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Deficiency of CKIP-1 aggravates high-fat diet-induced fatty liver in mice
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Deficiency of CKIP-1 aggravates high-fat diet-induced fatty liver in mice Yutao Zhana,1, Ping Xieb,1, Dongnian Lic,1, Li Lia, Jing Chena, Wei And, Lingqiang Zhange, ⁎ Chuan Zhanga, a
Department of Gastroenterology, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China Physical Examination Centre, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China Department of Biochemistry and Molecular Biology, Beijing Key Laboratory of Cancer & Metastasis Research, Capital Medical University, Beijing 100069, China d Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing 100069, China e State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China b c
A R T I C L E I N F O
A BS T RAC T
Keywords: Non-alcoholic fatty liver disease (NAFLD) Casein kinase 2 interacting protein-1 (CKIP-1) c-Jun NH2-terminal kinase 1 (JNK1) Insulin receptor substrate −1(IRS-1) High fat diet (HFD)
Casein kinase 2 interacting protein-1(CKIP-1) is widely expressed in a variety of tissues and cells, and plays an important role in various critical cellular and physiological processes including cell growth, apoptosis, differentiation, cytoskeleton and bone formation. Here, we found: (1) CKIP-1 deficient mice exhibited increased body weight, liver weight, number and size of lipid droplets, and TG content comparing with WT mice after being exposed to high fat diet (HFD); (2) the levels of serum insulin, liver glycogen, phosphorylated C-Jun-Nterminal kinase-1 (pJNK1) and phosphorylated insulin receptor substrate −1(pIRS1) in CKIP-1-/- mice were higher than those of WT mice; (3) CKIP-1 interacted with JNK1 in vitro. Our results indicate that CKIP-1 deficiency in mice aggravates HFD-induced fatty liver by upregulating JNK1 phosphorylation and further upregulating IRS-1 phosphorylation and RI.
1. Introduction Non-alcoholic fatty liver disease (NAFLD), with a 25% global prevalence, is the most common chronic liver disease in the world [1–3]. With an increase of the population with obesity and diabetes, the incidence of NAFLD will increase further in the future, with an expected 50% prevalence in the US by 2030 [4,5]. A growing body of evidence suggests that NAFLD is a potential independent risk factor for cardiovascular disease, which is the leading cause of mortality among NAFLD patients [6,7]. In addition, some forms of NAFLD (especially non-alcoholic steatohepatitis) can progress to fibrosis, cirrhosis and hepatocellular carcinoma (HCC) [8], and NALFD is predicted to be the leading cause of end-stage liver disease requiring liver transplantation by 2020 [9]. Despite the emergence of NAFLD and the health burden caused by this epidemic, no pharmacological therapy has been developed to treat NAFLD specifically partially because of our incomplete understanding of its pathogenesis. We therefore aimed to study NAFLD at the mechanistic level to provide a future framework for developing a potential effective treatment. Insulin resistance (IR) plays a key role in the pathogenesis of NAFLD by enhancing the uptake and synthesis of fatty acids and
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inhibiting the β-oxidation of fatty acids in hepatocytes [10,11]. IR is mediated by tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), the insulin receptor adapter protein, and C-Jun-N-terminal kinase-1 (JNK1), a member of the mitogen-activated protein kinase family. It is known that JNK1 deficiency protects mice against HFDinduced IR. JNK1 induces the phosphorylation of IRS-1 on Ser-307, which disrupts the interaction of the phosphotyrosine binding domain of IRS-1 with the tyrosine phosphorylated insulin receptor [12]. The PH-domain-containing casein kinase 2 interacting protein-1 (CKIP-1) was originally identified as an interacting partner of the α-subunit of casein kinase 2 [13]. Recently, we showed that the targeted deletion of CKIP-1 causes the upregulation of phosphorylated JNK1 without changing the total JNK1 in mouse embryonic fibroblasts [14]. We therefore speculated that CKIP-1 might play a role in the development of NAFLD via regulating JNK1 phosphorylation, IRS-1 phosphorylation, and IR. In this study, we demonstrated that CKIP-1 deficiency exacerbates fatty liver induction in mice fed with high-fat diet (HFD). In addition, the levels of serum insulin, liver glycogen, phosphorylated JNK1 (pJNK1), and phosphorylated IRS1 (pIRS1) also increased in CKIP-1 deficient mice. Finally, we found there was an interaction between CKIP-1 and JNK1 in hepatocytes.
Corresponding author. E-mail address: [email protected] (C. Zhang). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.yexcr.2017.03.033 Received 7 November 2016; Received in revised form 8 March 2017; Accepted 16 March 2017 0014-4827/ © 2017 Published by Elsevier Inc.
Please cite this article as: Zhan, Y., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.03.033
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2. Material and methods
2.7. Analysis of pJNK1 in isolated hepatocyte
2.1. Animals
Hepatocytes were isolated by collagenase digestion method. Briefly, 1 weeks old CKIP-1-/- and WT mice were killed to obtain the liver tissues. The liver sample was washed 3 times with PBS, then minced and digested by 0.02% IV collagenase for 20 min at 37 °C. The suspension was centrifuged at 1000 rpm for 10 min. The obtained hepatocytes were collected and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells were treated with anisomycin (10 ng/ml) for 48 h to induce JNK1 phosphorylation. Then, the cells were collected for testing pJNK1 protein by western blot analyses as above.
CKIP-1 knockout (CKIP-1−/−) mice in C57BL/6 background were prepared as previously described [14], and CKIP-1+/+(wild-type, WT) C57BL/6 mice were used as control. The mice were housed under pathogen-free conditions in a temperature-controlled animal facility with a 12 h light/dark illumination cycle. All procedures for handling animals were in accordance with protocols approved by Chinese Academy of Military Medicine Science. CKIP-1−/− mice and the WT control mice were fed with an HFD (D12492, 60% of kilocalories from fat; Research Diet) for 8 weeks. Their body weight was measured at the beginning and the end of the experiment. Mice were then sacrificed, their liver tissues were weighed, and blood serum samples were collected. The liver tissues were frozen in liquid nitrogen and maintained at −80 °C until further analyses. A portion of each liver was fixed in 10% buffered formalin for histopathology. The collected blood samples were kept on ice for 30 min and then separated into serum by centrifugation at 4◦C. The serum was then kept at −20 °C until the examination.
