1. Center for Integrated Animal Genomics Research Experience in Molecular Biotechnology & Genomics Summer 2008 Samir M. A’agha 1 ; Dr. Diane Moody Spurlock 1 ; Dawn Elkins 1 ;Hyelee Park 1 1 Department of Animal Science, Iowa State University, Ames, Iowa 50011 Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371 Variation Analyses in a Holstein population in three genes: ATGL, CG1-58 and Perilipin for Lipolysis During early lactation in dairy cows a negative energy balance is created. This negative energy balance occurs when energy used for milk production exceeds the amount of energy consumed. To balance this deficiency, energy substrates stored in adipose tissue are mobilized. In our study we are looking at four genes, perilipin, beta-adrenergic receptor 1, CGI-58 and ATGL, which are related to the mobilization of energy from adipose tissue. We hypothesize that sequence variation in these genes is associated with energy balance. Perilipin modulates adipocyte lipid metabolism. When unphosphorylated, perilipin coats lipid droplets and prevents the breakdown of triacylglycerols by hormone-sensitive lipase. Beta-adrenergic receptors are activated when lipolysis is stimulated. Activation of these receptors leads to a cascade of events that result in phosphorylation of hormone sensitive lipase and perilipin. Perilipin that has been phosphorylated acts together with hormone-sensitive lipase to mobilize triacylgycerols from adipose tissue. ATGL catalyzes the initial step in triglyceride hydrolysis in adipocyte and non-adipocyte lipid droplets. ATGL also has acylglycerol transacylase activity, regulates adiposome size, and may be involved in the degradation of adiposomes. ATGL plays an important role in energy homeostasis and response to starvation, enhancing hydrolysis of triglycerides and providing free fatty acids to other tissues to be oxidized in situations of energy depletion. CGI-58 interacts with both perilipin and ATGL, although its role in regulating lipolysis is not well defined. Selection of dairy cattle has focused largely on milk production traits for decades. Current trends suggest fitness traits, including health and reproduction, are compromised in favor of production traits. We are looking for variation genes so that we may select cows to promote the balance of energy mobilization for milk production as well as fitness traits. During early lactation in dairy cows a negative energy balance is created. This negative energy balance occurs when energy used for milk production exceeds the amount of energy consumed. To balance this deficiency, energy substrates stored in adipose tissue are mobilized. In our study we are looking at four genes, perilipin, beta-adrenergic receptor 1, CGI-58 and ATGL, which are related to the mobilization of energy from adipose tissue. We hypothesize that sequence variation in these genes is associated with energy balance. Perilipin modulates adipocyte lipid metabolism. When unphosphorylated, perilipin coats lipid droplets and prevents the breakdown of triacylglycerols by hormone-sensitive lipase. Beta-adrenergic receptors are activated when lipolysis is stimulated. Activation of these receptors leads to a cascade of events that result in phosphorylation of hormone sensitive lipase and perilipin. Perilipin that has been phosphorylated acts together with hormone-sensitive lipase to mobilize triacylgycerols from adipose tissue. ATGL catalyzes the initial step in triglyceride hydrolysis in adipocyte and non-adipocyte lipid droplets. ATGL also has acylglycerol transacylase activity, regulates adiposome size, and may be involved in the degradation of adiposomes. ATGL plays an important role in energy homeostasis and response to starvation, enhancing hydrolysis of triglycerides and providing free fatty acids to other tissues to be oxidized in situations of energy depletion. CGI-58 interacts with both perilipin and ATGL, although its role in regulating lipolysis is not well defined. Selection of dairy cattle has focused largely on milk production traits for decades. Current trends suggest fitness traits, including health and reproduction, are compromised in favor of production traits. We are looking for variation genes so that we may select cows to promote the balance of energy mobilization for milk production as well as fitness traits. Introduction Objective Identify DNA