FAM3A Activates PI3K p110a/Akt Signaling to Ameliorate Hepatic Gluconeogenesis and Lipogenesis Chunjiong Wang,1* Yujing Chi,2* Jing Li,3* Yifei Miao,1 Sha Li,1 Wen Su,1 Shi Jia,1 Zhenzhen Chen,1 Shengnan Du,1 Xiaoyan Zhang,1 Yunfeng Zhou,1 Wenhan Wu,4 Mingyan Zhu,5 Zhiwei Wang,5 Huaqian Yang,6 Guoheng Xu,1 Shiqiang Wang,6 Jichun Yang,1 and Youfei Guan1,7 FAM3A belongs to a novel cytokine-like gene family, and its physiological role remains largely unknown. In our study, we found a marked reduction of FAM3A expression in the livers of db/db and high-fat diet (HFD)-induced diabetic mice. Hepatic overexpression of FAM3A markedly attenuated hyperglycemia, insulin resistance, and fatty liver with increased Akt (pAkt) signaling and repressed gluconeogenesis and lipogenesis in the livers of those mice. In contrast, small interfering RNA (siRNA)-mediated knockdown of hepatic FAM3A resulted in hyperglycemia with reduced pAkt levels and increased gluconeogenesis and lipogenesis in the livers of C57BL/6 mice. In vitro study revealed that FAM3A was mainly localized in the mitochondria, where it increases adenosine triphosphate (ATP) production and secretion in cultured hepatocytes. FAM3A activated Akt through the p110a catalytic subunit of PI3K in an insulin-independent manner. Blockade of P2 ATP receptors or downstream phospholipase C (PLC) and IP3R and removal of medium calcium all significantly reduced FAM3A-induced increase in cytosolic free Ca21 levels and attenuated FAM3A-mediated PI3K/Akt activation. Moreover, FAM3A-induced Akt activation was completely abolished by the inhibition of calmodulin (CaM). Conclusion: FAM3A plays crucial roles in the regulation of glucose and lipid metabolism in the liver, where it activates the PI3K-Akt signaling pathway by way of a Ca21/CaM-dependent mechanism. Up-regulating hepatic FAM3A expression may represent an attractive means for the treatment of insulin resistance, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD). (HEPATOLOGY 2014;59:1779-1790)
T
ype 2 diabetes has become one of the most prevalent and debilitating chronic diseases, with a global prevalence 6.4%, affecting about 285 million adults in the year 2010.1 Hepatic insulin resistance and fatty liver play a crucial role in the development and progression of type 2 diabetes. Liver is the key tissue regulating release of glucose into circulation during the fasting state, and hepatic insulin resistance is a decisive factor causing fasting hypergly-
cemia and type 2 diabetes. The liver is also one of the major organs regulating triglyceride (TG) and cholesterol (CHO) metabolism.2 Hepatic insulin resistance is mainly described as the failure of insulin to repress the expression of gluconeogenic genes through the PI3K/ Akt signaling pathway and is closely associated with the dysregulation of glucose and lipid metabolism in the liver.2 Although the underlying mechanisms remain largely unknown, increasing evidence points to
Abbreviations: ANOVA, analysis of variance; ATP, adenosine triphosphate; CaM, calmodulin; CHO, cholesterol; HFD, high-fat diet; NAFLD, nonalcoholic fatty liver disease; PANDER, pancreatic derived factor; PLC, phospholipase C; TG, triglyceride. From the 1Department of Physiology and Pathophysiology, Key Laboratory of Molecular Cardiovascular Science of the Ministry of Education, Peking University Health Science Center, Beijing, China; 2Institute of Clinical Molecular Biology & Central Laboratory, Peking University People’s Hospital, Beijing, China; 3 Department of Gastroenterology, Peking University People’s Hospital, Beijing, China; 4Department of Surgery, Peking University First Hospital, Beijing, China; 5 Department of Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China; 6State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing, China; 7Shenzhen University Diabetes Center, Shenzhen University Health Science Center, Shenzhen, China. Received May 29, 2013; accepted November 18, 2013. Supported by grants from the Ministry of Science and Technology (2012CB517504/2009CB941603/2010CB912503) and the Natural Science Foundation (81170791/2011ZX09102/81322011). Support for this project was also provided by the "111 Project" of China and a grant from Beijing Natural Science Foundation (7122107). *These authors contributed equally to this work. 1779
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a close association between reduced hepatic adenosine triphosphate (ATP) synthesis and the progression of hepatic insulin resistance, steatosis, and type 2 diabetes in both human and rodents.3 The FAM3 gene family is a novel cytokine-like gene family, which includes four members designated as FAM3A, FAM3B, FAM3C, and FAM3D.4 Due to the high abundance of FAM3B in pancreatic islets, it is also called pancreatic-derived factor (PANDER).5 We and others have previously shown that PANDER negatively regulates pancreatic b cell function and hepatic insulin sensitivity.5-9 More recently, liver-derived PANDER is found to be involved in the progression of nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes.10,11 Increased hepatic PANDER expression in insulinresistant mice promotes gluconeogenesis and lipogenesis in the liver by way of the inhibition of Akt and adenosine monophosphate kinase (AMPK) and the activation of FOXO1.11 In contrast, PANDER deficiency protects against high-fat diet (HFD)-induced hyperglycemia in mice.12 Collectively, these findings suggest that dysregulated PANDER may promote the pathogenesis of type 2 diabetes by decreasing pancreatic b cell function and hepatic insulin sensitivity.13 To date, the physiological role of FAM3A remains largely unknown. In the present study, we report that FAM3A is constitutively expressed in the liver, where its expression is significantly decreased in db/db and HFD-induced diabetic mice. Hepatic overexpression of FAM3A significantly attenuates hyperglycemia, insulin resistance, and fatty liver in those mice by way of insulin-independent, Ca21/CaM-mediated activation of PI3K/Akt signaling. These findings reveal a vital role of FAM3A in the regulation of glucose and lipid metabolism in the liver.
Materials and Methods Antibodies. Anti-FAM3A antibody for western blot or immunofluorescence was purchased from Sigma. All other antibodies used in the study were obtained from Santa Cruz or Cell Signaling. The ade-
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novirus expressing a full-length murine FAM3A cDNA (Ad-FAM3A) was constructed using the FAM3A cDNA coding sequence kindly provided by Dr. Bryan A. Wolf (University of Pennsylvania).4 Anti-phosphorylated proteins detected in this study were as follows: pAkt(Ser473), pIRS1(Tyr895), pAMPK(Thr172), pGSK3a(Ser21), and pGSK3b(Ser9). Cell Culture. The human hepatocarcinoma cell line (HepG2) was purchased from the American Type Culture Collection (Rockville, MD), infected with 50 multiplicity of infection (MOI) of Ad-GFP or AdFAM3A for 48 hours. For insulin stimulation, the cells were serum-starved for 12 hours, followed by treatment with 100 nM insulin (NOVO) for 5 minutes.9 For inhibition of PI3K or the P2 receptor or calcium signaling, the cells were treated with indicated concentrations of wortmannin, LY294002, PIK75, TGX221, PPADS, suramin, U73122, 2-APB, and CPZ for 30 minutes to 1 hour before experimental assay. Experimental Animals and Human Liver Specimens. Eight-week-old male C57BL/6 mice or 8 to 12-week-old male db/db mice on a BKS background were used in this study. Eight-week-old male db/db mice were forced to swim in a water bath at a temperature of 35 6 2 C for 40 minutes per day for 7 weeks. Eight-week-old C57BL/6 mice were fed a 45% HFD14 for 12 weeks before experimental assays. Liver specimens were obtained from volunteers with simple steatosis diagnosed by histological examination. All procedures involving experimental animals were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center. Overexpression or Knockdown of FAM3A in Mouse Liver. To overexpress FAM3A in the liver, 1.0 3 109 plaque-forming units (pfu) Ad-FAM3A or Ad-GFP was injected into db/db mice (male, 8 to 12-week-old) by way of the tail vein as previously described.11,14,15 At the 4th and 7th day post-virus injection, glucose tolerance tests were performed. On the 8th day, the fed animals were sacrificed for experimental analysis. To knockdown hepatic FAM3A
Address reprint requests to: Youfei Guan, M.D, Ph.D., Department of Physiology and Pathophysiology, Shenzhen University Health Science Center, Shenzhen 518060, China. E-mail: [email protected] or Jichun Yang, Ph.D., Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100191, China. E-mail: [email protected]. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.26945 Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article.
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expression in normal C57BL/6 mice, a mixture of three sets of stealth small interfering RNA (siRNA) against mouse FAM3A cDNA coding sequence was synthesized by Invitrogen (sense1, 50 -UGAACUUCA AGAGAUCAUUGACAUC-30 , antisense1, 50 -GAUG UCAAUGAUCUCUUGAAGUUCA-30 ; sense2, 50 -U AUUCGAAAGCUCAGGUGCUCUUCA-30 , antisense2, 50 -UGAAGAGCACCUGAGCUUUCGAAU A-30 ; sense3, 50 -AUAAUGAUGAUUAGGGCCACG AUGC-30 , antisense3, 50 -GCAUCGUGGCCCUAAU CAUCAUUAU-30 ). The siRNA mixture was administrated to C57BL/6 mice by way of tail vein injection at 2.5 mg/kg body weight in 100 lL of sterile saline (Scrambled sequences as control). Then, 72 hours later glucose tolerance tests were performed. Hepatic and serum triglyceride (TG) and cholesterol (CHO) levels and serum lipid profile were analyzed as described previously.11,16 Oral Glucose Tolerance Testing. Mice were fasted for 6 hours (8 AM to 2 PM) were gavaged with 3 g/kg of glucose, and blood glucose levels were measured at timepoints of 0, 15, 30, 60, 90, and 120 minutes by blood collected from the tail vein using a Freestyle brand glucometer (Roche). Real-Time Polymerase Chain Reaction (PCR) Assays of Target Gene Messenger RNA (mRNA) Levels. The protocol for real-time PCR assays was detailed previously.11,17 Each sample was measured in duplicate or triplicate. The relative expression levels of target genes were calculated using 2-DDCt methodology as detailed previously.11,17 Sequences of the primers used for real-time PCR or PCR assays were listed in Supporting Table 1. Immunoprecipitation and Immunoblot Assays. The immunoprecipitation was performed using Protein G Agarose bead as described previously.9 For immunoblot analysis, 50-200 lg of liver protein was separated by 10%-12% sodium dodecyl sulfate (SDS) gel. Immunoblot was performed and the membrane was developed with enhanced chemiluminescence (ECL). Determination of ATP Content. 100 mg of frozen liver tissues or cultured cells were lysed in a lysis buffer provided by ATP-Lite Assay Kit (Vigorous Biotechnology Beijing). The medium of cultured cells was also collected for ATP determination. The ATP content was measured (nmol) and normalized by protein concentration (nmol/mg.protein) in the same sample and presented as a percentage of the control. Statistical Analysis. Data are presented as mean 6 SEM. Statistical significance of differences between groups was analyzed by unpaired t test or by one-way analysis of variance (ANOVA) when more
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than two groups were compared. Statistical significance was set at P < 0.05.
