Leptin signaling in the rainbow trout central nervous system is modulated by a truncated leptin receptor isoform.

ENERGY

BALANCE-OBESITY

Leptin Signaling in the Rainbow Trout Central Nervous System Is Modulated by a Truncated Leptin Receptor Isoform Ningping Gong and Björn Thrandur Björnsson Fish Endocrinology Laboratory, Department of Biological and Environmental Sciences, University of Gothenburg, S-40590 Gothenburg, Sweden

Central leptin (Lep) signaling is important in control of appetite and energy balance in mammals, but information on Lep signaling and physiological roles in early vertebrates is still lacking. To elucidate fish Lep signaling activation and modulation, a long-form Lep receptor (LepRL) and a truncated LepR (LepRT) are functionally characterized from rainbow trout. The receptors generated in alternatively splicing events have identical extracellular and transmembrane domains but differ in the intracellular sequence, both in length and identity. Gene transfection experiments show that LepRL is expressed as a 125-kDa protein in rainbow trout hepatoma cell line RTH-149, whereas LepRT is 100 kDa; both receptors specifically bind Lep. Homogenous Lep induces tyrosine phosphorylation of Janus kinase 2 and signal transducer and activation of transcription 3 in LepRLexpressing RTH-149 cells. This response is diminished in cells coexpressing LepRL and LepRT, suggesting that the LepRT which lacks these kinase-associated motifs competes with the LepRL for Lep availability, thus attenuating the Lep signal. Both receptor genes are highly expressed in the central nervous system. The mRNA levels of LepRT in hypothalamus, but not LepRL, change postprandially, with decreased transcription at 2 hours postfeeding and then elevated at 8 hours, concomitant with changes in proopiomelanocortin-A1 transcription. However, both receptors have no change in mRNA levels during 3 weeks of fasting. These data indicate that LepRT transcription is more likely a mechanism for modulating Lep effects on short-term feed intake than in regulating energy balance in the long term. In vitro and physiological characterization of LepR isoforms indicates divergent Lep signaling modulation patterns among vertebrates with different life histories and metabolic profiles. (Endocrinology 155: 2445–2455, 2014)

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eptin (Lep) is a peptide hormone with important roles in control of appetite and energy homeostasis in mammals (1, 2). Lep action in the central nervous system (CNS) is mediated through the long-form leptin receptor (LepRL) also termed obese receptor isoform b (ObRb) in mammals. The ObRb is highly expressed in hypothalamic arcuate, dorsomedial, ventromedial, and ventral premamillary nuclei, indicating the importance of Lep in the control of energy homeostasis (3–5). The activation of ObRb stimulates anorexigenic neurons and inhibits orexigenic neurons in the hypothalamus (6 – 8). The signaling activation

through different pathways leads to inhibition of neuropeptide Y (NPY) and agouti-related protein expression, whereas it stimulates cocaine- and amphetamine-regulated transcript and proopiomelanocortin (POMC) transcription (9 –11). Moreover, in the extrahypothalamic area, lower ObRb transcription has been found in Purkinje and granular cell layers of the cerebellum, thalamus, and hippocampus (4, 5). It has been suggested that extrahypothalamic Lep signaling through classic pathways is related to, eg, feed intake behavior and energy homeostasis (12–14).

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received December 12, 2013. Accepted April 28, 2014. First Published Online May 5, 2014

Abbreviations: aa, amino acid; CDS, coding sequence; CNS, central nervous system; DNase, deoxyribonuclease; EF, elongation factor; FBS, fetal bovine serum; GH, growth hormone; hpf, hour postfeeding; Jak, Janus kinase; Lep, leptin; LepBP, Lep binding protein; LepRL, long-form Lep receptor; LepRT, truncated LepR; NPY, neuropeptide Y; ObR, obese receptor; PBST, phosphate-buffered saline with 0.05% Tween 20; pJak2, phosphorylated Jak2; POMC, proopiomelanocortin; qPCR, quantitative polymerase chain reaction; RACE, rapid amplification of cDNA ends; Stat3, signal transducer and activation of transcription 3.

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Endocrinology, July 2014, 155(7):2445–2455

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Full-Length and Truncated LepRs in Trout CNS

