Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
GENETIC ANALYSIS OF NHLH2 AND ITS ROLE IN MARBLING AND BODY COMPOSITION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0214411
Grant No.
(N/A)
Project No.
VA-135842
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2008
Project End Date
Jun 30, 2013
Grant Year
(N/A)
Project Director
Good, DE, J.
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Human Nutrition & Foods
Non Technical Summary
Breeding programs have responded to consumer demands for meat quality for years, but it is only recently that breeders have been able to use genetic markers to select for meat quality. This proposal directly addresses the topic of Animal Genomics in "identifying, mapping and understanding the function and control of genes (to) permit the development of new genetic technologies (that will) increase the ability (of breeders) to realize the full genetic potential for improvement of agriculturally important animal species." The strength in this project is that a known gene that regulates body fat has been mapped to a region of bovine chromosome 3 shown to have two linked genes predisposing to increased marbling in cattle. Experiments in this proposal will determine if the NHLH2 candidate gene is responsible for differences in marbling in different breeds of cattle. Functional analyses of polymorphisms identified during the SNP analysis will be performed. The last objective will help basic biologists, as well as the cattle industry to understand and correlate single nucleotide polymorphisms with the function of bNHLH2 in regulating body fat, marbling and fertility.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023840101033%
3043399109034%
3053399104033%
Knowledge Area
302 - Nutrient Utilization in Animals; 305 - Animal Physiological Processes; 304 - Animal Genome;

Subject Of Investigation
3840 - Laboratory animals; 3399 - Beef cattle, general/other;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1010 - Nutrition and metabolism;
Goals / Objectives
My laboratory studies the role of the basic helix-loop-helix transcription factor, Nhlh2 in body weight and reproduction. We have found that targeted deletion of Nhlh2 in mice leads to adult-onset obesity and infertility. In mice we can show that muscle structure and metabolism is affected by deletion of Nhlh2. We have cloned the bovine Nhlh2 gene (bNHLH2) and have compared the sequence of the bNHLH2 gene in different breeds of cattle to detect polymorphisms that could be related to body fat, marbling or fertility phenotypes. The bNHLH2 gene maps to within 100,000 bp of a QTL for marbling. We have identified polymorphisms in the Wagyu breed of cattle that is well known for its highly marbled muscle. In this proposal, we will determine if polymorphisms in the bNHLH2 gene are present at a high frequency in Wagyu cattle. We will also functionally characterize the protein products from different alleles of bNHLH2, and finally will analyze muscle-specific phenotypes in WT and N2KO mice. Specifically, we have the following objectives: Objective 1: To determine whether polymorphisms in bNHLH2 are present at a high frequency in the Wagyu strain of cattle Objective 2: To determine whether the polymorphisms identified in Wagyu cattle affect normal function of the bNHLH2 protein. Objective 3: To determine if Nhlh2 knockout mice have altered fatty acid oxidation and metabolism, or increased marbling in their skeletal muscle. The studies could lead to novel genetic approaches to improve meat quality. This includes selected for animals with the potential to show increased marbling (i.e. increased economic quality and price per pound) or decreased fat content (i.e. leaner, healthier meat products). The implications of these studies are important for the state of Virgina, and to the nation as a whole.
Project Methods
Objective 1: To determine whether polymorphisms in bNHLH2 are present at a high frequency in the Wagyu strain of cattle. For this objective, Wagyu semen or tissue preparations (N = 50, different parental lines) will be obtained from Dr. Kristen Johnson and Dr. Charles Gaskins, University of Washington, Pullman. Angus, Hereford and Charolais animals will be sampled at the Virginia Tech Beef Center in collaboration with Dr. Dan Eversole, and will be obtained through the ABS Global Company. DNA will be prepared from live sperm separated from the extenders and dead sperm in the straw using a discontinuous gradient. PCR amplied DNA will be sequenced and compared using BioEdit Sequence Alignment Editor. SNPs will be compared using chi-square analysis. These experiments will allow us to determine if bNHLH2 polymorphisms have been selected for in a particular breed of cattle or with marbling score. For determination of whether different alleles of the bNHLH2 gene are correlated with marbling, the effect of genotype will be analyzed by two-way analysis of variance (ANOVA) with one between effect (genotype) and one within effect (marbling score). Objective 2: To determine whether the polymorphisms identified in Wagyu cattle affect normal function of the bNHLH2 protein. bHLH transcription factors can (1), form homo- and heterodimers with other bHLH proteins, (2) bind DNA and (3) transactivate target promoters. Mutant proteins obtained from Wagyu or other cattle will be used in our standard functional testing assays to determine if the mutations affect the ability of the protein to dimerize, bind DNA or transactivate known NHLH2 transcriptional targets. The experiments outlined in objective 2 are very powerful in that they allow us to analyze the functional consequence of mutations in the bNHLH2 gene. Without this objective, we would still be able to identify mutations in the bovine gene (objectives 1), but will not know if the mutations are silent or if they change the ability of the bNHLH2 protein to perform its normal functions. Objective 3: To determine if Nhlh2 knockout mice have altered fatty acid oxidation and metabolism, or increased marbling in their skeletal muscle. The Nhlh2 knockout mice and their matched WT mice will be used in these studies. Muscle tissue isolated from WT and N2KO mice will be used to examine fatty acid metabolism, and the phenotype of skeletal muscle using histological assays including fiber typing stains and oil red O stains. The experiments proposed will determine whether deficiencies in muscle mitochondria fatty acid oxidation and/or intramuscular lipid accumulation are present in N2KO mice. If positive, these data will be directly relevant to studies linking marbling in cattle to polymorphisms in bNHLH2. If these data demonstrate no difference in mice, but we still identify linked polymorphisms in cattle, they will suggest a divergence of function for the Nhlh2/bNHLH2 protein in cattle versus mice.