Results Hepatic FAM3A Expression Is Reduced in db/db and HFD-Induced Diabetic Mice. Quantitative RTPCR assays revealed a ubiquitous expression of FAM3A mRNA in all mouse tissues including the liver (Supporting Fig. 1A,B). FAM3A protein was diffusely expressed in mouse livers, but selectively expressed in pancreatic islets (Fig. 1A). To study the potential role of FAM3A in glucose and lipid metabolism, its expression in the main glucose- or lipid-metabolizing tissues of db/db and HFD-fed mice was determined. Compared with the control mice, both the mRNA and protein levels of FAM3A were significantly reduced in the livers of db/db mice (Fig. 1B) and HFD-fed mice (Fig. 1C). Constitutive expression of FAM3A was also observed in human livers (Fig. 1D), and its expression were markedly reduced in patients with NAFLD (Fig. 1E,F). The human liver biopsies were performed with patient consent within the diagnostic workup of NAFLD. Oil Red O staining revealed that lipid deposition were markedly increased in the livers of patients with NAFLD (Supporting Fig. 2A). Masson and F4/ 80 staining assays revealed that there were no significant fibrosis and inflammation in the livers of NAFLD patients involved in the present study. These findings support the diagnosis of simple steatosis (Supporting Fig. 2B,C, Supporting Table 2). Interestingly, reduced hepatic FAM3A expression was restored by a 7-week swimming exercise with a marked amelioration of hyperglycemia and fatty liver11 in db/db mice (Supporting Fig. 1B,D). In contrast, a 7-week swimming exercise failed to affect FAM3A expression in db/m mouse livers with little effect on fasting blood glucose and hepatic lipid deposition (Supporting Fig. 3A-D). Hepatic Overexpression of FAM3A Ameliorates Hyperglycemia and Fatty Liver in Type 2 Diabetic Mice. FAM3A was overexpressed in the livers of db/ db mice to evaluate its role in glucose and lipid metabolism (Supporting Fig. 4A,B). Compared with Ad-GFP-treated mice, Ad-FAM3A-treated mice exhibited a significant reduction in fasting blood glucose levels. Glucose intolerance was also significantly improved by hepatic overexpression of FAM3A (Fig. 2A-D). Morphological and Oil Red O staining analyses revealed a significant reduction in hepatic lipid deposition after FAM3A overexpression (Fig. 2E), which was consistent with a significant reduction in hepatic TG content (Fig. 2E). Serum insulin levels
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Fig. 1. FAM3A expression was reduced in the livers of obese diabetic mice and patients with NAFLD. (A) Immunohistochemical and immunofluorescent analyses of FAM3A localization in the liver and pancreas. (B) The mRNA (upper panel) and protein (lower panel) levels of FAM3A were reduced in the livers of db/db mice. *P < 0.05 versus db/m mice (n 5 5). (C) The mRNA (upper panel) and protein (down panel) levels of FAM3A were decreased in the livers of mice fed an HFD for 12 weeks. ND, normal diet; HFD, high-fat diet. *P < 0.05 versus ND mice (n 5 57). (D) FAM3A mRNA was expressed in human livers. L1-4 represent four healthy human liver samples; Neg: negative control. (E,F) The mRNA (E) and protein (F) levels of FAM3A were reduced in the livers of patients with NAFLD when compared to healthy subjects. Liver specimens were obtained from volunteers with simple steatosis diagnosed by histological examination. *P < 0.05 versus healthy subjects (n 5 4).
were reduced after FAM3A overexpression (Fig. 2F). Serum TG levels were slightly increased by hepatic FAM3A overexpression, with little change in serum cholesterol levels (Supporting Fig. 4C,D). Plasma lipid profile analysis revealed an increase in very low density lipoprotein (VLDL)-TG levels in mice with hepatic FAM3A overexpression (Supporting Fig. 4E). Consistently, hepatic FAM3A overexpression increased the mRNA level of microsomal triglyceride transfer protein (MTP), the rate-limiting enzyme controlling VLDL assembly and secretion (Supporting Fig. 4F). Similarly, hepatic overexpression of FAM3A also markedly attenuated hyperglycemia, hyperinsulinemia, glucose intolerance, and steatosis in HFD-fed mice (Supporting Figs. 5, 6). Pyruvate tolerance test clearly indicated that FAM3A overexpression for 7 days repressed hepatic glucose production (Supporting Fig. 7A,B). Euglycemic-hyperinsulinemic clamp assay demonstrated that FAM3A overexpression significantly improved the global insulin sensitivity of db/db mice
(Supporting Fig. 7C). Moreover, injection of AdFAM3A had little effect on FAM3A protein levels in skeletal muscle and the pancreas of db/db mice (Supporting Fig. 8A,B). Hepatic FAM3A Overexpression Activates Akt and Suppresses Glucogenesis and Lipogenesis. the phosphorylated Akt (pAkt) level was elevated about 5-fold in the livers of db/db mice after FAM3A overexpression (Fig. 3A,B). Levels of phosphorylated GSK3a/b (pGSK3a/b) were also increased (Fig. 3A,B). The protein levels of gluconeogenic enzymes G6Pase and PEPCK was reduced (Fig. 3A,B). Consistently, their mRNA levels were also repressed (data not shown). In addition, the protein levels of genes involved in the regulation of insulin sensitivity including adiponectin receptor 1 (AdipoR1), phosphorylated AMPK (pAMPK), and uncoupling protein 2 (UCP2) were up-regulated, whereas lipogenic enzyme FAS was repressed in livers with FAM3A overexpression (Fig. 3C,D). Although the expression of FXR was decreased
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Fig. 2. Hepatic overexpression of FAM3A markedly ameliorated hyperglycemia and steatosis of db/db mice. (A) Oral glucose tolerance test (OGTT) of mice before Ad-GFP or Ad-FAM3A injection. (B,C) OGTT of mice at 4th and 7th day post-virus injection, respectively. (D) Area under curves (AUC) for the OGTT data shown in A-C. (E) Morphological examination and Oil Red O staining revealed a marked reduction of lipid accumulation in the livers with FAM3A overexpression. Quantitative assay of triglyceride content in the livers is shown in the lower panel. (F) Hepatic overexpression of FAM3A ameliorated hyperinsulinemia of db/db mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus control mice (n 5 10).
and the expression of LXRa was increased, little change was observed in PPARa, PPARb, PPARc, and LXRb expression levels in livers (Fig. 3E,F). Similarly, hepatic FAM3A overexpression also increased the protein levels of pAkt, pGSK, AdipoR1, and UCP2 and reduced the protein levels of PEPCK, G6Pase, and FAS in the livers of HFD-fed mice (Supporting Fig. 9A,B).
siRNA-Knockdown of Hepatic FAM3A Results in Hyperglycemia and Hepatic Lipid Accumulation in C57BL/6 Mice. Three days after tail vein injection of siFAM3A, the mRNA and protein levels of FAM3A were reduced by about 40%-60% in mouse livers (Supporting Fig. 10A,B). siFAM3A-treated mice exhibited fasting hyperglycemia when compared to
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Fig. 3. Effect of FAM3A overexpression on the protein levels of glucose- and lipid-metabolizing genes in the livers of db/db mice. (A,B) FAM3A overexpression increased Akt and GSK3a/b phosphorylation and decreased gene expression of gluconeogenic PEPCK and G6Pase. (C,D) FAM3A overexpression increased AdipoR1, pAMPK, and UCP2 expression and decreased FAS expression. (E,F) Effect of hepatic FAM3A overexpression on the expression of nuclear receptors. Representative gel images were shown in (A,C,E) and quantitative data shown in (B,D,F). *P < 0.05, **P < 0.01 versus control mouse livers (n 5 6-8).
scrambled siRNA-treated mice (Fig. 4A). Although morphological examination and Red Oil O staining did not show massive lipid accumulation (Fig. 4B), hepatic TG content was significantly increased (Fig. 4C), with little change in hepatic CHO levels after siFAM3A treatment (data not shown). Moreover, serum TG and CHO levels were not significantly affected by siFAM3A treatment (data not shown). Western blot analysis revealed that pAkt and AdipoR1 levels were decreased, with increased protein (Fig. 4D,E) and mRNA levels (Fig. 4F) of G6Pase in siFAM3A-treated livers. Hepatic silencing of FAM3A increased mRNA expression of CD36 and ELOVL6 (Supporting Fig. 10C) and protein levels of cleaved SREBP-1 (Fig. 4D,E) and CD36 (Supporting Fig. 10D). Moreover, siFAM3A injection did not alter FAM3A protein levels in skeletal muscle and the pancreas of wild-type mice (Supporting Fig. 8C,D). FAM3A Activates Akt by Way of PI3K-Mediated, Insulin-Independent Pathway. In Ad-GFP-treated HepG2 cells, insulin elevated pAkt levels by 3-fold (P < 0.05). In Ad-FAM3A-treated cells, basal levels of pAkt were 4-fold higher than that in Ad-GFP-treated cells in the absence of insulin. Insulin treatment failed to further increase pAkt levels (Fig. 5A,B). FAM3A
overexpression had little effect on tyrosine phosphorylated IRS-1 (pIRS-1) levels and its association with the p85 subunit of PI3K despite the presence of insulin (Supporting Fig. 11A). Consistently, FAM3A had little effect on pIRS-1 levels and its association with p85 in db/db mouse livers (Supporting Fig. 11B). Similarly, FAM3A also increased pAkt levels in cultured mouse primary hepatocytes independent of insulin (Supporting Fig. 11C). Pretreatment of HepG2 cells with the PI3K inhibitors wortmannin or LY294002 completely abolished FAM3A-induced Akt activation independent of insulin (Fig. 5C,D). Furthermore, inhibition of PI3K p110a subunit reduced FAM3A-induced Akt activation by more than 85%, while inhibition of PI3K p110b subunit had little effect on FAM3Ainduced Akt activation (Fig. 5C,D). The protein levels of PTEN, p110a, and p110b were not affected by FAM3A in HepG2 cells or in livers of db/db mice (Supporting Fig. 11D,E). Clearly, FAM3A-mediated Akt phosphorylation requires PI3K p110a subunit activation but is independent of insulin. In support, hepatic FAM3A overexpression improved hyperglycemia, activated Akt, and repressed G6Pase and PEPCK expression in streptozotocin (STZ)-induced type 1 diabetic mouse livers (Supporting Fig. 12A-D). In
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Fig. 4. siRNA-mediated knockdown of hepatic FAM3A increased fasting glucose levels in normal C57BL/6 mice. (A) siFAM3A treatment elevated fasting blood glucose levels in normal C57BL/6 mice. (B) Morphological and Oil Red O staining assays of lipid deposition in mouse livers treated with siFAM3A. (C) Triglyceride contents were significantly increased in livers with siFAM3A-mediated FAM3A gene silencing. (D) FAM3A knockdown reduced pAkt and AdipoR1 levels, and increased the protein levels of gluconeogenic G6Pase and cleaved SREBP1 in the livers. (E) Quantitative assays of the protein levels in D. (F) The mRNA levels of G6Pase and PEPCK were increased in the livers with FAM3A knockdown. Scram, mice treated with scrambled siRNAs; siFAM3A, mice treated with FAM3A siRNAs. *P < 0.05, **P < 0.01 versus control mouse livers (n 5 5).
