Progress 04/01/07 to 03/31/12
Outputs
OUTPUTS: Adiponectin is a 30 kilo-Dalton adipokine hormone affecting carbohydrate and lipid metabolism in humans and rodent animal models. Adiponectin is assembled into homomultimers in the adipose tissue and secreted as heavy (HMW), medium, and light molecular weight isoforms. We have cloned and characterized the genes that encode for chicken adiponectin and its receptors (AdipoR1 and AdipoR2). A polyclonal antisera against chicken adiponectin was generated and affinity purified. Using gel filtration column chromatography and Western blot analysis, adiponectin in chicken plasma and adipose tissue was found to be predominantly a multimeric HMW isoform that is larger than 669 kilo-Dalton mass. Under reducing conditions and heating, a majority of the multimeric adiponectin in chicken plasma and adipose tissue was reduced to oligomeric and/or monomeric forms. Immunoprecipitation and elution under neutral pH preserved the HMW adiponectin multimer, while brief exposure to acidic pH led to dissociation of HMW multimer into multiple oligomers. Mass spectrometric analysis of chicken adiponectin revealed the amino acid sequence of a part of the adiponectin collagenous domain that contained a characteristic 22 triplets of glycine-proline-Y repeats and hydroxylated and glycosylated proline and lysine residues. An enzyme immunoassay was developed and validated for quantifying plasma adiponectin in chickens. Plasma adiponectin levels were found to be significantly lower in 8-week-old compared to 4-week-old broiler male chickens and inversely related to abdominal fat pad mass. We found that mammalian adiponectin is not biologically active in chicken hepatocytes and therefore, we produced recombinant chicken adiponectin (rcADN) in Escherichia coli and in Chinese hamster ovarian (CHO) cells in a biochemical form similar to that produced by chicken adipose tissue. Escherichia coli derived rcADN was found to be a mixture of oligomeric forms with molecular weights ranging from 30-150 kilo-Dalton under non-reducing and non-denaturing gel electrophoresis and did not form 720 kilodalton- HMW isoform found in chickens. A stable CHO cell line was established that robustly secretes rcADN under serum-free conditions. Highly purified CHO cell-derived rcADN was found to be a single species of 720 kilo-Dalton HMW isoform. In vitro studies using hepatocytes revealed that CHO-derived rcADN increased the abundance of phosphorylated adenosine monophosphate (AMP) -activated protein kinase (AMPK), phosphorylated acetyl coenzyme A carboxylase (pACC) as well as increased glucose uptake. We found that both rcADN and metformin, anti-diabetic drug, increased the abundance of phospho-extracellular signal related kinases (pErk 1/2) in a dose-dependent manner. Thus, the inclusion of metformin in the diet of broilers or broiler breeder parent chickens may represent a simple and practical approach to favorably altering metabolism for better carcass quality. Further studies are required to determine the biological effects of chicken adiponectin and the factors that would enhance adiponectin secretion and signaling in chickens. PARTICIPANTS: Drs. Ramachandran and Vasilatos-Younken contributed to the design and execution of the project. A part of this project was conducted by research technicians (Ms. Jill Hadley and Dr. Hendricks III) in the laboratories of the PD and Co-PD. In addition, this research project trained graduate students (Ms. Olga Ocon-Grove, Ms. Susan Krzysik-Walker and Ms. Tiwari). TARGET AUDIENCES: Scientists in academic institutions and in commercial broiler chicken breeder farms. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our results indicate that adiponectin is likely to affect overall body metabolism and, therefore, nutrient utilization. Feed intake and energy metabolism largely determine the profitability of broiler chicken production and, therefore, any improvement in nutrient utilization will enhance meat productivity. Our research findings, while characterizing the effect of adipose tissue hormones on metabolism, may lead to devising methods for manipulating metabolic processes towards improving nutrient utilization and carcass composition in broiler chickens.
Publications
- Ramachandran, R., G. L. Hendricks, III, R. Vasilatos-Younken, and J. A. Hadley. 2012. Adiponectin signaling in chicken growth and reproduction. General and Comparative Endocrinology (Pending).