Progress 07/01/08 to 06/30/13

Outputs
Target Audience: Scientific Communities Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Sabbatical--Max Plank, Bad Nauhem Gemany 2012 How have the results been disseminated to communities of interest? The results have been presented in seminars, poster presentations, and published in peer-reviewed journal articles. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Although the rate of obesity in the Commonwealth of Virginia (25.0%) stands at slightly below the national average of 26.7%,according to the Center for Disease Control (CDC), no state in the nation met the Healthy People 2010 target of a 15% obesity rate. Signals from the gut and fat stores signal neurons in the brain to initiate physical activity and control food intake. An understanding of the molecular mechanisms that control the response to brain neurons to these signals is imperative in identifying gene regulatory pathways that control adult body weight. However, very little is known about the complex transcriptional mechanisms involved in body weight control by hypothalamic neurons. Three newly identified human polymorphisms, one in the coding region of Nhlh2, one in the 3’ tail region of Nhlh2, and one in the promoter of MC4R, can be used to understand how these mutations affect body weight and to elucidate transcriptional control by NHlh2. While numerous studies have identified genetic loci affecting body fat and muscle in mice and humans, there are fewer studies on the genetics of body fat in meat-producing organisms. Marbling scores (the visible fat found between muscle fibers within the ribeye muscle, determined visually or by ultrasound measurements), and intramuscular fat content (the fat found in the muscle fibers, determined chemically) are two highly heritable suggesting that these traits may be influenced by alleles of specific genes. We are studying three bovine polymorphisms that affect the TFAM gene, two of which are within a putative Nhlh2 DNA binding site in the TFAM promoter. In the early years of the proposal, we were able to show that Angus steers contain a single nucleotide polymorphism (SNP) in the Nhlh2 gene, compared to Holstein and Wagyu cattle. Of the 65 animals from the SVARC herd genotyped, 44.6% of these were heterozygous at each locus. Slightly fewer than 5% (4.6%) of the animals were homozygous for all three SNPs, while 35.5% of steers are homozygous for the wild-type (WT) alleles. While we found no direct linkage to marbling in the yearling steers, heterozygosity at the 3’ E-box (T to C transition based on reference sequence NW_001494479.1; p = 0.029) or the presence of a single heterozygous CpG methylation site (p = 0.047) is associated with reduced central adiposity, as measured by a significant reduction in kidney, pelvic, and heart (KPH) fat estimates. Reduced KPH values in animals carrying genotypes that predispose them to higher marbling potential suggests that carriers of either of these polymorphisms have a unique and desired phenotype in cattle of reduced central adiposity accompanied by increased marbling potential. In examining Nhlh2 using mice and humans, we have shown that human carriers of two different mutations make either less NHLH2 protein, or make a non-functional protein. Specifically, human carriers of the A1568G mutation make an unstable RNA, and consequently about half as much NHLH2 protein as individuals with the normal allele. We are working with collaborators at Cambridge University to identify additional carriers of this polymorphism. Humans with the A83P polymorphism make a mutant form of NHLH2 protein which is unable to bind to DNA at E-box motifs on known NHLH2-regulated promoters. Two obese individuals have been shown to carry this polymorphism, and along with our collaborators, we are trying to identify more carriers to study the resultant phenoetypes. This work was published in 2013, with a publication date of 2013. This work completes and complements Objective 1. For Objective 2, we were unable to complete this aspect of the project, due to a lack of suffient resources. For Objective 3, studies on Nhlh2 expression in skeletal muscle have revealed the presence of a smaller transcript. We are trying to clone and sequence this transcript to determine if it represents a splicing variation of the Nhlh2 mRNA. We are also examining expression of Nhlh2 in response to exercise levels. We have recently submitted a paper showing that metabolic rate is lower in the N2KO line, and there is a disruption of mitochondrial energy production. In addition, Nhlh2 knockout animals do not run in a forced treadmill running paradigm. We are continuing to explore fatty acid oxidation and mitochondrial energetics in this link of animal in hopes of understanding energy availability and energy usage control by the hypothalamus.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Good, D.J., and Braun, T. (2013) NHLH2: At the intersection of obesity and fertility, Trends in Endocrinology and Metabolism, 24(8):385-90 Available online 17 May 2013
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Good, D.J., McIntyre, C.M., and Marchant, M.A. (2013) The USDA Scholars Program: Innovations in a Summer Undergraduate Research Program, NACTA Journal 57:62-70.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: 7. Kim, J-H, Park, Y., Kim, D., Good, D.J. and Park, Y., (2013) Dietary Conjugated Nonadecadienoic Acid Prevents Adult-onset Obesity in Nescient Basic Helix-Loop-Helix 2 Knockout Mice, J. Nutritional Biochemistry, 24: 556-566
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: 8. Al_Rayyan, N., Wankhade, U.D., Bush, K., and Good, D.J. (2013) Two Single Nucleotide Polymorphisms in the Human Nescient Helix Loop Helix 2 (NHLH2) Gene Reduce mRNA Stability and DNA Binding, Gene 512: 134-142.