addition, FAM3A also up-regulated the protein levels of AdipoR1 and UCP2 in STZ-treated mouse livers (Supporting Fig. 12B,C). FAM3A Overexpression Increases Intracellular and Extracellular ATP Levels in Liver Cells. Immunofluorescence assay revealed that FAM3A protein was mainly localized in the mitochondria and endoplasmic reticulum (ER) in HepG2 cells without (Supporting Fig. 13A,B) or with the transfection of an HA-tagged FAM3A plasmid (Supporting Fig. 13C,D). FAM3A protein was detected in isolated mitochondria of HepG2 cells (Supporting Fig. 13E) and mouse livers (Supporting Fig. 13F) by western blot assays. The FAM3A protein level was increased in isolated mitochondria from mouse livers FAM3A overexpression
(Supporting Fig. 13G). Little FAM3A protein was found to be present in the nucleus of cultured hepatocytes and liver cells (Supporting Fig. 13A,D), or medium of HepG2 cells with FAM3A overexpression (Supporting Fig. 13H). Given the critical role of the mitochondria in ATP production, the impact of FAM3A on cellular ATP content was evaluated in mouse liver and HepG2 cells. FAM3A overexpression increased ATP content in db/db, HFD-fed, and STZtreated mouse livers (Fig. 6A), whereas FAM3A knockdown slightly reduced hepatic ATP levels in C57BL/6 mice (data not shown). FAM3A overexpression elevated intracellular and extracellular ATP levels in HepG2 cells and primary mouse hepatocytes (Fig. 6B).
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Fig. 5. FAM3A activated Akt by way of a p110a-dependent mechanism in HepG2 cells. (A) FAM3A activated Akt in the absence of insulin stimulation. Infected HepG2 cells (48 hours) were serum starved and stimulated with or without insulin (100 nM) for 5 minutes before pAkt was assayed. (B) Quantitative assay of pAkt levels by western blot shown in (A). (C) FAM3A induced Akt activation by way of a PI3K-dependent manner. After serum starvation, infected cells were treated with wortmannin (1 lM), LY294002 (50 lM), p110a inhibitor PIK75 (100 nM), or p110b inhibitor TGX221 (100 nM) for 30 minutes before pAkt was assayed. D: DMSO, W: wortmannin, LY: LY294002, PIK: PIK75, TGX: TGX221. (D) Quantitative assays of pAkt levels shown in (C). *P < 0.05 versus DMSO-treated Ad-GFP-infected cells, #P < 0.05 versus Ad-FAM3A-infected cells treated with DMSO (n 5 5).
FAM3A Activates Akt Through the ATP/P2 Receptor Pathway. ATP can function as a signaling molecule.18 Treatment of HepG2 cells with 100 lM ATP for 5 minutes activated Akt by way of a p110adependent mechanism (Supporting Fig. 14A). ATPinduced Akt phosphorylation was completely abolished by the ATP receptor P2 antagonist PPADS (Supporting Fig. 14B). Treatment of ATP significantly increased cytosolic free calcium in HepG2 cells (Supporting Fig. 14C), which was blocked by either the phospholipase C (PLC) inhibitor, U73122, or the inositol 1,4,5-trisphosphate receptor (IP3R) antagonist, 2-APB (Supporting Fig. 14C,D). FAM3A-induced Akt activation was inhibited by two P2 receptor antagonists, PPADS (Fig. 6C) and suramin (Supporting Fig. 14E), in HepG2 cells. FAM3A overexpression increased cytosolic free calcium levels, which was blocked by PPADS, suramin, and 2APB (Supporting Fig. 15A-C). FAM3A-induced Akt activation was repressed by the IP3R antagonist 2-APB (Fig. 6D) and the PLC inhibitor U73122 (data not shown) in HepG2 cells. FAM3A-induced Akt activation was also partially dependent on the presence of extracellular Ca21 (Fig. 6E). Importantly, the calmodulin (CaM) antagonist CPZ completely abolished
FAM3A-induced Akt activation in HepG2 cells (Fig. 6F). These results suggest that FAM3A-induced Akt activation is dependent on ATP-activated P2 receptor signaling, which increases free cytosolic calcium possibly by way of the activation of the P2Y-PLC pathway and P2X calcium channel.
Discussion Since the discovery of the FAM3 gene family, the roles of FAM3B (PANDER) in glucose and lipid metabolism have been extensively investigated.4,5 However, the biological function of FAM3A remains poorly understood. In this study, we found FAM3A expression was reduced in the livers of patients with NAFLD and obese diabetic mice. Physical exercise, which attenuated hyperglycemia and fatty liver in db/db mice,11 increased hepatic FAM3A expression levels. These findings implicate that deregulated FAM3A expression might be associated with the pathogenesis of fatty liver and hepatic insulin resistance. Regarding possible mechanisms underlying the connections between exercise and FAM3A, we have recently demonstrated that FAM3A expression in liver cells was down-regulated by fatty acids, but up-
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Fig. 6. FAM3A activated Akt through the ATP/P2 receptor/CaM pathway. (A) FAM3A overexpression increased hepatic ATP contents of db/db, HFD-, and STZ-treated mice. *P < 0.05 versus control mice in each group (n 5 5-10). (B) FAM3A overexpression elevated intracellular and extracellular ATP levels in HepG2 cells (upper panel) and primary cultured mouse hepatocytes (lower panel). *P < 0.05 versus control cells (n 5 5-10). (C,D) Western blot analysis demonstrated that FAM3A-induced Akt activation was partially inhibited by the ATP receptor P2 antagonist PPADS (C) and the IP3R antagonist 2-APB (D). Infected HepG2 cells were with PPADS (50 lM) or 2-APB (10 lM) for 1 hour. *P < 0.05 versus control cells without treatment, #P < 0.05 versus Ad-FAM3A infected cells without treatment (n 5 5). (E) FAM3A-induced Akt activation was partially dependent on the presence of extracellular calcium. Infected cells were treated with a calcium free medium plus 0.5 mM EGTA for 2 hours before pAkt levels were assayed. *P < 0.05 versus control cells, #P < 0.05 versus Ad-FAM3A infected cells with calcium (n 5 5). (F) FAM3A-induced Akt activation was completely abolished by the CaM antagonist CPZ. Infected HepG2 cells were treated with CPZ (100 lM) for 1 hour before pAkt levels was measured. *P < 0.05 versus control cells, #P < 0.05 versus Ad-FAM3A infected cells without CPZ treatment (n 5 5).
regulated by PPARc activation.19 We also found that chronic exposure to high concentrations of insulin repressed FAM3A expression in HepG2 cells (data not shown). Therefore, swimming exercise may restore FAM3A expression in diabetic mouse livers by way of reducing hepatic lipid accumulation and attenuating hyperinsulinemia. OGTT, pyruvate tolerance test, and hyperinsulinemic-euglycemic clamp assays indicated that hepatic overexpression of FAM3A markedly attenuated hyperglycemia and insulin resistance, suggesting suppressed hepatic gluconeogenesis and improved insulin signaling may be responsible for metabolic improvement in db/db mice. FAM3A overexpression also attenuated hepatic steatosis, possibly by way of repressing fatty acid synthesis and increasing fatty acid oxidation and VLDL-TG secretion. Hepatic overex-
pression of FAM3A improved global insulin sensitivity and attenuated fatty liver with increased pAkt levels in the livers of diabetic mice. In contrast, siRNAmediated knockdown of hepatic FAM3A resulted in a fasting hyperglycemia and increased hepatic lipid accumulation, with reduced pAkt levels and increased gluconeogenesis in the livers of normal mice. Mechanistically, FAM3A increases mitochondrial ATP production and secretion, which activates the PI3KAkt signaling pathway by way of an insulinindependent, ATP receptor-mediated mechanism. FAM3A may thus represent an attractive target for the treatment of insulin resistance, type 2 diabetes, and NAFLD. In addition to blocking gluconeogenesis and glycogenolysis, Akt has also been found to play a critical role in the regulation of liver lipid metabolism. Activation of Akt represses lipogenesis and prevents
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Fig. 7. Proposed mechanism for FAM3Ainduced activation of the PI3K-Akt signaling pathway in liver cells. FAM3A enhances ATP synthesis and elevates extracellular ATP levels, which increases cytosolic free calcium concentrations through mechanisms involving the P2 receptors. Finally, elevated cytosolic calcium activates the PI3K-Akt signaling pathway in a CaM-dependent manner, leading to a suppression of hepatic gluconeogenesis and lipogenesis. PLC, phospholipase; CaM, calmodulin; IP3, inositol (1,4,5) triphosphate.