2.8. Co-immunoprecipitation (Co-IP) The human embryonic kidney (HEK293T) cells were transfected with CKIP-1 plasmid. The CKIP-1 plasmids were constructed as described previously [14]. After 48 h, cells were harvested and lysed in HEPES (N-(2hydroxyethyl) piperazine-N′-2-ethanesulfonic acid) lysis buffer (20 mM HEPES, 50 mM NaCl, 0.5% Triton X-100, 1 mM NaF and 1 mM dithiothreitol). Lysates were incubated with either anti-CKIP-1 or anti-JNK1 antibody overnight at 4 °C in the presence of 50 µl Protein-G/A beads. Beads were collected, washed, and resuspended in equal volumes of 5×SDS loading buffer. The immunoprecipitated proteins were separated by SDSPAGE and transferred onto PVDF membrane. The membrane was blocked with 5% skim milk, incubated at 4 °C overnight with indicated antibodies, and followed by detection with the related secondary antibody and the Super Signal chemiluminescence kit (ThermoFisher).
2.2. Analysis of blood insulin Serum insulin was measured using Ultra Sensitive Mouse Insulin ELISA Kit (Shanghai Elisa Biotech Co., Ltd., China) according to manufacturer's instructions. 2.3. Liver histopathology test
2.9. Confocal microscopy, immuno-fluorescence analysis
After embedded in paraffin, the live tissues were cut into 5micrometer thin sections and processed for hematoxylin and eosin staining to evaluate the extent of lipid accumulation in hepatocytes.
For immuno-staining of endogenous JNK-1, HEK293T cells transfected with GFP-CKIP-1 were fixed in 4% PFA (paraformaldehyde) for 10 min. Images of endogenous JNK-1 were then fixed in 0.1% PBST (containing 0.5% Triton X-100) for 15 min. Further processing included incubating cells in 5% BSA for 30 min before incubations with primary for 3 h at 37 °C and with secondary antibody for 1 h at room temperature. Cells were analyzed in PBS when the nucleus was stained with 0.1 g/ml DAPI. Images of fixed cells were acquired on a confocal microscope using LaserSharp software.
2.4. Analysis of Liver glycogen content 400 µl of 5% trichloroacetic acid was added into 150 mg of liver tissue. After a centrifugation at 4000 rpm for 3 min, the supernatant was mixed with 400 µl of 95% ethanol. The precipitate was then removed by centrifugation at 4000 rpm for 5 min. The amount of glycogen in the digested samples was measured using a Glycogen Assay Kit (Sigma), following the manufacturer's instructions.
2.10. Statistical Analysis 2.5. Analysis of liver triglyceride (TG) content The results were expressed as the mean ± SD. The significance of differences was determined by t-test using the SPSS 17.0 software (SPSS, Chicago, IL, USA). Difference with a p value of less than 0.05 was considered statistically significant.
50 mg frozen liver tissue was homogenized in 1 ml of tissue lysis solution (20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1% Triton). After centrifugation at 2000 rpm for 5 min at 37◦C, the supernatants were used to determinate the liver TG levels using an EnzyChrom™ TG assay kit (Kampenhout, Belgium) and normalized by the protein concentration, according to the protocol provided by the manufacturer. Data were expressed as milligrams of TG per gram of liver.
3. Results 3.1. CKIP-1 deficiency increases body weight and liver weight in mice fed with HFD
2.6. Western blot analyses At the end of experiment, the body weight of CKIP-1-/- mice significantly increased compared to those of WT mice (p < 0.05) (Table 1), showing that the deficiency of the CKIP-1 gene enhances the degree of obesity in mice fed with HFD. The liver weight of CKIP1-/- mice also significantly increased compared to those of WT mice (p < 0.05) (Table 1), suggesting that the CKIP-1 deficiency probably increases the degrees of fatty liver.
Liver lysates were prepared by homogenization of liver samples with RIPA buffer containing 1% phosphatase inhibitor. Protein concentrations were measured using the BCA assay. Protein from each sample was separated by 10% SDS-PAGE and electro-transferred to nitrocellulose filter membranes. The membranes were blocked with 5% skim milk, incubated at 4 °C overnight using the indicated primary antibodies, followed by detection with the related secondary antibody and the Super Signal chemiluminescence kit (Thermo Fisher). Primary antibodies used in this study are anti-CKIP-1 (Santa Cruz, anti-JNK1, anti-pJNK1 (Abcam), antipIRS1 (Ser307) (Abcam) and anti-GAPDH (MBL). GAPDH was used as an internal control. The relative band intensity in the scanned images was analyzed with Scion Image software.
3.2. CKIP-1 deficiency aggravates the degree of fatty liver in mice fed with HFD After 8 weeks of HFD consumption, lipid droplets were observed in liver tissues for both CKIP-1-/- and CKIP-1+/+ mice by hematoxilin & eosin 2
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Table 1. Effects of CKIP-1 deficiency on body weight and liver weight in mice fed with HFD.
Body weight Liver weight
WT
CKIP-1-/-
P
1.961 0.238
2.412 0.316
0.038 0.035
All mice were fed with HFD for 8 weeks. Data are expressed as means ± SD (n =6). The body weight and liver weight of CKIP-1-/- mice are higher than those of WT (p < 0.05).
staining. The number and size of lipid droplets in the liver of CKIP-1-/- mice are more and bigger compared to those in the WT mice (Fig. 1A). Biochemical analysis showed that the liver TG level was significantly higher in the CKIP-1-/- mice than that of WT mice (Fig. 1B). These results demonstrate that CKIP-1 deficiency can aggravate the degree of fatty liver in mice feed with HFD. Fig. 2. Serum insulin level and liver glycogen content in CKIP-1-/- and WT mice fed with HFD for 8 weeks. A: Serum insulin level. B: liver glycogen content. Data are presented as mean ± SD (n=6). The Serum insulin level and liver glycogen content of CKIP-1-/- are higher than those of WT mice (p < 0.05, respectively).
3.3. CKIP-1 deficiency increases serum insulin level and liver glycogen content in mice fed with HFD IR exhibits hyperinsulinemia and hepatic glycogen synthesis impairment. To determine the effect of CKIP-1 deficiency on RI in mice induce by HDF, we tested the serum insulin level and liver glycogen content in CKIP1-/- and WT mice. As shown in Fig. 2, CKIP-1-/- mice showed a dramatic increase in their serum insulin level compared to WT mice (Fig. 2A). The glycogen level in the liver of CKIP-1-/- mice also significantly increased compared to WT mice (Fig. 2B). These results suggest that CKIP-1 deficiency exacerbates RI induced by HFD.