sequence variation (SNP, single nucleotide polymorphisms) for genes that participate in the regulation of lipolysis. Ultimately, associations between alternate alleles of these genes and energy balance traits will be evaluated. Identify DNA sequence variation (SNP, single nucleotide polymorphisms) for genes that participate in the regulation of lipolysis. Ultimately, associations between alternate alleles of these genes and energy balance traits will be evaluated. Materials and Methods Nine Holstein cows DNA were used in this study. Blood for DNA was collected via jugular vein in two, 10 ml tubes with EDTA. Buffy coat was extracted from blood. DNA was extracted from buffy coat. Working solutions were prepared for use in PCR. PCR products were designed. PCR was used to amplify a region of the genes of interest. Agarose gel electrophoresis was done to evaluate PCR products. PCR product was prepared for sequencing. DNA was sequenced in the DNA Sequencing Facility of Iowa State University. Nine Holstein cows DNA were used in this study. Blood for DNA was collected via jugular vein in two, 10 ml tubes with EDTA. Buffy coat was extracted from blood. DNA was extracted from buffy coat. Working solutions were prepared for use in PCR. PCR products were designed. PCR was used to amplify a region of the genes of interest. Agarose gel electrophoresis was done to evaluate PCR products. PCR product was prepared for sequencing. DNA was sequenced in the DNA Sequencing Facility of Iowa State University. Primers ATGL FORWARD 5’- GCA CCC TTC CTT CAA CAT GG- 3’ ATGL REVERSE 5’-ATC CCT GTA GCC CTG TTT GC-3’ ATGL FORWARD 5’- GCA CCC TTC CTT CAA CAT GG- 3’ ATGL REVERSE 5’-ATC CCT GTA GCC CTG TTT GC-3’ Results No variation was found in the targeted regions of perilipin or beta-adrenergic receptor. A SNP resulting in an amino acid change has been reported in the Ensembl database for ATGL. However, our primers failed to amplify this targeted region of ATGL. No other SNP were identified in ATGL. CGI-58 was not successfully amplified by the primers. No variation was found in the targeted regions of perilipin or beta-adrenergic receptor. A SNP resulting in an amino acid change has been reported in the Ensembl database for ATGL. However, our primers failed to amplify this targeted region of ATGL. No other SNP were identified in ATGL. CGI-58 was not successfully amplified by the primers. Discussion The SNP in ATGL described in the Ensembl database would be of great interest in investigating the function of ATGL in lipolysis. Although the PCR primers used amplified a region of ATGL, the size of the PCR product was smaller than expected and did not include the potential SNP. Further work with additional PCR primers is needed to validate the presence of this SNP in Holstein cattle. Figure 1. Assiting in bleeding a cow via the jugular vein. Figure 2. Extracting serum from blood. References Figure 6. SNP not being reached by the amplification of primers during PCR. Figure 3. ATGL PCR bands in gel. Figure 4. Perilipin PCR bands in gel. Figure 5. Beta andregenic receptor PCR bands in gel. James G. Granneman, Hsiao-Ping H. Moore, Rachel L. Granneman, Andrew S. Greenberg, Martin S. Obin, and Zhengxian Zhu. 2006. Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes. D. Elkins, H. Park and D. Spurlock. Phosphorylation of Perilpin is Associated with Lipolysis in Holstein Cows. Journal of Dairy Science Vol. 91 No. 7, 2008. pg 2919 James G. Granneman, Hsiao-Ping H. Moore, Rachel L. Granneman, Andrew S. Greenberg, Martin S. Obin, and Zhengxian Zhu. 2006. Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes. D. Elkins, H. Park and D. Spurlock. Phosphorylation of Perilpin is Associated with Lipolysis in Holstein Cows. Journal of Dairy Science Vol. 91 No. 7, 2008. pg 2919 Acknowledgements I would like to thank the NSF for funding my research, the REU program and staff, Max Rothschild for giving me the chance to participate in this program. My mentor, Dr. Diane Spurlock, for leading my way and bringing me support in this research. Also Dawn Elkins, graduate student, for helping me day by day on this process, Hyelee Park for always being available when I needed during my experiments. Primers for Perilipin and beta andregenic receptor were provided by Dr. Diane Spurlock lab. ATGL primers were design by myself and made by Integrated DNA Technologies.