excessive lipid deposition in the liver by way of the inactivation of GSK-3, PGC-1a, and FOXO111,20-22 and increased hepatic pAkt levels in the liver is associated with the amelioration of steatosis in diabetic mice.23 As expected, hepatic overexpression of FAM3A significantly attenuated steatosis in db/db mice and HFD-induced diabetic mice. In addition, hepatic FAM3A overexpression increases, while hepatic knockdown of FAM3A reduces, AdipoR1 expression, suggesting FAM3A may attenuate gluconeogenesis and lipogenesis and increase fatty acid oxidation by way of activation of AdipoR/AMPK signaling in the liver.24 In the present study, we found that FAM3A activates AMPK activity regardless of increased ATP production. Multiple mechanisms are involved in AMPK activation. It has been recently demonstrated that AdipoR1 also plays an important role in the regulation of AMPK activation. AdipoR1 gene-deficient mouse livers exhibit blunted AMPK activation induced by adiponectin, while overexpression of AdipoR1 increases AMPK activity in db/db mouse livers.25 Given the important role of AdipoR1 in the activation of AMPK, it is likely that up-regulation of AdipoR1 contributes to FAM3A-induced AMPK activation in diabetic mouse livers. We further showed that FAM3A activates Akt by way of a PI3K p110a-dependent pathway in liver cells, which is consistent with recent report that p110a predominantly mediates Akt activation in the liver,26 where selective inhibition of p110a increases lipid deposition.27 Interestingly, FAM3A can activate Akt in the absence of insulin, with little effect on pIRS-1 levels and the association of p85 with IRS-1, suggesting the FAM3A-activated p110a/Akt signaling pathway is
insulin-independent. In support, hepatic FAM3A overexpression also activated Akt and attenuated of hyperglycemia and hepatic gluconeogenesis in type 1 diabetic mice. For the first time, we found that FAM3A protein is present in the mitochondria of mouse hepatocytes and HepG2 cells. Furthermore, FAM3A protein was increased in the mitochondria of mouse livers and HepG2 cells treated with Ad-FAM3A. These observations demonstrate that FAM3A is a novel mitochondrial protein. In vivo, hepatic FAM3A overexpression increased, while hepatic FAM3A knockdown reduced ATP levels in mouse livers. In vitro, FAM3A overexpression increased intracellular and extracellular ATP levels in both HepG2 cells and primary cultured mouse hepatocytes. These findings strongly support the possibility that increased expression of mitochondrial FAM3A enhances ATP production, thereby increasing intracellular and extracellular levels of ATP in hepatocytes. However, the mechanism by which FAM3A increases mitochondrial ATP synthesis warrants further investigation. It has been previously reported that ATP can be released and function as a signaling molecule to activate the PI3K/Akt signaling pathway by way of ATP receptors in many organs or cell types.18,28,29 The receptors mediating such action include P2 purinoceptors, i.e., P2X and P2Y receptors. P2X receptors are ligand-gated ion channels that are permeable to calcium,28 while P2Y receptors are G-protein-coupled receptors that stimulate PLC to increase IP3 production, resulting in calcium release from internal stores.28 As a result of increased cytosolic free calcium level upon extracellular ATP treatment, CaM is activated,
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leading to the activation of the PI3K/Akt pathway.30,31 In the present study, we confirmed that treatment of HepG2 cells with exogenous ATP elevated cytosolic free calcium and activated Akt in a p110a-dependent manner, which was attenuated by the inhibition of the P2 receptors or their downstream signaling molecules, including PLC and IP3R. FAM3A-induced intracellular calcium level increases and Akt activation were significantly blocked by P2 receptor antagonists, PLC inhibitor, IP3R antagonist, and depletion of extracellular calcium in HepG2 cells. Furthermore, the finding that the CaM antagonist CPZ completely abolished FAM3Ainduced Akt activation reveals a critical role of increased cellular calcium level in this process. Collectively, these findings demonstrate that both P2 receptor isoforms are involved in FAM3A-induced increases in cytosolic free calcium and Akt activation in liver cells. Interestingly, although blockage of the P2 receptors completely inhibited Akt activation induced by exogenous ATP, it did not completely abolish FAM3A-induced Akt activation, suggesting that other minor mechanism(s) may also be involved. One possibility is that FAM3A-elicited increase in intracellular ATP level leads to the closure of the KATP channels, resulting in increased calcium influx through L-type calcium channel.17 The findings that exogenous ATP can elicit intracellular calcium signaling and Akt activation through P2 receptors in liver cells suggests a novel mechanism involved in hepatic glucose and lipid homeostasis regulation (Fig. 7). Increasing evidence has revealed that impaired energy metabolism in the liver could be an early defect in the pathogenesis of type 2 diabetes in patients.32,33 It has been also reported that reduced hepatic ATP levels are associated with the development of NAFLD in rats.34 These observations support a possibility that defective ATP synthesis and P2 receptor-mediated signaling may underlie hepatic insulin resistance and elevated liver lipid contents. In summary, the present study demonstrates that FAM3A plays a crucial role in hepatic glucose and lipid metabolism. FAM3A enhances ATP synthesis and elevates extracellular ATP levels, which activate P2 receptors to increase cytosolic free calcium level, leading to activation PI3K-Akt signaling pathway in a CaM-dependent manner and suppression of hepatic gluconeogenesis and lipogenesis. In addition, FAM3A may also activate the AdipoR1-AMPK signaling pathway and enhance fatty acid b oxidation in the liver by way of a yet unknown mechanism. In conclusion, to increase hepatic ATP synthesis and signaling by way of up-regulation of FAM3A might represent a promising means for treatment of insulin resistance and NAFLD.
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References 1. Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract 2010;87:4-14. 2. Biddinger SB, Kahn CR. From mice to men: insights into the insulin resistance syndromes. Annu Rev Physiol 2006;68:123-158. 3. Turner N, Heilbronn LK. Is mitochondrial dysfunction a cause of insulin resistance? Trends Endocrinol Metab 2008;19:324-330. 4. Zhu Y, Xu G, Patel A, McLaughlin MM, Silverman C, Knecht K, et al. Cloning, expression, and initial characterization of a novel cytokine-like gene family. Genomics 2002;80:144-150. 5. Cao X, Yang J, Burkhardt BR, Gao Z, Wong RK, Greene SR, et al. Effects of overexpression of pancreatic derived factor (FAM3B) in isolated mouse islets and insulin-secreting betaTC3 cells. Am J Physiol Endocrinol Metab 2005;289:E543-550. 6. Burkhardt BR, Yang MC, Robert CE, Greene SR, McFadden KK, Yang J, et al. Tissue-specific and glucose-responsive expression of the pancreatic derived factor (PANDER) promoter. Biochim Biophys Acta 2005;1730:215-225. 7. Yang J, Gao Z, Robert CE, Burkhardt BR, Gaweska H, Wagner A, et al. Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells. Biochemistry 2005; 44:11342-11352. 8. Yang J, Robert CE, Burkhardt BR, Young RA, Wu J, Gao Z, et al. Mechanisms of glucose-induced secretion of pancreatic-derived factor (PANDER or FAM3B) in pancreatic beta-cells. Diabetes 2005;54: 3217-3228. 9. Yang J, Wang C, Li J, Burkhardt BR, Robert-Cooperman CE, Wilson C, et al. PANDER binds to the liver cell membrane and inhibits insulin signaling in HepG2 cells. FEBS Lett 2009;583:3009-3015. 10. Robert-Cooperman CE, Carnegie JR, Wilson CG, Yang J, Cook JR, Wu J, et al. Targeted disruption of pancreatic-derived factor (PANDER, FAM3B) impairs pancreatic {beta}-cell function. Diabetes 2010; 59:2209-2218. 11. Li J, Chi Y, Wang C, Wu J, Yang H, Zhang D, et al. Pancreaticderived factor promotes lipogenesis in the mouse liver: role of the Forkhead box 1 signaling pathway. HEPATOLOGY 2011;53:1906-1916. 12. Robert-Cooperman CE, Wilson CG, Burkhardt BR. PANDER KO mice on high-fat diet are glucose intolerant yet resistant to fasting hyperglycemia and hyperinsulinemia. FEBS Lett 2011;585:1345-1349. 13. Wang C, Burkhardt BR, Guan Y, Yang J. Role of pancreatic-derived factor in type 2 diabetes: evidence from pancreatic beta cells and liver. Nutr Rev 2012;70:100-106. 14. Wu J, Wang C, Li S, Li S, Wang W, Li J, et al. Thrsp promotes hepatic lipogenesis, and its expression LXRalpha through an SREBP1cdependent mechanism. HEPATOLOGY 2013;58:617-628. 15. Qin X, Xie X, Fan Y, Tian J, Guan Y, Wang X, et al. Peroxisome proliferator-activated receptor-delta induces insulin-induced gene-1 and suppresses hepatic lipogenesis in obese diabetic mice. HEPATOLOGY 2008;48:432-441. 16. Zhou Y, Zhang X, Chen L, Wu J, Dang H, Wei M, et al. Expression profiling of hepatic genes associated with lipid metabolism in nephrotic rats. Am J Physiol Renal Physiol 2008;295:F662-671. 17. Yang J, Wong RK, Park M, Wu J, Cook JR, York DA, et al. Leucine regulation of glucokinase and ATP synthase sensitizes glucose-induced insulin secretion in pancreatic beta-cells. Diabetes 2006;55:193-201. 18. Gonzales E, Julien B, Serriere-Lanneau V, Nicou A, Doignon I, Lagoudakis L, et al. ATP release after partial hepatectomy regulates liver regeneration in the rat. J Hepatol 2010;52:54-62. 19. Zhou Y, Jia S, Wang C, Chen Z, Chi Y, Li J, et al. FAM3A is a target gene of peroxisome proliferator-activated receptor gamma. Biochim Biophys Acta 2013;1830:4160-4170. 20. Li X, Monks B, Ge Q, Birnbaum MJ. Akt/PKB regulates hepatic metabolism by directly inhibiting PGC-1alpha transcription coactivator. Nature 2007;447:1012-1016. 21. Kim JJ, Li P, Huntley J, Chang JP, Arden KC, Olefsky JM. FoxO1 haploinsufficiency protects against high-fat diet-induced insulin
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22.
23.
24.
25.
26.
27.
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resistance with enhanced peroxisome proliferator-activated receptor gamma activation in adipose tissue. Diabetes 2009;58:1275-1282. Matsumoto M, Han S, Kitamura T, Accili D. Dual role of transcription factor FoxO1 in controlling hepatic insulin sensitivity and lipid metabolism. J Clin Invest 2006;116:2464-2472. Bijl N, Sokolovic M, Vrins C, Langeveld M, Moerland PD, Ottenhoff R, et al. Modulation of glycosphingolipid metabolism significantly improves hepatic insulin sensitivity and reverses hepatic steatosis in mice. HEPATOLOGY 2009;50:1431-1441. Hasenour CM, Berglund ED, Wasserman DH. Emerging role of AMP-activated protein kinase in endocrine control of metabolism in the liver. Mol Cell Endocrinol 2012;366:152-162. Yamauchi T, Nio Y, Maki T, Kobayashi M, Takazawa T, Iwabu M, et al. Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions. Nat Med 2007;13:332339. Sopasakis VR, Liu P, Suzuki R, Kondo T, Winnay J, Tran TT, et al. Specific roles of the p110alpha isoform of phosphatidylinsositol 3kinase in hepatic insulin signaling and metabolic regulation. Cell Metab 2010;11:220-230. Jackson LN, Larson SD, Silva SR, Rychahou PG, Chen LA, Qiu S, et al. PI3K/Akt activation is critical for early hepatic regeneration after partial hepatectomy. Am J Physiol Gastrointest Liver Physiol 2008;294: G1401-1410.
