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TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK Ye-Kuan Wu a, b, c, d, 1, Lin-Feng Hu b, c, d, 1, De-Shuai Lou c, d, 1, Bo-Chu Wang a, b, *, 1, Jun Tan c, d, ** a
Postdoctoral Research Station of Biology, Chongqing University, Chongqing, 400030, China Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400030, China c Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of Education, Chongqing, 400067, PR China d Research Center of Brain Intellectual Promotion and Development for Children Aged 0-6 Years, Chongqing University of Education, Chongqing, 400067, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 December 2019 Accepted 3 January 2020 Available online xxx
Nonalcoholic fatty liver disease (NAFLD) is featured by hepatic steatosis, insulin resistance, lipid deposition and inflammation. However, the pathogenic mechanism of NAFLD is still poorly understood. Dualspecificity phosphatase 16 (DUSP16), a c-Jun N-terminal kinase-specific phosphatase, has been reported to negatively modulate the mitogen-activated protein kinases (MAPKs) signaling, and it has never been investigated in NAFLD progression. In the study, we identified that DUSP16 could directly interact with TAK1 in human hepatocytes. DUSP16 knockdown in the isolated primary hepatocytes stimulated by palmitate (PA) showed accelerated lipid deposition and inflammatory response, along with the exacerbated activation of c-Jun NH2-terminal kinase (JNK), Transforming growth factor b (TGF-b)-activated kinase (TAK1) and nuclear factor-kB (NF-kB) signaling pathways; however, the opposite results were detected in PA-treated hepatocytes with DUSP16 over-expression. The in vivo experiments confirmed that DUSP16 knockout significantly aggravated the metabolic disorder and insulin resistance in high fat diet (HFD)-challenged mice. In addition, HFD-provoked hepatic lipid accumulation and inflammation were further promoted in mice with DUSP16 knockout through the same molecular mechanism as detected in vitro. Herein, these findings demonstrated that DUSP16 could directly interact with TAK1 and negatively regulate JNK signaling to alleviate metabolic stress-induced hepatic steatosis, and thus could be considered as a promising new molecular target for NAFLD treatment. © 2020 Published by Elsevier Inc.
Keywords: NAFLD DUSP16 TAK1 JNK Lipid deposition and inflammation
1. Introduction Nonalcoholic fatty liver disease (NAFLD) is ranged from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH),
* Corresponding author. Postdoctoral Research Station of Biology, Chongqing University, Chongqing, 400030, China. ** Corresponding author. Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of Education, Chongqing, 400067, PR China. E-mail addresses: [email protected] (B.-C. Wang), [email protected] (J. Tan). 1 These authors contributed equally to this work.
which is the leading cause of chronic liver disease [1]. About 6e30% of patients with NAFLD progress from simple steatosis to NASH, characterized by significant lipid deposition, inflammatory response and fibrosis [2]. The most common liver-associated complications of NASH that occur over time are cirrhosis and hepatocellular carcinoma (HCC) [3]. NASH and its complications, such as insulin resistance, hyperglycemia and obesity, also promote the risk of cardiovascular diseases, increasing the cardiovascular morbidity and mortality eventually [1,4,5]. However, treatments aimed to alleviate the progression of NAFLD have advanced at a slow pace, which is associated with the insufficient understanding of the signaling revealing NAFLD pathogenesis [6]. Thus, it is still necessary to investigate the potential molecular mechanisms
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Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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contributing to the NAFLD progression, as well as the associated abnormal metabolism. Dual-specificity phosphatases (DUSPs) have been reported as a group of proteins that dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within one substrate, which are critical regulators of central signaling pathways [7,8]. DUSP MAPK phosphatases contain a MAPK-binding domain, which determines their selectivity for different substrate MAPKs. For instance, DUSP1 could be strongly induced by Toll-like Receptor (TLR) signals and reduces p38 activation, thus regulating the expression of a subset of TLR-triggered inflammatory genes [9]. Although effects of DUSPs on tumor, immune response and metabolic disorders are well documented, they are still not fully understood [8,10]. DUSP16, as a member of DUSPs family, could be inducible in macrophages [11]. Some transcript variants of DUSP16 has been indicated to encode the MAPK-binding and catalytic domains of the protein, and suggested to negatively regulate JNK activity [12]. JNK plays a critical role in regulating insulin resistance, lipid deposition and inflammatory response during the progression of NAFLD [13,14]. Therefore, we hypothesized that DUSP16 might be involved in the pathogenesis of nonalcoholic steatohepatitis. However, the effects of DUSP16 on NAFLD need further exploration. In this study, we found that DUSP16 could directly interact with TAK1 in hepatocytes, and reduced the activation of TAK1. The in vitro analysis showed that DUSP16 knockdown significantly accelerated lipid accumulation and inflammation in PA-treated hepatocytes, along with markedly promoted activation of TAK1 and JNK. The contrast results were observed in PA-incubated hepatocytes with DUSP16 over-expression. The in vivo results confirmed that DUSP16 knockout significantly exacerbated hepatic steatosis by accelerating dyslipidemia and inflammatory response. Therefore, DUSP16/TAK1 signaling might be a promising approach for the treatment of metabolic stress-induced hepatic steatosis.
antibodies (Abcam, USA). The protein expression levels were analyzed using the NIH Image J software (Bethesda, USA) and normalized to the expression of GAPDH. 2.3. Real time-quantitative PCR (RT-qPCR) Total RNA was isolated from cells and liver samples using Trizol reagent (Invitrogen) following the manufacturer’s procedures. The reverse transcription reaction of mRNAs was then prepared. The primers used in the study were shown in Supplementary Table 2. The reverse transcription reaction products were amplified using RT-qPCR with the iTaqTM Universal SYBR Green Supermix (Bio-Rad, USA) and each primer. Data were recorded and expressed as a function of threshold cycle (Ct). Relative expression of genes was calculated using the Ct (2 DDCt) method. The mRNAs were normalized to GAPDH. 2.4. Plasmid constructs The sequences of DUSP16 and TAK1 were amplified using PCR with human cDNA as a template. The PCR-amplified fragments were then inserted into the pcDNA5 expression vector. GST-tagged DUSP16 (GST-DUSP16) and GST-TAK1 were obtained through cloning DUSP16or TAK1 cDNA into the pGEX-4T-1 vector. 2.5. Co-immunoprecipitation (Co-IP) analysis IP was performed as previously described [16]. In brief, after transient transfection with plasmid, L02 cells were lysed in ice-cold IP buffer (Thermo Fisher Scientific, USA). The cell lysates were incubated with the indicated antibody-conjugated beads (Thermo Fisher Scientific) at 4 C overnight. The immune complexes were then subjected to immunoblotting with the indicated primary and corresponding secondary antibodies.
