Progress 01/01/10 to 12/31/13
Outputs
Target Audience: Scientists in the field of mammary gland biology and in dairy production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Our laboratory studies the molecular genetics of enzymes involved in the synthesis of triacylglycerols. We use genetically engineered mice to study the physiological functions of these enzymes from the perspective of human health and disease. The proposed work provides interdisciplinary training, as the student experiences approaches used in molecular and cellular biology, genetics, nutritional biochemistry, and physiology, carrying out studies at organismal, organ system, cellular and molecular levels. Ms. Nicole Spencer, who led the initial phase of this project, has graduated from the Interdepartmental Graduate Program in Nutritional Sciences at the University of Wisconsin, Madison. Ms. Ting-Ni Huang, a second-year PhD student of the same program is now to leading the effort. In addition, Ting-Ni is involved in mentoring our undergraduate researchers as integrated activities of this project. For example, Ms. Tessa Warner, an undergraduate researcher who carried out part of the project as her Hilldale Undergraduate/Faculty Research Fellowship, has also graduated in December 2012. She has accepted a scholarship to attend the School of Medicine and Public Health at the University of Wisconsin-Madison. Partly because of this research experience, Tessa plans to become a pediatrician, specializing in neonatal nutrition. We have since recruited and trained two juniors, Ms. Ariel Johnson and Ms. Fengchun Miao, to participate in the proposed studies. How have the results been disseminated to communities of interest? We have discussed our findings thus far in scientific conferences and are preparing manuscripts to report the requirement of MGAT2 enzyme in determining the macronutrient levels in milk. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Specific Aim 1: To determine if expression of TG synthesis enzymes in mice correlates with lactation. mRNA expression levels of several TG synthesis genes were measured in whole mammary gland isolated from four different groups; chow fed virgin mice, chow fed lactating mice, high-fat (HF) fed virgin mice, and HF fed lactating mice. Lactating mammary glands were collected fourteen days postpartum. The genes measured include glycerol-3-phosphate acyltransferase (Gpat) 1, 2, and 3; 1-acylglycerol-3-phosphate O-acyltransferase (Agpat) 1, 2,3 and 4; diacylglycerol Oacyltransferase (Dgat) 1 and 2, and monoacylglycerol O-acyltransferase (Mogat) 1 and 2. In the whole glands of mice fed a chow diet, the levels of mRNA for Gpat3, Agpat2, Agpat3, and Mogat1 were all decreased during lactation. Similar trends were seen in Gpat1, Agpat4, Gpat2, Mogat2, Dgat1 and Dgat2, but the differences did not reach statistical significance. Likewise, in the whole mammary glands of mice fed a high-fat diet, the levels of mRNA for Gpat1 and Gpat3 were decreased during lactation. Gpat2, Agpat1, Agpat2, Agpat3, Agpat4, Mogat1 and 2, and Dgat1 and 2 showed similar, but not statistically significant trends. No significant effect of diet was observed on the expression of measured genes. In addition, the expression of these genes in the epithelial cells isolated from mammary glands at various stages, including virgin, day 18 pregnancy, day 9 and 14 lactation, and day 10 post weaning. With limited number of samples, no statistical differences were detected for any genes except Gpat4 with the highest expression detected at day 9 of lactation. Specific Aim 2: To determine which TG synthesis enzymes are expressed in bovine milk somatic cells. Somatic cell isolation was attempted from bovine milk. Cell yield was confirmed by cell counting on a hemocytometer. Cells were fixed and stained to identify epithelial cells. The results revealed that epithelial cells were a very minor population with an abundance of immune cells. Thus, we concluded the protocol was insufficient to measure TG synthesis gene in isolated epithelial cells from bovine milk. Specific Aim 3 our project has focused on determining if the production of milk fat requires any of the three enzymes involved in triacylglycerol synthesis: monoacylglycerol acyltranserase 1 (Mogat1), monoacylglycerol acyltranserase 2 (Mogat2), or diacylglycerol acyltransferase 1 (Dgat1). We use