3.4. CKIP-1 deficiency increases hepatic pJNK1 and pIRS1 in mice fed with HFD Western blot analysis demonstrated that there was no difference in the protein levels of hepatic JNK1 and IRS1 between CKIP-1-/- mice and WT mice. But the protein levels of pJNK1 and pIRS1 in CKIP-1-/-
Fig. 1. Liver histopathology and hepatic triglyceride concentrations in CKIP-1-/- and WT mice fed with HFD for 8 weeks. A: Representative histology graphs of hematoxylin and eosinstained hepatic sections. B: Mean hepatic triglyceride concentrations. Data are expressed as means ± SD (n =6). The hepatic triglyceride level of CKIP-1-/- mice is higher than that of WT mice (P < 0.05).
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Fig. 3. Hepatic JNK1, pJNK1 and pIRS1 in CKIP-1-/- and WT mice fed with HFD for 8 weeks. Data were presented as mean ± SD (n=6). There is no significant difference in the protein levels of hepatic JNK1 and IRS1 between CKIP-1-/- mice and WT mice (P > 0.05). The protein levels of pJNK1 and pIRS1 in CKIP-1-/- mice were significantly increased compared to those in WT mice (P < 0.05).
mice markedly increased compared to those in WT mice (Fig. 3). These results suggest that CKIP-1 deficiency affects the phosphorylation of JNK1 and IRS1. 3.5. CKIP-1 deficiency enhances JNK1 phosphorylation in hepatocytes in vitro To further confirm the effect of CKIP-1 deficiency on JNK1 phosphorylation, we isolated hepatocytes from CKIP-1-/- and WT mice and used anisomycin to stimulate the JNK1 phosphorylation. We found that the JNK1 phosphorylation of hepatocytes from CKIP-1-/- mice was higher than that of hepatocytes from WT mice (Fig. 4), further suggesting the role of CKIP-1 in regulating the JNK1 phosphorylation signaling pathway. Fig. 5. Western blot analysis of co-immunoprecipitation of CKIP-1 and JNK1 in HEK293T cells. Myc-CKIP-1 plasmids were transfected into HEK293T cells and cells were harvested after 48 h. Anti-Myc antibody was used to pull down Myc-CKIP-1 and the immunoprecipitated lysate was further blotted using anti-JNK1 antibody. IgHC represents the heavy chain of the antibody used.
3.6. Interaction between CKIP-1 and JNK1 To determine whether there is an interaction between CKIP-1 and JNK1, Co-IP was carried out using HEK293T cell lysate and specific antibodies against CKIP-1 and JNK1. As shown in Fig. 5, the CKIP-1 protein was co-immunoprecipitated with JNK1 protein by the antiJNK1 antibody. The findings suggest that there is an interaction between CKIP-1 and JNK1 in cells.
Fig. 6, CKIP-1 and JNK1 were co-localized in the cytoplasm and nucleus of HEK293T cells. The results further supported that there is an interaction between CKIP-1 and JNK1 in cells.
3.7. Location of CKIP-1 and JNK1 in cells 4. Discussion To assess the subcellular localization of CKIP-1 and JNK1, HEK293T cells were transfected with GFP-CKIP-1. As shown in
Accumulating evidence indicates that CKIP-1 is widely expressed in a variety of tissues and cells [15], and plays an important role in various critical cellular and physiological processes including cell growth, apoptosis, differentiation, cytoskeleton and bone formation [16], and has a potential therapeutic effect on cardiac hypertrophy, colonic cancer and diabetic renal disease [17–19]. Gene knockout mice have been recognized as the “gold standard” for determining whether a gene's function is essential in vivo [20]. A HFD is widely used to induce NAFLD in experimental animals [21]. Recently, we found that CKIP-1deficient mice display increased body weight and adipose tissue gains upon HFD feeding [22]. In the current study, we investigated the effect of CKIP-1 gene knockout on liver histology and the TG level using an HFD-induced NAFLD mouse model. We found that the CKIP-1 deficiency increased hepatic lipid droplet and hepatic TG level. Our results suggested that CKIP-1 has an inhibitive effect on the development of NAFLD. NAFLD is characterized mainly by the excessive accumulation of hepatocyte TG [23], which is formed from the esterification of free fatty acids (FFAs) and glycerol. Several mechanisms may lead to the
Fig. 4. CKIP-1 deficiency enhances JNK1 phosphorylation in hepatocytes in vitro. Hepatocytes were isolated from CKIP-1-/- and WT mice. The phosphorylation of JNK1 was induced by the addition of anisomycin (10 ng/ml) for 48 h. The pJNK1 protein was determined by western blot analysis. Data were presented as mean ± SD (n=6). *P < 0.05, CKIP-1-/- mice versus WT mice.
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Fig. 6. HEK293T cells were transfected with GFP-CKIP-1. At 36 h post-transfection, cells were fixed by PFA and incubated with JNK-1 antibody for immuno-fluorescence analysis. Then the cells were visualized with a confocal fluorescence microscope. CKIP-1and JNK-1 co-localize in the cytoplasm and nucleus of HEK293T cells. Green represents the CKIP-1 proteins, red represents the JNK-1 proteins and nuclei were stained with DAPI.
obesity-induced insulin resistance [42], and regulates the development of fatty liver disease [43,44]. Studies have showed that JNK1 could bind to IRS-1 [45], and JNK1 deficiency mice have decreased phosphorylation of IRS-1 at Ser307 in their liver compared to WT mice [39]. As a result, the mechanism for JNK1-mediated IR is considered to be mediated by phosphorylation of the insulin receptor adapter protein IRS1 on Ser307, a phosphorylation site that disrupts the interaction of the IRS1 phosphotyrosine binding domain with the tyrosine phosphorylated insulin receptor [12]. The activation of JNK requires dual phosphorylation at Thr183 and Tyr185 residues [46]. The activation of JNK could lead to a multitude of downstream changes in phosphorylation and transcriptional activation within cells [47]. In this study, we found that the phosphorylated JNK1 protein level in liver tissue was significantly increased in CKIP-1-/- mice compared to WT mice. We also found that with anisomycin stimulation in vitro, the phosphorylated JNK1 protein level in hepatocytes separated from the CKIP-1-/mice liver was significantly increased compared to that in WT hepatocytes. CKIP-1 deficiency did not affect the expression of total JNK1 protein in liver of mice fed with HDF or in hepatocyte treated with anisomycin. Thus, these data suggested that CKIP-1 deficiency increased JNK1 phosphorylation in liver, which possibly further leads to increased IRS-1 Ser307 phosphorylation. Activation of the JNK pathway relies on the coordinated interaction of the scaffold proteins belonging to the JNK activation complex [48]. CKIP-1 is a 46-kDa molecular scaffold protein [49]. CO-IP is a classical approach to show an interaction between two proteins [50]. We demonstrated the interaction between CKIP-1 and JNK1 by CO-IP. The finding that CKIP-1 and JNK1 co-localized in cultured cells also supports that there is an interaction between the two molecules. Taken together, we think that CKIP-1 can interact with JNK1 and further inhibit phosphorylation of JNK1. The exact mechanism of how CKIP-1 functions warrants further investigation.
