Avian blood induced intranuclear translocation of STAT3 via the chicken leptin receptor

doi:10.1016/j.cbpb.2014.05.001

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology

Volume 174, August 2014, Pages 9-14

https://doi.org/10.1016/j.cbpb.2014.05.001 Get rights and content

Abstract

Leptin is a multi-functional adipokine in vertebrates. The leptin gene and protein are found in many vertebrates; however, the existence of leptin in birds remains controversial. Here we detected leptin-like activity in avian blood using chicken leptin receptor (chLEPR) and green fluorescent protein (GFP)-fused chicken signal transducer and activator of transcription (chSTAT3) co-expressed in CHO-K1 cells (CHO-chLEPR/STAT3). We validated that rat serum specifically induces intranuclear migration of GFP-fused chSTAT3 (GFP-chSTAT3) in CHO-chLEPR/STAT3 cells, but not in CHO-K1 cells expressing GFP-STAT3 (CHO-chSTAT3) before testing the avian blood samples. Blood of chickens (Gallus gallus), wild jungle crows (Corvus macrorhynchos), and carrion crows (Corvus corone) accumulated the GFP signal into nuclei, and frequency varied in each blood sample. Western blotting showed that chicken and crow blood samples specifically phosphorylated GFP-chSTAT3 in the chLEPR-transfected cells. These results indicate that avian blood contains a leptin-like molecule that specifically binds to LEPR, suggesting that the leptin system is conserved across all vertebrate classes.

Introduction

Leptin is primarily secreted by adipocytes and has multiple physiological actions including energy homeostasis and food intake in mammals (Zhang et al., 1994, Campfield et al., 1995, Halaas et al., 1995, Pelleymounter et al., 1995, Kuo et al., 2005, Huising et al., 2006, Paolucci et al., 2006). Leptin and its receptor have been cloned in mammals and non-mammalian vertebrates, indicating that the leptin system may be conserved in vertebrates. Chicken leptin receptor (chLEPR) cDNA has been cloned by us and other groups (Horev et al., 2000, Ohkubo et al., 2000) as the first LEPR cloned from a non-mammalian vertebrate. In addition, the chLEPR gene has been mapped to chromosome 8 of the chicken genome (Dunn et al., 2000). Thereafter, LEPR cDNA was identified in other avian species (Richards and Poch, 2003, Liu et al., 2007, Wang et al., 2011). The LEPR protein is expressed in the chicken brain and in a chicken hepatoma cell line (Ohkubo et al., 2007). Furthermore, chLEPR activates the Janus kinase (JAK)-signal transducers and activator of transcription (STAT) signaling pathway in vitro (Adachi et al., 2008, Hen et al., 2008). Evidence indicates that the physiological actions displayed by injecting mammalian leptin such as food intake, immune response, and ovarian function (Ohkubo and Adachi, 2008, Ohkubo, 2014, review) are likely mediated through LEPR expressed in avian tissues. Although the LEPR gene and protein have been found and are functional in birds, the existence of a chicken leptin gene remains controversial (Friedman-Einat et al., 1999, Amills et al., 2003, Richards and Proszkowiec-Weglarz, 2007).

Therefore, a bioassay using chLEPR-expressing mammalian cells and the luciferase gene was established in separate groups to detect leptin-like activity in avian-derived samples (Adachi et al., 2008, Hen et al., 2008), but leptin-like activity was not detected in avian blood (Adachi et al., 2008, Hen et al., 2008, Yosefi et al., 2010). Because the leptin-like activity was considered to be below the detection threshold of the luciferase gene bioassay, we recently developed a qualitative leptin bioassay by monitoring the migration of activated green fluorescent protein (GFP)-fused chicken STAT3 (GFP-chSTAT3) into the nucleus, which made it possible to detect physiological doses of mammalian leptin (Adachi et al., 2012). However, we did not test whether the newly established bioassay could detect leptin-like activity in avian blood. Accordingly, in the present study, we monitored intranuclear transition of GFP-chSTAT3 after treatment with chicken serum, wild jungle crow (Corvus macrorhynchos) plasma, and carrion crow (Corvus corone) plasma using a qualitative bioassay. In addition, GFP-chSTAT3 phosphorylation was stimulated using avian blood samples and determined by Western blot analysis.

