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postgraduate thesis: Functional studies and expression regulation of two leptin isoforms in grass carp

TitleFunctional studies and expression regulation of two leptin isoforms in grass carp
Authors
Issue Date2012
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Chen, T. [陈廷]. (2012). Functional studies and expression regulation of two leptin isoforms in grass carp. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5016245
AbstractLeptin, the protein product of obese gene, is a 16-kD adipokine with regulatory functions on food intake and energy metabolism. At present, limited information is available on leptin functions and regulation in lower vertebrates mainly due to the fact that the primary structure of leptin is highly diversified from fish to mammals. Leptin in teleost fish is even more complicated as leptin isoforms have been reported presumably as a result of whole-genome duplication that occurred during the evolution of modern-day bony fish. Using grass carp (Ctenopharyngodon idella) as a model, I sought to investigate the physiological functions and endocrine regulation of leptin in bony fishes. As a first step, the structural identities of two leptins, namely leptin A and B, were established by 5’/3’RACE. The two isoforms share low levels of amino acid sequence homology with mammalian leptins but their deduced 3D-protein models are highly comparable to that of the human counterpart. In grass carp, leptin A and B are widely expressed with highest levels of expression detected in the liver with leptin A as the dominant form. To study the biological actions of grass carp leptins, recombinant proteins of leptin A and B were produced in Escherichia coli and found to inhibit both basal and NPY-stimulated food consumption and feeding behavior in goldfish by both intraperitoneal and intracerebroventricular injection. In addition to the anorexic effects observed, the effects of leptin on pituitary hormone secretion and synthesis were also examined in primary culture of carp pituitary cells. Using reverse transcription-polymerase chain reaction coupled to laser captured microdissection, leptin receptor expression was detected in somatotrophs, gonadotrophs and lactotrophs. Furthermore, leptin A and B were both effective in increasing basal secretion, cell content and transcript expression of growth hormone, luteinizing hormone and prolactin in carp pituitary cells. In the same study, parallel rises in somatolactin α and β mRNA levels without major changes in transcript expression of other pituitary hormones were also noted. These stimulatory effects were mediated by differential coupling with Janus kinase-2 (JAK2)/ signal transducers and activators of transcription (STATs), mitogen-activated protein kinase (MAPK) and/or phosphoinositide 3-kinase (PI3K)/Akt pathways. Although leptin A and B exhibited similar effects on feeding and pituitary hormone expression, their endocrine regulation appears to be quite different. In primary culture of carp hepatocytes, insulin could reduce leptin A but not leptin B mRNA levels through MAPK but not PI3K/Akt pathway. Glucagon, in contrast, could trigger leptin A but not leptin B mRNA expression via the cAMP/PKA cascades and this stimulatory effect could be negated by co-treatment with insulin. At the hepatic level, SLα could also induce leptin A but not leptin B mRNA expression via JAK2 activation of PI3K/Akt cascades. Parallel treatment with SLβ, however, was found to up-regulate leptin B but not leptin A transcription by MAPK coupling to JAK2. These results suggest that the two leptin isoforms identified in grass carp are responsible for similar biological functions but under differential regulation by various endocrine factors coupled to a number of post-receptor signaling mechanisms.
DegreeDoctor of Philosophy
SubjectCarp - Molecular genetics
Leptin
Dept/ProgramBiological Sciences
Persistent Identifierhttp://hdl.handle.net/10722/196085

 

DC FieldValueLanguage
dc.contributor.authorChen, Ting-
dc.contributor.author陈廷-
dc.date.accessioned2014-03-28T07:05:48Z-
dc.date.available2014-03-28T07:05:48Z-
dc.date.issued2012-
dc.identifier.citationChen, T. [陈廷]. (2012). Functional studies and expression regulation of two leptin isoforms in grass carp. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5016245-
dc.identifier.urihttp://hdl.handle.net/10722/196085-
dc.description.abstractLeptin, the protein product of obese gene, is a 16-kD adipokine with regulatory functions on food intake and energy metabolism. At present, limited information is available on leptin functions and regulation in lower vertebrates mainly due to the fact that the primary structure of leptin is highly diversified from fish to mammals. Leptin in teleost fish is even more complicated as leptin isoforms have been reported presumably as a result of whole-genome duplication that occurred during the evolution of modern-day bony fish. Using grass carp (Ctenopharyngodon idella) as a model, I sought to investigate the physiological functions and endocrine regulation of leptin in bony fishes. As a first step, the structural identities of two leptins, namely leptin A and B, were established by 5’/3’RACE. The two isoforms share low levels of amino acid sequence homology with mammalian leptins but their deduced 3D-protein models are highly comparable to that of the human counterpart. In grass carp, leptin A and B are widely expressed with highest levels of expression detected in the liver with leptin A as the dominant form. To study the biological actions of grass carp leptins, recombinant proteins of leptin A and B were produced in Escherichia coli and found to inhibit both basal and NPY-stimulated food consumption and feeding behavior in goldfish by both intraperitoneal and intracerebroventricular injection. In addition to the anorexic effects observed, the effects of leptin on pituitary hormone secretion and synthesis were also examined in primary culture of carp pituitary cells. Using reverse transcription-polymerase chain reaction coupled to laser captured microdissection, leptin receptor expression was detected in somatotrophs, gonadotrophs and lactotrophs. Furthermore, leptin A and B were both effective in increasing basal secretion, cell content and transcript expression of growth hormone, luteinizing hormone and prolactin in carp pituitary cells. In the same study, parallel rises in somatolactin α and β mRNA levels without major changes in transcript expression of other pituitary hormones were also noted. These stimulatory effects were mediated by differential coupling with Janus kinase-2 (JAK2)/ signal transducers and activators of transcription (STATs), mitogen-activated protein kinase (MAPK) and/or phosphoinositide 3-kinase (PI3K)/Akt pathways. Although leptin A and B exhibited similar effects on feeding and pituitary hormone expression, their endocrine regulation appears to be quite different. In primary culture of carp hepatocytes, insulin could reduce leptin A but not leptin B mRNA levels through MAPK but not PI3K/Akt pathway. Glucagon, in contrast, could trigger leptin A but not leptin B mRNA expression via the cAMP/PKA cascades and this stimulatory effect could be negated by co-treatment with insulin. At the hepatic level, SLα could also induce leptin A but not leptin B mRNA expression via JAK2 activation of PI3K/Akt cascades. Parallel treatment with SLβ, however, was found to up-regulate leptin B but not leptin A transcription by MAPK coupling to JAK2. These results suggest that the two leptin isoforms identified in grass carp are responsible for similar biological functions but under differential regulation by various endocrine factors coupled to a number of post-receptor signaling mechanisms.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.lcshCarp - Molecular genetics-
dc.subject.lcshLeptin-
dc.titleFunctional studies and expression regulation of two leptin isoforms in grass carp-
dc.typePG_Thesis-
dc.identifier.hkulb5016245-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiological Sciences-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b5016245-
dc.date.hkucongregation2013-

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