Orexin system is expressed in avian liver and regulates hepatic lipogenesis via ERK1/2 activation

Orexins are originally characterized as orexigenic hypothalamic neuropeptides in mammals. Subsequent studies found orexin to be expressed and perform pleiotropic functions in multiple tissues in mammals. In avian (non-mammalian) species, however, orexin seemed to not affect feeding behavior and its physiological roles are poorly understood. Here, we provide evidence that orexin and its related receptors are expressed in chicken hepatocytes. Double immunofluorescence staining showed that orexin is localized in the ER, Golgi, and in the lysosomes in LMH cells. Brefeldin A treatment reduced orexin levels in the culture media, but increased it in the cell lysates. Administration of recombinant orexins upregulated the expression of orexin system in the liver of 9-day old chicks, but did not affect feed intake. Recombinant orexins increased fatty acid synthase (FASN) protein levels in chicken liver, activated acetyl-CoA carboxylase (ACCα), and increased FASN, ATP citrate lyase(ACLY), and malic enzyme (ME) protein expression in LMH cells. Blockade ERK1/2 activation by PD98059 attenuated these stimulating effects of orexin on lipogenic factors. Overexpression of ERK1/2 increased the expression of lipogenic genes, and orexin treatment induced the phosphorylated levels of ERK1/2Thr202/Tyr204, but not that of p38 Thr180/Tyr182 or JNK1/2 Thr183/Tyr185 in chicken liver and LMH cells. Taken together, this is the first report evidencing that orexin is expressed and secreted from chicken hepatocytes, and that orexin induced hepatic lipogenesis via activation of ERK1/2 signaling pathway.


Materials and methods
Animals. Day-old male broilers (Cobb 500, Cobb-Vantress, Siloam Springs, AR) were divided into 3 weightmatched groups (39.75 ± 0.26 g), reared for 8 days, and have ad libitum access to clean water and feed (4.090 Mcal ME kg −1 and 22% CP). The ambient temperature was reduced gradually (1 °C every 2 days) from 32 to 29 °C, and the relative humidity was maintained at 55 ± 2%. Feed and water intake were recorded hourly. On day 9, birds received an intraperitoneal (i.p.) injection of recombinant human orexin-A or -B (rORX-A or -B; Alpha Diagnostic International, San Antonio, TX, 100 ng/100 g BW, n = 12). The control group received a saline solution. Three hours after administration, birds were euthanized by cervical dislocation. Blood was collected and 250 µL added to Trizol LS reagent (Life Technologies, Carlsbad, CA) for mRNA extraction. Liver samples were immediately collected, snap frozen in liquid nitrogen and stored at − 80 °C for further analysis. The present study was conducted in accordance with the recommendations in the guide for the care and use of laboratory animals of the National Institutes of Health and the protocols were approved by the University of Arkansas Institutional Animal Care and Use Committee under protocol # 16084.
Leghorn Male Hepatoma (LMH) cell culture and treatments. LMH cells (ATCC CRL-2117) or spontaneously immortalized chicken embryonic hepatocytes (siCEH 22 ) were grown on 0.1% gelatin-coated culture dishes in Waymouth's media (Life Technologies, Carlsbad, CA), supplemented with 10% FBS (Life Technologies, Carlsbad, CA), and 1% penicillin-streptomycin (BioBasic, Amherst, NY) at 37 °C, in a humidified atmosphere of 5% CO 2 and 95% air. At ~ 80% confluence, cells were synchronized with serum-free media overnight and treated with different doses (0, 10, and 100 nM) of recombinant human ORX-A or ORX-B (Alpha Diagnostic International, San Antonio, TX) for 24 h. The dose of orexins was selected based on pilot and previous studies 16,20,23,24 . 30 min before orexin treatments, LMH cells were incubated with brefeldin A (0.3 µg/mL) for 24 h and lysates and medium were subjected to immunoblot analyses. All culture experiments were performed with cells from passages 10-15 and repeated at least twice.