- Tiwari, A, S. M. Krzysik-Walker, and R. Ramachandran. 2012. Cloning and characterization of chicken fat mass and obesity associated (Fto) gene: Fasting affects FTO expression. Domestic Animal Endocrinology 42(1):1-10.
- Yuan, P, H. Zhang, R. Ramachandran, H. Miao, L. Yu, J. Song. 2012. Adiponectin and its receptors are involved in herpesvirus-related inbred chicken lines. PLoS ONE (Submitted).
- Yuan, P, Y. Yu, J. Luo, F. Tian, H, Zhang, S. Chang, R. Ramachandran, and J. Song. 2012. Comparative study of lipoprotein metabolism in Marek's disease susceptible and resistant chickens. Poultry Science (In Press).
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Progress 04/01/10 to 03/31/11
Outputs
OUTPUTS: Adiponectin, an adipokine hormone, exerts pleiotropic beneficial effects on carbohydrate and lipid metabolism in humans and rodent animal models. Our laboratories were the first to investigate the potential benefits of adiponectin in broiler chicken growth and nutrient utilization. We have earlier cloned the genes encoding chicken adiponectin and its receptors, AdipoR1 and AdipoR2. We found that mammalian adiponectin is not biologically active in chicken hepatocytes and therefore, we produced recombinant chicken adiponectin (rcADN) in Chinese hamster ovarian cells in a biochemical form similar to that produced by chicken adipose tissue. We earlier found that rcADN increased glucose uptake and increased the abundance of phospho-adenosine monophophophate-activated protein kinase (pAMPK) and phospho-acetyl coenzyme A carboxylase (pACC) in chicken liver hepatoma cell line (LMH). Recent reports suggest that glucose uptake in skeletal muscle cells is affected by extracellular signal related kinases (Erk 1/2). We tested the hypothesis that rcADN and metformin, a biguanide anti-diabetic drug that increases glucose uptake, would affect the abundance of phospho-Erk 1/2 (pErk 1/2) in LMH cells. Our results indicated that both rcADN and metformin increased the abundance of pErk 1/2 in LMH in a dose-dependent manner. Thus, the inclusion of metformin in the diet of broilers or broiler breeder parent chickens may represent a simple and practical approach to favorably altering metabolism for better carcass quality or for increased reproductive efficiency, respectively. APPL1, a pleckstrin homology domain containing protein, was identified as an adapter protein that is linked to activation of AdipoR1 and adiponectin-mediated increases in glucose uptake. We cloned the chicken gene that encodes for APPL1 and found that it was highly homologous to human APPL1. We found that both rcADN and metformin treatments resulted in dose-dependent decreases in the abundance of APPL1 protein in LMH cells suggesting increased turnover and utilization of APPL1 due to rcADN and metformin treatments. To further confirm association of rcADN signaling with APPL1, we performed several co-immunoprecipitation experiments using antibodies against APPL1, AdipoR1, or AdipoR2. The results indicate that rcADN treatment, in a dose-dependent manner, increased association of the adaptor protein APPL1 with AdipoR1 but not with AdipoR2 in LMH cells. These data suggest that the ubiquitously expressed adiponectin receptors (AdipoR1 and AdipoR2) could possibly use different signaling mechanisms to exert pleiotropic beneficial effects on carbohydrate and lipid metabolism. We are continuing to determine the biological effects of chicken adiponectin and the factors that would enhance adiponectin secretion and signaling. PARTICIPANTS: Drs. Ramachandran and Vasilatos-Younken contributed to the design and execution of the project. A part of this project was conducted by research technicians (Ms. Jill Hadley and Dr. Hendricks III) in the laboratories of the PD and Co-PD. In addition, this research project trains graduate students (Ms. Anupama Tiwari). TARGET AUDIENCES: Scientists in academic institutions and in commercial broiler breeder farms. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our results indicate that adiponectin is likely to affect overall body metabolism and, therefore, nutrient utilization. We have also identified metformin as a compound that mimics some of the beneficial effects of adiponectin signaling and therefore provides a practical solution for ameliorating excess lipid accretion in commercial broilers or broiler breeder parents. Feed intake and energy metabolism largely determine the profitability of broiler chicken production and, therefore, any improvement in nutrient utilization will enhance meat productivity. Our research findings, while characterizing the effect of adipose tissue hormones on metabolism, may lead to devising methods for manipulating metabolic processes towards improving nutrient utilization and carcass composition in broiler chickens.
