- Email: [email protected]
Pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes to the pathogenesis of nonalcoholic steatohepatitis
Accepted Manuscript Pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes to the pathogenesis of nonalcoholic steatohepatitis Ming Zhang, Yujie Zhao, Zhen Li, Congying Wang PII:
S0006-291X(17)32232-5
DOI:
10.1016/j.bbrc.2017.11.054
Reference:
YBBRC 38847
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 5 November 2017 Accepted Date: 8 November 2017
Please cite this article as: M. Zhang, Y. Zhao, Z. Li, C. Wang, Pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes to the pathogenesis of nonalcoholic steatohepatitis, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.11.054. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes
to
the
pathogenesis
of
Nonalcoholic
Steatohepatitis
RI PT
Ming Zhang1, Yujie Zhao1, Zhen Li1, Congying Wang2* 1
Intensive Care Unit, The Affiliated Hospital of Weifang Medical University,
Weifang, Shandong, 261031, China 2
SC
Department of Medical Equipment, Weifang People’s Hospital, Weifang,
*Corresponding
author:
M AN U
Shandong, 261000, China Dr.
Congying
Wang,
email:
[email protected]; Department of Medical Equipment, Weifang People’s Hospital, No. 151, Guangwen Street, Kuiwen District, Weifang,
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EP
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Shandong, 261000, China
ACCEPTED MANUSCRIPT Abstract Nonalcoholic steatohepatitis (NASH) is a progressive disease and poses a high risk of severe liver damage. However, the pathogenesis of NASH is still
RI PT
unclear. Accumulation of lipid droplets and insulin resistance is the hallmark of NASH. Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) plays key role in glucose metabolism via regulating the activity of pyruvate dehydrogenase
SC
complex (PDC). Here, we demonstrated a novel of PDK4 in NASH by
M AN U
regulating hepatic steatosis and insulin signaling pathway in methionine and choline deficient (MCD) diet induced NASH model. Hepatic PDK4 levels were highly induced in human patients with NASH and MCD diet fed mice, as well as in hepatocytes treated with oleic acid. The glucose and lipid metabolism
TE D
were impaired in Pdk4-/- mice. Pdk4 deficiency ameliorated the hepatic steatosis significantly in NASH mice. Pdk4-/--MCD mice had reduced liver weights and triglyceride (TG) levels. And Pdk4 deficiency dramatically reduced
EP
the expression of genes related to fatty acid uptake, synthesis and
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gluconeogenesis. In addition, elevated phosphorylated AMPK (p-AMPK), p-SAPK/JNK and diminished p-ERK, p-P38, p-Akt and p-mTOR/p-4EBP1 proteins were observed. In conclusion, our data indicated that PDK4 potentially contributes to the hepatic steatosis in NASH via regulating several signaling pathway and PDK4 may be a new therapeutic strategy against NAFLD. Key Words: PDK4; NASH; hepatic steatosis.
ACCEPTED MANUSCRIPT
Introduction Hepatic steatosis, the accumulation of fat in the liver, is a common feature of
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nonalcoholic fatty liver disease (NAFLD), which is known to be a risk factor for nonalcoholic steatohepatitis (NASH), ultimately culminating in liver cirrhosis, portal hypertension, and liver failure[1]. Hepatic steatosis, obesity, insulin
SC
resistance, and type 2 diabetes mellitus are all closely linked[2, 3]. Indeed,
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most obese, type 2 diabetic patients also have hepatic steatosis[4]. The reason why fatty acids are retained in the liver in NAFLD is poorly understood. Learning how to prevent NAFLD and its progression to NASH depends upon a better understanding of why fat accumulates in tissues.
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Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) plays key role in the regulation of the activity of pyruvate dehydrogenase complex (PDC), which is essential for the catalyzation of pyruvate to acetyl-CoA[5]. PDK4 is highly
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expressed in the liver, heart and skeletal muscle[6]. And PDK4 is a gatekeeper
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regulator for glucose metabolism[7]. The mRNA and protein levels of PDK4 are elevated in livers of diabetic, fasted and insulin resistant animals[8, 9]. Further, PDK4 knock out mice have lower blood glucose levels, better glucose tolerance, and greater insulin sensitivity than wild type mice[10]. Further, the expression of PDK4 is upregulated by glucocorticoids, long chain fatty acids and fibrates, while its expression is inhibited by insulin [11, 12]. However, If PDK4 also participates in the pathogenesis of NAFLD is still unknown.
ACCEPTED MANUSCRIPT In this study, we revealed a new function of PDK4 in the hepatic lipid metabolism.
