THE ROLE OF FETUIN -A ON ADIPOSE TISSUE LIPID MOBILIZATION IN DAIRY COWS By Clarissa Strieder Barboza A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Comparative Medicine and Int egrative Biology Ð Doctor of Philosophy 2018 ABSTRACT THE ROLE OF FETUIN -A ON ADIPOSE TISSUE LIPID MOBILIZATION IN DAIRY COWS By Clarissa Strieder Barboza Adipose tissue (AT) is a major modulator of metabolic functions by regulating energy storage and act ing as an endocrine organ. In periparturient dairy cows, increased AT mobilization of free fatty acids (FFA) is one a major adaptive mechanism to cope with higher energy demand for rapid fetal growth and the onset of lactation. As lactation progresses, lip olysis rates decrease, and lipogenesis replenishes triacylglycerol (TAG) stores in adipocytes. However, dysregulated metabolic responses, characterized by altered AT sensitivity to hormonal and endocrine changes around parturition, lead to a massive releas e of FFA into circulation and an increased susceptibility of cows to disease. These maladaptive responses are underlined by an altered secretory pattern of adipokines and a marked unbalance in lipolysis and lipogenesis rates, favoring TAG breakdown in adip ocytes. Thus, identifying adipokines that modulate AT function in periparturient dairy cows can facilitate the development of novel management, nutritional, or pharmaceutical interventions to reduce disease incidence. Fetuin -A (FetA; alpha -2-Heremans -Schmi d glycoprotein, AHSG) is an adipokine that functions as a carrier of FFA in plasma and is associated with insulin -mediated inhibition of lipolysis and stimulation of lipogenesis in humans. FetA increases the incorporation of fatty acids (FA) into intracell ular lipids and enhances cellular TAG in human cells. However, the mechanisms by which FetA induces TAG synthesis are not defined. FetA has also anti -inflammatory properties by inhibiting the production of pro -inflammatory cytokines and acting as a negativ e acute -phase protein (APP) in acute inflammation. These findings s uggest that FetA may also be involved in lipid mobilization and inflammation in AT of dairy cows. In our first in vivo study with periparturient dairy cows, we observed that serum and AT Fe tA expression decreased at the onset of lactation when lipogenesis was downregulated and plasma FFA was increased. FetA expression dynamics in AT were analogous to the patterns of lipogenic markers suggesting its link with lipid mobilization in AT of dairy cows. We also demonstrated that FetA is negative -APP locally in AT of dairy cows. These results suggest that FetA could support physiological adaptations to NEB in AT of periparturient dairy cows. To explore the potential roles of FetA on AT lipid mobiliz ation of dairy cows, we developed an in vitro model for culturing bovine adipocytes that closely mimics the in vivo AT environment. For the first time, we report ed an abundant expression and secretion of FetA by primary bovine adipocytes, thus suggesting a potential autocrine effect of FetA in AT of dairy cows. We observed that FetA attenuates lipolytic responses and enhances both, FA uptake and TAG accumulation in bovine adipocytes. Our results reveal that the upregulation of the expression and activity of 1-acylglycerol -3-phosphate acyltransferase (AGAPT2), a rate limiting lipogenic enzyme for TAG synthesis, may be a potential mechanism by which FetA enhances lipogenic function of bovine adipocytes. Overall, our results indicate that FetA is a lipogenic ad ipokine with anti -inflammatory function in the AT of dairy cows. Our findings provide evidence that FetA could buffer increased plasma FFA during negative energy balance by stimulating AGAPT2 activity and the use of excess FFA for TAG synthesis in AT of da iry cows. The genetic selection of cows by variations of the FetA coding gene associated with its anti -lipolytic and pro -lipogenic functions (already known in humans), the identification of dietary supplements (i.e. FA) that enhance FetA function, as well as the parenteral use of FetA to stimulate AGAPT2 activity, could serve as potential strategies to be tested and implemented in dairy cows. Copyright by CLARISSA STRIEDER BARBOZA 2018 v ACKNOWLEDGEMENTS I would like to thank everybody that b elieved on my potential and supported me throughout this journey making me stronger as a scientist and as a person. Thank you, Dr. Andres Contreras . Your mentorship guided me throughout the years helping me to develop as a scientist. I will be forever grat eful and will never forget your lessons as a researcher, as an advisor, and as a friend. I am very proud of being your first PhD student. Thank you, Dr. Lorraine Sordillo, Dr. Adam Lock, and Dr. Richard Ehrhardt, for your willingness to help with my resea rch ideas, for the guidance, and for giving me suggestio ns and support throughout my Ph D. Dr. Sordillo, thank you for our long talks, advice and caring. You have my deepest appreciation. Special thanks to my dear fri end Jeff Gandy, whose expertise, genero us help and care made my life much easier and happier! Thanks to Kyan Thelen for her assistance and friendship; you will always be my Òlab sisterÓ! Thanks to Dr. William Raphael, Dr. Jenne De Koster, Dr. Rahul Nelli and all of the graduate, veterinary and undergraduate students in the Dr. Contreras Laboratory, MSU Dairy Group and CMIB Program for assisting my research and youÕre your friendship. Thank you to all my friends that made me feel like home! Thanks to my family, especially my mom, Dolores Barboza , for all the love, support and care no matter the distance. You are the reason why I am who I am. Gracias, mi amor, Oscar Benitez for alway s being by my side, encouraging, supporting , and loving me. In the happiest and in the most difficult days, you were a lways there . You are my home, my safe haven. Thank you! vi TABLE OF CONTENT S LIST OF TABLES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ É. viii LIST OF FIGURES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉ... ix KEY TO ABBREVIATIONS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ É... . x CHAPTER 1 ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ É... 1 INTRODUCTION ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... ....... 1 REFERENCES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ É... 4 CHAPTER 2 ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ É... 7 LITERATURE REVIEW ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ É... 7 ADIPOKINES AS MODULATORS OF METABOLIC FUNCTION IN PERIPARTURIENT DAIRY COWS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 7 CONCLUSIONS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 21 FETUIN -A AS A LINK BETWEEN LIPID MOBILIZATION AND INFLAMMATION ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 23 CONCLUSIONS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 27 REFERENCES ÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... ..... 29 CHAPTER 3 ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ É. 40 FETUIN -A: A NEGATIVE ACUTE -PHASE PROTEIN LINKED TO ADIPOSE TISSUE FUNCTION IN PERIPARTURIENT DAIRY COWS É ÉÉÉÉÉÉÉ ÉÉÉÉÉ 40 ABSTRACT É ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 40 CHAPTER 4 ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ É. 42 IN VITRO ADIPOGENIC DIFFERENTIATION OF BOVINE PREADIPOCYTES: A CO-CULTURE MODEL ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... ... 42 ABSTRACT ÉÉ ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ... 42 TECHNICAL NOT E ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉ 44 REFERENCES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... .. 58 CHAPTER 5 ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... .. 61 FETUIN -A MODULATES LIPID MOBILIZATION IN BOVINE ADIPOSE TISSUE BY ENHANCING L IPOGENIC ACTIVITY OF ADIPOCYTES ÉÉÉÉÉÉÉÉ ÉÉ. 61 ABSTRACT É ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉÉÉÉÉÉÉ 61 INTROD UCTION ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ É 63 MATERIALS AND METHODS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ .. 65 RESULTS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ . 73 DISCUSSION ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 78 CONCLUSIONS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ .. 85 REFERENCES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉ. 86 vii CHAPTER 6 ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... .. 91 CONCLUSIONS AND FUTURE DIRECTIONS ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ É 91 REFERENCES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 96 viii LIST OF TABLES Table 2. 1 Metabolic functions, plasma concentrations and known clinical relevance of adipokines in periparturient dairy cows. ...................................................................................................... 10 Supplemental Table 4. 1 mRNA probes by product and NCBI accession numbers ÉÉÉÉ... 48 Supplemental Table 5. 1 mRNA probes by product and NCBI accession numbers ÉÉÉÉÉ. 71 ix LIST OF FIGURES Figure 2. 1. Adipokines and metabolic function in adipose tissue of periparturient dairy cows. ... 9 Figure 4. 1 Viability of pr imary bovine adipocytes placed in inse rts during co -culture protocol. ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉ.É.. 50 Figure 4. 2 Effect of culture conditions on gene expression of adipogenic markers in bovine adipocytes from subcutaneous adipose tissue depot. .......................................................... 51!Figure 4. 3 Effect of culture conditions on gene expression of adipogenic markers in bovine adipocytes from visceral adipose tissue depot. ................................................................... 52!Figure 4. 4 Lipid accumulation in cultured bovine adipocytes derived from subcutaneous adipose tissue depot in dairy cows. ................................................................................................. 55!Figure 4. 5 Lipid accumulation in cultured bovine adipocytes derived from omental vis ceral adipose tissue in dairy cows. .............................................................................................. 56!Figure 4. 6 Responsiveness to !-adrenergic stimulation in subcutaneous and visceral adipocytes differentiated in different culture conditions. ..................................................................... 57!Figure 5. 1 Fetuin -A expression in cultured bovine adipocytes and subcutaneou s adipose tissue of dairy cows. ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 74 Figure 5. 2 Effect o f Fetuin -A on !-adrenergic induced -lipolysis in bovine adipocytes. ............. 75 Figure 5. 3 Effect of Fetuin -A on fatty acid (FA) uptake by bovine adipocytes in vitro .............. 75 Figure 5. 4 Effect of Fetuin -A on triacylglycerol accumulation in cultured bovine adipocytes. .. 77 Figure 5. 5 Effect of Fetuin -A on gene expression of lipogenic ma rkers and phosphatidic acid concentrations in bovine adipocytes. ......................................................................................... 79 x KEY TO ABBREVIATIONS ADIPOQ Adiponectin AdipoR1 Adiponectin receptor -1 AdipoR2 Adiponectin receptors -2 AGAPT2 1-acylglycerol -3-phosphate acyltransferase AHSG Alph a-2-Heremans -Schmid glycoprotein AL ad libitum AMPK 5'-adenosine monophosphate -activated protein kinase ANGTLP4 Angiopoietin -like protein -4 aP2 Adipocyte fatty acid -binding protein APP Acute -phase protein ARG1 Arginase -1 AT Adipose tissue AUC Area under the curve B2M Beta -2-microglobulin BCS Body condition score BHB Beta -hydroxybutyrate BME Mercaptoethanol BSA Bovine serum albumin Ca Calcium CC7 7 d in co -culture CCL2 Chemoattractant protein -1 xi CD36 Fatty acid translocase CD44 Cluster differentiation 44 CD68 Cluster differentiation 68 CEBPA CCAAT/enhancer -binding protein alpha CEBPB CCAAT/enhancer -binding protein beta COL6A2 Collagen alpha -2 CP Crude protein CU Close -up DGAT Diacylglycerol acyltransferases DGAT1 Diacyl glycerol O -Acyltransferase -1 DGAT2 Diacylglycerol O -Acyltransferase -2 DM Dry matter DTT Dithiothreitol EIF3K Eukaryotic translation initiation factor 3 subunit K EL Early lactation ELOVL6 Fatty Acid Elongase 6 FA Fatty acids FABP4 Fatty acid bindi ng protein 4 FASN Fatty acid synthase FASN Fatty acid synthase FATP1 Fatty acid transporter -1 FBS Fetal bovine serum Fet A Fetuin -A xii FFA Free fatty acids FO Far off FR Feed-restricted GLUT4 Glucose transporter -4 GPAT Glycerol phosphate acyltra nsferases GPAT1 Glycerol -3-Phosphate Acyltransferase -1 GPAT2 Glycerol -3-Phosphate Acyltransferase -2 HBCS High body condition score HCAR1 Hydroxycarboxylic acid receptors -1 HDL High density lipoprotein HSL Hormone lipase IBMX 2 isobutyl -1-methyl axanthine IFN -" Interferon gamma IL1 Interleukin 1 IL10 Interleukin -10 IL6 Interleukin 6 ISO Isoproterenol KRBB Krebs -Ringer Bicarbonate Buffer LIPE Hormone sensitive lipase LIPIN1 Phosphatidate phosphatase -1 LPL Lipoprotein lipase LPS Lipopolysacchari de MBCS Moderate body condition score xiii MCP -1 Monocyte chemoattractant protein -1 NDF Neutral detergent fiber NE Net energy of lactation NEB Negative energy balance P Phosphorus PA Phosphatidic acid PAI -1 Plasminogen activator inhibitor 1 PPARG Peroxisome proliferator -activated receptor gamma PPARGC1A PPAR " co -activator 1 # PPAR # Peroxisome proliferator -activated receptor alpha PPAR " Peroxisome proliferator -activated receptor -" PRE Preadipocytes RBP Retinol -binding protein RPLO 50S ribosomal protein L15 RPS9 40S ribosomal protein S9 RPS9 40S ri bosomal protein S9 SCD1 Stearoyl -CoA desaturase -1 SD14 14 d in standard differentiation SD7 7 d in standard differentiation SIRPA Signal regulatory protein # SIRT1 Sirtuin -1 SNP Single nucleotide polymorphism SPP1 Osteopontin -1 xiv SVF Stromal vascul ar fraction TAG Triacylglycerol TBS Tris -buffered saline solution TNF Tumor necrosis alpha 1 CHAPTER 1 INTRODUCTION Periparturient dairy cows are at higher risk of disease due to intense lipid mobilization rates in the adipose tissues (AT) trigger ed by increased energy demands around parturition (Ospina et al., 2010). Increased lipolysis and decreased lipogenesis are major adaptive mechanisms to cope with negative energy balance (NEB) in periparturient cows. However, dysregulated adaptive responses result in excessive circulating FFA and lead to impaired immune and inflammatory responses (Contreras and Sordillo, 2011). Thus, adequate AT function is essential for a successful transition from gestation to lactation in dairy cattle (McNamara, 2012). Despite the advances on the management of periparturient dairy cows, at least 30% of early lactation cows develop metabolic and inflammatory diseases leading to high economic losses to the dairy industry (Van Saun and Sniffen, 2014). Strategies for effectiv ely modulating AT function and assure an adequate balance between lipolysis and lipogenesis in AT are required. Adipokines are signaling proteins produced by the cellular components of AT including adipocytes and cells of the stromal vascular fraction, suc h as immune and vascular cells. Adipokines have crucial autocrine and endocrine functions regulating energy homeostasis, insulin sensitivity and inflammatory pathways, thus being suitable targets for the modulation of AT and whole -body metabolic function i n dairy cows (Kusminski et al., 2016). In dairy cows, more than 500 adipokines have been identified in subcutaneous AT, but just few of them have been characterized in the context of the periparturient period (Zachut, 2015). Especially during this period, identifying adipokines involved in the autocrine regulation of lipid mobilization and inflammatory responses in AT are particularly relevant for the development of novel strategies to reduce diseases around parturition (Lehr et al., 2012, Zachut, 2015). 2 In humans, Fetuin -A (FetA; alpha -2-Heremans -Schmid glycoprotein, AHSG) is a recently reported adipokine and an attractive candidate gene for disturbed adipocyte lipolytic function during metabolic diseases, such as obesity and insulin resistance (Dahlman e t al., 2004). FetA is involved on insulin -dependent and independent inhibition of lipolysis and stimulation of lipogenesis in human adipocytes (Dahlman et al., 2004). Attenuating lipolysis independently of insulin would be particularly important in early l actation when dairy cows develop hypoinsulinemia concurrently with a state of insulin resistance, thus releasing even higher FFA concentrations into circulation (Contreras et al., 2017). However, whether FetA plays the same roles in AT of dairy cows and th e specific mechanisms by which this adipokine modulates lipid mobilization have not yet been described. Because of its adipogenic and lipogenic properties inducing incorporation of extracellular lipids into intracellular TAG (Cayatte et al., 1990), increas ed circulating concentrations and AT expression of FetA have been associated with obesity and obesity -related metabolic diseases, such as metabolic syndrome (Jialal et al., 2015, P”rez -Sotelo et al., 2016). Fetuin -A is also involved in acute inflammatory r esponses acting as a negative acute -phase protein (APP) in cases of infection, sepsis and trauma in non -ruminant models (Li et al., 2011, Wang and Sama, 2012, Zhang et al., 2014). Based on these antecedents, FetA could play a crucial role in the AT functio n of periparturient dairy cows as a modulator of lipid mobilization and inflammatory responses. However, little is known about this adipokine in dairy cows. The first report of FetA abundancy in AT of dairy cows indicates that its expression is downregulat ed during environmental metabolic stress when cows had increased plasma FFA and were in a pro -inflammatory status (Zachut et al., 2017). If acting similarly to non -ruminants, FetA might help buffering the increased circulating FFA concentrations during NEB in periparturient dairy cows, by potentially stimulating the use of FFA for TAG synthesis. The 3 better understanding of the roles of FetA in AT function of dairy cows could facilitate its use as, for example, a disease predictor or biomarker, a target for genetic selection, or even as a potential drug for improving AT function of dairy cows. We hypothesized that FetA is a pro -lipogenic and anti -inflammatory role in the AT of periparturient dairy cows and enhances TAG synthesis in bovine adipocytes by augmen ting FA uptake and the expression and activity of lipogenic enzymes. Therefore, our objectives were to (1) determine the roles of FetA on AT lipid mobilization and inflammation in AT of periparturient dairy cows, (2) to evaluate how FetA affects lipogenic and lipolytic functions in bovine adipocytes, and (3) to identify mechanisms and key cellular components underlying the effects of FetA on lipogenic activity of bovine adipocytes. 4 REFERENCES 5 REFERENCES Cayatte, A. J., L. Kumbla, and M. T. Subbiah. 1990. Marked acceleration of exogenous fatty acid incorporation into cellular triglycerides by fetuin. Journal of Biological Chemistry 265(10):5883 -5888. Contreras, G. A. and L. M. Sordillo. 2011. Lipid mobilization and inflammatory responses during the transition period of dairy cows. Comparative Immunology, Microbiology and Infectious Diseases 34(3):281 -289. Contreras, G. A., C. Strieder -Barboza, and W. Raphael. 2017. Adipose tissue lipolysis and remodeling during the transition period of dairy cows. Journal of Animal Science and Biotechnology 8(1):41. Dahlman, I., P. Eriksson, M. Kaaman, H. Jiao, C. Lindg ren, J. Kere, and P. Arner. 2004. # 2-Heremans ÐSchmid glycoprotein gene polymorphisms are associated with adipocyte insulin action. Diabetologia 47(11):1974 -1979. Jialal, I., S. Devaraj, A. Bettaieb, F. Haj, and B. Adams -Huet. 2015. Increased adipose tissue secretion of Fetuin -A, lipopolysaccharide -binding protein and high -mobility group box protein 1 in metabolic syndrome. Atherosclerosis 241(1):130 -137. Kusminski, C. M., P. E. Bickel, and P. E. Scherer. 2016. Targeting adipose tissue in the treatment of ob esity -associated diabetes. Nature Reviews Drug Discovery 15(9):639. Lehr, S., S. Hartwig, and H. Sell. 2012. Adipokines: a treasure trove for the discovery of biomarkers for metabolic disorders. PROTEOMICS -Clinical Applications 6(1 !2):91 -101. Li, W., S. Zhu, J. Li, Y. Huang, Z. Rongrong, X. Fan, H. Yang, X. Gong, N. T. Eissa, W. Jahnen -Dechent, P. Wang, K. J. Tracey, A. E. Sama, and H. Wang. 2011. A Hepatic Protein, Fetuin -A, Occupies a Protective Role in Lethal Systemic Inflammatio n. PLoS ONE 6(2):e16945. McNamara, J. 2012. Ruminant Nutrition Symposium: A systems approach to integrating genetics, nutrition, and metabolic efficiency in dairy cattle. Journal of Animal Science 90(6):1846 -1854. Ospina, P., D. Nydam, T. Stokol, and T. Ov erton. 2010. Evaluation of nonesterified fatty acids and beta -hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases. J Dairy Sci 93(2):546 -554. P”rez -Sotelo, D., A. Roca -Rivada , M. Larrosa -Garc™a, C. Castelao, I. Baamonde, J. Baltar, A. B. Crujeiras, L. M. Seoane, F. F. Casanueva, and M. Pardo. 2016. Visceral and subcutaneous adipose tissue express and secrete functional alpha2hsglycoprotein (fetuin a) especially in obesity. End ocrine:1 -12. 6 Van Saun, R. J. and C. J. Sniffen. 2014. Transition cow nutrition and feeding management for disease prevention. Veterinary Clinics of North America: Food Animal Practice 30(3):689 -719. Wang, H. and A. E. Sama. 2012. Anti -inflammatory role of Fetuin -A in Injury and Infection. Curr Mol Med 12(5):625 -633. Zachut, M. 2015. Defining the Adipose Tissue Proteome of Dairy Cows to Reveal Biomarkers Related to Peripartum Insulin Resistance and Metabolic Status. Journal of Proteome Research 14(7):2863 -2871. Zachut, M., G. Kra, L. Livshitz, Y. Portnick, S. Yakoby, G. Friedlander, and Y. Levin. 2017. Seasonal heat stress affects adipose tissue proteome toward enrichment of the Nrf2 -mediated oxidative stress response in late -pregnant dairy cows. Journal of P roteomics 158:52 -61. Zhang, P., H. Shen, J. Huang, H. Wang, B. Zhang, R. Zhou, B. Zhong, and X. Fan. 2014. Intraperitoneal Administration of Fetuin -A Attenuates d -Galactosamine/Lipopolysaccharide -Induced Liver Failure in Mouse. Dig Dis Sci 59(8):1789 -1797. 