2. Materials and methods 2.6. Glutathione S-transferase (GST) pull-down analysis 2.1. Cells and culture Human hepatocyte cell line L02 was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Primary hepatocytes were isolated from 6-week-old male mice through liver perfusion as previously indicated [15]. L02 cells and the obtained hepatocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin in a 5% CO2/water-saturated incubator at 37 C. Palmitate (PA, SigmaAldrich, USA) was subjected to cells to establish the in vitro model of lipid accumulation in hepatocytes. Lipid droplet accumulation in cells was determined using Oil Red O solution (Solarbio, Beijing, China) according to the manufacturers’ protocols. DUSP16 siRNA (si-DUSP16) and the negative control siRNA (si-NC) were purchased from Santa Cruz Biotechnology (USA). The DUSP16 expression plasmid (pcDNA3.1-DUSP16) and the empty vector were bought from RiboBio (Guangzhou, China). All si-DUSP16 and pcDNA3.1-DUSP16 were transfected to cells utilizing Lipofectamine® 2000 (Invitrogen) following the manufacturers’ advice. 2.2. Western blotting Proteins were isolated from cells or liver tissues using RIAP lysis buffer (Beyotime. Nanjing, China), separated by the 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). After blocking in 5% skim milk, the membranes were incubated with primary antibodies (Supplementary Table 1) at 4 C overnight, followed by incubation with the corresponding secondary
Rosetta (DE3) Escherichia coli were separately transformed with HA-GST-DUSP16- and HA-GST-TAK1-encoding plasmids, which were induced using isopropyl-b-D-thiogalactopyranoside (0.5 mM) after the culture reached an optical density (OD) of 0.8 at 600 nm. Then, the isolated proteins were eluted, resolved through SDSPAGE and analyzed using western blotting with the displayed antibodies. 2.7. Biochemical parameters calculation in vitro and in vivo Liver functions were measured in mice by calculating the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). TG and TC levels in cells or liver samples were measured using commercial kits also purchased from Serum blood glucose levels were measured with o-toluidine reagent (Sigma Aldrich) following the instructions recommended by the manufacturer. Serum insulin levels were measured with an enzyme-linked immunosorbent assay (ELISA) kit (Sigma Aldrich) specific for mouse insulin according to the manufacturer’s protocols. Homeostasis model assessment (HOMA)-IR was determined from fasting glucose and insulin levels in serum as previously indicated [17]. 2.8. Animals and treatments All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, issued by the
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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National Institutes of Health in 1996. The Institutional Animal Care and Use Committee at Chongqing University approved the in vivo animal study procedures. The wild type (WT), male, 6-week-old, C57BL/6 mice (20 ± 2 g body weight) were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China). The DUSP16 knockout (KO), male, 6-week-old, mice (20 ± 2 g body weight) with C57BL/6 background were generated as previously described [18,19]. 8-week-old male genetically obese mice (ob/ob, Beijing Vital River Laboratory Animal Technology) were severed as another fatty liver model. All mice were fed in a specific pathogen (SPF)-free barrier facility under controlled environment (25 ± 2 C temperature, 50 ± 5% humidity) with a standard 12 h light-dark cycle and free access to standard chow and water ad libitum in their cages. Standard normal chow contained the most important nutrients. After adaption for 1 week, all mice were randomly divided into 4 groups with 12 in each: 1) WT mice fed with normal chow diet (NCD/WT); 2) KO mice fed with NCD (NCD/KO); 3) WT mice fed with HFD (HFD/WT); and 4) KO mice fed with HFD (HFD/ KO). The model group of mice was fed a HFD (D12492; Research Diets, New Brunswick, USA) for 16 weeks. Mice fed with NCD (D12450B; Research Diets) were served as controls. To establish a fatty liver model, ob/ob mice were fed with a HFHC diet (TP 26304; TrophicDiet, Nantong, China) for 12 weeks. During the experiments, body weight and fasting blood glucose levels were measured at the indicated time points.
2.9. Metabolic indicators measurements OGTT and ITT assays were conducted to determine the in vivo insulin resistance. After fasting for 12 h, each mouse were orally given 2 g/kg of glucose. Then, blood samples were collected from the tail vein at the exhibited time. Blood glucose levels were then assessed with o-toluidine reagent (Sigma Aldrich). For ITT, mice were fasted for 8 h before intraperitoneally injected with insulin (1 U/kg body weight, Sigma Aldrich). Then, the blood glucose levels were measured at 0, 15, 30, 60 and 90 min postinjection.
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2.10. Histochemical analysis Liver sections were embedded in paraffin and stained using hematoxylin and eosin (H&E) to observe the lipid deposition and the inflammatory condition in the tissues. Oil Red O staining was used to visualize lipid droplets in the frozen liver sections. Images were acquired with a light microscope (Olympus). 2.11. Statistical analysis Data were presented as the mean ± standard error of mean (SEM). Student’s two-tailed t-test was applied to compare the means of two-group samples, and a one-way analysis of variance (ANOVA) was performed for comparison of multiple groups. P values < 0.05 were considered significant by GraphPad Prism 6.0 (San Diego, USA). 3. Results 3.1. DUSP16 directly interacts with TAK1 to restrain TAK1 activity DUSP family has been reported to directly interact with TAK1 in mammalian cells to regulate various cellular processes [20]. Through Co-IP assays, we also found that DUSP16 could physically interact with TAK1 (Fig. 1A), which was confirmed by the GST pulldown analysis (Fig. 1B). We also found that DUSP16 showed an inhibitory effect on TAK1 activation, and the suppressive effect of DUSP16 on TAK1 activation was dose-dependent (Fig. 1C). Therefore, DUSP16 could interact with TAK1, and it might be a potent inhibitor of TAK1 activity. 3.2. DUSP16 suppression accelerates dyslipidemia and inflammation in palmitate-treated hepatocytes Considering that TAK1 activation has been widely implicated in hepatic steatosis [21], we supposed that DUSP16/TAK1 signaling might be also associated with the progression of nonalcoholic steatohepatitis. PA is commonly used to establish in vitro hepatic
Fig. 1. DUSP16 directly interacts with TAK1. (A) Co-IP analysis in L02 cells transfected with Flag-tagged TAK1 and Myc-tagged DUSP16. Anti-Flag and anti-Myc antibodies were used as western blotting probes. (B) GST precipitation analysis showing direct DUSP16-TAK1 binding. Purified GST was used as a control. (C) Western blotting of p-TAK1 and total TAK1 in L02 cells transfected with different amounts of Flag-tagged DUSP16. The values were expressed as the mean ± SEM.
