betatrophin alleviates insulin resistance via the Akt-GSK3β or Akt-FoxO1 pathway in HepG2 cells

Author’s Accepted Manuscript ANGPTL8/betatrophin Alleviates insulin resistance via the Akt-GSK3β or Akt-FoxO1 pathway in HepG2 Cells Xing Rong Guo, Xiao Li Wang, Yun Chen, Ya Hong Yuan, Yong Mei Chen, Yan Ding, Juan Fang, Liu Jiao Bian, Dong Sheng Li www.elsevier.com/locate/yexcr

PII: DOI: Reference:

S0014-4827(15)30095-1 http://dx.doi.org/10.1016/j.yexcr.2015.09.012 YEXCR10054

To appear in: Experimental Cell Research Received date: 24 June 2015 Revised date: 17 August 2015 Accepted date: 16 September 2015 Cite this article as: Xing Rong Guo, Xiao Li Wang, Yun Chen, Ya Hong Yuan, Yong Mei Chen, Yan Ding, Juan Fang, Liu Jiao Bian and Dong Sheng Li, ANGPTL8/betatrophin Alleviates insulin resistance via the Akt-GSK3β or AktFoxO1 pathway in HepG2 Cells, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2015.09.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ANGPTL8/betatrophin Alleviates Insulin Resistance via the Akt-GSK3ȕ or Akt-FoxO1 Pathway in HepG2 Cells

Xing Rong Guo1

Xiao Li Wang1, Yun Chen1,5, Ya Hong Yuan1, Yong Mei Chen3, Yan

Ding1, Juan Fang4, Liu Jiao Bian2 , Dong Sheng Li1

1

Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China.

2

College of Life Sciences, Northwest University, Xi’an, Shanxi 710069, China.

3

Clinical Laboratory, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China.

4

Department of Pathology, Academic College, Hubei University of Medicine, Shiyan, Hubei 442000, China.

5

Institute of Genomic Medicine, Wenzhou Medical University, Wenzhou, China.

*

Corresponding author: Dong Sheng Li, E-mail: [email protected]

Co-Corresponding author: Liu Jiao Bian, E-mail: [email protected]

Abstract Angiopoietin-like protein 8 (ANGPTL8)/betatrophin, a newly identified protein, is primarily expressed in the liver and regulates the glucose metabolic transition during fasting and re-feeding in mice with or without insulin resistance. These findings strongly suggest that ANGPTL8/betatrophin could be a novel glucose-lowering candidate medicine for type 2 diabetes. However, the molecular mechanisms by which ANGPTL8/betatrophin regulates glucose metabolism are poorly understood in human. Two

sub-clones

of

HepG2

cells,

ANGPTL8/betatrophin

knockouts

and

ANGPTL8/betatrophin over-expressors, were established using TALENs (transcription activator-like effector nucleases) and through stable transfection, respectively. 1

Over-expression of ANGPTL8/betatrophin enhanced the insulin-stimulated activation of the Akt-GSK3ȕ or Akt-FoxO1 pathway, no matter whether the cells were preset with insulin resistance or not. In contrast, knockout of ANGPTL8/betatrophin did not affect the Akt-GSK3ȕ or Akt-FoxO1 pathway unless the HepG2 cells were preset with insulin resistance. Our results suggest that ANGPTL8/betatrophin might play an important role in glucose metabolism in the context of insulin resistance. Keywords: ANGPTL8/betatrophin, Akt, GSK3ȕ, FoxO1, insulin resistance, HepG2 cells

Introduction Insulin resistance refers to a physiological condition in which cells are unable to use insulin effectively, leading to hyperglycemia and type 2 diabetes mellitus (T2D), which is a major health burden worldwide (1, 2). The identification of novel molecules involved in regulating glycometabolism might provide potential new targets for novel therapeutic interventions in the treatment of T2D.

Recently, several groups independently studied a previously uncharacterized gene, officially designated C19 or f80 (human), or Gm6484 (mouse), but more commonly known as ANGPTL8/betatrophin, although it is also referred to as TD26 (3), RIFL (4), Lipasin (5, 6), an ANGPTL8 (7). ANGPTL8/betatrophin is a protein secreted by the liver and white adipose tissue under conditions of insulin resistance and can improve glucose tolerance by promoting beta cell proliferation in the pancreas in mice (8-10). These findings have raised hope for the rapid development of a novel therapeutic approach for the treatment of diabetes. However, recent studies found that ANGPTL8/betatrophin had no effect on beta cell growth in mice (11, 12). This result raises the question of how ANGPTL8/betatrophin improves glucose tolerance.