genetically engineered mice lacking each of the genes coding for the enzymes to determine if any of the enzymes is indispensible for normal milk fat concentration and fatty acid (FA) profile. (Heterozygous Dgat1 mice were used because mice lacking both Dgat1 alleles fail to lactate.) Other macronutrients, namely lactose and protein, were also measured as controls. In carrying out the project, we found that litters born to Mogat2-/- mothers had approximately a 40% lower survival rate as compared to wild type. We had to increase the number of Mogat2-/- dams to collect sufficient number of samples. Our results showed that the concentrations of protein and lactose in addition to triacylglycerol were all lower in milk expressed from mice lacking MGAT2 (Mogat2–/– ) as compared to that from wildtype mice. There are also changes in the fatty acid profiles in milk from Mogat2–/– dams, with lower percentage of medium-chain saturated fatty acids and more long-chain unsaturated fatty acids comparing to milk from wildtype mice. In contrast, milk from Dgat1+/– mice had a TG level lower than controls but no differences in the other two macronutrients. Milk from Mogat1–/– mice was not different from control in any of the measurements.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Nelson, D. W., Y. Gao, N. M.
Spencer, T. Banh, and C-L. E. Yen. Defi ciency of MGAT2
increases energy expenditure without high-fat feeding and
protects genetically obese mice from excessive weight gain.
J. Lipid Res . 2011. 52: 17231732.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Our overall goal is to advance the understanding of milk fat production at the molecular level both in mice and in dairy cattle. Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in most living organisms and account for more than 95% of dietary fat, including milk fat. Our laboratory studies the molecular genetics of enzymes involved in the synthesis of triacylglycerols. We use genetically engineered mice lacking DGAT1, MGAT1, or MGAT2 to study the physiological functions of these enzymes from the perspective of human health and disease. In this line of proposed research, we started studies to examine the role of TG synthesis enzymes in the production of milk fat. Ms. Nicole Spencer, who led the initial phase of this project, has graduated from the Interdepartmental Graduate Program in Nutritional Sciences at the University of Wisconsin, Madison. Ms. Ting-Ni Huang, a first-year graduate student of the same program has been recruited now to lead the effort. Ms. Tessa Warner, an undergraduate researcher who carried out part of the project as her Hilldale Undergraduate/Faculty Research Fellowship, has also graduated in December 2012. She has accepted a scholarship to attend the School of Medicine and Public Health at the University of Wisconsin-Madison. Partly because of this research experience, Tessa plans to become a pediatrician, specializing in neonatal nutrition. Nicole and Tessa have collected virgin as well as lactating whole mammary glands and isolated mammary epithelial cells from mice fed a chow or a high-fat diet. Nicole and Tessa have purified mRNA and prepared cDNA to measure expression levels of genes of interest using quantitative PCR from whole mammary glands; however, the effort to isolate mammary epithelial cells was not successful. They have completed the preliminary study determining triacylglycerol content of mouse milk collected from wild type, MGAT2 knockouts, and DGAT1 heterozygotes, when maintained on a chow diet. Ting-Ni is now completing milk collection from all four genotypes (wildtype, MGAT2 knockouts, MGAT1 knockouts, DGAT1 heterozygotes). She has extracting the lipid from each milk sample using the Folch method and created methyl esters from the lipids using boron trifluoride and begun analyzing them using gas chromatography. Nicole has also isolated somatic cells from raw bovine milk obtained from the UW herd. Sufficient cell yield was confirmed by cell counting on a hemocytometer. Cells were diluted and an aliquot was spun onto a slide using a cytospin centrifuge. She has fixed the cells in 4% formalin and stained them with hematoxylin and eosin to identify epithelial cells. She found that most cells obtained with this method were lymphocytes and other cells of immune system. Mammary epithelia, which we aimed to examine, accounted for only a minor fraction. Pursuing this aim with bovine milk would not likely to be fruitful. Thus, based on the results, we plan to focus on murine mammary glands only for the remaining project period. PARTICIPANTS: Ting-Ni Huang, a first-year graduate student, was recruited to carry out the project. Nicole Spencer, a research assistant on the project, graduated in August 2012. Tessa Warner, an undergraduate researcher, also graduated in December 2012. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