Lep signaling in the mammalian CNS is modulated by shorter ObR isoforms (15). Three prime end alternative splicing event generates at least 6 different isoforms in rodents: the full-length ObRb and 5 shorter isoforms, ObRa, ObRc, ObRd, ObRe, and ObRf (3, 16). The signaling capacities of ObR isoforms differ due to different truncations of the intracellular receptor segment (15). The ObRb has 302 cytoplasmic amino acid (aa) residues, which include 2 boxes for associating Janus kinase (Jak) family tyrosine kinase (Jak2), 2 tyrosine phosphorylation sites, and a box for binding signal transducer and activation of transcription 3 (Stat3) (17). The truncated isoforms ObRa, ObRc, ObRd, and ObRf have cytoplasmic segments of only 32 to 40 aa (3, 18), of which 29 aa are identical with ObRb (3). The truncated receptors have a conserved Jak box 1 and reduced signaling capabilities in mediating tyrosine phosphorylation of Jak2 (15). ObRb is mainly expressed in the CNS, whereas the truncated receptor isoforms have wider tissue distribution (3, 16). The physiological functions of the truncated receptors have been proposed to involve in the production of circulating Lep binding proteins (LepBPs), transportation of Lep across the blood-brain barrier, and the degradation and clearance of circulating Lep (19 –21). Amino acid identity is low between fish and mammalian Leps, being around 20% between rainbow trout and human Lep (22). In fish, including salmonids, the liver appears to be the primary Lep expression site (22, 23). Lep has been proposed to participate in the control of energy homeostasis in fish, because liver lep expression and plasma Lep levels change in relation to nutritional status (24 –27). In contrast to mammals, zebrafish, and sea bass (28, 29), plasma Lep levels increase during fasting in salmonids as well as in the fine flounder (24, 26). Because peripheral Lep treatment decreases food intake in salmonids (22), it has been hypothesized that the Lep response to fasting is an anorexia-inducing mechanism during periods of such low food availability that foraging activity would be energetically wasteful (26). Plasma Lep levels in fish can change rapidly in response to nutritional status, decreasing significantly within 2 hours of feeding of previously fasted fine flounder (26), and in ad libitum-fed Arctic charr, plasma Lep levels are higher in individuals with empty gut at the time of sampling (Arnason T., S. Gunnarsson, A.K. Imsland, H. Thorarensen, H. Smaradottir, A. Steinarsson, A. Gustavsson, M. Johansson, B.Th. Björnsson, unpublished data). Intraperitoneal Lep injection inhibits food intake of rainbow trout for about 2 hours, at which point changes in hypothalamic expression of NPY and POMC isoforms also occur (22). Human Lep treatment of rainbow trout hypothalamus slice culture in vitro similarly decreases NPY


expression, but it did not affect POMC expression (30). Together, these data suggest rapid anorexigenic effects of Lep in the CNS through changes in neuropeptide transcription. However, at present, less is known about the ligand-receptor interactions and signaling activation, eg, in relation to the various existing LepR isoforms. The aim of the current study was therefore to elucidate Lep signaling action in fish through characterization of LepR signaling system in rainbow trout using homologous molecular tools and in vitro as well as in vivo approaches. The cytoplasmic sequences of the LepRL and the truncated LepR (LepRT) were analyzed through sequence alignment comparison with other vertebrates. The sequence information was used to establish methods for quantifying mRNA levels and then to determine the tissue distribution patterns of LepRL and LepRT. The role of Lep in Jak2/ Stat3 activation was then examined in vitro by expressing LepRL and LepRT in a rainbow trout cell line and determining the intracellular effects of Lep stimulation, using recombinant rainbow trout Lep. To increase understanding the role of the LepR signaling system in energy homeostasis, mRNA levels of LepRL, LepRT, and the neuropeptides NPY and POMC-A1 were assessed in the hypothalamus of fasting and feeding rainbow trout.

Materials and Methods Fish and experimental design Juvenile rainbow trout (⬃250 g) were purchased from a local hatchery (Laxodling AB), transferred to the recirculating aquarium facilities at the Department of Biological and Environmental Sciences, University of Gothenburg, and acclimated to an ad libitum feeding regime in circulating aerated freshwater at 11°C for 1 week before the start of the experiments. The experiments were approved by the Ethical Committee for Animal Research in Gothenburg (license 85–2012) and comply with current Swedish legislation.

Effects of fasting A total of 200 fish were distributed randomly among 4 experimental tanks, with 50 fish in a tank. At the start of the experiment, food was withheld from fish in 2 tanks for up to 3 weeks, whereas fish in 2 tanks were fed ad libitum by hand once a day. Eight fish from each tank without regard to sex were sampled after 1, 2, and 3 weeks, and for the fed group, food was withheld for 24 hours before sampling. Fish were anesthetized by 2-phenoxyethanol (0.4 mL/L) and killed. The body weight and fork length were measured for calculation of condition factor. Hypothalamus was dissected and preserved in liquid nitrogen for RNA extraction.

Postprandial changes A total of 100 fish were distributed among 5 tanks (20 fish in a tank) and acclimated to an ad libitum feeding regiment for 1


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week. After being given a single meal to satiation, 8 fish were sampled as described above at 0, 2, 4, 8, and 24 hours postfeeding (hpf).

Gene cloning and sequencing The lepr genes were obtained from 3⬘- and 5⬘-rapid amplification of cDNA ends (RACE)-PCRs. Total RNA was isolated from rainbow trout brain with RNeasy Mini kit (QIAGEN). RACE-ready cDNAs were synthesized from the total RNA using SMARTer RACE cDNA amplification kit (CLONTECH Laboratories). Advantage 2 polymerase mix kit (CLONTECH Laboratories) was used in RACE-PCR. After RACE-PCR, the coding region of lepr genes was amplified by RT-PCR using Platinum Taq DNA polymerase high-fidelity PCR reagents (Invitrogen) and the primer sets (5⬘-gttatctcactcacagaggccatg-3⬘ and 5⬘-tcatgtgcttatgctgtacagtc-3⬘) for leprl and (5⬘-gttatctcactcacagaggccatg-3⬘ and 5⬘-gggacattcattgacaagtgaaggaagg-3⬘) for leprt. All PCR products were analyzed on agarose gel electrophoresis, ligated into pGEM-T vector (Promega Corp), and subcloned in DH5␣ competent cells (Invitrogen) grown on selective agar plates. The positive colonies were screened by colony PCR using universal primers (SP6 and T7). Three colonies with the same insert DNA were chosen for plasmid extraction, and the plasmids were sequenced by Eurofins MWG Operon Co.