To investigate the molecular mechanism by which ANGPTL8/betatrophin improves glucose

tolerance,

we

(ANGPTL8/betatrophin−/−)

established and

ANGPTL8/betatrophin

ANGPTL8/betatrophin 2

knockouts

over-expressors

(ANGPTL8/betatrophin++) using TALENs and through stable transfection, respectively, in HepG2 cells. We compared these altered cells with their wild-type counterparts in terms of the expression and phosphorylation status of certain key factors involved in insulin-mediated glucose metabolism. The results consistently indicated that ANGPTL8/betatrophin potentiates the insulin-mediated synthesis of glycogen, gluconeogenesis and alleviates insulin resistance via the Akt-GSK3ȕ or the Akt-FoxO1 pathway in HepG2 cells.


  
  Cell culture and animals The HepG2 cell line was cultured in Į-minimum essential medium with low glucose (5.5 mM), containing glutamine and pyruvate (Invitrogen, USA), and supplemented with 10% fetal bovine serum, penicillin, and streptomycin. BALB/c mice (males, 6-8 weeks, 18–20 g) were used for the hydrodynamic tail vein over-expression experiment. Knockout of ANGPTL8/betatrophin in HepG2 cells ANGPTL8/betatrophin TALEN plasmid construction and cell transfection: The paired ANGPTL8/betatrophin TALEN arms were designed according to the manufacturer’s instructions (SIDANSAI, China). TALEN plasmids were constructed through one-step ligation using the Fast TALETM TALEN Assembly Kit (SIDANSAI, China) [13]. Approximately 16 ȝg of the paired ANGPTL8/betatrophin TALEN plasmids (8 ȝg each) was mixed with 1×106 HepG2 cells in a cuvette with 100 ȝl of Opti-MEM. Transfection was performed at 135 V for 5 s using NEPA21 (Japan). The transfected cells were transferred to one well of a 6-well plate and cultured at 37°C with puromycin at 3 ȝg/ml. After 3 days, the cells were refreshed with puromycin-free medium and then maintained at 37°C for 2-3 days before harvesting for DNA extraction and single-cell culture to select the ANGPTL8/betatrophin-disrupted clones.

T7 endonuclease 1 (T7E1) assay and sequencing: Genomic DNA was prepared from both ANGPTL8/betatrophin-disrupted and HepG2 wild-type cells with a Blood 3

Genomic DNA Extraction Mini Kit (TIANGEN). The genomic region encompassing the TALEN target site was amplified via PCR, and the resulting products were denatured and annealed to form heteroduplex DNA. The annealed DNA was treated with 5 units of T7E1 (Viewsolid Biotech) at 37°C for 15 min and then separated in a 2.5% agarose gel. TALEN targeting rates were calculated according to the brightness of the DNA bands, which was proportionally measured according to the grayscale technique, followed by gene sequencing. The RT-PCR primers for the TALEN target site were as follows: forward, 5’- CTGTGGCTATACCTTAGACCCTC-3’, and reverse, 5’- GAATATCCTCCTCCATCTGCTTA-3’.

Monoclonal cell genomes with mutations detected through the T7EN1 cleavage assay were sub-cloned into the T vector (Takara, D103A). For each sample, colonies were randomly picked and sequenced using the M13-T7 primer. The biallelic ANGPTL8/betatrophin-disrupted

HepG2

cell

clone,

designated

ANGPTL8/betatrophin-/-, was selected for further study. Over-expression of ANGPTL8/betatrophin in HepG2 cells The human ANGPTL8/betatrophin (containing a Flag tag) coding sequence was cloned and subcloned into pcDNA3.1-b-(-) to obtain hbeta-Flag-pcDNA3.1-b-(-). For transfection, HepG2 cells were seeded in six-well plates (105 cells/well). At 60%-80% confluence.

The

cells

were

then

ANGPTL8/betatrophin-Flag-pcDNA3.1-b-(-)

transfected

through

with

liposomal

human transfection

(Invitrogen, USA). After 10-14 days of selection with 800 ȝg/mL G418 (Invitrogen, USA), the transfected cells were dissociated and transferred to a 96-well plate (one cell/well). PCR and western blot assays were performed separately for each clone, and one of the clones showing the strongest expression of ANGPTL8/betatrophin-Flag (ANGPTL8/betatrophin++) was selected for further experiments. The primers used for RT-PCR

were

as

(ANGPTL8/betatrophin

follows:

forward,

cDNA

5’-ATGCCAGTGCCTGCTCTGTG-3’ sequence),

5’-TTACTTATCGTCGTCATCCTT-3’ (Flag-tag cDNA sequence). 4

and

reverse,

Treatment of HepG2 cells with insulin and other reagents All chemicals employed for the treatment of cultured cells were obtained from Sigma-Aldrich.

