in Fig. 5A, protein level of FBXO2 was dramatically

increased while IR was decreased in the liver, but not in other tissues,

including white adipose tissues and skeletal muscles (data not shown).

Overexpression of hepatic FBXO2 did not affect body weight and food

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intake (Supplementary Fig. 4A-B), but significantly increased circulating

levels of glucose and insulin, indicating of insulin resistance (Fig. 5B-C). A

dramatic reduction in insulin sensitivity was also revealed by glucose and

insulin tolerance tests (Fig. 5D). These changes was accompanied at a

molecular level by phosphorylation of insulin receptor substrate 1

(p-IRS1) and AKT (p-AKT), two crucial molecules in the insulin-signaling

pathway, in response to acute intraperitoneal insulin injection (Fig. 5E).

Moreover, the mRNA expression of PEPCK and G6Pase was up-regulated

by FBOX2 overexpression (Fig. 5F).

Ablation of FBXO2 enhances insulin sensitivity in db/db mice

To further confirm the effects of FBXO2 in an independent setting, we

disrupted its expression in the liver of db/db mice by delivering

adenoviruses expressing FBXO2-specific shRNA or a nonspecific control

shRNA. FBXO2 shRNA treatment significantly reduced hepatic FBXO2

protein levels and increased IR protein expression compared with

negative control shRNA-injected littermates (Fig. 6A). As a result, loss of

FBXO2 dramatically improved hyperglycemia, hyperinsulinemia, glucose

tolerance and insulin resistance (Fig. 6B-D). The well-improved insulin

signaling and down-regulation of gluconeogenic enzymes were also

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observed in db/db mice with FBXO2 deficiency (Fig. 6E-F). Similar effects

on glucose homeostasis were also observed in HFD-induced obese mice

transduced with FBXO2 shRNA (Supplementary Fig. 5A-D), suggesting

that knockdown of FBXO2 in the liver could alleviate diabetic phenotype

in obese mice.

Regulation of hepatic FBXO2 in obesity

The results above demonstrated that FBXO2 was upregulated in obese

livers, and manipulation of FBXO2 could modulate insulin sensitivity.

Finally, we sought to determine the signaling pathway that regulates

FBXO2 expression. T2DM is tightly associated high circulating levels of

glucose, fatty acids, insulin and pro-inflammatory cytokines. Therefore,

we performed a screen to assess whether these cellular factors and

hormones could affect FBXO2 expression. As a result, TNF-α and IL-1β,

but not high glucose, fatty acids, insulin or dexamethasone, induced

FBXO2 expression in MPHs (Fig. 7A, Supplementary Fig. 6A-D), suggesting

that inflammation might be responsible for the up-regulation of FBXO2

in obese mice. To confirm this point, we deleted Kupffer cells in HFD

mice by administration of gadolinium chloride (Gdcl3) (19). Consistent

with previous reports that Kupffer cells are the primary source for the

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hepatic inflammation in obesity (19-21), Gdcl3 treatment significantly

reduced expression of pro-inflammatory markers including TNFα, IL-1β

and F4/80 in liver tissues (Supplementary Fig. 6E). Under this condition,

there was a marked decrease in FBXO2 expression in the liver of obese

mice compared with controls (Fig. 7B).

Growing evidence has noted the roles of inflammation-mediated

JNK1 and IKKβ/NF-κB signaling pathways on the regulation of liver

metabolic homeostasis (20; 22-24). Hence, it is interesting to decide

whether JNK1 and/or IKKβ/NF-κB activation may underlie the

up-regulation of FBXO2. As shown in Supplementary Fig. 7A, FBXO2

mRNA expression showed similar changes after TNFα treatment in JNK1

knockout MPHs compared with JNK1 wild-type MPHs, suggesting that

JNK1 might not be essential for the regulation of FBXO2 expression. In

agreement, the induction of FBXO2 was largely blocked by BAY 11-7082

(NF-κB inhibitor), but not SP600125 (JNK inhibitor) or U0126 (ERK

inhibitor) (Supplementary Fig. 7B), suggesting that the canonical

IKKβ/NF-κB pathway mediates the effects of pro-inflammatory cytokines

to induce FBXO2 expression.

Next, we speculate that FBXO2 is a molecular target of IKKβ/NF-κB.

To do this, we examined the promoter region of FBXO2 and found that a

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canonical NF-κB-DNA-binding motif (5’-GGGRNNYYCC-3’) exists in the

proximal promoter region of FBXO2 gene (Fig. 7C). We then created

luciferase plasmids controlled by FBXO2 promoter, and found that IKKβ

increased the transcriptional activities of these promoters when

transfected into HEK293T cells (Fig. 7D). On the other hand, mutagenesis

of the NF-κB-DNA-binding motif abrogated the effect of IKKβ/NF-κB in

activating the transcriptional activities of these promoters (Fig. 7D).