We
demonstrated
that
Pdk4
deficiency
ameliorated
hepatosteatosis and insulin resistance in NASH. Our results could help in the
RI PT
effort to develop pharmaceuticals for the prevention of NAFLD and NASH. Material and method Human sample
SC
Informed written consent was obtained from every patient. The study protocol
M AN U
was approved by the Ethics Committee of Weifang People’s Hospital. The liver tissues were previously collected from patients of benign focal hepatic lesions undergoing liver surgery. The samples were immediately shock-frozen and stored at −80 °C. Blood samples were collected from all subjects following
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overnight fasting. Serum was isolated by centrifugation at 3,000 rpm for 15 min and stored at −80°C. Mouse model
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Pdk4-/- mice and Pdk4+/+ littermates were generated on a C57BL/6J
AC C
background through heterozygote mating from our laboratory. All the mice were maintained on a 12-hour light-dark cycle in a temperature (22°C) and humidity controlled (45%–55%) environment with ad libitum access to food and water. Eight-week-old Pdk4-/- and Pdk4+/+ littermates’ male mice were fed with either a control chow diet (CD) or a methionine-choline deficient (MCD) diet for 2 weeks induce the NASH phenotype. After 2 weeks of the feeding, the mice were sacrificed, serum and livers were collected.
ACCEPTED MANUSCRIPT Cell culture The human hepatocarcinoma cell line (HepG2) was cultured in DMEM medium containing glucose (4.5 g/l) and 10% FBS. Primary mouse hepatocytes were
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isolated from 10-week-old mouse by two-step perfusion method described previously[13]. Primary hepatocytes were plated onto collagen-coated dishes and maintained in EMEM medium with 10% FBS. All the cells were incubated
SC
at 37°C in 5% CO2 humidity.
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Statistical analysis
Statistical analysis was performed using SPSS 19.0. All Data are presented as mean ± SEM. A Student’s unpaired t test was used to determine differences between two groups. P < 0.05 was considered significant.
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Results
The expression of PDK4 is increased in NAFLD human and mice To determine the role of PDK4 in NASH, we analyzed both the mRNA and
EP
protein levels of PDK4 in human NASH liver specimens. Compared with normal control livers, the mRNA and protein levels of PDK4 were upregulated
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in NASH livers (Figure 1A). In addition, liver PDK4 levels were also significantly increased in wild type (WT) mice fed the MCD diet compared with the chow diet (CD) (Figure 1B). Furthermore, Oleic acids are the most abundant fatty unsaturated acids in liver triglycerides (TG) in both normal subjects and patients with NAFLD[14]. Oleic acid treatment significantly increased intracellular TG level and the expression of PDK4 in HepG2 and primary mouse hepatocytes (Fig. 1C&D). Taken together, our studies indicated that the expression of PDK4 is induced in NASH in both humans and mice.
ACCEPTED MANUSCRIPT Pdk4 deficiency results in impaired glucose and lipid metabolism in the liver. To investigate the role of PDK4 in liver metabolism, we initially analyzed
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changes in glucose and lipid homeostasis in the livers of Pdk4–/– mice. The fasting blood glucose (FBG) and serum triglyceride (TG) levels, as well as hepatic TG levels were decreased in Pdk4–/– compared with Pdk4+/+ mice
SC
(Figure 2A &B). Furthermore, gene expression analyses indicated that mRNA
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level of some genes related to lipd and glucose metabolism such as Srebp-1c, Chrebp, Pklr, Acaca, Fasn, and Scd1 were also decreased in Pdk4–/– mice (Figure 2C). Moreover, consistently with the mRNA levels, the protein levels of FASN, ACACA, and GCK were also decreased in in the livers of Pdk4–/– mice
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compared with Pdk4+/+ mice (Figure 2D). Taken together, Pdk4 deficiency impaired glucose and lipid metabolism in the liver. Pdk4 deficiency ameliorated hepatic steatosis in NASH mice
EP
Given the role of PDK4 in the hepatic lipid and glucose metabolism, and Insulin
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resistance and hyperglycemia are common complications and paly central role in the pathogenesis of NASH[15]. We established NASH mice model by MCD diet feeding to elucidate the effect of PDK4 in the pathogenesis of NASH. Althougt the body weights were similar between Pdk4+/+ and Pdk4–/– mice after the 2-week CD or MCD diet feeding regime (Figure 3A). The absolute and relative liver weights were significantly reduced in Pdk4–/–-MCD mice compared with Pdk4+/+-MCD mice, while no differences were detected
ACCEPTED MANUSCRIPT between groups on the CD (Fig. 3A). Moreover, H&E and Oil Red O staining revealed a marked reduction in hepatic lipid accumulation in Pdk4–/–-MCD vs Pdk4+/+-MCD mice (Fig. 3B&C), which was accompanied by the decreased
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levels of liver TG content (Fig. 3D). Accordingly, the mRNA levels of the major lipogenic related factors Srebp-1c, Fatp1, Fas, Accα and Pparγ were significantly decreased in Pdk4-/--MCD mice vs Pdk4+/+-MCD mice mice (Fig.