qPCR

analysis

of

insulin

and

glucose

regulation

by

ANGPTL8/betatrophin was performed as described previously(4). To induce insulin resistance to a high concentration of glucose (30 mM) in HepG2 cells, growth medium with normal glucose (5.5 mM) was switched to a hyperglycemic medium for 24 h. After the induction of insulin resistance, the cells were treated with insulin (100 nM) for 20 min (14). PAS staining PAS staining was performed as described previously with some modifications (15). HepG2 cells were fixed with 4.0% formalin (Beyotime) for 10 min, then treated with 1% periodic acid for 15 min, followed by Schiff's reagent for 1 h in the dark. The samples were finally mounted and examined under a microscope. RNA isolation and real-time quantitative RT-PCR (RT-qPCR) Total RNA was extracted using TRIzol (Invitrogen) following the manufacturer’s recommendations and quantified through UV spectroscopy. To prepare RNA for PCR analysis, 2 ȝg of total RNA was converted to cDNA using the FastQuant RT Kit with gDNase (TIANGEN). RT-qPCR was performed using a standard SYBR Green PCR kit (Invitrogen) protocol on the StepOneTM Real-Time PCR System (Applied Biosystems). Each sample was analyzed in triplicate. For both types of gene expression, the target genes were quantified via relative quantification using the comparative CT method. PCR products were amplified using the following primers: ANGPTL8/betatrophin forward,

5’-GCCTGTTGGAGACTCAGATGGA-3’,

and

reverse,

5’-

CGCTGTCCCGT AGCACCTTC-3’; ȕ-actin forward, 5’- CTGGAACGGTGAAGGTGACA-3’, and reverse, 5’-AAGGGACTTCCTGTAACAACGCA-3’. Western blotting Cells were washed with PBS three times and collected with cell lysis buffer (Beyotime) according to the manufacturer’s protocol. Equal amounts of protein (50 ȝg) were loaded into each lane, separated through 12% SDS polyacrylamide gel electrophoresis, 5

and electrotransferred to nitrocellulose membranes. The nitrocellulose membranes were blocked in blocking buffer for 1 h at RT, followed by incubation with the following antibodies overnight at 4°C: mouse anti-Flag (1:1000, Sigma) and anti-Actin (1:1000, Abcam); rabbit anti-AKT, anti-phospho-AKT (Ser473), anti-GSK3-ȕ, anti-phospho-GSK-3ȕ (Ser9), anti-FoxO1, and anti-phospho-FoxO1 (Ser256; 1:1000, Santa Cruz Biotechnology); and goat anti-ANGPTL8/betatrophin (1:800 Aviscera Bioscience). The blots were subsequently rinsed with TBST three times and incubated with anti-rabbit, anti-mouse or anti-goat horseradish peroxide-conjugated secondary antibodies (1:1000) for 60 min. The resultant signals were detected through chemiluminescence using ECL Hyperfilm. Hydrodynamic tail vein injection Six- to seven-week-old male BALB/c mice were used for hydrodynamic tail vein injection due to the ease of identifying the tail vein (16-18). The injections were performed as described previously, with some modifications. Twenty to thirty micrograms (1 ȝg/g body weight) of pcDNA-3.1-LUC, pcDNA-3.1-mouse or pcDNA3.1-human ANGPTL8/betatrophin expression plasmid DNA in sterile saline was injected into the tail vein (3-5 mice per group). The volume of sterile saline solution was 10% of body weight, and the injection time was between 8 and 10 s. The control mice were observed for LUC expression every 24 h using an in vivo imaging system (IVIS® Spectrum, Caliper). Glucose tolerance test Glucose tolerance: Mice were fasted for 12 h and then injected with D-glucose (1 mg/g body weight, Sigma) intraperitoneally. Blood glucose levels were measured from the tail tip using One Touch Sure StepTM glucose monitoring (LifeScan, Inc.) at 0, 60, 120 and 240 min post-injection. The area under the curve was calculated using standard methods. Transfection

of

HepG2

cells

with

in

vitro-synthesized

ANGPTL8/betatrophin-FLAG mRNA ANGPTL8/betatrophin-Flag mRNA was synthesized using previously described methods (19). RNA transfection was performed with TransIT-mRNA (Mirus). RNA 6

was diluted in Opti-MEM basal medium (Gibco), and the Boost reagent and TransIT-mRNA were then added sequentially. After 2 min of incubation at room temperature (RT), the RNA-lipid complexes were delivered to the culture medium in culture

plates.