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: As in the previous reporting period, we continue to focus on Objective 3. Very little is known about the complex transcriptional mechanisms involved in body weight control by hypothalamic neurons and peripheral tissues. Specifically, we have studied three newly identified human polymorphisms, using mouse models. We are also studying Nhlh2 knockout mice to assess the role of Nhlh2 in muscle function and metabolism, and this work is part of both a collaboration with other Human Nutrition, Foods and Exercise department faculty, as well as part of a sabbatical project by Dr. Good. Identification of genes that directly contribute to differences in body fat content and muscle function, especially through physical activity directly addresses the College of Agriculture and Life Sciences Initiative in food, nutrition and health. During the reporting period, we have published 6 papers and presented 2 abstracts at International Meetings. PARTICIPANTS: Haiyan Zhang (left in December 2011) and Jinhua Zhang (began in January 2012) are the laboratory specialists assigned to this project. Hao Jiang (Graduate Student) works on Objective 3. We collaborate with Dr. Matt Hulver and Dr. Rob Grange on metabolism and physical activity studies. Dr. Good is currently on sabbatical at the Max Planck Institute in Bad Nauheim, Germany, where she collaborates with Dr. Thomas Braun on studying the genetics of physical activity. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: We are no longer working on the bovine model as there is not funding available nor significant advances to warrent another grant proposal.

Impacts
We have shown that human carriers of two different mutations make either less NHLH2 protein, or make a non-functional protein. Specifically, human carriers of the A1568G mutation make an unstable RNA, and consequently about half as much NHLH2 protein as individuals with the normal allele. We are working with collaborators at Cambridge University to identify additional carriers of this polymorphism. Humans with the A83P polymorphism make a mutant form of NHLH2 protein which is unable to bind to DNA at E-box motifs on known NHLH2-regulated promoters. Two obese individuals have been shown to carry this polymorphism, and along with our collaborators, we are trying to identify more carriers to study the resultant phenoetypes. This work was accepted in 2012, with a publication date of 2013. Dr. Good is currently on a sabbatical at the Max Planck Institute in Bad Nauheim, Germany. There she is using mice with targeted deletions of Nhlh2 in specific hypothalamic neurons to identify the neuron types that control physical activity. Using forced treadmill running, we show that Nhlh2 knockout mice, with a deletion in all cells of the body run an average of only 17 minutes on the treadmill, compared to 60 minutes (entire length of test) for WT mice. Likewise, mice with a targeted deletion of Nhlh2 in POMC neurons, or in neurons of the peripheral nervous system show reduced running times, while mice with a deletion of Nhlh2 in GnRH neurons run like or more than WT mice. Thus, Nhlh2 mice display a lack of both spontaneous and forced physical activity. Studies on Nhlh2 expression in skeletal muscle have revealed the presence of a smaller transcript. We are trying to clone and sequence this transcript to determine if it represents a splicing variation of the Nhlh2 mRNA. To determine if metabolic rate is lower in the N2KO line, whole body energy expenditure measurements (calorimetry) were performed for WT and N2KO mice at 8 weeks of age (pre-obese) and 20 weeks of age (obese). Surprisingly, although there is an age-related decline in energy expenditure for both genotypes, there is no significant difference between energy expenditure for WT and N2KO mice. There is a difference in energy partitioning by N2KO mice, which we are exploring.