Section snippets

Blood samples

Blood samples were collected from the wing vein of juvenile chickens (Gallus gallus) (12–16 weeks old, both sexes) and laying White Leghorn hens. Rat (adult male Wistar; Rattus norvegicus) blood was taken from the abdominal aorta under light anesthesia. Sera from the chickens and rat were obtained by centrifugation and stored at − 20 °C. Carrion crows (C. corone) (450–630 g, three males and five females) and jungle crows (C. macrorhynchos) (600–770 g, three females and five males) were caught in

Assessment of qualitative bioassay validity using rat serum

Translocation of the GFP signal into the nuclei was observed 5 min after stimulation of rat serum, and this increased by 10 min in CHO-chLEPR/STAT3 cells. However, rat serum did not induce nuclear accumulation of the GFP signal in CHO-chSTAT3 cells (Fig. 1).

Furthermore, we found in a previous study that recombinant mouse leptin specifically induces transition of the GFP signal in CHO-chLEPR/STAT3 but not CHO-chSTAT3 cells (Adachi et al., 2012).

Analysis of leptin activity in avian blood

Because we confirmed that nuclear condensation of

Discussion

A homolog of mammalian LEPR has been identified in several species of birds (Horev et al., 2000, Ohkubo et al., 2000, Richards and Poch, 2003, Liu et al., 2007, Wang et al., 2011); however, the existence of avian leptin has not been identified since a long time (Friedman-Einat et al., 1999, Pitel et al., 2000, Amills et al., 2003, Richards and Proszkowiec-Weglarz, 2007, Pitel et al., 2010). Earlier approaches to identify avian leptin based on comparative genomics revealed no sequences in the

Acknowledgments

The present study was supported, in part, by a Grant-in-Aid for Scientific Research (C) to TO (No. 24580386) and a Grant-in-Aid for JSPS Fellows to HA (No. 09J08801) from the Japan Society for the Promotion of Science.

References (37)

H. Adachi et al.
Chicken leptin receptor is functional in activating JAK–STAT pathway in vitro
J. Endocrinol.
(2008)
H. Adachi et al.
Detection of leptin activity in living cells expressing chicken leptin receptor and STAT3
J. Poult. Sci.
(2012)
H. Adachi et al.
Inhibitory mechanism of signal transduction through chicken leptin receptor by suppressor of cytokine signaling 3 (SOCS3)
J. Poult. Sci.
(2013)
M. Amills et al.
Identification of three single nucleotide polymorphisms in the chicken insulin-like growth factor 1 and 2 genes and their associations with growth and feeding traits
Poult. Sci.
(2003)
A.S. Banks et al.
Activation of downstream signals by the long form of the leptin receptor
J. Biol. Chem.
(2000)
C.P. Briscoe et al.
Leptin receptor long-form signalling in a human liver cell line
Cytokine
(2001)
L.A. Campfield et al.
Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks
Science
(1995)
P. Cano et al.
Effect of aging on 24-hour pattern of stress hormones and leptin in rats
Life Sci.
(2008)
I.C. Dunn et al.
Mapping of the leptin receptor gene (LEPR) to chicken chromosome 8
Anim. Genet.
(2000)
M. Friedman-Einat et al.
The chicken leptin gene: has it been cloned?
Gen. Comp. Endocrinol.
(1999)

M. Friedman-Einat et al.

Serum leptin activity in obese and lean patients

Regul. Pept.

(2003)

J.L. Halaas et al.

Weight-reducing effects of the plasma protein encoded by the obese gene

Science

(1995)

G. Hen et al.

Monitoring leptin activity using the chicken leptin receptor

J. Endocrinol.

(2008)

G. Horev et al.

Molecular cloning and properties of the chicken leptin-receptor (CLEPR) gene

Mol. Cell. Endocrinol.

(2000)

M.O. Huising et al.

Increased leptin expression in common Carp (Cyprinus carpio) after food intake but not after fasting or feeding to satiation

Endocrinology

(2006)

S.K. Ju et al.

Determination of rat leptin activity in vitro using a novel luciferase reporter assay

Mol. Cells

(2001)

A.K. Kretzschmar et al.

Analysis of Stat3 (signal transducer and activator of transcription 3) dimerization by fluorescence resonance energy transfer in living cells

Biochem. J.

(2004)

A.Y. Kuo et al.

Leptin effects on food and water intake in lines of chickens selected for high or low body weight

Physiol. Behav.