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3E). Collectively, these results demonstrated that Pdk4 deficiency ameliorated
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hepatic steatosis in NASH mice.
Multiple hepatic signaling pathways are altered by Pdk4 deficiency. To elucidate the potential mechanism of PDK4 involved the pathogenesis of NASH, we assessed several signaling pathways which are reported to be
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major links to the pathology of NASH. AMPK is a central regulator of energy balance[16]. Increased AMPK phosphorylation was observed in Pdk4-/--MCD mice compared
with
Pdk4+/+-MCD
mice
(Fig.
4A).
The
increased
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phosphorylation SAPK/JNK, which is known to be involved in the pathogenesis
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of NAFLD[17], were also observed in Pdk4-/--MCD mice. However, the phosphorylation of ERK1/2 and P38 in Pdk4-/--MCD mice were reduced compared with Pdk4+/+-MCD mice (Fig. 4B). Furthermore, the impairment of hepatic insulin signaling in NASH was markedly reversed in the Pdk4-/--MCD mice, as compared to that in their corresponding controls (Fig. 4C). mTOR signaling pathway was also reported to promote de novo lipogenesis through activation of SREBP1. The expression levels of p-mTOR
ACCEPTED MANUSCRIPT and its target protein p-4EBP1 was also reduced in Pdk4-/--MCD mice (Fig. 4D). Discussion
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In the current study, we identified the role of PDK4 contributed to the pathogenesis of NAFLD and NASH via regulating hepatic lipid and glucoase metabolism. Pdk4 deficiency can ameliorated the hepatic steatosis in MCD
SC
induced NASH mice via several signaling pathway. This newly identified role of
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PDK4 provides a novel clue to the pathogenesis of NASH and PDK4 might become a new target for the treatment of NAFLD and NASH. The pathways associated with steatosis are interconnected to form an intricate regulatory network and can mutually enhance each other, forming a
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vicious cycle. Pdk4 deficiency in mice reduced TG accumulation via AMPK activation, which may promote PPARα-mediated fatty acid β-oxidation. Additionally, mTOR pathway was reported to participate in the pathogenesis of
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NAFLD and NASH[18], which was found to be inactivated in Pdk4 deficiency
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mice. So AMPK may also reduce lipogenesis via negatively regulating mTOR signaling pathway, which need more experiment to confirm. Our study also has some limitations. First, we only used male mice in this
study, further studies will incorporate the use of female mice to determine whether sex differences are observed in the pathogenesis of NASH. Second, we used Pdk4 whole-body knockout mice which has the limitations in terms of compensatory mechanisms. Ideally, tissue-specific knockout mice would be
ACCEPTED MANUSCRIPT required. Third, the MCD diet has been reported to have some limitation to study steatohepatitis, additional diet models would be useful to further explore the role of PDK4 in NASH development.
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In summary, our study reveals a novel and important role of PDK4 in regulating hepatic steatosis and insulin signaling pathway. Our findings also provide strong evidence that PDK4 has therapeutic potential for NAFLD and
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NASH.
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Financial Interests All authors report no conflicts of interest. References
[1] Z. Younossi, Q.M. Anstee, M. Marietti, T. Hardy, L. Henry, M. Eslam, J. George, E.
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Bugianesi, Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention, Nature reviews. Gastroenterology & hepatology, (2017). [2] Y. Tokita, Y. Maejima, K. Shimomura, S. Takenoshita, N. Ishiyama, M. Akuzawa, Y.
