Medical academic journalMedical academic journal1608-41012687-1378Eco-Vector6289210.17816/MAJ62892Research ArticleMechanisms of the influence of adiponectin on apolipoproteins A-1 and B production by human hepatocytesTanyanskiyDmitriy A.<p>MD, PhD (Medicine), Head of Laboratory of Lipoproteins, Department of Biochemistry</p>dmitry.athero@gmail.comhttps://orcid.org/0000-0002-5321-8834DizheElla B.<p>MD, PhD (Biology), Leading Researcher, Department of Biochemistry</p>dizhe@iem.sp.ruhttps://orcid.org/0000-0001-5147-4749OleinikovaGalina N.<p>Research Assistant, Department of Biochemistry</p>galina@iem.sp.ruShavvaVladimir S.<p>MD, PhD (Biology), Senior Researcher, Department of Biochemistry</p>vssreinard.fox@gmail.comDenisenkoAleksandr D.<p>MD, PhD, DSc (Medicine), Professor, Head of Department of Biochemistry</p>add@iem.sp.ruhttps://orcid.org/0000-0003-1613-0654Institute of Experimental Medicine1006202121139451003202123032021Copyright © 2021, Tanyanskiy D.A., Dizhe E.B., Oleinikova G.N., Shavva V.S., Denisenko A.D.2021<p><strong><em>The aim of the study</em></strong> was to find out the mechanisms of the adiponectin effect on apolipoproteins (apo) A-1 and B production by human hepatocytes.</p>
<p><strong><em>Materials and methods.</em></strong> The study was performed on the human hepatoma cell line HepG2. The expression of the <em>apoA-1</em> gene was evaluated at the mRNA level by quantitative PCR with reverse transcription, and the production of apoB by ELISA method. The activity of lipogenesis was assessed by the inclusion of labeled <sup>14</sup>C-acetate in triglycerides, as well as by mRNA expression of lipogenesis genes, and by the estimation of total triglycerides content in cells. To determine the involvement of signaling pathways, the RNA interference method was used.</p>
<p><strong><em>Results.</em></strong> Knockdown of genes, coding the specific receptors, AMP-activated protein kinase, and its regulated transcription factors inhibited adiponectin-dependent stimulation of <em>apoA-1</em> gene expression in hepatocytes. Adiponectin had no effect on lipogenesis and apoB production under basal conditions, but suppressed these processes induced by the addition of oleate.</p>
<p><strong><em>Conclusion.</em></strong> Adiponectin stimulates the production of apoA-1 in hepatocytes by inducing the transcription of the <em>apoA-1</em> gene and suppresses the secretion of apoB by affecting lipogenesis. These effects may underlie the effect of adiponectin on lipoproteins metabolism.</p>adiponectinapolipoproteinshepatocytesnuclear receptorslipogenesismetabolic syndromeадипонектинаполипопротеиныгепатоцитыядерные рецепторылипогенезметаболический синдром<p>Abbreviations</p>
<p>apo apolipoprotein; RT-PCR reverse transcription polymerase chain reaction; TG triglycerides; AdipoRs adiponectin receptors; AMPK AMP-activated protein kinase; BSA bovine serum albumin; FCS fetal calf serum; LXR liver X receptor alpha; PPAR peroxisome proliferator-activated receptor alpha.</p>
<h3>Introduction</h3>
<p>One of the primary risk factors for atherosclerosis is metabolic syndrome, a complex of pathogenetically interrelated disorders, such as obesity, insulin resistance, dyslipoproteinemia, and hypertension [1]. The adipose tissue proteins (adipokines) are involved in the formation of these disorders [2]. Adiponectin increases the sensitivity of tissues to insulin and stimulates the oxidation of fatty acids. Thus, it favorably affects plasma lipoproteins spectrum and is of greatest interest among all adipokines [3, 4]. The metabolic effects of adiponectin are realized by activating type 1 and type 2 adiponectin receptors (AdipoR1/2). They transmit a signal to AMP-activated protein kinase (AMPK) and nuclear peroxisome proliferator-activated receptors alpha (PPAR) [3].</p>