(2005)

Cited by (10)

Delayed access to feed alters gene expression associated with hormonal signaling, cellular differentiation, and protein metabolism in muscle of newly hatch chicks
2020, General and Comparative Endocrinology
Citation Excerpt :
Based on its expression pattern and that of the LEPR, as well as an inability to detect circulating leptin activity from chicken or quail serum, it has been suggested that avian leptin may act in an autocrine or paracrine manner in the central nervous system (i.e. brain, hypothalamus, and pituitary) rather than an endocrine manner (Seroussi et al., 2016). However, serum from these species was only tested during late juvenile development; furthermore, another study did find that plasma collected from chickens of a similar age (3–4 months) was able to activate LEPR in their bioassay (Ohkubo et al., 2014). Therefore, while the presence of leptin in avian species does appear indisputable, the role it plays and physiological processes it may be involved in throughout development have not been fully characterized and appear to differ from physiological roles in mammals.
Birds rely solely on utilization of the yolk sac as a means of nutritional support throughout embryogenesis and early post-hatch, before first feeding occurs. Newly hatched broiler (meat-type) chickens are frequently not given immediate access to feed, and this can result in numerous alterations to developmental processes, including those that occur in muscle. The objective of this study was to characterize the gene expression profile of newly hatched chicks’ breast muscle with regards to hormonal regulation of growth and metabolism and development and differentiation of muscle tissue, and determine impacts of delayed access to feed on these profiles. Within 3 h of hatch, birds were placed in battery pens and given immediate access to feed (Fed) or delayed access to feed for 48 h (Delayed Fed). Breast muscle collected from male birds at hatch, or 4 h, 1 day (D), 2D, 4D, and 8D after hatch was used for analysis of mRNA expression by reverse transcription-quantitative PCR. Under fully fed conditions, insulin-like growth factor receptor and leptin receptor mRNA expression decreased as birds aged; however, delayed access to feed resulted in prolonged upregulation of these genes so their mRNA levels were higher in Delayed Fed birds at 2D. These expression profiles suggest that delayed feed access alters sensitivity to hormones that may regulate muscle development. Myogenin, a muscle differentiation factor, showed increasing mRNA expression in Fed birds through 2D, after which expression decreased. A similar expression pattern in Delayed Fed birds was deferred until 4D. Levels of myostatin, a negative regulator of muscle growth, increased in Fed birds starting at 2D, while levels in Delayed Fed birds began to increase at 4D. In Fed birds, levels of transcripts for two genes associated with protein catabolism, F-box protein 32 and forkhead box O3, were lower at 2D, while Delayed Fed mRNA levels did not decrease until 4D. Mechanistic target of rapamycin mRNA levels decreased from 1D through 8D in both treatments, except for a transient increase in the Delayed Fed birds between 1D and 2D. These data suggest that within breast muscle, delayed feeding alters hormonal signaling, interrupts tissue differentiation, postpones onset of growth, and may lead to increased protein catabolism. Together, these processes could ultimately contribute to a reduction in proper growth and development of birds not given feed immediately after hatch, and ultimately hinder the long-term potential of muscle accretion in meat type birds.
Avian Leptin: Bird's-Eye View of the Evolution of Vertebrate Energy-Balance Control
2019, Trends in Endocrinology and Metabolism
Citation Excerpt :
These doves also had high liver leptin expression (Box 2), which may be the source of the rare indication of circulating leptin. Other reports showing leptin activity in blood samples of birds [34,57–59] used nonverified assay systems, and therefore await further verification (Box 1). In summary, it appears that avian leptin expression is operating at low levels in an autocrine/paracrine fashion, and is usually undetectable in circulation.
Discovery of the satiety hormone leptin in 1994 and its characterization in mammals provided a key tool to deciphering the complex mechanism governing adipose tissue regulation of appetite and energy expenditure. Surprisingly, despite the perfectly logical notion of an energy-storing tissue announcing the amount of fat stores using leptin signaling, alternate mechanisms were chosen in bird evolution. This conclusion emerged based on the recent discovery and characterization of genuine avian leptin – after it had been assumed missing by some, and erroneously identified by others. Critical evaluation of the past and present indications of the role of leptin in Aves provides a new perspective on the evolution of energy-balance control in vertebrates; proposing a regulation strategy alternative to the adipostat mechanism.
Anti-leptin receptor antibodies strengthen leptin biofunction in growing chickens
2018, General and Comparative Endocrinology
Citation Excerpt :
Therefore, it was hypothesized that there was no leptin or leptin binding capacity in chicken blood. However, other studies had reported that the leptin-like substances are found in chicken and crow blood and are capable of phosphorylating GFP-chSTAT3 in the cLEPR- transfected cells (Ohkubo et al., 2014). Indeed, the present study confirmed the presence of leptin or leptin-like molecules in the chicken blood, which was supported by the results of active immunization against the cLEPR ECD in hens and growing chickens (Lei et al., 2014, 2015).
Antibodies against the extracellular domains of the chicken leptin receptor were used to study the biological function of leptin in growing chickens. Both polyclonal and monoclonal anti-LEPR antibodies were administered intramuscularly to 30-d-old Chinese indigenous Gushi pullets. Both antibody preparations increased feed intake for 6 h after injection and reduced plasma concentrations of glucose, triglycerides, and both high- and low-density lipoproteins. The antibody treatments also upregulated agouti-related peptide and neuropeptide Y in the hypothalamus and downregulated proopiomelanocortin, melanocortin 4 receptor, and leptin receptor. The treatments also upregulated leptin receptor, acetyl CoA carboxylase beta, and acyl-CoA oxidase in the liver, abdominal fat, and breast muscle and downregulated sterol regulatory element-binding protein-1 and fatty acid synthase. Furthermore, even though the anti-leptin receptor antibodies failed to affect leptin receptor signaling transduction when administered alone, they did augment the induction of leptin receptor signaling transduction by leptin. These results demonstrate that antibodies against the extracellular domains of leptin-specific receptor enhance, but do not mimic, the ability of leptin to activate receptors. Furthermore, the enhanced leptin bioactivity observed after the intramuscular injection of anti-LEPR antibodies confirmed the occurrence of de novo leptin in the peripheral tissues and blood of treated chickens.