7 CHAPTER 2 LITERATURE REVIEW ADIPOKINES AS MODULATORS OF METABOLIC FUNCTION IN PERIPARTURIENT DAIRY COWS Hormonal and endocrine changes and the abrupt increase in energy requirements associated with parturition and the onset of lactation lead to a pron ounced negative energy balance (NEB) in dairy cows (McNamara and Hillers, 1986) . The major metabolic adaptation to NEB is the increment in the use of lipids as an energy substrate (Bell and Bauman, 1997) . Within adipose tissue (AT), enhanced lipolysis and reduced lipogenesis increase FFA flux into circulation. These AT responses are coupled with reduced insulin sensitivity within AT and in other peripheral tissues (i.e. muscle) that prioritize the use of glucose by the mammary gland (Bauman and Currie, 1980b). These metabolic adaptations maintain energy supply for the successful transition from gestation to lactation, however, when dysregulated, trigger an excessive release of FFA that leads to inadequate inflammatory responses and alterations in AT met abolic function (Contreras and Sordillo, 2011a, Contreras et al., 2017b) . Currently, the mechanisms that lead to AT dysfunction in periparturient dairy cows are poorly understood and may involve not only alteratio ns in lipid mobilization but also changes in its endocrine function. Classic literature defined fat depots as connective tissues in which lipids were deposited and FFA were identified as its only secretory products (Wells, 1940) . AT is now recognized as a complex organ with sympathetic and sensory innervation that plays a fundamental role in energy homeostasis, and endocrine and immune functions. AT fulfills its function in part through the synthesis and secreti on of signaling proteins that are collectively termed adipokines (Blr, 2012). These signaling proteins are produced by the cellular components of AT including 8 adipocytes and cells of the stromal vascular fraction (SVF), such as immune , vascular, and adipocyte progenitor cells. Adipokines regulate glucose and lipid metabolism, insulin signaling, and inflammatory pathways through autocrine, paracrine and endocrine actions [Fig. 1]. Although dairy cows express hundreds of adipokines in AT (Zachut, 2015) , only a few have been characterized in the context of the periparturient period. Around parturition, the secretory pattern of adipokines is altered by the overall metabolic status and by within -AT depot factors such as infiltration of immune cells (Contreras et al., 2015, H−ussler et al., 2015) . The central role of adipokines as modulato rs of energy homeostasis and AT function opens the possibility of using these signaling proteins as targets in the prevention and treatment of metabolic diseases in periparturient dairy cows (Lehr et al., 2012, Zachut, 2015) [Table 1]. However, only a few studies have analyzed the association of adipokine s and diseases in dairy cows (Kasimanickam et al., 2013, Fadden and Bobe, 2016) . This review summarizes current knowledge on the dynamics and roles of main adipokines involved on lipid mobilization and inflammatory responses around parturition in dairy cows, and their relationship with disease and metabolic dysfunction. 9 Figure 2. 1. Adipokines and metabolic function in adipose tissue of periparturient dairy cows. Hormonal and endocrin e changes around parturition stimulate changes in lipid mobilization, inflammatory responses, and the endocrine function of adipose tissue through the synthesis of adipokines. Hormone - and cytokine -like adipokines derived from adipose tissue participate in autocrine/paracrine and endocrine functions. Adipokines mediate crosstalk among different adipose tissue depots and cell populations within each depot (i.e. immune cells) and reach other organs/systems such as liver, muscle, and brain to regulate systemic energy metabolism . 10 Table 2. 1 Metabolic functions, plasma concentrations and known clinical relevance of adipokines in periparturient dairy cows. Metabolic functions Plasma concentrations Clinical relevance Adiponectin Thro ugh AMPK and PPAR # activation (Kadowaki and Yamauchi, 2005): !!$ glucose uptake and FA oxidation in liver, muscle and AT !!$ food intake acting through CNS !!$ insulin sensitivity in AT !!% NF-"B activation in AT (Sauerwein and H−u§ler, 2016) !!%TNF -# expression in bovine monocytes (Kabara et al., 2014) -3 to +3 wk from parturition: ~35 ug/mL Calving: ~20 ug/mL (Singh et al., 2014b) !!Plasma concentrations are: o!Negatively associated with plasma FFA concentrations (Kabara et al., 2014, Mellouk et al., 2017) o!Negatively associated with adiposity (De Koster et al., 2017) o!Positively associated with the insulin responsiveness of the glucose and FFA metabolism (De Koster et al., 2017) o!Low adipocyte -derived adiponectin increase inflammatory -based disease development during the periparturient period in cattle (Kabara et al., 2014) Leptin !!% lipogenesis by inhibiting de novo synthesis of FA (William et al., 2002) !!$ lipolysis and FFA oxidation (William et al., 2002) !!$ lipid oxidation (Wang et al., 1999) !!$conservation of glucose and energy at the onset of lactation (Ehrhardt et al., 2016) Prepartum: 7.83 ± 2.84 ng/mL Postpartum: 4.64 ± 2.24 ng/mL (Lemor et al. 2009) !!Exp erimental parental infusion of leptin (Ehrhardt et al., 2016) : o!Attenuates accumulation of TAG in the liver o!Increases plasma concentrations of T4 and T3 o!Increases gluc ose disposal during an insulin tolerance test !!Plasma leptin is directly correlated with fat mass (Leury et al., 2003) 11 Table 2. 1 (contÕd) RBP !!Transport system for retinol in circulation (Rezamand et al., 2012a) !!% adipogenes is and glucose uptake by adipocytes (Klıting et al., 2007) 1 wk prepartum: 50.7±5.3 mg/ml Calving: 29.8±5.4 mg/ml 2 w ks postpartum: 52.1±2.1 mg/ml (Eldaim et al., 2010) !!Negatively associated with hyperketonemia (Grıhn and Lindberg, 1985) !!Indicator of the liver activity index (Trevisi et al., 2001) !!Visceral AT RBP expression is positively associated with that of TNF -# (Rezamand et al., 2012a) ANGTLP4 !!Inhibits adipocyte uptake of FA for esterification controlled by PPAR -" (Kersten, 2005) !!$ lipolysis by increasing cAMP and enhancing the phosphorylation of PKA (Gray et al., 2012) !!Inhibits LPL -mediated lipolytic processing of TAG -rich lipoprotein (Preedy and Hunter, 2016) Lactating cows: 3.22 Ð 4.00 ng/mL (Li, 2011) !!May signal for lipolysis induction and contribute to the sustained release of FFA and lipid accumu lation in the liver (Loor et al., 2007) Fetuin -A !!$ Lipogensesis and adipogenesis factor (Cayatte et al. , 1990) !!Lipid transporter in plasma (Kumbla et al., 1989) !!Negative acute phase protein systemically and in the AT (Strieder -Barbo za et al., 2017b, Zachut et al., 2017) 30d prepartum: 0.89 ± 0.13 mg/mL 10d prepartum: 0.96 ± 0.13 mg/mL 10d postpartum: 0.77 ± 0.13 mg/mL (Strieder -Barboza et al., 2017b) !!Expression in adipose tissue: o!Decreased during heat stress and lipolysis in late gestation (Zachut et al., 2017) o!Negatively associated with pro -inflammatory markers in AT of periparturient cows (Strieder -Barboza et al., 2017b) !!Circulating concentrations (Strieder -Barboza et al., 2017b) : o!Positively associated with serum albumin and calcium o!Positively associated with BCS 12 Table 2. 1 (contÕd) Resistin !!$ lipolysis in subcutaneous AT (Reverchon et al., 2014). !!Influence the insulin -dependent glucose uptake in mammary epithelial cells (Komatsu et al., 2003) !!May inhibit GLUT4 translocation in AT (Komats u et al., 2003) 4-2 wk prepartum: 43.25 ng/mL 7-14d postpartum: 75.10 ng/mL - 4-6 wk postpartum: 36.42 ng/mL (Reverchon et al., 2014) . !!In adipose tissue: o!May contribute to insulin resistance (Komatsu et al., 2003) !!Circulating concentrations: o!Positively correlated with subcutaneous AT HCAR1 gene expression, an insulin -induced anti -lipolyt ic factor (Weber et al., 2016a) o!Positively associated with plasma FFA (Reverchon et al., 2014). o!Positively associated with adiposity (Mellouk et al., 2017) . o!Negatively associated with plasma leptin (Mellouk et al ., 2017). Visfatin !!Induces insulin secretion and promotes glucose uptake (Fukuhara et al., 2005) !!Induces inflammation response through cytokine secretion (Moschen et al., 2007) !!$ cell survival (Rongvaux et al., 2008) -3 to +3 weeks to calving: median 6.59 µg/L; interquartile range: 5.48 to 7.86 µg/L (Fadden and Bobe, 2016) !!Plasma concentrations are increased during periparturient disease (Fadden and Bobe, 2016) !!Early predictive indicator of retained placenta and other diseases (metritis, mastitis, ketosis, or laminitis) in peripa rturient dairy cows (Fadden and Bobe, 2016) TNF -# !!Inhibits adipogenesis by downregulating LPL, FATP and ACS ( Arner, 2003) !!Regulates the formation of leptin, PAI -1, aP2 and GLUT4 (Hotamisligil and Spiegelman, 1994) !!$ lipolysis (Arner, 2003) !!Indu ces AT insulin resistance (Sethi and Hotamisligil, 1999) !!Activates inflammation systemically and in the AT (Sadri et al., 2010, Contreras et al., 2015) 1d postpartum: 92 ±13 pg/ml 7 weeks portpartum: 85 ± 12 pg/ml (Sadri et al., 2010) !!Serum TNF activity is associated with insulin resistance in cows with fatty liver (Ohtsuka et al., 2001) !!Increased expression in the visceral and subcutaneous AT of cows with displaced abomasum (Contreras et al., 2015) !!Administration of TNF -# promoted lipolysis and insulin resistance in AT of dairy steers (Kushibiki et al., 2001) ANGTLP4, Angiopoietin -like protein -4; LPL, lipoprotein lipase; FetA, Fetuin -a; TNF -# , tumor necrosis factor alpha; RBP, retinol -bind ing -protein. 13 Adiponectin Adiponectin is a hormone -like adipokine exclusively produced by adipocytes and secreted directly into the circulation without further tissue accumulation (Sauerwein and H−u§ler, 2016 ). Adiponectin is synthesized as a monomer (30 kDa) and then assembled to oligomers detectable as low molecular weight trimers, medium molecular weight hexamers and high molecular weight oligomers (Waki et al., 2003) . This adipokine regulates glucose and FA metabolism and has insulin -sensitizing and anti -inflammatory properties in different tissues (Singh et al., 2014a, De Koster et al., 2017) . Adiponectin binds to its receptors in the skeletal muscle (AdipoR1) and liver (AdipoR2) to enhance glucose uptake and FA oxidation through the activation of 5' -adenosine monophosph ate -activated protein kinase (AMPK) and the peroxisome proliferator -activated receptor alpha (PPAR # ) (Yamauchi et al., 2003) . Adiponectin also acts in the in hypothalamic neurons increasing food intake and dec reasing peripheral energy expenditure through the activation of AMPK (Kadowaki and Yamauchi, 2005) . In periparturient da iry cows, adiponectin expression in subcutaneous AT reaches its nadir around parturition and doubles by day 21 postpartum (Singh et al., 2014b) , and its expression is greater in cows with high prepartum BCS (Vailati -Riboni et al., 2016) . Adiponectin actions are modulated by the expression of its receptors, AdipoR1 and AdipoR2 acros s different tissues in dairy cows (Sauerwein and H−u§ler, 2016) . AdipoR1 and AdipoR2 expression in AT are downregulated during the first 3 weeks after calving and increases steadily to peak at around 100 DIM in dairy cows (Saremi et al., 2014, Weber et al., 2016b) . Decreased AdipoR1 and AdipoR2 mRNA abundance in AT might be the result of a complex regulatory system where expression of Sirtuin -1 (SIRT1) PPAR " co -activ ator 1 # (PPARGC1A) axis modulates the abundance of adiponectin receptors (Weber et al., 2016b) . However, the role of this axis on the modulation of 14 adiponectin signaling is a matter of debate as Giesy et al. (2012) did not observe any changes in the expression of AT adiponectin mRNA and its receptors AdipoR1 and AdipoR2 in mature dairy cows around parturition . Changes in A T adiponectin expression in periparturient cows are reflected in its plasma concentrations that reach their nadir at calving (Singh et al., 2014a) . At 3 weeks prepartum, plasma adiponectin concentrations are ~35 #g/mL and decrease to about 20 #g/mL at the time of parturition. By the third week of lactation plasma adiponectin returns to pre -calving values and is mainly influenc ed by visceral AT secretion of adiponectin (Singh et al., 2014b) . Plasma adiponectin is negatively associated with FFA concentrations and therefore with lipolysis intensity in periparturient cows (Kaba ra et al., 2014, Mellouk et al., 2017) . Adiponectin circulates predominantly in high and medium molecular weight forms (Giesy et al., 2012) , and this oligomer distribution is independent of parity and the dietary supplementation with conjugated linoleic acid (Singh et al., 2014b) . In dairy cows during late gestation, serum adiponectin was negatively associated with adiposity and positively associated with the insulin responsive ness of the glucose and FFA metabolism (De Koster et al., 2017) . Adiponectin modulates inflammation by suppressing nuclear factor -& § (NF & §) activation (Sauerwein and H−u§ler, 2016) . Accordingly, adiponectin exposure decreased TNF -# expression in bovine monocyte after LPS -stimulation (Kabara et al., 2014) . Therefore, decreased adiponectin concentrations could be determinant for increased inflammatory -based disease susceptibility in periparturient cattle (Kabara et al., 2014) . In summary, adiponectin acts as an autocrine, par acrine, and endocrine modulator of the AT homeorhetic adaptations and in part of the inflammatory responses that may increase disease susceptibility in periparturient cows. All 15 together, these data indicate that adiponectin as a biomarker for metabolic fun ction and insulin resistance in dairy cows has great potential for clinical use. Leptin Leptin is a 16 -kDa adipokine encoded by the OB gene and synthesized by white AT (Leury et al., 2003) . In ruminants, leptin is also expressed in t he rumen, abomasum, duodenum, mammary gland, skeletal muscle and pituitary gland (Chilliard et al., 2001) . In adipocytes, l eptin reduces lipogenesis by inhibiting de novo synthesis of FA and increases lipolysis and FFA oxidation (William et al., 2002) . These effects are induced by modulating the expression of enzymes involved in lipid oxidation such as acyl -CoA oxidase, uncoupling protein 2, and PPA R-# (Wang et al., 1999) . Leptinemia dynamics reflect AT leptin gene expression (Block et al., 2001) and are directly correlated with fat mass (Leury et al., 2003) . Plasma leptin peaks during the dry period and then decreases from 7.83 ± 2.84 ng/mL two weeks before parturition to 4.64 ± 2.24 ng/mL at 20 days postpartum (Block et al., 2001, Lemor et al., 2009) . The reduction in leptinemia during periparturient NEB was implicated as one of the signals promoting conservation of glucose and energy (Ehrhardt et al., 2016) and precedes significant depletion of lipid reserves mediated by the hypoinsulinemia (Block et al., 2003, Leury et al., 2003) . Leptin acts on the hypothalamus to regulate energy metabolism by decreasing food intake and increasing energy expenditure (Ingvartsen and Boisclair, 2001) . A fall in plasma leptin concentrations signals to the CNS that a state of energy insufficiency prevai ls in the periphery (Leury et al., 2003) . In early lactation cows, cerebrospinal fluid leptin concentrations decline as NEB and lipolysis increase suggesting that central leptin signals to promote hyperphagia (Laeger et al., 2013) . Reduced AT leptin transcription and secretion may also promote a rapid return to 16 normal feed intake after parturition (Ehrhardt et al., 2016) . However, voluntary feed intake becomes unresponsive to variations in plasma leptin during NEB in early lactation indicating that if leptin resistance exists in dairy co ws, reduced plasma leptin may not promote higher voluntary feed intake (Leury et al., 2003, Ehrhardt et al., 2016) . Therefore, leptin acts in autocrine and endocrine manners to modulate energy expenditure and is e specially important during NEB as one of the signals promoting conservation of glucose. Besides the recent report that circulating concentrations of leptin could be used to assess subclinical uterine diseases (Kasimanickam et al., 2013) , a more recent study demonstrated how the manipulation of leptinemia could benefit metabolic function in periparturient cows. By reverting hypoleptinemia in early lactation co ws, Ehrhardt et al. (2016) increased the glucose response during an insulin tolerance test, decreased hepatic lipid accumulation in early lactation, and increased (~45%) plasma concentrations of both T4 and T3 (Ehrhardt et al., 2016) . These studies revealed that leptin has an important clinical relevance not only as a marker or predictors of diseases, but as a target in the treatment of metabolic diseases in periparturien t dairy cows. Angiopoietin -like protein -4 Angiopoietin -like protein -4 (ANGTLP4), also known as fasting -induced adipose factor, is an adipokine secreted during NEB that under transcriptional control by PPAR " inhibits FA uptake by adipocytes (Kersten, 2005) . ANGPTL4 enhances catecholamine -induced lipolysis in adipocytes by increasing cAMP and enhancing the phosphorylation of PKA (Gray et al., 2012) . ANGPTL4 inhibits lipoprotein lipase (LPL) -lipolytic activity at the same time that induces intracellular lipolysis and FFA release into circulation (Preedy and Hunter, 2016) . In dairy cattle, AT ANGPTL4 expression increased with the transition from pregnancy to lactation (Sumner -Thomson et al., 2011) . ANGPTL4 transcription was inversely associated with the degree of NEB 17 during lip olytic states as induced by GH and feed restriction in dairy cows (Koltes and Spurlock, 2012). The relative importance of circulating concentration vs local expression of ANGPTL4 in AT depots is currently unknown, and ANGPTL4 protein content in AT from periparturient cows has not been determined (Koltes and Spurlock, 2012) . Increased hepatic synthesis of ANGPTL4 during periods of NEB might serve as a signal for lipolysis and contribute to the sustained release of FFA and lipid accumulation in th e liver (Loor et al., 2007) . Therefore, ANGPTL4 could be a potential sensitive marker of AT lipid mobiliz ation due to its regulation by transcriptional factors of adipogenesis and its potent lipolytic activity in adipocytes. Retinol -bindi ng protein Retinol -binding protein (RBP) secreted by adipocytes serves as the main transport system for circulating retino l (vitamin A). RBP inhibits adipogenesis and glucose uptake by adipocytes by suppressing the insulin signaling (Klıting et al., 2007) . In visceral AT of cows, the expression of RBP was positively associated with that of TNF -# (Rezamand et al., 2012b) , which is a recognized inhib itor of adipocyte differentiation. In plasma, RBP concentrations decrease 3 to 6 days before parturition, peak at calving, and then return to pre -calving levels by the second (Eldaim et al., 2010) or third (Rezamand et al., 2012b) week of lactation when lipogenesis also increase. These data suggest that RBP supports homeorhetic mechanisms of metabolic adaptation during the onset of lactation through the downregulat ion of adipogenesis and lipogenesis in dairy cows. Reduced plasma RBP have been associated with metabolic disorders such as hyperketonemia (Grıhn and Lindberg, 1985), and has been used as part of an index to evaluate liver activity (Trevisi et al., 2001). The clinical relevance of RPB and its effects on AT metabolism in the dairy cow needs to be elucidated. 18 Tumor necrosis factor alpha Tumor necrosis factor -# was the first pro -inflammatory cytokine shown to be constitutively expressed in the adipocytes of insulin -resistant animals (Hotamisligil et al., 1993) , and is also secreted by AT macrophages (Gregoire et al., 1998) . Besides its pro -inflammatory role, TNF -# modulates AT glucose homeostasis and lipid mobilization in an autocrine manner (Hotamisligil et al., 1993, Vernon and Houseknecht, 2000) . These effects are mediated by downregulating the production and activity of LPL, fatty acid transport proteins (FATP) and acetyl CoA synthase (Arner, 2003) . TNF -# also regulates the synthesis of leptin and plasminogen activator inhibitor 1 (PAI -1), adipocyte fatty acid -binding protein (aP2) and glucose transporter 4 (GLUT4), and is a major contributor to the development of AT insulin resistance (Sethi and Hotamisligil, 1999) . Early studies imply that TNF -# is involved in the control of adipocyte hypertrophy since increasing adipocyte size led to increased production of TNF -# , which in turn inhibits adipogenesis (Hotamisligil and Spiegelman, 1994). The net effect of TNF -# is to decrease lipogenesis (FFA uptake and TAG synthesis) and to increase lipolysis (Arner, 2003) . Accordingly, in dairy cows during the first 5 weeks of lactation, higher expression of TNF -# in subcutaneous AT coincides with increased lipolysis activity (Sadri et al., 2010) . TNF -# action in AT appears to be more auto - and paracrine than endocrine as it is not released into circulation (Mohamed -Ali et al., 1998) . These results are supported by Sadri et al (2010) who described high abundance of TNFA mRNA in AT postpartum despite low plasma TNF -# . Therefore, in dairy cows, adipocyte -derived TNF -# may act locally in a similar way to what has been described for monogastric animals (Ronti et al., 2006, Mukesh et al., 2010) . AT TNF -# secretion is affected by proadipogenic PPAR -" agonist drugs. Administration of such as thiazolidinedione enhanced AT adipogenesis and lipogenesis and increased TNF -# plasma 19 concentrations in periparturient cows; however, AT TNF -# was not evaluated and the plasma concentrations of TNF -# could have derived from immune cells (Schoenberg et al., 2011) . Further investigation is needed to determine the actual importance of adipocyte -derived TNF -# in the end ocrine function in dairy cows. Systemic inflammatory responses can affect TNF -# secretion in AT and modify metabolic function as it inhibits the secretion of leptin and adiponectin (Fasshauer and Paschke, 2003, Vernon, 2005) . In visceral and subcutaneous AT of dairy cows, the expression of TNF -# and IL6 increased after a 2h -LPS challenge demonstrating that AT is capable of rapidly synthesizing pro -inflammatory cytokines when animals are exposed to inflammatory conditions arising from a pathogenic insult or because of parturition (Mukesh et al., 2010) . The same was observed in subcutaneous AT of heat stressed prepartum dairy cows (Zachut et al., 2017) . Besides contributing to AT inflammation, administration of TNF -# promoted lipolysis and insulin resistance in AT of dairy steers (Kushibiki et al., 2001) . In dairy cows, TNF -# serum activity was elevated during moderate to severe fatty liver and insulin resistance (Ohtsuka et al., 2001). These resu lts demonstrate that TNF -# not only activates inflammatory pathways in AT but also promotes peripheral insulin resistance and lipolysis to increase glucose flow to the mammary gland in dairy cows (Cawthorn and Sethi, 2008) and has the potential to be used as a biomarker of metabolic homeostasi s in periparturient dairy cows. Resistin Resistin is a small cysteine -rich secretory protein considered both hormone - and cytokine -like adipokine (Holcomb et al., 2000) . Resistin is overexpressed in white AT of murine models of obesity and downregulated by the insulin -sensitizin g agents thiazolidinediones (Steppan et al., 2001) . Komatsu et al. (2003) were the first to report resistin expression in bovine 20 AT and that its transcription increased in lactating compared with non -lactating dairy cows, while the inverse w as observed in the mammary gland. Resistin modulates lipid metabolism, glucose homeostasis, and feed intake in monogastrics (Tovar et al., 2005, V ⁄zquez et al., 2008) . In dairy cows, recombinant bovine resistin promotes lipolysis in subcutaneous AT explants from animals during the first two months of lactation, and its circulating concentrations are positively associated with plasma FFA (Reverchon et al., 2014) . Nevertheless, circulating resistin was positively associated with subcutaneous AT hydroxycarboxylic acid receptors -1 (HCAR1) expression which mediates insulin -induced anti -lipolytic effects in dairy cows; however, th is effect was not observed in retroperitoneal AT (Weber et al., 2016a) . Plasma resistin is also associated with increased adiposity, and negatively associated with plasma leptin in dairy cows fed a l ow-energy diet from 1 month before calving through lactation (Mellouk et al., 2017) . While some studies report an increase in AT and plasma resistin during the first week of lactation (Komatsu et al., 2003, Reverchon et al., 2014) , others report an increase in circulating resistin concentrations toward parturition and a decrease during the first week of lactation (Webe r et al., 2016a, Mellouk et al., 2017). During early lactation, AT resistin contributes to the development of insulin resistance via inhibition of GLUT4 translocation to the cytomembrane (Komatsu et al., 2003) , in a similar way to what was reported in monogastrics (Steppan et al., 2001) . Resistin stimulates the secretion of pro -inflammatory cytokines by macrophages such as TNF -#, IL -6, and IL -12 (Silswal et al., 2005) and is linked to systemic inflammation in cardiovascular diseases, diabetes, obesity and metabolic syndrome in humans [reviewed by (Abate et al., 2014) ]. The inflammatory effect of resistin in dairy cows has not yet been studied. Although conjecture, this adipokine could induce systemic and local AT inflammation through 21 the stimulation of cytokine production. These findings demonstrate that resistin content in plasma and AT are altered by the metabolic status, diet, and adiposity in periparturient cows. Therefore, the potential of resistin to be a marker of insulin resistance and AT inflammation in dairy cows should be further explored. Visfatin Visfatin is a cytokine -like adipokine and a multifunctional protein highly conserved across species. Visfatin is expressed ubiquitously (Adeghate, 2008) , and was demonstrated to promote cell survival and function by producing nicotinamide mononucleotide, a precursor to NAD+ (Rongvaux et al., 2008) . Like insulin, visfatin stimulates glucose uptake and homeostasis (Fuk uhara et al., 2005) . In dairy cows, AT visfatin mRNA abundance tended to decrease after parturition and was negatively correlated with serum FFA and BHB concentrations (Lemor et al., 2009). Serum visfatin concentrations decreased during the last three weeks before calving and increased back to concentrations observed 3 weeks before parturition by the first week of lactation (Fadden and Bobe, 2016) . Visfatin induces pro -inflammatory response through cytokine secre tion (Moschen et al., 2007) . In cattle, Fadden and Bobe (2016) reported that serum visfatin may s erve as chronic disease indicator and could assist in early detection of cows at increased risk for developing retained placenta, ketosis, and metritis (Fadden and Bobe, 2016) . These results open the possibility of the study of visfatin as a predictive biomarker for periparturient diseases in dairy cows; however, larger epidemiological studies are needed. CONCLUSIONS Adipose tissue functions go bey ond energy storage and supply. Through the secretion of adipokines, AT regulates lipid mobilization, insulin sensitivity, and immune and inflammatory functions in an autocrine, paracrine and endocrine manner. In human medicine, adipokines are 22 used in clini cal practice in preventive and diagnostic strategies for metabolic disorders (Blr, 2012), and are targets for drug development for treatment of metabolic dysfunction (Ouchi et al., 2010), lipodystrophy (Oral et al., 2002) , obesity, and diabetes (Blr, 2014) . In dairy cows, adipokines have potential clinical relevance as biomarkers of AT function, lipid mobilization, adiposity, insulin sensitivity, oxidative stress status, and inflammation, however few adipokines have been explored as biomarkers for health status or as disease predictors [Table 1]. In addition to that, providing that their mode of action is fully delineated, and the key molecular targets are identified, adipokines are promising targets for developing novel diagnostic tools, nutritional strategies, and therapies to improve dairy cowsÕ wellbeing and productivity. 