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28. Franke H, Illes P. Involvement of P2 receptors in the growth and survival of neurons in the CNS. Pharmacol Ther 2006;109:297-324. 29. Ishikawa M, Iwamoto T, Nakamura T, Doyle A, Fukumoto S, Yamada Y. Pannexin 3 functions as an ER Ca(21) channel, hemichannel, and gap junction to promote osteoblast differentiation. J Cell Biol 2011; 193:1257-1274. 30. Nishimatsu H, Suzuki E, Nagata D, Moriyama N, Satonaka H, Walsh K, et al. Adrenomedullin induces endothelium-dependent vasorelaxation via the phosphatidylinositol 3-kinase/Akt-dependent pathway in rat aorta. Circ Res 2001;89:63-70. 31. Cheng A, Wang S, Yang D, Xiao R, Mattson MP. Calmodulin mediates brain-derived neurotrophic factor cell survival signaling upstream of Akt kinase in embryonic neocortical neurons. J Biol Chem 2003; 278:7591-7599. 32. Schmid AI, Szendroedi J, Chmelik M, Krssak M, Moser E, Roden M. Liver ATP synthesis is lower and relates to insulin sensitivity in patients with type 2 diabetes. Diabetes Care 2011;34:448-453. 33. Szendroedi J, Chmelik M, Schmid AI, Nowotny P, Brehm A, Krssak M, et al. Abnormal hepatic energy homeostasis in type 2 diabetes. HEPATOLOGY 2009;50:1079-1086. 34. Serviddio G, Bellanti F, Tamborra R, Rollo T, Romano AD, Giudetti AM, et al. Alterations of hepatic ATP homeostasis and respiratory chain during development of non-alcoholic steatohepatitis in a rodent model. Eur J Clin Invest 2008;38:245-252.
T
ype 2 diabetes has become one of the most prevalent and debilitating chronic diseases, with a global prevalence 6.4%, affecting about 285 million adults in the year 2010.1 Hepatic insulin resistance and fatty liver play a crucial role in the development and progression of type 2 diabetes. Liver is the key tissue regulating release of glucose into circulation during the fasting state, and hepatic insulin resistance is a decisive factor causing fasting hypergly-
cemia and type 2 diabetes. The liver is also one of the major organs regulating triglyceride (TG) and cholesterol (CHO) metabolism.2 Hepatic insulin resistance is mainly described as the failure of insulin to repress the expression of gluconeogenic genes through the PI3K/ Akt signaling pathway and is closely associated with the dysregulation of glucose and lipid metabolism in the liver.2 Although the underlying mechanisms remain largely unknown, increasing evidence points to
Abbreviations: ANOVA, analysis of variance; ATP, adenosine triphosphate; CaM, calmodulin; CHO, cholesterol; HFD, high-fat diet; NAFLD, nonalcoholic fatty liver disease; PANDER, pancreatic derived factor; PLC, phospholipase C; TG, triglyceride. From the 1Department of Physiology and Pathophysiology, Key Laboratory of Molecular Cardiovascular Science of the Ministry of Education, Peking University Health Science Center, Beijing, China; 2Institute of Clinical Molecular Biology & Central Laboratory, Peking University People’s Hospital, Beijing, China; 3 Department of Gastroenterology, Peking University People’s Hospital, Beijing, China; 4Department of Surgery, Peking University First Hospital, Beijing, China; 5 Department of Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China; 6State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing, China; 7Shenzhen University Diabetes Center, Shenzhen University Health Science Center, Shenzhen, China. Received May 29, 2013; accepted November 18, 2013. Supported by grants from the Ministry of Science and Technology (2012CB517504/2009CB941603/2010CB912503) and the Natural Science Foundation (81170791/2011ZX09102/81322011). Support for this project was also provided by the "111 Project" of China and a grant from Beijing Natural Science Foundation (7122107). *These authors contributed equally to this work. 1779
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a close association between reduced hepatic adenosine triphosphate (ATP) synthesis and the progression of hepatic insulin resistance, steatosis, and type 2 diabetes in both human and rodents.3 The FAM3 gene family is a novel cytokine-like gene family, which includes four members designated as FAM3A, FAM3B, FAM3C, and FAM3D.4 Due to the high abundance of FAM3B in pancreatic islets, it is also called pancreatic-derived factor (PANDER).5 We and others have previously shown that PANDER negatively regulates pancreatic b cell function and hepatic insulin sensitivity.5-9 More recently, liver-derived PANDER is found to be involved in the progression of nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes.10,11 Increased hepatic PANDER expression in insulinresistant mice promotes gluconeogenesis and lipogenesis in the liver by way of the inhibition of Akt and adenosine monophosphate kinase (AMPK) and the activation of FOXO1.11 In contrast, PANDER deficiency protects against high-fat diet (HFD)-induced hyperglycemia in mice.12 Collectively, these findings suggest that dysregulated PANDER may promote the pathogenesis of type 2 diabetes by decreasing pancreatic b cell function and hepatic insulin sensitivity.13 To date, the physiological role of FAM3A remains largely unknown. In the present study, we report that FAM3A is constitutively expressed in the liver, where its expression is significantly decreased in db/db and HFD-induced diabetic mice. Hepatic overexpression of FAM3A significantly attenuates hyperglycemia, insulin resistance, and fatty liver in those mice by way of insulin-independent, Ca21/CaM-mediated activation of PI3K/Akt signaling. These findings reveal a vital role of FAM3A in the regulation of glucose and lipid metabolism in the liver.
Materials and Methods Antibodies. Anti-FAM3A antibody for western blot or immunofluorescence was purchased from Sigma. All other antibodies used in the study were obtained from Santa Cruz or Cell Signaling. The ade-
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novirus expressing a full-length murine FAM3A cDNA (Ad-FAM3A) was constructed using the FAM3A cDNA coding sequence kindly provided by Dr. Bryan A. Wolf (University of Pennsylvania).4 Anti-phosphorylated proteins detected in this study were as follows: pAkt(Ser473), pIRS1(Tyr895), pAMPK(Thr172), pGSK3a(Ser21), and pGSK3b(Ser9). Cell Culture. The human hepatocarcinoma cell line (HepG2) was purchased from the American Type Culture Collection (Rockville, MD), infected with 50 multiplicity of infection (MOI) of Ad-GFP or AdFAM3A for 48 hours. For insulin stimulation, the cells were serum-starved for 12 hours, followed by treatment with 100 nM insulin (NOVO) for 5 minutes.9 For inhibition of PI3K or the P2 receptor or calcium signaling, the cells were treated with indicated concentrations of wortmannin, LY294002, PIK75, TGX221, PPADS, suramin, U73122, 2-APB, and CPZ for 30 minutes to 1 hour before experimental assay. Experimental Animals and Human Liver Specimens. Eight-week-old male C57BL/6 mice or 8 to 12-week-old male db/db mice on a BKS background were used in this study. Eight-week-old male db/db mice were forced to swim in a water bath at a temperature of 35 6 2 C for 40 minutes per day for 7 weeks. Eight-week-old C57BL/6 mice were fed a 45% HFD14 for 12 weeks before experimental assays. Liver specimens were obtained from volunteers with simple steatosis diagnosed by histological examination. All procedures involving experimental animals were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center. Overexpression or Knockdown of FAM3A in Mouse Liver. To overexpress FAM3A in the liver, 1.0 3 109 plaque-forming units (pfu) Ad-FAM3A or Ad-GFP was injected into db/db mice (male, 8 to 12-week-old) by way of the tail vein as previously described.11,14,15 At the 4th and 7th day post-virus injection, glucose tolerance tests were performed. On the 8th day, the fed animals were sacrificed for experimental analysis. To knockdown hepatic FAM3A
Address reprint requests to: Youfei Guan, M.D, Ph.D., Department of Physiology and Pathophysiology, Shenzhen University Health Science Center, Shenzhen 518060, China. E-mail: [email protected] or Jichun Yang, Ph.D., Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100191, China. E-mail: [email protected]. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.26945 Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article.
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expression in normal C57BL/6 mice, a mixture of three sets of stealth small interfering RNA (siRNA) against mouse FAM3A cDNA coding sequence was synthesized by Invitrogen (sense1, 50 -UGAACUUCA AGAGAUCAUUGACAUC-30 , antisense1, 50 -GAUG UCAAUGAUCUCUUGAAGUUCA-30 ; sense2, 50 -U AUUCGAAAGCUCAGGUGCUCUUCA-30 , antisense2, 50 -UGAAGAGCACCUGAGCUUUCGAAU A-30 ; sense3, 50 -AUAAUGAUGAUUAGGGCCACG AUGC-30 , antisense3, 50 -GCAUCGUGGCCCUAAU CAUCAUUAU-30 ). The siRNA mixture was administrated to C57BL/6 mice by way of tail vein injection at 2.5 mg/kg body weight in 100 lL of sterile saline (Scrambled sequences as control). Then, 72 hours later glucose tolerance tests were performed. Hepatic and serum triglyceride (TG) and cholesterol (CHO) levels and serum lipid profile were analyzed as described previously.11,16 Oral Glucose Tolerance Testing. Mice were fasted for 6 hours (8 AM to 2 PM) were gavaged with 3 g/kg of glucose, and blood glucose levels were measured at timepoints of 0, 15, 30, 60, 90, and 120 minutes by blood collected from the tail vein using a Freestyle brand glucometer (Roche). Real-Time Polymerase Chain Reaction (PCR) Assays of Target Gene Messenger RNA (mRNA) Levels. The protocol for real-time PCR assays was detailed previously.11,17 Each sample was measured in duplicate or triplicate. The relative expression levels of target genes were calculated using 2-DDCt methodology as detailed previously.11,17 Sequences of the primers used for real-time PCR or PCR assays were listed in Supporting Table 1. Immunoprecipitation and Immunoblot Assays. The immunoprecipitation was performed using Protein G Agarose bead as described previously.9 For immunoblot analysis, 50-200 lg of liver protein was separated by 10%-12% sodium dodecyl sulfate (SDS) gel. Immunoblot was performed and the membrane was developed with enhanced chemiluminescence (ECL). Determination of ATP Content. 100 mg of frozen liver tissues or cultured cells were lysed in a lysis buffer provided by ATP-Lite Assay Kit (Vigorous Biotechnology Beijing). The medium of cultured cells was also collected for ATP determination. The ATP content was measured (nmol) and normalized by protein concentration (nmol/mg.protein) in the same sample and presented as a percentage of the control. Statistical Analysis. Data are presented as mean 6 SEM. Statistical significance of differences between groups was analyzed by unpaired t test or by one-way analysis of variance (ANOVA) when more
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than two groups were compared. Statistical significance was set at P < 0.05.