Tissue distribution of lepr genes Three juvenile rainbow trout (⬃200 g) were used in the analysis. The tested tissues include brain, pituitary gland, bellyflap, kidney, and gill. Different brain regions were dissected and analyzed separately, using 30 mg or less, including telencephalon without the preoptic area, optic tectum without midbrain tegmentum, preoptic area with part of the anterior hypothalamus, basal hypothalamus, midbrain, cerebellum, and hind-brain (see figure 3 below). Total RNA was isolated with RNeasy Mini kit (QIAGEN) and treated with Turbo deoxyribonuclease (DNase) (Ambion, Applied Biosystems) to remove genomic DNA trace. First-strand cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad Laboratories) on 1-␮g total RNA template in 20 ␮L of the final volume. Meanwhile, another 1 ␮g of RNA mixed the cDNA synthesis reagents without adding iScript reverse transcriptase was used as negative control. No or very few quantitative polymerase chain reaction (qPCR) products were amplified from the negative control; 0.5- to 2-␮L cDNA samples were applied for each qPCR in analysis of leprl and leprt expression, depending on mRNA abundance in the tissues. The qPCR included 300nM primers, templates, and iQ SYBR Green supermix (Bio-Rad Laboratories) in 25 ␮L of the final volume. The qPCR primer sets are 5⬘-ccaccgtgcttctctctgac-3⬘ and 5⬘-cctgagaagttgccctcgtc-3⬘ for leprl and 5⬘-tgatcatcgccttcctctcc-3⬘ and 5⬘-ttgacctcctccttctccac-3⬘ for leprt. Data from the qPCR runs were collected with Bio-Rad iCycler iQ Optical System. The qPCR efficiencies were between 95% and 105%. Dilutions of the templates were served as the standards with the copies of 30 –300 000. Gene leprl and leprt mRNA copy numbers were calculated from the standard curve with the linearized plasmids of leprlpcDNA3.1 and leprt-pcDNA3.1 templates.


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qPCR analysis of leprl, leprt, pomc-a1, and npy expression Hypothalamic tissues collected during the fasting and postprandial in vivo studies were prepared as follows. Total RNA was isolated with RNeasy Mini kit (QIAGEN) and DNasetreated with Turbo DNase (Ambion). First-strand cDNA was synthesized using the reverse transcription kit Superscript III First-Strand Synthesis System (Invitrogen) on 3-␮g total RNA template. Oligo(dT) was chosen as primer. A negative control of pooled RNA from all of samples, and no reverse transcriptase was included. No signal was observed in the negative control qPCR. The mRNA levels of leprl, leprt, pomc-a1 (31), npy (GenBank accession number AF203902), and elongation factor (EF)1␣ gene (ef1␣; GenBank accession number AF498320.1) were quantified by qPCR with 0.5-␮L cDNA and iQ SYBR Green supermix (Bio-Rad Laboratories) in final volume of 25 ␮L. The qPCR primer sets are 5⬘-ctcgctgtcaagacctcaactct and 5⬘-gagttgggttggagatggacctc for pomc-a1, 5⬘-ctcgtctggacctttatatgc and 5⬘-gttcatcatatctggactgtg for npy, and 5⬘-caaggatatccgtcgtggca and 5⬘-acagcgaaacgaccaagagg for ef1␣. The standards for quantification of leprl and leprt mRNA copy numbers were dilutions of linearized plasmids of leprl-pcDNA3.1 and leprt-pcDNA3.1. The standards for pomc-a1, npy, and ef1␣ qPCR were the dilutions of pooled cDNA samples. Data were collected with Bio-Rad iCycler iQ Optical System. A melting curve analysis was performed for each qPCR assay and showed a single peak, confirming PCR specificity. The PCR efficiency of the standards was above 95%. The relative expression values of all samples were normalized by ef1␣. One-way ANOVA followed by a Tukey post hoc test was used to identify temporal postfeeding changes. Two-way ANOVA followed by a Bonferroni post hoc test was used to analyze data from the 3-week fasting experiment. Differences were considered significant at P ⬍ .05.

Construction of LepR expression vectors, leprlpcDNA3.1 and leprt-pcDNA3.1 The coding sequence (CDS) for LepRL containing 3399 nucleotides was hardly ligated directly in expression vector. Because it includes a BamHI restriction site at nucleotide 1865 in the CDS, 2 fragments encoding the nucleotide 1–1865 and 1866 –3399 were inserted to vector in sequence. The DNA fragments were amplified by RT-PCR using Platinum Taq DNA polymerase high-fidelity PCR reagents (Invitrogen) and confirmed by DNA sequencing. The DNA, including Kozak sequence and the first 1865 nucleotides with the start codon, was inserted into pcDNA3.1/V5-His expression vector (Invitrogen) through the restriction sites of HindIII and BamHI. Next, DNA sequencing nucleotides 1866 –3399 was inserted to the constructed vector through the restriction sites of BamHI and XhoI. By this way, the CDS for LepRL was constructed in the expression vector as leprl-pcDNA3.1. Similarly, the CDS for LepRT containing 2622 nucleotides was constructed to the vector as leprt-pcDNA3.1.