<p>Another way that adiponectin affects lipoprotein metabolism is through hepatic apolipoprotein (apo) production. Adiponectin increases apoA-1 (the main protein of high-density lipoproteins) production and decreases apoB (the main protein of low-density lipoproteins) production by hepatocytes [46]. At the same time, the mechanisms of these adiponectin effects remain poorly understood.</p>
<p>In this regard, the aim of the study was to identify the mechanisms of adiponectins effects on apoA-1 and apoB production by human hepatocytes.</p>
<h3>Materials and methods</h3>
<p>This study was performed on cells of the human hepatoma cell line HepG2 (Russian collection of cell cultures, Institute of Cytology, Russian Academy of Sciences). The cells were cultured in Dulbeccos modified Eagle medium (DMEM) supplemented with 4 mM glutamine, 0.1 mg/ml gentamicin (all-Biolot, Russia), 10% fetal calf serum (FCS) (Hyclone, USA) in an atmosphere of 5% CO<sub>2</sub> at 37C. The cells were seeded on 96-well culture plates (Sarstedt, Germany) with a density of 1 10<sup>4</sup> cells/cm<sup>2</sup> and grown in complete medium for 23 days until subconfluence (~70%80%). Further, the culture medium was replaced with FCS-free medium supplemented with adiponectin (catalog number RD172023100-C, Biovendor, Czech Republic) at concentrations of 10 or 30 g/ml or 1 mM AICAR (5-aminoimidazole-4-carboxamide-1--D-ribofuranoside) (Calbiochem, USA) or phosphate-buffered saline for 24 hours. In several experiments, a complex of bovine serum albumin (BSA) with 290 M oleate (all-Sigma, USA) or fatty acids free BSA at a final concentration of 5 g/l was added. By the end of the incubation, cells were harvested for RNA isolation (Evrogen, Russia) or intracellular protein determination (BCA Protein Assay, ThermoScientific, USA) and triglycerides (TG) (Randox enzymatic kits, UK) content. The culture media of HepG2 cells were collected to determine apoB concentration using an enzyme-linked immunosorbent assay.</p>
<p>Transfection of HepG2 cells with siRNA was performed using the Lipofectamine RNAiMAX reagent (Invitrogen, USA), according to the manufacturers instructions. The cells were transfected for 72 h and kept in DMEM with 10% FCS. On the last day of transfection, cells were incubated with 10 g/ml adiponectin or 1 mM AICAR or phosphate-buffered saline under serum-free conditions. The cells were then lysed for gene expression determination by reverse transcription polymerase chain reaction (RT-PCR). Transfection efficiency was assessed by using RT-PCR. For transfection, siRNAs were used against AdipoR1, AdipoR2 [7], 1/2-subunits of AMPK [8] (all-Syntol, Russia), PPAR (sc-36307), LXR (hepatic receptors-X) (sc-38828), and nonspecific siRNA (sc-37007) (all Santa Cruz, USA).</p>
<p>RNA isolation, reverse transcription, and real-time PCR were performed, as described previously [9, 10]. The relative mRNA content of the desired genes was normalized to the levels of the housekeeping (constitutive) genes (of -actin, ribosomal protein RPLP0, and cyclophilin A) expression.</p>