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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steatosis [22]. At first, RT-qPCR and western blot assays showed that DUSP16 expression both from the mRNA and protein levels were markedly reduced by PA incubation in a time-dependent manner (Fig. 2A and B), indicating that DUSP16 might have a potential regulatory role in hepatic steatosis. Then, DUSP16 was knocked down in primary hepatocytes (Fig. 2C). Obviously, Oil Red O staining showed that PA-induced lipid accumulation in cells was further accelerated when DUSP16 was inhibited (Fig. 2D). Consistently, DUSP16 knockdown further aggravated the TG
deposition in PA-incubated hepatocytes (Fig. 2E). The mRNA levels of key enzymes participating in lipid metabolism were then measured, showing significantly exacerbated mRNA levels of genes-associated with synthesis (SREBF1, FAS, SCD1, PPARg) and fatty acid uptake (FATP1, FABP1, CD36) but further reduced transcriptions of genes related to fatty acid b-oxidation (PPARa, MCAD, CPT-1a, UCP2) in DUSP16-knockdown cells in response to PA (Fig. 2F). DUSP16 could inactivate JNK signaling, which is closely related to various metabolic events [12,13]. We found that PA-
Fig. 2. DUSP16 suppression accelerates dyslipidemia and inflammation in palmitate-treated hepatocytes. (A,B) The isolated primary hepatocytes were treated with PA (0.4 mM) for the indicated time, followed by RT-qPCR and western blot assays of DUSP16. (C) The isolated primary hepatocytes were transfected with DUSP16 siRNA for 24 h to inhibit the expression of DUSP16, and western blot analysis was used for transfection efficacy analysis. (DeI) The isolated primary hepatocytes were transfected with si-DUSP16 for 24 h, and then were treated with PA (0.4 mM) for another 24 h. All cells were collected for the subsequent assays. (D) Oil Red O staining of cells. (E) TG contents in cells. (F) RT-qPCR analysis was used calculate the mRNA expression levels of genes associated with fatty acid synthesis, uptake and b-oxidation. (G) Western blot for p-JNK. (H) RT-qPCR analysis for pro-inflammatory factors in cells. (I) Western blot analysis of p-TAK1, p-IKKa, p-IkBa and peNFekB (p65) in cells. The values were expressed as the mean ± SEM. *p < 0.05 and **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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induced activation of JNK was further promoted by si-DUSP16 (Fig. 2G). Then, DUSP16 knockdown markedly enhanced the expression of genes associated with pro-inflammatory response, including TNF-a, IL-1b, IL-6, IL-4, MCP1 and CXCL1 in PAstimulated hepatocytes (Fig. 2H). Western blot assays showed that PA treatment led to significant increases in p-TAK1, p-IKKa, pIkBa and peNFekB (p65) in cells, while being further enhanced bysi-DUSP16 (Fig. 2I). Collectively, these findings demonstrated that DUSP16 suppression might be involved in lipid accumulation and inflammation in PA-treated hepatocytes. 3.3. DUSP16 over-expression improves dyslipidemia and inflammatory response in hepatocytes treated with palmitate To confirm the effects of DUSP16 on hepatic steatosis, we overexpressed DUSP16 in hepatocytes (Fig. 3A). Oil Red O staining showed that PA-induced lipid deposition in hepatocytes was
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apparently alleviated when DUSP16 was over-expressed, and these results were accompanied with reduced TG contents (Fig. 3B and C). In addition, PA-stimulated mRNA levels of genes-associated with synthesis (SREBF1, FAS, SCD1, PPARg) and fatty acid uptake (FATP1, FABP1, CD36) were markedly abrogated by DUSP16 overexpression, but the contrast transcriptions of genes associated with fatty acid b-oxidation (PPARa, MCAD, CPT-1a, UCP2) were observed (Fig. 3D). Subsequently, DUSP16 over-expression significantly abolished the activation of JNK in PA-incubated hepatocytes (Fig. 3E). The mRNA expression levels of TNF-a, IL-1b, IL-6, IL-4, MCP1 and CXCL1 were highly reduced by DUSP16 over-expression in PA-treated cells when compared to the oe-EV group (Fig. 3F). As displayed in Fig. 3G, PA-potentiated expression of p-TAK1, p-IKKa, p-IkBa and peNFekB (p65) was obviously attenuated in hepatocytes transfected with oe-DUSP16. Collectively, the findings here showed that DUSP16 exhibited protective effects against dyslipidemia and inflammation induced by metabolic stress.
Fig. 3. DUSP16 over-expression improves dyslipidemia and inflammatory response in hepatocytes treated with palmitate. (A) The isolated primary hepatocytes were transfected with pcDNA3.1-DUSP16 for 24 h to over-express DUSP16, and then the western blotting was used for transfection efficiency. (BeG) The isolated primary hepatocytes were transfected with pcDNA3.1-DUSP16 for 24 h, followed by PA (0.4 mM) treatment for another 24 h. Then, all cells were collected for the following studies. (B) Oil Red O staining of cells. (C) TG levels in cells were measured. (D) RT-qPCR assays for the calculation of genes related to fatty acid synthesis, uptake and b-oxidation. (E) Western blot analysis of pJNK in cells. (F) RT-qPCR assays of genes associated with inflammatory response. (G) Western blot assay of p-TAK1, p-IKKa, p-IkBa and peNFekB (p65) in cells. The values were expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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3.4. DUSP16 knockout enhances metabolic disorder and hepatic steatosis in HFD-fed mice In this regard, the mouse model with HFD-induced nonalcoholic
steatohepatitis was established to further investigate the regulatory role of DUSP16. As displayed in Fig. 4A, HFD-challenged mice had weaker DUSP16 expression in liver tissues than that of the NCD mice. Consistently, obviously reduced expression of DUSP16 was
Fig. 4. DUSP16 knockout enhances metabolic disorder and hepatic steatosis in HFD-fed mice. (A) Western blot analysis of DUSP16 in liver of HFD-fed mice (up panel), and in liver samples of ob/ob mice (down panel). (B) Body weight of mice. Calculation of (C) fasting blood glucose levels, (D) fasting insulin levels and (E) HOMA-IR. (F) OGTT (left panel) and ITT (right panel) analysis for insulin resistance evaluation. (G) H&E staining (up panel) and Oil Red O staining (down panel) of liver sections. (H) Ratio of liver weight (LW) to body weight (BW). (I) ALT and AST contents in serum. (J) TG and TC levels in liver of mice. (K) RT-qPCR assays for the calculation of genes associated with fatty acid synthesis, uptake and b-oxidation. (L) RT-qPCR analysis for pro-inflammatory factors in liver tissues. (M) Western blot assay of p-TAK1, p-JNK, p-IKKa, p-IkBa and peNFekB in hepatic samples. (N) Schematic diagram of the molecular mechanisms revealing DUSP16-modulated hepatic steatosis. The values were expressed as the mean ± SEM. *p < 0.05 and **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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detected in hepatic tissues of ob/ob mice compared to the lean mice. Then, the loss-function-approach was used in animals treated with or without HFD. We found that HFD treatment resulted in a significant increase in the body weight change of mice, and notably HFD-mice with DUSP16 deletion displayed more body weight (Fig. 4B). Fasting blood glucose and insulin levels up-regulated by HFD were further accelerated in mice with DUSP16 knockout, along with markedly elevated HOMA-IR (Fig. 4CeE), demonstrating the elevated insulin resistance in mice. OGTT and ITT assays confirmed that DUSP16 deletion exacerbated HFD-induced insulin resistance (Fig. 4F). H&E and Oil Red O staining displayed accelerated steatosis, hepatocyte ballooning and enhanced lipid accumulation in the hepatic samples from HFD-fed mice (Fig. 4G). DUSP16-KO mice showed elevated ratio of LW to BW following HFD consumption (Fig. 4H). Serum ALT and AST contents induced by HFD were markedly enhanced in mice with DUSP16 deletion (Fig. 4I). Consistently, DUSP16 knockout led to severer TG and TC deposition in liver of HFD-fed mice (Fig. 4J). In response to HFD, DUSP16 deletion caused deteriorated changes in lipid metabolismassociated gene expressions (Fig. 4K). Moreover, hepatic inflammatory factors were significantly enhanced in HFD-challenged DUSP16-KO mice compared to the HFD group (Fig. 