During the current reporting period, our project has focused on determining if the production of milk fat requires any of the three enzymes involved in triacylglycerol synthesis: monoacylglycerol acyltranserase 1 (Mogat1), monoacylglycerol acyltranserase 2 (Mogat2), or diacylglycerol acyltransferase 1 (Dgat1). We use genetically engineered mice lacking each of the genes coding for the enzymes to determine if any of the enzymes is indispensible for normal milk fat concentration and fatty acid (FA) profile. (Heterozygous Dgat1 mice were used because mice lacking both Dgat1 alleles fail to lactate.) Other macronutrients, namely lactose and protein, were also measured as controls. Our preliminary data indicate that protein and TG concentrations were lower in milk from mice lacking MGAT2 (Mogat2-/-) as compared to that from wild type mice. Another interesting phenotype we found was that the litters born to Mogat2-/-mothers had a much lower survival rate than wild type mother. Currently, we are exploring possible causes of the high Mogat2-/- litter mortality rate. To determine if Mogat2-/- had altered milk macronutrient content on a low-fat chow diet, the protein, lactose, and triacylglycerol concentrations were measured on days 2, 9 and 12 of lactation. In carrying out the project, we found that litters born to Mogat2-/- mothers had approximately a 40% lower survival rate as compared to wild type. We had to increase the number of Mogat2-/- dams to collect sufficient number of samples. We found that protein concentrations were lower in milk from Mogat2-/- mice than that from wild type mice. Lactose concentrations showed no difference between genotypes. Like protein concentrations, triacylglycerol concentrations were lower in Mogat2-/- milk on days 2 and 12 of lactation as compared to WT milk. Whether these differences in protein and triacylglycerol concentrations led to the low survival rate of pups born to Mogat2-/- mice is not clear. The genotype of the pups is not a likely explanation, as we breed Mogat2-/- females with wild type males and wild type females with Mogat2-/- males; therefore, all pups born are of the same heterozygous genotype. In order to determine if the mortalities are due to the Mogat2-/- mother's inability to nurse her litter sufficiently, a cross fostering experiment will be conducted in which pups from wildtype and Mogat2-/- dams will be pooled and an equal number of pups will be assigned to each dam. We will determine if these pups can survive. In addition, we are examining if mammary gland development is impaired in virgin and lactating Mogat2-/- mice.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in most living organisms and account for more than 95% of dietary fat, including milk fat. Our laboratory studies the molecular genetics of enzymes involved in the synthesis of triacylglycerols. We use genetically engineered mice lacking DGAT1, MGAT1, or MGAT2 to study the physiological functions of these enzymes from the perspective of human health and disease. With this funding support, we are exploring agronomic implications of our research to fulfill our mission as part of a land-grant university. Specifically, we started studies to examine the role of TG synthesis enzymes in the production of milk fat. Our overall goal is to advance the understanding of milk fat production at the molecular level both in mice and in dairy cattle. We propose 1) to determine if expression of TG synthesis enzymes in mice correlates with lactation, 2) to determine if the expression levels of these enzymes in bovine milk somatic cells correlate with the levels of milk fat, 3) to determine if the expression of DGAT1 or MGAT2 regulates milk fat production, and 4) to determine if polymorphisms in the bovine MGAT2 gene, and/or other candidate genes identified, are associated with differences in milk fat production. We