Jill A. Hadley

Unique Profile of Chicken Adiponectin, a Predominantly Heavy Molecular Weight Multimer, and Relationship to Visceral Adiposity

Adiponectin, a 30-kDa adipokine hormone, circulates as heavy, medium, and light molecular weight isoforms in mammals. Plasma heavy molecular weight (HMW) adiponectin isoform levels are inversely correlated with the incidence of type 2 diabetes in humans. The objectives of the present study were to characterize adiponectin protein and quantify plasma adiponectin levels in chickens, which are naturally hyperglycemic relative to mammals. Using gel filtration column chromatography and Western blot analysis under nonreducing and non-heat-denaturing native conditions, adiponectin in chicken plasma, and adipose tissue is 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, whereas brief exposure to acidic pH led to dissociation of HMW multimer into multiple oligomers. Mass spectrometric analysis of chicken adiponectin revealed the presence of hydroxyproline and differential glycosylation of hydroxylysine residues in the collagenous domain. An enzyme immunoassay was developed and validated for quantifying plasma adiponectin in chickens. Plasma adiponectin levels were found to be significantly lower in 8- compared with 4-wk-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.

Expression of adiponectin and its receptors in avian species.

Adipose tissue is a dynamic endocrine organ secreting a variety of hormones that affect physiological functions within the central nervous system, cardiovascular system, reproductive, and immune systems. The endocrine role of avian adipose tissue remains enigmatic as many of the classical hormones found in mammalian adipose tissue have not been found in avians. This mini-review summarizes our current knowledge on avian adiponectin, one of the most abundant adipose tissue hormones, and its receptors. We cloned the genes encoding chicken adiponectin and its receptors, AdipoR1 and AdipoR2. Using anti-chicken adiponectin antibody, we found that chicken adipose tissue and plasma predominantly contain a unique polymer of adiponectin with a mass greater than 669kDa, unlike mammalian adiponectin which is found as three distinct oligomers. Mass spectrometric analyses of chicken adiponectin revealed certain post-translational modifications that are likely to favor the unique multimerization of adiponectin in chickens. Unlike adiponectin, the nucleotide sequences of chicken AdipoR1- and AdipoR2 cDNA are highly similar to that of mammalian adiponectin receptors. Both adiponectin and adiponectin receptors are widely expressed in several tissues in the chicken. Herein, we review the unique biochemistry of adiponectin as well as expression of adiponectin and its receptors in the chicken. Future studies should focus on elucidating the role of adiponectin, AdipoR1, and AdipoR2 on metabolism, steroidogenesis, and adipose tissue remodeling during growth and reproduction in birds.

Characterization of Ascites-Derived Ovarian Tumor Cells from Spontaneously Occurring Ovarian Tumors of the Chicken: Evidence for E-Cadherin Upregulation

Ovarian cancer, a highly metastatic disease, is the fifth leading cause of cancer-related deaths in women. Chickens are widely used as a model for human ovarian cancer as they spontaneously develop epithelial ovarian tumors similar to humans. The cellular and molecular biology of chicken ovarian cancer (COVCAR) cells, however, have not been studied. Our objectives were to culture COVCAR cells and to characterize their invasiveness and expression of genes and proteins associated with ovarian cancer. COVCAR cell lines (n = 13) were successfully maintained in culture for up to19 passages, cryopreserved and found to be viable upon thawing and replating. E-cadherin, cytokeratin and α-smooth muscle actin were localized in COVCAR cells by immunostaining. COVCAR cells were found to be invasive in extracellular matrix and exhibited anchorage-independent growth forming colonies, acini and tube-like structures in soft agar. Using RT-PCR, COVCAR cells were found to express E-cadherin, N-cadherin, cytokeratin, vimentin, mesothelin, EpCAM, steroidogenic enzymes/proteins, inhibin subunits-α, βA, βB, anti-müllerian hormone, estrogen receptor [ER]-α, ER-β, progesterone receptor, androgen receptor, and activin receptors. Quantitative PCR analysis revealed greater N-cadherin, vimentin, and VEGF mRNA levels and lesser cytokeratin mRNA levels in COVCAR cells as compared with normal ovarian surface epithelial (NOSE) cells, which was suggestive of epithelial-mesenchymal transformation. Western blotting analyses revealed significantly greater E-cadherin levels in COVCAR cell lines compared with NOSE cells. Furthermore, cancerous ovaries and COVCAR cell lines expressed higher levels of an E-cadherin cleavage product when compared to normal ovaries and NOSE cells, respectively. Cancerous ovaries were found to express significantly higher ovalbumin levels whereas COVCAR cell lines did not express ovalbumin thus suggesting that the latter did not originate from oviduct. Taken together, COVCAR cell lines are likely to improve our understanding of the cellular and molecular biology of ovarian tumors and its metastasis.

Aquaporin 5 expression is altered in ovarian tumors and ascites-derived ovarian tumor cells in the chicken model of ovarian tumor.