Leptin activates chicken growth hormone promoter without chicken STAT3 in vitro
2016, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology
Citation Excerpt :
Recently, we established CHO cell stably expressing chicken LEPR (chLEPR) and in which mouse leptin dose dependently activated leptin responsive reporter gene (Adachi et al., 2008). Moreover, intranuclear migration of chicken STAT3 (chSTAT3) was observed, when CHO cell expressing chLEPR and green fluorescent protein (GFP)-fused chSTAT3 was stimulated by mouse leptin, but such nuclear translocation of chSTAT3 was not detected by GH and prolactin treatment (Ohkubo et al., 2014). The results indicate that the chLEPR expressing cell is a valuable tool to analyze leptin signaling through chLEPR.
Leptin is an adipocyte-derived hormone that not only regulates food intake and energy homeostasis but also induces growth hormone (GH) mRNA expression and release, thereby controlling growth and metabolism in mammals. The molecular mechanism of leptin-induced regulation of GH gene transcription is unclear. The current study investigated the effects of leptin on the chicken GH (cGH) promoter and the molecular mechanism underlying leptin-induced cGH gene expression in vitro. Leptin activated the cGH promoter in the presence of chPit-1α in CHO cells stably expressing the chicken leptin receptor. Promoter activation did not require STAT-binding elements in the cGH promoter or STAT3 activity. However, JAK2 activation was required for leptin-dependent activity. JAK2-dependent pathways include p42/44 MAPK and PI3K, and inhibition of these pathways partially blocked leptin-induced cGH gene transcription. Although CK2 directly activates JAK2, a CK2 inhibitor blocked leptin-dependent activation of the cGH gene without affecting JAK2 phosphorylation. The CK2 inhibitor suppressed Erk1/2 and Akt phosphorylation. Additional data implicate Src family kinases in leptin-dependent cGH gene activation. These results suggest that leptin activates the cGH gene in the presence of chPit-1α via several leptin-activated kinases. Although further study is required, we suggest that the leptin-induced JAK2/p42/44 MAPK and JAK2/PI3K cascades are activated by Src-meditated CK2, leading to CBP phosphorylation and interaction with chPit-1α, resulting in transactivation of the cGH promoter.
Regulation of the avian central melanocortin system and the role of leptin
2015, General and Comparative Endocrinology
The avian central melanocortin system is well conserved between birds and mammals in terms of the component genes, the localisation of their expression in the hypothalamic arcuate nucleus, the effects on feeding behaviour of their encoded peptides and the sensitivity of agouti-related protein (AGRP) and pro-opiomelanocortin (POMC) gene expression to changes in energy status. Our recent research has demonstrated that AGRP gene expression precisely differentiates between broiler breeder hens with different histories of chronic food restriction and refeeding. We have also shown that the sensitivity of AGRP gene expression to loss of energy stores is maintained even when food intake has been voluntarily reduced in chickens during incubation and in response to a stressor. However, the similarity between birds and mammals does not appear to extend to the way AGRP and POMC gene expression are regulated. In particular, the preliminary evidence from the discovery of the first avian leptin (LEP) genes suggests that LEP is more pleiotropic in birds and may not even be involved in regulating energy balance. Similarly, ghrelin exerts inhibitory, rather than stimulatory, effects on food intake. The fact that the importance of these prominent long-term regulators of AGRP and POMC expression in mammals appears diminished in birds suggests that the balance of regulatory inputs in birds may have shifted to more short-term influences such as the tone of cholecystokinin (CCK) signalling. This is likely to be related to the different metabolic fuelling required to support flight.
Creating leptin-like biofunctions by active immunization against chicken leptin receptor in growing chickens
2015, Domestic Animal Endocrinology
Citation Excerpt :
The chicken genomic database also lacks the sequence reported by Taouis et al (1998) [20] in the chicken genome [23]. But leptin-like molecule was recently suggested to exist in avian blood [24]. Furthermore, recent findings revealed the existence of a leptin gene in other avian species such as the Peregrine falcon [25], and a partial gene fragment of leptin-like protein in dove [26] and zebra finch [27].
In this study, immunization against chicken leptin receptor (cLEPR) extracellular domain (ECD) was applied to investigate leptin regulation and LEPR biofunction in growing chicken pullets. A recombinant protein (cLEPR ECD) based on the cLEPR complemenary DNA sequence corresponding to the 582nd to 796th amino acid residues of cLEPR mature peptide was prepared and used as antigen. Immunization against cLEPR ECD in growing chickens increased anti-cLEPR ECD antibody titers in blood, enhanced proportions of phosphorylated janus kinase 2 (JAK2) and served as signal transducer and activator of transcription 3 (STAT3) protein in liver tissue. Chicken live weight gain and abdominal fat mass were significantly decreased (P < 0.05), but feed intake was stimulated by cLEPR ECD immunization (P < 0.05). The treatment also upregulated the gene expression levels of lepR, AMP-activated protein kinase (AMPK), acetyl CoA carboxylase-2 (ACC2), and uncoupling protein 3 (UCP3) in liver, abdominal fat, and breast muscle (P < 0.05) but decreased fasn expression levels (P < 0.01). Apart from that of lepR, the expression of appetite-regulating genes, such as orexigenic genes, agouti-related peptide (AgRP) and neuropeptide Y (NPY), were upregulated (P < 0.01), whereas the anorexigenic gene proopiomelanocortin (POMC) was downregulated in the hypothalamic tissue of cLEPR-immunized pullets (P < 0.01). Blood concentrations of metabolic molecules, such as glucose, triglycerides, and very-low-density lipoprotein, were significantly decreased in cLEPR-immunized pullets but those of cholesterol, high-density lipoprotein, and low-density lipoprotein increased. These results demonstrate that antibodies to membrane proximal cLEPR ECD enhance cLEPR signal transduction, which stimulates metabolism and reduces fat deposition in chickens.