hepatocyte TG accumulation: (1) increased FFAs supply due to increased lipolysis from both visceral/subcutaneous adipose tissue and/or increased intake of dietary fat; (2) decreased free fatty oxidation; (3) increased de novo hepatic lipogenesis (DNL) and (4) decreased hepatic very low density lipoprotein–triglyceride secretion [24]. IR is defined as an inadequate response to the physiologic effects of circulating insulin in specific target tissues, such as skeletal muscle, liver, and adipose tissue [25] and it has been suggested to a central role in the pathogenesis of NAFLD via the following three pathways: (1) increases the release of FFAs from white adipose tissues due to lipolysis, which results in high levels of circulating FFAs and enhanced hepatic uptake; (2) activates sterol regulatory element binding protein 1c that promote de novo lipogenesis in the liver; (3) inhibits the βoxidation of FFAs, thus further promotes hepatic triglyceride synthesis [26–29]. The liver is the key target organ for insulin activity and plays a primary role in the development of IR [30]. IR causes a compensatory hyperinsulinemia due to insulin hypersecretion [31], and leads to impaired glycogen synthesis in the liver [32]. In the present study, we found that the serum insulin level and hepatic glycogen content of CKIP-1-/- mice increased markedly compared to WT control mice. These data suggested that CKIP-1 deficiency enhanced IR therefore exacerbating HFD-induced fatty liver formation. Normally, insulin triggers the downstream signaling by binding to insulin receptor on the surface of cells, which initiates the tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins and further activates phosphatidylinositol-3-kinase [33]. The phosphorylation of serine residue on IRS downregulates normal insulin-stimulated tyrosine phosphorylation and interferes with the normal physiologic function of insulin, leading to IR. The IRS family currently consists of four proteins: IRS-1, IRS-2, IRS-3 and IRS-4 [34]. IRS-1 and its phosphorylation play a pivotal role in the insulin signaling pathway [35]. IRS-1 has more than 70 serine phosphorylation sites. Studies have demonstrated hyper-serine phosphorylation of IRS-1 on Ser302, Ser307, Ser612, and Ser632 in several insulin-resistant rodent models [36]. One serine residue located near the phosphotyrosine-binding domain on IRS-1 is Ser307 in mice, rats, and humans [37]. It was reported that the serine phosphorylation at Ser307 of IRS-1 is associated with human hepatic insulin resistance and NAFLD [38,39]. The results of the present study showed that the extent of IRS-1 Ser307 phosphorylation in liver tissue significantly increased in CKIP-1-/- mice comparing to WT mice. Our results suggested that CKIP-1 deficiency increased IRS-1 Ser307 phosphorylation in liver, which could also contribute to the increased IR. JNK is a member of the mitogen-activated protein kinase family [40]. It has two ubiquitously expressed isoforms, JNK1 and JNK2, and a tissue-specific isoform, JNK3 [41]. JNK1 plays a central role in
5. Conclusions The deficiency of CKIP-1 could aggravate HFD-induced fatty liver by upregulating JNK1 phosphorylation, IRS-1 phosphorylation, and enhancing IR (Fig. 7). This study provides insights into the effect and mechanisms of the important role of CKIP-1 on fatty liver development and can serve as a future framework to develop a better strategy to treat NAFLD.
Competing interests Those authors declare that they have no competing interests. 5
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Fig. 7. Proposed model of CKIP-1 deficiency aggravating HFD-induced fatty liver in mice. CKIP-1 inhibits the phosphorylation of JNK1. CKIP-1 deficiency increases the levels of JNK1 phosphorylation. Elevated phosphorylaed JNK1 caused IRS-1 serine phosphorylation, which inhibits the IRS-1 tyrosine phosphorylation. The inhibition of the IRS-1 tyrosine phosphorylation results in insulin resistance and subsequently aggravates fatty liver.