Transient overexpression and inhibition of ERK1/2. At 50-60% confluence, LMH cells were pretreated with PD98059 (10 µM, ThermoFisher Scientific, Waltham, MA) 30 min before orexin administration. Cells were transiently transfected with pEGFP-ERK1, pEGFP-ERK2, and pEGFP empty vector (mock) using Lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's instructions. Six to eight hours post transfection, complete medium was added and cells were maintained for additional 16 h and were processed for immunoblotting.

RNA isolation and quantitative real-time PCR.
For chicken liver and cell lysates, total RNA was extracted using Trizol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions, and concentration and quality were determined by using the Take3 micro-volume plate and the Synergy HT multimode microplate reader (BioTek, Winooski, VT). The ratio of absorbance (A260/280) as well as agarose gel electrophoresis were used to assess RNA integrity and quality. RNA was reverse transcribed using qScript cDNA Sythesis Supermix (Quanta Biosciences, Gaithersburg, MD), and amplified by qPCR (Applied Biosystems 7500 Real Time System) with SYBR green master mix (Life Technologies, Carlsbad, CA) as previously described [25][26][27] . Relative expression of the target genes was determined using the 2 −ΔΔCT method, with normalization to r18S expression 28 . Oligonucleotide primers specific for chicken are summarized in Table 1.
To assess protein loading, the expression of the housekeeping GAPDH protein was determined using rabbit anti-GAPDH antibody (Santa Cruz Biotechnology, Dallas, TX Statistical analysis. Data were analyzed by one-way ANOVA. Significant differences among individual group means were determined by Student-Newman-Keul's multiple range test with GraphPad Prism v. 6.00 (La Jolla, CA). Significance was set at α = 0.05. All data are represented as means ± SEM. www.nature.com/scientificreports/

Results
Orexin system is expressed in avian liver cells. By using immunoblot with antibody that cross-reacts with chicken orexin system 20 , we amplified a strong signal of orexin with only one band at the predicted size (16 kDa) in chicken liver as well as in LMH and siCEH cells (Fig. 1a). However, we observed two bands with molecular weight of 16 and 230 kDa in the hypothalamus (positive control, Fig. 1a). We amplified also a strong signal of orexin receptors in chicken hepatocytes (Fig. 1a). Orexin protein levels are significantly higher in the chicken liver compared to the hypothalamus (Fig. 1b), however its mRNA abundance are significantly lower (Fig. 1d). The expression (mRNA and protein) of orexin receptor (ORXR2) did not differ between the chicken liver and the hypothalamus (Fig. 1c,e,f). Immunofluorescence staining showed a strong reactivity of ORX and its related receptors (ORXR1 and ORXR2) in the cytoplasm of LMH cells (Fig. 1g). The predominant cytoplasmic localization of chicken orexin system was further confirmed by cell fractionation and Western blot analysis (Fig. 1h). To further determine the sub-cellular localization and distribution of orexin in LMH cells, doublelabeling immunofluorescence was performed. Chicken orexin was stained in combination with the well characterized ER fluorescent tracking dye, ER-Tracker, and shows to be predominantly localized in the ER of LMH cell line (yellow arrows, Fig. 2a-c). Immunofluorescence localization of Golgi markers suggests that chicken orexin is also localized, in the cis side (co-staining with ERGIC 53), and in the trans side of Golgi complex (co-staining with TGN38) in LMH cells (yellow arrows, Fig. 2d-i). The co-staining of chicken orexin with the lysosome tracking dye, lyso-Tracker deep red, suggests that chicken orexin is also localized in the lysosome (yellow arrows, Fig. 2j-l). Analysis of conditioned medium by Western blot showed specific secretion of orexin in LMH cells at 48 h (Fig. 2m-o), and treatment of cells with brefeldin A (BFA, 0.3 µg/mL), partially but significantly blocked orexin secretion which is demonstrated by a ~ 26% decrease of ORX levels in the medium and a significant accumulation in cell lysate (Fig. 2p,q).