Cell transfection Adherent rainbow trout hepatoma cell line RTH-149 was grown in DMEM/F12 Ham (Sigma-Aldrich) with 2mM L-glutamine, 25mM HEPES, and 10% fetal bovine serum (FBS) (Sigma-Aldrich). When cell growth reached about 80% confluence


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in 35-mm petri dishes, the cells were transfected with the plasmids of leprl-pcDNA3.1 or leprt-pcDNA3.1, using Effectene transfection reagent (QIAGEN) according to the manufacture’s instruction. After 36 hours, the culture medium were changed to DMEM/F12 with or without 2% charcoal-stripped FBS (Gibco). After 12 hours, the cells were treated with recombinant rainbow trout Lep in concentration of 0.2nM, 2nM, 20nM, and 50nM for 15– 60 minutes. The recombinant Lep was prepared by the method described by Murashita et al (22). Cells were washed once with cold PBS and scraped from the petri dishes. Collected cells were suspended in cold lysis buffer (50mM Tris-HCl [pH 7.4], 0.5% sodium chloride, 1% IGEPAL CA-630 [Sigma-Aldrich], 1% Triton X-100, 50mM sodium fluoride, 0.2mM sodium orthovanadate, 1mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktails [Sigma-Aldrich]) and incubated on ice for 30 minutes. After centrifugation, the supernatant with the total protein was collected and saved at ⫺20°C.


20 (PBST) for 2 hours, followed by the antibodies against LepR (LepR-Antibody 1) (32), Jak2, phosphorylated Jak2 (pJak2), Stat3, or pStat3 (pStat3), diluted in 3% BSA, PBST overnight at 4°C. The antibodies against Jak2 and pJak2 were purchased from Merck, Millipore. The antibodies against Stat3 and pStat3 (Tyr705) were from Santa Cruz Biotechnology, Inc. The membrane was washed with PBST for 30 minutes and incubated with the second antibody diluted in 3% BSA, PBST at room temperature for 1 hour. The second antibody was antirabbit IgG horseradish peroxidase-linked whole antibody (GE Healthcare). The information of the antibodies is listed in Supplemental Table 1. Imaging was carried out using enhanced chemiluminescence plus Western-blotting detection reagents and high performance chemiluminescence film (GE Healthcare).

Results

125

I-labeled Lep binding assay

The recombinant Lep (20 ␮g) was iodinated [125I] using 600-␮g chloramines T to catalyze the reaction, which was stopped after 55 seconds with sodium metabisulfide. The radiolabeled protein was separated from free iodine with size exclusion chromatography (Sephadex G75) in a 0.7 ⫻ 20 cm column (Bio-Rad) using 0.1M Tris-HCl with 0.1% Triton X-100 as elution buffer. Fractions in 500 ␮L were collected into a 50-␮L elution buffer containing 2% BSA. The 125I-labeled Lep was adjusted to be approximately 550 cpm pM⫺1. RTH-149 cells were transfected with Effectene transfection reagent and the plasmids of leprt-pcDNA3.1 or empty vector. After 48 hours, the cells were serum deprived overnight. The cells (3 ⫻ 106) were then detached by 1mM EDTA in PBS, suspended in HEPES buffer (pH 7.4), and divided into aliquots. A saturation isotherm was produced by incubating cells with increasing concentrations of 125I-labeled Lep between 50pM and 550pM, at room temperature for 2 hours. Control cells were incubated with 125Ilabeled Lep as above plus 150nM Lep. After incubation, the cells were precipitated by centrifugation at room temperature and resuspended in buffer (50mM Tris-HCl, 0.5% NaCl, 0.1% sodium dodecyl sulfate, 1% IGEPAL CA-630, and 0.5% sodium deoxycholate [pH 7.4]). Radioactivity of the cell lysates was counted in a ␥-counter (PerkinElmer). The specific binding was calculated as total binding minus unspecific binding. The protein concentrations of cell lysates were measured by Pierce BCA protein assay kit (Thermo Scientific) with BSA as standards.

Sequences of LepRL and LepRT The sequences of LepR were obtained from RACEPCR. The longest transcript LepRL contains a 3399-bp open reading frame for a predicted 1132-aa long protein (GenBank accession number JX878485) with a 29-aa signal peptide, a 782-aa extracellular segment, a 21-aa transmenbrane domain, and a 300-aa intracellular segment (Figure 1). A shorter LepR transcript contains a 2622-bp open reading frame for a predicted 873-aa protein and a 414-bp 3⬘ untranslated region (GenBank accession number JX878488). The first 835 aa, which are predicted to be involved in the extracellular segment and transmembrane domain, are identical to those of the LepRL. A unique sequence is spliced in the 3⬘ end and contains the sequence