4L). Activation of TAK1, JNK, IKKa, IkBa and NF-kB signaling following HFD was also accelerated by DUSP16-knockout (Fig. 4M). Therefore, DUSP16 deletion exacerbated metabolic disorder, lipid depostion and inflammation in liver of HFD-fed mice. 4. Discussion The pathogenesis of NAFLD is complex, including a complicated reprogrammed molecular network [4,5]. However, presently, there are no effective therapeutic strategies for NAFLD, which is largely attributed to the incomplete understanding of the mechanisms of such disease. In the present study, our findings demonstrated the protective effects of DUSP16 on metabolic stress-induced hepatic steatosis, abnormal metabolism and inflammation in mice. Promoting DUSP16 expression could alleviate lipid accumulation and inflammatory response in PA-incubated hepatocytes through directly interacting with TAK1 and negatively regulating JNK activation. Herein, DUSP16 might be novel protective factor of NAFLD that inhibited dyslipidemia and inflammation. TAK1 is detrimental in various maladies, such as metabolic disorders, insulin resistance, inflammation and apoptosis in liver and hepatocytes [21,23]. Previous studies have reported that restraining TAK1 activation could attenuate hepatic steatosis, lipid deposition and inflammatory response in animals with HFDinduced NAFLD [24]. In our study, we found that DUSP16 could directly interact with TAK1, and promoting DUSP16 expression markedly repress TAK1 activity in hepatocytes induced by PA. Thus, DUSP16 might be effective for maintaining liver homeostasis, protecting against hepatic steatosis. TAK1 is a up-streaming kinase that activates NF-kB and MAPK signaling during NAFLD progression [25,26]. DUSP16 has been reported as an inactivator of JNK [12]. In our study, we also found that DUSP16 knockdown accelerated JNK activation in PA-incubated hepatocytes, while its over-expression markedly inhibited JNK, further indicating that DUSP16 showed a negative role in regulating JNK activity. Accumulating studies before have suggested that JNK suppression is protective in NAFLD [27], which strongly supported our findings. Therefore, DUSP16/ TAK1/JNK regulatory axis might be critical under these pathological conditions, which might be a novel target for developing effective therapeutic strategies against NAFLD or associated diseases. The dyslipidemia and inflammatory response play essential roles in the pathogenesis of NAFLD, and suppressing lipid accumulation and inflammation may be ideal strategy for the
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prevention or intervention of hepatic steatosis [28,29]. In our study, we found that DUSP16 inhibition significantly promoted lipid accumulation in hepatocytes and liver tissues through accelerating fatty acid synthesis and up-take, while restraining fatty acid boxidation. A previous study showed that DUSP16-deficient mice exhibited severer release of pro-inflammatory factors induced by endotoxin [30]. The inflammatory response regulated by NF-kB and JNK signaling pathways plays a pivotal role in meditating NAFLD progression [31,32]. The secretion of TNF-a, MCP-1, IL-1b, IL-6 and CXCL-1 is significantly induced by metabolic stress both in vitro and in vivo, contributing to the progression of hepatic steatosis [22,30]. Increasing studies report that JNK activation could potentiate NF-kB signaling, promoting the inflammatory response under various disease conditions, including NAFLD [33,34]. Identifying and characterizing the key regulator of NF-kB and JNK signaling pathways may elucidate the molecular mechanism underlying the pathogenesis of NAFLD and provide targets for intervention. In this study, we also found that PA- and HFD-induced inflammatory response were highly accelerated by DUSP16 suppression, as evidenced by the releases of TNF-a, IL-1b, IL-6, IL-4, MCP1 and CXCL1 through blocking NF-kB signaling, which was consistent with the expression change of p-JNK. In contrast, promoting DUSP16 expression could markedly alleviate inflammation in PA-treated hepatocytes. Therefore, we identified DUSP16 as an efficient meditator of NF-kB signaling in response to hepatic steatosis. In summary, our study demonstrated that the DUSP16 might be a novel therapeutic target for clinical intervention of NAFLD. DUSP16 could target TAK1 and negatively regulate JNK activation to modulate dyslipidemia and inflammatory response (Fig. 4N). Thus, DUSP16/TAK1/JNK axis is a potential target for the alleviation of hepatic steatosis or associated metabolic disorders. However, further studies are still necessary in future to comprehensively reveal the relationship between these signals. Acknowledgment This work was supported by Chongqing Research Program of Basic Research and Frontier Technology (cstc2018jcyjAX0371); Science and Technology Research Program of Chongqing Education Commission of China (No. KJ1601402) and Chongqing Professional Talents Plan for Innovation and Entrepreneurship Demonstration Team (CQCY201903258). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.037 Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.037. References [1] Z. Younossi, et al., Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention, Nat. Rev. Gastroenterol. Hepatol. 15 (1) (2018) 11. [2] Y. Sumida, et al., Current and future pharmacological therapies for NAFLD/ NASH, J. Gastroenterol. 53 (3) (2018) 362e376. [3] R. de Franchis, et al., Ruling out esophageal varices in NAFLD cirrhosis: can we do without endoscopy? J. Hepatol. 69 (4) (2018) 769e771. [4] G. Targher, et al., Diabetes and NAFLD. Diabetes Complications, Comorbidities and Related Disorders, 2018, pp. 1e27. [5] G. Targher, et al., Nonalcoholic fatty liver disease and chronic vascular complications of diabetes mellitus, Nat. Rev. Endocrinol. 14 (2) (2018) 99. [6] C.Y. Chang, et al., Therapy of NAFLD: antioxidants and cytoprotective agents, J. Clin. Gastroenterol. 40 (2006) S51eS60. [7] A.M. Bennett, DUSPs, twists and turns in the journey to vascular inflammation,
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK Ye-Kuan Wu a, b, c, d, 1, Lin-Feng Hu b, c, d, 1, De-Shuai Lou c, d, 1, Bo-Chu Wang a, b, *, 1, Jun Tan c, d, ** a
Postdoctoral Research Station of Biology, Chongqing University, Chongqing, 400030, China Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400030, China c Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of Education, Chongqing, 400067, PR China d Research Center of Brain Intellectual Promotion and Development for Children Aged 0-6 Years, Chongqing University of Education, Chongqing, 400067, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 December 2019 Accepted 3 January 2020 Available online xxx
Nonalcoholic fatty liver disease (NAFLD) is featured by hepatic steatosis, insulin resistance, lipid deposition and inflammation. However, the pathogenic mechanism of NAFLD is still poorly understood. Dualspecificity phosphatase 16 (DUSP16), a c-Jun N-terminal kinase-specific phosphatase, has been reported to negatively modulate the mitogen-activated protein kinases (MAPKs) signaling, and it has never been investigated in NAFLD progression. In the study, we identified that DUSP16 could directly interact with TAK1 in human hepatocytes. DUSP16 knockdown in the isolated primary hepatocytes stimulated by palmitate (PA) showed accelerated lipid deposition and inflammatory response, along with the exacerbated activation of c-Jun NH2-terminal kinase (JNK), Transforming growth factor b (TGF-b)-activated kinase (TAK1) and nuclear factor-kB (NF-kB) signaling pathways; however, the opposite results were detected in PA-treated hepatocytes with DUSP16 over-expression. The in vivo experiments confirmed that DUSP16 knockout significantly aggravated the metabolic disorder and insulin resistance in high fat diet (HFD)-challenged mice. In addition, HFD-provoked hepatic lipid accumulation and inflammation were further promoted in mice with DUSP16 knockout through the same molecular mechanism as detected in vitro. Herein, these findings demonstrated that DUSP16 could directly interact with TAK1 and negatively regulate JNK signaling to alleviate metabolic stress-induced hepatic steatosis, and thus could be considered as a promising new molecular target for NAFLD treatment. © 2020 Published by Elsevier Inc.