The

plates

ANGPTL8/betatrophin-Flag

were

then

expression

returned was

to

the

analyzed

incubator, 12

h

and later.

ANGPTL8/betatrophin-Flag in the medium was detected after the supernatant samples were filtered through a 0.22-ȝm membrane and equally concentrated using 10,000 MWCO (cat # 42406; Millipore, Billerica, MA, USA). Immunohistochemical assays Immunohistochemical assays were performed as previously described (19). Cells were incubated with anti-Flag secondary antibodies to analyze ANGPTL8/betatrophin-Flag expression following transfection of ANGPTL8/betatrophin-Flag mRNA. Statistical analyses The results are presented as the mean±standard deviation (SD). The significance of the differences was tested with Student’s t-test in Microsoft Office Excel. A value of p<0.05 (*) was considered significant.

RESULTS Over-expression of ANGPTL8/betatrophin in the liver of mice improves glucose tolerance Three

different

plasmids,

expressing

mouse

ANGPTL8/betatrophin,

human

ANGPTL8/betatrophin or LUC, were separately transfected into the livers of mice by rapidly injecting the plasmids into the tail veins. The LUC proteins were expressed well in the liver for at least 7 days, peaking at 48 h (Fig. 1A), which confirmed that the so-called hydrodynamic transfection performed in this study worked well in vivo. Then, glucose tolerance tests were performed in the mice transfected with both mouse and human ANGPTL8/betatrophin plasmids immediately after a 12-h fast. Significantly better glucose tolerance was observed in the mice transfected with either mouse or human ANGPTL8/betatrophin compared with those transfected with LUC (Fig. 1B).

7

ANGPTL8/betatrophin mRNA is dramatically up-regulated by insulin in the presence of glucose Based on the observation that ANGPTL8/betatrophin expression is enriched in the liver, especially during re-feeding (7, 20), we speculated that different nutrients might have different influences on ANGPTL8/betatrophin expression. We cultured HepG2 cells in medium supplemented with insulin, glucose, or both. ANGPTL8/betatrophin was dramatically up-regulated only when insulin was added together with glucose into the medium. Neither of these two reagents alone affected ANGPTL8/betatrophin expression (Fig. 2A). Furthermore, the increase in ANGPTL8/betatrophin transcript levels reached significance approximately 4 h after insulin and glucose were added (Fig.

2B),

and

the

higher

the

insulin

concentration,

the

higher

the

ANGPTL8/betatrophin transcript level (Fig. 2C). Establishment of ANGPTL8/betatrophin knockouts and over-expressors in HepG2 cells using TALENs and through stable transfection, respectively After two rounds of transfection with ANGPTL8/betatrophin-TALENs, genomic DNA was

prepared

from

the

transfected

HepG2

cells.

The

target

region

of

ANGPTL8/betatrophin was amplified via PCR and then digested with the T7E1 enzyme (Fig. 3A and B). Following single-cell culture, we randomly selected 10 monoclonal cells for T7E1 enzyme analyses (Fig. S1), and we predicted that clones 5, 7, and 10 might be homozygous. Further gene sequencing, RT-PCR and western blot analyses indicated that clones 5, 7 and

10 were

homozygous biallelic

ANGPTL8/betatrophin disruptions cell lines (Fig. 3C-E and Fig. S2). One of the three clones was selected as the ANGPTL8/betatrophin knockout (ANGPTL8/betatrophin-/-) for the subsequent experiments. ANGPTL8/betatrophin-over-expressing HepG2 cells were established through stable transfection of human ANGPTL8/betatrophinFlag-pcDNA3.1-b-(-) plasmids. After 3 weeks of selection by neomycin, four cell clones were collected separately for RT-PCR and western blot assays. The results indicated that the ANGPTL8/betatrophin-Flag plasmid was successfully integrated into the HepG2 genome and expressed well at the mRNA (Fig. 4A) and protein (Fig. 4B) levels. Clone 3 was selected as the ANGPTL8/betatrophin over-expressor 8