23 FETUIN -A AS A LINK BETWEEN LIPID MOBILIZATION AND I NFLAMMATION Fetuin -A Structure, Biosynthesis and General Properties Fetuin -A (FetA, AHSG, alpha -2-Heremans -Schmid glycoprotein in humans) is a 64 -kDa acidic glycoprotein with 3 cystatin -like domains that was first isolated from newborn calf serum in 1944 (Pedersen, 1944) . Fetuin -A molecule goes thr ough posttranslational modifications, such as proteolytic processing, glycosylation, phosphorylation (Ser and Thr), and sulfation (Jahnen -Dechent et al., 2011) , which may regulate its protein expression levels, stability, and biological activity (Jahnen -Dechent et al., 2011) . For example, while bovine FetA is both N - and 0 -glycosylated and contains nearly 30% of carbohydrates, that of human contains only 13%. The variation in the degree of glycosylation between species may also be asso ciated with differential activities among humans and bovine FetA (Brown et al., 1992) . Fetuin -A is mainly synthesized and secreted by the liver and to a lesser extent the kidneys, placenta and the tongue (Denecke et al., 2003) . In addition to be a hepatokine, FetA has been recently consider ed an adipokine (Chatterjee et al., 2013, Jialal et al., 2015, P”rez -Sotelo et al., 2016) . In these studies, FetA secretion from subcutaneous adipose tissue (AT) was increased in patients with the metabolic syndrom e (Jialal et al., 2015) , while in rats, FetA was increased in visceral and subcutaneous AT during obesity and high fat diet (P”rez -Sotelo et al., 2016) . Besides its involvement in lipid metabolism, as indicated by these studies demonstrating variations in AT abundance depending on AT dysfu nction, fat mass, and amounts of fat in the diet, a number of studies suggest that FetA is a multifunctional protein. Among its main functions, FetA is an inhibitor of ectopic calcification (Sch−fer et al., 2003) and has a primarily role as a binding and carrier protein like albumin (Jahn en-Dechent et al., 2011) . Fetuin -A binds to matrix metalloproteinases (particularly MMP -9) and protects this enzyme from autolytic 24 degradation (Ker et al., 2007) . Upon its rapid calcium -dependent uptake by cells (Chen et al., 2007), FetA also mediates critical roles for cellsÕ metabolism including the activation of PI3 kinase/Akt (Kundranda et al., 2004) and the mediation of grow th signaling in tumor cells (Sakwe et al., 2010) . Notably, FetA is an antagonist of TGF -§ in vivo, in that it inhibited intestinal tumor progression (Swallow et al., 2004) . FetA also bin ds and carries lipids in plasma, thus being associated with strong lipogenic activity (Kumbla et al., 1989, Kumbla et al., 1991) . Although FetA binding and carrier properties have been consistently described, a specific receptor for FetA on plasma membrane of cells has not yet been identified. Fet uin -A and lipid metabolism Early studies have characterized bovine FetA as a lipoprotein -like particle corresponding to the density of a high density lipoprotein (HDL) (Kumbla et al., 1989, Kumbla et al., 1991) . Accordingly, FetA binds and transports considerable amounts of lipids including choles terol, cholesteryl esters, triacylglycerol (TAG) , and fatty acids ( FA) that nearly account for 33% of the its molecule (Kumbla et al., 1991, Subbiah, 1991, Jialal et al., 2016) . Like FA binding proteins (i.e. FABP4 , FATP1), FetA reversibly binds hydrophobic ligands, including saturated and unsaturated long -chain FA, and other lipids with high affinity (Furuhashi and Hotamisligil, 2008). Others studies suggest that FetA may also remove cholesterol from cells (Kumbla et al., 1991). In this study, the incubation of FetA with cells increased FetA -binding with cholesterol and cholesteryl ester, but had no changes in cellular cholesterol content, while the cellular TAG content increased markedly (Kumbla et al., 1991) . Potent adipogenic and lipogenic activities have been attributed to FetA. An early in vitro study with 1246 adipogenic cell line and 3T3 -L1 cells reported the existence of three adipogenic factors in crude FetA preparation (Zaitsu and Serrero, 1990) . In adipogenic models with rabbit 25 and humanÕs cells, FetA increased incorporation of exogenous FA into cellular TAG by 50 times compared with albumin Ñthe best characterized FA transpor ter Ñ (Kumbla et al., 1989, Cayatt e et al., 1990) . However, the mechanisms by which FetA increased TAG accumulation have not yet been identified. Whether the lipogenic activity of FetA is due to the facilitation of FA entry into cells or whether FetA might directly stimulate lipogenic enz ymes in TAG synthesis pathway is not known. According to its lipogenic properties, FetA has been associated with increased fat mass, obesity, high plasma FFA concentrations in rodents and humans, and with lipid -induced insulin resistance (Chen et al., 2009, Pal et al., 2012, P”rez -Sotelo et al., 2016) . Notably, loss of fat mass induces a reduction in plasma FetA content i n obese humans (Brix et al., 2010, Choi et al., 2013) . These studies highlight not only the involvement of FetA in lipogenic pathways, but also an effect of TAG breakdown on FetA synthesis, and potentially, on its function in AT. Fetuin -A has been associated with differential regulation of lipolysis. A common variation, single nucleotide polymorphism (SNP) rs4917 (Thr230Met), in the FetA gene was associated with a marked increase in ! 2-adrenoceptor sensitivity. This variation affects the sensitivity for lipolysis in adipocytes, and may be of importance in body weight regulation in humans (Lavebratt et al., 2005a) . In fact, another common variant of FetA gene ( AHSG ; homozygosity for the rs2593813:G -230:Met238:Ser haplotype) associated with a lower FetA concentrations, was more common among lean than obese and overweight Swedish men (Lavebratt et al., 2005b) . In a different study, two FetA SNPs ( -469T>G and IVS6+98C>T) were connected to dyslipidemia in human subjects and -469T>G SNP was associated wit h insulin -mediated inhibition of lipolysis and stimulation of lipogenesis in adipocytes (Dahlman et al., 2004, Andersen et al., 2008) . Dahlman et al. (2004) suggested that FetA may control insulin 26 signaling in AT, thus being an attractive candid ate gene for the treatment of disturbed adipocyte lipolytic function in obesity and insulin resistance in humans (Dahlman et al., 2004) . These results consistently demonstrate the roles of FetA on the enhancement of lipogenic activity and modulation of lipolytic responses in adipocytes. However, whether FetA plays similar roles on lipid metabolism of dairy cows and may contribute to the improvement of the lipogenic function during periods of intense lipid mobilization in bovine adipocytes are completely unknown. Fetuin -A and inflammatory responses Fetuin -A is traditionally regarded as one of the few negative acute - phase proteins (APP) (Wang et al., 1997, Ombrellino et al., 2001b, Wang and Sama, 2012) . While FetA is known to inhibit TNF - # production by immune cells during acute inflammation (Wang and Sama, 2012) , this protein is also downregulated by TNF - #, IL -6, IL -1, and interferon gamma (IFN -") (Daveau et al., 1988, Li et al., 2011) . Low serum FetA concentrations have been strongly associated with inflammatory markers like C -reactive protein (Wang et al., 2005, Metry et al., 2008) . The strong anti -inflammatory effects of FetA were verified in vivo using several models of inflammation, including lipopolysaccharide -induced miscarriage in rats (Dziegielewska and Andersen, 1998) , carrageenan injection (Ombrellino et al., 2001b) , cerebral ischemic in jury in rodents (Wang and Sama, 201 2), and cecal ligation and puncture in mice (Li et al., 2011) . In all these reports, FetA was associated with reduced inflammatory response and increased survival and administer ing additional FetA generally improved outcome. Accordingly, FetA deficiency is linked to features of the malnutrition -inflammation -atherosclerosis syndrome and to cardiovascular events and mortality in dialysis patients (Wang et al., 2005) . 27 Few studies have focused on the roles of FetA on AT inflammation. During chronic obesity -induced inflammation, FetA appears to upregulate pro -inflammatory mediators and impairs the response to insulin in cells of AT, liver and skeletal muscle (Stefan et al., 2008, Dasgupta et al., 2010) . Adipose tissue expression of FetA wa s upregulated during chronic inflammation in obese dyslipidemic humans and rodents (Heinrichsdorff and Olefsky, 2012, Jialal et al., 2015, P”rez -Sotelo et al., 2016) . In fact, FetA was identified as an endogenous l igand between FFA and toll -like receptor -4 in adipocytes that triggers lipid -induced inflammation resulting in insulin resistance in AT (Heinrichsdorff and Olefsky, 2012, Pal et al., 2012) . In contrast to these res ults, but in agreement with other reports classifying FetA as a negative APP, in the first report of FetA abundance in AT of dairy cows, FetA expression decreased 1.5 times in the subcutaneous AT of cows in late gestation during environmental heat stress (Zachut et al., 2017). This decrease on AT FetA abundance was associated with a decrease in other negative APP such as albumin, hemopexin, serotransferrin and apolipoprotein A -II (Zachut et al., 2017) . One of the potential explanations for this finding is that FetA is downregulated by different pro -inflammatory mediators such as TNF - # and IL6, (Wang and Sama, Zhang et al., 2014) , which are known to be upregulated in AT of periparturient dairy cows during intense lipid mobilization (Contreras et al., 2015) . While periparturient dairy cows have increased plasma FFA concentrations, which lead to a systemic and local AT inflammation (Saremi et al., 2014, Contreras et al., 2015, Mann et al., 2016), the potential effects of FetA supporting anti -inflammatory responses during exacerbated FFA release around parturition and through early lactation in dairy cows remain unknown. CONCLUSIONS Based on the importance of FetA in lipid metabolism and inflammatory responses, FetA 28 has emerged as a predictor and biomarker of diseases and metabolic dysfunction in several clinical epidemiological studies in human patients. Evidence from in vitro and in vivo studies with non -ruminants, and from the single report describing decreased FetA abundance in AT of periparturient cows during a pro -inflammatory and pro -lipolytic state (Zachut et al., 2017) , support the possibility that FetA could also be involved in the modulation of AT functions in dairy cows, especially during the periparturient period when lipid mobilization and inflammatory responses are intensified. Based on previous reports, FetA may have the potential to buffer excessive release of FFA from AT of periparturient dairy cows by (1) stimulating the use of circulating FFA for intracellular TAG synthesis in AT, (2) mediating insulin -inhibition of lipolysis, and (3) preventing dysregulated pro -inflammatory responses, thus also attenuating exacerbated lipolytic activity in AT. However, very little is known about FetA dynamics and roles in dairy cows. A better understanding of the role of FetA o n modulating the met abolic and AT function of periparturient dairy cows could lead to the development of novel management, nutritional, or pharmaceutical interventions to reduce around parturition. Acknowledgements This work was supported in part by USDA -National Institute o f Food and Agriculture (Washington, DC) grant 2015 -67015-23207, Department of Large Animal Clinical Sciences (East Lansing, MI), and the Michigan Alliance for Animal Agriculture (East Lansing, MI). The authors are grateful to Oscar Benitez for his assistan ce with graphical design. 29 REFERENCES 30 REFERENCES Abate, N., H. S Sallam, M. Rizzo, D. Nikolic, M. Obradovic, P. Bjelogrlic, and E. R Isenovic. 2014. Resistin: an inflammatory cytokine. Role in cardiovascular diseases, diabetes and the metabol ic syndrome. Current pharmaceutical design 20(31):4961 -4969. Adeghate, E. 2008. Visfatin: structure, function and relation to diabetes mellitus and other dysfunctions. Current medicinal chemistry 15(18):1851 -1862. Andersen, G., K. S. Burgdorf, T. Spars¿, K . Borch -Johnsen, T. J¿rgensen, T. Hansen, and O. Pedersen. 2008. AHSG tag single nucleotide polymorphisms associate with type 2 diabetes and dyslipidemia: studies of metabolic traits in 7,683 white Danish subjects. Diabetes 57(5):1427 -1432. Arner, P. 2003. The adipocyte in insulin resistance: key molecules and the impact of the thiazolidinediones. Trends in Endocrinology & Metabolism 14(3):137 -145. Bauman, D. E. and W. B. Currie. 1980. Partitioning of nutrients during pregnancy and lactation: a review of me chanisms involving homeostasis and homeorhesis. J Dairy Sci 63(9):1514 -1529. Bell, A. W. and D. E. Bauman. 1997. Adaptations of glucose metabolism during pregnancy during pregnancy and lactation. J Mammary Gland Biol Neoplasia 2(3):265 -278. Block, S., W. B utler, R. Ehrhardt, A. Bell, M. Van Amburgh, and Y. Boisclair. 2001. Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance. Journal of Endocrinology 171(2):339 -348. Block, S., R. Rhoads, D. Bauman, R. Eh rhardt, M. McGuire, B. Crooker, J. Griinari, T. Mackle, W. Weber, and M. Van Amburgh. 2003. Demonstration of a role for insulin in the regulation of leptin in lactating dairy cows. Journal of dairy science 86(11):3508 -3515. Blr, M. 2012. Clinical releva nce of adipokines. Diabetes & metabolism journal 36(5):317 -327. Blr, M. 2014. Adipokines Ðremoving road blocks to obesity and diabetes therapy. Molecular metabolism 3(3):230 -240. Brix, J. M., H. Stingl, F. Hıllerl, G. H. Schernthaner, H. -P. Kopp, and G. Schernthaner. 2010. Elevated Fetuin -A Concentrations in Morbid Obesity Decrease after Dramatic Weight Loss. J Clin Endocrinol Metab 95(11):4877 -4881. Brown, W., K. Dziegielewska, N. Saunders, and K. M¿sllg„rd. 1992. Fetuin !an old friend revisited. Bioessays 14(11):749 -755. Cawthorn, W. P. and J. K. Sethi. 2008. TNF -# and adipocyte biology. FEBS letters 582(1):117 -131. 31 Cayatte, A. J., L. Kumbla, and M. T. Subbiah. 1990. Marked acceleration of exogenous fatty acid incorporati on into cellular triglycerides by fetuin. Journal of Biological Chemistry 265(10):5883 -5888. Chatterjee, P., S. Seal, S. Mukherjee, R. Kundu, S. Mukherjee, S. Ray, S. Mukhopadhyay, S. S. Majumdar, and S. Bhattacharya. 2013. Adipocyte Fetuin -A Contributes t o Macrophage Migration into Adipose Tissue and Polarization of Macrophages. J Biol Chem 288(39):28324 -28330. Chen, H. -Y., Y. -L. Chiu, S. -P. Hsu, M. -F. Pai, C. -F. Lai, Y. -S. Peng, T. -W. Kao, K. -Y. Hung, T.-J. Tsai, and K. -D. Wu. 2009. Association of serum f etuin A with truncal obesity and dyslipidemia in non -diabetic hemodialysis patients. Eur J Endocrinol 160(5):777 -783. Chen, N. X., K. D. O'Neill, X. Chen, D. Duan, E. Wang, M. S. Sturek, J. M. Edwards, and S. M. Moe. 2007. Fetuin -A uptake in bovine vascula r smooth muscle cells is calcium dependent and mediated by annexins. American Journal of Physiology -Renal Physiology 292(2):F599 -F606. Chilliard, Y., M. Bonnet, C. Delavaud, Y. Faulconnier, C. Leroux, J. Djiane, and F. Bocquier. 2001. Leptin in ruminants. Gene expression in adipose tissue and mammary gland, and regulation of plasma concentration. Domestic Animal Endocrinology 21(4):271 -295. Choi, K. M., K. A. Han, H. J. Ahn, S. Y. Lee, S. Y. Hwang, B. -H. Kim, H. C. Hong, H. Y. Choi, S. J. Yang, H. J. Yoo, S . H. Baik, D. S. Choi, and K. W. Min. 2013. The effects of caloric restriction on Fetuin -A and cardiovascular risk factors in rats and humans: a randomized controlled trial. Clin Endocrinol 79(3):356 -363. Contreras, G. A., E. Kabara, J. Brester, L. Neuder, and M. Kiupel. 2015. Macrophage infiltration in the omental and subcutaneous adipose tissues of dairy cows with displaced abomasum. Journal of Dairy Science 98(9):6176 -6187. Contreras, G. A. and L. M. Sordillo. 2011. Lipid mobilization and inflammatory re sponses during the transition period of dairy cows. Comp Immunol Microbiol Infect Dis 34(3):281 -289. Contreras, G. A., C. Strieder -Barboza, and W. Raphael. 2017. Adipose tissue lipolysis and remodeling during the transition period of dairy cows. Journal of Animal Science and Biotechnology 8(1):41. Dahlman, I., P. Eriksson, M. Kaaman, H. Jiao, C. Lindgren, J. Kere, and P. Arner. 2004. # 2-Heremans ÐSchmid glycoprotein gene polymorphisms are associated with adipocyte insulin action. Diabetologia 47(11):1974 -1979. Dasgupta, S., S. Bhattacharya, A. Biswas, S. S. Majumdar, S. Mukhopadhyay, and S. Ray. 2010. NF-kappaB mediates lipid -induced fetuin -A expression in hepatocytes that impairs adipocyte function effecting insulin resistance. Biochemical Journal 429(3):451 -462. Daveau, M., D. Christian, N. Julen, M. Hiron, P. Amaud, and J. -P. Lebreton. 1988. The synthesis of human #-2-HS glycoprotein is down -regulated by cytokines in hepatoma HepG2 cells. FEBS Letters 241(1):191 -194. 32 De Koster, J., C. Urh, M. Hostens, W. Van den Broeck, H. Sauerwein, and G. Opsomer. 2017. Relationship between serum adiponectin concentration, body condition score, and peripheral tissue insulin response of dairy cows during the dry period. Domestic Animal Endocrinology 59:100 -104. Denecke, B., S. Gr −ber, C. Sch−fer, A. Heiss, M. Wıltje, and W. Jahnen -Dechent. 2003. Tissue distribution and activity testing suggest a similar but not identical function of fetuin -B and fetuin -A. Biochemical Journal 376(1):135 -145. Dziegielewska, K. and N. Andersen. 1998. The fetal glycoprotein, fetuin, counteracts ill -effects of the bacterial endotoxin, lipopolysaccharide, in pregnancy. Neonatology 74(5):372 -375. Ehrhardt, R. A., A. Foskolos, S. L. Giesy, S. R. Wesolowski, C. S. Krumm, W. R. Butler, S. M. Quirk, M. R. Wal dron, and Y. R. Boisclair. 2016. Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows. Journal of Endocrinology 229(2):145 -157. Eldaim, M. A. A., A. Kamikawa, M. M. Soliman, M. M. Ahmed, Y. Okamatsu -Ogura, A. Terao, T. Miyam oto, and K. Kimura. 2010. Retinol binding protein 4 in dairy cows: its presence in colostrum and alteration in plasma during fasting, inflammation, and the peripartum period. Journal of dairy research 77(1):27 -32. Fadden, A. and G. Bobe. 2016. Serum Visfat in is a Predictive Indicator of Retained Placenta and Other Diseases in Dairy Cows. Journal of Veterinary Science & Medical Diagnosis 2016. Fasshauer, M. and R. Paschke. 2003. Regulation of adipocytokines and insulin resistance. Diabetologia 46(12):1594 -1603. Fukuhara, A., M. Matsuda, M. Nishizawa, K. Segawa, M. Tanaka, K. Kishimoto, Y. Matsuki, M. Murakami, T. Ichisaka, and H. Murakami. 2005. Visfatin: a protein secreted by visceral fat that mimics the effects of insulin. Science 307(5708):426 -430. Furuhas hi, M. and G. S. Hotamisligil. 2008. Fatty acid -binding proteins: role in metabolic diseases and potential as drug targets. Nature reviews Drug discovery 7(6):489. Giesy, S. L., B. Yoon, W. B. Currie, J. W. Kim, and Y. R. Boisclair. 2012. Adiponectin Defic it During the Precarious Glucose Economy of Early Lactation in Dairy Cows. Endocrinology 153(12):5834 -5844. Gray, N. E., L. N. Lam, K. Yang, A. Y. Zhou, S. Koliwad, and J. -C. Wang. 2012. Angiopoietin -like 4 (Angptl4) protein is a physiological mediator of intracellular lipolysis in murine adipocytes. Journal of Biological Chemistry 287(11):8444 -8456. Gregoire, F. M., C. M. Smas, and H. S. Sul. 1998. Understanding adipocyte differentiation. Physiological reviews 78(3):783 -809. Grıhn, Y. and L. -A. Lindberg. 1 985. Ultrastructural changes of the liver in spontaneously ketotic cows. Journal of comparative pathology 95(3):443 -452. 33 H−ussler, S., C. Sacr”, K. Friedauer, S. D−nicke, and H. Sauerwein. 2015. Short communication: Localization and expression of monocyte chemoattractant protein -1 in different subcutaneous and visceral adipose tissues of early -lactating dairy cows. Journal of Dairy Science 98(9):6278 -6283. Heinrichsdorff, J. and J. M. Olefsky. 2012. Fetuin -A: the missing link in lipid -induced inflammation. Nat Med 18(8):1182 -1183. Holcomb, I. N., R. C. Kabakoff, B. Chan, T. W. Baker, A. Gurney, W. Henzel, C. Nelson, H. B. Lowman, B. D. Wright, and N. J. Skelton. 2000. FIZZ1, a novel cysteine !rich secreted protein associated with pulmonary inflammation, defin es a new gene family. The EMBO journal 19(15):4046 -4055. Hotamisligil, G. S., N. S. Shargill, and B. M. Spiegelman. 1993. Adipose expression of tumor necrosis factor -alpha: direct role in obesity -linked insulin resistance. Science 259(5091):87 -91. Ingvarts en, K. L. and Y. Boisclair. 