Publications

  • Davis, G., Jacob J. and Good, D.J. (2012) Effects of Price and Genetics on Food Choices and Obesity-Related Phenotypes in Mice, Open Neuroendocrinology Journal, 5: 13-21 (Open source journal)
  • Good, D.J. (2012) Extending the Reach of Exendin-4: New Pathways in the Control of Body Weight and Glucose Homeostasis, Endocrinology, 153: 2051-2053, impact factor 5.1.
  • Jiang, H., Modise, T., Bowen, R., Helm, R., Jensen, R., and Good, D.J. (2012) Differential Regulation of Hypothalamic mRNA, but not miRNA Implicated in Response to Short-Term Fasting. Genomics 2012 Meeting, Boston, MA, April 2012. (poster)
  • Jiang, H., Modise, T., Bowen, R., Helm, R., Jensen, R., and Good, D.J. (2012) Differential Expression of mRNA, MicroRNA and Protein During Experimentally-Induced Changes in Hypothalamic Energy Homeostasis, Endocrine Reviews, in press (published poster abstract)
  • Good, D.J., McIntyre, C.M., and Marchant, M.A. (2012) The USDA Scholars Program: Innovations in a Summer Undergraduate Research Program., NACTA Journal, accepted
  • Kim, J-H., Gilliard, D., Good, D.J., and Park, Y., (2012) Preventive effects of conjugated linoleic acid on obesity by improved physical activity in nescient basic helix-loop-helix 2 knockout mice during growth period, Food and Function, In press
  • Kim, J-H, Park, Y., Kim, D., Good, D.J. and Park, Y., (2012) Dietary Conjugated Nonadecadienoic Acid Prevents Adult-onset Obesity in Nescient Basic Helix-Loop-Helix 2 Knockout Mice, J. Nutritional Biochemistry, In press
  • Al_Rayyan, N.,Wankhade, U.D., Bush, K., and Good, D.J. (2013) Two Single Nucleotide Polymorphisms in the Human Nescient Helix Loop Helix 2 (NHLH2) Gene Reduce mRNA Stability and DNA Binding, Gene 512: 134-142


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: We continue to focus on Objective 3 for this reporting period. Very little is known about the complex transcriptional mechanisms involved in body weight control by hypothalamic neurons and peripheral tissues. Three newly identified human polymorphisms, one in the coding region of Nhlh2, one in the 3' tail region of Nhlh2, and one in the promoter of MC4R, can be used to understand how these mutations affect body weight and to elucidate transcriptional control by NHlh2. While numerous studies have identified genetic loci affecting body fat and muscle in mice and humans, there are fewer studies on the genetics of body fat in meat-producing organisms. Marbling scores (the visible fat found between muscle fibers within the ribeye muscle, determined visually or by ultrasound measurements), and intramuscular fat content (the fat found in the muscle fibers, determined chemically) are two highly heritable suggesting that these traits may be influenced by alleles of specific genes. We are studying three bovine polymorphisms that affect the TFAM gene, two of which are within a putative Nhlh2 DNA binding site in the TFAM promoter. Identification of genes that directly contribute to differences in body fat content between different strains of cattle and humans directly addresses the College of Agriculture and Life Sciences Initiative in food, nutrition and health. We are also studying Nhlh2 knockout mice to assess the role of Nhlh2 in muscle function and metabolism. During the reporting period, we have published 4 papers and presented 4 abstracts at International Meetings. We submitted a grant proposal to NIH to fund future studies in this area, but the proposal was not funded. For Objectives 1 and 2, we a proposal was submitted to the joint USDA-NIH program on large animal studies, but was not funded. PARTICIPANTS: Risa Pesapane (left in December 2009) and Haiyan Zhang (began in January 2010) are the laboratory specialists assigned to this project. Risa was responsible for the bovine project (Objectives 1 and 2) while Haiyan has been working on the metabolism project (Objective 3). Collaborators on the bovine project include Dr. Zhihua Jiang (Washington State University) and Dr. William S. Swecker, Jr (Virginia Tech, College of Veterinary Medicine) who has provided steer ear biospies for DNA isolation,and help in collecting muscle biopsies for TFAM expression analysis. Also on the bovine project, Dr. Susan Duckett (Clemson University) has been directly responsible for providing the carcass composition data (including marbling and KPH values) for the steers and Dr. Madlyn Frisard (Virginia Tech) has worked with our laboratory and Dr. William Swecker to make primary bovine cell lines from adult animals. Dr. Umesh Wankhade, who graduated in May 2010, was the graduate student responsible for experiment in Objective 3. Dr. Numan Al_Raayan, who graduated in May 2011, was the graduate student responsible for analysis of Nhlh2 polymorphisms and regulation. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Using forced treadmill running, we show that Nhlh2 knockout mice run an average of only 17 minutes on the treadmill, compared to 60 minutes (entire length of test) for WT mice. Thus, Nhlh2 mice display a lack of both spontaneous and forced physical activity. Studies on Nhlh2 expression in skeletal muscle have revealed the presence of a smaller transcript. We are trying to clone and sequence this transcript to determine if it represents a splicing variation of the Nhlh2 mRNA. To determine if metabolic rate is lower in the N2KO line, whole body energy expenditure measurements (calorimetry) were performed for WT and N2KO mice at 8 weeks of age (pre-obese) and 20 weeks of age (obese). Surprisingly, although there is an age-related decline in energy expenditure for both genotypes, there is no significant difference between energy expenditure for WT and N2KO mice.