(ANGPTL8/betatrophin++) for the subsequent experiments because it showed the highest ANGPTL8/betatrophin-Flag expression level. ANGPTL8/betatrophin promotes the phosphorylation of Akt, FoxO1 and GSK-3ȕ in the insulin signaling pathway Because ANGPTL8/betatrophin mRNA is dramatically up-regulated by insulin in the presence of glucose, we speculated that ANGPTL8/betatrophin plays a role in insulin signaling. Both total Akt protein and phospho-Akt (Ser473; pAkt) were measured using western blot assays. As shown in Fig. 5A, total Akt levels did not change in any of the cells in the presence or absence of insulin in the medium (containing 5.5 mM glucose); however, pAkt levels in ANGPTL8/betatrophin++ cells were significantly increased when insulin was added to the medium. Interestingly, pAkt levels were increased the most compared with those in ANGPTL8/betatrophin−/− and wild-type cells. Next, we investigated the expression and phosphorylation of JNK (Thr183/Tyr185; pJNK), IRS-1(Ser307; pIRS-1(307)) and IRS-1(Ser302; pIRS-1(302)) upstream and FoxO1 (Ser256; pFoxO1), GSK-3ȕ (Ser9; pGSK-3ȕ) and mTOR (Ser2448, pmTOR) downstream. There were no significant differences between the three types of cells in terms of total protein expression, even after insulin was added to the medium (Fig. 5B and C). However, their phosphorylation was altered. pFoxO1 and pGSK-3ȕ of ANGPTL8/betatrophin++ cells were increased after insulin was added to the media, but there was no significant change in pJNK, pIRS-1(302), pIRS-1(307) and pmTOR, which are upstream regulators of Akt or are regulated by Akt. Taking these results together, it appears that high expression ANGPTL8/betatrophin participates in the insulin signaling pathway by regulating the phosphorylation of crucial proteins involved in the pathway, but ANGPTL8/betatrophin deficiency or low expression may not affect the signaling pathway. ANGPTL8/betatrophin

promotes

glycogen

synthesis

and

suppresses

gluconeogenesis in HepG2 cells The Akt-GSK3ȕ/-FoxO1 signaling pathway is required for insulin-mediated glycogen synthesis

and

gluconeogenesis.

(21,

22).

We

further

examined

whether

ANGPTL8/betatrophin affects glycogen synthesis and gluconeogenesis. After 9

treatment with insulin and/or glucose in the three cell lines, glycogen accumulation was detected through PAS staining. Glycogen synthesis was greatly increased in ANGPTL8/betatrophin++ cells when glucose, insulin or both were added, but there was no significant decrease in glycogen synthesis in ANGPTL8/betatrophin-/- cells compared

with

control

cells

(Fig.

6A).

In

addition,

overexpression

of

ANGPTL8/betatrophin enhanced the effect of insulin on suppressing the expression of PEPCK and G6Pase, the two key enzymes involved in gluconeogenesis but there were no significant changes in ANGPTL8/betatrophin-/- cells compared with control cells (Fig. 6B). The above results suggest that ANGPTL8/betatrophin potentiates the hepatic actions of insulin on promotes glycogen synthesis and suppresses gluconeogenic genes via Ak activation

Effects of ANGPTL8/betatrophin on insulin resistance in HepG2 cells Because ANGPTL8/betatrophin potentiated insulin-mediated Akt activation, we evaluated whether ANGPTL8/betatrophin could ameliorate insulin resistance in HepG2 cells. As shown in Fig. 7A, incubation of cells with hyperglycemic medium induced a dramatic suppression of insulin-mediated Akt activation. However, when insulin was present in the medium, insulin-mediated Akt, FoxO1 and GSK-3ȕ phosphorylation

showed

no

significant

differences

between

the

ANGPTL8/betatrophin++ and control cell lines. Compared with the control cell lines, insulin-mediated Akt, GSK-3ȕ and FoxO1 phosphorylation was significantly down-regulated in ANGPTL8/betatrophin-/- cells. Hence, we asked why the levels of pAkt, pFoxO1 and pGSK-3ȕ showed no significant differences between ANGPTL8/betatrophin++ and control cell lines under insulin resistance.
We speculated that this might be related to the expression level of ANGPTL8/betatrophin. As shown in Fig. 7B, compared with the control cell lines, the expression of ANGPTL8/betatrophin was higher in ANGPTL8/betatrophin++ cells in growth medium (containing 5.5 mM glucose). However, under insulin resistance (growth medium with normal glucose was switched to hyperglycemic medium for 24 h),

the

expression

of

ANGPTL8/betatrophin 10

was

similarly

increased

in

ANGPTL8/betatrophin++ and control cell lines, possibly induced by the high concentrations glucose and insulin (the growth medium contains insulin). The above results demonstrate that high expression ANGPTL8/betatrophin could alleviate the process of insulin resistance, whereas ANGPTL8/betatrophin deficiency could accelerate the process of insulin resistance.