Immunoprecipitation and immmunoblotting LepR and Jak2 were isolated from the cell extracts by immunoprecipitation. Briefly, the antibodies against LepR or Jak2 were added to the cell extracts and incubated at 4°C for 4 hours. Afterwards, Protein A Sepharose beads (GE Healthcare) were added in order to precipitate antigen-antibody complex and incubated for 2 hours at 4°C. After wash, the beads were suspended in 2⫻ SDS-PAGE buffer and heated at 95°C for 6 minutes. After centrifugation, the supernatants from immunoprecipitation were applied for immunoblotting. Proteins from immunoprecipitation supernatants or crude cell extracts were separated in 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane (Bio-Rad). The membranes were incubated with 5% skimmed milk in phosphate-buffered saline with 0.05% Tween

Figure 1. Amino acid sequences of the long-form (LepRL) and truncated (LepRT) LepR of rainbow trout. Both isoforms have the same first 835 aa. The upstream sequence of 833Ser is predicted to represent the extracellular segment (indicated as extracellular) and the transmembrane domain (indicated as TM). The intracellular sequences of LepRL and LepRT differ in length, being 300 and 39 aa long, respectively, as well as in sequence identity. Two proline-rich motifs putatively for Jak binding are identified in LepRL, together with 7 tyrosine residues. The 1115Tyr is involved in the motif putatively recruiting Stat. The Jak-Stat motifs are indicated by white letters on black background.


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for 39 intracellular aa and the 3⬘ untranslated region (Figure 1). Thus, this isoform is defined as a LepRT. The gene sequence shows that LepRL has full functionality for ligand binding, membrane anchoring, and intracellular signaling. The aa sequence of the rainbow trout LepR intracellular domain has been aligned with those of other fish species, human, chicken, and frog (Figure 2). The LepRL contains the proline-rich motifs, including DVPNP and PLLL residues, which have been implicated in Jak binding box 1 and 2 of human ObRb (33) and also conserved in chicken, frog, and other fish species. Moreover, 3 tyrosine residues (950Y, 1051Y, and 1115Y) are conserved in LepRL, as potential tyrosine phosphorylation sites. The 1115Y in LepRL has the similar location as 1141Y in Stat3 binding box of ObRb (34). Thus, the functional motifs that have been implicated in Jak-Stat binding in ObRb are predicted in rainbow trout LepRL. In contrast


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with LepRL, LepRT has much shorter intracellular segment with 39 aa, including 2 serine residues that may be phosphorylated. No common intracellular sequences (except a tryptophan residue) are found in LepRL and LepRT (Figure 2). The proline-rich motifs and tyrosine residues are not present in LepRT. It appears that LepRT has no signaling capability through the Jak2-Stat3 pathways. Tissue distribution of LepRL and LepRT Distribution of leprl and leprt transcripts was determined by qPCR in the CNS and some peripheral tissues (Figure 3). Absolute quantification of qPCR showed that leprl mRNA had around 4 ⫻ 104 copies in 1-␮g total RNA extraction from the hypothalamus, whereas copy number in other brain areas ranged from 1 ⫻ 104 to 2 ⫻ 104 (Figure 3B). The leprt mRNA copy number was generally 10% of leprl number (Figure 3C). Both leprl and leprt had high

Figure 2. Alignment of aa sequences of LepR intracellular domain in various vertebrates. Asterisks indicate aa that are conserved in all sequences, whereas colons and dots indicate decreasing levels of aa similarity. The predicted Jak-Stat motifs are indicated by white letters on black background. GenBank accession numbers: rainbow trout Oncorhynchus mykiss, RT LepRL: AGC55253.1 and RT LepRT: AGC55256.1; Atlantic salmon Salmo salar, BAI23197; crucian carp Carassius carassius, ADZ75460.1; human Homo sapiens, AAB09673.1; chicken Gallus gallus, AAF31355.2; Western clawed frog Xenopus laevis, ABD63000.2; and Japanese medaka Oryzias latipes, NP_001153915.1.


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Figure 4. Expression of LepRL and LepRT in RTH-149 cells transfected by leprl-pcDNA3.1 (v⫹leprl) and leprt-pcDNA3.1 (v⫹leprt). The vehicle control (v) was RTH-149 cells transfected by the vector without insert. LepRL is about 125 kDa. LepRT is about 100 kDa. RTH-149 cells express LepRL but not LepRT.

fected cells, but no LepRT was found in the vehicle-control cells (Figure 4). Thus, RTH-149 transfected by leprlpcDNA3.1 are LepRL-expressing cells. The cells transfected by leprt-pcDNA3.1 express both LepRL and LepRT.

Figure 3. The quantification of leprl and leprt mRNA copies in 1-␮g total RNA extracts from the various tissues. In B and C, the leprl and leprt mRNA copy numbers were calculated from the standard curves with the linearized plasmids of leprl-pcDNA3.1 and leprt-pcDNA3.1 templates. Error bars represent SEM (n ⫽ 3). The examined tissues are the brain, pituitary gland (p), bellyflap (bf), kidney (ki), and gill (gi). Different brain areas were dissected and analyzed separately, including telencephalon without the preoptic area (t), optic tectum without midbrain tegmentum (ot), preoptic area with part of anterior hypothalamus (po), basal hypothalamus (hy), cerebellum (c), midbrain (mb), and hind brain (hb). The optic nerve (on) and the spinal cord (sc) were not analyzed. The drawling of rainbow trout brain (A) was modified from the picture in Doyon et al (44).