Publications

  • Wankhade, U. D., Vella, K.R., Fox, D. L., and Good, D.J. (2010) Deletion of Nhlh2 Results in a Defective Torpor Response and Reduced Beta Adrenergic Receptor Expression in Adipose Tissue, PlosOne, 5(8). pii: e12324, PubMed, impact factor 4.4,
  • Davis, G.C. and Good D.J. (2011) The Interaction of Obesity Related Genotypes, Phenotypes, and Economics: The Importance of Food Choice, Experimental Biology Annual Meeting, Federation of American Societies for Experimental Biology, April 2011, Washington D.C. invited oral presentation
  • Good, D. J., Davis, G.C., and Jacob, J.C. (2011) Economic and Genetic Influences on Food Choice in the Tubby Obese Mice Endocrine Society Annual Meeting, Boston, MA, June 2011, Endocrine Reviews 32: P2-297
  • Al_Rayyan, N. and Good, D.J. (2011) Transcriptional and Post-Transcriptional Regulation of the Nescient Helix-Loop-Helix 2 (Nhlh2) Gene, Endocrine Society Annual Meeting, Boston, MA June 2011, Endocrine Reviews 32: P2-53
  • Davis, G.C. and Good, D.J. (2011) "The Interaction of Obesity Related Genotypes, Phenotypes, and Economics: An Experimental Economics Approach with Mice", The 2011 AAEA & NAREA Joint Annual Meeting in Pittsburgh, July 2011, invited oral presentation
  • Zhang, H. and Good, D.J. (2011) Comparison of Hypothalamic mRNA Levels in Animals Euthanized by CO2 Asphyxiation or Focused-Beam Microwave Irradiation, Lab Animal impact factor 0.6, in press
  • Wankhade, U.D. and Good, D.J. (2011) Melanocortin 4 Receptor is a Transcriptional Target of Nescient Helix-Loop-Helix-2, Molecular and Cellular Endocrinology, 341: 39-47, PubMed, impact factor 4.1
  • Boyle, K. J., Davis, G., Estabrooks, P.A., Good, D.J., King-Casas, B., Marathe, A., and You, W. (2011) Translational Economics Research, Association of Environmental and Resource Economists, 31(1): 16-25.


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: This reporting period we have focused our efforts on Objective 3. Nhlh2 knockout mice (N2KO) have a targeted deletion in the neuronal basic helix-loop-helix transcription factor, nescient helix-loop-helix 2 (Nhlh2). We have previously shown that these animals lack high levels of spontaneous physical activity but have also suspected that metabolism is aberrant in the N2KO mice. Differences in body temperature and serum analysis of leptin were performed in fasted and ad lib fed wild-type (WT) and N2KO mice. Histological analysis of white (WAT) and brown adipose tissue (BAT) was performed. Gene and protein level expression of inflammatory and metabolic markers were compared between the two genotypes. To determine if metabolic rate is lower in the N2KO line, whole body energy expenditure measurements (calorimetry) were performed for WT and N2KO mice at 8 weeks of age (pre-obese) and 20 weeks of age (obese). Others have shown that mutations in the melanocortin 4 receptor gene lead to reduced energy expenditure in both humans (MC4R) and mice (Mc4r). During this reporting period, we have studied the role of Nhlh2 in transcriptional regulation of this gene. For Objective 3 related projected, we have published 3 papers, and have one paper under review, related to Nhlh2 control of metabolism. We have also submitted a grant proposal to NIH to fund future studies in this area. For Objectives 1 and 2, we have published one book chapter related to the work, and submitted a proposal to NIH for future studies. PARTICIPANTS: Deborah J. Good (PI) oversaw all experiments and in some cases performed the experiments. She has been responsible for all paper write-ups and assists the graduate students with poster and oral presentations. Risa Pesapane (left in December 2009) and Haiyan Zhang (began in January 2010) are the laboratory specialist assigned to this project. Risa was responsible for the bovine project (Objectives 1 and 2) while Haiyan has been working on the metabolism project (Objective 3). Umesh Wankhade began the energy expenditure studies, and completed the melanocortin-4 receptor studies and the adipose tissue analysis. Collaborators on the bovine project include Dr. Zhihua Jiang (Washington State University) and Dr. William S. Swecker, Jr (Virginia Tech, College of Veterinary Medicine) who has provided steer ear biospies for DNA isolation,and help in collecting muscle biopsies for TFAM expression analysis. Also on the bovine project, Dr. Susan Duckett (Clemson University) has been directly responsible for providing the carcass composition data (including marbling and KPH values) for the steers and Dr. Madlyn Frisard (Virginia Tech) has worked with our laboratory and Dr. William Swecker to make primary bovine cell lines from adult animals. Dr. Umesh Wankhade, who graduate in May 2010, was the graduate student responsible for experiment in Objective 3. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We report significant differences in serum leptin levels and body temperature in N2KO mice compared with WT mice exposed to a 24-hour fast, suggestive of a defect in both white (WAT) and brown adipose tissue (BAT) function. As compared to WT mice, N2KO mice showed increased serum IL-6 protein and WAT IL-6 mRNA levels. This was accompanied by slight elevations of mRNA for several macrophage markers, including expression of macrophage specific protein F4/80 in adipose, suggestive of macrophage infiltration of WAT in the mutant animals. The mRNAs for beta3-adrenergic receptors (beta3-AR), beta2-AR and uncoupling proteins were significantly reduced in WAT and BAT from N2KO mice compared with WT mice. These studies implicate Nhlh2 in the central control of WAT and BAT function, with lack of Nhlh2 leading to adipose inflammation and altered gene expression, impaired leptin response to fasting, all suggestive of a deficient torpor response in mutant animals. Ongoing studies examining energy expenditure in the female N2KO mice show that even at 8 weeks of age (pre-obese animals) there is a significant reduction in resting energy expenditure, compared to age and gender-matched WT mice. These studies are ongoing with males and with older-aged animals. New research just submitted for publication show that the Mc4r gene is a direct transcriptional target of Nhlh2. We also show that a polymorphism in the promoter of the human MC4R gene, which is implicated in human obesity, completely abrogates transcription from the mutant promoter, suggesting that Nhlh2 regulation of Mc4r is a key step in body weight control. Overall the results link hypothalamic transcriptional regulation by Nhlh2 to peripheral tissue and whole body metabolism, critical for the regulation of body weight.