Results Hepatic FAM3A Expression Is Reduced in db/db and HFD-Induced Diabetic Mice. Quantitative RTPCR assays revealed a ubiquitous expression of FAM3A mRNA in all mouse tissues including the liver (Supporting Fig. 1A,B). FAM3A protein was diffusely expressed in mouse livers, but selectively expressed in pancreatic islets (Fig. 1A). To study the potential role of FAM3A in glucose and lipid metabolism, its expression in the main glucose- or lipid-metabolizing tissues of db/db and HFD-fed mice was determined. Compared with the control mice, both the mRNA and protein levels of FAM3A were significantly reduced in the livers of db/db mice (Fig. 1B) and HFD-fed mice (Fig. 1C). Constitutive expression of FAM3A was also observed in human livers (Fig. 1D), and its expression were markedly reduced in patients with NAFLD (Fig. 1E,F). The human liver biopsies were performed with patient consent within the diagnostic workup of NAFLD. Oil Red O staining revealed that lipid deposition were markedly increased in the livers of patients with NAFLD (Supporting Fig. 2A). Masson and F4/ 80 staining assays revealed that there were no significant fibrosis and inflammation in the livers of NAFLD patients involved in the present study. These findings support the diagnosis of simple steatosis (Supporting Fig. 2B,C, Supporting Table 2). Interestingly, reduced hepatic FAM3A expression was restored by a 7-week swimming exercise with a marked amelioration of hyperglycemia and fatty liver11 in db/db mice (Supporting Fig. 1B,D). In contrast, a 7-week swimming exercise failed to affect FAM3A expression in db/m mouse livers with little effect on fasting blood glucose and hepatic lipid deposition (Supporting Fig. 3A-D). Hepatic Overexpression of FAM3A Ameliorates Hyperglycemia and Fatty Liver in Type 2 Diabetic Mice. FAM3A was overexpressed in the livers of db/ db mice to evaluate its role in glucose and lipid metabolism (Supporting Fig. 4A,B). Compared with Ad-GFP-treated mice, Ad-FAM3A-treated mice exhibited a significant reduction in fasting blood glucose levels. Glucose intolerance was also significantly improved by hepatic overexpression of FAM3A (Fig. 2A-D). Morphological and Oil Red O staining analyses revealed a significant reduction in hepatic lipid deposition after FAM3A overexpression (Fig. 2E), which was consistent with a significant reduction in hepatic TG content (Fig. 2E). Serum insulin levels
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Fig. 1. FAM3A expression was reduced in the livers of obese diabetic mice and patients with NAFLD. (A) Immunohistochemical and immunofluorescent analyses of FAM3A localization in the liver and pancreas. (B) The mRNA (upper panel) and protein (lower panel) levels of FAM3A were reduced in the livers of db/db mice. *P < 0.05 versus db/m mice (n 5 5). (C) The mRNA (upper panel) and protein (down panel) levels of FAM3A were decreased in the livers of mice fed an HFD for 12 weeks. ND, normal diet; HFD, high-fat diet. *P < 0.05 versus ND mice (n 5 57). (D) FAM3A mRNA was expressed in human livers. L1-4 represent four healthy human liver samples; Neg: negative control. (E,F) The mRNA (E) and protein (F) levels of FAM3A were reduced in the livers of patients with NAFLD when compared to healthy subjects. Liver specimens were obtained from volunteers with simple steatosis diagnosed by histological examination. *P < 0.05 versus healthy subjects (n 5 4).
were reduced after FAM3A overexpression (Fig. 2F). Serum TG levels were slightly increased by hepatic FAM3A overexpression, with little change in serum cholesterol levels (Supporting Fig. 4C,D). Plasma lipid profile analysis revealed an increase in very low density lipoprotein (VLDL)-TG levels in mice with hepatic FAM3A overexpression (Supporting Fig. 4E). Consistently, hepatic FAM3A overexpression increased the mRNA level of microsomal triglyceride transfer protein (MTP), the rate-limiting enzyme controlling VLDL assembly and secretion (Supporting Fig. 4F). Similarly, hepatic overexpression of FAM3A also markedly attenuated hyperglycemia, hyperinsulinemia, glucose intolerance, and steatosis in HFD-fed mice (Supporting Figs. 5, 6). Pyruvate tolerance test clearly indicated that FAM3A overexpression for 7 days repressed hepatic glucose production (Supporting Fig. 7A,B). Euglycemic-hyperinsulinemic clamp assay demonstrated that FAM3A overexpression significantly improved the global insulin sensitivity of db/db mice
(Supporting Fig. 7C). Moreover, injection of AdFAM3A had little effect on FAM3A protein levels in skeletal muscle and the pancreas of db/db mice (Supporting Fig. 8A,B). Hepatic FAM3A Overexpression Activates Akt and Suppresses Glucogenesis and Lipogenesis. the phosphorylated Akt (pAkt) level was elevated about 5-fold in the livers of db/db mice after FAM3A overexpression (Fig. 3A,B). Levels of phosphorylated GSK3a/b (pGSK3a/b) were also increased (Fig. 3A,B). The protein levels of gluconeogenic enzymes G6Pase and PEPCK was reduced (Fig. 3A,B). Consistently, their mRNA levels were also repressed (data not shown). In addition, the protein levels of genes involved in the regulation of insulin sensitivity including adiponectin receptor 1 (AdipoR1), phosphorylated AMPK (pAMPK), and uncoupling protein 2 (UCP2) were up-regulated, whereas lipogenic enzyme FAS was repressed in livers with FAM3A overexpression (Fig. 3C,D). Although the expression of FXR was decreased
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Fig. 2. Hepatic overexpression of FAM3A markedly ameliorated hyperglycemia and steatosis of db/db mice. (A) Oral glucose tolerance test (OGTT) of mice before Ad-GFP or Ad-FAM3A injection. (B,C) OGTT of mice at 4th and 7th day post-virus injection, respectively. (D) Area under curves (AUC) for the OGTT data shown in A-C. (E) Morphological examination and Oil Red O staining revealed a marked reduction of lipid accumulation in the livers with FAM3A overexpression. Quantitative assay of triglyceride content in the livers is shown in the lower panel. (F) Hepatic overexpression of FAM3A ameliorated hyperinsulinemia of db/db mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus control mice (n 5 10).
and the expression of LXRa was increased, little change was observed in PPARa, PPARb, PPARc, and LXRb expression levels in livers (Fig. 3E,F). Similarly, hepatic FAM3A overexpression also increased the protein levels of pAkt, pGSK, AdipoR1, and UCP2 and reduced the protein levels of PEPCK, G6Pase, and FAS in the livers of HFD-fed mice (Supporting Fig. 9A,B).
siRNA-Knockdown of Hepatic FAM3A Results in Hyperglycemia and Hepatic Lipid Accumulation in C57BL/6 Mice. Three days after tail vein injection of siFAM3A, the mRNA and protein levels of FAM3A were reduced by about 40%-60% in mouse livers (Supporting Fig. 10A,B). siFAM3A-treated mice exhibited fasting hyperglycemia when compared to
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Fig. 3. Effect of FAM3A overexpression on the protein levels of glucose- and lipid-metabolizing genes in the livers of db/db mice. (A,B) FAM3A overexpression increased Akt and GSK3a/b phosphorylation and decreased gene expression of gluconeogenic PEPCK and G6Pase. (C,D) FAM3A overexpression increased AdipoR1, pAMPK, and UCP2 expression and decreased FAS expression. (E,F) Effect of hepatic FAM3A overexpression on the expression of nuclear receptors. Representative gel images were shown in (A,C,E) and quantitative data shown in (B,D,F). *P < 0.05, **P < 0.01 versus control mouse livers (n 5 6-8).
scrambled siRNA-treated mice (Fig. 4A). Although morphological examination and Red Oil O staining did not show massive lipid accumulation (Fig. 4B), hepatic TG content was significantly increased (Fig. 4C), with little change in hepatic CHO levels after siFAM3A treatment (data not shown). Moreover, serum TG and CHO levels were not significantly affected by siFAM3A treatment (data not shown). Western blot analysis revealed that pAkt and AdipoR1 levels were decreased, with increased protein (Fig. 4D,E) and mRNA levels (Fig. 4F) of G6Pase in siFAM3A-treated livers. Hepatic silencing of FAM3A increased mRNA expression of CD36 and ELOVL6 (Supporting Fig. 10C) and protein levels of cleaved SREBP-1 (Fig. 4D,E) and CD36 (Supporting Fig. 10D). Moreover, siFAM3A injection did not alter FAM3A protein levels in skeletal muscle and the pancreas of wild-type mice (Supporting Fig. 8C,D). FAM3A Activates Akt by Way of PI3K-Mediated, Insulin-Independent Pathway. In Ad-GFP-treated HepG2 cells, insulin elevated pAkt levels by 3-fold (P < 0.05). In Ad-FAM3A-treated cells, basal levels of pAkt were 4-fold higher than that in Ad-GFP-treated cells in the absence of insulin. Insulin treatment failed to further increase pAkt levels (Fig. 5A,B). FAM3A
overexpression had little effect on tyrosine phosphorylated IRS-1 (pIRS-1) levels and its association with the p85 subunit of PI3K despite the presence of insulin (Supporting Fig. 11A). Consistently, FAM3A had little effect on pIRS-1 levels and its association with p85 in db/db mouse livers (Supporting Fig. 11B). Similarly, FAM3A also increased pAkt levels in cultured mouse primary hepatocytes independent of insulin (Supporting Fig. 11C). Pretreatment of HepG2 cells with the PI3K inhibitors wortmannin or LY294002 completely abolished FAM3A-induced Akt activation independent of insulin (Fig. 5C,D). Furthermore, inhibition of PI3K p110a subunit reduced FAM3A-induced Akt activation by more than 85%, while inhibition of PI3K p110b subunit had little effect on FAM3Ainduced Akt activation (Fig. 5C,D). The protein levels of PTEN, p110a, and p110b were not affected by FAM3A in HepG2 cells or in livers of db/db mice (Supporting Fig. 11D,E). Clearly, FAM3A-mediated Akt phosphorylation requires PI3K p110a subunit activation but is independent of insulin. In support, hepatic FAM3A overexpression improved hyperglycemia, activated Akt, and repressed G6Pase and PEPCK expression in streptozotocin (STZ)-induced type 1 diabetic mouse livers (Supporting Fig. 12A-D). In
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Fig. 4. siRNA-mediated knockdown of hepatic FAM3A increased fasting glucose levels in normal C57BL/6 mice. (A) siFAM3A treatment elevated fasting blood glucose levels in normal C57BL/6 mice. (B) Morphological and Oil Red O staining assays of lipid deposition in mouse livers treated with siFAM3A. (C) Triglyceride contents were significantly increased in livers with siFAM3A-mediated FAM3A gene silencing. (D) FAM3A knockdown reduced pAkt and AdipoR1 levels, and increased the protein levels of gluconeogenic G6Pase and cleaved SREBP1 in the livers. (E) Quantitative assays of the protein levels in D. (F) The mRNA levels of G6Pase and PEPCK were increased in the livers with FAM3A knockdown. Scram, mice treated with scrambled siRNAs; siFAM3A, mice treated with FAM3A siRNAs. *P < 0.05, **P < 0.01 versus control mouse livers (n 5 5).
addition, FAM3A also up-regulated the protein levels of AdipoR1 and UCP2 in STZ-treated mouse livers (Supporting Fig. 12B,C). FAM3A Overexpression Increases Intracellular and Extracellular ATP Levels in Liver Cells. Immunofluorescence assay revealed that FAM3A protein was mainly localized in the mitochondria and endoplasmic reticulum (ER) in HepG2 cells without (Supporting Fig. 13A,B) or with the transfection of an HA-tagged FAM3A plasmid (Supporting Fig. 13C,D). FAM3A protein was detected in isolated mitochondria of HepG2 cells (Supporting Fig. 13E) and mouse livers (Supporting Fig. 13F) by western blot assays. The FAM3A protein level was increased in isolated mitochondria from mouse livers FAM3A overexpression
(Supporting Fig. 13G). Little FAM3A protein was found to be present in the nucleus of cultured hepatocytes and liver cells (Supporting Fig. 13A,D), or medium of HepG2 cells with FAM3A overexpression (Supporting Fig. 13H). Given the critical role of the mitochondria in ATP production, the impact of FAM3A on cellular ATP content was evaluated in mouse liver and HepG2 cells. FAM3A overexpression increased ATP content in db/db, HFD-fed, and STZtreated mouse livers (Fig. 6A), whereas FAM3A knockdown slightly reduced hepatic ATP levels in C57BL/6 mice (data not shown). FAM3A overexpression elevated intracellular and extracellular ATP levels in HepG2 cells and primary mouse hepatocytes (Fig. 6B).