2001. Leptin and the regulation of food intake, energy homeostasis and immunity with special focus on periparturient ruminants. Domestic animal endocrinology 21(4):215 -250. Jahnen -Dechent, W., A. Heiss, C. Sch−fer, and M. Kettel er. 2011. Fetuin -A regulation of calcified matrix metabolism. Circulation research 108(12):1494 -1509. Jialal, I., S. Devaraj, and B. Adams -Huet. 2016. Plasma fetuin -A does not correlate with monocyte TLR4 in humans. Diabetologia 59(1):222 -223. Jialal, I., S. Devaraj, A. Bettaieb, F. Haj, and B. Adams -Huet. 2015. Increased adipose tissue secretion of Fetuin -A, lipopolysaccharide -binding protein and high -mobility group box protein 1 in metabolic syndrome. Atherosclerosis 241(1):130 -137. Kabara, E., L. M. Sord illo, S. Holcombe, and G. A. Contreras. 2014. Adiponectin links adipose tissue function and monocyte inflammatory responses during bovine metabolic stress. Comp Immunol Microbiol Infect Dis 37(1):49 -58. Kadowaki, T. and T. Yamauchi. 2005. Adiponectin and a diponectin receptors. Endocr Rev 26. Kasimanickam, R. K., V. R. Kasimanickam, J. R. Olsen, E. J. Jeffress, D. A. Moore, and J. P. Kastelic. 2013. Associations among serum pro - and anti -inflammatory cytokines, metabolic mediators, body condition, and uterin e disease in postpartum dairy cows. Reproductive Biology and Endocrinology 11(1):103. Kersten, S. 2005. Regulation of lipid metabolism via angiopoietin -like proteins. Portland Press Limited. Klıting, N., T. E. Graham, J. Berndt, S. Kralisch, P. Kovacs, C. J. Wason, M. Fasshauer, M. R. Schın, M. Stumvoll, M. Blr, and B. B. Kahn. 2007. Serum Retinol -Binding Protein Is More Highly Expressed in Visceral than in Subcutaneous Adipose Tissue and Is a Marker of Intra -abdominal Fat Mass. Cell Metab 6(1):79 -87. 34 Koltes, D. and D. Spurlock. 2012. Adipose tissue angiopoietin -like protein 4 messenger RNA changes with altered energy balance in lactating Holstein cows. Domestic animal endocrinology 43(4):307 -316. Komatsu, T., F. Itoh, S. Mikawa, and K. Hodate. 2003. Gene expression of resistin in adipose tissue and mammary gland of lactating and non -lactating cows. Journal of Endocrinology 178(3):R1 -R5. Ker, D., D. Gosenca, M. Wind, H. Heid, I. Friedberg, W. Jahnen -Dechent, and W. D. Lehmann. 2007. Proteolytic processi ng by matrix metalloproteinases and phosphorylation by protein kinase CK2 of fetuin -A, the major globulin of fetal calf serum. Biochimie 89(3):410 -418. Kumbla, L., S. Bhadra, and M. T. Subbiah. 1991. Multifunctional role for fetuin (fetal protein) in lipid transport. The FASEB Journal 5(14):2971 -2975. Kumbla, L., A. Cayatte, and M. Subbiah. 1989. Association of a lipoprotein -like particle with bovine fetuin. The FASEB Journal 3(9):2075 -2080. Kundranda, M. N., S. Ray, M. Saria, D. Friedman, L. M. Matrisian, P. Lukyanov, and J. Ochieng. 2004. Annexins expressed on the cell surface serve as receptors for adhesion to immobilized fetuin -A. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1693(2):111 -123. Kushibiki, S., K. Hodate, H. Shingu, Y. Ueda, M. Shinoda, Y. Mori, T. Itoh, and Y. Yokomizo. 2001. Insulin resistance induced in dairy steers by tumor necrosis factor alpha is partially reversed by 2,4 Ðthiazolidinedione. Domestic Animal Endocrinology 21(1):25 -37. Laeger, T., H. Sauerwein, A. Tuchscher er, O. Bellmann, C. C. Metges, and B. Kuhla. 2013. Concentrations of hormones and metabolites in cerebrospinal fluid and plasma of dairy cows during the periparturient period. Journal of Dairy Science 96(5):2883 -2893. Lavebratt, C., E. Dungner, and J. Hoff stedt. 2005a. Polymorphism of the AHSG gene is associated with increased adipocyte ! 2-adrenoceptor function. Journal of lipid research 46(10):2278 -2281. Lavebratt, C., S. Wahlqvist, L. Nordfors, J. Hoffstedt, and P. Arner. 2005b. AHSG gene variant is assoc iated with leanness among Swedish men. Human Genetics 117(1):54 -60. Lehr, S., S. Hartwig, and H. Sell. 2012. Adipokines: a treasure trove for the discovery of biomarkers for metabolic disorders. PROTEOMICS -Clinical Applications 6(1 !2):91 -101. Lemor, A., A. Hosseini, H. Sauerwein, and M. Mielenz. 2009. Transition period -related changes in the abundance of the mRNAs of adiponectin and its receptors, of visfatin, and of fatty acid binding receptors in adipose tissue of high -yielding dairy cows. Domestic Animal Endocrinology 37(1):37 -44. 35 Leury, B. J., L. H. Baumgard, S. S. Block, N. Segoale, R. A. Ehrhardt, R. P. Rhoads, D. E. Bauman, A. W. Bell, and Y. R. Boisclair. 2003. Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows. Am J P hysiol Regul Integr Comp Physiol 285:1107 -1115. Li, S. 2011. Angiopoietin -like protein 4 in bovine physiology. Kansas State University. Li, W., S. Zhu, J. Li, Y. Huang, Z. Rongrong, X. Fan, H. Yang, X. Gong, N. T. Eissa, W. Jahnen -Dechent, P. Wang, K. J. T racey, A. E. Sama, and H. Wang. 2011. A Hepatic Protein, Fetuin -A, Occupies a Protective Role in Lethal Systemic Inflammation. PLoS ONE 6(2):e16945. Loor, J. J., R. E. Everts, M. Bionaz, H. M. Dann, D. E. Morin, R. Oliveira, S. L. Rodriguez -Zas, J. K. Drac kley, and H. A. Lewin. 2007. Nutrition -induced ketosis alters metabolic and signaling gene networks in liver of periparturient dairy cows. Physiological Genomics 32(1):105 -116. McNamara, J. P. and J. K. Hillers. 1986. Adaptations in lipid metabolism of bov ine adipose tissue in lactogenesis and lactation. Journal of Lipid Research 27(2):150 -157. Mellouk, N., C. Rame, J. L. Touz”, E. Briant, L. Ma, D. Guillaume, D. Lomet, A. Caraty, T. Ntallaris, P. Humblot, and J. Dupont. 2017. Involvement of plasma adipokin es in metabolic and reproductive parameters in Holstein dairy cows fed with diets with differing energy levels. Journal of Dairy Science 100(10):8518 -8533. Metry, G., P. Stenvinkel, A. R. Qureshi, J. J. Carrero, M. I. Yilmaz, P. B⁄r⁄ny, S. Snaedal, O. Heim ger, B. Lindholm, and M. E. Suliman. 2008. Low serum fetuin -A concentration predicts poor outcome only in the presence of inflammation in prevalent haemodialysis patients. Eur J Clin Invest 38(11):804 -811. Mohamed -Ali, V., J. Pinkney, and S. Coppack. 19 98. Adipose tissue as an endocrine and paracrine organ. International Journal of Obesity & Related Metabolic Disorders 22(12). Moschen, A. R., A. Kaser, B. Enrich, B. Mosheimer, M. Theurl, H. Niederegger, and H. Tilg. 2007. Visfatin, an Adipocytokine with Proinflammatory and Immunomodulating Properties. The Journal of Immunology 178(3):1748 -1758. Mukesh, M., M. Bionaz, D. E. Graugnard, J. K. Drackley, and J. J. Loor. 2010. Adipose tissue depots of Holstein cows are immune responsive: Inflammatory gene expre ssion in vitro. Domestic Animal Endocrinology 38(3):168 -178. Ohtsuka, H., M. Koiwa, A. Hatsugaya, K. Kudo, F. Hoshi, N. Itoh, H. Yokota, H. Okada, and S. -i. Kawamura. 2001. Relationship between Serum TNF Activity and Insulin Resistance in Dairy Cows Affect ed with Naturally Occurring Fatty Liver. Journal of Veterinary Medical Science 63(9):1021 -1025. Ombrellino, M., H. Wang, H. Yang, M. Zhang, J. Vishnubhakat, A. Frazier, L. A. Scher, S. G. Friedman, and K. J. Tracey. 2001. Fetuin, a negative acute phase pro tein, attenuates TNF synthesis and the innate inflammatory response to carrageenan. Shock 15(3):181 -185. 36 Oral , E. A., V. Simha , E. Ruiz , A. Andewelt , A. Premkumar , P. Snell , A. J. Wagner , A. M. DePaoli , M. L. Reitman , S. I. Taylor , P. Gorden , an d A. Garg 2002. Leptin -Replacement Therapy for Lipodystrophy. New England Journal of Medicine 346(8):570 -578. Ouchi, N., A. Higuchi, K. Ohashi, Y. Oshima, N. Gokce, R. Shibata, Y. Akasaki, A. Shimono, and K. Walsh. 2010. Sfrp5 is an anti -inflammatory adipo kine that modulates metabolic dysfunction in obesity. Science 329(5990):454 -457. Pal, D., S. Dasgupta, R. Kundu, S. Maitra, G. Das, S. Mukhopadhyay, S. Ray, S. S. Majumdar, and S. Bhattacharya. 2012. Fetuin -A acts as an endogenous ligand of TLR4 to promote lipid -induced insulin resistance. Nature Medicine 18(8):1279 -1285. Pedersen, K. O. 1944. Fetuin, a new globulin isolated from serum. Nature 154(3914):575. P”rez -Sotelo, D., A. Roca -Rivada, M. Larrosa -Garc™a, C. Castelao, I. Baamonde, J. Baltar, A. B. Cruj eiras, L. M. Seoane, F. F. Casanueva, and M. Pardo. 2016. Visceral and subcutaneous adipose tissue express and secrete functional alpha2hsglycoprotein (fetuin a) especially in obesity. Endocrine:1 -12. Preedy, V. R. and R. J. Hunter. 2016. Adipokines. CRC P ress. Reverchon, M., C. Rame, J. Cognie, E. Briant, S. Elis, D. Guillaume, and J. Dupont. 2014. Resistin in dairy cows: plasma concentrations during early lactation, expression and potential role in adipose tissue. PloS one 9(3):e93198. Rezamand, P., J. Wa tts, K. Hunt, B. Bradford, L. Mamedova, and S. Morey. 2012a. Bovine hepatic and adipose retinol -binding protein gene expression and relationship with tumor necrosis factor -# . Journal of dairy science 95(12):7097 -7104. Rezamand, P., J. S. Watts, K. M. Hunt, B. J. Bradford, L. K. Mamedova, and S. D. Morey. 2012b. Bovine hepatic and adipose retinol -binding protein gene expression and relationship with tumor necrosis factor -# . Journal of Dairy Science 95(12):7097 -7104. Rongvaux, A., M. Galli, S. Denanglaire, F. Van Gool, P. L. Dreze, C. Szpirer, F. Bureau, F. Andris, and O. Leo. 2008. Nicotinamide phosphoribosyl transferase/pre -B cell colony -enhancing factor/visfatin is required for lymphocyte development and cellular resistance to genotoxic stress. The Journal of Immunology 181(7):4685 -4695. Ronti, T., G. Lupattelli, and E. Mannarino. 2006. The endocrine function of adipose tissue: an update. Clinical endocrinology 64(4):355 -365. Sadri, H., R. M. Bruckmaier, H. R. Rahmani, G. R. Ghorbani, I. Morel, and H. A. Van Dorland. 2010. Gene expression of tumour necrosis factor and insulin signalling -related factors in subcutaneous adipose tissue during the dry period and in early lactation in dairy cows. Journal of Animal Physiology and Animal Nutrition 94(5):e194 -e202. 37 Sakwe, A. M., R. Koumangoye, S. J. Goodwin, and J. Ochieng. 2010. Fetuin -A ( # 2HS -glycoprotein) is a major serum adhesive protein that mediates growth signaling in breast tumor cells. Journal of Biological Chemistry 285(53):41827 -41835. Saremi, B., S. Winand , P. Friedrichs, A. Kinoshita, J. Rehage, S. Danicke, S. Haussler, G. Breves, M. Mielenz, and H. Sauerwein. 2014. Longitudinal profiling of the tissue -specific expression of genes related with insulin sensitivity in dairy cows during lactation focusing on different fat depots. PLoS One 9(1):e86211. Sauerwein, H. and S. H−u§ler. 2016. Endogenous and exogenous factors influencing the concentrations of adiponectin in body fluids and tissues in the bovine. Domestic Animal Endocrinology 56:S33 -S43. Sch−fer, C., A. Heiss, A. Schwarz, R. Westenfeld, M. Ketteler, J. Floege, W. Mler -Esterl, T. Schinke, and W. Jahnen -Dechent. 2003. The serum protein # 2ÐHeremans -Schmid glycoprotein/fetuin -A is a systemically acting inhibitor of ectopic calcification. J Clin Investig 112(3):357 -366. Schoenberg, K., K. Perfield, J. Farney, B. Bradford, Y. Boisclair, and T. Overton. 2011. Effects of prepartum 2, 4 -thiazolidinedione on insulin sensitivity, plasma concentrations of tumor necrosis factor -# and leptin, and adipose tissue gen e expression. Journal of dairy science 94(11):5523 -5532. Sethi, J. K. and G. S. Hotamisligil. 1999. The role of TNF # in adipocyte metabolism. Pages 19 -29 in Proc. Seminars in cell & developmental biology. Elsevier. Silswal, N., A. K. Singh, B. Aruna, S. Mu khopadhyay, S. Ghosh, and N. Z. Ehtesham. 2005. Human resistin stimulates the pro -inflammatory cytokines TNF -# and IL -12 in macrophages by NF-&B-dependent pathway. Biochemical and biophysical research communications 334(4):1092 -1101. Singh, S. P., S. H−uss ler, J. J. Gross, F. J. Schwarz, R. M. Bruckmaier, and H. Sauerwein. 2014a. Short communication: Circulating and milk adiponectin change differently during energy deficiency at different stages of lactation in dairy cows. Journal of Dairy Science 97(3):153 5-1542. Singh, S. P., S. H−ussler, J. F. L. Heinz, B. Saremi, B. Mielenz, J. Rehage, S. D−nicke, M. Mielenz, and H. Sauerwein. 2014b. Supplementation with conjugated linoleic acids extends the adiponectin deficit during early lactation in dairy cows. Gener al and Comparative Endocrinology 198:13 -21. Stefan, N., A. Fritsche, C. Weikert, H. Boeing, H. -G. Joost, H. -U. H−ring, and M. B. Schulze. 2008. Plasma Fetuin -A Levels and the Risk of Type 2 Diabetes. Diabetes 57(10):2762 -2767. Steppan, C. M., S. T. Bailey, S. Bhat, and E. J. Brown. 2001. The hormone resistin links obesity to diabetes. Nature 409(6818):307. 38 Strieder -Barboza, C., J. de Souza, W. Raphael, A. L. Lock, and G. A. Contreras. 2017. Fetuin -A: A negative acute -phase protein linked to adipose tissue f unction in periparturient dairy cows. Journal of Dairy Science In Press. Subbiah, M. R. 1991. Newly recognized lipid carrier proteins in fetal life. Exp Biol Med 198(1):495 -499. Sumner -Thomson, J. M., J. L. Vierck, and J. P. McNamara. 2011. Differential ex pression of genes in adipose tissue of first -lactation dairy cattle1. Journal of Dairy Science 94(1):361 -369. Swallow, C. J., E. A. Partridge, J. C. Macmillan, T. Tajirian, G. M. DiGuglielmo, K. Hay, M. Szweras, W. Jahnen -Dechent, J. L. Wrana, and M. Redst on. 2004. # 2HS -glycoprotein, an antagonist of transforming growth factor ! in vivo, inhibits intestinal tumor progression. Cancer research 64(18):6402 -6409. Tovar, S., R. Nogueiras, L. Y. Tung, T. R. Castaneda, M. J. V⁄zquez, A. Morris, L. M. Williams, S. L. Dickson, and C. Di”guez. 2005. Central administration of resistin promotes short -term satiety in rats. European Journal of Endocrinology 153(3):R1 -R5. Trevisi, E., L. Calamari, and G. Bertoni. 2001. Definition of liver activity index in the dairy cow and its relationship with the reproductive performance. Pages 118 -119 in Proc. Proceedings X International Symposium of Veterinary Laboratory Diagnosticians, Salsomaggiore -Parma, Italy. Vailati -Riboni, M., M. Kanwal, O. Bulgari, S. Meier, N. V. Priest, C. R. Burke, J. K. Kay, S. McDougall, M. D. Mitchell, C. G. Walker, M. Crookenden, A. Heiser, J. R. Roche, and J. J. Loor. 2016. Body condition score and plane of nutrition prepartum affect adipose tissue transcriptome regulators of metabolism and inflammation in grazing dairy cows during the transition period. Journal of Dairy Science 99(1):758 -770. V⁄zquez, M. a. J., C. R. Gonz⁄lez, L. Varela, R. Lage, S. Tovar, S. Sangiao -Alvarellos, L. M. Williams, A. Vidal -Puig, R. Nogueiras, and M. LŠpez. 2008. Central resistin regulates hypothalamic and peripheral lipid metabolism in a nutritional -dependent fas hion. Endocrinology 149(9):4534 -4543. Vernon, R. and K. Houseknecht. 2000. Adipose tissue: beyond an energy reserve. Ruminant physiology: digestion, metabolism, growth and reproduction (ed. PB Cronj”):171 -186. Vernon, R. G. 2005. Lipid metabolism during la ctation: a review of adipose tissue -liver interactions and the development of fatty liver. Journal of dairy research 72(4):460. Waki, H., T. Yamauchi, J. Kamon, Y. Ito, S. Uchida, S. Kita, K. Hara, Y. Hada, F. Vasseur, and P. Froguel. 2003. Impaired multim erization of human adiponectin mutants associated with diabetes molecular structure and multimer formation of adiponectin. Journal of Biological Chemistry 278(41):40352 -40363. Wang, A. Y. -M., J. Woo, C. W. -K. Lam, M. Wang, I. H. -S. Chan, P. Gao, S. -F. Lui, P. K. -T. Li, and J. E. Sanderson. 2005. Associations of serum fetuin -A with malnutrition, inflammation, 39 atherosclerosis and valvular calcification syndrome and outcome in peritoneal dialysis patients. Nephrology Dialysis Transplantation 20(8):1676 -1685. Wang, H. and A. E. Sama. 2012. Anti -inflammatory role of Fetuin -A in Injury and Infection. Curr Mol Med 12(5):625 -633. Wang, H., M. Zhang, K. Soda, A. Sama, and K. J. Tracey. 1997. Fetuin protects the fetus from TNF. Lancet 350(9081):861 -862. Wang, M. -Y., Y . Lee, and R. H. Unger. 1999. Novel form of lipolysis induced by leptin. Journal of Biological Chemistry 274(25):17541 -17544. Weber, M., L. Locher, K. Huber, ç. Ken”z, J. Rehage, R. Tienken, U. Meyer, S. D−nicke, H. Sauerwein, and M. Mielenz. 2016a. Longit udinal changes in adipose tissue of dairy cows from late pregnancy to lactation. Part 1: The adipokines apelin and resistin and their relationship to receptors linked with lipolysis. Journal of Dairy Science 99(2):1549 -1559. Weber, M., L. Locher, K. Huber, J. Rehage, R. Tienken, U. Meyer, S. D−nicke, L. Webb, H. Sauerwein, and M. Mielenz. 2016b. Longitudinal changes in adipose tissue of dairy cows from late pregnancy to lactation. Part 2: The SIRT -PPARGC1A axis and its relationship with the adiponectin syst em. Journal of Dairy Science 99(2):1560 -1570. Wells, H. G. 1940. Adipose tissue, a neglected subject. Journal of the American Medical Association 114(22):2177 -2183. William, W., R. Ceddia, and R. Curi. 2002. Leptin controls the fate of fatty acids in isola ted rat white adipocytes. Journal of endocrinology 175(3):735 -744. Yamauchi, T., J. Kamon, Y. Ito, A. Tsuchida, T. Yokomizo, S. Kita, T. Sugiyama, M. Miyagishi, K. Hara, M. Tsunoda, K. Murakami, T. Ohteki, S. Uchida, S. Takekawa, H. Waki, N. H. Tsuno, Y. S hibata, Y. Terauchi, P. Froguel, K. Tobe, S. Koyasu, K. Taira, T. Kitamura, T. Shimizu, R. Nagai, and T. Kadowaki. 2003. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423. Zachut, M. 2015. Defining the Adipose Tissue Proteome of Dairy Cows to Reveal Biomarkers Related to Peripartum Insulin Resistance and Metabolic Status. Journal of Proteome Research 14(7):2863 -2871. Zachut, M., G. Kra, L. Livshitz, Y. Portnick, S. Yakoby, G. Friedlander, and Y. Levin. 2017. Seasonal h eat stress affects adipose tissue proteome toward enrichment of the Nrf2 -mediated oxidative stress response in late -pregnant dairy cows. Journal of Proteomics 158:52 -61. Zaitsu, H. and G. Serrero. 1990. Pedersen fetuin contains three adipogenic factors wit h distinct biochemical characteristics. J Cell Physiol 144(3):485 -491. Zhang, P., H. Shen, J. Huang, H. Wang, B. Zhang, R. Zhou, B. Zhong, and X. Fan. 2014. Intraperitoneal Administration of Fetuin -A Attenuates d -Galactosamine/Lipopolysaccharide -Induced Li ver Failure in Mouse. Dig Dis Sci 59(8):1789 -1797. 40 CHAPTER 3 FETUIN -A: A NEGATIVE ACUTE -PHASE PROTEIN LINKED TO ADIPOSE TISSUE FUNCTION IN PERIPARTURIENT DAIRY COWS Clarissa Strieder -Barboza, Jonas de Souza, William Raphael, Adam L. Lock, G. Andres Contr eras J. Dairy Sci. 101:2602 Ð2616. https://doi.org/10.3168/jds.2017 -13644 ABSTRACT Fetuin -A ( FetA ) is a free fatty acid ( FFA) transporter and an acute -phase protein ( APP ) that enhances cellular lipid uptake and lipogenesis. In non -ruminants, FetA is invol ved in lipid -induced inflammation. Despite FetA importance in lipid metabolism and inflammation, its expression and dynamics in adipose tissue ( AT) of dairy cows are unknown. The objectives of this study were to: 1) Determine serum and AT FetA dynamics ove r the periparturient period and in mid -lactation cows in negative energy balance ( NEB ) after a feed restriction protocol. 2) Characterize how an inflammatory challenge affects adipocyte FetA expression. Blood and subcutaneous AT were collected from 16 cows with high ( ' 3.75, n=8) or moderate BCS ( ( 3.5, n=8) at -26±7 (far off, FO) and -8±5 (close -up, CU) days before calving, and at 10±2 d after parturition (early lactation, EL), and from 14 non -pregnant mid -lactation cows (>220 DIM ) after a feed restriction p rotocol . Serum FetA concentrations were 0.89±0.13 at FO, 0.96±0.13 at CU, and 0.77±0.13 mg/mL at EL, and 1.09±0.09 and 1.17±0.09 mg/mL in feed -restricted and control cows, respectively. Serum and AT FetA contents decreased at the onset of lactation when lipolysis was higher. No changes in AT and serum FetA were observed after feed restriction induced -NEB in mid -lactation cows. Prepartum BCS had no effect on serum FetA, but AT expression of AHSG, the gene encoding FetA, was reduced in periparturient cows wit h high BCS at dry off throughout all time points. Circulating FetA was positively associated with serum 41 albumin and calcium, and with BCS variation over the periparturient period. The dynamics of AHSG expression were analogous to the patterns of lipogenic markers ABDH5, ELOVL6, FABP4, FASN , PPARy and SCD1. AHSG and FetA protein expression in AT were inversely correlated with AT pro -inflammatory markers CD68, CD44, SPP1 , and CCL2 . In vitro, bovine adipocytes challenged with lipopolysaccharide ( LPS ) downregul ated FetA protein expression. Adipocytes treated with FetA had lower CCL2 expression compared to those exposed to LPS. Overall, FetA is a systemic and local (AT) negative APP linked to AT function in periparturient cows. Furthermore, FetA may support physi ological adaptations to NEB in periparturient cows. Key words: adipose tissue, biomarker, inflammation, lipolysis, transition cow 42 CHAPTER 4 IN VITRO ADIPOGENIC DIFFERENTIATION OF BOVINE PREADIPOCYTES: A CO -CULTURE MODEL ABSTRACT Reductionists studies o f adipose tissue ( AT) biology require reliable in vitro adipocyte culturing models. Current protocols for adipogenesis induction in stromal vascular fraction (SVF )-derived preadipocytes require extended culturing periods and have low adipogenic rates, espe cially in visceral SVF. We compared the adipogenic efficiency of direct co -culture of adipocytes with preadipocytes and the standard adipocyte differentiation in visceral and subcutaneous bovine adipocytes. SVF -derived preadipocytes and mature adipocytes w ere obtained by collagenase digestion from subcutaneous and visceral AT of dairy cows ( n = 6). Primary adipocytes were retained for use in co -culture. Confluent preadipocytes were induced to differentiate with medium containing insulin, acetate, troglitazo ne, 2 -isobutyl -1-methylaxanthine (IBMX) and dexamethasone for 48 h, and then maintained in this medium, excluding the dexamethasone and IBMX, for 7 d in co -culture ( CC7), and for 7 d ( SD7 ) or 14 d ( SD14 ) in standard differentiation. For CC7, 900 primary ad ipocytes/cm 2 were added to 0.4 µm transwell inserts and placed in the wells with preadipocyte cultures for the first 5 d of differentiation. Adipogenic efficiency was evaluated by gene expression of adipogenesis markers, triacylglycerol ( TAG ) accumulation, and glycerol release upon § -adrenergic stimulation. CC7 and SD14 similarly stimulated higher gene expression of ADIPOQ, CEBPA, CEBPB , AHSG , FABP4 and LIPIN1 in visceral and subcutaneous cells compared with preadipocytes ( P < 0.05). Mature adipocytes viabi lity in CC7 decreased 50% from the d 1 to d 5 of induction. TAG accumulation in subcutaneous and visceral adipocytes in CC7 incremented 40 and 16 -fold compared with 22 and 4 -fold increment in SD14, respectively. In the same period as SD7, CC7 43 incremented T AG accumulation by 8 times in subcutaneous and visceral adipocytes ( P < 0.05). As measured by glycerol concentrations, responsiveness to § -adrenergic stimulation was higher in CC7 and SD14 than SD7 in subcutaneous adipocytes ( P < 0.05); in visceral adipocy tes, CC7 had higher glycerol release than SD7 ( P < 0.05), however SD7 and SD14 were similar ( P > 0.05). Overall, CC7 is more efficient inducing adipogenesis in visceral and subcutaneous bovine adipocytes than SD14 since it stimulated similar responses in a shorter period of time. This protocol will facilitate the use of reductionist models to study adipocyte physiology in periparturient dairy cows and the assessment of pharmacological or nutritional interventions in conditions closer to those observed in vi vo. Key words : Adipocytes, adipogenesis, bovine, co -culture, in vitro 44 TECHNICAL NOTE During the periparturient period, dairy cows rely on adipocyte triacylglycerol ( TAG ) reserves to offset the effects of negative energy balance (Contreras and Sordillo, 2011b) . Through its secretome, adipose tissue ( AT) regulates energy balance and storage, nutrient transport, glucose homeostasis, lipid meta bolism, and immune function (Trayhurn, 2013). In order to expand our knowledge about the underlying mechanisms of adipocyte functions in periparturient dairy cows, including lipid mobilization and adipokine roles, it is necessary to use reductionist approa ches such as in vitro adipocyte culturing. Primary adipocyte isolation and culturing is difficult because these cells are buoyant and lack sufficient matrix proteins to facilitate in vitro adherence (Sugihara et al., 1987, Picot et al., 2005) . An alternate approach is to isolate stromal vascular fraction ( SVF ) and induce the differentiation of those cells into adipocytes. Across species, in vi tro adipocyte differentiation is induced by preadipocyte exposure to a mixture of hormones that includes glucocorticoids, an agent to increase cAMP activity, and high concentrations of insulin (MacDougald and Mandrup, 2002) . In bovines, adipocyte differentiation is complemented by supplementation of individual unbound fatty acids such as acetate and octanoate, partially characterized bovine serum lipids, and pharmacological peroxisomal prolifera tion activated receptor gamma ligands such as rosiglitazone and troglitazone (Grant et al., 2008, Smith et al., 2009, Lengi and Corl, 2010) . To increase adipogenic and lipogenic efficiency, addition of phosphatid ylcholine, TAG, lipoproteins derived from human plasma, and fatty acids (i.e. oleic and linoleic acids) have been used as alternative approaches to the standard pharmacological induction (Wu et al., 2001, Yanting e t al., 2018) . However, even lipid -enriched adipocyte differentiation protocols take 12 d to 14 d differentiating preadipocytes into adipocytes. Although sources of lipids and extended 45 differentiation protocols have been used to develop suitable reductioni st models for the study of bovine AT, low efficiency of adipogenesis is still an issue, mainly when using visceral SVF. Visceral adipocytes differentiate poorly, only 30% Ð40%, compared with the robust differentiation seen in subcutaneous cells (Macotela et al., 2012) . As a result, information on visceral -specific regulatory mechanisms and adipokine secretion in bovine are limited. Another limiting charac teristic of standard differentiation protocol is the lack on emulating important conditions of in vivo AT environment, such as cell by cell communication (Stacey et al., 2009) . In adipogenic induction of human adipocytes, the direct co -culture of primary adipoc ytes with preadipocytes using a transwell insert induces adipogenesis more effectively when compared to standard pharmacological differentiation protocol while maintaining the expression of adipogenic markers (Stacey et al., 2009) . It was suggested that mature adipocytes may release factors that promote preadipocyte differentiation and maturation, thus promoting a closer mimic of in vivo environment (Considine et al., 1996) . Given the need for developing a more efficient and physiological adipogenic model for bovine cells that could also be suitable for the on the differentiation of both, visceral and subcutaneous SVF, the goa l of this study was to compare the efficiency of two different culture conditions in stimulating adipogenic differentiation: Direct co -culture of adipocytes with preadipocytes in a transwell insert and the standard adipocyte differentiation medium. Subcut aneous and omental AT samples were obtained from 6 different dry cows at a local abattoir. Stromal -vascular fraction was isolated as described previously (Contreras et al., 2015, Strieder -Barboza et al., 2018) . Bri efly, AT samples were collected during carcass processing from the flank (subcutaneous) and omental (visceral) region. Specimens were transported to the laboratory while in Krebs -Ringer Bicarbonate Buffer (KRBB) supplemented 46 with HEPES 10 mM (pH = 7.4) and gentamicin solution (50 ) /mL) at 37¡C. Once in the laboratory, 500 mg of AT were washed three times in KRBB and minced in 2 to 3 mm sections in 5 mL collagenase type II solution (2 mg/mL; Worthington Biochemical, Lakewood, NJ) in KRBB with 4% bovine serum albumin (BSA, Millipore -Sigma, USA). Samples were incubated in a 37¡C water bath with inversion of the vials every 5 min for 15 min and then transferred to an incubator for further digestion with shaking for 45 min at 37¡C and 230 rpm. After incubation, digested material was centrifuged for 10 min at 300 x g to separate primary adipocytes (upper layer) from SVF (lower layer). Primary mature adipocytes were filtered through a 250 µm mesh while washed in 5 mL of KRBB with 4% BSA, then centrifuged for 5 min at 300 x g. Final primary adipocyte population was retained for culture in transwell insert in the co -culture condition. The viability of freshly isolated adipocytes was measured by Vybrant¨ MTT Cell Proliferation Assay Kit (Thermo Fisher, Waltham, MA). Afte r digestion, SVF was sequentially filtered through 100 µm and 40 µm cell strainers (Falcon, Corning, NY) and centrifuged for 10 min at 800 x g. Resulting cell pellet was resuspended and incubated in erythrocyte lysis buffer for 5 min at room temperature. A fter another centrifugation, resultant cells were resuspended in 9 mL of basal medium containing DulbeccoÕs modified EagleÕs medium: F12 (DMEM:F12, Corning, Corning, NY), 10% fetal bovine serum (FBS; Corning), 2 mmol/L of L -glutamine (Corning), 1% (vol/vol ) antibiotic -antimycotic (Corning), 44.05 mmol/L of sodium bicarbonate (Sigma -Aldrich, St. Louis, MO), 100 µmol/L of ascorbic acid (Sigma -Aldrich), 33 µmol/L of biotin (Sigma -Aldrich), 17 µmol/L of pantothenate (Sigma -Aldrich), and 20 mmol/L of HEPES (Corn ing), and incubated at 37¡C in a humidified atmosphere of 95% air and 5% CO 2.. Growth medium replacement was performed every 2 d (Strieder -Barboza et al., 2018) . Preadipocytes were obtained by outgrowth of plastic adherent cells from the SVF cells after 2 serial passages in 47 culture flasks (Corning). Ex panded preadipocytes populations were seeded in 24 - and 12 -well plates (Costar ¨, Corning) and allowed to proliferate to confluency. Confluent preadipocytes were induced to differentiate with basal medium supplemented with 5 ) M (Cayman Chemical, Ann Arbor, MI), 0.5 mmol/L isobutyl -1-methylaxanthine (IBMX; AdipoGen Life Sciences, San Diego, CA) and the following reagents from Sigma -Aldrich: 5 µg/mL insulin, 10 mM acetate, and 1 µmol/L dexamethasone . IBMX and dexamethasone were used only during the first 48 h of induction . For the co -culture condition, a differentiation protocol of 7 d was used ( CC7). Briefly, 0.4 µ m transwell inserts (Greiner Bio -One, Kremsm nster, Austria) were used to generate a co -culture system of adipocytes and preadipocytes. Freshly iso lated mature adipocytes (900 cell/cm 2) were added to the inserts and then placed into the wells of 24 - or 12 -well plates containing preadipocyte cultures for the first 5 d of adipogenic induction. After removing inserts from wells at d 5, attached adipocyt es were kept in differentiation media for 2 extra days. For standard differentiation condition, preadipocytes were induced to differentiate using the same medium as for CC7 during 7 d ( SD7 ) or 14 d ( SD14 ). SD7 served as a control for CC7, whichÕs protocol duration was also 7 d. After treatments, the adipogenic differentiation adipocytes was analyzed via the quantification of gene expression of adipogenic and lipogenic markers, TAG accumulation, and release of glycerol upon § -adrenergic stimulation. Gene exp ression of adipocyte activity markers adiponectin ( ADIPOQ), fatty acid translocase (CD36), CCAAT/enhancer -binding protein beta and alpha ( CEBPB, CEBPA), diacylglycerol O -Acyltransferase -1 and -2 ( DGAT1, DGAT2), fatty acid binding protein 4 ( FABP4), fatty acid transporter -1 ( FATP1), glycerol -3-phosphate acyltransferase -1 and -2 ( GPAT2, GPAT2), and peroxisome proliferator -activated receptor gamma (PPARG) were evaluated (Supplemental Table 1). RNA extraction, purification, conversion to cDNA and quantitative PCR analysis were 48 performed as described previously by our laboratory (Contreras et al ., 2017a) . The most stable reference genes eukaryotic translation initiation factor 3 subunit K ( EIF3K), 50S ribosomal protein L15 ( RPL0), and 40S ribosomal protein S9 (RPS9) were selected using geNorm (Vandeso mpele et al., 2002) . The Cq values were converted to normalized relative gene expression as described by Hellemans et al. (2007b) . The calibrator was set as the preadipocytes (PRE) cultured in growth media. AdipoRedª Adipogenesis Assay (Lonza, Allendale, NJ) and HCS LipidToxª (Life Technologies, Carlsbad, CA) staining were performed to quantitatively examine TAG accumulation. Supplemental Table 4. 1 mRNA probes by product and NCBI accession numbers Gene Product 1 RefSeq AHSG 2 Bt.23250 NM_173984.2 ADIPOQ Bt03292341_s1 NM_174742.2 CD36 Bt0 3234878_m1 NM_001046239.1. CEBPA Bt03224529_s1 NM_176784.2 DGAT1 Bt03251718_g1 NM_174693.2 DGAT2 Bt03259837_m1 NM_001253891.1 EIF3K Bt03226565_m1 NM_001034489.2 FABP4 Bt03213820_m1 NM_174314.2 FATP1 Hs01587911_m1 NM_198580.2 GPAT1 APU63EN - PPARG Bt03217547_m1 NM_181024.2 RPL0 Bt03218086_m1 NM_001012682.1 RPS9 Bt03272016_m1 NM_001101152.2 1Thermo Fisher, Waltham, MA, USA. 2 Integrated DNA Technologies, Coralville, IA, USA. AHSG : Fetuin -A; ADIPOQ: Adiponectin; CD36: Fatty acid translocas e; CEBPB: CCAAT/enhancer -binding protein beta; CEBPA: CCAAT/enhancer -binding protein alpha; DGAT1: Diacylglycerol O -Acyltransferase -1; DGAT2: Diacylglycerol O -Acyltransferase -2; EIF3K: Eukaryotic translation initiation factor 3 subunit K; FABP4: fatty acid binding protein 4; FATP1: Fatty acid transporter -1; GPAT1: Glycerol -3-Phosphate Acyltransferase -1; GPAT2: Glycerol -3-Phosphate Acyltransferase -2, PPARG: Peroxisome proliferator -activated receptor gamma; RPL0: 50S ribosomal protein L15; RPS9 : 40S ribosomal protein S9. Confocal microscopy imaging was performed in HCS LipidToxª stained cells (red fluorescence) using an Olympus FluoView 1000 Confocal Laser Scanning Microscope (Olympus America, Inc., Center Valley, PA) configured on an IX81 inverted microscope and FV10 -ASW software (version 4.2.3.6) using a UPLFLN 20 * /0.50 dry objective. Glycerol release was evaluated after a 2 h -!-adrenergic stimulation with 1 ) M isoproterenol (ISO, Sigma -Aldrich), a 49 !-adrenergic receptor agonist. Media alone [KRBB -HEPES conta ining 3% FA free BSA (Millipore -Sigma)] served as negative control (CON). Glycerol concentrations in the supernatant were analyzed in triplicates (cat. no. MAK117 -1KT, Millipore -Sigma) and corrected by the number of cells. Data were analyzed using JMP Stat istical Software (SAS Institute Inc., Cary, NC). Normality of the variables was checked using the Kolmogorov -Smirnov test ( P < 0.05). Non -normally distributed variables were ln transformed. One -way ANOVA Pairwise comparisons were performed using the TukeyÕ s post hoc test. Results are presented as MEAN ± SEM unless otherwise stated. Mean differences were considered significant when P ( 0.05 and tendencies when P < 0.10. Adipocyte differentiation or adipogenesis is the result of sequential changes from a fibroblast -like cell to a lipid -filled cell, with the expression of transcription factors, genes, and enzymes indicative of a mature f at cell (Grant et al., 2008) . The hallmark of adipogenesis is the end of cellular replication and the beginning of biosynthesis of specialized proteins required for cytoplasmic accumulation of TAG (MacDougald and Mandrup, 2002) . Limited information is available on the culture conditions to improve adipogenic efficiency and developing reductionist models that closely emulate the AT conditions in dairy cows. In this study, preadipocytes were differentiated into adipocytes under two different culture conditions and evaluated for adipogenic efficiency: Standard differentiation and co -culture containing mature adipocytes in transwell inserts. This co -culture method has been previously used in th e differentiation of human subcutaneous adipocytes (Considine et al., 1996, Janke et al., 2002, Stacey et al., 2009) , but has not yet been used in the culture of visceral adipocytes, nor with bovine adipocytes. The refore, this is the first study to evaluate the adipogenic efficiency of a short 7 -d co -culture differentiation protocol between bovine adipocytes and preadipocytes as a model for culturing visceral and subcutaneous adipocytes. In the present study, even t hough the 50 viability of adipocytes placed on inserts decreased 50% from d 0 to d5 of differentiation in CC7 (P < 0.05; Fig. 4.1), CC7 did demonstrate better adipogenic efficiency than SD7 and SD14. Figure 4. 1 Viability of primary bovine adipocytes placed in inserts during co -culture protocol. Samples (100 uL) from transwell inserts with primary adipocytes were assayed in triplicates at d 0 and d 5 after adipogenesis induction using Vybrant¨ MTT Cell Proliferation Assay Kit (Thermo Fisher) following directions of the manufacturer. Values are expressed as mean ± SEM. *Statistically significant ( P < 0.05). By the end of the differentiation protocol, C C7 and SD14 similarly stimulated an increase in the gene expression of ADIPOQ, CEBPA , CEBPB and AHSG (P > 0.05; Fig. 4.2 and Fig. 4.3) compared with their corresponding preadipocytes ( P < 0.05). Subcutaneous adipocytes in CC7 and SD14 also increased gene e xpression of the FA transporter FABP4, while in visceral cells, CC7 and SD14 increased LIPIN1, a required lipogenic enzyme for TAG synthesis ( P <0 .05). While CEBPB and CEBPA expression directs the differentiation process, expression of ADIPOQ, FABP4, AHSG and LIPIN1 promote FA transport and TAG accumulation and therefore are highly expressed in lipid -filled mature adipocytes (Arimochi et al., 2016) . These results demonstrate that in a shorter period of time, CC7 efficiently stimulated the transcription of critical adipogenic and lipogenic regulators in subcutaneous and visceral cells as induced by a 14-d standard differentiation protocol. Adipose tissue function during the periparturient period Day 0Day 50.000.050.100.150.200.25Day of induction*Relative Viability of Primary Adicpocytes 51 supplies the energy necessary to support the rapid fetal growth and the lactation onset, thus depending on TAG reserves in adipocytes to offset negative energy balance. The development of functional and efficient cell culture models of AT is extremely beneficial to in vitro assays that would assess adipocyte lipolytic and lipogenic responses during this critical time in dairy cows, thus improving our knowledge of mechanistic adaptations to metaboli c challenges. Figure 4. 2 Effect of culture conditions on gene expression of adipogenic markers in bovine adipocytes from subcutaneous adipose tissue depot. Gene expression of adipogenic and lipogenic markers in subcutaneous bov ine adipocytes cultured with a standard differentiation protocol of 14 d (SD14) or using a co -culture model (CC7). Preadipocytes (PRE) from tailhead subcutaneous adipose tissue depot were used as controls. The relative gene expression of (A) Adiponectin (ADIPOQ ), (B) CCAAT/enhancer - binding protein alpha ( CEBPA ), (C) CCAAT/enhancer -binding protein beta ( CEBPB), (D) Peroxisome proliferator -activated receptor gamma (PPARG), (E) Fetuin -A (AHSG), and (F) Fatty acid binding protein -4 (FABP4) were normalized by control genes 40S ribosomal protein S9 ( RPS9), Eukaryotic translation initiation factor 3 subunit K ( EIF3K) , and 50S ribosomal protein L15 ( RPLO) .Values are shown as 2 (+,, CT) (where CT = cycle threshold). Results represent means ± SEM. PRE SD14 CC70.000.010.020.030.51.01.5ADIPOQ Relative gene expression (2-ddct)baaPRE SD14 CC701!10-42!10-43!10-44!10-40.51.01.5AHSG Relative gene expression (2-ddct)baaPRE SD14 CC70.000.010.020.030.51.01.5CEBPA Relative gene expression (2-ddct)baaPRE SD14 CC70.00.51.01.5CEBPBRelative gene expression (2-ddct)baaPRE SD14 CC70.00.51.01.5PPARG Relative gene expression (2-ddct)PRE SD14 CC70.00.51.01.52.0FABP4 Relative gene expression (2-ddct)baaABCDEF 52 Figure 4. 3 Effect of culture conditions on gene expression of adipogenic markers in bovine adipocytes from visceral adipose tissue depot. Gene expression of adipogenic and lipogenic markers in visceral bovine adipocytes cultured with a standard differentiation protocol of 14 d (SD14) or using a co-culture model (CC7). Preadipocytes (PRE) from omental visceral adipose tissue depot were used as controls. The relative gene expression of (A) Adiponectin ( ADIPOQ ), (B) CCAAT/enhancer -binding protein alpha ( CEBPA ), (C) CCAAT/enhancer -binding protein be ta ( CEBPB), (D) Peroxisome proliferator -activated receptor gamma (PPARG), (E) Fetuin -A (AHSG), and (F) phosphatidate phosphatase -1 (LIPIN1) were normalized by control genes 40S ribosomal protein S9 ( RPS9), Eukaryotic translation initiation factor 3 subunit K ( EIF3K) , and 50S ribosomal protein L15 (RPLO) .Values are shown as 2( +,, CT) (where CT = cycle threshold). Results represent means ± SEM. In this study, we quantitatively evaluated adipogenic efficiency through the measurement of cytoplasmic TAG accumulation in adipocytes. Even though CC7 and SD14 had similar TAG accumulation (P > 0.05; Fig. 4.4 and Fig. 4.5), respectively, CC7 was more efficient than SD14 in both, visceral and subcutaneous adipocytes, because CC7 synthetized similar amounts of cytoplasmic TAG in the half of time took by SD14. Relative to preadipocytes TAG accumulation, CC7 incremented in 40 - and 16 -fold the total lipid accumulation in subcutaneous and visceral adipocytes compared with an increment of 22 and 4 -fold in SD14, respectively. In the same differentiation time -point (d 7), CC7 incremented by 8 times the TAG accumulation in PRE SD14 CC70.00.51.01.52.0CEBPA Relative gene expression (2-ddct)aabPRE SD14 CC70.00.51.01.5LIPIN1Relative gene expression (2-ddct)aabPRE SD14 CC70.00.20.40.60.81.0PPARG Relative gene expression (2-ddct)P = 0.06PRE SD14 CC70.0000.0010.0020.0030.004246AHSGRelative gene expression (2-ddct)aabPRE SD14 CC70.000.020.040.060.080.1012345ADIPOQRelative gene expression (2-ddct)aabPRE SD14 CC70.000.020.040.060.080.100.51.01.52.0CEBPBRelative gene expression (2-ddct)aabABCDEF 53 subcutaneous and visceral adipocytes compared with SD7 ( P < 0.05; Fig. 4.4N, 4 .5N). The augmentation of TAG accumulation in co -culture conditions compared with standard differentiation was also previously obs erved in subcutaneous human adipocytes (Stacey et al., 2009). Based on previous reports and on our results, the secretome of the mature adipocytes present in the inserts during co -culture may aid in the differentiation of adjacent cells enhancing lipogenic acti vity of adipocytes (Considine et al., 1996) . As important as the capacity of synthetizing and accumulating TAG, is the ability of responding to lipolytic stimulus to release glycerol and free fatty acids during periods of negative energy balance as the peripartum in dairy cows. As exacerbat ed lipolysis in closely related with metabolic disorders and high economic losses in the dairy industry, the development of reliable reductionist models for the study of lipolytic pathways and strategies to modulate them are crucial. In our study, we evalu ated the functionality of co -culture and standard protocols through the quantification of glycerol release upon the adipocytes stimulation with a § -adrenergic receptor agonist. In subcutaneous adipocytes, responsiveness to § -adrenergic stimulation was high er in CC7 and SD14 compared with SD7, as demonstrated by higher glycerol release in ISO over CON ( P < 0.05; Fig. 4.6A). Total glycerol released by subcutaneous adipocytes upon stimulation of lipolysis were 272 ± 131 nM, 987 ± 303 nM, and 1362 ± 208 nM for SD7, CC7 and SD14, respectively, and were similar between CC7 and SD14 ( P > 0.05), and higher in SD14 compared with SD7 ( P < 0.05). Visceral adipocytes released approximately half of glycerol concentrations compared with subcutaneous cells (Fig. 4.6C, 4.6D) and did not differ between SD7, SD14 and CC7 ( P > 0.05). Upon ISO -stimulation, visceral adipocytes released 300 ± 79 nM, 621 ± 80 nM, and 480 ± 135 nM glycerol for SD7, SD14 and CC7, respectively. Two possible reasons for these findings are (1) the lower TAG accumulation in visceral 54 adipocytes compared with subcutaneous, and (2) lower § -agonist effectivity of ISO in the stimulation of lipolysis in mesenteric adipocytes, as previously reported (Umekawa et al., 1997) . Notably, while SD7 and SD14 responses to lipolysis stimulation were similar in visceral adipo cytes ( P > 0.05), CC7 had greater responsiveness than SD7 ( P < 0.05, Fig. 4.6B). These results suggest that co -culture may be a suitable model for evaluating lipolytic responses and pathways in visceral adipocytes. This is particularly important because ma ny in vitro studies have been performed with only subcutaneous adipocytes due to the low adipogenic efficiency of visceral SFV, potentially missing visceral -specific regulatory mechanisms and differential physiological functions compared with other AT depo ts (Macotela et al., 2012) . In this study, we demonstrated that a 7 -d co -culture protocol with visceral or subcutaneous bovine adipocytes improves adipogenic efficiency by promoting similar responses than a 14 -d standard differentiation protocol in a shorter period of time. Co -culture model may better mimic the complex differentiation stimuli provided in vivo and may be a more suitable model for studying visceral adipocyte functions compared with standard model. Therefore, co -culture is physiologically relevant for reductionist models of adipocyte biology studies in periparturient dairy cows and the assessment of pharmacological or nutritional interventions in conditions closer to those obser ved in vivo . 55 Figure 4. 4 Lipid accumulation in cultured bovine adipocytes derived from subcutane ous adipose tissue depot in dairy cows. Images A -L: Laser scanning confocal microscopy imaging of lipid droplets in bovine adipocytes derived from subcutaneous AT using (A -D) standard induction for 7 d (SD7); (E -H) standard induction for 14 d (SD14); and (I-L) a co -culture model for 7 d (CC7). (M) Relative fluorescence intensity measured through confocal microscopy images using ImageJ software (means ± SEM). (N) Lipid accumulation fold change of visceral adipocytes over subcutaneous preadipocytes (AdipoRed ª assay). For confocal microscopy images, from left to right: 1 st column: Bovine adipocytes co -stained with the lipid droplet stain HCS LipidToxª and nuclei stain NucBlueª. SD7 SD14 CC701!1052!1053!1054!105Subcutaneous Relative fluorescence intensityaabMSD7 SD14 CC70204060aabbSubcutaneous Fold change / preadipocytes N 56 Figure 4. 4 (cont Õd) 2nd: HCS LipidToxª red fluorescence. 3 rd: HCS LipidToxª and NucBlueª fluorescence overlaid on differential interference contrast image. 4 th: Differential interference contrast image of bovine adipocytes. Scale bars: 100 ) m. Bars with different letters (a Ðc) are significantly different ( P ( 0.05). Figure 4. 5 Lipid accumulation in cultured bovine adipocytes derived from omental visceral adipose tissue in dairy cows. Images A -L: Laser scanning confocal microscopy imaging of lipid droplets in bovine adipocyt es derived from omental visceral adipose depot using (A -D) standard induction for 7 d (SD7); SD7 SD14 CC70.05.0!1041.0!1051.5!105Visceral Relative fluorescence intensity aabbMSD7 SD14 CC70102030Visceral Fold change / preadipocytesP = 0.07ababN 57 Figure 4. 