Acknowledgments This research was supported by the Scientific Research Common Program of Beijing Municipal Commission of Education (NO: km201510025017) and the National Natural Science Foundation of China (NO: 81570515; NO: 31470035). References [1] A. Asgharpour, S.C. Cazanave, T. Pacana, M. Seneshaw, R. Vincent, B.A. Banini, D.P. Kumar, K. Daita, H.K. Min, F. Mirshahi, P. Bedossa, X. Sun, Y. Hoshida, S.V. Koduru, D. Contaifer Jr, U.O. Warncke, D.S. Wijesinghe, A.J. Sanyal, A dietinduced animal model of non-alcoholic fatty liver disease and hepatocellular cancer, J. Hepatol. 65 (3) (2016) 579–588. [2] J.H. Ngu, G.B. Goh, Z. Poh, R. Soetikno, Managing non-alcoholic fatty liver disease, Singap. Med. J. 57 (7) (2016) 368–371. [3] Z.M. Younossi, A.B. Koenig, D. Abdelatif, Y. Fazel, L. Henry, M. Wymer, Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes, Hepatology 64 (1) (2016) 73–84. [4] Y. Zhan, F. Zhao, P. Xie, L. Zhong, D. Li, Q. Gai, L. Li, H. Wei, L. Zhang, W. An, Mechanism of the effect of glycosyltransferase GLT8D2 on fatty liver, Lipids Health Dis. 14 (1) (2015) 43. [5] M.W. Fleischman, M. Budoff, I. Zeb, D. Li, T. Foster, NAFLD prevalence differs among hispanic subgroups. The multi-ethnic study of atherosclerosis, World J. Gastroenterol. 20 (17) (2014) 4987–4993. [6] L.S. Bhatia, N.P. Curzen, P.C. Calder, C.D. Byrne, Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor?, Eur. Heart J. 33 (10) (2012) 1190–1200. [7] A. Lonardo, S. Sookoian, C.J. Pirola, G. Targher, Non-alcoholic fatty liver disease and risk of cardiovascular disease, Metabolism 65 (8) (2016) 1136–1150. [8] Y.T. Zhan, C. Zhang, L. Li, C.S. Bi, X. Song, S.T. Zhang, Non-alcoholic fatty liver disease is not related to the incidence of diabetic nephropathy in type 2 diabetes, Int. J. Mol. Sci. 13 (2012) 14698–14706. [9] G.B. Goh, C. Kwan, S.Y. Lim, N.K. Venkatanarasimha, R. Abu-Bakar, T.L. Krishnamoorthy, H.H. Shim, K.H. Tay, W.C. Chow, Perceptions of nonalcoholic fatty liver disease - an Asian community-based study, Gastroenterol. Rep. (Oxf.) 4 (2) (2016) 131–135. [10] R. Lomonaco, C. Ortiz-Lopez, B. Orsak, A. Webb, J. Hardies, C. Darland, J. Finch, A. Gastaldelli, S. Harrison, F. Tio, K. Cusi, Effect of adipose tissue insulin resistance on metabolic parameters and liver histology in obese patients with nonalcoholic fatty liver disease, Hepatology 55 (5) (2012) 1389–1397. [11] M. Demir, S. Lang, H.M. Steffen, Nonalcoholic fatty liver disease - current status and future directions, J. Dig. Dis. 16 (10) (2015) 541–557. [12] G. Sabio, R.J. Davis, c-Jun NH2-terminal kinase 1 (JNK1): roles in metabolic
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Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr
Deficiency of CKIP-1 aggravates high-fat diet-induced fatty liver in mice Yutao Zhana,1, Ping Xieb,1, Dongnian Lic,1, Li Lia, Jing Chena, Wei And, Lingqiang Zhange, ⁎ Chuan Zhanga, a
Department of Gastroenterology, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China Physical Examination Centre, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China Department of Biochemistry and Molecular Biology, Beijing Key Laboratory of Cancer & Metastasis Research, Capital Medical University, Beijing 100069, China d Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing 100069, China e State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China b c
A R T I C L E I N F O
A BS T RAC T
Keywords: Non-alcoholic fatty liver disease (NAFLD) Casein kinase 2 interacting protein-1 (CKIP-1) c-Jun NH2-terminal kinase 1 (JNK1) Insulin receptor substrate −1(IRS-1) High fat diet (HFD)
Casein kinase 2 interacting protein-1(CKIP-1) is widely expressed in a variety of tissues and cells, and plays an important role in various critical cellular and physiological processes including cell growth, apoptosis, differentiation, cytoskeleton and bone formation. Here, we found: (1) CKIP-1 deficient mice exhibited increased body weight, liver weight, number and size of lipid droplets, and TG content comparing with WT mice after being exposed to high fat diet (HFD); (2) the levels of serum insulin, liver glycogen, phosphorylated C-Jun-Nterminal kinase-1 (pJNK1) and phosphorylated insulin receptor substrate −1(pIRS1) in CKIP-1-/- mice were higher than those of WT mice; (3) CKIP-1 interacted with JNK1 in vitro. Our results indicate that CKIP-1 deficiency in mice aggravates HFD-induced fatty liver by upregulating JNK1 phosphorylation and further upregulating IRS-1 phosphorylation and RI.
1. Introduction Non-alcoholic fatty liver disease (NAFLD), with a 25% global prevalence, is the most common chronic liver disease in the world [1–3]. With an increase of the population with obesity and diabetes, the incidence of NAFLD will increase further in the future, with an expected 50% prevalence in the US by 2030 [4,5]. A growing body of evidence suggests that NAFLD is a potential independent risk factor for cardiovascular disease, which is the leading cause of mortality among NAFLD patients [6,7]. In addition, some forms of NAFLD (especially non-alcoholic steatohepatitis) can progress to fibrosis, cirrhosis and hepatocellular carcinoma (HCC) [8], and NALFD is predicted to be the leading cause of end-stage liver disease requiring liver transplantation by 2020 [9]. Despite the emergence of NAFLD and the health burden caused by this epidemic, no pharmacological therapy has been developed to treat NAFLD specifically partially because of our incomplete understanding of its pathogenesis. We therefore aimed to study NAFLD at the mechanistic level to provide a future framework for developing a potential effective treatment. Insulin resistance (IR) plays a key role in the pathogenesis of NAFLD by enhancing the uptake and synthesis of fatty acids and
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inhibiting the β-oxidation of fatty acids in hepatocytes [10,11]. IR is mediated by tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), the insulin receptor adapter protein, and C-Jun-N-terminal kinase-1 (JNK1), a member of the mitogen-activated protein kinase family. It is known that JNK1 deficiency protects mice against HFDinduced IR. JNK1 induces the phosphorylation of IRS-1 on Ser-307, which disrupts the interaction of the phosphotyrosine binding domain of IRS-1 with the tyrosine phosphorylated insulin receptor [12]. The PH-domain-containing casein kinase 2 interacting protein-1 (CKIP-1) was originally identified as an interacting partner of the α-subunit of casein kinase 2 [13]. Recently, we showed that the targeted deletion of CKIP-1 causes the upregulation of phosphorylated JNK1 without changing the total JNK1 in mouse embryonic fibroblasts [14]. We therefore speculated that CKIP-1 might play a role in the development of NAFLD via regulating JNK1 phosphorylation, IRS-1 phosphorylation, and IR. In this study, we demonstrated that CKIP-1 deficiency exacerbates fatty liver induction in mice fed with high-fat diet (HFD). In addition, the levels of serum insulin, liver glycogen, phosphorylated JNK1 (pJNK1), and phosphorylated IRS1 (pIRS1) also increased in CKIP-1 deficient mice. Finally, we found there was an interaction between CKIP-1 and JNK1 in hepatocytes.
Corresponding author. E-mail address: [email protected] (C. Zhang). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.yexcr.2017.03.033 Received 7 November 2016; Received in revised form 8 March 2017; Accepted 16 March 2017 0014-4827/ © 2017 Published by Elsevier Inc.