BackgroundAquaporin 5 (AQP5), a member of the aquaporin family of transmembrane channel proteins, is involved in water transport and cellular proliferation in various tumors. The objective of this study was to determine cellular localization of aquaporin 5 (AQP5) in the ovarian tumors of chicken, a preclinical model for human ovarian tumor and to determine if AQP5 mRNA and protein expression levels in cancerous chicken ovaries and in ascites-derived chicken ovarian cancer (COVCAR) cell lines are different from normal ovaries and normal ovarian surface epithelial (NOSE) cells, respectively.MethodsImmunohistochemical staining was performed to determine the localization of AQP5-immunoreactive (ir) cells in normal and cancerous ovaries. To determine AQP5 mRNA and protein concentrations in cancerous ovaries and COVCAR cell lines, quantitative real time PCR and Western blotting analysis were performed, respectively. Students t-test was performed to compare the levels of AQP5 mRNA or protein in cancerous ovaries and COVCAR cell lines with that of normal ovaries and NOSE cells, respectively.ResultsAQP5-ir cells were localized in granulosa and theca layers of normal ovarian follicles whereas cancerous ovaries showed AQP5 immunostaining in the surface epithelium, fibroblast cells of the stroma, and in the cells lining tumor cysts and acini. AQP5 mRNA concentration were significantly lesser while AQP5 protein concentrations were significantly greater in cancerous ovaries compared to that in normal ovaries (P < 0.05). Whereas AQP5 mRNA concentrations were significantly greater while AQP5 protein concentrations were lesser (P < 0.05) in COVCAR cell lines compared with that in NOSE cells.ConclusionAQP5 is differentially expressed in ovarian tumor and in COVCAR cell lines suggesting a potential involvement of AQP5 in ovarian tumorigenesis, metastasis, and survival of ovarian tumor cells in ascites.

Nampt/visfatin/PBEF affects expression of myogenic regulatory factors and is regulated by interleukin-6 in chicken skeletal muscle cells.

Nicotinamide phosphoribosyltransferase (Nampt/visfatin/PBEF) has been identified as a rate-limiting NAD(+) biosynthetic enzyme and an adipokine found in the circulation. Human and chicken skeletal muscles are reported to have the highest level of Nampt expression among various tissues whose functional significance remains undetermined. Expression of Nampt is regulated by interleukin-6 (IL-6), an essential cytokine for postnatal muscle growth in mammals. The objective of the current study was to characterize expression of Nampt in chicken (Gallus gallus) myogenic cells and to determine the effect of Nampt on expression of IL-6, myogenic transcription factors, and glucose uptake. We also sought to determine the effect of IL-6 on Nampt expression in chicken myogenic cells. Nampt mRNA and protein were identified in both myoblasts and myocytes, although expression did not differ between the two cell types. Treatment with recombinant human Nampt was found to decrease myoD and mrf4 expression but to increase myf5 expression in myocytes, while glucose uptake was unaffected. In response to treatment with recombinant Nampt, IL-6 expression in myocytes was increased at 24h but decreased when treated for 48 or 72 h. Forced over-expression of chicken Nampt cDNA significantly decreased myf5 expression in myoblasts. Treatment of myogenic cells with lower levels (1 ng.mL(-1)) of recombinant IL-6 increased Nampt expression, whereas a higher IL-6 concentration (100 ng.mL(-1)) decreased Nampt mRNA abundance. Collectively, these results demonstrate that Nampt, regulated in part by IL-6, alters the expression of key myogenic transcription factors and thereby may influence postnatal myogenesis.

Bone sialoprotein keratan sulfate proteoglycan (BSP-KSPG) and FGF-23 are important physiological components of medullary bone

Medullary bone is a specialized bone found in the marrow cavity of laying birds. It provides a significant contribution to the calcium supply for egg shell formation. Medullary bone is distinguished from cortical bone by the presence of large amounts of a keratan sulfate proteoglycan (KSPG). The aims of the present experiment are to confirm the identity of the core protein of KSPG, identify a marker of medullary bone metabolism, and determine whether changes in keratan sulfate (KS) concentration in blood are associated with the egg-laying cycle. Using two different isolation techniques- one specific for bone and another for blood- we have identified bone sialoprotein (BSP) to be the core protein of this KSPG. We also determined that the amount of keratan sulfate (KS) in laying hen blood fluctuates in synchrony with the egg-laying cycle, and thus can serve as a specific marker for medullary bone metabolism. During the course of this investigation, we also found FGF-23 (phosphatonin) to be expressed in medullary bone, in synchrony with the egg-laying cycle. Western blotting was used to demonstrate the presence of this peptide in both laying hen blood and medullary bone extracts. The importance of FGF-23 (phosphatonin) and parathyroid hormone in normalizing the dramatic changes in plasma calcium and phosphorus during the 24h egg-laying cycle is discussed.