Similarly, inhibition of NF-κB activation, by BAY 11-7082, abolished the

TNFα-induced activity of FBXO2 promoter (Fig. 7E), further suggesting

that hepatic inflammation regulates FBXO2 through the NF-κB signaling.

The association of p65 with FBXO2 promoter was also confirmed by

chromatin immunoprecipitation assays (Fig. 7F). Taken together, we

speculate that chronic hepatic inflammation-mediated IKKβ/NF-κB

activation may be an important mechanism leading to upregulation of

FBXO2 in obesity.

Discussion

Via the Cre-loxP system, previous studies have created mice with

tissue-specific disruption of the IR gene. Intriguingly, hyperglycemia and

insulin resistance were only exhibited in liver-specific IR knockout mice,

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but not in skeletal muscle- or fat-specific IR knockout mice (25-27),

suggesting that a critical role of hepatic IR in regulating glucose

homeostasis and insulin sensitivity. Although down-stream signaling

pathways of insulin have been well-established, molecular determinants

that directly regulate IR expression remain poorly elucidated. In the

present study, we provide in vitro and in vivo evidence showing a critical

role of FBXO2 as a post-transcriptional regulator of hepatic insulin

signaling. Firstly, a protein purification approach combined with

HPLC/MS/MS assay was used to identify IR as a novel interacting protein

of FBXO2, which was further confirmed by co-immunoprecipitation

assays. FBXO2 interacts with IR to enhance its ubiquitination-mediated

protein degradation. Secondly, the physiological role of FBXO2 is further

revealed by both gain-of-function and loss-of-function studies in mice.

Overexpression of FBXO2 in the liver led to hyperglycemia,

hyperinsulinmia, glucose intolerance and insulin resistance in healthy

mice, while selective knockdown of FBXO2 in obese mice improved these

symptoms. Thirdly, FBXO2 was up-regulated in obese livers, suggesting

that inhibiting the expression or activity of FBOX2 might represent a

potential therapeutic target for enhancing insulin sensitivity.

Decades of years ago, several studies have reported the abnormal

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number and function of IR in various tissues of insulin-resistant mice,

including liver, adipose tissue, skeletal muscle, leukocytes and

endothelial cells, while its mRNA levels are not decreased (28-31). These

results suggest that the low receptor number could be due to

post-transcriptional levels. Indeed, it has been shown that protein

expression of IR could be targeted and inhibited by several MicroRNAs in

adipocytes, heart and liver (32-34). Besides, Song et al. demonstrated

that IR is ubiquitinated by Mitsugumin 53 (MG53) in skeletal muscle

because that IR ubiquitination and insulin-elicited downstream signaling

are inversely changed in MG53 transgenic mice and MG53 knockout

mice (35). Moreover, a recent study identified nuclear ubiquitous casein

and cyclin-dependent kinase substrate (NUCKS) as a regulator of IR

expression thereby regulating energy homeostasis and glucose

metabolism (36). Therefore, together with these studies, molecular

interventions that selectively increase IR expression might provide an

attractive avenue to treat with T2DM. Although both we and other group

found that proteasome inhibitor administration could efficiently prevent

the degradation of IR by different E3 ligases (35), it remains unclear how

IR gets into the proteasome for degradation. Moreover, our

bioinformatics analyses showed that the glycoproteins interacted

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exclusively with FBXO2WT were highly enriched in membrane,

endoplasmic reticulum and lysosome, suggesting others membrane

glycoproteins might also be ubiquitinated by FBXO2. Membrane proteins

are subject to a complex series of sorting, trafficking, quality control, and

quality maintenance systems, which are largely controlled by

ubiquitination (37). Besides, retrotranslocation of misfolded membrane

proteins from the endoplasmic reticulum (ER) into the cytoplasm and

processive cleavage by the 26S proteasome also participates in the

ubiquitination-mediated degradation (38). Interestingly, it has been

reported that FBXO2 ubiquitinates N-glycosylated proteins that are

translocated from the ER to the cytosol and functions in ER-associated

degradation pathway (14). Therefore, the degradation of IR might take

place in the ER via retrotranslocation, which needs to be determined in

the future studies.

In addition, our data indicate that aberrant expression of FBXO2 is

attributed to, at least in part, activation of IKKβ/NF-κB by

pro-inflammatory factors. Numerous studies have demonstrated that

low-grade and chronic inflammation plays a positive role in the glucose

intolerance and insulin resistance seen in obesity (39). While several

potential mechanisms have been proposed (39), our results may provide

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a novel insight whereby inflammation inhibits hepatic actions of insulin.

In addition, whether FBXO2 expression could be regulated by other

factors, such as ER stress and autophagy, remains to be determined.