Please cite this article as: Zhan, Y., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.03.033
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2. Material and methods
2.7. Analysis of pJNK1 in isolated hepatocyte
2.1. Animals
Hepatocytes were isolated by collagenase digestion method. Briefly, 1 weeks old CKIP-1-/- and WT mice were killed to obtain the liver tissues. The liver sample was washed 3 times with PBS, then minced and digested by 0.02% IV collagenase for 20 min at 37 °C. The suspension was centrifuged at 1000 rpm for 10 min. The obtained hepatocytes were collected and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells were treated with anisomycin (10 ng/ml) for 48 h to induce JNK1 phosphorylation. Then, the cells were collected for testing pJNK1 protein by western blot analyses as above.
CKIP-1 knockout (CKIP-1−/−) mice in C57BL/6 background were prepared as previously described [14], and CKIP-1+/+(wild-type, WT) C57BL/6 mice were used as control. The mice were housed under pathogen-free conditions in a temperature-controlled animal facility with a 12 h light/dark illumination cycle. All procedures for handling animals were in accordance with protocols approved by Chinese Academy of Military Medicine Science. CKIP-1−/− mice and the WT control mice were fed with an HFD (D12492, 60% of kilocalories from fat; Research Diet) for 8 weeks. Their body weight was measured at the beginning and the end of the experiment. Mice were then sacrificed, their liver tissues were weighed, and blood serum samples were collected. The liver tissues were frozen in liquid nitrogen and maintained at −80 °C until further analyses. A portion of each liver was fixed in 10% buffered formalin for histopathology. The collected blood samples were kept on ice for 30 min and then separated into serum by centrifugation at 4◦C. The serum was then kept at −20 °C until the examination.
2.8. Co-immunoprecipitation (Co-IP) The human embryonic kidney (HEK293T) cells were transfected with CKIP-1 plasmid. The CKIP-1 plasmids were constructed as described previously [14]. After 48 h, cells were harvested and lysed in HEPES (N-(2hydroxyethyl) piperazine-N′-2-ethanesulfonic acid) lysis buffer (20 mM HEPES, 50 mM NaCl, 0.5% Triton X-100, 1 mM NaF and 1 mM dithiothreitol). Lysates were incubated with either anti-CKIP-1 or anti-JNK1 antibody overnight at 4 °C in the presence of 50 µl Protein-G/A beads. Beads were collected, washed, and resuspended in equal volumes of 5×SDS loading buffer. The immunoprecipitated proteins were separated by SDSPAGE and transferred onto PVDF membrane. The membrane was blocked with 5% skim milk, incubated at 4 °C overnight with indicated antibodies, and followed by detection with the related secondary antibody and the Super Signal chemiluminescence kit (ThermoFisher).
2.2. Analysis of blood insulin Serum insulin was measured using Ultra Sensitive Mouse Insulin ELISA Kit (Shanghai Elisa Biotech Co., Ltd., China) according to manufacturer's instructions. 2.3. Liver histopathology test
2.9. Confocal microscopy, immuno-fluorescence analysis
After embedded in paraffin, the live tissues were cut into 5micrometer thin sections and processed for hematoxylin and eosin staining to evaluate the extent of lipid accumulation in hepatocytes.
For immuno-staining of endogenous JNK-1, HEK293T cells transfected with GFP-CKIP-1 were fixed in 4% PFA (paraformaldehyde) for 10 min. Images of endogenous JNK-1 were then fixed in 0.1% PBST (containing 0.5% Triton X-100) for 15 min. Further processing included incubating cells in 5% BSA for 30 min before incubations with primary for 3 h at 37 °C and with secondary antibody for 1 h at room temperature. Cells were analyzed in PBS when the nucleus was stained with 0.1 g/ml DAPI. Images of fixed cells were acquired on a confocal microscope using LaserSharp software.
2.4. Analysis of Liver glycogen content 400 µl of 5% trichloroacetic acid was added into 150 mg of liver tissue. After a centrifugation at 4000 rpm for 3 min, the supernatant was mixed with 400 µl of 95% ethanol. The precipitate was then removed by centrifugation at 4000 rpm for 5 min. The amount of glycogen in the digested samples was measured using a Glycogen Assay Kit (Sigma), following the manufacturer's instructions.
2.10. Statistical Analysis 2.5. Analysis of liver triglyceride (TG) content The results were expressed as the mean ± SD. The significance of differences was determined by t-test using the SPSS 17.0 software (SPSS, Chicago, IL, USA). Difference with a p value of less than 0.05 was considered statistically significant.
50 mg frozen liver tissue was homogenized in 1 ml of tissue lysis solution (20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1% Triton). After centrifugation at 2000 rpm for 5 min at 37◦C, the supernatants were used to determinate the liver TG levels using an EnzyChrom™ TG assay kit (Kampenhout, Belgium) and normalized by the protein concentration, according to the protocol provided by the manufacturer. Data were expressed as milligrams of TG per gram of liver.
3. Results 3.1. CKIP-1 deficiency increases body weight and liver weight in mice fed with HFD
2.6. Western blot analyses At the end of experiment, the body weight of CKIP-1-/- mice significantly increased compared to those of WT mice (p < 0.05) (Table 1), showing that the deficiency of the CKIP-1 gene enhances the degree of obesity in mice fed with HFD. The liver weight of CKIP1-/- mice also significantly increased compared to those of WT mice (p < 0.05) (Table 1), suggesting that the CKIP-1 deficiency probably increases the degrees of fatty liver.
Liver lysates were prepared by homogenization of liver samples with RIPA buffer containing 1% phosphatase inhibitor. Protein concentrations were measured using the BCA assay. Protein from each sample was separated by 10% SDS-PAGE and electro-transferred to nitrocellulose filter membranes. The membranes were blocked with 5% skim milk, incubated at 4 °C overnight using the indicated primary antibodies, followed by detection with the related secondary antibody and the Super Signal chemiluminescence kit (Thermo Fisher). Primary antibodies used in this study are anti-CKIP-1 (Santa Cruz, anti-JNK1, anti-pJNK1 (Abcam), antipIRS1 (Ser307) (Abcam) and anti-GAPDH (MBL). GAPDH was used as an internal control. The relative band intensity in the scanned images was analyzed with Scion Image software.
3.2. CKIP-1 deficiency aggravates the degree of fatty liver in mice fed with HFD After 8 weeks of HFD consumption, lipid droplets were observed in liver tissues for both CKIP-1-/- and CKIP-1+/+ mice by hematoxilin & eosin 2
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Table 1. Effects of CKIP-1 deficiency on body weight and liver weight in mice fed with HFD.