To determine whether the effect of ANGPTL8/betatrophin in ameliorating insulin resistance is dose dependent, we synthesized ANGPTL8/betatrophin-Flag mRNA, which was highly expressed in the cytoplasm of HepG2 cells (Fig. 8A). Western blots showed that the expression of ANGPTL8/betatrophin-Flag in both whole cells and supernatants was dose dependent (Fig. 8B). ANGPTL8/betatrophin-/- cells were incubated in the presence of 30 mM glucose for 24 h and then transfected with different

concentrations

of

ANGPTL8/betatrophin

mRNA.

Remarkably,

the

phosphorylation of Akt, GSK-3ȕ and FoxO1 increased with increasing amounts of transfected ANGPTL8/betatrophin mRNA (Fig. 8C).

Discussion ANGPTL8/betatrophin is a protein derived from the liver that plays dual roles in glucose homeostasis and triglyceride metabolism in mice (4, 8, 20). However, the intracellular roles of ANGPTL8/betatrophin are poorly understood in the liver, as are the molecular mechanisms involved. In this study, we sought to characterize the molecular mechanisms underlying the regulation of glucose metabolism by ANGPTL8/betatrophin in humans. First, glucose tolerance was improved by over-expressing ANGPTL8/betatrophin in the livers of mice, which is consistent with a report by Yi et al. (8). The expression of ANGPTL8/betatrophin mRNA was dramatically up-regulated by insulin in the presence of glucose in HepG2 cells, which was also observed in adipocytes by another group (4). These results indicate that ANGPTL8/betatrophin might play an important role in glucose metabolism. Next, using the human liver-derived cell line HepG2, we found

that over-expressing ANGPTL8/betatrophin 11

promoted insulin-mediated

glycogen synthesis in HepG2 cells by regulating the Akt-GSK3ȕ or Akt-FoxO1 signaling pathway, which are important pathways through which insulin maintains glucose homeostasis (23-25). ANGPTL8/betatrophin knockout only affected these pathways if the cells were insulin resistant. Therefore, we provide the first evidence that insulin-induced ANGPTL8/betatrophin expression may promote glucose tolerance through Akt-GSK3ȕ or Akt-FoxO1 pathway phosphorylation and hepatic glycogen synthesis in HepG2 cells.

The above findings could explain why ANGPTL8/betatrophin expression was significantly increased in mice with insulin resistance or during re-feeding, as the insulin level is dramatically elevated under these two physiological conditions. The results also explain why the serum concentrations of ANGPTL8/betatrophin measured by different researchers are inconsistent in T2DM (26-28). In the early stage of insulin resistance, the normal insulin concentration does not produce an adequate, normal insulin response in target tissues, such as the liver, adipose tissue and muscle. Under these conditions, pancreatic beta cells secrete more insulin (i.e., hyperinsulinemia) to overcome hyperglycemia in these individuals, and ANGPTL8/betatrophin expression is elevated. Over time, the inability of the pancreatic beta cells to produce sufficient insulin to compensated for worsening tissue insulin resistance leads to hyperglycemia and overt T2D (29), after which the concentration of ANGPTL8/betatrophin progressively decreases. Therefore, the inconsistency is actually caused by varying degrees of insulin resistance. Recently, Hu et al. discovered that the concentration of circulating ANGPTL8/betatrophin is significantly increased in newly diagnosed T2DM patients, and serum ANGPTL8/betatrophin is positively correlated with post-oral glucose tolerance test (OGTT) glucose (2 hPG) and postprandial serum insulin (PSI), but negatively correlated with insulin sensitivity in T2DM patients (30). These correlations indirectly support our results. ANGPTL8/betatrophin-knockout mice exhibit disrupted triglyceride metabolism without impaired glucose homeostasis (20), which suggests that ANGPTL8/betatrophin might not play a role in glucose metabolism. However, the authors of this previous 12

study did not investigate ANGPTL8/betatrophin-knockout mice with insulin resistance. In the present study, we found that ANGPTL8/betatrophin deficiency did not affect the Akt-GSK3ȕ or Akt-FoxO1 signaling pathway only in the HepG2 cells without insulin resistance. Interestingly, the insulin-mediated phosphorylation of Akt, GSK3ȕ and FoxO1 was sharply suppressed in ANGPTL8/betatrophin-/- cells with insulin resistance, and the effect of ANGPTL8/betatrophin in ameliorating insulin resistance was dose dependent. All of the results suggest that ANGPTL8/betatrophin deficiency accelerates the process of insulin resistance.