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Fig. 5. FAM3A activated Akt by way of a p110a-dependent mechanism in HepG2 cells. (A) FAM3A activated Akt in the absence of insulin stimulation. Infected HepG2 cells (48 hours) were serum starved and stimulated with or without insulin (100 nM) for 5 minutes before pAkt was assayed. (B) Quantitative assay of pAkt levels by western blot shown in (A). (C) FAM3A induced Akt activation by way of a PI3K-dependent manner. After serum starvation, infected cells were treated with wortmannin (1 lM), LY294002 (50 lM), p110a inhibitor PIK75 (100 nM), or p110b inhibitor TGX221 (100 nM) for 30 minutes before pAkt was assayed. D: DMSO, W: wortmannin, LY: LY294002, PIK: PIK75, TGX: TGX221. (D) Quantitative assays of pAkt levels shown in (C). *P < 0.05 versus DMSO-treated Ad-GFP-infected cells, #P < 0.05 versus Ad-FAM3A-infected cells treated with DMSO (n 5 5).
FAM3A Activates Akt Through the ATP/P2 Receptor Pathway. ATP can function as a signaling molecule.18 Treatment of HepG2 cells with 100 lM ATP for 5 minutes activated Akt by way of a p110adependent mechanism (Supporting Fig. 14A). ATPinduced Akt phosphorylation was completely abolished by the ATP receptor P2 antagonist PPADS (Supporting Fig. 14B). Treatment of ATP significantly increased cytosolic free calcium in HepG2 cells (Supporting Fig. 14C), which was blocked by either the phospholipase C (PLC) inhibitor, U73122, or the inositol 1,4,5-trisphosphate receptor (IP3R) antagonist, 2-APB (Supporting Fig. 14C,D). FAM3A-induced Akt activation was inhibited by two P2 receptor antagonists, PPADS (Fig. 6C) and suramin (Supporting Fig. 14E), in HepG2 cells. FAM3A overexpression increased cytosolic free calcium levels, which was blocked by PPADS, suramin, and 2APB (Supporting Fig. 15A-C). FAM3A-induced Akt activation was repressed by the IP3R antagonist 2-APB (Fig. 6D) and the PLC inhibitor U73122 (data not shown) in HepG2 cells. FAM3A-induced Akt activation was also partially dependent on the presence of extracellular Ca21 (Fig. 6E). Importantly, the calmodulin (CaM) antagonist CPZ completely abolished
FAM3A-induced Akt activation in HepG2 cells (Fig. 6F). These results suggest that FAM3A-induced Akt activation is dependent on ATP-activated P2 receptor signaling, which increases free cytosolic calcium possibly by way of the activation of the P2Y-PLC pathway and P2X calcium channel.
Discussion Since the discovery of the FAM3 gene family, the roles of FAM3B (PANDER) in glucose and lipid metabolism have been extensively investigated.4,5 However, the biological function of FAM3A remains poorly understood. In this study, we found FAM3A expression was reduced in the livers of patients with NAFLD and obese diabetic mice. Physical exercise, which attenuated hyperglycemia and fatty liver in db/db mice,11 increased hepatic FAM3A expression levels. These findings implicate that deregulated FAM3A expression might be associated with the pathogenesis of fatty liver and hepatic insulin resistance. Regarding possible mechanisms underlying the connections between exercise and FAM3A, we have recently demonstrated that FAM3A expression in liver cells was down-regulated by fatty acids, but up-
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Fig. 6. FAM3A activated Akt through the ATP/P2 receptor/CaM pathway. (A) FAM3A overexpression increased hepatic ATP contents of db/db, HFD-, and STZ-treated mice. *P < 0.05 versus control mice in each group (n 5 5-10). (B) FAM3A overexpression elevated intracellular and extracellular ATP levels in HepG2 cells (upper panel) and primary cultured mouse hepatocytes (lower panel). *P < 0.05 versus control cells (n 5 5-10). (C,D) Western blot analysis demonstrated that FAM3A-induced Akt activation was partially inhibited by the ATP receptor P2 antagonist PPADS (C) and the IP3R antagonist 2-APB (D). Infected HepG2 cells were with PPADS (50 lM) or 2-APB (10 lM) for 1 hour. *P < 0.05 versus control cells without treatment, #P < 0.05 versus Ad-FAM3A infected cells without treatment (n 5 5). (E) FAM3A-induced Akt activation was partially dependent on the presence of extracellular calcium. Infected cells were treated with a calcium free medium plus 0.5 mM EGTA for 2 hours before pAkt levels were assayed. *P < 0.05 versus control cells, #P < 0.05 versus Ad-FAM3A infected cells with calcium (n 5 5). (F) FAM3A-induced Akt activation was completely abolished by the CaM antagonist CPZ. Infected HepG2 cells were treated with CPZ (100 lM) for 1 hour before pAkt levels was measured. *P < 0.05 versus control cells, #P < 0.05 versus Ad-FAM3A infected cells without CPZ treatment (n 5 5).
regulated by PPARc activation.19 We also found that chronic exposure to high concentrations of insulin repressed FAM3A expression in HepG2 cells (data not shown). Therefore, swimming exercise may restore FAM3A expression in diabetic mouse livers by way of reducing hepatic lipid accumulation and attenuating hyperinsulinemia. OGTT, pyruvate tolerance test, and hyperinsulinemic-euglycemic clamp assays indicated that hepatic overexpression of FAM3A markedly attenuated hyperglycemia and insulin resistance, suggesting suppressed hepatic gluconeogenesis and improved insulin signaling may be responsible for metabolic improvement in db/db mice. FAM3A overexpression also attenuated hepatic steatosis, possibly by way of repressing fatty acid synthesis and increasing fatty acid oxidation and VLDL-TG secretion. Hepatic overex-
pression of FAM3A improved global insulin sensitivity and attenuated fatty liver with increased pAkt levels in the livers of diabetic mice. In contrast, siRNAmediated knockdown of hepatic FAM3A resulted in a fasting hyperglycemia and increased hepatic lipid accumulation, with reduced pAkt levels and increased gluconeogenesis in the livers of normal mice. Mechanistically, FAM3A increases mitochondrial ATP production and secretion, which activates the PI3KAkt signaling pathway by way of an insulinindependent, ATP receptor-mediated mechanism. FAM3A may thus represent an attractive target for the treatment of insulin resistance, type 2 diabetes, and NAFLD. In addition to blocking gluconeogenesis and glycogenolysis, Akt has also been found to play a critical role in the regulation of liver lipid metabolism. Activation of Akt represses lipogenesis and prevents
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Fig. 7. Proposed mechanism for FAM3Ainduced activation of the PI3K-Akt signaling pathway in liver cells. FAM3A enhances ATP synthesis and elevates extracellular ATP levels, which increases cytosolic free calcium concentrations through mechanisms involving the P2 receptors. Finally, elevated cytosolic calcium activates the PI3K-Akt signaling pathway in a CaM-dependent manner, leading to a suppression of hepatic gluconeogenesis and lipogenesis. PLC, phospholipase; CaM, calmodulin; IP3, inositol (1,4,5) triphosphate.