5 (cont Õd) (E-H) standard induction for 14 d (SD14); and (I -L) a co -culture model for 7 d (CC7). (M) Relative fluorescence intensity measured through confocal microscopy imag es using ImageJ software (means ± SEM). (N) Lipid accumulation fold change of visceral adipocytes over omental preadipocytes (AdipoRedª assay). For confocal microscopy images, from left to right: 1st column: Bovine adipocytes co -stained with the lipid drop let stain HCS LipidToxª and nuclei stain NucBlueª. 2 nd: HCS LipidToxª red fluorescence. 3 rd: HCS LipidToxª and NucBlueª fluorescence overlaid on differential interference contrast image. 4 th: Differential interference contrast image of bovine adipocytes. S cale bars: 100 ) m. Bars with different letters (a Ðc) are significantly different ( P ( 0.05). Figure 4. 6. Responsiveness to !-adrenergic stimulation in subcutaneous and visceral adipocy tes differentiated in different culture conditions. Concentrations of glycerol (nM) released in the medium during 2 h -stimulation with (ISO) or without (CON) isoproterenol (1 uM). Glycerol concentration fold change increase over CON in (A) subcutaneous an d (B) visceral adipocytes induced to differentiate using a standard protocol for 7 (SD7, control) or 14 d (SD), and a co -culture (CC) in vitro model. Glycerol concentrations were calibrated by the number of cells per well and are expressed as mean fold cha nge over CON ± SEM. Bars with different letters (a Ðc) are significantly different ( P ( 0.05). Acknowledgments This research was funded by USDA -National Institute of Food and Agriculture (Washington, DC) grants 2014 -68004-21972 and 2015 -67015-23207 and the Department of Large Animal Clinical Sciences (East Lansing, MI). The authors are grateful to Jennifer Dominguez and the staff at the Michigan State University Meat Laboratory (East Lansing, MI) and West Michigan Beef for proving samples . SD7 SD14 CC701234Fold change nM glycerol ISO / CONaabSubcutaneous adipocytesASD7 SD14 CC701234Fold change nM glycerol ISO / CONVisceral adipocytes aabbB 58 REFERENCES 59 REFERENCES Arimochi, H., Y. Sasaki, A. Kitamura, and K. Yasutomo. 20 16. Differentiation of preadipocytes and mature adipocytes requires PSMB8. Scientific Reports 6:26791. Considine, R. V., M. R. Nyce, L. M. Morales, S. A. Magosin, M. K. Sinha, T. L. Bauer, E. L. Rosato, J. Colberg, and J. F. Caro. 1996. Paracrine stimulati on of preadipocyte -enriched cell cultures by mature adipocytes. American Journal of Physiology -Endocrinology And Metabolism 270(5):E895 -E899. Contreras, G. A., E. Kabara, J. Brester, L. Neuder, and M. Kiupel. 2015. Macrophage infiltration in the omental an d subcutaneous adipose tissues of dairy cows with displaced abomasum. Journal of Dairy Science 98(9):6176 -6187. Contreras, G. A. and L. M. Sordillo. 2011. Lipid mobilization and inflammatory responses during the transition period of dairy cows. Comparative Immunology, Microbiology and Infectious Diseases 34(3):281 -289. Contreras, G. A., C. Strieder -Barboza, J. de Souza, J. Gandy, V. Mavangira, A. L. Lock, and L. M. Sordillo. 2017. Periparturient lipolysis and oxylipid biosynthesis in bovine adipose tissues. PLOS ONE 12(12):e0188621. Grant, A. C., G. Ortiz -Colon, M. E. Doumit, and D. D. Buskirk. 2008. Optimization of in vitro conditions for bovine subcutaneous and intramuscular preadipocyte differentiation. J. Anim Sci. 86(1):73 -82. Hellemans, J., G. Mortier, A. De Paepe, F. Speleman, and J. Vandesompele. 2007. qBase relative quantification framework and software for management and automated analysis of real -time quantitative PCR data. Genome Biol. 8(2):R19. Janke, J., S. Engeli, K. Gorzelniak, F. C. Luft, and A. M. Sharma. 2002. Mature adipocytes inhibit in vitro differentiation of human preadipocytes via angiotensin type 1 receptors. Diabetes 51(6):1699 -1707. Lengi, A. J. and B. A. Corl. 2010. Factors influencing the differentiation of bovine preadipocytes in vitro. J. Anim Sci. 88(6):1999 -2008. MacDougald, O. A. and S. Mandrup. 2002. Adipogenesis: forces that tip the scales. Trends in Endocrinology and Metabolism 13(1):5 -11. Macotela, Y., B. Emanuelli, M. A. Mori, S. Gesta, T. J. Schulz, Y. -H. Tseng, and C. R . Kahn. 2012. Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots. Diabetes 61(7):1691 -1699. 60 Picot, J., V. Harmelen, T. Skurk, and H. Hauner. 2005. Primary Culture and Differentiation of Human Adipocyte Precursor Cells. Pages 125-135 in Human Cell Culture Protocols. Vol. 107. Humana Press. Smith, S. B., H. Kawachi, C. B. Choi, C. W. Choi, G. Wu, and J. E. Sawyer. 2009. Cellular regulation of bovine intramuscular adipose tissue development and composition. Journal of Animal Sci ence 87(14 suppl):E72 -E82. Stacey, D. H., S. E. Hanson, G. Lahvis, K. A. Gutowski, and K. S. Masters. 2009. In vitro adipogenic differentiation of preadipocytes varies with differentiation stimulus, culture dimensionality, and scaffold composition. Tissue Engineering Part A 15(11):3389 -3399. Strieder -Barboza, C., J. de Souza, W. Raphael, A. L. Lock, and G. A. Contreras. 2018. Fetuin -A: A negative acute -phase protein linked to adipose tissue function in periparturient dairy cows. Journal of Dairy Science 101 (3):2602 -2616. Sugihara, H., N. Yonemitsu, S. Miyabara, and S. Toda. 1987. Proliferation of unilocular fat cells in the primary culture. Journal of Lipid Research 28(9):1038 -1045. Umekawa, T., T. Yoshida, N. Sakane, and M. Kondo. 1997. Effect of CL316, 243 , a Highly Specific ! 3-Adrenoceptor Agonist, on Lipolysis of Epididymal, Mesenteric and Subcutaneous Adipocytes in Rats. Endocrine journal 44(1):181 -185. Vandesompele, J., K. De Preter, F. Pattyn, B. Poppe, N. Van Roy, A. De Paepe, and F. Speleman. 2002. A ccurate normalization of real -time quantitative RT -PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3(7):1. Wu, P., K. SATO, F. SUZUTA, Y. HIKASA, and K. KAGOTA. 2001. Effects of lipid -related factors on adipocyte differenti ation of bovine stromal -vascular cells in primary culture. Journal of Veterinary Medical Science 62(9):933 -939. Yanting, C., Q. Y. Yang, G. L. Ma, M. Du, J. H. Harrison, and E. Block. 2018. Dose - and type -dependent effects of long -chain fatty acids on adip ogenesis and lipogenesis of bovine adipocytes. Journal of Dairy Science 101(2):1601 -1615. 61 CHAPTER 5 FETUIN -A MODULATES LIPID MOBILIZATION IN BOVINE ADIPOSE TISSUE BY ENHANCING LIPOGENIC ACTIVITY OF ADIPOCYTES. ABSTRACT Fetuin -A ( FetA ) is an adipokine a nd free fatty acids ( FFA) transporter in plasma linked to adipose tissue ( AT) function in transition dairy cows. Plasma and AT FetA decrease after parturition coinciding with reduced lipogenesis and increased lipolysis. In monogastrics, FetA enhances lipog enesis, but its role on lipid mobilization of ruminants is unclear. We hypothesized that FetA modulates lipid mobilization by enhancing lipogenic activity in bovine adipocytes. Our objective was to determine the effects of FetA on lipogenesis and lipolysis in cultured primary adipocytes from dairy cows. Preadipocytes from tailhead subcutaneous AT depot of dairy cows were induced to differentiate in a 7 -day co -culture in vitro model. Lipolytic responses of adipocytes were evaluated after 2h -!-adrenergic stim ulation with 1 ) M isoproterenol ( ISO ) alone or combined with 0.1 mg/mL of FetA ( FETA+ISO ), and in cells treated with media alone (CON ) or with 0.1 mg/mL of FetA ( FETA ). Lipogenic responses of adipocytes treated with CON or FETA from day 5 to 7 of different iation were assessed by FFA uptake quantification and triacylglycerol ( TAG ) accumulation, and the gene and protein expression of lipogenic markers. Cultured bovine adipocytes abundantly expressed AHSG and FetA protein, and secreted 48 ± 3.5 ng FetA. Adrene rgic stimulation with ISO increased lipolysis compared with CON, as reflected in the release of glycerol (12±0.04 vs 0.04±0.02 nM) and FFA (15±13 vs 6.2±2.4 nM). Lipolysis induced by ISO was attenuated by the addition of FetA (FETA+ISO) as reflected by a l ower glycerol (0.06±0.04 nM) and FFA (5.7±2.7 nM) release. The treatment with FetA enhanced lipogenic responses compared with CON as demonstrated by an increment in FFA uptake and 1.7 times in the accumulation of TAG. Exposure to FetA upregulated the AGAPT 2 gene 62 expression and protein content, as well as its activity as demonstrated by increased concentrations of phosphatidic acid, the final product of its activity in adipocytesÕ synthesis of TAG. In conclusion, FetA attenuates lipolytic responses and enhan ces lipogenesis in bovine adipocytes. The upregulation of the rate -limiting lipogenic enzyme AGAPT2 by FetA suggests a potential pathway by which this adipokine promotes TAG synthesis in adipocytes. FetA is a potential target for lipid mobilization modulat ion in AT of periparturient dairy cows. Key words: adipokine, adipocytes, dairy cow, lipogenesis, lipolysis 63 INTRODUCTION Increased lipid mobilization in adipose tissues ( AT) is the major metabolic adaptation to negative energy balance in periparturient dairy cows. Lipid reserves are used to meet the energy needs of milk synthesis thus favoring lipolysis over lipogenesis in AT (Contrer as and Sordillo, 2011). During lipolysis, one molecule of triacylglycerol ( TAG ) is broken down into 3 free fatty acids ( FFA) molecules and 1 molecule of glycerol through the activity of adipose triglyceride lipase (ATGL) and hormone -sensitive lipase (HSL ). Released FFA are either re -esterified into TAG within the adipocytes or used as energy source elsewhere in the body (Vernon and Pond, 1997). Lipogenesis involves the biosynthesis of TAG from fatty acids ( FA) either synthesized from FFA released from blood TAG or de novo within the adipocytes. Glycerol phosphate pathway is the major pathway for de n ovo synthesis of TAG by adipocytes, which consists in stepwise addition of fatty acyl groups catalyzed by distinct enzymes: glycerol -3-phosphate acyltransferase (GPAT), 1 -acylglycerol -3-phosphate acyltransferase (AGPAT), lipins (phosphatidate phosphatases) , and diacylglycerol acyltransferase (DGAT) to finally form TAG (Takeuchi and Reue, 2009 ). Adipocyte TAG reserves serve as the main source of energy to offset the effects of negative energy balance in periparturient cows (McNamara, 1995 ). In peripart urient cows, the main endocrine factors enhancing lipolysis and diminishing lipid synthesis in adipocytes include decreased plasma insulin and glucose concentrations, impaired insulin sensitivity in AT and other peripheral tissues, and increased concentrat ions of catecholamines, growth hormone and glucocorticoids (Bauman and Currie, 1980 ). In addition, a decrease in the transcription and activity of key lipogenic enzymes lead to a marked decrease in the ability of adipocytes to synthesize FA and TAG thus enhancing FFA release from AT (McNamara, 1995 ). 64 The relative balance between lipolysis and lipogene sis rates in AT controls the release of FFA from adipocytes and is critical to assure a successful transition from gestation to lactation in dairy cows. Dysregulated lipid mobilization leads to a massive release of FFA and a pro -inflammatory response that are major underlying factors of health disorders in periparturient dairy cows (Vernon and Houseknecht, 2000 , Contreras et al., 2018 ). Currently, a major challenge in the field of periparturient dairy cowsÕ management is the adequate regulation of AT lipolysis and lipogenesis rates to ensure that released FFA will be fully metabolized for energy needs, thus avoiding the detrimental FFA acc umulation in blood and tissues. We have recently determined the dynamics of a novel adipokine, Fetuin -A ( FetA ; alpha -2-Heremans -Schmid glycoprotein), in serum and AT of dairy cows during the periparturient period (Strieder -Barboza et al., 20 18). Our results indicated that FetA is a negative acute -phase adipokine in the subcutaneous AT and is potentially involved in AT lipid mobilization in periparturient dairy cows (Strieder -Barboza et al., 2018 ). We also reported the gene and protein expression of FetA in bovine adipocytes suggesting a potential autocrine role for this adipokine; however, its secretion has not yet been reported (Strieder -Barboza et al., 2018 ). In humans and mice, FetA secretion by visceral an d subcutaneous adipocytes is augmented during obesity and in animals fed a high fat diet (Jialal et al., 2015 , P”rez -Sotelo et al., 2016 ). This effect is probably related with lipogenic properties of FetA. Fetuin -A carries lipids in plasma and facilitates the incorporation of exogenous FA into intracellular TAG in non -ruminantsÕ adipogenic models (Kumbla et al., 1989 , Cayatte et al., 1990 ). Variation in AHSG , the gene encoding FetA, was strongly associated with stimulation of lipogenesis and insulin -mediated inhibition of lipolysis in women (Dahlman et al., 2004 ). Because we previously observed that decreased FetA coincided with low lipogenesis and high lipolysis rates, and given the fact that FetA was associated with 65 pro -lipogenic states in non -ruminant species, we hypothesized that F etA modulates lipid mobilization by enhancing lipogenic activity in bovine adipocytes. Our objective was to determine the effect of FetA on lipogenesis and lipolysis and identify potential mechanisms by which this adipokine modulates lipid mobilization of bovine adipocytes. MATERIALS AND METHODS All animal procedures were approved by the Michigan State University Animal Care and Use Committee. Tissue Collection and Processing Subcutaneous AT from the tailhead depot from seven dairy cows were collected in KRBB supplemented with HEPES 10 mM (pH = 7.4) at a local slaughterhouse as previously described (Strieder -Barboza et al., 2018 ). In brief, AT (500 mg) was digested with 5 mL collagenase type II solution (2 mg/mL; Worthington Biochemical, Lak ewood, NJ) and then centrifuged to separate the primary adipocytes from the stromal vascular fraction (SVF). Primary mature adipocytes were washed in 5 mL of KRBB with 4% bovine serum albumin (BSA, Millipore -Sigma, USA) and centrifuged. Final adipocyte pop ulation was retained for use in transwell inserts for inductions using a co -culture protocol. The SVF was then sequentially filtered through 100 µm and 40 µm cell strainers (Falcon, Corning, NY) and centrifuged. Resulting cell pellet was resuspended and in cubated in erythrocyte lysis buffer. After another centrifugation, resultant cells were resuspended in basal preadipocyte media containing DulbeccoÕs modified EagleÕs medium: F12 (Corning, Corning, NY), 10% fetal bovine serum (FBS; Corning), 2 mmol/L of L -glutamine (Corning), 1% (vol/vol) antibiotic -antimycotic (Corning), 44.05 mmol/L of sodium bicarbonate (Sigma -Aldrich, St. Louis, MO), 100 µmol/L of ascorbic acid (Sigma -Aldrich), 33 µmol/L of biotin (Sigma -Aldrich), 17 µmol/L of pantothenate 66 (Sigma -Aldric h), and 20 mmol/L of HEPES (Corning) with replacement every 2 d as described previously (Strieder -Barboza et al., 2018 ). Preadipocytes were obtained by outgrowth of plastic adherent cells from the SVF cells after 2 serial passages in cultur e flasks (Corning). Cell Induction and Differentiation Expanded preadipocytes populations were seeded in 6 -well plates (Corning) and allowed to proliferate to confluency. Preadipocytes were induced to differentiate after 48 h at 100% confluency (d 0) usin g a co -culture model. Nine hundred mature adipocytes /cm2 were placed in 0.4 µ m transwell inserts (Greiner Bio -One, Kremsm nster, Austria) over the attached preadipocytes for the first 5 days of differentiation. Induction basal media was supplemented with 10% FBS, 5 µmol/L troglitazone (Cayman Chemical, Ann Arbor, MI), 0.5 mmol/L 2 isobutyl -1-methylaxanthine (IBMX; AdipoGen Life Sciences, San Diego, CA) and the following reagents from Sigma Aldrich: 5 µg/mL insulin, 10 mM acetate, and 1 µmol/L dexamethasone . IBMX and dexamethasone were used only during the first 48 h of induction and media changes were performed every 48 h for 7 d. Adipocyte lipid accumulation was assessed quantitatively in triplicates per experimental unit using a 96 -well plate for the Adip oRedª assay (Lonza, Allendale, NJ) using a Synergy H1 Microplate Reader (Biotek, Vermont, MA). Lipolysis -induction Assay !-adrenergic induction of lipolysis was performed using isoproterenol (ISO, Sigma -Aldrich), an agonist of !-adrenergic receptors in ad ipocytes. Briefly, cultured adipocytes ( n = 6) were removed from 6 -well plates (Corning) using Trypsin (ThermoFisher, Waltham, MA), seeded in triplicate at 1x10 5 cells/well in black wall 96 -well plates (Nunc, Roskilde, Denmark) and allowed to attach for 4 h at 37 ¡C in a humidified atmosphere with 5% CO 2. Adipocytes were then starved for 4 h with serum free -basal preadipocyte media supplemented with 0.1% FA 67 free BSA (Millipore -Sigma, USA). Lipolytic responses of adipocytes were evaluated after 2 h incubatio n at 37 ¡C with Krebs Ringer Bicarbonate HEPES buffer (KRBH, pH 7.4) containing 3% FA free BSA (Millipore -Sigma) supplemented with 1 ) M isoproterenol alone (ISO), 0.1 mg/mL of FetA alone (FETA; cat. no. 341506; Millipore, Darmstadt, Germany) or the combina tion of 0.1 mg/mL of FetA and 1 ) M isoproterenol (ISO+FETA). Basal lipolysis was determined without addition of any reagent (CON). FetA dose was stablished based on previous studies with adipocytes (Heinrichsdorff and Olefsky, 2012 , Pal et al., 2012 ). All reagents were prepared fresh on the day of the experiment. Lipolytic responses were assessed by the con centrations of glycerol (cat. no. MAK117 -1KT, Millipore -Sigma) and FFA (HR Series NEFA-HR (2), FUJIFILM Wako Diagnostics U.S.A) released in the culture medium during 2 h -assay. To evaluate whether FetA is secreted by bovine adipocytes, we analyzed FetA con centrations in CON cell culture media. FetA concentrations were determined by ELISA following the manufacturerÕs guidelines (cow AHSG/fetuin A, cat. no. LS -F6106; LSBio, Seattle, WA) as reported previously (Strieder -Barboza et al., 2018 ). Concentrations of glycerol, FFA and FetA were adjusted by the number of cells/well as determined by CyQUANT Assay (Life Technologies, Carlsbad, CA) post functional analysis. All conditions were performed in triplicate. Statistical analysis was performed usi ng the average glycerol and FFA concentrations of the triplicates corrected by the number of cells. Fatty Acid Uptake Assay Fatty acid uptake analysis was performed using the kinetic QBT Fatty Acid Uptake Assay (cat. no. R8132; Molecular Devices, Sunnyval e, CA). Cultured adipocytes ( n = 6) were detached and seeded as described for lipolysis assay. Adipocytes were then serum -starved overnight and pre -incubated with 0.1mg/mL of FetA, 10 nM insulin (INSULIN, positive control; 68 Sigma -Aldrich, St. Louis, MO) or serum -free basal preadipocyte media (CON; basal FFA uptake) during 30 min at 37 ¡C in a humidified atmosphere with 5% CO 2. Reagent from the QBT Fatty Acid Uptake assay containing a fluorescent labelled FA analog was added to the cells (100 µL/well) and kine tic uptake was measured every 20 sec for 1 h using a Synergy H1 Microplate Reader (Biotek). After the assay, to calculate area under the curve for each treatment, triplicate values per sample were averaged, subtracted from blank and calibrated by basal val ues (CON). Values are expressed as fold change over CON. Triacylglycerol Accumulation Intracellular TAG accumulation in adipocytes was assessed by Adipogenesis Assay Reagent (AdipoRedª, Lonza) and the lipid droplet staining HCS LipidToxª (Life Technologies , Carlsbad, CA) analyzed through confocal microscopy imaging. On d 5 of differentiation, cultured adipocytes were incubated with basal adipocyte differentiation media supplemented with 0.1 mg/mL of FetA (FETA) for 48 h (d 5 to d 7). Basal TAG accumulation was determined in adipocytes incubated with basal differentiation media without addition of FetA (CON). Lipid Droplets Staining and Confocal Microscopy Imaging . On d 5 of differentiation, cultured adipocytes ( n = 8) were seeded in a glass bottom 24 well plate (Corning) at a concentration of 20,000 cells/cm 2 and incubated with or without FetA for 48 h. DAPI (NucBlueª, Life Technologies) and Alexa Fluor¨ 594 (HCS LipidToxª, Life Technologies) were used to visualize adipocytesÕ nuclei and intracellular TAG, respectively, through confocal laser scanning microscopy. These dyes were utilized according to the manufacturer's protocols. Briefly, adipocytes were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in phosphate -buffered saline (1X PBS). 200 µL of HCS LipidTOXª neutral lipid 69 stain 1:200 in 1X PBS was added to each well (Corning) and incubated at room temperature for 30 min. Buffer was removed and 200 µL DAPI (300 nM in 1X PBS) was added to each well and incubated at room tempera ture for 5 min. Images were acquired using an Olympus FluoView 1000 Confocal Laser Scanning Microscope (Olympus America, Inc., Center Valley, PA) configured on an IX81 inverted microscope and FV10 -ASW software (version 4.2.3.6) using a PLAPON 60 * /1.42 oil objective. Alex Fluor¨ 594 fluorescence (577 nm excitation/609 nm emission) was excited with the 543 nm HeNe gas laser, detected using a BP 560IF emission filter, and displayed in red color. DAPI fluorescence (358nm excitation/461nm emission) was excited u sing the 405nm diode laser, detected using a 430 -470nm band pass emission filter, and displayed in blue color. Images were obtained using sequential single confocal XY scan mode. Control images for TAG accumulation included preadipocytes and non -stained ad ipocytes. Relative fluorescence intensity of intracellular TAG per cell was determined by ImageJ software. Adipogenesis Assay Reagent. AdipoRedª is a solution of Nile Red, a hydrophilic stain, that specifically partitions into the lipid droplets of differ entiated adipocytes. On d 7 of differentiation, the assay was performed in triplicates in 24 well plates (Corning). Briefly, adipocytes ( n = 5) were washed once with 1X PBS, incubated with AdipoRedª assay reagent for 20 min at room temperature, and then an alyzed for fluorescence intensity at 572 nm using a Synergy H1 Microplate Reader (Biotek). Preadipocytes served as negative controls. Relative fluorescence units (RFU) data of adipocytes was calibrated by their correspondent preadipocytes RFU and are prese nted as fold change over preadipocytes lipid accumulation. Gene Expression Analysis of Adipogenesis and Lipogenesis Markers After treating adipocytes ( n = 5) for 48 h with or without FetA, culture media was removed, and cells were rinsed twice with ice -cold 1X PBS. AdipocytesÕ RNA was extracted 70 using Promega simplyRNA Cells Kits (Cat# AS1390, Promega, Madison, WI) in the Maxwell ¨ RSC Instrument (Promega, Madison, WI) as described previously (Strieder -Barboza et al., 2018). 200 µL of 1 -thiog lycerol/homogenization solution were added to each well of 6 -well plates with adipocytes and then transferred to a microfuge tube. Next, 200 µL of lysis buffer were added and homogenate was vortexed and then placed in Maxwell¨ RSC Cartridges, which were pr eviously loaded with 10 µL of DNase I. Purity, concentration, and integrity of AT and cellsÕ RNA were evaluated using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). All samples had a RNA integrity number ' 6. Conversion to cDNA was per formed using the Applied Biosystems High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Transcriptional studies were performed on the cDNA samples using qPCR reactions on QuantStudio 7 Flex (Applied Biosystems). All qPCR reactions were pe rformed in duplicates and no template controls (NTC) were included on each plate for each TaqMan gene expression assay. Gene expression data of 5 endogenous control genes ( B2M, EIF3K, GAPDH, RPLP0 and RPS9 ) were analyzed using qBase+ analysis software, which calculates the stability of endo genous control genes (M -value). Following qBase+ analysis of gene expression data, endogenous control genes EIF3K and RPS9 , were ranked best. The Cq values of the target genes (AHSG, FABP4, CD36, FATP1, DGAT1, DGAT2, GPAT1, GPAT2, AGAPT2, PPARG, ADIPOQ and CEBPB ) were converted to normalized relative gene expression as described previously (Hellemans et al., 2007 )(30). The quantitative PCR assays were conducted with TaqMan gene expression assays from Applied Biosystems, with the exception of FetA (AHSG) which was provided by Integrated DNA Technologies (IDT; Coralville , IA) as described previously (Strieder -Barboza et al., 2018 ) (Supplemental Table 1). 