Keywords: NAFLD DUSP16 TAK1 JNK Lipid deposition and inflammation
1. Introduction Nonalcoholic fatty liver disease (NAFLD) is ranged from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH),
* Corresponding author. Postdoctoral Research Station of Biology, Chongqing University, Chongqing, 400030, China. ** Corresponding author. Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of Education, Chongqing, 400067, PR China. E-mail addresses: [email protected] (B.-C. Wang), [email protected] (J. Tan). 1 These authors contributed equally to this work.
which is the leading cause of chronic liver disease [1]. About 6e30% of patients with NAFLD progress from simple steatosis to NASH, characterized by significant lipid deposition, inflammatory response and fibrosis [2]. The most common liver-associated complications of NASH that occur over time are cirrhosis and hepatocellular carcinoma (HCC) [3]. NASH and its complications, such as insulin resistance, hyperglycemia and obesity, also promote the risk of cardiovascular diseases, increasing the cardiovascular morbidity and mortality eventually [1,4,5]. However, treatments aimed to alleviate the progression of NAFLD have advanced at a slow pace, which is associated with the insufficient understanding of the signaling revealing NAFLD pathogenesis [6]. Thus, it is still necessary to investigate the potential molecular mechanisms
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Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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contributing to the NAFLD progression, as well as the associated abnormal metabolism. Dual-specificity phosphatases (DUSPs) have been reported as a group of proteins that dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within one substrate, which are critical regulators of central signaling pathways [7,8]. DUSP MAPK phosphatases contain a MAPK-binding domain, which determines their selectivity for different substrate MAPKs. For instance, DUSP1 could be strongly induced by Toll-like Receptor (TLR) signals and reduces p38 activation, thus regulating the expression of a subset of TLR-triggered inflammatory genes [9]. Although effects of DUSPs on tumor, immune response and metabolic disorders are well documented, they are still not fully understood [8,10]. DUSP16, as a member of DUSPs family, could be inducible in macrophages [11]. Some transcript variants of DUSP16 has been indicated to encode the MAPK-binding and catalytic domains of the protein, and suggested to negatively regulate JNK activity [12]. JNK plays a critical role in regulating insulin resistance, lipid deposition and inflammatory response during the progression of NAFLD [13,14]. Therefore, we hypothesized that DUSP16 might be involved in the pathogenesis of nonalcoholic steatohepatitis. However, the effects of DUSP16 on NAFLD need further exploration. In this study, we found that DUSP16 could directly interact with TAK1 in hepatocytes, and reduced the activation of TAK1. The in vitro analysis showed that DUSP16 knockdown significantly accelerated lipid accumulation and inflammation in PA-treated hepatocytes, along with markedly promoted activation of TAK1 and JNK. The contrast results were observed in PA-incubated hepatocytes with DUSP16 over-expression. The in vivo results confirmed that DUSP16 knockout significantly exacerbated hepatic steatosis by accelerating dyslipidemia and inflammatory response. Therefore, DUSP16/TAK1 signaling might be a promising approach for the treatment of metabolic stress-induced hepatic steatosis.
antibodies (Abcam, USA). The protein expression levels were analyzed using the NIH Image J software (Bethesda, USA) and normalized to the expression of GAPDH. 2.3. Real time-quantitative PCR (RT-qPCR) Total RNA was isolated from cells and liver samples using Trizol reagent (Invitrogen) following the manufacturer’s procedures. The reverse transcription reaction of mRNAs was then prepared. The primers used in the study were shown in Supplementary Table 2. The reverse transcription reaction products were amplified using RT-qPCR with the iTaqTM Universal SYBR Green Supermix (Bio-Rad, USA) and each primer. Data were recorded and expressed as a function of threshold cycle (Ct). Relative expression of genes was calculated using the Ct (2 DDCt) method. The mRNAs were normalized to GAPDH. 2.4. Plasmid constructs The sequences of DUSP16 and TAK1 were amplified using PCR with human cDNA as a template. The PCR-amplified fragments were then inserted into the pcDNA5 expression vector. GST-tagged DUSP16 (GST-DUSP16) and GST-TAK1 were obtained through cloning DUSP16or TAK1 cDNA into the pGEX-4T-1 vector. 2.5. Co-immunoprecipitation (Co-IP) analysis IP was performed as previously described [16]. In brief, after transient transfection with plasmid, L02 cells were lysed in ice-cold IP buffer (Thermo Fisher Scientific, USA). The cell lysates were incubated with the indicated antibody-conjugated beads (Thermo Fisher Scientific) at 4 C overnight. The immune complexes were then subjected to immunoblotting with the indicated primary and corresponding secondary antibodies.