Characterization of 125I-labeled Lep binding to LepRL and LepRT-expressing cells Cell surface expression of LepRT on leprt-pcDNA3.1transfected RTH-149 was confirmed with binding assays using 125I-labeled Lep. Specific Lep binding to the cells increased with increased addition of 125I-labeled Lep, and the radioactivity approached a plateau at concentrations of 200pM–550pM (Figure 5). Higher specific binding was obtained in the LepRT-expressing cells than in the vehiclecontrol cells (Figure 5), and the maximum number of binding site values, calculated by GraphPad software using nonlinear regression, were approximately 62.3 and 16.3 fm mg⫺1 in the LepRT-expressing cells and the control cells, respectively. The dissociation constant values are 200pM and 183pM in the LepRT-expressing cells and the control cells, respectively. Effects of Lep on phosphorylation of Jak2 and Stat3 in LepRL- and LepRT-expressing cells Immunoprecipitation and immunoblotting analysis showed that Lep (200pM to 20nM) stimulated tyrosine

mRNA expression in the hypothalamus. Peripheral tissues, such as bellyflap and gill, moderately expressed leprl and leprt transcripts, which had around 2 ⫻ 104 and 2 ⫻ 103 mRNA copies per 1-␮g total RNA, respectively. Significantly lower leprt mRNA levels were observed in the pituitary gland and kidney (Figure 3C). Expression of LepRL and LepRT in RTH-149 cells RTH-149 cells were transfected by leprl-pcDNA3.1, leprt-pcDNA3.1, and vector without insertion. Immunoblotting analysis of cell extracts confirmed that LepRL was expressed as a 125-kDa protein in the leprl-pcDNA3.1 and vehicle-transfected cells (Figure 4). LepRT was expressed as a 100-kDa protein in leprt-pcDNA3.1-trans-

Figure 5. 125I-labeled Lep binding to RTH-149 cells transfected by leprt-pcDNA3.1 (f) and empty vector (●). Specific Lep binding to the cells was increased when more 125I-labeled Leps were added and approached a plateau between 200pM and 550pM. Higher specific binding levels (fm mg⫺1 total proteins) were obtained in leprtpcDNA3.1-transfected cells than the vehicle-control cells.


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phosphorylation of Jak2 in LepRL-expressing RTH-149 cells within 15 minutes (Figure 6A). Jak2 phosphorylation in the cells transfected by leprl-pcDNA3.1 was induced by Lep in a low concentration (200pM), whereas only minimal phosphorylation was observed in leprt-pcDNA3.1transfected cells. However, Jak2 phosphorylation in leprtpcDNA3.1-transfected cells was stimulated when higher concentrations of Lep (2nM and 20nM) were used (Figure 6A). Stat3 was tyrosine phosphorylated in 50nM Lep stimulation within 1 hour in both cell types (Figure 6B). Although cells had a trace of phosphorylated Stat3 when cultured in the medium with 2% charcoal-stripped FBS, the phosphorylation was significantly elevated by the addition of 50nM Lep for 15–30 minutes, after which it started to decrease, especially in the leprt-pcDNA3.1transfected cells (Figure 6B). Postprandial expression of leprl, leprt, pomc-a1, and npy in the hypothalamus No significant postprandial changes were observed in leprl mRNA levels in the hypothalamus after a meal (Figure 7A), whereas postprandial leprt transcription changed from being barely detectable at 2 and 4 hpf to being significantly elevated at 8 hpf (Figure 7B). Subsequently, a trend toward decreased leprt expression was observed at 24 hpf, and the mRNA levels were not significantly different from the ones at 0 hpf. The pomc-a1 mRNA levels were increased after meal at 2 hpf, after which this was significantly decreased at 8 and 24 hpf (Figure 7C). The npy mRNA levels appeared to have no significant postprandial change (Figure 7D). Expression of leprl, leprt, pomc-a1, and npy in the hypothalamus within 3 weeks of fasting Three-week fasting had no significant effects on leprl and leprt mRNA levels in the hypothalamus (Figure 8, A


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and B). The pomc-a1 mRNA levels increased in the fasted group at week 3 compared with the fed group (Figure 8C). A tendency towards increased npy expression in fasted group was observed at week 3 compared with the fed group (P ⫽ .2) (Figure 8D).

Discussion Gene sequencing and aa sequence alignment shows that the rainbow trout long-form receptor LepRL has low aa similarity to the mammalian ObRb. However, the Jak and Stat binding boxes in the intracellular region of ObRb appear to be well conserved in the LepRL. After transfection of the rainbow trout cell line RTH-149, Lep stimulation of the LepRL-expressing cells caused a rapid tyrosine phosphorylation of Jak2 and Stat3, with strong phosphorylation of Jak2 in 15 minutes. These data demonstrate that the LepRL in rainbow trout is a fully functional receptor, mediating the ligand binding of Lep through the Jak2-Stat3 intracellular signaling pathways in a similar manner as has been elucidated previously for mammalian species (15, 17). The 3⬘ alternatively splicing events are known to generate multiple lepr transcripts in mammals (3) and fish species (23, 32, 35). The splicing sites are located in the intracellular and extracellular regions and generate truncated receptors or soluble LepBPs. Only 1 shorter isoform containing the predicted transmembrane domain, LepRT, was currently identified in rainbow trout by RACE-PCR. It would be expected to bind Lep in similar affinity as LepRL but not expected to activate Jak2, as its absence from the predicted Jak-Stat motifs. 125I-labeled Lep binding assay indicates that the LepRT is present on the RTH149 cell surface and specifically binds Lep. The dissocia-