Orexin modulates its own expression in chicken liver and LMH cells. Intraperitoneal (IP) admin-
istration of recombinant orexin B, but not orexin A, significantly increases ORX mRNA abundances in chick blood (Fig. 3a). Neither recombinant orexin A nor B affect body weight (BW), feed intake (FI), or water consumption (WI) in chicks ( Fig. 3b-d). Hepatic ORX and ORXR2 protein levels were significantly increased following administration of recombinant orexins (ORX-A and ORX-B) (Fig. 3e,f). Although, neither hepatic ORX nor ORXR1 mRNA abundances were affected by recombinant orexin treatments, hepatic ORXR2 gene expression was significantly down regulated by rORX-B administration (Fig. 3g). In LMH cells, treatment for 24 h with 100 nM of rORX-A significantly down regulated prepro-ORX and ORXR1, but increased ORXR2 protein levels ( Fig. 3h,i). Low dose of rORX-A increased both ORXR1 and ORXR2 protein expression and upregulated ORX and ORXR1 gene expression in LMH cells (Fig. 3h-j). Treatment with rORX-B at high dose, however, increased ORX and ORXR1 protein levels and down regulated ORX mRNA abundance in LMH cells (Fig. 3k-m). Recombinant ORX-B at low dose (10 nM) induced both ORXR1 and ORXR2 protein expression and only ORXR2 mRNA levels in LMH cells (Fig. 3k-m).

Orexin regulates lipogenic program in chicken hepatocytes. Administration of recombinant orexin
A or B significantly decreased phosphorylated ACCα Ser79 and increased FASN protein levels, the rate-limiting enzymes involved in lipogenesis (Fig. 4a-c) and down regulated the expression of ACCα, FASN, ACLY, and SCD-1 genes in chicken liver (Fig. 4d,e). Recombinant orexin A significantly upregulated the hepatic expression of the transcription factors SREBP-1 and SREBP-2 (Fig. 4f), however recombinant orexin B significantly increased only SREBP-2 mRNA abundances (Fig. 4g). In vitro studies using LMH cells showed that both recombinant orexins at low and high dose significantly reduced the phosphorylated levels of ACCα at Ser79 site ( Fig. 5a-c). As shown in Fig. 5d, immunofluorescence staining confirmed the reduced levels of ACCα phosphorylation in LMH cells following recombinant orexin B treatment. Orexin treatments also significantly increased the protein levels of FASN, ACLY, and ME in LMH cells (Fig. 5a-c). At transcriptional levels, recombinant orexin A down regulated ACCα and upregulated ME and SCD-1 gene expression in LMH cells (Fig. 5e). Administration of high dose of recombinant orexin B significantly increased the mRNA levels of ACCα, FASN, ACLY, and ME (Fig. 5f). These changes were accompanied with a significant down regulation of SREBP-1 and an upregulation of SREBP-2, INSIG-1, and PPARγ with recombinant orexin A treatment, and an upregulation of SREBP-1 and a down regulation of INSIG-1 and SCAP with the administration of recombinant orexin B (Fig. 5g, h).