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Shimomura, K. Nakajima, Non-alcoholic Fatty Liver Disease Is a Risk Factor for Type 2
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Diabetes in Middle-aged Japanese Men and Women, Internal medicine, 56 (2017) 763-771. [3] S. Lallukka, H. Yki-Jarvinen, Non-alcoholic fatty liver disease and risk of type 2 diabetes, Best practice & research. Clinical endocrinology & metabolism, 30 (2016) 385-395. [4] M. Yi, R.P. Chen, R. Yang, H. Chen, Increased prevalence and risk of non-alcoholic fatty liver disease in overweight and obese patients with Type 2 diabetes in South China, Diabetic medicine : a journal of the British Diabetic Association, 34 (2017) 505-513. [5] R.A. Harris, M.M. Bowker-Kinley, B. Huang, P. Wu, Regulation of the activity of the
ACCEPTED MANUSCRIPT pyruvate dehydrogenase complex, Advances in enzyme regulation, 42 (2002) 249-259. [6] S. Connaughton, F. Chowdhury, R.R. Attia, S. Song, Y. Zhang, M.B. Elam, G.A. Cook, E.A. Park, Regulation of pyruvate dehydrogenase kinase isoform 4 (PDK4) gene expression by
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glucocorticoids and insulin, Molecular and cellular endocrinology, 315 (2010) 159-167. [7] X. Liu, R. Zuo, Y. Bao, X. Qu, K. Sun, H. Ying, Down-regulation of PDK4 is Critical for the Switch of Carbohydrate Catabolism during Syncytialization of Human Placental Trophoblasts,
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Scientific reports, 7 (2017) 8474.
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[8] P. Wu, P.V. Blair, J. Sato, J. Jaskiewicz, K.M. Popov, R.A. Harris, Starvation increases the amount of pyruvate dehydrogenase kinase in several mammalian tissues, Archives of biochemistry and biophysics, 381 (2000) 1-7.
[9] R.A. Harris, B. Huang, P. Wu, Control of pyruvate dehydrogenase kinase gene expression,
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Advances in enzyme regulation, 41 (2001) 269-288.
[10] N.H. Jeoung, R.A. Harris, Pyruvate dehydrogenase kinase-4 deficiency lowers blood glucose and improves glucose tolerance in diet-induced obese mice, American journal of
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physiology. Endocrinology and metabolism, 295 (2008) E46-54.
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[11] B. Huang, P. Wu, M.M. Bowker-Kinley, R.A. Harris, Regulation of pyruvate dehydrogenase kinase expression by peroxisome proliferator-activated receptor-alpha ligands, glucocorticoids, and insulin, Diabetes, 51 (2002) 276-283. [12] M.J. Holness, K. Bulmer, N.D. Smith, M.C. Sugden, Investigation of potential mechanisms regulating protein expression of hepatic pyruvate dehydrogenase kinase isoforms 2 and 4 by fatty acids and thyroid hormone, The Biochemical journal, 369 (2003) 687-695. [13] Y. Zhang, N. Xu, J. Xu, B. Kong, B. Copple, G.L. Guo, L. Wang, E2F1 is a novel fibrogenic
ACCEPTED MANUSCRIPT gene that regulates cholestatic liver fibrosis through the Egr-1/SHP/EID1 network, Hepatology, 60 (2014) 919-930. [14] H. Wu, T. Zhang, F. Pan, C.J. Steer, Z. Li, X. Chen, G. Song, MicroRNA-206 prevents
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hepatosteatosis and hyperglycemia by facilitating insulin signaling and impairing lipogenesis, Journal of hepatology, 66 (2017) 816-824.
[15] S. Kim, J. Choi, M. Kim, Insulin resistance, inflammation, and nonalcoholic fatty liver
SC
disease in non-obese adults without metabolic syndrome components, Hepatology
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international, 7 (2013) 586-591.
[16] Y. Li, S. Xu, M.M. Mihaylova, B. Zheng, X. Hou, B. Jiang, O. Park, Z. Luo, E. Lefai, J.Y. Shyy, B. Gao, M. Wierzbicki, T.J. Verbeuren, R.J. Shaw, R.A. Cohen, M. Zang, AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis
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in diet-induced insulin-resistant mice, Cell metabolism, 13 (2011) 376-388. [17] K. Miura, E. Seki, H. Ohnishi, D.A. Brenner, Role of toll-like receptors and their downstream
molecules
in
the
development
of
nonalcoholic
Fatty
liver
disease,
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Gastroenterology research and practice, 2010 (2010) 362847.
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[18] V. Sapp, L. Gaffney, S.F. EauClaire, R.P. Matthews, Fructose leads to hepatic steatosis in zebrafish that is reversed by mechanistic target of rapamycin (mTOR) inhibition, Hepatology, 60 (2014) 1581-1592.