Body weight Liver weight
WT
CKIP-1-/-
P
1.961 0.238
2.412 0.316
0.038 0.035
All mice were fed with HFD for 8 weeks. Data are expressed as means ± SD (n =6). The body weight and liver weight of CKIP-1-/- mice are higher than those of WT (p < 0.05).
staining. The number and size of lipid droplets in the liver of CKIP-1-/- mice are more and bigger compared to those in the WT mice (Fig. 1A). Biochemical analysis showed that the liver TG level was significantly higher in the CKIP-1-/- mice than that of WT mice (Fig. 1B). These results demonstrate that CKIP-1 deficiency can aggravate the degree of fatty liver in mice feed with HFD. Fig. 2. Serum insulin level and liver glycogen content in CKIP-1-/- and WT mice fed with HFD for 8 weeks. A: Serum insulin level. B: liver glycogen content. Data are presented as mean ± SD (n=6). The Serum insulin level and liver glycogen content of CKIP-1-/- are higher than those of WT mice (p < 0.05, respectively).
3.3. CKIP-1 deficiency increases serum insulin level and liver glycogen content in mice fed with HFD IR exhibits hyperinsulinemia and hepatic glycogen synthesis impairment. To determine the effect of CKIP-1 deficiency on RI in mice induce by HDF, we tested the serum insulin level and liver glycogen content in CKIP1-/- and WT mice. As shown in Fig. 2, CKIP-1-/- mice showed a dramatic increase in their serum insulin level compared to WT mice (Fig. 2A). The glycogen level in the liver of CKIP-1-/- mice also significantly increased compared to WT mice (Fig. 2B). These results suggest that CKIP-1 deficiency exacerbates RI induced by HFD.
3.4. CKIP-1 deficiency increases hepatic pJNK1 and pIRS1 in mice fed with HFD Western blot analysis demonstrated that there was no difference in the protein levels of hepatic JNK1 and IRS1 between CKIP-1-/- mice and WT mice. But the protein levels of pJNK1 and pIRS1 in CKIP-1-/-
Fig. 1. Liver histopathology and hepatic triglyceride concentrations in CKIP-1-/- and WT mice fed with HFD for 8 weeks. A: Representative histology graphs of hematoxylin and eosinstained hepatic sections. B: Mean hepatic triglyceride concentrations. Data are expressed as means ± SD (n =6). The hepatic triglyceride level of CKIP-1-/- mice is higher than that of WT mice (P < 0.05).
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Fig. 3. Hepatic JNK1, pJNK1 and pIRS1 in CKIP-1-/- and WT mice fed with HFD for 8 weeks. Data were presented as mean ± SD (n=6). There is no significant difference in the protein levels of hepatic JNK1 and IRS1 between CKIP-1-/- mice and WT mice (P > 0.05). The protein levels of pJNK1 and pIRS1 in CKIP-1-/- mice were significantly increased compared to those in WT mice (P < 0.05).
mice markedly increased compared to those in WT mice (Fig. 3). These results suggest that CKIP-1 deficiency affects the phosphorylation of JNK1 and IRS1. 3.5. CKIP-1 deficiency enhances JNK1 phosphorylation in hepatocytes in vitro To further confirm the effect of CKIP-1 deficiency on JNK1 phosphorylation, we isolated hepatocytes from CKIP-1-/- and WT mice and used anisomycin to stimulate the JNK1 phosphorylation. We found that the JNK1 phosphorylation of hepatocytes from CKIP-1-/- mice was higher than that of hepatocytes from WT mice (Fig. 4), further suggesting the role of CKIP-1 in regulating the JNK1 phosphorylation signaling pathway. Fig. 5. Western blot analysis of co-immunoprecipitation of CKIP-1 and JNK1 in HEK293T cells. Myc-CKIP-1 plasmids were transfected into HEK293T cells and cells were harvested after 48 h. Anti-Myc antibody was used to pull down Myc-CKIP-1 and the immunoprecipitated lysate was further blotted using anti-JNK1 antibody. IgHC represents the heavy chain of the antibody used.
3.6. Interaction between CKIP-1 and JNK1 To determine whether there is an interaction between CKIP-1 and JNK1, Co-IP was carried out using HEK293T cell lysate and specific antibodies against CKIP-1 and JNK1. As shown in Fig. 5, the CKIP-1 protein was co-immunoprecipitated with JNK1 protein by the antiJNK1 antibody. The findings suggest that there is an interaction between CKIP-1 and JNK1 in cells.
Fig. 6, CKIP-1 and JNK1 were co-localized in the cytoplasm and nucleus of HEK293T cells. The results further supported that there is an interaction between CKIP-1 and JNK1 in cells.
3.7. Location of CKIP-1 and JNK1 in cells 4. Discussion To assess the subcellular localization of CKIP-1 and JNK1, HEK293T cells were transfected with GFP-CKIP-1. As shown in
Accumulating evidence indicates that CKIP-1 is widely expressed in a variety of tissues and cells [15], and plays an important role in various critical cellular and physiological processes including cell growth, apoptosis, differentiation, cytoskeleton and bone formation [16], and has a potential therapeutic effect on cardiac hypertrophy, colonic cancer and diabetic renal disease [17–19]. Gene knockout mice have been recognized as the “gold standard” for determining whether a gene's function is essential in vivo [20]. A HFD is widely used to induce NAFLD in experimental animals [21]. Recently, we found that CKIP-1deficient mice display increased body weight and adipose tissue gains upon HFD feeding [22]. In the current study, we investigated the effect of CKIP-1 gene knockout on liver histology and the TG level using an HFD-induced NAFLD mouse model. We found that the CKIP-1 deficiency increased hepatic lipid droplet and hepatic TG level. Our results suggested that CKIP-1 has an inhibitive effect on the development of NAFLD. NAFLD is characterized mainly by the excessive accumulation of hepatocyte TG [23], which is formed from the esterification of free fatty acids (FFAs) and glycerol. Several mechanisms may lead to the
Fig. 4. CKIP-1 deficiency enhances JNK1 phosphorylation in hepatocytes in vitro. Hepatocytes were isolated from CKIP-1-/- and WT mice. The phosphorylation of JNK1 was induced by the addition of anisomycin (10 ng/ml) for 48 h. The pJNK1 protein was determined by western blot analysis. Data were presented as mean ± SD (n=6). *P < 0.05, CKIP-1-/- mice versus WT mice.