have recruited Ms. Nicole Spencer, a graduate student of the Interdepartmental Graduate Program in Nutritional Sciences, to lead this project. The team for this project also includes Dr. Gao Yu, a postdoctoral fellow in our group and Ms. Tessa Warner, an undergraduate researcher. During the second year of the support, we focused on the two Aims involving mice and attempted to isolate bovine somatic cells from milk. Nicole and Tessa have collected virgin as well as lactating whole mammary glands and isolated mammary epithelial cells from mice fed a chow or a high-fat diet. Nicole and Tessa have purified mRNA and prepared cDNA to measure expression levels of genes of interest using quantitative PCR. They have completed the preliminary study determining triacylglycerol content of mouse milk collected from wild type, MGAT2 knockouts, and DGAT1 heterozygotes, when maintained on a chow diet. Additionally, Nicole has isolated the lipid from each milk sample using the Folch method and seperated lipid species using thin-layer chromatography (TLC). She has created methyl esters from the lipids using boron trifluoride and begun analyzing them using gas chromatography. Nicole has also isolated somatic cells from raw bovine milk obtained from the UW herd. Sufficient cell yield was confirmed by cell counting on a hemocytometer. Cells were diluted and an aliquot spun onto a slide using a cytospin centrifuge. She has fixed the cells in 4% formalin and stained them with hematoxylin and eosin g to identify epithelial cells. Nicole has presented this line of research in progress in the Departmental seminar series call Tuesday Noon. PARTICIPANTS: Ms. Nicole Spencer, a graduate student of the Interdepartmental Graduate Program in Nutritional Sciences,leads this project, as part of her thesis. The team for this project also includes Dr. Yu Gao, a postdoctoral fellow in our group and Ms. Tessa Warner, an undergraduate researcher. To isolate bovine somatic cells, Nicole obtained fresh raw milk with the help of Mike Peters in the Department of Dairy Science. Nicole has presented this line of research in progress in the Departmental seminar series call Tuesday Noon. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: We have attempted to isolate somatic cell from bovine milk. We have obtained sufficient cell yield as confirmed by cell counting on a hemocytometer. Cells were diluted to a concentration of 250,000 cells/mL, and 5ul were spun onto a slide using a cytospin centrifuge. Cells were fixed with 4% formalin and washed in 1x PBS. Cells were then stained using hematoxylin and eosin stain to identify epithelial cells. The staining revealed that epithelial cells were a very minor population with an abundance of immune cells. We concluded the protocol was not appropriate to measure TG synthesis genes in isolated epithelial cells from bovine milk. Thus, we propose to change the Aim to examine if the triacylglycerol synthesis enzyme, MGAT1, MGAT2, or DGAT1 is involved in converting dietary to milk fat, using mice deficient in one of the enzyme. Milk fat content of these mice fed a high-fat diet will be determined.
Impacts
Specific Aim 1: To determine if expression of TG synthesis enzymes in mice correlates with lactation. mRNA expression levels of several TG synthesis genes were measured in whole mammary gland isolated from four different groups; chow fed virgin mice (n=5), chow fed lactating mice (n=3), high-fat (HF) fed virgin mice (n=4), and HF fed lactating mice (n=2). Lactating mammary glands were collected fourteen days postpartum. The genes measured include glycerol-3-phosphate acyltransferase (Gpat) 1, 2, and 3; 1-acylglycerol-3-phosphate O-acyltransferase (Agpat) 1, 2,3 and 4; diacylglycerol O-acyltransferase (Dgat) 1 and 2, and monoacylglycerol O-acyltransferase (Mogat) 1 and 2. In the whole glands of mice fed a chow diet, the levels of mRNA for Gpat3, Agpat2, Agpat3, and Mogat1 were all decreased during lactation. Similar trends were seen in Gpat1, Agpat4, Gpat2, Mogat2, Dgat1 and Dgat2, but the differences did not reach statistical significance. Likewise, in the whole mammary glands of mice fed a high-fat diet, the levels of mRNA for Gpat1 and Gpat3 were decreased during lactation. Gpat2, Agpat1, Agpat2, Agpat3, Agpat4, Mogat1 and 2, and Dgat1 and 2 showed similar, but not statistically significant trends. No significant