71 Supplemental Table 5. 1 mRNA probes by product and NCBI accession numbers Gene Product 1 RefSeq AHSG 2 Bt.23250 NM_173984.2 ADIPOQ Bt03292341_s1 NM_174742.2 AGAPT2 Bt03244182_m1 NM_001012727.1 B2M Bt03251628_m1 NM_173893.3 CD36 Bt03234878_m1 NM_001046239.1. DGAT1 Bt03251 718_g1 NM_174693.2 DGAT2 Bt03259837_m1 NM_001253891.1 EIF3K Bt03226565_m1 NM_001034489.2 FABP4 Bt03213820_m1 NM_174314.2 FATP1 Hs01587911_m1 NM_198580.2 GAPDH Bt03210913_g1 NM_001034034.2 GPAT1 APU63EN - GPAT2 APGZFWZ - PPARG Bt03217547_m 1 NM_181024.2 RPL0 Bt03218086_m1 NM_001012682.1 RPS9 Bt03272016_m1 NM_001101152.2 1Thermo Fisher, Waltham, MA, USA. 2 Integrated DNA Technologies, Coralville, IA, USA. AHSG : Fetuin -A; ADIPOQ: Adiponectin; AGAPT: 1-acylglycerol -3-phosphate acyltransferas e-2; B2M : beta -2-microglobulin; CD36: Fatty acid translocase; CEBPB: CCAAT/enhancer -binding protein beta ( CEBPB ); DGAT1: Diacylglycerol O -Acyltransferase -1; DGAT2: Diacylglycerol O -Acyltransferase -2; EIF3K: Eukaryotic translation initiation factor 3 subuni t K; FABP4: fatty acid binding protein 4; FATP1: Fatty acid transporter -1; GAPDH: Glyceraldehyde -3-phosphate dehydrogenase; GPAT1: Glycerol -3-Phosphate Acyltransferase -1; GPAT2: Glycerol -3-Phosphate Acyltransferase -2, PPARG: Peroxisome proliferator -activat ed receptor gamma; RPL0: 50S ribosomal protein L15; RPS9 : 40S ribosomal protein S9. Western Blotting Western blots were performed as previously described (Strieder -Barboza et al., 2018 ). Protein extraction from cultured a dipocytes ( n = 8) were extracted using ice -cold RIPA buffer (Teknova, Hollister, CA) containing protease (Roche, San Francisco, CA) and phosphatase (Thermo Scientific, Waltham, MA) inhibitors. Estimation of protein content was carried out using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). FetA content was analyzed using reducing conditions. Briefly, samples were added to a reducing buffer containing 10 mmol/L dithiothreitol (DTT) and 5% -mercaptoethanol (BME) and denatured at 95¡C for 4 min. Equal amounts of total protein (20 µg) were electrophoresed on a 4 -20% SDS -PAGE gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in tris - 72 buffered saline solution (TBS) with 0.01% tween -20 (TBST) and 5% fat free m ilk. Membranes were incubated with a conjugated polyclonal rabbit anti -bovine AGAPT2 antibody (C -terminal region cat. no. ARP44636_P050; Aviva Systems Biology Corporation, San Diego, CA) in 1ug/mL concentration in TBST -5% milk for 16 h at 8 ¡C. #! -tubulin (1:1,000; Cat# 2148; Cell Signaling Technology, Danvers, MA) served as loading controls for adipocytes protein. Membranes were then exposed to horseradish peroxidase substrate ( cat. no. WBLUR0100; Millipore, Darmstadt, Germany), and visualized by chemilumi nescence using the ChemiDoc TM Touch Imaging System (Bio -Rad, Hercules, CA). Band densitometry was determined using the Image Lab software (Bio -Rad, Hercules, CA). Values of AGAPT2 protein content are expressed as means of relative band density using #! -tub ulin band intensity values as calibrator. Phosphatidic Acid Analysis Phosphatidic acid ( PA) concentrations in adipocytes were measured using the enzymatic fluorometric PicoProbeª Phosphatidic Acid Assay Kit (cat. no. K748, BioVision Incorporated, Milpitas , CA) following the manufacturer instructions. Briefly, after treating adipocytes ( n = 5) with or without 0.1 mg/mL of FetA from d 5 to d 7 of differentiation, culture media was removed, and cells were rinsed twice with ice -cold 1X PBS, scraped from cell c ulture plates, and collected in 1X PBS. For lipid extraction, 1x10 6 adipocytes were mixed with 1 mL of assay buffer (BioVision) and sonicated using an ultrasonic liquid processor (Misonix; Farmingdale, NY) for 2 min. Protein content was estimated at this p oint using a BCA Protein Assay Kit (Thermo Scientific). Following, 3.75 ml of chloroform/methanol/12N HCl (2 : 4 : 0.1 v/v) were added to the cell homogenate and mixed thoroughly. Then, 1.25 ml of chloroform were added to the solution, vortexed for 30 sec and added to 1.25 ml of 1 M NaCl. Followed by centrifugation, the lower organic layer containing solubilized lipids was collected and transferred to a glass tube. 73 Chloroform was evaporated in a vacuum concentrator (Savant SPD121P, Thermo Fisher) for 2 h at 40¡C. Before performing the analysis, samples were solubilized in 50 ) l 5% Triton X -100 solution. For PA measurement, 10 ) l of sample were added to a 96 -well plate and incubated for 1 h at 45¡C with a compound that hydrolyzes PA to form an intermediate. F ollowing, samples were incubated for 30 min at 37¡C in the presence of a developer and enzyme mix that converts a non-fluorescent probe to a fluorescent product (Ex/Em= 535/587 nm) that can be quantified (Synergy H1 Microplate Reader; Biotek). Statistical Analysis Data were analyzed using JMP Statistical Software (SAS Institute Inc., Cary, NC). Normality of the variables was checked using the Kolmogorov -Smirnov test ( P < 0.05). Non -normally distributed variables (FFA concentrations in lipolysis assay, FA up take assay, adipocytes AGAPT2 protein content) were ln transformed. One -way ANOVA Pairwise comparisons were performed using the TukeyÕs post hoc test. Mean differences were considered significant when P ( 0.05 and tendencies when P < 0.10. RESULTS Bovine Cultured Adipocytes Abundantly Express and Secrete Fetuin -A Based in a potential autocrine effect of FetA in AT lipid mobilization, we first evaluated the capacity of bovine adipocytes to express and sec rete FetA. We observed that cultured bovine adipocytes abundantly express AHSG and FetA protein at similar level as AT of periparturient cows ( P > 0.0 5; Fig. 5.1) and secrete 48 ± 3.5 ng of FetA (mean ±SEM). 74 Figure 5. 1 Fetuin -A expression in cultured bovine adipocytes and subcutaneous adipose tissue of dairy cows. (A) Fetuin -A gene ( AHSG ) and (B) protein expression in cultured bovine adipocytes (n=5) at 7th day of differentiation in vitro and subcutaneous adipose tissue of dairy cows (n=5) at 8 ± 3 d after parturition. Cultured adipocytes were derived from tailhead subcutaneous adipose depot of dairy cows. For qPCR results, values are shown as relative gene expression 2( +,, CT) (where CT = cycle threshold), normalized by control genes RPS9 and EIF3K, and represent mean ± SEM. For Western blot analysis, #! -tubulin was used as a loading control, and values are expressed as a ratio between Fetuin -A and #! -tubulin adjusted band intensity (mm2). Results represent means ± SEM. Fetuin -A Attenuates Lipolytic Responses in Bovine Adipocytes After assessing the secretory ability of bovine adipocytes to secrete FetA, we were interested in evaluating the effect of FetA on the modulation of lipid mobiliza tion. Therefore, we first assessed the effect of FetA on lipolytic responses of bovine adipocytes. !-adrenergic stimulation with ISO increased lipolysis compared with CON, as reflected in the release of glycerol (12±0.04 vs 0.04±0.02 nM, P = 0.003; Fig. 5.2A) and FFA (15±13 vs 6.2±2.4 nM, P = 0.04; Fig. 5.2B). Lipolysis induced by ISO was attenuated by FetA (FETA+ISO) as reflected by a lower glycerol (0.06±0.04 nM, P = 0.02) and FFA (5.7±2.7 nM, P = 0.01) release compared with ISO alone. ABAdipocytes Adipose tissue 0123P = 0.31Fetuin-A!"TubulinFetuin-A / !"Tubulin Relative ExpressionAdipocytes Adipose tissue 0123AHSG Relative Gene Expression (2-!!ct)P = 0.59 75 Figure 5. 2 Effect of Fetuin -A on !-adrenergic induced -lipolysis in bovine adipocytes. (A) !Concentrations of glycerol and (B) FFA released in the supernatant during 2 h -lipolysis induction. Values were calibrated by the number of cells per well and are expressed as mean ± SEM. *Statistically different ( P < 0.05). Figure 5. 3 Effect of Fetuin -A on fatty acid (FA) uptake by bovine adipocytes in vitro . (A) !Example of Kinetic QBT FA uptake output from b ovine adipocytes over 1 h -assay. (B) Area under the curve (AUC) from FA uptake assays of bovine adipocytes treated with 0 (CON) or 0.1 mg/mL FetA (FETA) or 10 nM insulin (INSULIN). Bovine adipocytes were treated and pre -incubated with treatments for 30 min utes at 37C before the assay. Values are expressed as AUC using CON FA uptake as the calibrator (means ± SEM). Bars with different letters (a Ðc) are significantly different ( P ( 0.05). Fetuin -A enhances lipogenesis in bovine adipocytes Since lipolysis and lipogenesis are continuous processes occurring simultaneously within adipocytes, after evaluating lipolysis, we were interested on evaluatin g the effect of FetA on lipogenic activity. We assessed lipogenesis by evaluating FFA uptake and accumulation of 76 intracellular TAG. Exposure of adipocytes to FetA during 1.5 h (pre -incubation and 1 h -kinetic assay; Fig. 5.3A) incremented FFA uptake by 1.5 times ( P = 0.02; Fig. 5.3B) compared with CON and stimulated similar response as induced by INSULIN ( P = 0.65; Fig. 5.3B). Insulin increased FFA uptake by 1.69 times compared with CON ( P = 0.004; Fig. 5.3B). In agreement with the higher incorporation of F FA, we observed that cells treated with 0.1 mg/mL of FetA during 48 h (d 5 to d 7 of differentiation) increased their adipogenic capacity as demonstrated by higher accumulation of TAG compared with non -treated adipocytes (CON), analyzed by both, lipid drop lets staining with HCS LipidTOXª ( P = 0.003; Fig. 5.4A-C) and AdipoRedª assay ( P = 0.04; Fig. 5.4D-E). There was 1.7 times increment in the accumulation of TAG in FETA compared with CON ( P = 0.04; Fig. 5.4D-E) and 37 times compared with preadipocytes ( P <0.0001). CON increased TAG accumulation by 22 times compared with preadipocytes ( P = 0.006). Fetuin -A Upregulates AGAPT2 Expression and Activity in Bovine Adipocytes To explore potential drivers of FetA lipogenic function, we evaluated the effect of FetA treatment on key regulators of adipogenesis, such as CEBPB, ADIPOQ and PPARG , on components of FA uptake cascade including FABP4, FATP1 and FAT/CD36, and on lipogenic enzymes controlling de novo TAG synthesis in adipocytes, such as AGAPT2, DGAT1, DGAT2, GPAT1, GPAT2 and LIPIN1. We observed that the treatment with 0.1 mg/mL of FetA increa sed the gene expression of AGAPT2 (P = 0.02; Fig. 5.5A) and AHSG (P = 0.05; Fig. 5.5B) and tended to increase the expression of CEBPB (P = 0.07; Fig. 5.5C) and FATP1 (P = 0.07; Fig. 5.5D). There was no difference on the gene expression of PPARG, ADIPOQ, DG AT1, DGAT2, GPAT1, GPAT2, LIPIN1, FAT/CD36 and FABP4 between FETA and CON ( P > 0.05). Given the specific upregulation of AGAPT2 gene expression by FetA, we also analyzed protein content of 77 AGAPT2 treated with FetA. In agreement with the increase on gene ex pression, protein content of AGAPT2 was also increased in FETA compared with CON ( P = 0.04; Fig. 5.5B). Figure 5. 4 Effect of Fetuin -A on triacylglycerol accumulation in cultured bovine adipocytes. CONFETA Cow 1Cow 2Cow 3Cow 4PreadipocytesCONFETA 02!1054!1056!1058!105Relative Fluorescence Unit*Non-stained adipocytes ABCPREADIPO CONFETA 0100200300400Relative Fluorescence UnitabcCONFETA DPREADIPOE 78 Figure 5. 4 (cont Õd) (A) Laser scanning confocal microscopy imaging (60 X) of lipid droplets in cultured bovine adipocytes ( n= 8) treated w ith treated with 0 (CON; bottom panel) or 0.1 mg/mL FetA (FETA; upper panel). (B) Preadipocytes and non -stained cells served as controls. Lipid droplets of bovine adipocytes were stained with Alexa Fluor 594 (HCS LipidToxª; red fluorescence) and nuclei wer e stained with DAPI (NucBlueª; blue fluorescence). (C) Plotted relative fluorescence intensity measured through ImageJ software (means ± SEM). Scale bars: 20 ) m. (D) Relative fluorescent unit of AdipoRedª assay analysis in cultured bovine adipocytes ( n= 5) treated with with 0 (CON) or 0.1 mg/mL FetA (FETA). (E) Microscopy imaging (20 X) of lipid droplets (dark red dots) in bovine adipocytes and preadipocytes (PREADIPO) incubated with AdipoRedª for 20 minutes. Scale bars: 100 ) m. Bars wi th different letters (a Ðc) are significantly different ( P ( 0.05). The enzymatic activity of AGAPT2 during TAG synthesis corresponds to transferring an additional FA to lyso -phosphatidic acid (LPA) to produce phosphati dic acid (PA). We assessed the effect of FetA on AGAPT2 activity by quantifying the concentrations PA in bovine adipocytes treated or not with 0.1 mg/mL of FetA from d5 to d7 of differentiation. Adipocytes treated with FetA synthetized more PA (0.27 ± 0.00 2 nmol) compared with non -treated CON adipocytes (0.20 ± 0.01 nmol; P = 0.060; Fig. 5.5 F). DISCUSSION The balance between lipogenesis and lipolysis rates in AT of periparturient dairy cows is critical to assure adequate adaptive responses and a healthy and successful transition to lactation. In our previous study (Strieder -Barboza et al., 2018 ), we identified a negative association between FetA and markers of lipolysis, and similar dynamics between AT FetA and other lipogenic mar kers. In addition to that, previous in vivo and in vitro studies in non -ruminants have consistently reported strong lipogenic properties of FetA (Cayatte et al., 1990 , P”rez -Sotelo et al., 2016 ), however, the mechanisms by which FetA increases TAG synthesis in adipocytes have not yet been studies. In this study, for the first time, we report that FetA modulates lipid mobilization in bovine adipocytes and identify A GAPT2 activity as a novel potential driver of lipogenic function of FetA in bovine adipocytes. 79 Figure 5. 5 Effect of Fetuin -A on gene expression of lipogenic markers and phosphatidic acid concentrations in bovine adipocytes. Bovin e adipocytes were treated with 0 (CON) or 0.1 mg/mL of Fetuin -A (FETA) for 48 h during late differentiation in vitro . (A) Gene expression and (B) protein content ( n=8) of 1 -acylglycerol -3-phosphate acyltransferase -2 (AGAPT2). Gene expression of (C) Fetuin -A ( AHSG ), (D) adipogenesis promoter CCAAT/enhancer -binding protein beta ( CEBPB ), and (E) fatty acid transporter -1 ( FATP1 ). (F) Phosphatidic acid concentrations calibrated by total protein CONFETA 0.00.51.01.5AGAPT2 relative expression (2-ddct)A*CONFETA 0123AGAPT2/ !"-Tubulin Relative expressionAGAPT2!"-Tubulin *BCONFETA 0.00.10.20.30.4P = 0.06Phosphatidic acid nmol/ng FCONFETA 0.00.51.01.52.0P = 0.07FATP1 relative expression (2-ddct)ECONFETA 0.00.51.01.52.02.53.0AHSG relative expression (2-ddct)C*CONFETA 0.00.51.01.5CEBP"Relative Expression (2-ddct)P = 0.07D 80 Figure 5. 5 (cont Õd) concentrations (mg) in sampled bovine adipocytes. For qPCR results ( n=6), values are shown as relative gene expression 2( +,, CT) (where CT = cycle threshold), normalized by control genes RPS9 and EIF3K, and represent mean ± SEM. For Western blot analysis, #! -tubulin was used as a loading control, and values are expressed as a ratio between Fetuin -A and #! -tubulin adjusted band intensity (mm2). Results represent means ± SEM. In our study, cultured bovine adipocytes derived from subcutaneous AT of dairy cows abundantly express and secrete FetA. Previous secretome studies using viscer al and gonadal AT of rats detected significant amounts of FetA (Roca -Rivada et al., 2011 ). More recently, the same authors reported an abundant secretion of FetA by C3H10T1/2 cells as a murine model of pre -adipocyte differentiation, but only the secretom e from mature differentiated adipocytes contained FetA (P”rez -Sotelo et al., 2016 ). We have recentl y reported an abundant mRNA and protein expression of FetA in subcutaneous and omental AT of periparturient and mid -lactation dairy cows, as well as in cultured bovine adipocytes (Strieder -Barboza et al., 2018 ).In a proteomics study, Zachut et al. (2017) also reported an abundant FetA in subcutaneous AT of dairy cows in late gestation. However, this is the first study reporting FetA secretion by adipocytes. Secretion of FetA was demonstrated to decrease during fasting, weight loss and anorex ia and to be increased during weight gain and obesity in rodents and humans (Jialal et al., 2015 , P”rez -Sotelo et al., 2016 ). Similarly, serum concentrations and AT gene and protein expression of FetA decreased during a marked negative energy balance in early lactation dairy cows (Strieder -Barboza et al., 2018 ). The changes in the FetA secr etion pattern by the AT of animals and humans going through lipid mobilization suggest the participation of FetA in adipocytesÕ lipogenesis and lipolysis. In the present study, FetA attenuated lipolytic responses stimulated by !-adrenergic activation with ISO. In humans, lipolysis in adipocytes appears to be a phenotype that is 81 particularly sensitive to variation in AHSG , the gene encoding FetA (Dahlman et al., 2004 , Lavebratt et al., 2005 ). A common variation (Thr230Met) in the AHSG is associated with a marke d incre ase in ! 2-adrenoceptor sensitivity in subcutaneous adipocytes, which may be of importance in body weight regulation (Lavebratt et al., 2005 ). In a large study screening adipocyte phenotype in obese and non -obese healthy women, single -nucleotide polymorphisms ( + 469T>G) in AHSG was strongly associated with insulin inhibition of lipolysis (Dahlman et al., 2004). Our results suggest that the effect of FetA during induced -lipolysis in bovine adipocytes was insulin -independent since no insulin was added to the medium during stimulated lipolysis. Previous studies suggest that FetA may control insulin signaling in AT and is an attractive candidate gene for disturbed adipocyte lipolytic function in obesity and insulin resistance in humans (Dahlman et al., 2004 ). Adipose tissue lipolytic responsivenes s and sensitivity to adrenergic agents, including the natural catecholamines, are increased in dairy cows around parturition (Bell, 1995 ), and are one of the mechanisms leading to the very hig h plasma concentrations of FFA and disease development during the periparturient period (Grummer, 1993). Therefore, attenuating lipolysis and/or regulating insulin function in early lactation would be parti cularly important in dairy cows that develop hypoinsulinemia concurrently with a state of insulin resistance (Contreras et al., 2017 ). Even though FetA seems to be involved in the pathways that control !-adrenergic lipolytic responses in bovine adipocytes, the mechanisms by which this adipokine may play an anti -lipolytic roles, how to modulate them, and the identification of potential AHSG alleles that could be particularly involved in AT lipolytic responses in dairy cows remain to be stablished. In the present study, we observed that incubation of bovine adipocytes with FetA increased FA uptake compared with non -treated cells, and induced similar responses as cells 82 treated with insulin, a strong lipogenic factor (Stahl et al., 2002 ). Although speculative at this time, FetA could have stimulated t he translocation of plasma membrane FA transporters (i.e. FATP1 and CD36), similar to known effects of insulin (Stahl et al., 2002 ). Variants of FetA gene had a local impact in AT on insulin regulation of lipogenesis and lipolysis in humans (Dahlman et al., 2004 ). Other p ossibility is that FetA could have bound FA and translocated them into the intracellular compartment. FA transporters, such as albumin and FetA, are known by carrying lipids in plasma and facilitating FA uptake by cells (Glatz et al., 2010 ). Previously, FetA was reported to translocate into 3T3 -L1 a nd human adipocytes (Dasgupta et al., 2010 ), and to accumulate in vesicles in the cytosol (Reynolds et al., 2005 ); nevertheless, its relationship with FA transport was not reported by these studies. Fetuin -A carries high amounts of chole sterol, cholesteryl esters, TAG, and FA in plasma, which correspond to nearly 33% of its molecule (Kumbla et al., 1989 , Kumbla et al., 1991 ). In rabbit and humanÕs cells, FetA increased the incorporation of exogenous FA into intracellular TAG by nearly 50 -fold compared with albumin (Cayatte et al., 1990 ). Similarly, we observed that FetA increases TAG accumulation in cultured bovine adipocytes compared with non -treated cells. This suggests that increased FetA -stimulated FA uptake by adipocytes might have enhanced the intracellular synthesis of TAG. Due to its lipogenic properties, increased serum concentrations and AT expression and secretion of have been associated with obesity and obesity -rel ated disorders in humans and animals, such as metabolic syndrome (Chen et al., 2009 , Jialal et al., 2015 , P”rez -Sotelo et al., 2016 ). In dairy cows, we previously reported a decrease in FetA serum concentrations and in AT expression from 2 wks prepartum to 10 DIM, and was more pronounced in cows that lost m ore fat mass from dry off to early lactation (Strieder -Barboza et al., 2018 ). This period of decreased FetA coincided with the downregulation in the transcription of lipogenic enzymes in the subcutaneous 83 AT and with a marked increase of pla sma FFA concentrations in periparturient cows (Strieder -Barboza et al., 2018 ). Similarly, FetA protein abundancy in subcutaneous AT of dairy cows during late gestation also decreased during environmental heat stress and increased circulati ng FFA (Zachut et al ., 2017 ). Together, these results provide evidence that FetA may promote lipogenesis in vitro and in vivo in the AT of dairy cows. Although it is clear that FetA stimulates the synthesis of TAG, the mechanisms involved are yet to be studied. Whether the stimulation of TAG accumulation in bovine adipocytes is mainly due to the entry of FA into cells or whether FetA might directly stimulate lipogenic enzymes is not known. To start addressing these questions, we evaluated the gene expression of key enzymes involved in the TAG synthesis through the glycerol -3-phosphate pathway in adipocytes: (1) Glycerol phosphate acyltransferases (GPAT), which is involved in glycero -P synthesis; (2) 1-acylglycerol -3-phosphate acyltransferase (AGPAT), catalyst for the first s tep in the formation of phosphotidic acid (PA); (3) phosphatidate phosphatase (Lipin), which forms diacylglycerol; and diacylglycerol acyltransferases (DGAT) (Cole man and Lee, 2004 , Takeuchi and Reue, 2009 ). In our study, we observed that FetA upregulated the gene expression of AGAPT2 in bovine adipocytes. In agreement with that, we also observed an increased AGAPT2 protein content in FETA. AGPAT2 is the predominant AGAPT isoform in AT and catalyzes acylation of its strict substrate lysophosphatidic acid to PA (Takeuchi and Reue, 2009 ). In cows, AGAPTs play an important role in the TAG synthesis in the mammary gland (Mistry and Medrano, 2002 ), but have not yet been reported in AT. AGAPT2 is strongly induced by PPAR " agonists (Blanchard et al., 2016 ) and is required for TAG mass accumulation in mature adipocytes (Gale et al., 2006 ). Knocking down AGPAT2 decreased gene expression of adipogenesis regulators such as PPAR ! and C/EBPB , delayed induction of mature adipocyte 84 markers such as FABP4 and GLUT4 , and reduced TAG accumulati on in adipocytes (Blanchard et al., 2016 ). The crucial function of AGAPT2 for adipogenesis is highlighted by the near complete absence of AT and a range of metabolic changes, such as ex treme insulin resistance, in humans with c ongenital generalized lipodystrophy as a consequence of AGAPT2 deficiency (Agarwal and Garg, 2006 ). These result s demonstrate that other AGAPT family members (AGAPT1, and AGAPT3 -10) cannot compensate AGAPT2Õs activity for the synthesis of TAG, thus being a specific and rate limiting enzyme during TAG synthesis (Agarwal et al., 2002 , Takeuchi and Reue, 2009 ). AGAPT2 controls adipogenesis through modulation of the synthesis of phospholipids and TAG precursors, especially PA (Gale et al., 2006 ). Impaired AGPAT2 activity affects availability of PA for TAG synthesis but not overall PA synthesis nor utilization of PA for phospholipid synt hesis (Gale et al., 2006 ). In our study, we indirectly evaluated the effect of FetA on AGAPT2 activity by measuring adipocyte concentrations of PA. We observed that FetA increased not only gene expression and protein content of AGAPT2 in bovine adipocytes, but also PA concentrations. Phosphaditic acid was highlighted as one of the main lipid regulators of the size of lipid droplets, an important lipid -storage organell e (Fei et al., 2011 ). Our results suggest that FetA increases TAG accumulation by increasing AGAPT2 activity, and therefore PA concentrations for TAG synthesis in bovin e adipocytes. Knowing that FetA may modulate TAG synthesis precursors in adipocytes provide valuable insights into potential targets for modulating flux of lipids in dairy cowsÕ AT. Our study haa limitations. First, we analyzed the effect of FetA on a lim ited pool of genes involved on adipogenesis, FA uptake and lipogenesis. Although we identified an effect of FetA on a few of these selected genes, it is possible that other key lipogenic markers could be driving 85 the lipogenic function of FetA. Second, we c annot conclusively affirm that AGPAT2 signaling is required for FetA lipogenic effect. To do so, further studies using AGAPT2 and FetA knockout models are necessary to evaluate the potential interdependent function of these components on TAG synthesis in b ovine adipocytes. CONCLUSIONS Results from this study demonstrate that FetA modulates lipid mobilization by attenuating lipolytic responses and enhancing lipogenesis in bovine adipocytes. FetA upregulates the expression and activity of AGAPT2, a rate -limiting lipogenic enzyme, and suggests a potential mechanism by which this FetA promotes TAG synthesis in adipocytes. Our study provides novel knowledge on how FetA promotes TAG synthesis in adipocytes and opens the possibility of using FetA as a potential th erapeutic target for the modulation of lipid mobilization in AT of periparturient dairy cows and humans with similar AT disorders. Acknowledgments This research was supported by USDA -National Institute of Food and Agriculture (Washington, DC) grant 2015 -67015-23207, Department of Large Animal Clinical Sciences (East Lansing, MI) , Research and Educational Grants Program (2015 -2016), and Global Agri -Trade Corporation (Gardena, CA). The authors are grateful to Kyan Thelen, Jenne De Koster and Connor Lewicki. The staff at the Michigan State University Meat Laboratory (East Lansing) and West Michigan Beef for providing samples, and Jeff Gandy for technical assistance. 