Publications
- No publications reported this period
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Progress 04/01/09 to 03/31/10
Outputs
OUTPUTS: Adiponectin, a 30-kDa adipokine hormone, is known to affect carbohydrate and lipid metabolism in humans and rodent animal models. We have earlier cloned the genes encoding chicken adiponectin and its receptors, AdipoR1 and AdipoR2. Using custom-generated antibodies against chicken adiponectin, we reported that chicken adiponectin is a unique heavy molecular weight (HMW) isoform both in circulation and in adipose tissue. We also reported that plasma adiponectin levels are lower in 8-week-old compared to 4-week-old broiler chickens, a time-frame marked by rapid body growth and visceral adipose accretion. We produced recombinant chicken adiponectin (rcADN) using prokaryotic expression systems by cloning chicken adiponectin complementary deoxyribonucleic acid (cDNA) into bacterial expression vectors. Escherichia coli derived (rcADN) was found to be a mixture of oligomeric forms with molecular weights ranging from 30-150-kDa under non-reducing and non-denaturing gel electrophoresis. Bacterially derived rcADN did not form the 720-kDa HM isoform found in chickens. This observation highlights the importance of post-translational modifications in multimerization and stability of adiponectin in chickens. Furthermore, we found that bacterially expressed rcADN did not increase glucose uptake in hepatocytes, possibly indicating that multimerization and post-translational modifications as occurring in eukaryotic systems are necessary for biological functions as well. Consequently, we developed a eukaryotic expression system to produce rcADN by cloning chicken adiponectin cDNA into a mammalian expression plasmid vector. A stable eukaryotic cell line was established that robustly secretes rcADN under serum-free conditions. Chinese hamster ovarian cell (CHO)-derived rcADN was found to be a single species of 720-kDa HMW isoform. In vitro studies using hepatocytes revealed that CHO-derived rcADN increased the abundance of phosphorylated adenosine monophosphate (AMP)-activated protein kinase (AMPK) and phosphorylated acetyl coenzyme A carboxylase (pACC) and also increased glucose uptake. Phosphorylation of AMPK, a nutrient sensing molecule, is critical for energy homeostasis while phosphorylation of ACC would lead to decreased lipid synthesis. We are currently scaling up production of eukaryotically expressed rcADN for further biological studies. PARTICIPANTS: Drs. Ramachandran and Vasilatos-Younken contributed to the design and execution of the project. A part of this project was conducted by research technicians (Ms. Jill Hadley and Dr. Hendricks III) in the laboratories of the PD and Co-PD. In addition, this research project trains graduate students (Ms. Susan Krzysik-Walker and Ms. Anupama Tiwari). TARGET AUDIENCES: Scientists in academic institutions and the broiler and broiler breeder industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our results indicate that adiponectin is likely to affect overall body metabolism and, therefore, nutrient utilization. Feed intake and energy metabolism largely determine the profitability of broiler chicken production and, therefore, any improvement in nutrient utilization will enhance meat production. Our research findings, while characterizing the effect of adipose tissue hormones on metabolism, may lead to devising methods for manipulating metabolic processes towards improving nutrient utilization and carcass composition in broiler chickens.