Publications

  • Wankhade, U.D., Rudd, A., McMillan R.P., Hulver M.W. and Good, D.J. (2010) The role of Nescient Helix-Loop-Helix 2 in transcriptional regulation of the melanocortin pathway, and peripheral energy expenditure and metabolism, Endocrine Society Annual Meeting, San Diego, CA, June 2010
  • Wankhade, U.D. and Good, D.J. (2010) Melanocortin 4 Receptor is a Transcriptional Target of Nescient Helix-Loop-Helix-2, Molecular Endocrinology, impact factor 5.2, submitted
  • Wankhade, U. D., Vella, K.R., Fox, D. L., and Good, D.J. (2010) Deletion of Nhlh2 Results in a Defective Torpor Response and Reduced Beta Adrenergic Receptor Expression in Adipose Tissue, PlosOne Biology, 5(8). pii: e12324, impact factor 4.3,
  • Good, D.J., (2010) Examining Polymorphisms Affecting Transcription and Translation of Genes Linked to Marbling in Beef Cattle, Animal Genetics, Rechi , chapter 7, L.J. Ed, Nova Scientific Publishers, ISBN 978-1-60741-844-3,
  • Good, D.J. (2010) Transcriptional Regulation of Energy Availability Sensing Within Hypothalamic Neurons, Open Neuroendorinol. J., Vol. 3, 1-5.
  • Vella, K.R., and Good, D.J. (2010) Nhlh2 is a Cold-Responsive Gene, Open Neuroendorinol. J. Vol 3, 38-44.
  • Al_Rayyan, N. and Good, D.J. (2010) Leptin-induced Transcriptional Regulation of the Nescient helix-loop-helix 2 (Nhlh2) Gene, Endocrine Society Annual Meeting, San Diego, CA, June 2010


Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: As reported last year, polymorphisms in the bovine NHLH2 do not appear to affect marbling or fat content in Angus cattle. However, new evidence from our laboratory shows a significant association of fat content in cattle with polymorphisms in an Nhlh2-binding site in the promoter region for transcription factor A of mitochondria (TFAM). TFAM is a nuclear encoded mitochondrial gene that has been widely reported to regulate mitochondrial DNA (mtDNA) copy number and stimulate transcription of mtDNA in mammals. Our collaborator, Dr. Zhihua Jiang (Washington State) has identified three single nucleotide polymorphisms (SNPs) in the bovine TFAM promoter region which are linked to increased marbling and fat deposition in beef cattle (Jiang et al., 2005; 2006). These polymorphisms are of direct relevance to our studies, as two of the SNPs flank a putative E-box site that we show binds to NHLH2. The third SNP creates a new methylation site in the 5' promoter region, which has the potential to reduce TFAM expression. Data from our collaborator, Dr. Jiang shows that each of the SNPs confer altered transcription on the TFAM promoter, in vitro. For this reporting period, we obtained 65 ear notch samples from the Shenandoah Valley Agriculture Research and Extension (SVAREC) Herd. These animals have been genotyped and the steers have been sent to our collaborator, Dr. Susan Duckett for carcass analysis. In addition, in a collaborative activity with Dr. Madlyn Frisard (Virginia Tech), we have taken cheek muscle biopsies from cattle to attempt to establish adult primary bovine skeletal muscle cells. This would be the first published account of primary bovine skeletal muscle cells. To date we have not disseminated the results of the project, except as a proposal to the USDA in July 2009. PARTICIPANTS: Risa Pesapane was the laboratory specialist assigned to this project. She completed some of the data described in the report with Dr. Deborah Good completing the final sequencing and data analysis for the 65 steers. Dr. Zhihua Jiang (Washington State University) is a collaborator on the project. Dr. William S. Swecker, Jr (Virginia Tech, College of Veterinary Medicine) is a collaborator for the Angus project, providing steer ear biospies for DNA isolation,and help in collecting muscle biopsies for TFAM expression analysis. Dr. Susan Duckett (Clemson University) is directly responsible for providing the carcass composition data (including marbling and KPH values) for the steers. Dr. Madlyn Frisard has worked with our laboratory and Dr. William Swecker to make primary bovine cell lines from adult animals. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Of the 65 animals from the SVARC herd genotyped, 44.6 percent of these were heterozygous at each locus. Slightly fewer than 5 percent (4.6 percent) of the animals were homozygous for all three SNPs, while 35.5 percent of steers are homozygous for the wild-type (WT) alleles. While we found no direct linkage to marbling in the yearling steers, heterozygosity at the 3 foot E-box (T to C transition based on reference sequence NW_001494479.1; p = 0.029) or the presence of a single heterozygous CpG methylation site (p = 0.047) is associated with reduced central adiposity, as measured by a significant reduction in kidney, pelvic, and heart (KPH) fat estimates. Reduced KPH values in animals carrying genotypes that predispose them to higher marbling potential suggests that carriers of either of these polymorphisms have a unique and desired phenotype in cattle of reduced central adiposity accompanied by increased marbling potential. In other studies related to TFAM expression, we can show that mice with a deletion of Nhlh2 have a significant elevation in TFAM gene expression in hypothalamic and skeletal muscle tissues, supporting our contention that Nhlh2 regulates TFAM through the E-box motifs in its promoter. In order to begin to study TFAM expression and molecular regulation by Nhlh2, we have isolated primary bovine skeletal muscle cell lines from adult cattle. These cell lines express muscle specific markers (medium-chain acyl coA dehydrogenase [MCAD]; myogenic transcription factor [MyoD]; and peroxisome-proliferator activated receptor alpha [PPARa]) to the same level as the established and widely used mouse skeletal muscle cell line, C2C12 cells. To the best of our knowledge, these data represent the first time that primary bovine muscle cells have been cultured and differentiated from adult animals for use in molecular- and cellular-based studies. We plan to use skeletal muscle cells lines from animals of different genotypes to study gene expression and mitochondrial function in vitro.