excessive lipid deposition in the liver by way of the inactivation of GSK-3, PGC-1a, and FOXO111,20-22 and increased hepatic pAkt levels in the liver is associated with the amelioration of steatosis in diabetic mice.23 As expected, hepatic overexpression of FAM3A significantly attenuated steatosis in db/db mice and HFD-induced diabetic mice. In addition, hepatic FAM3A overexpression increases, while hepatic knockdown of FAM3A reduces, AdipoR1 expression, suggesting FAM3A may attenuate gluconeogenesis and lipogenesis and increase fatty acid oxidation by way of activation of AdipoR/AMPK signaling in the liver.24 In the present study, we found that FAM3A activates AMPK activity regardless of increased ATP production. Multiple mechanisms are involved in AMPK activation. It has been recently demonstrated that AdipoR1 also plays an important role in the regulation of AMPK activation. AdipoR1 gene-deficient mouse livers exhibit blunted AMPK activation induced by adiponectin, while overexpression of AdipoR1 increases AMPK activity in db/db mouse livers.25 Given the important role of AdipoR1 in the activation of AMPK, it is likely that up-regulation of AdipoR1 contributes to FAM3A-induced AMPK activation in diabetic mouse livers. We further showed that FAM3A activates Akt by way of a PI3K p110a-dependent pathway in liver cells, which is consistent with recent report that p110a predominantly mediates Akt activation in the liver,26 where selective inhibition of p110a increases lipid deposition.27 Interestingly, FAM3A can activate Akt in the absence of insulin, with little effect on pIRS-1 levels and the association of p85 with IRS-1, suggesting the FAM3A-activated p110a/Akt signaling pathway is
insulin-independent. In support, hepatic FAM3A overexpression also activated Akt and attenuated of hyperglycemia and hepatic gluconeogenesis in type 1 diabetic mice. For the first time, we found that FAM3A protein is present in the mitochondria of mouse hepatocytes and HepG2 cells. Furthermore, FAM3A protein was increased in the mitochondria of mouse livers and HepG2 cells treated with Ad-FAM3A. These observations demonstrate that FAM3A is a novel mitochondrial protein. In vivo, hepatic FAM3A overexpression increased, while hepatic FAM3A knockdown reduced ATP levels in mouse livers. In vitro, FAM3A overexpression increased intracellular and extracellular ATP levels in both HepG2 cells and primary cultured mouse hepatocytes. These findings strongly support the possibility that increased expression of mitochondrial FAM3A enhances ATP production, thereby increasing intracellular and extracellular levels of ATP in hepatocytes. However, the mechanism by which FAM3A increases mitochondrial ATP synthesis warrants further investigation. It has been previously reported that ATP can be released and function as a signaling molecule to activate the PI3K/Akt signaling pathway by way of ATP receptors in many organs or cell types.18,28,29 The receptors mediating such action include P2 purinoceptors, i.e., P2X and P2Y receptors. P2X receptors are ligand-gated ion channels that are permeable to calcium,28 while P2Y receptors are G-protein-coupled receptors that stimulate PLC to increase IP3 production, resulting in calcium release from internal stores.28 As a result of increased cytosolic free calcium level upon extracellular ATP treatment, CaM is activated,
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leading to the activation of the PI3K/Akt pathway.30,31 In the present study, we confirmed that treatment of HepG2 cells with exogenous ATP elevated cytosolic free calcium and activated Akt in a p110a-dependent manner, which was attenuated by the inhibition of the P2 receptors or their downstream signaling molecules, including PLC and IP3R. FAM3A-induced intracellular calcium level increases and Akt activation were significantly blocked by P2 receptor antagonists, PLC inhibitor, IP3R antagonist, and depletion of extracellular calcium in HepG2 cells. Furthermore, the finding that the CaM antagonist CPZ completely abolished FAM3Ainduced Akt activation reveals a critical role of increased cellular calcium level in this process. Collectively, these findings demonstrate that both P2 receptor isoforms are involved in FAM3A-induced increases in cytosolic free calcium and Akt activation in liver cells. Interestingly, although blockage of the P2 receptors completely inhibited Akt activation induced by exogenous ATP, it did not completely abolish FAM3A-induced Akt activation, suggesting that other minor mechanism(s) may also be involved. One possibility is that FAM3A-elicited increase in intracellular ATP level leads to the closure of the KATP channels, resulting in increased calcium influx through L-type calcium channel.17 The findings that exogenous ATP can elicit intracellular calcium signaling and Akt activation through P2 receptors in liver cells suggests a novel mechanism involved in hepatic glucose and lipid homeostasis regulation (Fig. 7). Increasing evidence has revealed that impaired energy metabolism in the liver could be an early defect in the pathogenesis of type 2 diabetes in patients.32,33 It has been also reported that reduced hepatic ATP levels are associated with the development of NAFLD in rats.34 These observations support a possibility that defective ATP synthesis and P2 receptor-mediated signaling may underlie hepatic insulin resistance and elevated liver lipid contents. In summary, the present study demonstrates that FAM3A plays a crucial role in hepatic glucose and lipid metabolism. FAM3A enhances ATP synthesis and elevates extracellular ATP levels, which activate P2 receptors to increase cytosolic free calcium level, leading to activation PI3K-Akt signaling pathway in a CaM-dependent manner and suppression of hepatic gluconeogenesis and lipogenesis. In addition, FAM3A may also activate the AdipoR1-AMPK signaling pathway and enhance fatty acid b oxidation in the liver by way of a yet unknown mechanism. In conclusion, to increase hepatic ATP synthesis and signaling by way of up-regulation of FAM3A might represent a promising means for treatment of insulin resistance and NAFLD.
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References 1. Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract 2010;87:4-14. 2. Biddinger SB, Kahn CR. From mice to men: insights into the insulin resistance syndromes. Annu Rev Physiol 2006;68:123-158. 3. Turner N, Heilbronn LK. Is mitochondrial dysfunction a cause of insulin resistance? Trends Endocrinol Metab 2008;19:324-330. 4. Zhu Y, Xu G, Patel A, McLaughlin MM, Silverman C, Knecht K, et al. Cloning, expression, and initial characterization of a novel cytokine-like gene family. Genomics 2002;80:144-150. 5. Cao X, Yang J, Burkhardt BR, Gao Z, Wong RK, Greene SR, et al. Effects of overexpression of pancreatic derived factor (FAM3B) in isolated mouse islets and insulin-secreting betaTC3 cells. Am J Physiol Endocrinol Metab 2005;289:E543-550. 6. Burkhardt BR, Yang MC, Robert CE, Greene SR, McFadden KK, Yang J, et al. Tissue-specific and glucose-responsive expression of the pancreatic derived factor (PANDER) promoter. Biochim Biophys Acta 2005;1730:215-225. 7. Yang J, Gao Z, Robert CE, Burkhardt BR, Gaweska H, Wagner A, et al. Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells. Biochemistry 2005; 44:11342-11352. 8. Yang J, Robert CE, Burkhardt BR, Young RA, Wu J, Gao Z, et al. Mechanisms of glucose-induced secretion of pancreatic-derived factor (PANDER or FAM3B) in pancreatic beta-cells. Diabetes 2005;54: 3217-3228. 9. Yang J, Wang C, Li J, Burkhardt BR, Robert-Cooperman CE, Wilson C, et al. PANDER binds to the liver cell membrane and inhibits insulin signaling in HepG2 cells. FEBS Lett 2009;583:3009-3015. 10. Robert-Cooperman CE, Carnegie JR, Wilson CG, Yang J, Cook JR, Wu J, et al. Targeted disruption of pancreatic-derived factor (PANDER, FAM3B) impairs pancreatic {beta}-cell function. Diabetes 2010; 59:2209-2218. 11. Li J, Chi Y, Wang C, Wu J, Yang H, Zhang D, et al. Pancreaticderived factor promotes lipogenesis in the mouse liver: role of the Forkhead box 1 signaling pathway. HEPATOLOGY 2011;53:1906-1916. 12. Robert-Cooperman CE, Wilson CG, Burkhardt BR. PANDER KO mice on high-fat diet are glucose intolerant yet resistant to fasting hyperglycemia and hyperinsulinemia. FEBS Lett 2011;585:1345-1349. 13. Wang C, Burkhardt BR, Guan Y, Yang J. Role of pancreatic-derived factor in type 2 diabetes: evidence from pancreatic beta cells and liver. Nutr Rev 2012;70:100-106. 14. Wu J, Wang C, Li S, Li S, Wang W, Li J, et al. Thrsp promotes hepatic lipogenesis, and its expression LXRalpha through an SREBP1cdependent mechanism. HEPATOLOGY 2013;58:617-628. 15. Qin X, Xie X, Fan Y, Tian J, Guan Y, Wang X, et al. Peroxisome proliferator-activated receptor-delta induces insulin-induced gene-1 and suppresses hepatic lipogenesis in obese diabetic mice. HEPATOLOGY 2008;48:432-441. 16. Zhou Y, Zhang X, Chen L, Wu J, Dang H, Wei M, et al. Expression profiling of hepatic genes associated with lipid metabolism in nephrotic rats. Am J Physiol Renal Physiol 2008;295:F662-671. 17. Yang J, Wong RK, Park M, Wu J, Cook JR, York DA, et al. Leucine regulation of glucokinase and ATP synthase sensitizes glucose-induced insulin secretion in pancreatic beta-cells. Diabetes 2006;55:193-201. 18. Gonzales E, Julien B, Serriere-Lanneau V, Nicou A, Doignon I, Lagoudakis L, et al. ATP release after partial hepatectomy regulates liver regeneration in the rat. J Hepatol 2010;52:54-62. 19. Zhou Y, Jia S, Wang C, Chen Z, Chi Y, Li J, et al. FAM3A is a target gene of peroxisome proliferator-activated receptor gamma. Biochim Biophys Acta 2013;1830:4160-4170. 20. Li X, Monks B, Ge Q, Birnbaum MJ. Akt/PKB regulates hepatic metabolism by directly inhibiting PGC-1alpha transcription coactivator. Nature 2007;447:1012-1016. 21. Kim JJ, Li P, Huntley J, Chang JP, Arden KC, Olefsky JM. FoxO1 haploinsufficiency protects against high-fat diet-induced insulin
1790
22.
23.
24.
25.
26.
27.
WANG, CHI, ET AL.
resistance with enhanced peroxisome proliferator-activated receptor gamma activation in adipose tissue. Diabetes 2009;58:1275-1282. Matsumoto M, Han S, Kitamura T, Accili D. Dual role of transcription factor FoxO1 in controlling hepatic insulin sensitivity and lipid metabolism. J Clin Invest 2006;116:2464-2472. Bijl N, Sokolovic M, Vrins C, Langeveld M, Moerland PD, Ottenhoff R, et al. Modulation of glycosphingolipid metabolism significantly improves hepatic insulin sensitivity and reverses hepatic steatosis in mice. HEPATOLOGY 2009;50:1431-1441. Hasenour CM, Berglund ED, Wasserman DH. Emerging role of AMP-activated protein kinase in endocrine control of metabolism in the liver. Mol Cell Endocrinol 2012;366:152-162. Yamauchi T, Nio Y, Maki T, Kobayashi M, Takazawa T, Iwabu M, et al. Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions. Nat Med 2007;13:332339. Sopasakis VR, Liu P, Suzuki R, Kondo T, Winnay J, Tran TT, et al. Specific roles of the p110alpha isoform of phosphatidylinsositol 3kinase in hepatic insulin signaling and metabolic regulation. Cell Metab 2010;11:220-230. Jackson LN, Larson SD, Silva SR, Rychahou PG, Chen LA, Qiu S, et al. PI3K/Akt activation is critical for early hepatic regeneration after partial hepatectomy. Am J Physiol Gastrointest Liver Physiol 2008;294: G1401-1410.
HEPATOLOGY, May 2014
28. Franke H, Illes P. Involvement of P2 receptors in the growth and survival of neurons in the CNS. Pharmacol Ther 2006;109:297-324. 29. Ishikawa M, Iwamoto T, Nakamura T, Doyle A, Fukumoto S, Yamada Y. Pannexin 3 functions as an ER Ca(21) channel, hemichannel, and gap junction to promote osteoblast differentiation. J Cell Biol 2011; 193:1257-1274. 30. Nishimatsu H, Suzuki E, Nagata D, Moriyama N, Satonaka H, Walsh K, et al. Adrenomedullin induces endothelium-dependent vasorelaxation via the phosphatidylinositol 3-kinase/Akt-dependent pathway in rat aorta. Circ Res 2001;89:63-70. 31. Cheng A, Wang S, Yang D, Xiao R, Mattson MP. Calmodulin mediates brain-derived neurotrophic factor cell survival signaling upstream of Akt kinase in embryonic neocortical neurons. J Biol Chem 2003; 278:7591-7599. 32. Schmid AI, Szendroedi J, Chmelik M, Krssak M, Moser E, Roden M. Liver ATP synthesis is lower and relates to insulin sensitivity in patients with type 2 diabetes. Diabetes Care 2011;34:448-453. 33. Szendroedi J, Chmelik M, Schmid AI, Nowotny P, Brehm A, Krssak M, et al. Abnormal hepatic energy homeostasis in type 2 diabetes. HEPATOLOGY 2009;50:1079-1086. 34. Serviddio G, Bellanti F, Tamborra R, Rollo T, Romano AD, Giudetti AM, et al. Alterations of hepatic ATP homeostasis and respiratory chain during development of non-alcoholic steatohepatitis in a rodent model. Eur J Clin Invest 2008;38:245-252.