Figure Legends
Figure 1. PDK4 expression is increased in NASH mice and humans. (A) The mRNA and protein levels of PDK4 in human liver specimens. (B) The mRNA and protein levels of PDK4 in livers of mice fed the control diet (CD) and
ACCEPTED MANUSCRIPT methionine and choline–deficient (MCD) diet. The mRNA and protein levels of PDK4 in (C) HepG2 cells and (D) primary mouse hepatocytes treated with oleic acid or vehicle. Data are shown as mean ± SEM (triplicate assays). OA:
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oleic acid. ** P<0.01 vs con or CD. Figure 2. Pdk4-/- mice showed a decrease liver glucose and lipid metabolism. (A) The serum FBG and TG levels in Pdk4-/- and Pdk4+/+ mice. (B)
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The liver TG levels in Pdk4-/- and Pdk4+/+ mice. (C) Relative mRNA levels of
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Pdk4 and relevant glycolytic and lipogenic genes in the livers of Pdk4-/- and Pdk4+/+ mice. (D) Western blot analyses of the expression of the indicated proteins in Pdk4-/- and Pdk4+/+ mice. Data are shown as mean ± SEM (triplicate assays). * P<0.05, ** P<0.01 vs Pdk4+/+.
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Figure 3. Pdk4 deficiency ameliorated hepatic steatosis in NASH mice. (A) The body and liver weight in Pdk4+/+ and Pdk4-/- fed with CD or MCD diet. (B) H&E and (C) oil red staining in Pdk4+/+ and Pdk4-/- fed with CD or MCD diet. (D)
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Hepatic TG content in Pdk4+/+ and Pdk4-/- fed with MCD diet. (E) mRNA levels
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of genes related to lipid metabolism were detected by qPCR in Pdk4+/+ and Pdk4-/- fed with MCD diet. Data are shown as mean ± SEM (triplicate assays). * P<0.05, ** P<0.01 vs Pdk4+/+-CD diet or Pdk4+/+. # P<0.05 vs Pdk4+/+-MCD. Figure 4. Pdk4 deficiency affects proteins in multiple signaling pathways. Immunoblotting analysis of proteins in (A) p-AMPK and AMPK; (B) ERK, SAPK/JNK, and p38; (C) p-AKT, Akt; (D) mTOR signaling pathways in Pdk4+/+ and Pdk4-/- mice fed with the MCD diet.
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights: 1) The expression of PDK4 is increased in both NAFLD human and mice; 2) Pdk4 deficiency results in impaired glucose and lipid metabolism in the liver;
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3) Pdk4 deficiency ameliorated hepatic steatosis in NASH mice;
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4) Multiple hepatic signaling pathways are altered by Pdk4 deficiency.
S0006-291X(17)32232-5
DOI:
10.1016/j.bbrc.2017.11.054
Reference:
YBBRC 38847
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 5 November 2017 Accepted Date: 8 November 2017
Please cite this article as: M. Zhang, Y. Zhao, Z. Li, C. Wang, Pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes to the pathogenesis of nonalcoholic steatohepatitis, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.11.054. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
pyruvate dehydrogenase kinase 4 mediates lipogenesis and contributes
to
the
pathogenesis
of
Nonalcoholic
Steatohepatitis
RI PT
Ming Zhang1, Yujie Zhao1, Zhen Li1, Congying Wang2* 1
Intensive Care Unit, The Affiliated Hospital of Weifang Medical University,
Weifang, Shandong, 261031, China 2
SC
Department of Medical Equipment, Weifang People’s Hospital, Weifang,
*Corresponding
author:
M AN U
Shandong, 261000, China Dr.
Congying
Wang,
email:
[email protected]; Department of Medical Equipment, Weifang People’s Hospital, No. 151, Guangwen Street, Kuiwen District, Weifang,
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EP
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Shandong, 261000, China
ACCEPTED MANUSCRIPT Abstract Nonalcoholic steatohepatitis (NASH) is a progressive disease and poses a high risk of severe liver damage. However, the pathogenesis of NASH is still
RI PT
unclear. Accumulation of lipid droplets and insulin resistance is the hallmark of NASH. Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) plays key role in glucose metabolism via regulating the activity of pyruvate dehydrogenase
SC
complex (PDC). Here, we demonstrated a novel of PDK4 in NASH by
M AN U
regulating hepatic steatosis and insulin signaling pathway in methionine and choline deficient (MCD) diet induced NASH model. Hepatic PDK4 levels were highly induced in human patients with NASH and MCD diet fed mice, as well as in hepatocytes treated with oleic acid. The glucose and lipid metabolism
TE D
were impaired in Pdk4-/- mice. Pdk4 deficiency ameliorated the hepatic steatosis significantly in NASH mice. Pdk4-/--MCD mice had reduced liver weights and triglyceride (TG) levels. And Pdk4 deficiency dramatically reduced
EP
the expression of genes related to fatty acid uptake, synthesis and
AC C
gluconeogenesis. In addition, elevated phosphorylated AMPK (p-AMPK), p-SAPK/JNK and diminished p-ERK, p-P38, p-Akt and p-mTOR/p-4EBP1 proteins were observed. In conclusion, our data indicated that PDK4 potentially contributes to the hepatic steatosis in NASH via regulating several signaling pathway and PDK4 may be a new therapeutic strategy against NAFLD. Key Words: PDK4; NASH; hepatic steatosis.