Taken together, for the first time to our knowledge, we identified

FBXO2 as a functional E3 ligase for IR in the liver. Several recent reports

have shown that FBXO2 plays an important role in brain by controlling

the abundance of NMDA receptor and amyloid precursor protein (40; 41).

However, its role in other biological events remains largely unexplored.

Therefore, future studies directed at understanding its tissue-specific

downstream targets are still needed.

Acknowledgements We are grateful to Professor Xiaoying Li from

(Zhongshan Hospital, Fudan University, Shanghai) for helpful discussion

of the manuscript. This study is supported by grants from the Natural

Science Foundation of China (Nos 81402478, 31401185 and 81570769),

Shanghai Rising-Star Program (No.16QA1402900) and Research

Foundation of Hubei Polytechnic University for Talented Scholars (No.

9666).

Author Contributions

ZW and YL conceived the research ideas, supervised the project and

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wrote the manuscript. BL, HL and LG performed animal and cellular

experiments and analyzed the data. DL and XX provided technical advice

on the cellular studies. YL is the guarantor of this work and, as such, had

full access to all the data in the study and take responsibility for the

integrity of the data and the accuracy of the data analysis.

Conflict of interest

The authors have declared that no conflict of interest exists.

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Figure legends

Figure 1 FBXO2 expression in the liver.

(A-B) Relative mRNA and representative protein levels of FBXO2,

determined by quantitative real-time PCR and western blot, in livers of

C57BL/6 mice. 8-week-old mice were fed ND or HFD for 12 weeks. (n = 6).

(C-D) Hepatic mRNA and protein levels of FBXO2 in db/db mice. (n=8).

Figure 2 Identification of IR as a novel interacting protein for FBXO2.

(A) Venn diagram of the proteins identified from wild-type (WT) and

mutant (MUT) FBXO2 interacting proteins.

(B) KEGG analysis of the glycoproteins exclusively identified from cells

overexpressing WT FBXO2.

(C) Gene ontology analysis of the glycoproteins exclusively identified

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from cells overexpressing WT FBXO2.

(D) Spectra-counting based quantification analysis of IR protein from WT

and MUT FBXO2 interacting proteins. R1 and R2 represent two

replicates.

Figure 3 FBXO2 negatively regulates the stability of IR.

(A) Western blots of coimmunoprecipitated FBXO2 from HEK293T cells

transfected with Flag-tagged FBXO2 and HA-tagged IR. Cells were

pretreated with MG132 for 4 hr.

(B) FBXO2 was immumoprecipitated from mouse primary hepatocytes

(MPHs) using anti-FBXO2 or IgG antibody. Whole-cell extracts and

immunoprecipitations were separated by SDS-PAGE and immunoblotted

for the proteins indicated.

(C) Endogenous protein expression of IR, IRS-1, IRS-2, Glut1, Glut4 and

IGF1R were determined in MPHs overexpressing FBXO2 or GFP for 48 hr.

(D) Relative mRNA level of IR in MPHs.

(E) IR ubiquitination in MPHs overexpressing FBXO2 or GFP. Cells were

pretreated with MG132 for 4 hr.

(F) Time course of IR levels in cycloheximide (CHX)-treated MPHs with or

without FBXO2 overexpression, with quantification shown on the right.

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Figure 4 The inhibitory effects of insulin on glucose production and

gluconeogenic gene expression are blocked by FBXO2 overexpression

(A-B) Glucose production (A) and genes expression (B) in MPHs

overexpressing FBXO2 or GFP. The effects of insulin on

cAMP/Dex-induced glucose production were measured with a

colorimetric glucose assay kit. The mRNA expression of PEPCK and

G6Pase was quantified by real-time PCR.

(C) Representative protein levels of IR and FBXO2 in MPHs

overexpressing FBXO2 or GFP. Cells were treated with MG132 or

Leupeptin for 6 hr before harvest.

(D-E) Relative glucose production (D) and genes expression (E) in MPHs.

Cells were treated with MG132 for 6 hr before harvest.

Figure 5 Overexpression of FBXO2 impairs the hepatic actions of insulin

and induces hyperglycemia in C57BL/6 Mice.

(A) Representative western blots showing protein levels of FBXO2 in the

liver of C57BL/6 mice at day 14 after infection with adenoviruses

encoding FBXO2 or GFP control.

(B-D) Blood glucose (B) and insulin (C) levels, glucose and insulin

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tolerance tests (D) in C57BL/6 mice. Data were obtained on day 5 (B, C),

day 8 (D, GTT) and day 11 (D, ITT) after virus administration. For insulin

levels, aliquots of blood (30 µL) were collected at 9:00AM from

individual mice. (n = 8).