<p>The synthesis of TG in cells was assessed by inclusion of <sup>14</sup>C-acetate into TG. For this, 1 Ci of <sup>14</sup>C sodium acetate (specific radioactivity = 20,000 cpm/nmol) in the presence of 10 or 30 g/ml adiponectin or 10 g/ml BSA (negative control) was added to HepG2 cells for 5 h under serum-free conditions. Then, lipids were extracted from the cells with a mixture of hexaneisopropanol (3:2 by volume) and separated by thin-layer chromatography in the system heptaneisopropyl etheracetic acid (15:10:1 by volume) on Kieselgel 60 aluminum plates (Merck, Germany). After developing with iodine (J<sub>2</sub>), spots corresponding to the TG fraction were excised, placed in vials, and filled with scintillation liquid for radioactivity counting (RakBeta, LKB, Sweden). Delipidated cell pellets were solubilized with 0.2 M NaOH to determine the protein concentration.</p>
<p>The results are presented as mean values standard error of mean (mean SEM) of three or four independent experiments. The statistical analysis of differences between the control and experimental groups was performed using Dunnetts test. Differences were considered significant at <em>p</em> 0.05. Statistical analysis was performed using the GraphPad Prism v.6 software (USA).</p>
<h3>Results and discussion</h3>
<p>The production of apoA-1 by hepatocytes is primarily regulated at the transcriptional level with the participation of transcription factors that interact with specific sites located in the 5-regulatory region of this gene. The transcriptional activators of the <em>apoA-1</em> gene are PPAR and HNF4 (hepatocyte nuclear factor 4), whereas LXR suppress the expression of this gene [9]. In turn, the activity of PPAR and LXRin hepatocytes is controlled by AMPK [11, 12].</p>
<p>We used the RNA interference method to elucidate the participation of these signaling molecules in adiponectin-dependent activation of the <em>apoA-1</em> gene expression in hepatocytes. The knockdown of genes encoding AdipoRs, AMPK kinase, and nuclear receptors, PPARand LXR, led to the cancelation of the effect of adiponectin on <em>apoA-1</em> gene expression at the mRNA level (Table). Like adiponectin, the AMPK activator AICAR stimulated <em>apoA-1</em> gene expression in hepatocytes. This effect was also canceled after the knockdown of genes encoding AMPK and both nuclear receptors (Table). Upon these data we suggest the involvement of both types of adiponectin receptors, AMPK and nuclear receptors PPAR and LXR in regulation of <em>apoA-1</em> gene expression under adiponectin action.</p>
<p></p>
<p><strong><em>Table / </em><em>Таблица</em></strong></p>
<p><strong>Effect of adiponectin (10 mkg/ml) on the expression of the <em>apoA-1</em> gene in human hepatoma HepG2 cells upon the knockdown of the <em>AdipoRs</em>, <em>AMPK</em>, <em>PPAR</em>, and <em>LXR</em>genes</strong></p>
<p><strong>Влияние адипонектина (10 мкг/мл) на экспрессию гена <em>apoA-1</em> в клетках гепатомы человека линии HepG2 на фоне нокдауна генов <em>AdipoRs</em>, <em>AMPK</em>, <em>PPAR</em>и <em>LXR</em></strong></p>
<table>
<tbody>
<tr>
<td>
<p>Suppressed gene</p>
</td>
<td>
<p>Active agent</p>
</td>
<td>
<p>apoA-1 siRNA, percent (%) of control</p>
</td>
</tr>
<tr>
<td rowspan="3">
<p></p>
</td>
<td>
<p>Control</p>
</td>
<td>
<p>100.0 1.1</p>
</td>
</tr>
<tr>
<td>
<p>Adiponectin</p>
</td>
<td>
<p>150.5 3.5*</p>
</td>
</tr>
<tr>
<td>
<p>AICAR</p>
</td>
<td>
<p>140.3 3.3*</p>
</td>
</tr>
<tr>
<td rowspan="2">
<p><em>AdipoR1</em></p>
</td>
<td>
<p>Control</p>