2. Materials and methods 2.6. Glutathione S-transferase (GST) pull-down analysis 2.1. Cells and culture Human hepatocyte cell line L02 was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Primary hepatocytes were isolated from 6-week-old male mice through liver perfusion as previously indicated [15]. L02 cells and the obtained hepatocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin in a 5% CO2/water-saturated incubator at 37 C. Palmitate (PA, SigmaAldrich, USA) was subjected to cells to establish the in vitro model of lipid accumulation in hepatocytes. Lipid droplet accumulation in cells was determined using Oil Red O solution (Solarbio, Beijing, China) according to the manufacturers’ protocols. DUSP16 siRNA (si-DUSP16) and the negative control siRNA (si-NC) were purchased from Santa Cruz Biotechnology (USA). The DUSP16 expression plasmid (pcDNA3.1-DUSP16) and the empty vector were bought from RiboBio (Guangzhou, China). All si-DUSP16 and pcDNA3.1-DUSP16 were transfected to cells utilizing Lipofectamine® 2000 (Invitrogen) following the manufacturers’ advice. 2.2. Western blotting Proteins were isolated from cells or liver tissues using RIAP lysis buffer (Beyotime. Nanjing, China), separated by the 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). After blocking in 5% skim milk, the membranes were incubated with primary antibodies (Supplementary Table 1) at 4 C overnight, followed by incubation with the corresponding secondary
Rosetta (DE3) Escherichia coli were separately transformed with HA-GST-DUSP16- and HA-GST-TAK1-encoding plasmids, which were induced using isopropyl-b-D-thiogalactopyranoside (0.5 mM) after the culture reached an optical density (OD) of 0.8 at 600 nm. Then, the isolated proteins were eluted, resolved through SDSPAGE and analyzed using western blotting with the displayed antibodies. 2.7. Biochemical parameters calculation in vitro and in vivo Liver functions were measured in mice by calculating the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). TG and TC levels in cells or liver samples were measured using commercial kits also purchased from Serum blood glucose levels were measured with o-toluidine reagent (Sigma Aldrich) following the instructions recommended by the manufacturer. Serum insulin levels were measured with an enzyme-linked immunosorbent assay (ELISA) kit (Sigma Aldrich) specific for mouse insulin according to the manufacturer’s protocols. Homeostasis model assessment (HOMA)-IR was determined from fasting glucose and insulin levels in serum as previously indicated [17]. 2.8. Animals and treatments All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, issued by the
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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National Institutes of Health in 1996. The Institutional Animal Care and Use Committee at Chongqing University approved the in vivo animal study procedures. The wild type (WT), male, 6-week-old, C57BL/6 mice (20 ± 2 g body weight) were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China). The DUSP16 knockout (KO), male, 6-week-old, mice (20 ± 2 g body weight) with C57BL/6 background were generated as previously described [18,19]. 8-week-old male genetically obese mice (ob/ob, Beijing Vital River Laboratory Animal Technology) were severed as another fatty liver model. All mice were fed in a specific pathogen (SPF)-free barrier facility under controlled environment (25 ± 2 C temperature, 50 ± 5% humidity) with a standard 12 h light-dark cycle and free access to standard chow and water ad libitum in their cages. Standard normal chow contained the most important nutrients. After adaption for 1 week, all mice were randomly divided into 4 groups with 12 in each: 1) WT mice fed with normal chow diet (NCD/WT); 2) KO mice fed with NCD (NCD/KO); 3) WT mice fed with HFD (HFD/WT); and 4) KO mice fed with HFD (HFD/ KO). The model group of mice was fed a HFD (D12492; Research Diets, New Brunswick, USA) for 16 weeks. Mice fed with NCD (D12450B; Research Diets) were served as controls. To establish a fatty liver model, ob/ob mice were fed with a HFHC diet (TP 26304; TrophicDiet, Nantong, China) for 12 weeks. During the experiments, body weight and fasting blood glucose levels were measured at the indicated time points.
2.9. Metabolic indicators measurements OGTT and ITT assays were conducted to determine the in vivo insulin resistance. After fasting for 12 h, each mouse were orally given 2 g/kg of glucose. Then, blood samples were collected from the tail vein at the exhibited time. Blood glucose levels were then assessed with o-toluidine reagent (Sigma Aldrich). For ITT, mice were fasted for 8 h before intraperitoneally injected with insulin (1 U/kg body weight, Sigma Aldrich). Then, the blood glucose levels were measured at 0, 15, 30, 60 and 90 min postinjection.
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2.10. Histochemical analysis Liver sections were embedded in paraffin and stained using hematoxylin and eosin (H&E) to observe the lipid deposition and the inflammatory condition in the tissues. Oil Red O staining was used to visualize lipid droplets in the frozen liver sections. Images were acquired with a light microscope (Olympus). 2.11. Statistical analysis Data were presented as the mean ± standard error of mean (SEM). Student’s two-tailed t-test was applied to compare the means of two-group samples, and a one-way analysis of variance (ANOVA) was performed for comparison of multiple groups. P values < 0.05 were considered significant by GraphPad Prism 6.0 (San Diego, USA). 3. Results 3.1. DUSP16 directly interacts with TAK1 to restrain TAK1 activity DUSP family has been reported to directly interact with TAK1 in mammalian cells to regulate various cellular processes [20]. Through Co-IP assays, we also found that DUSP16 could physically interact with TAK1 (Fig. 1A), which was confirmed by the GST pulldown analysis (Fig. 1B). We also found that DUSP16 showed an inhibitory effect on TAK1 activation, and the suppressive effect of DUSP16 on TAK1 activation was dose-dependent (Fig. 1C). Therefore, DUSP16 could interact with TAK1, and it might be a potent inhibitor of TAK1 activity. 3.2. DUSP16 suppression accelerates dyslipidemia and inflammation in palmitate-treated hepatocytes Considering that TAK1 activation has been widely implicated in hepatic steatosis [21], we supposed that DUSP16/TAK1 signaling might be also associated with the progression of nonalcoholic steatohepatitis. PA is commonly used to establish in vitro hepatic
Fig. 1. DUSP16 directly interacts with TAK1. (A) Co-IP analysis in L02 cells transfected with Flag-tagged TAK1 and Myc-tagged DUSP16. Anti-Flag and anti-Myc antibodies were used as western blotting probes. (B) GST precipitation analysis showing direct DUSP16-TAK1 binding. Purified GST was used as a control. (C) Western blotting of p-TAK1 and total TAK1 in L02 cells transfected with different amounts of Flag-tagged DUSP16. The values were expressed as the mean ± SEM.