effect of diet was observed on the expression of measured genes. In addition, the expression of these genes in the epithelial cells isolated from mammary glands at various stages, including virgin, day 18 pregnancy, day 9 and 14 lactation, and day 10 post weaning. With limited number of samples, no statistical differences were detected for any genes except Gpat4 with the highest expression detected at day 9 of lactation. Specific Aim 2: To determine which TG synthesis enzymes are expressed in bovine milk somatic cells. Somatic cell isolation was attempted from bovine milk. Cell yield was confirmed by cell counting on a hemocytometer. Cells were fixed and stained to identify epithelial cells. The results revealed that epithelial cells were a very minor population with an abundance of immune cells. Thus, we concluded the protocol was insufficient to measure TG synthesis gene in isolated epithelial cells from bovine milk. Specific Aim 3: To determine if deficiency of TG synthesis enzymes in mice, DGAT1 or MGAT2, reduces milk fat production. Milk samples were collected from eight wild type, 4 MGAT1 knockout (KO), 10 MGAT2 KO, and 5 DGAT1 heterozygous (Het) mice at mid-lactation. The average TG concentration in the WT mice was 90.5, 94.3, 83.7, and 74.1 mg/mL for WT, M1KO, M2KO, and DGAT1 Het mice respectively. Additionally, lipid from each milk sample was extracted, and TG was isolated using thin-layer chromatography (TLC), scraped into a glass vial, and isolated from the silica. The TG sample will be analyzed by creating butyl esters and analyzing using gas chromatography. We next decided to determine if these TG synthesis enzymes are necessary when the nursing dam is fed a high-fat diet. We designed a 45E% fat diet using corn oil as the major lipid source. We switched the dam to the high-fat diet at day fourteen of pregnancy and maintained on this diet until day fourteen of lactation.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in most living organisms and account for more than 95% of dietary fat, including milk fat. Our laboratory studies the molecular genetics of enzymes involved in the synthesis of triacylglycerols. We use genetically engineered mice lacking DGAT1 or MGAT2 to study the physiological functions of these enzymes from the perspective of human health and diseases. With this funding support, we explore agronomic implications of our research to fulfill our mission as part of a land-grant university. Specifically, we started studies to examine the role of TG synthesis enzymes in the production of milk fat. Our overall goal is to advance the understanding of milk fat production at the molecular level both in mice and in dairy cattle. We propose 1) to determine if expression of TG synthesis enzymes in mice correlates with lactation, 2) to determine if the expression levels of these enzymes in bovine milk somatic cells correlate with the levels of milk fat, 3) to determine if the expression of DGAT1 or MGAT2 regulates milk fat production, and 4) to determine if polymorphisms in the bovine MGAT2 gene, and/or other candidate genes identified, are associated with differences in milk fat production. We have recruited Ms. Nicole Spencer, a first year Ph.D. student of the Interdepartmental Graduate Program in Nutritional Sciences, to carry out the milk fat project. The team for this project also includes Dr. Yu Gao, a postdoctoral fellow in our group and Ms. Tessa Warner, an undergraduate researcher. During the first year of the support, we focused on establishing methods for completing the two Aims involving rodent before working with precious samples from dairy cattle. We have obtained a protocol from Dr. Caroline Alexander to isolate mammary epithelial cells, and Nicole and Tessa have practiced several methods needed for accomplishing this aim. We have successfully measured expression levels of several genes in whole mammary gland. During the next year, we will focus on separating mammary epithelia from stroma cells, especially adipocytes. Yu has completed a preliminary study, determining triacylglycerol content mice milk. The data validated the methods and protocols used. For the next funding period, we will increases and sample sizes and expand the study to include DGAT1-deficient mice. In addition, Yu has established a method using gas chromatography/mass spectrometry to determine if these enzymes