</td>
<td>
<p>117.5 11.2</p>
</td>
</tr>
<tr>
<td>
<p>Adiponectin</p>
</td>
<td>
<p>120.3 12.8</p>
</td>
</tr>
<tr>
<td rowspan="2">
<p><em>AdipoR</em><em>2</em></p>
</td>
<td>
<p>Control</p>
</td>
<td>
<p>138.0 15.6</p>
</td>
</tr>
<tr>
<td>
<p>Adiponectin</p>
</td>
<td>
<p>131.6 16.8</p>
</td>
</tr>
<tr>
<td rowspan="3">
<p>Subunits <em>1-</em><em>AMPK</em> and <em>2-</em><em>AMPK</em></p>
</td>
<td>
<p>Control</p>
</td>
<td>
<p>44.3 1.9*</p>
</td>
</tr>
<tr>
<td>
<p>Adiponectin</p>
</td>
<td>
<p>35.7 1.3</p>
</td>
</tr>
<tr>
<td>
<p>AICAR</p>
</td>
<td>
<p>30.1 2.1</p>
</td>
</tr>
<tr>
<td rowspan="3">
<p><em>PPAR</em></p>
</td>
<td>
<p>Control</p>
</td>
<td>
<p>131.8 4.0</p>
</td>
</tr>
<tr>
<td>
<p>Adiponectin</p>
</td>
<td>
<p>124.3 2.5</p>
</td>
</tr>
<tr>
<td>
<p>AICAR</p>
</td>
<td>
<p>122.0 2.1</p>
</td>
</tr>
<tr>
<td rowspan="3">
<p><em>LXR</em></p>
</td>
<td>
<p>Control</p>
</td>
<td>
<p>191.4 7.0*</p>
</td>
</tr>
<tr>
<td>
<p>Adiponectin</p>
</td>
<td>
<p>186.9 1.3</p>
</td>
</tr>
<tr>
<td>
<p>AICAR</p>
</td>
<td>
<p>148.5 10.2</p>
</td>
</tr>
</tbody>
</table>
<p>Note. The results of real-time RT-PCR of the relative content of apoA-1 mRNA, mean SEM (<em>n</em> = 1216). * <em>p</em> 0.05 versus control with nonspecific siRNA.</p>
<p></p>
<p>Unlike apoA-1, the production of apoB is regulated mainly at the post-translational level by stabilizing this protein in the lipid environment [13]. In this regard, it is most likely that adiponectin affects the production of apoB-containing lipoproteins, influencing the TG synthesis. Although adiponectin reduces lipogenesis activity in rat and bovine hepatocytes [14, 15], these data are not confirmed in studies performed on human hepatocytes [6]. These discrepancies may be due to the different species of hepatocytes and differences in the activity of lipogenesis in the studied cell models. According to our data, adiponectin does not affect basal lipogenesis in HepG2 cells (diagrams in Figures, <em>a</em> and <em>b</em>). However, upon loading cells with oleate, adiponectin, at a concentration of 30 mkg/ml, decreases TG content in HepG2 cells (Diagram <em>c</em>). The described effects of adiponectin on TG synthesis in cells were accompanied by changes in apoB secretion by the cells (Diagram <em>d</em>). These data support the hypothesis that adiponectin interferes with hepatic apoB production by the effect of this adipokine on lipogenesis.</p>
<p></p>
<center>
<div class="preview fancybox" style="text-align: center;"><a title="Figure. Effect of adiponectin on TG synthesis and apoB secretion in human hepatoma HepG2 cells. a synthesis of TG was evaluated by the inclusion of 14C-acetate into TG. The results are presented as counts per minutes, normalized for the content of cellular protein, relative to the average value in the control, taken as 100%. b The expression level of lipogenesis genes ACC-1 (acetyl-CoA-carboxylase) and FASN (fatty acid synthase), measured by the reverse transcription PCR assay. TO-901317 LXR agonist, the activator of lipogenesis [12], positive control. c TG content in cell lysates (enzymatic method), normalized for the level of intracellular protein. N. d. TG were not detected by this method. d apoB concentrations in hepatocytes culture media (ELISA assay), normalized for the content of intracellular protein, relative to the control taken as 100% (the absolute values of apoB concentrations were ~5-50 ng/mkg of cellular protein). Mean values SEM are given (a n = 8, b d n = 1216). * p 0.05, ** p 0.005 versus the control, # p 0.05 versus control with oleate treatment. Adipo adiponectin, TG triglycerides, apoB apolipoprotein B" href="/files/journals/21/articles/62892/supp/62892-152872-1-SP.jpg" rel="simplebox"><img style="max-height: 300px; max-width: 300px;" src="/files/journals/21/articles/62892/supp/62892-152872-1-SP.jpg" /></a></div>