ACCEPTED MANUSCRIPT
Introduction Hepatic steatosis, the accumulation of fat in the liver, is a common feature of
RI PT
nonalcoholic fatty liver disease (NAFLD), which is known to be a risk factor for nonalcoholic steatohepatitis (NASH), ultimately culminating in liver cirrhosis, portal hypertension, and liver failure[1]. Hepatic steatosis, obesity, insulin
SC
resistance, and type 2 diabetes mellitus are all closely linked[2, 3]. Indeed,
M AN U
most obese, type 2 diabetic patients also have hepatic steatosis[4]. The reason why fatty acids are retained in the liver in NAFLD is poorly understood. Learning how to prevent NAFLD and its progression to NASH depends upon a better understanding of why fat accumulates in tissues.
TE D
Pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) plays key role in the regulation of the activity of pyruvate dehydrogenase complex (PDC), which is essential for the catalyzation of pyruvate to acetyl-CoA[5]. PDK4 is highly
EP
expressed in the liver, heart and skeletal muscle[6]. And PDK4 is a gatekeeper
AC C
regulator for glucose metabolism[7]. The mRNA and protein levels of PDK4 are elevated in livers of diabetic, fasted and insulin resistant animals[8, 9]. Further, PDK4 knock out mice have lower blood glucose levels, better glucose tolerance, and greater insulin sensitivity than wild type mice[10]. Further, the expression of PDK4 is upregulated by glucocorticoids, long chain fatty acids and fibrates, while its expression is inhibited by insulin [11, 12]. However, If PDK4 also participates in the pathogenesis of NAFLD is still unknown.
ACCEPTED MANUSCRIPT In this study, we revealed a new function of PDK4 in the hepatic lipid metabolism.
We
demonstrated
that
Pdk4
deficiency
ameliorated
hepatosteatosis and insulin resistance in NASH. Our results could help in the
RI PT
effort to develop pharmaceuticals for the prevention of NAFLD and NASH. Material and method Human sample
SC
Informed written consent was obtained from every patient. The study protocol
M AN U
was approved by the Ethics Committee of Weifang People’s Hospital. The liver tissues were previously collected from patients of benign focal hepatic lesions undergoing liver surgery. The samples were immediately shock-frozen and stored at −80 °C. Blood samples were collected from all subjects following
TE D
overnight fasting. Serum was isolated by centrifugation at 3,000 rpm for 15 min and stored at −80°C. Mouse model
EP
Pdk4-/- mice and Pdk4+/+ littermates were generated on a C57BL/6J
AC C
background through heterozygote mating from our laboratory. All the mice were maintained on a 12-hour light-dark cycle in a temperature (22°C) and humidity controlled (45%–55%) environment with ad libitum access to food and water. Eight-week-old Pdk4-/- and Pdk4+/+ littermates’ male mice were fed with either a control chow diet (CD) or a methionine-choline deficient (MCD) diet for 2 weeks induce the NASH phenotype. After 2 weeks of the feeding, the mice were sacrificed, serum and livers were collected.
ACCEPTED MANUSCRIPT Cell culture The human hepatocarcinoma cell line (HepG2) was cultured in DMEM medium containing glucose (4.5 g/l) and 10% FBS. Primary mouse hepatocytes were
RI PT
isolated from 10-week-old mouse by two-step perfusion method described previously[13]. Primary hepatocytes were plated onto collagen-coated dishes and maintained in EMEM medium with 10% FBS. All the cells were incubated
SC
at 37°C in 5% CO2 humidity.
M AN U
Statistical analysis
Statistical analysis was performed using SPSS 19.0. All Data are presented as mean ± SEM. A Student’s unpaired t test was used to determine differences between two groups. P < 0.05 was considered significant.