Publications

  • Good, D.J., (2009) Examining Polymorphisms Affecting Transcription and Translation of Genes Linked to Marbling in Beef Cattle, Animal Genetics, Rechi , L.J. Ed, Nova Scientific Publishers, ISBN 978-1-60741-844-3, in press
  • Good, D.J. (2009) Transcriptional Regulation of Energy Availability Sensing Within Hypothalamic Neurons, Open Neuroendorinol. J., Vol. 2, in press.
  • Vella, K.R., and Good, D.J. (2009) Nhlh2 is a Cold-Responsive Gene, Open Neuroendorinol. J. Vol 2, in press.
  • Wankhade, U.D. and Good, D.J. (2009) Transcriptional Regulation of Hypothalamic Genes Controlling Energy Balance, Endocrine Society Annual Meeting, Washington, DC, June 2009, invited oral presentation
  • Wankhade U.D. , Fawcett, K. and Good D.J., (2009) Transcriptional Regulation of Genes Involved in Hypothalamic Melanocortin Signaling, Keystone Symposium on "Obesity: Novel Aspects in the Regulation of Body Weight", Banff, Alberta, Canada (January 2009).
  • Wankhade, U.D., Fawcett, K., Rudd, A., McMillan R.P., Hulver M.W. and Good, D.J., (2008) Nhlh2 regulates MC4R transcription and peripheral fatty acid metabolism Neuroscience, Washington DC (November 2008).


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: We have obtained DNA from 6 purebred Wagyu steers obtained from our collaborators at Washington State (Dr. Charles Gaskins and Dr. Zhihua Jiang) and 38 purebred Angus steers from our collaborator at Virginia Tech (Dr. William S. Swecker, Jr). The bNHLH2 coding region has been sequenced from all 6 Wagyu and 36/38 Angus individuals. To accomplish this, new primers had to be developed that would allow us to perform a single polymerase chain reaction (PCR) amplification, with sequencing in both directions from the same primers. Based on our collaboration with Dr. Jiang, we have expanded our analysis of genetic control of marbling to include the promoter region "Transcription Factor A of Mitochondria" gene (TFAM). Dr. Jiang and colleagues identified two single nucleotide polymorphisms (SNPs) in the TFAM proximal promoter region which are linked to marbling in Wagyu cattle. These SNPs flank a DNA binding motif for the bNHLH2 transcription factor. The DNA binding motif (A/C-CAGATGA-T/C-C) matches the predicted binding site motif for NHLH2 (NCANNTGNN). Based on the published data from Jiang and colleagues we have designed new primers for the TFAM promoter and begun sequencing the TFAM promoter from our Angus samples, as Angus cattle show a high to intermediate level of marbling. We have also used electrophoretic mobility shift assays to determine if bNHLH2 can bind to the motif in the TFAM promoter. Finally, we have analyzed TFAM expression levels in three different muscle biopsies taken from one Angus and one Charolais breed animal. Angus steers have been sent for harvest and we are awaiting detailed body composition and marbling analysis on these animals. To date we have not disseminated the results of the project, but plan to submit the data for publication during the 2008-2009 reporting period. PARTICIPANTS: Risa Pesapane is a laboratory specialist assigned to this project. She has completed all of the data described in the report. Dr. Charles Gaskins and Dr. Zhihua Jiang (Washington State University) are collaborators for the Wagyu project, providing Wagyu ear biopsy samples for the isolation of DNA. Dr. William S. Swecker, Jr (Virginia Tech, College of Veterinary Medicine) is a collaborator for the Angus project, providing steer ear biospies for DNA isolation, and help in collecting muscle biopsies for TFAM expression analysis. Dr. Swecker is directly responsible for obtaining the carcass composition data (including marbling) for the steers. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: As reported in the objectives and outcomes, our analysis of the role of bNHLH2 in marbling in beef cattle has been expanded to include analysis of a possible bNHLH2 transcriptional target gene, TFAM. We have obtained significant results in identifying TFAM polymorphisms in a new breed of cattle. Future experiments will seek to link TFAM polymorphisms and TFAM expression with marbling data from carcass compositions, and with the ability of bNHLH2 to transcriptionally-regulate TFAM in skeletal muscle tissue.