</center>
<p><strong>Figure. Effect of adiponectin on TG synthesis and apoB secretion in human hepatoma HepG2 cells. a synthesis of TG was evaluated by the inclusion of 14C-acetate into TG. The results are presented as counts per minutes, normalized for the content of cellular protein, relative to the average value in the control, taken as 100%. b The expression level of lipogenesis genes ACC-1 (acetyl-CoA-carboxylase) and FASN (fatty acid synthase), measured by the reverse transcription PCR assay. TO-901317 LXR agonist, the activator of lipogenesis [12], positive control. c TG content in cell lysates (enzymatic method), normalized for the level of intracellular protein. N. d. TG were not detected by this method. d apoB concentrations in hepatocytes culture media (ELISA assay), normalized for the content of intracellular protein, relative to the control taken as 100% (the absolute values of apoB concentrations were ~5-50 ng/mkg of cellular protein). Mean values SEM are given (a n = 8, b d n = 1216). * p 0.05, ** p 0.005 versus the control, # p 0.05 versus control with oleate treatment. Adipo adiponectin, TG triglycerides, apoB apolipoprotein B</strong></p>
<p></p>
<p>Suppression of TG synthesis by adiponectin can be a result of AMPK activation with a further decrease in lipogenesis gene activity at the transcriptional and post-translational levels [3, 16] on the one hand, and by the activation of PPAR and the transcriptional coactivator PGC1, which increase fatty acids oxidation at the transcriptional level on the other hand [3, 17].</p>
<h3>Conclusion</h3>
<p>We conclude that adiponectins effects on apolipoproteins production in hepatocytes occur through the signaling pathways of adiponectin receptors, including AMPK activation and changes in the activity of nuclear receptors, PPAR and LXR. These effects, along with the activation of fatty acids oxidation and an increase in insulin sensitivity in peripheral tissues by adiponectin, can provide beneficial effects of this adipokine on lipoproteins blood levels and development of dyslipoproteinemia in metabolic syndrome.</p>
<h3>Additional information</h3>
<p><strong>Acknowledgments.</strong> The authors are grateful to senior researcher A.O. Sherstobitov (I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, RAS) for his help in working with the radioactive label.</p>
<p><strong>Funding.</strong> The study was carried out with the financial support of the Russian Foundation for Basic Research within the framework of project No. 12-04-01410-a.</p>
<p><strong>Compliance with ethical standards.</strong> This study is not related to work on animals and clinical material.</p>
