Aberrant Expression of Fbxo2 Disrupts Glucose Homeostasis ...
Transcript of 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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Diabetes Publish Ahead of Print, published online December 8, 2016
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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,
E-mail: [email protected]
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
E-mail: [email protected]
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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.
Supplementary Figure 2
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.
Supplementary Figure 3
(A-D) Relative mRNA and representative protein levels of IR in the liver
from lean and obese mice. n=6-8.
Supplementary Figure 4
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(A-B) Body weight (A) and food intake (B) in C57BL/6 mice administrated
with adenoviruses expressing FBXO2 or GFP.
Supplementary Figure 5
(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).
Supplementary Figure 6
(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.
Supplementary Figure 7
(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).
Supplementary Table 2
List of proteins identified from WT and MT FBXO2 interacting proteins.
Supplementary Table 3
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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