Melton and Kaestner found that ANGPTL8/betatrophin promotes pancreatic beta cell proliferation and improves glucose tolerance through transient over-expression of ANGPTL8/betatrophin in the liver [8, 9]. However, this phenomenon was not confirmed by Gusarova et al. (11). Melton later explained that they agreed with the main conclusion of Gusarova et al., that deletion of ANGPTL8/betatrophin itself did not support the notion that ANGPTL8/betatrophin alone was capable of inducing pancreatic beta cell proliferation (31). Additionally, they considered the discrepancy from their previous paper to possibly have been caused by the high variability in hydrodynamic

tail

vein

injection,

which

was

employed

to

increase ANGPTL8/betatrophin expression in the liver. Interesting, Chen recently observed that ANGPTL8/betatrophin induced adult and aged beta cell regeneration in a rat model by employing ultrasound-targeted microbubble destruction (UTMD), rather than

hydrodynamic

tail

vein

injection,

to

deliver

plasmids carrying

the

human ANGPTL8/betatrophin gene to the pancreas, liver and skeletal muscle of normal adult rats (10). Both exciting and conflicting results have been obtained, and there is significant ongoing controversy. Although we did not study the function of ANGPTL8/betatrophin in promoting beta cell proliferation, our findings may provide some

new

evidence

to

support

the

hypothesis

that

over-expressing

ANGPTL8/betatrophin might affect beta cell mass. As we found that over-expression ANGPTL8/betatrophin could promote the insulin-mediated regulation of the activity of AKT2, GSK3ȕ or FoxO1 in HepG2 cells, and many studies have reported that 13

increased expression or activity of AKT2(32), GSK3ȕ(33) or FoxO1(34-36) affects the replication and survival of pancreatic ȕ cells, we speculate that ANGPTL8/betatrophin, as a secretory protein secreted from the liver and adipose tissue, might promote the insulin-mediated phosphorylation of AKT2, GSK3ȕ or FoxO1 in other tissues, and especially in the pancreas, to control pancreatic ȕ cell proliferation.

Conclusion In summary, the data presented in this report consistently suggest that ANGPTL8/betatrophin

could

potentiate

insulin-mediated

glycogen

synthesis,

gluconeogenesis, alleviate insulin resistance and supplement the insulin signaling pathway, as shown in Fig. 9. Our research has implications for the management of insulin-resistant type 2 diabetes, indicating that ANGPTL8/betatrophin may be useful as a glucose-lowering agent. Acknowledgements This study was financially supported by the Major Science and Technology Projects of China (No. 2013ZX10001-004-002-005), the National Natural Science Foundation of Hubei province (2012FFA037), the National Foundation of College Students' Innovative Entrepreneurial Training Program (201410929005) and the Hubei Province Health and Family Planning Scientific Research Project (WJ2015MB223). Competing interests The authors declare no competing or financial interests. Authors’ contributions Xing Rong Guo performed the experiments, analyzed the data, and wrote and revised the manuscript. Yun Chen and Yan Ding generated the TALEN plasmids. Xiao Li Wang contributed to the synthesis of ANGPTL8/betatrophin-Flag mRNA. Ya Hong Yuan performed the hydrodynamic tail vein injections. Juan Fang contributed to the analysis of LUC expression in vivo. Dong Sheng Li and Liu Jiao Bian designed the study, analyzed the data and drafted, revised and edited the manuscript. All authors approved the final version of the manuscript. 14

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Figure legends: Figure 1 Over-expression of ANGPTL8/betatrophin in the liver improves glucose tolerance. A. Seven-week-old male BALB/c mice were subjected to hydrodynamic tail vein injection. LUC expression in the liver was measured with an in vivo imaging system. B. Results of the glucose tolerance test in mice on day 3 after transfection with LUC (n=3 mice) or mouse or human ANGPTL8/betatrophin (each, n=5 mice) DNA via hydrodynamic tail vein injection. Figure 2 ANGPTL8/betatrophin expression induced by insulin in the presence of glucose in HepG2 cells. A. Analysis of ANGPTL8/betatrophin expression induced by insulin or glucose alone or in combination under serum-free conditions through RT-PCR (a) and qPCR (b). Con: control; I: insulin; G: glucose; I+G: insulin+glucose. B. ANGPTL8/betatrophin expression induced by different doses of insulin (medium with 5.5 mM glucose). HepG2 cells were exposed to 0 to 1,000 nM insulin, and the samples were analyzed via RT- PCR (a) and qPCR (b) 24 h later. C. Time-course of ANGPTL8/betatrophin expression induced by insulin (medium with 5.5 mM glucose). HepG2 cells were exposed to 100 nM insulin for various times up to 48 h, and the samples were analyzed by RT- PCR (a) and qPCR (b). Data were presented as mean ± SEM. * p < 0.05 vs. control. Figure 3 Knockout of ANGPTL8/betatrophin in HepG2 cells by TALEN A. Schematic representation of the ANGPTL8/betatrophin gene and the target sites of TALENs. B. T7E1 analysis of the efficiency of TALEN-induced ANGPTL8/betatrophin mutation after two rounds of targeting. M: 100-bp DNA ladder; Con: control, 17