Orexin B modulates hepatic lipogenesis via ERK1/2 pathway. Treatment with both doses of recombinant orexin B specifically and significantly induced phospho-ERK1/2 Thr202/Tyr204 but not that of phospho-p38 Thr180/Tyr182 or phospho-JNK1/2 Thr183/Tyr185 in LMH cells (Fig. 6a-c). High dose of recombinant orexin B significantly up regulated the expression of ERK1, ERK2, JNK, and P38 genes in LMH cells (Fig. 6e). Administration of recombinant orexin A, however, did not elicit any changes to ERK1/2, p38, or JNK1/2 at both mRNA and protein levels ( Fig. 6a,b,d). Overexpression of ERK1 or ERK2 (Fig. 6f,g) significantly decreased phospho-ACCα Ser79 and increased ME protein levels in LMH cells (Fig. 6f,g). ERK1 and ERK2 overexpression significantly upregulated the expression of ACCα, FASN, ACLY, and SREBP-1 (Fig. 6h). Pharmacological inhibition of ERK1/2 activation by PD98059 (Fig. 7a,b) blockaded the effects of recombinant orexin B on lipogenic program in LMH cells (increased phospho-ACCα Ser79 and decreased FASN protein levels and down regulated ACCα and ME mRNA abundances) (Fig. 7c-e). Recombinant orexins specifically activated ERK1/2 at Thr202/Tyr204 site, but not P38 or JNK, in chicken liver (Fig. 8a-c). Recombinant orexins did not affect the hepatic expression of ERK1/2, P38, or JNK1/2 genes (Fig. 8d,e). www.nature.com/scientificreports/ (d-f) Total RNA was reverse transcribed and subjected to RT-qPCR as described in the "Materials and methods" with chicken hypothalamus served as a calibrator, and relative expression was determined by 2 −ΔΔCt method 28 . (g) Distribution of intracellular orexin system was determined by immunofluorescence staining with secondary antibody conjugated with AlexaFluor 488 (green) and DAPI (blue). (h) Proteins of cellular components (nucleus, cytoplasmic, and total) were obtained by cell fractionation and submitted to Western blot analysis as described above. Nucleolin was used as a positive control for the nuclear fraction. *Significant difference from the hypothalamus, P < 0.05. Cyto cytoplasmic, LMH leghorn male hepatoma cells, Nuc nuclear, siCEH spontaneously immortalized chicken embryonic hepatocytes, Tot total. Full-length blots/gels are presented in Supplementary Fig. S1. protein (e,f) and mRNA levels (g). Orexin treatment affects orexin, ORXR1, and ORXR2 at protein (h,i,k,l) and mRNA levels (j,m) in LMH cells. mRNA abundances and protein levels were determined by qPCR and Western blot, respectively as described in "Materials and methods". Data are presented as mean ± SEM (n = 12 birds/group). *Significant difference from controls, P < 0.05. Western blot figures are representative of 4 replicates. BW body weight, FI feed intake, WI water intake. Full-length blots/gels are presented in Supplementary Fig. S1.

Discussion
Orexin A and B neuropeptides have attracted much recent attention due to their pleiotropic physiological functions. They were initially characterized in the brain and first emerged as appetite inducers and regulators of Overexpression of ERK1 or ERK2 increased p-ERK1/2 Thr202/Tyr204 , activated ACC and increased ME protein levels (f,g) as well as the mRNA abundances of ACC, FASN, ME, ACLY and SREBP-1 (h). Data are presented as mean ± SEM (n = 6/group). *Indicates significant difference from controls at P < 0.05. ACC acetyl-CoA carboxylase, ACLY ATP citrate lyase, ERK extracellular signal-regulated kinase, FASN fatty acid synthase, JNK c-Jun N-terminal kinase, ME malic enzyme, P38 P38 mitogen-activated protein kinase, SREBP-1 sterol regulatory element-binding protein 1. Full-length blots/gels are presented in Supplementary Fig. S1. www.nature.com/scientificreports/ feeding behaviors and energy homeostasis in mammals 4 . Numerous studies showed that orexins and their two known G-protein-coupled receptors are expressed in many peripheral tissues and play crucial roles in sleep and wakefulness cycle 6-8 , energy metabolism 9,10 , stress response 13,14 , and glucose and lipid metabolism 23,29 in mammalian species. Although orexins were found to be expressed not only in the hypothalamus of chickens (nonmammalian species) 17 , but also in gastrointestinal tract 18 , reproductive organs 19 , and muscle 20 , their functions are still not well defined. The objective of the current study was to investigate the potential role of the orexin system in avian hepatic lipid metabolism. As in humans, chicken liver is the primary site of de novo fatty acid synthesis 21 . www.nature.com/scientificreports/ Additionally, commercial broiler (meat-type) chicken are often hyperphagic and prone to fat accumulation 30 .