<p><strong>Conflict of interest.</strong> The authors declare no conflict of interest.</p>[Mychka VB, Vertkin AL, Vardaev LI, et al. Experts’ consensus on the interdisciplinary approach towards the management, diagnostics, and treatment of patients with metabolic syndrome. Cardiovascular therapy and prevention. 2013;12 (6):41–81. (In Russ.)][Denisenko AD, Tanyansky DA. Adipokines in the pathogenesis of atherosclerosis in metabolic syndrome. In: Metabolic syndrome. Ed. by A.V. Shabrov. Saint Petersburg; 2020. P. 105–139. (In Russ.)][Yamauchi T, Kamon J, Ito Y, et al. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature. 2003;423(6941):762–769. DOI: 10.1038/nature01705][Qiao L, Zou C, van der Westhuyzen DR, Shao J. Adiponectin reduces plasma triglyceride by increasing VLDL triglyceride catabolism. Diabetes. 2008;57(7):1824–1833. DOI: 10.2337/db07-0435][Matsuura F, Oku H, Koseki M, et al. Adiponectin accelerates reverse cholesterol transport by increasing high density lipoprotein assembly in the liver. Biochem Biophys Res Commun. 2007;358(4):1091–1095. DOI: 10.1016/j.bbrc.2007.05.040][Wanninger J, Liebisch G, Eisinger K, et al. Adiponectin isoforms differentially affect gene expression and the lipidome of primary human hepatocytes. Metabolites. 2014;4(2):394–407. DOI: 10.3390/metabo4020394][Wanninger J, Neumeier M, Weigert J, et al. Adiponectin-stimulated CXCL8 release in primary human hepatocytes is regulated by ERK1/ERK2, p38 MAPK, NF-kappaB, and STAT3 signaling pathways. Am J Physiol Gastrointest Liver Physiol. 2009;297(3):G611–G618. DOI: 10.1152/ajpgi.90644.2008][Shavva VS, Bogomolova AM, Nikitin AA, et al. FOXO1 and LXRα downregulate the apolipoprotein A-I gene expression during hydrogen peroxide-induced oxidative stress in HepG2 cells. Cell Stress Chaperones. 2017;22(1):123–134. DOI: 10.1007/s12192-016-0749-6][Mogilenko DA, Dizhe EB, Shavva VS, et al. Role of the nuclear receptors HNF4 alpha, PPAR alpha, and LXRs in the TNF alpha-mediated inhibition of human apolipoprotein A-I gene expression in HepG2 cells. Biochemistry. 2009;48(50):11950–11960. DOI: 10.1021/bi9015742][Nekrasova EV, Danko KV, Shavva VS, et al. Effect of the insulin on the apolipoprotein A-I gene expression in human macrophages. Medical Academic Journal. 2020;20(1):65–74. (In Russ.). DOI: 10.17816/MAJ16437][Lee J, Hong SW, Park SE, et al. AMP-activated protein kinase suppresses the expression of LXR/SREBP-1 signaling-induced ANGPTL8 in HepG2 cells. Mol Cell Endocrinol. 2015;414:148–155. DOI: 10.1016/j.mce.2015.07.031][Hwahng SH, Ki SH, Bae EJ, et al. Role of adenosine monophosphate-activated protein kinase-p70 ribosomal S6 kinase-1 pathway in repression of liver X receptor-alpha-dependent lipogenic gene induction and hepatic steatosis by a novel class of dithiolethiones. Hepatology. 2009;49(6):1913–1925. DOI: 10.1002/hep.22887][Fazio S, Linton MF. Regulation and clearance of apolipoprotein B-containing lipoproteins. In: Clinical lipidology: a companion to Braunwald’s heart disease. Ed by C.M. Ballantyne. 2nd ed. Sauders Elsevier; 2015: 11–24. DOI: 10.1016/B978-141605469-6.50006-8][Awazawa M, Ueki K, Inabe K, et al. Adiponectin suppresses hepatic SREBP1c expression in an AdipoR1/LKB1/AMPK dependent pathway. Biochem Biophys Res Commun. 2009;382(1):51–56. DOI: 10.1016/j.bbrc.2009.02.131][Chen H, Zhang L, Li X, et al. Adiponectin activates the AMPK signaling pathway to regulate lipid metabolism in bovine hepatocytes. J Steroid Biochem Mol Biol. 2013;138:445–454. DOI: 10.1016/j.jsbmb.2013.08.013][Garcia D, Shaw RJ. AMPK: Mechanisms of cellular energy sensing and restoration of metabolic balance. Mol Cell. 2017;66(6):789–800. DOI: 10.1016/j.molcel.2017.05.032][Iwabu M, Yamauchi T, Okada-Iwabu M, et al. Adiponectin and AdipoR1 regulate PGC-1alpha and mitochondria by Ca(2+) and AMPK/SIRT1. Nature. 2010;464(7293):1313–1319. DOI: 10.1038/nature08991]