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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steatosis [22]. At first, RT-qPCR and western blot assays showed that DUSP16 expression both from the mRNA and protein levels were markedly reduced by PA incubation in a time-dependent manner (Fig. 2A and B), indicating that DUSP16 might have a potential regulatory role in hepatic steatosis. Then, DUSP16 was knocked down in primary hepatocytes (Fig. 2C). Obviously, Oil Red O staining showed that PA-induced lipid accumulation in cells was further accelerated when DUSP16 was inhibited (Fig. 2D). Consistently, DUSP16 knockdown further aggravated the TG
deposition in PA-incubated hepatocytes (Fig. 2E). The mRNA levels of key enzymes participating in lipid metabolism were then measured, showing significantly exacerbated mRNA levels of genes-associated with synthesis (SREBF1, FAS, SCD1, PPARg) and fatty acid uptake (FATP1, FABP1, CD36) but further reduced transcriptions of genes related to fatty acid b-oxidation (PPARa, MCAD, CPT-1a, UCP2) in DUSP16-knockdown cells in response to PA (Fig. 2F). DUSP16 could inactivate JNK signaling, which is closely related to various metabolic events [12,13]. We found that PA-
Fig. 2. DUSP16 suppression accelerates dyslipidemia and inflammation in palmitate-treated hepatocytes. (A,B) The isolated primary hepatocytes were treated with PA (0.4 mM) for the indicated time, followed by RT-qPCR and western blot assays of DUSP16. (C) The isolated primary hepatocytes were transfected with DUSP16 siRNA for 24 h to inhibit the expression of DUSP16, and western blot analysis was used for transfection efficacy analysis. (DeI) The isolated primary hepatocytes were transfected with si-DUSP16 for 24 h, and then were treated with PA (0.4 mM) for another 24 h. All cells were collected for the subsequent assays. (D) Oil Red O staining of cells. (E) TG contents in cells. (F) RT-qPCR analysis was used calculate the mRNA expression levels of genes associated with fatty acid synthesis, uptake and b-oxidation. (G) Western blot for p-JNK. (H) RT-qPCR analysis for pro-inflammatory factors in cells. (I) Western blot analysis of p-TAK1, p-IKKa, p-IkBa and peNFekB (p65) in cells. The values were expressed as the mean ± SEM. *p < 0.05 and **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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induced activation of JNK was further promoted by si-DUSP16 (Fig. 2G). Then, DUSP16 knockdown markedly enhanced the expression of genes associated with pro-inflammatory response, including TNF-a, IL-1b, IL-6, IL-4, MCP1 and CXCL1 in PAstimulated hepatocytes (Fig. 2H). Western blot assays showed that PA treatment led to significant increases in p-TAK1, p-IKKa, pIkBa and peNFekB (p65) in cells, while being further enhanced bysi-DUSP16 (Fig. 2I). Collectively, these findings demonstrated that DUSP16 suppression might be involved in lipid accumulation and inflammation in PA-treated hepatocytes. 3.3. DUSP16 over-expression improves dyslipidemia and inflammatory response in hepatocytes treated with palmitate To confirm the effects of DUSP16 on hepatic steatosis, we overexpressed DUSP16 in hepatocytes (Fig. 3A). Oil Red O staining showed that PA-induced lipid deposition in hepatocytes was
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apparently alleviated when DUSP16 was over-expressed, and these results were accompanied with reduced TG contents (Fig. 3B and C). In addition, PA-stimulated mRNA levels of genes-associated with synthesis (SREBF1, FAS, SCD1, PPARg) and fatty acid uptake (FATP1, FABP1, CD36) were markedly abrogated by DUSP16 overexpression, but the contrast transcriptions of genes associated with fatty acid b-oxidation (PPARa, MCAD, CPT-1a, UCP2) were observed (Fig. 3D). Subsequently, DUSP16 over-expression significantly abolished the activation of JNK in PA-incubated hepatocytes (Fig. 3E). The mRNA expression levels of TNF-a, IL-1b, IL-6, IL-4, MCP1 and CXCL1 were highly reduced by DUSP16 over-expression in PA-treated cells when compared to the oe-EV group (Fig. 3F). As displayed in Fig. 3G, PA-potentiated expression of p-TAK1, p-IKKa, p-IkBa and peNFekB (p65) was obviously attenuated in hepatocytes transfected with oe-DUSP16. Collectively, the findings here showed that DUSP16 exhibited protective effects against dyslipidemia and inflammation induced by metabolic stress.
Fig. 3. DUSP16 over-expression improves dyslipidemia and inflammatory response in hepatocytes treated with palmitate. (A) The isolated primary hepatocytes were transfected with pcDNA3.1-DUSP16 for 24 h to over-express DUSP16, and then the western blotting was used for transfection efficiency. (BeG) The isolated primary hepatocytes were transfected with pcDNA3.1-DUSP16 for 24 h, followed by PA (0.4 mM) treatment for another 24 h. Then, all cells were collected for the following studies. (B) Oil Red O staining of cells. (C) TG levels in cells were measured. (D) RT-qPCR assays for the calculation of genes related to fatty acid synthesis, uptake and b-oxidation. (E) Western blot analysis of pJNK in cells. (F) RT-qPCR assays of genes associated with inflammatory response. (G) Western blot assay of p-TAK1, p-IKKa, p-IkBa and peNFekB (p65) in cells. The values were expressed as the mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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3.4. DUSP16 knockout enhances metabolic disorder and hepatic steatosis in HFD-fed mice In this regard, the mouse model with HFD-induced nonalcoholic
steatohepatitis was established to further investigate the regulatory role of DUSP16. As displayed in Fig. 4A, HFD-challenged mice had weaker DUSP16 expression in liver tissues than that of the NCD mice. Consistently, obviously reduced expression of DUSP16 was
Fig. 4. DUSP16 knockout enhances metabolic disorder and hepatic steatosis in HFD-fed mice. (A) Western blot analysis of DUSP16 in liver of HFD-fed mice (up panel), and in liver samples of ob/ob mice (down panel). (B) Body weight of mice. Calculation of (C) fasting blood glucose levels, (D) fasting insulin levels and (E) HOMA-IR. (F) OGTT (left panel) and ITT (right panel) analysis for insulin resistance evaluation. (G) H&E staining (up panel) and Oil Red O staining (down panel) of liver sections. (H) Ratio of liver weight (LW) to body weight (BW). (I) ALT and AST contents in serum. (J) TG and TC levels in liver of mice. (K) RT-qPCR assays for the calculation of genes associated with fatty acid synthesis, uptake and b-oxidation. (L) RT-qPCR analysis for pro-inflammatory factors in liver tissues. (M) Western blot assay of p-TAK1, p-JNK, p-IKKa, p-IkBa and peNFekB in hepatic samples. (N) Schematic diagram of the molecular mechanisms revealing DUSP16-modulated hepatic steatosis. The values were expressed as the mean ± SEM. *p < 0.05 and **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037