Since obesity is a worldwide epidemic, these peculiarities make the chicken a high relevant animal model for further understanding hepatic lipid metabolism in both poultry and humans. By using different techniques (qPCR, immunoblot, and immunofluorescence staining), we first demonstrated that orexin and its related receptors ORXR1 and ORXR2 are expressed in chicken liver and LMH cells. Cell fractionation showed that orexin system is mainly localized in the cytoplasm. Further subcellular analyses by double-labeling immunofluorescence indicated that orexin is localized in ER, Golgi apparatus, and in the lysosome. Although the intracellular distribution of orexin system has not been reported previously, several studies showed that orexin system couples to Ca 2+ influx and release [31][32][33] . As the ER plays a key role in calcium homeostasis and storage 34 , together these studies suggested that orexin might be localized in the ER which is supported by COMPARTMENTS, the subcellular localization database (https ://compa rtmen ts.jense nlab.org). Additionally, orexin contains an ER signal sequence 35 . The presence of orexin in ER and Golgi as well as in the culture media indicated that orexin is secreted from chicken hepatocytes. This is confirmed by brefeldin A treatment, the potent inhibitor of secretion via disruption and disassembly of the Golgi leading to the accumulation of orexin in the cell lysate 36 . The localization of orexin in the lysosome suggested that orexin might be involved in autophagosome-lysosome fusion process and autophagy machinery 37,38 .

Figure 8.
Orexins activate ERK1/2 signaling cascade in chicken liver. Intraperitoneal administration of recombinant orexins increased p-ERK1/2 Thr202/Tyr204 levels, but not that of p-P38 Thr180/Tyr182 or p-JNK1/2 Thr183/ Tyr185 in chicken liver as demonstrated by Western blot analyses (a-c). The relative expression of MAPK genes was measured by qPCR (d,e). Data are presented as mean ± SEM (n = 12 birds/group). *Indicates significant difference from controls at P < 0.05; ERK extracellular signal-regulated kinase, JNK c-Jun N-terminal kinase, P38 P38 mitogen-activated. Full-length blots/gels are presented in Supplementary Fig. S1 www.nature.com/scientificreports/ Although both recombinant orexins upregulated the hepatic orexin system in vivo, rORX-A and rORX-B had differential effects on ORX protein expression in LMH cells. These differences might be due to structural differences between ORX-A and -B (ORX-A contains two disulfide bonds as compared to one in ORX-B) and/ or binding affinity of the orexins to their receptors 39 . In fact, mammalian ORXR1 preferentially binds ORX-A, while ORXR2 has similar affinity for both ORX-A and -B 1 . Additionally, heterodimerization with other G-protein coupled receptors is known to affect orexin receptor activation 40 . Determination of the crystal structure of the chicken orexin receptors as well as their ligand-binding properties may help to further elucidate the differential actions of orexin-A and -B 41 . Similarly, changes in mRNA and protein expression of orexin system are not concomitant. For instance, orexin administration upregulated ORX and ORXR2 protein levels without affecting their mRNA abundances. This lack of correlation between mRNA and protein levels is not surprising 42 and it might be explained by the involvement of varied post-transcriptional and post-translational mechanisms and/ or protein and RNA half-lives 43 . The hepatic expression and secretion of orexin system as well as its ability to regulate its own system suggested that orexin might have autocrine, paracrine, and/or endocrine function 44 .