(E) Phosphorylation of IRS-1 and AKT in response to acute insulin

injection in C57BL/6 mice. Mice were fasted overnight and injected

intraperitoneally with insulin (0.75 U insulin/kg body weight) or saline.

10 min after injection, liver tissues were harvested for homogenization.

(F) Relative mRNA levels of PEPCK and G6Pase from two groups of mice.

(n = 8).

Figure 6 Knockdown of FBXO2 alleviates diabetic phenotype in db/db

obese mice.

(A) qPCR and western blot analysis to detect the mRNA and protein

levels of IR and FBXO2 in the liver of db/db mice at day 15 after infection

with adenoviral FBXO2 shRNA or LacZ shRNA. (n = 8-9).

(B-D) Blood glucose (B) and insulin (C) levels, glucose and insulin

tolerance tests (D) in db/db mice. Data were obtained on day 5 (B, C), day

8 (D, GTT) and day 12 (D, ITT) after virus administration. (n = 8-9).

(E) Phosphorylation of IRS-1 and AKT in response to acute insulin

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injection in db/db mice. Mice were fasted overnight and injected

intraperitoneally with insulin (0.75 U insulin/kg body weight) or saline for

10 min.

(F) Relative mRNA levels of PEPCK and G6Pase from two groups of db/db

mice. (n = 8-9).

Figure 7 Regulation of FBXO2 by activation of IKKββββ/NF-κκκκB pathway.

(A) Relative mRNA levels of FBXO2 in MPHs treated with TNFα (10 ng/ml)

or IL-1β (10 ng/ml), for the indicated time.

(B) Relative mRNA and representative protein levels of FBXO2 in HFD-fed

mice. Mice were fed with high-fat-diet for 12 weeks and then treated

with GdCl3 or NaCl for another 2 weeks. (n = 6).

(C) Proximal promoter region of mouse FBXO2 gene contains a potential

binding site for NF-κB.

(D-E) Luciferase reporter assays. HEK293T cells were transfected with

luciferase reporter plasmids containing wild-type (WT-Luc) or mutant

(Mut-Luc) binding site of NF-κB. Cells were treated with vehicle control

(DMSO) or BAY 11-7082, an inhibitor of NF-κB activation.

(F) Chromatin immunoprecipitation assays showing representative p65

binding to the FBXO2 promoter in MPHs. Cells were treated with TNFα

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or PBS for 2 hr and then subjected to ChIP assays.

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Online Appendix

Supplementary Figure 1

(A) Relative mRNA levels of FBXO2 in the liver, skeletal muscle (SM),

white adipose tissue (WAT), heart and kidney from C57BL/6 mice. n=4.

(B) Real-time PCR quantification of FBXI2 in the hepatocyte versus

non-hepatocyte fraction of C57BL/6 mice. The expression of PEPCK was

used as a positive control. n=4.


Purification of the protein complexes from HEK293T cells stably

expressing WT and MUT FBXO2. The whole purification procedures were

monitored by Coomassie Brilliant Blue (CBB) staining as well as Western

blots with anti-Flag antibody. The glycoproteins were visualized with

ConA-HRP.


(A-D) Relative mRNA and representative protein levels of IR in the liver

from lean and obese mice. n=6-8.


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(A-B) Body weight (A) and food intake (B) in C57BL/6 mice administrated

with adenoviruses expressing FBXO2 or GFP.


(A-D) Glucose (A) and insulin tolerance tests (B), blood glucose (C) and

insulin (D) levels in HFD-induced obese mice administrated with

adenoviral FBXO2 shRNA or LacZ shRNA. (n = 6-7).


(A-D) Relative mRNA levels of FBXO2 in MPHs treated with high glucose

(25mM), palmitate (100µM), insulin (100nM) and dexamethasone

(100nM) for the indicated time.

(E) Relative mRNA levels of TNFα, IL-1β and F4/80 in HFD mice treated

with GdCl3 or NaCl.


(A) Relative mRNA levels of FBXO2 in JNK1 wild-type (WT) or knockout

(KO) MPHs. Cells were treated with TNFα or PBS for 6 hr.

(B) Relative mRNA levels of FBXO2 in MPHs treated with TNFα. Cells were

pre-treated with SP600125 (SP), U0126 (U) or BAY 11-7082 (BAY) for 4 hr

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and then administrated with TNFα for another 6 hr.

Supplementary Table 1

Dys-regulated FBPs in livers from C57BL/6 mice fed a normal diet (ND) or

high-fat-diet (HFD).


List of proteins identified from WT and MT FBXO2 interacting proteins.


GO analysis of glycoproteins identified from WT FBXO2 interacting

proteins.

All of the three Supplementary Tables were available online:

http://pan.baidu.com/s/1qY5zpHq

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Aberrant Expression of Fbxo2 Disrupts Glucose Homeostasis ...

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