C. Gene sequencing of the target region in 3 selected ANGPTL8/betatrophin-/- cell clones. a. Sequences of the target regions of 3 the selected clones; b-d. peaks observed in the 3 selected clones. D. Western blot of ANGPTL8/betatrophin expression detected with the ANGPTL8/betatrophin antibody in clone 5, clone 7 and clone 10. Figure 4. Over-expression of ANGPTL8/betatrophin in HepG2 cells. Analysis of ANGPTL8/betatrophin-Flag expression via RT-PCR (A) and western blotting with an Flag-tag antibody (B). M: 100-bp DNA ladder; Con: control; 1-4: four different cell clones. Figure 5. Comparison of the expression of Akt, FoxO1 and GSK-3ȕ in ANGPTL8/betatrophin-/-, ANGPTL8/betatrophin++ and wild-type HepG2 cells Three different groups of HepG2 cells were stimulated with or without 100 nM insulin for 20 min, followed by western blotting. A. Total Akt and pAkt. B. Total GSK-3ȕ, pGSK-3ȕ, total FoxO1, and pFoxO1. The results are representative of 3 independent experiments. Figure 6. Analysis of glycogen synthesis and the expression of gluconeogenic genes in HepG2 cells A. Analysis of glycogen synthesis by PAS staining. Three different HepG2 cell lines were cultured in glucose-free and insulin-free medium with 0.5% BSA for 24 h, after which 5.5 nM glucose or 5.5 nM glucose+100 nM insulin was added to the medium. PAS staining was performed after 12 h (100x). B. Analysis of the expression of gluconeogenic genes by Real-time RT-PCR. Three different HepG2 cell lines were treated with growth medium or containing 100 mM insulin medium for 6 h. Data were presented as mean ± SEM. * p < 0.05 vs. control at the same medium.

Figure 7. Effects of ANGPTL8/betatrophin on insulin resistance A. Western blots of pAkt, pGSK-3ȕ and pFoxO1 in HepG2 cells with insulin resistance. Three different HepG2 cell lines were treated with a high concentration of glucose (30 mM) for 24 h and were subsequently treated with or without 100 nM insulin for 20 min. B. Western blots of ANGPTL8/betatrophin in HepG2 cells. Three different HepG2 cell lines were treated with growth medium (containing 5.5 mM glucose) or hyperglycemic medium (containing 30 mM glucose) for 24 h. Figure 8. The effects of ANGPTL8/betatrophin expression on pAkt, pGSK3ȕ and pFoxO1 are dose dependent under insulin resistance. 18

A. Transfection with ANGPTL8/betatrophin-Flag mRNA in HepG2 cells. The transfected cells were fixed at 12 h and stained with anti-Flag antibodies. Microphotographs were captured with a fluorescence microscope (200x). B. HepG2 cells were transfected with 0, 0.5, 1.0 or 1.5 ȝg of ANGPTL8/betatrophin-Flag mRNA, and the western blots show that the expression of ANGPTL8/betatrophin-flag in both whole cells and the supernatant at 12 h was dose dependent. C. ANGPTL8/betatrophin-/- HepG2 cells were preset with insulin resistance after treatment with 30 mM glucose for 24 h, then transfected with different doses of ANGPTL8/betatrophin-flag mRNA and incubated for another 12 h. The cells were finally treated with or without 100 nM insulin for 20 min. Western blots indicated that the effects of ANGPTL8/betatrophin on pAkt, pGSK3ȕ and pFoxO1 were very sensitive and dose dependent. Figure 9. Modified Akt-GSK3ȕ and Akt-FoxO1 signaling pathways. In the presence of glucose, insulin initiates IRS phosphorylation and, at the same time, up-regulates ANGPTL8/betatrophin, which in turn further increases Akt phosphorylation, resulting in increased phosphorylation of FoxO1 and GSK3ȕ. pFoxO1 suppresses gluconeogenic genes, while pGSK3ȕ promotes glycogen synthesis. As a result, glycogen accumulation increases. Thus, ANGPTL8/betatrophin participates in glucose metabolism and alleviates insulin resistance. (“ĺ” indicates up-regulation, and “ ” indicates down-regulation).

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