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Results
The expression of PDK4 is increased in NAFLD human and mice To determine the role of PDK4 in NASH, we analyzed both the mRNA and
EP
protein levels of PDK4 in human NASH liver specimens. Compared with normal control livers, the mRNA and protein levels of PDK4 were upregulated
AC C
in NASH livers (Figure 1A). In addition, liver PDK4 levels were also significantly increased in wild type (WT) mice fed the MCD diet compared with the chow diet (CD) (Figure 1B). Furthermore, Oleic acids are the most abundant fatty unsaturated acids in liver triglycerides (TG) in both normal subjects and patients with NAFLD[14]. Oleic acid treatment significantly increased intracellular TG level and the expression of PDK4 in HepG2 and primary mouse hepatocytes (Fig. 1C&D). Taken together, our studies indicated that the expression of PDK4 is induced in NASH in both humans and mice.
ACCEPTED MANUSCRIPT Pdk4 deficiency results in impaired glucose and lipid metabolism in the liver. To investigate the role of PDK4 in liver metabolism, we initially analyzed
RI PT
changes in glucose and lipid homeostasis in the livers of Pdk4–/– mice. The fasting blood glucose (FBG) and serum triglyceride (TG) levels, as well as hepatic TG levels were decreased in Pdk4–/– compared with Pdk4+/+ mice
SC
(Figure 2A &B). Furthermore, gene expression analyses indicated that mRNA
M AN U
level of some genes related to lipd and glucose metabolism such as Srebp-1c, Chrebp, Pklr, Acaca, Fasn, and Scd1 were also decreased in Pdk4–/– mice (Figure 2C). Moreover, consistently with the mRNA levels, the protein levels of FASN, ACACA, and GCK were also decreased in in the livers of Pdk4–/– mice
TE D
compared with Pdk4+/+ mice (Figure 2D). Taken together, Pdk4 deficiency impaired glucose and lipid metabolism in the liver. Pdk4 deficiency ameliorated hepatic steatosis in NASH mice
EP
Given the role of PDK4 in the hepatic lipid and glucose metabolism, and Insulin
AC C
resistance and hyperglycemia are common complications and paly central role in the pathogenesis of NASH[15]. We established NASH mice model by MCD diet feeding to elucidate the effect of PDK4 in the pathogenesis of NASH. Althougt the body weights were similar between Pdk4+/+ and Pdk4–/– mice after the 2-week CD or MCD diet feeding regime (Figure 3A). The absolute and relative liver weights were significantly reduced in Pdk4–/–-MCD mice compared with Pdk4+/+-MCD mice, while no differences were detected
ACCEPTED MANUSCRIPT between groups on the CD (Fig. 3A). Moreover, H&E and Oil Red O staining revealed a marked reduction in hepatic lipid accumulation in Pdk4–/–-MCD vs Pdk4+/+-MCD mice (Fig. 3B&C), which was accompanied by the decreased
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levels of liver TG content (Fig. 3D). Accordingly, the mRNA levels of the major lipogenic related factors Srebp-1c, Fatp1, Fas, Accα and Pparγ were significantly decreased in Pdk4-/--MCD mice vs Pdk4+/+-MCD mice mice (Fig.
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3E). Collectively, these results demonstrated that Pdk4 deficiency ameliorated
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hepatic steatosis in NASH mice.
Multiple hepatic signaling pathways are altered by Pdk4 deficiency. To elucidate the potential mechanism of PDK4 involved the pathogenesis of NASH, we assessed several signaling pathways which are reported to be
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major links to the pathology of NASH. AMPK is a central regulator of energy balance[16]. Increased AMPK phosphorylation was observed in Pdk4-/--MCD mice compared
with
Pdk4+/+-MCD
mice
(Fig.
4A).