modulates fatty acid profiles. While continuing a related project, examining the role of MGAT2 in regulating metabolic responses to meals, Dr. David Nelson, our post-doctoral fellow, is mentoring Taylor Bahn, a fellow in the Undergraduate Research and Mentoring Program, and Adela Stieve, an undergraduate in the CALS honors program. Tessa Warner and Adela Stieve have presented posters in the University of Wisconsin-Madison Undergraduate Research Symposium. Dr. Eric Yen was invited to speak at the FASEB Summer Research symposium. In addition, David was selected to give an oral presentation at Experimental biology 2011 based on findings in his recently submitted abstract. PARTICIPANTS: We have recruited Ms. Nicole Spencer, a first year Ph.D. student of the Interdepartmental Graduate Program in Nutritional Sciences, to carry out the milk fat project. The team for this project also includes postdoctoral fellows, Dr. Yu Gao and Dr. David Nelson, and undergraduate researchers Ms. Adela Stieve, Tessa Warner, and Taylor Bahn in our group. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Specific Aim 1: To determine if expression of TG synthesis enzymes in mice correlates with lactation. Specific Aim 1: To determine if expression of TG synthesis enzymes in mice correlates with lactation. We have successfully measured expression levels of several genes in whole mammary gland. During the next year, we will focus on separating mammary epithelia from stroma cells, especially adipocytes. Specific Aim 3: To determine if deficiency of TG synthesis enzymes in mice, DGAT1 or MGAT2, reduces milk fat production. This aim will test the hypothesis that DGAT1 and/or MGAT2 regulates milk fat production in mice. Yu has completed a preliminary study, determining triacylglycerol content mice milk. In this study, the milk samples from two wild type mice and four mice lacking one copy of Mgat2 gene (Mogat2+/-) were collected at mid-lactation (about 11 days) after 3~4 hours of having the pups separated from dams. The dams were injected with oxytocin and anaesthetized with isoflurane. About 200 ul milk was obtained from each mouse. The milk triglyceride levels were assessed colorimetrically using a kit, following the manufacturer's instructions (ThermoFisher). Yu found that the average of TG concentration in wide type mice was 187 g/L (about 18%), whereas the average of TG concentration in Mogat2+/- mice was 127 g/L (about 12 %). These preliminary data support our hypothesis that MGAT2 regulates milk fat production, although, with the small sample sizes, the difference did not reach statistical significance. Nonetheless, these concentrations fall into the range of mouse milk fat concentrations reported in published studies, ranging from 13.5 to 21.4%, validating the methods and protocols used. For the next funding period, we will increases and sample sizes and expand the study to include DGAT1-deficient mice. Regarding a closely related project, MGAT2 is thought to be crucial for fat absorption. Indeed, mice lacking the enzyme (Mogat2-/-) are resistant to obesity and other metabolic disorders induced by high-fat feeding. However, these mice absorb normal quantities of fat. To explore the role of dietary fat in the underlying mechanism(s), we examined metabolic responses of Mogat2-/- mice to diets containing varying levels of fat. Mogat2-/- mice exhibited 8-15% increases in energy expenditure compared to wildtype littermates. While high levels of dietary fat exacerbated the differences, the increases occurred even on a fat-free diet. The differences were closely linked to meal consumption, and Mogat2-/- mice expend energy and lose weight to similar degrees as controls during fasting. To examine if MGAT2 deficiency protects against obesity in the absence of high-fat feeding, we crossed Mogat2-/- mice with hyperphagic and genetically obese Agouti mice. MGAT2 deficiency increased energy expenditure and prevented Agouti mice from gaining excess weight, even when fed a low-fat chow. Our results suggest that MGAT2 modulates energy expenditure through multiple mechanisms, including one independent of dietary fat consumption. Inhibiting MGAT2 may prove to be useful in combating obesity and related metabolic disorders resulted from excessive calories.
Publications
- No publications reported this period
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