86 REFERENCES 87 REFERENCES Agarwal, A. K., E. Arioglu, S. de Almeida , N. Akkoc, S. I. Taylor, A. M. Bowcock, R. I. Barnes, and A. Garg. 2002. AGPAT2 is mutated in congenital generalized lipodystrophy linked to chromosome 9q34. Nature genetics 31(1):21 -23. Agarwal, A. K. and A. Garg. 2006. Genetic basis of lipodystrophies a nd management of metabolic complications. Annu. Rev. Med. 57:297 -311. Bauman, D. E. and W. B. Currie. 1980. Partitioning of nutrients during pregnancy and lactation: a review of mechanisms involving homeostasis and homeorhesis. J Dairy Sci 63. Bell, A. W. 1995. Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation. Journal of Animal Science 73:2804 -2819. Blanchard, P. G., V. Turcotte, M. CŽt”, Y. G”linas, S. Nilsson, G. Olivecrona, Y. Deshaies, and W. T. Festucci a. 2016. Peroxisome proliferato r-activated receptor " activation favours selective subcutaneous lipid deposition by coordinately regulating lipoprotein lipase modulators, fatty acid transporters and lipogenic enzymes. Acta Physiologica 217(3):227 -239. Cayatte, A. J., L. Kumbla, and M. T . Subbiah. 1990. Marked acceleration of exogenous fatty acid incorporation into cellular triglycerides by fetuin. Journal of Biological Chemistry 265(10):5883 -5888. Chen, H. -Y., Y. -L. Chiu, S. -P. Hsu, M. -F. Pai, C. -F. Lai, Y. -S. Peng, T. -W. Kao, K. -Y. Hung , T.-J. Tsai, and K. -D. Wu. 2009. Association of serum fetuin A with truncal obesity and dyslipidemia in non -diabetic hemodialysis patients. Eur J Endocrinol 160(5):777 -783. Coleman, R. A. and D. P. Lee. 2004. Enzymes of triacylglycerol synthesis and their regulation. Progress in lipid research 43(2):134 -176. Contreras, G. A. and L. M. Sordillo. 2011. Lipid mobilization and inflammatory responses during the transition period of dairy cows. Comparative Immunology, Microbiology and Infectious Diseases 34(3):2 81-289. Contreras, G. A., C. Strieder -Barboza, and J. De Koster. 2018a. Modulating adipose tissue lipolysis and remodeling to improve immune function during the transition period and early lactation of dairy cows. Journal of Dairy Science In press. Contrer as, G. A., C. Strieder -Barboza, and J. De Koster. 2018b. Symposium review: Modulating adipose tissue lipolysis and remodeling to improve immune function during the transition period and early lactation of dairy cows. Journal of dairy science 101(3):2737 -2752. Contreras, G. A., C. Strieder -Barboza, and W. Raphael. 2017. Adipose tissue lipolysis and remodeling during the transition period of dairy cows. Journal of Animal Science and Biotechnology 8(1):41. 88 Dahlman, I., P. Eriksson, M. Kaaman, H. Jiao, C. Lindg ren, J. Kere, and P. Arner. 2004. # 2-Heremans ÐSchmid glycoprotein gene polymorphisms are associated with adipocyte insulin action. Diabetologia 47(11):1974 -1979. Dasgupta, S., S. Bhattacharya, A. Biswas, S. S. Majumdar, S. Mukhopadhyay, and S. Ray. 2010. NF-kappaB mediates lipid -induced fetuin -A expression in hepatocytes that impairs adipocyte function effecting insulin resistance. Biochemical Journal 429(3):451 -462. Fei, W., G. Shui, Y. Zhang, N. Krahmer, C. Ferguson, T. S. Kapterian, R. C. Lin, I. W. Dawe s, A. J. Brown, and P. Li. 2011. A role for phosphatidic acid in the formation of ÒsupersizedÓ lipid droplets. PLoS genetics 7(7):e1002201. Gale, S. E., A. Frolov, X. Han, P. E. Bickel, L. Cao, A. Bowcock, J. E. Schaffer, and D. S. Ory. 2006. A regulatory role for 1 -acylglycerol -3-phosphate -O-acyltransferase 2 in adipocyte differentiation. Journal of Biological Chemistry 281(16):11082 -11089. Glatz, J. F., J. J. Luiken, and A. Bonen. 2010. Membrane fatty acid transporters as regulators of lipid metabolism: i mplications for metabolic disease. Physiological reviews 90(1):367 -417. Grummer, R. R. 1993. Etiology of lipid -related metabolic disorders in periparturient dairy cows. Journal of Dairy Science 76(12):3882 -3896. Heinrichsdorff, J. and J. M. Olefsky. 2012. Fetuin -A: the missing link in lipid -induced inflammation. Nat Med 18(8):1182 -1183. Hellemans, J., G. Mortier, A. De Paepe, F. Speleman, and J. Vandesompele. 2007. qBase relative quantification framework and software for management and automated analysis of real -time quantitative PCR data. Genome Biology 8(2):R19 -R19. Jialal, I., S. Devaraj, A. Bettaieb, F. Haj, and B. Adams -Huet. 2015. Increased adipose tissue secretion of Fetuin -A, lipopolysaccharide -binding protein and high -mobility group box protein 1 in metabolic syndrome. Atherosclerosis 241(1):130 -137. Kumbla, L., S. Bhadra, and M. T. Subbiah. 1991. Multifunctional role for fetuin (fetal protein) in lipid transport. The FASEB Journal 5(14):2971 -2975. Kumbla, L., A. Cayatte, and M. Subbiah. 1989. Associ ation of a lipoprotein -like particle with bovine fetuin. The FASEB Journal 3(9):2075 -2080. Kusminski, C. M., P. E. Bickel, and P. E. Scherer. 2016. Targeting adipose tissue in the treatment of obesity -associated diabetes. Nature Reviews Drug Discovery 15(9 ):639. Lavebratt, C., S. Wahlqvist, L. Nordfors, J. Hoffstedt, and P. Arner. 2005. AHSG gene variant is associated with leanness among Swedish men. Human Genetics 117(1):54 -60. Lehr, S., S. Hartwig, and H. Sell. 2012. Adipokines: a treasure trove for the d iscovery of biomarkers for metabolic disorders. PROTEOMICS -Clinical Applications 6(1 !2):91 -101. 89 McNamara, J. P. 1995. Role and regulation of metabolism in adipose tissue during lactation. The Journal of Nutritional Biochemistry 6(3):120 -129. Mistry, D. H. and J. F. Medrano. 2002. Cloning and Localization of the Bovine and Ovine Lysophosph atidic Acid Acyltransferase (LPAAT) Genes that Codes for an Enzyme Involved in Triglyceride Biosynthesis. Journal of Dairy Science 85(1):28 -35. Pal, D., S. Dasgupta, R. Kundu, S. Maitra, G. Das, S. Mukhopadhyay, S. Ray, S. S. Majumdar, and S. Bhattacharya. 2012. Fetuin -A acts as an endogenous ligand of TLR4 to promote lipid -induced insulin resistance. Nature Medicine 18(8):1279 -1285. P”rez -Sotelo, D., A. Roca -Rivada, M. Larrosa -Garc™a, C. Castelao, I. Baamonde, J. Baltar, A. B. Crujeiras, L. M. Seoane, F. F . Casanueva, and M. Pardo. 2016. Visceral and subcutaneous adipose tissue express and secrete functional alpha2hsglycoprotein (fetuin a) especially in obesity. Endocrine:1 -12. Reynolds, J. L., J. N. Skepper, R. McNair, T. Kasama, K. Gupta, P. L. Weissberg, W. Jahnen -Dechent, and C. M. Shanahan. 2005. Multifunctional roles for serum protein fetuin -a in inhibition of human vascular smooth muscle cell calcification. Journal of the American Society of Nephrology 16(10):2920 -2930. Roca -Rivada, A., J. Alonso, O. Al-Massadi, C. Castelao, J. R. Peinado, L. M. Seoane, F. F. Casanueva, and M. Pardo. 2011. Secretome analysis of rat adipose tissues shows location -specific roles for each depot type. Journal of Proteomics 74(7):1068 -1079. Stahl, A., J. G. Evans, S. Pattel , D. Hirsch, and H. F. Lodish. 2002. Insulin causes fatty acid transport protein translocation and enhanced fatty acid uptake in adipocytes. Developmental cell 2(4):477 -488. Strieder -Barboza, C., J. de Souza, W. Raphael, A. L. Lock, and G. A. Contreras. 20 18. Fetuin -A: A negative acute -phase protein linked to adipose tissue function in periparturient dairy cows. Journal of Dairy Science 101(3):2602 -2616. Takeuchi, K. and K. Reue. 2009. Biochemistry, physiology, and genetics of GPAT, AGPAT, and lipin enzymes in triglyceride synthesis. American Journal of Physiology - Endocrinology and Metabolism 296(6):E1195 -E1209. Vernon, R. and K. Houseknecht. 2000. Adipose tissue: beyond an energy reserve. Ruminant physiology: digestion, metabolism, growth and reproduction (ed. PB Cronj”):171 -186. Vernon, R. G. and C. M. Pond. 1997. Adaptations of maternal adipose tissue to lactation. Journal of Mammary Gland Biology and Neoplasia 2(3):231 -241. Zachut, M. 2015. Defining the Adipose Tissue Proteome of Dairy Cows to Reveal Bi omarkers Related to Peripartum Insulin Resistance and Metabolic Status. Journal of Proteome Research 14(7):2863 -2871. 90 Zachut, M., G. Kra, L. Livshitz, Y. Portnick, S. Yakoby, G. Friedlander, and Y. Levin. 2017. Seasonal heat stress affects adipose tissue p roteome toward enrichment of the Nrf2 -mediated oxidative stress response in late -pregnant dairy cows. Journal of Proteomics 158:52 -61. 91 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS The main objective of the present dissertation was to evaluate the dynamic s of FetA systemically and in the AT of dairy cows during the periparturient period, and to elucidate the roles of FetA on adipocyte lipid mobilization. We demonstrated that serum and AT FetA decreases after parturition and has similar dynamics as other li pogenic markers in AT of periparturient dairy cows (Strieder -Barboza et al., 2018). Although FetA is increased during obesity in non -ruminants (Chen et al., 2009, Jialal et al., 2015), we were unable to detect an effect of adiposity on serum concentrations of FetA in periparturient cows. In contrast, we observed decreased gene expression of FetA in subcutaneous AT of overconditioned cows. These results may indicate a local effect of excess adiposity on the expression and secretion of FetA by AT and therefor e in the mechanisms of lipid mobilization in adipocytes. Previous studies demonstrated that FetA knockout mice have decreased body fat content, altered lipid metabolism and higher energy expenditure (Mathews et al., 2002). These findings suggest that FetA may play a significant role in regulating glucose disposal, insulin sensitivity, weight gain and fat accumulation. In dairy cows, whether lower FetA expression in AT of overconditioned animals is a cause or an effect of decreased lipogenesis or excessive l ipolysis is still unknown and requires further investigation. FetA has been reported as an anti -inflammatory factor during acute inflammation such as during infections, cerebral ischemic injury, and lethal endotoxemia in monogastrics (Wang and Sama, 2012) . Accordingly, we observed that serum FetA follows the dynamics of a negative acute -phase protein in periparturient dairy cows and its protein content in AT decreases upon an acute inflammatory stimulus (Strieder -Barboza et al., 2018). A potential anti -inf lammatory effect 92 of FetA on bovine adipocytes was also supported by the decreased CCL2 expression in cells treated with FetA compared with LPS (Strieder -Barboza et al., 2018). Following up on these observations, we generated initial epidemiological data th at may suggest a beneficial role of FetA on inflammatory responses in periparturient dairy cows: we observed that at time of dry off and 2 weeks before parturition, serum FetA concentrations were lower in cows that were later diagnosed with retained placen ta and metritis, respectively (data not shown). Even though these results suggest that FetA plays an anti -inflammatory role systemically and in the AT of dairy cows, further studies are needed. Future studies will evaluate the expression and secretion of inflammation markers (e.g., TNF, IFN -" , and IL -6) by bovine adipocytes in response to inflammatory stimulus followed by the treatment with different doses of FetA. Based on our results and previous studies, we also expect to find an anti -inflammatory effect of FetA in future studies with bovine cells. One of the main goals of this dissertation was to elucidate the effects of FetA on AT lipid mobilization in dairy cows. For this purpose, we developed a novel in vitro model for efficiently inducing adipogenes is in bovine adipocytes and reported, for the first time, that primary adipocytes not only express FetA gene and protein, but also secrete FetA. Our findings support that FetA is a pro -lipogenic and anti -lipolytic adipokine in AT of dairy cows. We demonstr ated potential modes of action for FetA during lipid mobilization and identified a potential molecular target for its pro -lipogenic effect. For the first time, we revealed that the upregulation of the expression and activity of AGAPT2, a rate limiting lipo genic enzyme, is a potential mechanism by which FetA enhances triacylglycerol synthesis in bovine adipocytes. When performing these in vitro assays, we chose to use a single dose of 0.1 mg/mL of FetA. This was based on previous studies with human and mice adipocytes (Dasgupta et al., 2010, Pal et al., 2012), on the 93 biological range of FetA concentrations in serum of dairy cows (Strieder -Barboza et al., 2018), and on the results of a dose -response to FetA using bovine adipocytes in our laboratory. When perfo rming a dose -response analysis on the effects of FetA (0, 0.01, 0.1, 0.25, 0.5 and 1 mg/mL) on the gene expression of adipogenesis and lipogenesis regulators, we observed a nonlinear effect with the expression of ADIPOQ, CD36 and FABP4 tending to decrease when treated with 0.25 mg/mL or higher doses of FetA. The effects of a wider dose range of FetA on the expression and secretion of lipogenesis regulators and other proteins involved on lipolysis regulation and inflammatory responses in the AT of dairy cows require further investigation. Besides the treatment of adipocytes with different doses of FetA, the analysis of the potential autocrine effects of FetA on adipocytes through silencing or inhibiting the transcription of FetA gene, could also reveal intrin sic effects of FetA on AT function in dairy cows. Based on our in vivo and in vitro results, we hypothesize that the lipogenic and negative acute phase protein role of FetA is beneficial for periparturient cowsÕ health. First, we observed a positive assoc iation of FetA with health biomarkers, such as serum albumin and calcium, and an inverse association with pro -inflammatory markers from 2 weeks prepartum to 10 DIM in dairy cows. Second, we observed that FetA dynamics are similar to a negative acute -phase protein with potential anti -inflammatory effects in AT. Third, we observed an anti -lipolytic and pro -lipogenic effect of FetA in adipocytes of dairy cows, suggesting that this adipokine could play a beneficial role during the intense lipid mobilization in periparturient dairy cows. Therefore, strategies to enhance FetA synthesis could potentially improve metabolic function and prevent diseases, mainly during periods of negative energy balance such as the peripartum. Previous studies demonstrated that high -fat diet induced an increase in circulating concentrations and adipose tissue secretion of FetA in rats (P”rez -Sotelo et al., 2016). In vitro, Dasgupta et al. 94 (2010) demonstrated a dose -dependent lipid -induced secretion of FetA by rat hepatocytes, being hig her in cells treated with palmitic, myristic and stearic fatty acids compared with oleic, linolenic and arachidonic acids (Dasgupta et al., 2010). These studies suggest that the secretion of FetA could be potentially augmented by the dietary supplementatio n with fatty acids. Determining whether feeding supplemental fats with diverse fatty acid profiles affects FetA concentrations and function, as well as dairy cowÕs health will be valuable for defining potential novel nutritional strategies for the peripart urient period. Genetic studies identified that variations on FetA gene (AHSG) are associated with anti -lipolytic and pro -lipogenic properties in adipocytes, and highlighted AHSG as an attractive candidate gene for disturbed adipocyte lipolytic function in obesity and insulin resistance in humans (Dahlman et al., 2004). In ruminants, evidence from global gene expression profiling studies revealed that AHSG may be involved in regulating energy metabolism in dairy cattle (Chen et al., 2011), and lipid accumul ation in several adipose tissue depots in beef cattle (Robinson and Oddy, 2004). Additionally, we found an anti -lipolytic and pro -lipogenic effect of FetA in adipocytes and observed that FetA is a negative acute phase protein in adipose tissue. We envision a genetic study identifying potential variations on AHSG associated with these valuable functions in adipose tissue of periparturient dairy cows. This will be important because AHSG could be used through genetic selection by dairy producers to help reduce disease incidence and improve profitability. Even though we were able to demonstrate the up -regulatory effect of FetA on AGAPT2 activity, we could not conclusively affirm that AGPAT2 signaling is required for FetA lipogenic effects. Further studying the potential interdependent relationship between FetA and AGAPT2 through the use of knockout models will be conclusive to unravel additional mechanisms and 95 potential key targets of FetA in the lipogenic pathways in adipocytes. Additionally, AGAPT2 deficiency causes a human disease characterized by impaired triacylglycerol synthesis in white adipose tissue and leads to the development of serious insulin resistance and other metabolic dysfunctions (Agarwal et al., 2002). Therefore, the better understanding of th e molecular signaling pathways connecting AGAPT2 and FetA could be comparatively important in developing therapies for human metabolic diseases. In this study, we were able to take advantage of in vivo studies with dairy cows to develop an in vitro model w ith bovine adipocytes to better understand the role of FetA on lipid mobilization and inflammatory responses in adipose tissue of dairy cows. Our research provided data that can be further integrated into nutritional, genetic, and even clinical studies to prevent metabolic diseases in dairy cows. Future understandings on pathways involved on FetA modulation of lipid mobilization can potentially be translated to humans with similar ad ipose tissue disorders. 96 REFERENCES 97 REFERENCES Agarwal, A. K., E. Arioglu, S. de Almeida, N. Akkoc, S. I. Taylor, A. M. Bowcock, R. I. Barnes, and A. Garg. 2002. AGPAT2 is mutated in congenital generalized lipodystrophy linked to chromosom e 9q34. Nature genetics 31(1):21 -23. Chen, H. -Y., Y. -L. Chiu, S. -P. Hsu, M. -F. Pai, C. -F. Lai, Y. -S. Peng, T. -W. Kao, K. -Y. Hung, T.-J. Tsai, and K. -D. Wu. 2009. Association of serum fetuin A with truncal obesity and dyslipidemia in non -diabetic hemodialys is patients. Eur J Endocrinol 160(5):777 -783. Chen, Y., C. Gondro, K. Quinn, R. Herd, P. Parnell, and B. Vanselow. 2011. Global gene expression profiling reveals genes expressed differentially in cattle with high and low residual feed intake. Animal geneti cs 42(5):475 -490. Dahlman, I., P. Eriksson, M. Kaaman, H. Jiao, C. Lindgren, J. Kere, and P. Arner. 2004. # 2-Heremans ÐSchmid glycoprotein gene polymorphisms are associated with adipocyte insulin action. Diabetologia 47(11):1974 -1979. Dasgupta, S., S. Bhatt acharya, A. Biswas, S. S. Majumdar, S. Mukhopadhyay, and S. Ray. 2010. NF-kappaB mediates lipid -induced fetuin -A expression in hepatocytes that impairs adipocyte function effecting insulin resistance. Biochemical Journal 429(3):451 -462. Jialal, I., S. Deva raj, A. Bettaieb, F. Haj, and B. Adams -Huet. 2015. Increased adipose tissue secretion of Fetuin -A, lipopolysaccharide -binding protein and high -mobility group box protein 1 in metabolic syndrome. Atherosclerosis 241(1):130 -137. Mathews, S. T., G. P. Singh, M. Ranalletta, V. J. Cintron, X. Qiang, A. S. Goustin, K. -L. C. Jen, M. J. Charron, W. Jahnen -Dechent, and G. Grunberger. 2002. Improved Insulin Sensitivity and Resistance to Weight Gain in Mice Null for the Ahsg Gene. Diabetes 51(8):2450 -2458. Pal, D., S. Dasgupta, R. Kundu, S. Maitra, G. Das, S. Mukhopadhyay, S. Ray, S. S. Majumdar, and S. Bhattacharya. 2012. Fetuin -A acts as an endogenous ligand of TLR4 to promote lipid -induced insulin resistance. Nature Medicine 18(8):1279 -1285. P”rez -Sotelo, D., A. Roc a-Rivada, M. Larrosa -Garc™a, C. Castelao, I. Baamonde, J. Baltar, A. B. Crujeiras, L. M. Seoane, F. F. Casanueva, and M. Pardo. 2016. Visceral and subcutaneous adipose tissue express and secrete functional alpha2hsglycoprotein (fetuin a) especially in obes ity. Endocrine:1 -12. Robinson, D. and V. Oddy. 2004. Genetic parameters for feed efficiency, fatness, muscle area and feeding behaviour of feedlot finished beef cattle. Livestock Production Science 90(2):255 -270. Strieder -Barboza, C., J. de Souza, W. Rapha el, A. L. Lock, and G. A. Contreras. 2018. Fetuin -A: A negative acute -phase protein linked to adipose tissue function in periparturient dairy cows. Journal of Dairy Science 101(3):2602 -2616. 98 Wang, H. and A. E. Sama. 2012. Anti -inflammatory role of Fetuin -A in Injury and Infection. Curr Mol Med 12(5):625 -633.