Figure 6. Lep signaling in RTH-149 cells expressing LepRL only (leprl-pcDNA3.1-transfected cells), or expressing LepRL and LepRT (leprt-pcDNA3.1transfected cells). A, Thirty-six hours after gene transfection, the cells were serum deprived overnight, then stimulated with 0.2nM, 2nM, or 20nM Lep for 15 minutes and exposed to lysis solution. Cell lysates were immunoprecipitated (IP) with Jak2 antibody and resolved by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes and immunoblotted (IB) with antibodies to Jak2 or pJak2. B, The cells were pretreated in basic medium plus 2% charcoal-stripped FBS overnight and stimulated with 50nM Lep for 15, 30, or 60 minutes. Crude cell lysates were directly applied to SDS-PAGE for IB with antibodies to Stat3 or pStat3.


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and thus act as a dominant negative inhibitor to modulate the functional amplitude of Lep signal. Such modulation in ligand availability by truncated receptor has been demonstrated in the fine flounder, in which high expression of a truncated growth hormone (GH) receptor in that tissue leads a GH resistance in muscle (36). The negative effect of LepRT expression on the Lep signaling is further detected in the present study as “low-dose” Lep stimulation (0.2nM) induces less pJak2 in RTH149 cells expressing both LepRL and LepRT than in cells expressing LepRL only. This indicates that the Figure 7. Postprandial mRNA expression of leprl (A), leprt (B), pomc-a1 (C), and npy (D) in the competition of Lep by LepRT inhibhypothalamus of rainbow trout after being fed a single meal. The sample mRNA expression was its LepRL signaling in a dose-depennormalized by ef1␣ copy numbers. Error bars represent SEM (n ⫽ 5). Significant differences dent fashion. Similar dose-depenbetween treatment groups are indicated by *, P ⬍ .05 and **, P ⬍ .01. dent inhibition of cytokine receptor tion constant of 125I-labeled Lep binding in the LepRL- and signaling by its truncated isoform has been observed in LepRT-coexpressing RTH-149 cells is similar to that of the cells coexpressing the full-length GH receptor and its trunLepRL-expressing cells, indicating similar binding affinity cated receptor in vitro (37). The degree of inhibition was for LepRT and LepRL. Although the LepR isoforms are more significant when low-dose GH was used in in vitro produced by alternately splicing of the common pre- treatment (37). All rainbow trout cell lines so far tested mRNA, it is currently unknown whether the LepRT and express the LepRL, and it has therefore not been possible LepRL are coexpressed in the same cells in vivo or not. to examine LepRT signaling capability separately at the However, at the tissue level, the ligand-binding ability of cellular level. However, it would be expected that ligand the LepRT indicates that it would compete with the LepRL binding to LepRT will not activate Jak2 in the same manner as occurs after ligand binding to the LepRL, due to the absence of JakStat motifs in the LepRT. The rainbow trout LepRT appears to attenuate Lep signaling through competitive ligand binding, thus function differently from the mouse truncated receptors ObRa, ObRc, and ObRd that have reduced signaling capabilities in mediating tyrosine phosphorylation of Jak2, due to the conserved Jak box 1 (15). Apart from the LepRT described in the current study, 3 shorter isoforms containing only the extracellular segment, similar to the ObRe isoform, have also been found to be generated through 3⬘ alternative splicing and expressed as soluble Figure 8. The mRNA expression of leprl (A), leprt (B), pomc-a1 (C), and npy (D) in the LepBPs in rainbow trout plasma hypothalamus of rainbow trout fasted (blank bar) or fed (filled bar) for 3 weeks. Error bars (32). Thus, 5 lepr transcripts have represent SEM (n ⫽ 5). The mRNA expression was normalized by ef1␣ copy number. An asterisk been identified in rainbow trout, indicates significant difference between fish groups (P ⬍ .05).