Though originally identified as a regulator of feeding behavior in mammals, orexin does not appear to have this effect in chicken in our study and that of others 45 , leading to questions regarding its role in avian physiology. Here we showed for the first time that orexin administration in vivo increased hepatic FASN protein levels, which is a rate-limiting enzyme of fatty acid synthesis via catalyzing the synthesis of palmitate from acetyl-CoA and malonyl-CoA into long-chain saturated fatty acids 46 . In support of the in vivo data, orexin treatments also induced the expression of key lipogenic proteins including FASN, ACLY and ME in LMH cells. Reduction of its phosphorylated levels indicated the activation of ACCα, which constitutes a key control point in the synthesis of long-chain fatty acids through catalyzing the carboxylation of acetyl-CoA in the generation of malonyl-CoA 47 . In line with our results, previous studies have shown that orexin stimulated lipid accumulation in primary rat adipocytes and 3T3-L1 cells 23 . Pruszynska-Oszmalek and co-workers reported that orexin stimulated proliferation and differentiation of porcine preadipocytes and regulated lipid metabolism 29,48 . It is noteworthy to mention that, as for orexin system and for the same reasons mentioned above, mRNA and protein levels of lipogenic factors were not correlated. Interestingly, in vivo administration of rORX-A up-regulated both hepatic SREBP-1 and SREBP-2, however rORX-B induced only SREBP-2 gene expression. In LMH cells, however, rORX-A downregulated SREBP-1 and up-regulated SREBP-2 and INSIG-1 gene expression, and rORX-B increased SREBP-1 and decreased INSIG-1 and SCAP mRNA abundances. Although the mechanisms contributing to the differential regulation of transcription factors SREBP-1/2 between rORX-A and rORX-B on one hand, and between in vivo and in vitro studies on the other hands, are not known, it is possible that other transcription factors might be involved. For instance, the up-regulation of PPARγ in LMH cells following rORX-A treatment might compensate for the down-regulation of SREBP-1. It has been shown that PPARγ is one of the downstream mediators of orexin's action 23 . The consistent increase in SREBP-2 mRNA levels following rORX-A administration in both chicken liver and LMH cells indicated that at least rORX-A regulate cholesterol metabolism 49 .
Orexin has been shown to activate various signaling pathways 4,50 and lipogenic enzymes have been reported to be regulated by diverse mechanisms 51 . In attempt to define the downstream mediators of orexin action on avian hepatic lipogenesis, we sought to determine the involvement of MAPK cascades. Orexin administration led to ERK1/2 phosphorylation at Thr202/Tyr204 residues, but not that of P38 and JNK1/2 in both chicken liver and LMH cells. Overexpression and activation of ERK1 or ERK2 up-regulated the expression of lipogenic genes (ACCα, FASN, ME, and ACLY), and their transcription factor SREBP-1, increased ME protein levels, and activated ACCα as demonstrated by its dephosphorylation. Pharmacological inhibition of ERK1/2 activation by PD98059 prevents orexin-induced lipogenesis. Orexin has been shown to activate ERK1/2 in multiple cell types and species, including CHO cells 52 , SGC-7901 gastric cancer cells 53 , H295R adrenocortical cells 54 , neuro-2a cells 55 , and 3T3-L1 cells 56 . ERK1/2 signaling pathway is involved in a wide variety of cellular processes. Roth and co-workers 57 have shown that ERK1/2 regulated the key lipogenic transcription factor SREBP-1 and several lines of evidence have implied a link between ERK1/2 and lipid metabolism in mammals [58][59][60][61] . Although further in-depth investigations are needed to delineate the mechanisms by which orexin activate ERK1/2 in chicken hepatocytes, we speculate that Gq/Gi, PLC, and PKC are potentially involved 54,62 .
In conclusion, this is the first report evidencing the hepatic expression of orexin system in avian (nonmammalian) species and its role in lipogenesis via ERK1/2 pathway. This is significant because it identified a new molecular signature that could open new vistas for mechanistic understanding of avian hepatic lipid metabolism.