Impacts
In previous work, we had found that 3/9 Wagyu purebreds harbored a SNP in the bNHLH2 gene. These SNPs all caused an amino acid change near the putative casein kinase phosphorylation domain in the amino-terminus of the bNHLH2 protein. The amino acid changes caused a shift in the hydrophobicity profiles of the protein, suggesting the protein function could be affected by the polymorphisms. However, sequencing of 6 additional purebred Wagyu cattle and 36 Angus cattle, which was completed during this reporting period failed to identify additional individuals with SNPs in their bNHLH2 gene. In expanding our analysis to a possible transcriptional regulatory target of bNHLH2, we have sequenced the TFAM promoter region containing the putative bNHLH2 binding site from 36 Angus cattle. The SNPs have been identified in the Angus breed with 39% (14/36) WT, 17% (6/36) 3' Heterozygous, 0% (0/36) 5' Heterozygous, 42% (15/36) double Heterozygous and 3% (1/36) 3' Mutant/5' Heterozygous. Thus, more than half of the animals of the Virginia Tech herd (61%) have at least one of the polymorphisms described by Jiang and colleagues to be associated with increased marbling in Wagyu cattle. Furthermore, we have shown that bNHLH2 can bind to the DNA motif(A/C-CAGATGA-T/C-C) using the electrophoretic mobility shift analysis method. Binding to the TFAM promoter motif is equivalent to the binding of a known bNHLH2 target gene, necdin. Finally, we find that we can detect TFAM RNA expression, using quantitative real-time PCR (QPCR) in muscle isolated from cheek, loin and rump from animals sent to slaughter. We plan to expand this to sample cheek muscle using punch biopsies from live animals so that TFAM expression and genotype can be compared in the same animals. Our long term goals are to characterize the SNPs in the TFAM and bNHLH2 genes that contribute to differences in cattle marbling, and to understand the biological mechanisms that factor into each SNPs contribution to skeletal muscle fat deposition. SNPs in the TFAM gene or bNHLH2 may influence marbling in other US breeds and could be tied to the inheritance of economically-important traits. Based on our results, commercial tests could be developed to aid in bull and heifer selection.

Publications

  • Hur, S.J., Whitcomb, F., Rhee, S., Park, Y., Good, D.J. and Park, Y. (2008) Effects of trans-10, cis-12 conjugated linoleic acid on body composition in genetically obese mice. J. Medicinal Food, in press.
  • Good, D.J., Coyle, C.A., and Fox, D.L. (2008) Nhlh2: A basic helix-loop-helix transcription factor controlling physical activity. Exercise and Sports Science Reviews, 36: 187-192
  • Coyle, C.A., Strand, S. C., and Good, D. J. (2008) Reduced physical activity contributes to obesity in Tub Mutant Mice, Physiology and Behavior.95 (1-2): 168-175
  • Fox, D.L. and Good, D.J. (2008) Nhlh2 Interacts with STAT3 to Regulate Transcription of Prohormone Convertase 1/3, Molecular Endocrinology, 22 (6): 1438-1448
  • Fox, D.L., Fox, D.L.G., Jensen R.V., and Good, D.J. (2007) Microarray Technology Uncovers Biological Pathways Involved in the Development of Obesity in Nhlh2 Knockout Mice, in Genes, Genomes and Genomics, Vol 2, Regency Publications
  • Vella, K.R., Burnside, A.S. Brennan, K.A. and Good, D.J. (2007)Expression of the Hypothalamic Transcription Factor Nhlh2 is Dependent on Energy Availability, J. Neuroendocrinology, 19: 499-510
  • Fox, D. L., Vella, K.R., and Good, D.J. (2007) Energy Balance Pathways Converging on the Nhlh2 Transcription Factor, in Frontiers in Bioscience, Peptides Controlling Energy Balance, 12: 3983-3993
  • Good, D.J. (2007) Mouse Models of Obesity and Overweight, in Sourcebook of Models for Biomedical Research, Humana Press. Totowa, NJ pp 683-702.