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Fig. 6. HEK293T cells were transfected with GFP-CKIP-1. At 36 h post-transfection, cells were fixed by PFA and incubated with JNK-1 antibody for immuno-fluorescence analysis. Then the cells were visualized with a confocal fluorescence microscope. CKIP-1and JNK-1 co-localize in the cytoplasm and nucleus of HEK293T cells. Green represents the CKIP-1 proteins, red represents the JNK-1 proteins and nuclei were stained with DAPI.
obesity-induced insulin resistance [42], and regulates the development of fatty liver disease [43,44]. Studies have showed that JNK1 could bind to IRS-1 [45], and JNK1 deficiency mice have decreased phosphorylation of IRS-1 at Ser307 in their liver compared to WT mice [39]. As a result, the mechanism for JNK1-mediated IR is considered to be mediated by phosphorylation of the insulin receptor adapter protein IRS1 on Ser307, a phosphorylation site that disrupts the interaction of the IRS1 phosphotyrosine binding domain with the tyrosine phosphorylated insulin receptor [12]. The activation of JNK requires dual phosphorylation at Thr183 and Tyr185 residues [46]. The activation of JNK could lead to a multitude of downstream changes in phosphorylation and transcriptional activation within cells [47]. In this study, we found that the phosphorylated JNK1 protein level in liver tissue was significantly increased in CKIP-1-/- mice compared to WT mice. We also found that with anisomycin stimulation in vitro, the phosphorylated JNK1 protein level in hepatocytes separated from the CKIP-1-/mice liver was significantly increased compared to that in WT hepatocytes. CKIP-1 deficiency did not affect the expression of total JNK1 protein in liver of mice fed with HDF or in hepatocyte treated with anisomycin. Thus, these data suggested that CKIP-1 deficiency increased JNK1 phosphorylation in liver, which possibly further leads to increased IRS-1 Ser307 phosphorylation. Activation of the JNK pathway relies on the coordinated interaction of the scaffold proteins belonging to the JNK activation complex [48]. CKIP-1 is a 46-kDa molecular scaffold protein [49]. CO-IP is a classical approach to show an interaction between two proteins [50]. We demonstrated the interaction between CKIP-1 and JNK1 by CO-IP. The finding that CKIP-1 and JNK1 co-localized in cultured cells also supports that there is an interaction between the two molecules. Taken together, we think that CKIP-1 can interact with JNK1 and further inhibit phosphorylation of JNK1. The exact mechanism of how CKIP-1 functions warrants further investigation.
hepatocyte TG accumulation: (1) increased FFAs supply due to increased lipolysis from both visceral/subcutaneous adipose tissue and/or increased intake of dietary fat; (2) decreased free fatty oxidation; (3) increased de novo hepatic lipogenesis (DNL) and (4) decreased hepatic very low density lipoprotein–triglyceride secretion [24]. IR is defined as an inadequate response to the physiologic effects of circulating insulin in specific target tissues, such as skeletal muscle, liver, and adipose tissue [25] and it has been suggested to a central role in the pathogenesis of NAFLD via the following three pathways: (1) increases the release of FFAs from white adipose tissues due to lipolysis, which results in high levels of circulating FFAs and enhanced hepatic uptake; (2) activates sterol regulatory element binding protein 1c that promote de novo lipogenesis in the liver; (3) inhibits the βoxidation of FFAs, thus further promotes hepatic triglyceride synthesis [26–29]. The liver is the key target organ for insulin activity and plays a primary role in the development of IR [30]. IR causes a compensatory hyperinsulinemia due to insulin hypersecretion [31], and leads to impaired glycogen synthesis in the liver [32]. In the present study, we found that the serum insulin level and hepatic glycogen content of CKIP-1-/- mice increased markedly compared to WT control mice. These data suggested that CKIP-1 deficiency enhanced IR therefore exacerbating HFD-induced fatty liver formation. Normally, insulin triggers the downstream signaling by binding to insulin receptor on the surface of cells, which initiates the tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins and further activates phosphatidylinositol-3-kinase [33]. The phosphorylation of serine residue on IRS downregulates normal insulin-stimulated tyrosine phosphorylation and interferes with the normal physiologic function of insulin, leading to IR. The IRS family currently consists of four proteins: IRS-1, IRS-2, IRS-3 and IRS-4 [34]. IRS-1 and its phosphorylation play a pivotal role in the insulin signaling pathway [35]. IRS-1 has more than 70 serine phosphorylation sites. Studies have demonstrated hyper-serine phosphorylation of IRS-1 on Ser302, Ser307, Ser612, and Ser632 in several insulin-resistant rodent models [36]. One serine residue located near the phosphotyrosine-binding domain on IRS-1 is Ser307 in mice, rats, and humans [37]. It was reported that the serine phosphorylation at Ser307 of IRS-1 is associated with human hepatic insulin resistance and NAFLD [38,39]. The results of the present study showed that the extent of IRS-1 Ser307 phosphorylation in liver tissue significantly increased in CKIP-1-/- mice comparing to WT mice. Our results suggested that CKIP-1 deficiency increased IRS-1 Ser307 phosphorylation in liver, which could also contribute to the increased IR. JNK is a member of the mitogen-activated protein kinase family [40]. It has two ubiquitously expressed isoforms, JNK1 and JNK2, and a tissue-specific isoform, JNK3 [41]. JNK1 plays a central role in
5. Conclusions The deficiency of CKIP-1 could aggravate HFD-induced fatty liver by upregulating JNK1 phosphorylation, IRS-1 phosphorylation, and enhancing IR (Fig. 7). This study provides insights into the effect and mechanisms of the important role of CKIP-1 on fatty liver development and can serve as a future framework to develop a better strategy to treat NAFLD.
Competing interests Those authors declare that they have no competing interests. 5
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Fig. 7. Proposed model of CKIP-1 deficiency aggravating HFD-induced fatty liver in mice. CKIP-1 inhibits the phosphorylation of JNK1. CKIP-1 deficiency increases the levels of JNK1 phosphorylation. Elevated phosphorylaed JNK1 caused IRS-1 serine phosphorylation, which inhibits the IRS-1 tyrosine phosphorylation. The inhibition of the IRS-1 tyrosine phosphorylation results in insulin resistance and subsequently aggravates fatty liver.
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