Source: UNIVERSITY OF VERMONT submitted to
INTERPRETING CATTLE GENOMIC DATA: BIOLOGY, APPLICATIONS, AND OUTREACH
 
PROJECT DIRECTOR: Zhao, F.
 
PERFORMING ORGANIZATION
ANIMAL SCIENCE
UNIVERSITY OF VERMONT
BURLINGTON,VT 05405
 
NON TECHNICAL SUMMARY: Glucose uptake by the milk-producing cells in the mammary gland is a rate-limiting step for milk production because glucose is the major precursor of milk lactose which controls milk yield. This project is to chracterize the expression and localization of glucose transporters GLUT8, SGLT1 and SGLT2 in bovine mammary gland.
 
OBJECTIVES: 1. Determine the location, structure, function and expression of genes affecting health, reproduction, production, and product quality in cattle. 2. Interpret and apply genomics and proteomics information by developing statistical/bioinformatics methods and utilizing molecular tools in cattle. 3. Develop and deliver educational materials about bovine genomics research to consumers and stakeholders.
 
APPROACH: 1. Generate specific anti-bSGLT1 antibody by Sigma-Genosys: The amino acid sequence of bovine SGLT1 will be analyzed by Sigma-Genosys technical team. The designed peptide will be synthesized, conjugated to carrier protein, and used to immunize two rabbits by the same company following the company standard protocol. The antisera will be purified by peptide specific affinity column. 2. Characterize anti-GLUT8 and anti-SGLT2 from Alpha Diagnostics and Santa Cruz and anti-bSGLT1 produced by Sigma-Genosys: The antibodies will be characterized by Western blot of in vitro transfected cells with FLAG epitope tagged and untagged transporter constructs. 2.1. Generate FLAG epitope tagged constructs: A FLAG epitope tagged bGLUT8 construct has been built by amplifying the full reading frame of bGLUT8 cDNA by PCR using pfu polymerase (Stratagene), a 3' primer located in 3' UTR of bGLUT8 cDNA, and a 5' primer which includes a consensus Kozak translation initiation sequence, a FLAG epitope tag, and the bGLUT8-specific primer at the putative translation initiation site. Using the same methodology, we will generate FLAG tagged SGLT1 and SGLT2 constructs. 2.2. Cell culture and transient transpfection: Bovine mammary gland epithelial cell line MAC-T cells will be cultured. Cells will be transiently transfected using FuGene (Roche) reagent with FLAG tagged or untagged transporter construct or control vector pcDNA3.1 plasmid DNA. 2.3. Western blot: Transfected cells will be lysed by sonication in ice-cold homogenization buffer (Pierce M-PEP) with proteinase inhibitors (Sigma). The lysates will be subjected to Western blot analysis following PI previously established procedures. 3. Examine protein expression of GLUT8, SGLT1 and SGLT2 expression in lactating bovine mammary gland: After the antibodies against GLUT8, SGLT1 and SGLT2 have been tested and characterized, the levels and pattern of protein expression of these transporters will be analyzed in mammary tissues from lactating cows by Western blot. The liver, kidney, spleen, skeletal muscle and intestine tissues from individual cows will also be used as controls and to examine the relative expression levels in the mammary gland compared to these tissues. 4. Determine the cellular and subcellular localization of GLUT8, SGLT1 and SGLT2 in lactating bovine mammary gland: The cellular and subcellular localization of GLUT8, SGLT1 and SGLT2 will be studied in the lactating bovine mammary gland by immunofluoresence staining and Western blot analysis of subcellular fractionation. Results from each method will be used to verify and complement the results from the other. 5. Study changes of expression and localization of GLUT1, GLUT8, GLUT12, SGLT1 and SGLT2 in bovine mammary gland during the transition period to support milk synthesis. Mammary gland tissue samples will be collected by biopsy at day -28, 0 and +7. The mRNA levels, protein levels and subcellular localization of GLUT1, GLUT8, GLUT12, SGLT1 and SGLT2 will be studied using quentative RT-PCR, Western blot and immunofluoresence staining, respectively.
 
CRIS NUMBER: 0205291 SUBFILE: CRIS
PROJECT NUMBER: VT-H01216MS SPONSOR AGENCY: CSREES
PROJECT TYPE: HATCH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: NC-1010
START DATE: Oct 1, 2005 TERMINATION DATE: Sep 30, 2008

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
302341010102.220%
302341010402.220%
302341010502.210%
304341010102.210%
305341010102.220%
305341010402.210%
305341010502.210%

CLASSIFICATION HEADINGS
KA302 - Nutrient Utilization in Animals
KA305 - Animal Physiological Processes
KA304 - Animal Genome
S3410 - Dairy cattle, live animal
F1050 - Developmental biology
F1010 - Nutrition and metabolism
F1040 - Molecular biology
G2.2 - Increase Efficiency of Production and Marketing Systems


RESEARCH EFFORT CATEGORIES
BASIC 80%
APPLIED 20%
DEVELOPMENTAL (N/A)%

KEYWORDS: glucose transport; mammary gland; milk synthesis; glucose uptake; glucose transporter; gene expression; nutrient utilization; glucose homeostasis; lactation

PROGRESS: Oct 1, 2006 TO Sep 30, 2007
The specific aims of this project are to characterize or generate antibodies to bovine GLUT8, SGLT1 and SGLT2 and study protein expression and localizations of these transporters in the lactating bovine mammary gland. We also study changes in expression and localization of glucose transporter proteins in the bovine mammary gland during the transition period of milk synthesis. In this year, we have studied the expression and regulation of GLUT1 and GLUT8 in vitro using a bovine mammary epithelial cell line, Mac-T. RT-PCR studies confirmed expression of all three transporters in the Mac-T cell. Immunofluorescence staining showed that in the Mac-T cells, GLUT1 and GLUT8 proteins were distributed in both the cytoplasm and plasma membrane. Preliminary studies indicated that the expression of GLUT8 protein appears to be insulin responsive; GLUT8 expression increases with increasing amounts of insulin. Results of this study are the first to demonstrate that Mac-T cells express these glucose transporter proteins. We also examined the localization of GLUT8 in mammary tissue of late pregnant cows. The anti-GLUT8 antibody was strongly detected in the basolateral membrane of mammary epithelial cells and relative weakly in the apical membrane. In addition, we have generated an antibody against bovine SGLT1. The antibody has been tested by immunofluorescent staining in the mammary tissues which has shown that SGLT1 is mainly located in the apical membrane of mammary epithelial cells in bovine.

IMPACT: 2006-10-01 TO 2007-09-30 Glucose uptake by the milk-producing cells in the mammary gland is a rate-limiting step for milk production because glucose is the major precursor of milk lactose which, in turn, controls milk yield. Regulation of glucose transport in the mammary gland and compensatory regulation in other tissues provides the key role in maintaining glucose homeostasis during lactation. Our work lays the groundwork for future studies aimed at unraveling the functional roles of bovine glucose transporter in supporting milk synthesis and maintaining glucose homeostasis during lactation. In a broader sense, our work will contribute to the knowledge base on glucose utilization, and, ultimately, to the improvement of dairy productivity and efficiency.

PUBLICATION INFORMATION: 2006-10-01 TO 2007-09-30
Finucane KA, Keating AF, Zhao, F-Q. (2006) Expression and regulation of glucose transporter gene expression in a bovine mammary epithelial cell line, Mac-T. J. Dairy Sci. 89 (Suppl. 1), 198

PROJECT CONTACT INFORMATION
NAME: Zhao, F.
PHONE: 802-656-0786
FAX: 802-656-8196