View all citing articles on Scopus

View full text

Avian blood induced intranuclear translocation of STAT3 via the chicken leptin receptor

Abstract

Introduction

Section snippets

Blood samples

Assessment of qualitative bioassay validity using rat serum

Analysis of leptin activity in avian blood

Discussion

Acknowledgments

Chicken leptin receptor is functional in activating JAK–STAT pathway in vitro

J. Endocrinol.

Detection of leptin activity in living cells expressing chicken leptin receptor and STAT3

J. Poult. Sci.

Inhibitory mechanism of signal transduction through chicken leptin receptor by suppressor of cytokine signaling 3 (SOCS3)

J. Poult. Sci.

Identification of three single nucleotide polymorphisms in the chicken insulin-like growth factor 1 and 2 genes and their associations with growth and feeding traits

Poult. Sci.

Activation of downstream signals by the long form of the leptin receptor

J. Biol. Chem.

Leptin receptor long-form signalling in a human liver cell line

Cytokine

Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks

Science

Effect of aging on 24-hour pattern of stress hormones and leptin in rats

Life Sci.

Mapping of the leptin receptor gene (LEPR) to chicken chromosome 8

Anim. Genet.

The chicken leptin gene: has it been cloned?

Gen. Comp. Endocrinol.

Serum leptin activity in obese and lean patients

Regul. Pept.

Weight-reducing effects of the plasma protein encoded by the obese gene

Science

Monitoring leptin activity using the chicken leptin receptor

J. Endocrinol.

Molecular cloning and properties of the chicken leptin-receptor (CLEPR) gene

Mol. Cell. Endocrinol.

Increased leptin expression in common Carp (Cyprinus carpio) after food intake but not after fasting or feeding to satiation

Endocrinology

Determination of rat leptin activity in vitro using a novel luciferase reporter assay

Mol. Cells

Analysis of Stat3 (signal transducer and activator of transcription 3) dimerization by fluorescence resonance energy transfer in living cells

Biochem. J.

Leptin effects on food and water intake in lines of chickens selected for high or low body weight

Physiol. Behav.