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detected in hepatic tissues of ob/ob mice compared to the lean mice. Then, the loss-function-approach was used in animals treated with or without HFD. We found that HFD treatment resulted in a significant increase in the body weight change of mice, and notably HFD-mice with DUSP16 deletion displayed more body weight (Fig. 4B). Fasting blood glucose and insulin levels up-regulated by HFD were further accelerated in mice with DUSP16 knockout, along with markedly elevated HOMA-IR (Fig. 4CeE), demonstrating the elevated insulin resistance in mice. OGTT and ITT assays confirmed that DUSP16 deletion exacerbated HFD-induced insulin resistance (Fig. 4F). H&E and Oil Red O staining displayed accelerated steatosis, hepatocyte ballooning and enhanced lipid accumulation in the hepatic samples from HFD-fed mice (Fig. 4G). DUSP16-KO mice showed elevated ratio of LW to BW following HFD consumption (Fig. 4H). Serum ALT and AST contents induced by HFD were markedly enhanced in mice with DUSP16 deletion (Fig. 4I). Consistently, DUSP16 knockout led to severer TG and TC deposition in liver of HFD-fed mice (Fig. 4J). In response to HFD, DUSP16 deletion caused deteriorated changes in lipid metabolismassociated gene expressions (Fig. 4K). Moreover, hepatic inflammatory factors were significantly enhanced in HFD-challenged DUSP16-KO mice compared to the HFD group (Fig. 4L). Activation of TAK1, JNK, IKKa, IkBa and NF-kB signaling following HFD was also accelerated by DUSP16-knockout (Fig. 4M). Therefore, DUSP16 deletion exacerbated metabolic disorder, lipid depostion and inflammation in liver of HFD-fed mice. 4. Discussion The pathogenesis of NAFLD is complex, including a complicated reprogrammed molecular network [4,5]. However, presently, there are no effective therapeutic strategies for NAFLD, which is largely attributed to the incomplete understanding of the mechanisms of such disease. In the present study, our findings demonstrated the protective effects of DUSP16 on metabolic stress-induced hepatic steatosis, abnormal metabolism and inflammation in mice. Promoting DUSP16 expression could alleviate lipid accumulation and inflammatory response in PA-incubated hepatocytes through directly interacting with TAK1 and negatively regulating JNK activation. Herein, DUSP16 might be novel protective factor of NAFLD that inhibited dyslipidemia and inflammation. TAK1 is detrimental in various maladies, such as metabolic disorders, insulin resistance, inflammation and apoptosis in liver and hepatocytes [21,23]. Previous studies have reported that restraining TAK1 activation could attenuate hepatic steatosis, lipid deposition and inflammatory response in animals with HFDinduced NAFLD [24]. In our study, we found that DUSP16 could directly interact with TAK1, and promoting DUSP16 expression markedly repress TAK1 activity in hepatocytes induced by PA. Thus, DUSP16 might be effective for maintaining liver homeostasis, protecting against hepatic steatosis. TAK1 is a up-streaming kinase that activates NF-kB and MAPK signaling during NAFLD progression [25,26]. DUSP16 has been reported as an inactivator of JNK [12]. In our study, we also found that DUSP16 knockdown accelerated JNK activation in PA-incubated hepatocytes, while its over-expression markedly inhibited JNK, further indicating that DUSP16 showed a negative role in regulating JNK activity. Accumulating studies before have suggested that JNK suppression is protective in NAFLD [27], which strongly supported our findings. Therefore, DUSP16/ TAK1/JNK regulatory axis might be critical under these pathological conditions, which might be a novel target for developing effective therapeutic strategies against NAFLD or associated diseases. The dyslipidemia and inflammatory response play essential roles in the pathogenesis of NAFLD, and suppressing lipid accumulation and inflammation may be ideal strategy for the
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prevention or intervention of hepatic steatosis [28,29]. In our study, we found that DUSP16 inhibition significantly promoted lipid accumulation in hepatocytes and liver tissues through accelerating fatty acid synthesis and up-take, while restraining fatty acid boxidation. A previous study showed that DUSP16-deficient mice exhibited severer release of pro-inflammatory factors induced by endotoxin [30]. The inflammatory response regulated by NF-kB and JNK signaling pathways plays a pivotal role in meditating NAFLD progression [31,32]. The secretion of TNF-a, MCP-1, IL-1b, IL-6 and CXCL-1 is significantly induced by metabolic stress both in vitro and in vivo, contributing to the progression of hepatic steatosis [22,30]. Increasing studies report that JNK activation could potentiate NF-kB signaling, promoting the inflammatory response under various disease conditions, including NAFLD [33,34]. Identifying and characterizing the key regulator of NF-kB and JNK signaling pathways may elucidate the molecular mechanism underlying the pathogenesis of NAFLD and provide targets for intervention. In this study, we also found that PA- and HFD-induced inflammatory response were highly accelerated by DUSP16 suppression, as evidenced by the releases of TNF-a, IL-1b, IL-6, IL-4, MCP1 and CXCL1 through blocking NF-kB signaling, which was consistent with the expression change of p-JNK. In contrast, promoting DUSP16 expression could markedly alleviate inflammation in PA-treated hepatocytes. Therefore, we identified DUSP16 as an efficient meditator of NF-kB signaling in response to hepatic steatosis. In summary, our study demonstrated that the DUSP16 might be a novel therapeutic target for clinical intervention of NAFLD. DUSP16 could target TAK1 and negatively regulate JNK activation to modulate dyslipidemia and inflammatory response (Fig. 4N). Thus, DUSP16/TAK1/JNK axis is a potential target for the alleviation of hepatic steatosis or associated metabolic disorders. However, further studies are still necessary in future to comprehensively reveal the relationship between these signals. Acknowledgment This work was supported by Chongqing Research Program of Basic Research and Frontier Technology (cstc2018jcyjAX0371); Science and Technology Research Program of Chongqing Education Commission of China (No. KJ1601402) and Chongqing Professional Talents Plan for Innovation and Entrepreneurship Demonstration Team (CQCY201903258). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.037 Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.037. References [1] Z. Younossi, et al., Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention, Nat. Rev. Gastroenterol. Hepatol. 15 (1) (2018) 11. [2] Y. Sumida, et al., Current and future pharmacological therapies for NAFLD/ NASH, J. Gastroenterol. 53 (3) (2018) 362e376. [3] R. de Franchis, et al., Ruling out esophageal varices in NAFLD cirrhosis: can we do without endoscopy? J. Hepatol. 69 (4) (2018) 769e771. [4] G. Targher, et al., Diabetes and NAFLD. Diabetes Complications, Comorbidities and Related Disorders, 2018, pp. 1e27. [5] G. Targher, et al., Nonalcoholic fatty liver disease and chronic vascular complications of diabetes mellitus, Nat. Rev. Endocrinol. 14 (2) (2018) 99. [6] C.Y. Chang, et al., Therapy of NAFLD: antioxidants and cytoprotective agents, J. Clin. Gastroenterol. 40 (2006) S51eS60. [7] A.M. Bennett, DUSPs, twists and turns in the journey to vascular inflammation,
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Please cite this article as: Y.-K. Wu et al., Targeting DUSP16/TAK1 signaling alleviates hepatic dyslipidemia and inflammation in high fat diet (HFD)-challenged mice through suppressing JNK MAPK, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2020.01.037