The
increased
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phosphorylation SAPK/JNK, which is known to be involved in the pathogenesis
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of NAFLD[17], were also observed in Pdk4-/--MCD mice. However, the phosphorylation of ERK1/2 and P38 in Pdk4-/--MCD mice were reduced compared with Pdk4+/+-MCD mice (Fig. 4B). Furthermore, the impairment of hepatic insulin signaling in NASH was markedly reversed in the Pdk4-/--MCD mice, as compared to that in their corresponding controls (Fig. 4C). mTOR signaling pathway was also reported to promote de novo lipogenesis through activation of SREBP1. The expression levels of p-mTOR
ACCEPTED MANUSCRIPT and its target protein p-4EBP1 was also reduced in Pdk4-/--MCD mice (Fig. 4D). Discussion
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In the current study, we identified the role of PDK4 contributed to the pathogenesis of NAFLD and NASH via regulating hepatic lipid and glucoase metabolism. Pdk4 deficiency can ameliorated the hepatic steatosis in MCD
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induced NASH mice via several signaling pathway. This newly identified role of
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PDK4 provides a novel clue to the pathogenesis of NASH and PDK4 might become a new target for the treatment of NAFLD and NASH. The pathways associated with steatosis are interconnected to form an intricate regulatory network and can mutually enhance each other, forming a
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vicious cycle. Pdk4 deficiency in mice reduced TG accumulation via AMPK activation, which may promote PPARα-mediated fatty acid β-oxidation. Additionally, mTOR pathway was reported to participate in the pathogenesis of
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NAFLD and NASH[18], which was found to be inactivated in Pdk4 deficiency
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mice. So AMPK may also reduce lipogenesis via negatively regulating mTOR signaling pathway, which need more experiment to confirm. Our study also has some limitations. First, we only used male mice in this
study, further studies will incorporate the use of female mice to determine whether sex differences are observed in the pathogenesis of NASH. Second, we used Pdk4 whole-body knockout mice which has the limitations in terms of compensatory mechanisms. Ideally, tissue-specific knockout mice would be
ACCEPTED MANUSCRIPT required. Third, the MCD diet has been reported to have some limitation to study steatohepatitis, additional diet models would be useful to further explore the role of PDK4 in NASH development.
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In summary, our study reveals a novel and important role of PDK4 in regulating hepatic steatosis and insulin signaling pathway. Our findings also provide strong evidence that PDK4 has therapeutic potential for NAFLD and
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NASH.
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Financial Interests All authors report no conflicts of interest. References
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Figure Legends
Figure 1. PDK4 expression is increased in NASH mice and humans. (A) The mRNA and protein levels of PDK4 in human liver specimens. (B) The mRNA and protein levels of PDK4 in livers of mice fed the control diet (CD) and
ACCEPTED MANUSCRIPT methionine and choline–deficient (MCD) diet. The mRNA and protein levels of PDK4 in (C) HepG2 cells and (D) primary mouse hepatocytes treated with oleic acid or vehicle. Data are shown as mean ± SEM (triplicate assays). OA:
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oleic acid. ** P<0.01 vs con or CD. Figure 2. Pdk4-/- mice showed a decrease liver glucose and lipid metabolism. (A) The serum FBG and TG levels in Pdk4-/- and Pdk4+/+ mice. (B)
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The liver TG levels in Pdk4-/- and Pdk4+/+ mice. (C) Relative mRNA levels of
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Pdk4 and relevant glycolytic and lipogenic genes in the livers of Pdk4-/- and Pdk4+/+ mice. (D) Western blot analyses of the expression of the indicated proteins in Pdk4-/- and Pdk4+/+ mice. Data are shown as mean ± SEM (triplicate assays). * P<0.05, ** P<0.01 vs Pdk4+/+.
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Figure 3. Pdk4 deficiency ameliorated hepatic steatosis in NASH mice. (A) The body and liver weight in Pdk4+/+ and Pdk4-/- fed with CD or MCD diet. (B) H&E and (C) oil red staining in Pdk4+/+ and Pdk4-/- fed with CD or MCD diet. (D)
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Hepatic TG content in Pdk4+/+ and Pdk4-/- fed with MCD diet. (E) mRNA levels
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of genes related to lipid metabolism were detected by qPCR in Pdk4+/+ and Pdk4-/- fed with MCD diet. Data are shown as mean ± SEM (triplicate assays). * P<0.05, ** P<0.01 vs Pdk4+/+-CD diet or Pdk4+/+. # P<0.05 vs Pdk4+/+-MCD. Figure 4. Pdk4 deficiency affects proteins in multiple signaling pathways. Immunoblotting analysis of proteins in (A) p-AMPK and AMPK; (B) ERK, SAPK/JNK, and p38; (C) p-AKT, Akt; (D) mTOR signaling pathways in Pdk4+/+ and Pdk4-/- mice fed with the MCD diet.
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ACCEPTED MANUSCRIPT Highlights: 1) The expression of PDK4 is increased in both NAFLD human and mice; 2) Pdk4 deficiency results in impaired glucose and lipid metabolism in the liver;
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3) Pdk4 deficiency ameliorated hepatic steatosis in NASH mice;
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4) Multiple hepatic signaling pathways are altered by Pdk4 deficiency.