Publications
- No publications reported this period
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Progress 04/01/08 to 03/31/09
Outputs
OUTPUTS: Adiponectin, a 30 kDa adipokine hormone, is known to affect carbohydrate and lipid metabolism in humans and rodent animal models. Adiponectin is assembled into homomultimers in the adipose tissue and secreted as heavy, medium, and light molecular weight isoforms. Plasma heavy molecular weight (HMW) adiponectin isoform levels are inversely correlated with the incidence of type 2 diabetes in humans. In order to characaterize adiponectin in broiler chickens, a polyclonal antisera against chicken adiponectin was generated in rabbits and affinity purified. Using gel filtration, column chromatography, and Western blot analysis under non-reducing and non-heat denaturing native conditions, adiponectin in chicken plasma and adipose tissue was found to be predominantly a multimeric HMW isoform that is larger than 669 kDa mass. Under reducing conditions and heating to 70-100 C, however, a majority of the multimeric adiponectin in chicken plasma and adipose tissue was reduced to oligomeric and/or monomeric forms. Immunoprecipitation and elution under neutral pH preserved the HMW adiponectin multimer, while brief exposure to acidic pH led to dissociation of HMW multimer into multiple oligomers. Mass spectrometric analysis of chicken adiponectin revealed the amino acid sequence of a part of the adiponectin collagenous domain that contained a characteristic 22 triplets of glycine-proline-Y repeats. There were twice as many lysine residues in the chicken adiponectin collagenous domain compared with human adiponectin that might be responsible, at least in part, for the unique multimerization of chicken adiponectin. Furthermore, proline and hydroxylysine residues in the collagenous domain were found to be hydroxylated and glycosylated, respectively. An enzyme immunoassay was developed and validated for quantifying plasma adiponectin in chickens. Plasma adiponectin levels were found to be significantly lower in 8-week-old compared to 4-week-old male chickens and inversely related to abdominal fat pad mass. Collectively, our results provide novel evidence that adiponectin in chicken plasma and tissues is predominantly a HMW multimer, suggesting the presence of unique multimerization and stabilization mechanisms in the chicken that favors preponderance of HMW adiponectin over other oligomers. Biological functions of adiponectin in the broiler chicken are currently explored. PARTICIPANTS: Drs. Ramachandran and Vasilatos-Younken contributed to the design and execution of the project. A part of this project was conducted by research technicians (Ms. Jill Hadley and Dr. Hendricks III) in the laboratories of the PD and Co-PD. In addition, this research project trains graduate students (Ms. Olga Ocon-Grove and Ms. Susan Krzysik-Walker). TARGET AUDIENCES: Scientists in academic institutions and in commercial broiler breeder farms. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our results indicate that adiponectin is likely to affect overall body metabolism and, therefore, nutrient utilization. Feed intake and energy metabolism largely determine the profitability of broiler chicken production and, therefore, any improvement in nutrient utilization will enhance meat productivity. Our research findings, while characterizing the effect of adipose tissue hormones on metabolism, may lead to devising methods for manipulating metabolic processes towards improving nutrient utilization and carcass composition in broiler chickens.
Publications
- Ocon-Grove, O.M, S.M. Krzysik-Walker, S.R. Maddineni, G.L. Hendricks III, and R. Ramachandran. 2008. Adiponectin and Its receptors are expressed in the chicken testis: Influence of sexual maturation on testicular AdipoR1 and AdipoR2 mRNA abundance. Reproduction 136:627-638.
- Hendricks III, G.L., J.A. Hadley, S. M. Krzysik-Walker, S.K. Prabhu, R. Vasilatos-Younken, and R. Ramachandran. 2009. Unique profile of chicken adiponectin, a predominantly heavy molecular weight multimer, and relationship to visceral adiposity. Endocrinology (In Print) doi:10.1210/en.2008-1558
- Ocon-Grove, O.M., S.M. Krzysik-Walker, S.R. Maddineni, G. L. Hendricks III and R. Ramachandran. 2009. Changes in Cellular Localization of Nampt/PBEF/Visfatin in the Chicken Testis: Influence of Sexual Maturation. Reproduction (Pending).