doi: 10.1210/en.2013-2131

similar to what is found in mammals. However, the present study indicates that there are differences in the alternative splicing sites and thus the LepR types generated by fish and mammals (3, 16). This is likely to be reflected in different modulation mechanisms in Lep signaling among vertebrate species. The leprl mRNA has extensive distribution in the rainbow trout brain. The absolute qPCR quantification data show that leprl mRNA levels are relatively higher in the hypothalamus, where the mammalian ObRb is mainly expressed (4). Moderate leprl mRNA expression is also found in telencephalon, optic tectum, and hindbrain, which are known to control feeding behavior and energy balance of teleost fish (38). The findings thus give strength to the notion that fish Lep acts mainly through central actions but not restrictively in the hypothalamus. Similar to leprl, the leprt mRNA is extensively expressed in the CNS, in particular in the hypothalamus. The similar distribution patterns for the 2 isoforms indicate that LepRT could have significant modulating effects on Lep stimulation by affecting the ligand availability to the LepRL and thus be involved in the regulation of energy homeostasis of rainbow trout. The potential importance of LepRT as a regulator of physiological responses to Lep is further indicated by the finding that after a single meal, there is a postprandial change in hypothalamic leprt expression, but not leprl, concomitant with a pomc-a1 transcriptional change. The postprandial elevation of pomc-a1 mRNA levels in rainbow trout at 2 hpf in the present study is similar to that earlier seen in Atlantic salmon at 3 hpf (39). In mammals, central ObRb activation mediated by Stat3 regulates pomc transcription in POMC neurons (10). Rainbow trout pomc-a1 expression responds to changes in energy balance and is up-regulated during ip Lep treatment (22, 31). Thus, pomc-a1 has been suggested as a target gene for Lep action. The current postprandial data show that leprt expression is lowest at 2 hpf, when high pomc-a1 mRNA levels are observed. In contrast, a significant up-regulation of leprt expression occurs at 8 hpf, whereas pomc-a1 mRNA levels decrease. Because LepRL expression in the hypothalamus is at least a magnitude higher than the LepRT expression, it is unlikely that Lep signaling can be fully blocked through competitive ligand binding by the LepRT. However, because the postprandial expression of the LepRL is stable, as well as the postprandial plasma LepBP levels (Einarsdottir I.E., M. Johansson, N. Gong, and B.Th. Björnsson, unpublished data), while the expression of the LepRT increases transiently, it appears that this mechanism can module ligand availability to LepRL and thus to affect signal transduction and subsequent gene expression, such as pomc-a1.


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In rainbow trout, appetite returns at 9 –12 hpf when 80%–90% of the meal has evacuated from the stomach to the intestine (40). The increased LepRT levels at 8 hpf seen in the current study may represent a mechanisms through which the anorexigenic effect of Lep is decreased, allowing appetite to return. Concurrently, low pomc-a1 mRNA levels are observed and likely to be a part of the regulatory mechanism for the return of appetite. Inhibition of npy expression is related to Lep activation in mammals (11). However, postprandial npy expression change is not statistically significant in the rainbow trout hypothalamus, similar to the observation of no change in npy expression in catfish hypothalamus after feeding (40). In contrast, whole brain npy mRNA levels change after feeding in Atlantic salmon (39), catfish (41), and goldfish (42). During the 3-week fast of the rainbow trout in the current study, no significant changes were observed in leprl and leprt mRNA levels. It is therefore likely that modulation of Lep signaling through changes in LepRT expression is related to short-term food intake regulation rather than long-term regulation of energy homeostasis. Previous studies on fasting rainbow trout have shown decreased plasma LepBP levels after 3 weeks (32), concomitant with elevated plasma Lep levels (24). Thus, it appears that the Lep system is highly activated during an extended period, in which no food is available to the fish, possibly resulting in the stimulation of pomc-a1 transcription observed after 3 weeks of fasting. It appears that Lep signaling can be regulated by LepRT or/and LepBPs levels by modulating Lep availability to the functional LepRL, depending on the physiological status. No significant npy expression was observed after 1-week fasting compared with the fed control, similar to the observation of no change in Atlantic salmon npy transcription after 6-day fasting (43). The present study shows that LepRL is the only isoform that can mediate Lep signaling through Jak2 and Stat3 pathways in rainbow trout. The Jak2-Stat3 binding motifs in the LepRL are highly conserved among those fish species examined, including Atlantic salmon and carp, and it thus appears likely that the classic Jak2-Stat3 signaling pathways is common among fish for Lep signaling activation. The LepRT isoform binds Lep but is likely unable to activate Jak2, due to the absence of its associating motifs. It is therefore concluded that the LepRT mostly acts as a modulator of Lep signaling through competing ligand binding and decreasing the ligand availability to the LepRL. Postprandial changes in the LepRT mRNA levels in hypothalamus, concomitant with pomc-a1 transcription changes, suggest a possible involvement of the LepRT in rainbow trout appetite regulation.


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Acknowledgments We thank Dr Ingibjörg E. Einarsdottir and Marcus Johansson for collaboration on the feeding and fasting experiments. Address all correspondence and requests for reprints to: Ningping Gong, PhD, Department of Biological and Environmental Sciences, University of Gothenburg, 413 90 Gothenburg, Sweden. E-mail: [email protected]. This work was supported by the European Community’s Seventh Framework Programme (FP7/2007–2013) Grant 222719, by the project LIFECYCLE, and by Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) Grants 229 –2009-298 and 223–2011-1356. Disclosure Summary: The authors have nothing to disclose.

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Leptin sustains spontaneous remyelination in the adult central nervous system.

Facilitation of breathing by leptin effects in the central nervous system.

Fatty acid-specific alterations in leptin, PPARα, and CPT-1 gene expression in the rainbow trout.

Leptin, the sympathetic nervous system and blood pressure: the tale is still without an end.

Kinetics of leptin binding to the Q223R leptin receptor.

ApoJ enhances central leptin signaling through Lrp2-mediated endocytosis.

PTPRJ Inhibits Leptin Signaling, and Induction of PTPRJ in the Hypothalamus Is a Cause of the Development of Leptin Resistance.

Fatty Acid Profile Is Modulated by Dietary Resveratrol in Rainbow Trout (Oncorhynchus mykiss).

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