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Progress 04/01/07 to 03/31/08
Outputs
OUTPUTS: Adiponectin is an adipocytokine hormone that is known to affect carbohydrate and lipid metabolism in mammals. We cloned and characterized the complementary deoxyribonucleic acids encoding adiponectin and its adiponectin receptors (AdipoR1 and AdipoR2). By reverse transcription-polymerase chain reaction (PCR), we found that adiponectin, AdipoR1, and AdipoR2 mRNAs are ubiquitously expressed in various tissues. Tissue adiponectin mRNA expression was quantified using real-time quantitative PCR and found to be highest in adipose tissue, followed by liver, anterior pituitary, diencephalon, kidney, and skeletal muscle. We also found that after a 48-hour period of feed deprivation, adiponectin mRNA quantity was significantly decreased in adipose tissue, liver, and anterior pituitary gland, but not in diencephalon. Our results provide novel evidence that, unlike mammals, the adiponectin gene is expressed in several tissues in the chicken and its expression is influenced by feed deprivation. In contrast to adiponectin gene expression, AdipoR1 mRNA expression was the highest in skeletal muscle, adipose tissue and diencephalon. AdipoR2 mRNA expression was the highest in adipose tissue, followed by skeletal muscle, liver, ovary, diencephalon, anterior pituitary gland, kidney, and spleen. We also found that a 48-hour period of feed deprivation significantly decreased AdipoR1 mRNA quantity in the chicken pituitary gland, while AdipoR2 mRNA quantity was significantly increased in adipose tissue (P<0.05). We have generated antisera against chicken adiponectin, AdipoR1, and AdipoR2 and are currently investigating the role of adiponectin in carbohydrate and lipid metabolism in broiler chickens. Using affinity-purified anti-chicken adiponectin antibody, we have developed an enzyme-linked immunosorbent assay (ELISA) for quantifying adiponectin and found that circulating adiponectin levels in broiler chickens range between 4 and 6 mcg/ml. Using non-reducing gel electrophoresis, we found that the circulating form of adiponectin, as well as the form associated with adipose tissue in the chicken is a 720 kDa multimer, possibly comprised of 26 monomeric adiponectin units. We are currently generating this multimeric adiponectin using a eukaryotic plasmid expression vector in Chinese hamster ovary (CHO) cells, in order to determine the effect of recombinant chicken adiponectin on carbohydrate and lipid metabolism. PARTICIPANTS: Drs. Ramachandran and Vasilatos-Younken contributed to the design and execution of the project. A part of this project was conducted by research technicians (Ms. Jill Hadley and Dr. Hendricks III) in the laboratories of the PD and Co-PD. In addition, this research project trains graduate students (Ms. Olga Ocon-Grove and Ms. Susan Krzysik-Walker) and undergraduate student (Mr. Pradeep Selvan). TARGET AUDIENCES: Scientists in academic research laboratories and in commercial broiler breeder farms. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our results indicate that adiponectin is likely to affect overall body metabolism and therefore, can affect nutrient utilization. Feed intake and energy metabolism largely determine the profitability of broiler chicken production and, therefore, any improvement in nutrient utilization will enhance meat productivity. Our research findings, while characterizing the effect of adipose tissue hormones on metabolism, will lead to devising methods for manipulating metabolic processes towards improving nutrient utilization and carcass composition in broiler chickens.
Publications
- Ramachandran, R., O.M. Ocon-Grove, and S. L. Metzger. 2007. Molecular cloning and tissue expression of chicken AdipoR1 and AdipoR2 complementary deoxyribonucleic acids. Domestic Animal Endocrinology 33:19-31.
- Selvan, P. K., G. L. Hendricks III, S. R. Maddineni, S. M. Krzysik-Walker, O. M. Ocon-Grove, and R. Ramachandran. 2007. Cloning and characterization of chicken nucleobindin-2 (NUCB2) cDNA: The precursor for a putative anorexigenic peptide, Nesfatin-1. Poultry Science, 86 (Suppl.1):318.
- Ocon-Grove, O. M., S. M. Krzysik-Walker, S. R. Maddineni, G. L. Hendricks III, and R. Ramachandran. 2007. Expression of Adiponectin and Its Receptors, AdipoR1 and AdipoR2, in the Chicken Testis. Proceedings of 40th Annual Meeting of Society for Study of Reproduction, San Antonio, TX. Abstract #380, page 181.
- Maddineni, S., S. Metzger, O. Ocon, G. L. Hendricks 3rd, and R. Ramachandran. 2005. Adiponectin gene is expressed in multiple tissues in the chicken: food deprivation influences adiponectin messenger ribonucleic acid expression. Endocrinology, 146:4250-4256.
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