Molecular and Cellular Physiology Laboratory Ramaley C373 Department of Integrative Physiology University of Colorado Boulder, CO 80309-0354 phone: 303-735-6712 fax: 303-492-0811

Research Interests

The primary research focus of the Molecular and Cellular Physiology Laboratory is on understanding the molecular mechanisms underlying changes in gene expression during adaptation of fat and muscle cells to adaptation and disease. In particular, we are interested in the following topics:

• Transcriptional regulation of skeletal muscle gene expression in response to exercise and disuse.


• Transcriptional regulation of adipocyte gene expression.


• The role of various signaling pathways involved in muscle hypertrophy and atrophy.


• The effects of psychological stress and obesity on muscle and skeletal muscle and adipose gene expression.


• Molecular mechanisms underlying exercise-induced muscle damage and repair.

We employ a broad range of integrative techniques and approaches to study these questions. The techniques include genetic cloning of regulatory regions, untranslated regions, and coding regions for cellular and in vivo over-expression studies; mutagenesis and deletional analysis of genes and their regulatory regions; regular and real-time RT-PCR; immunohistochemistry, ELISA and Western blotting; cell culture; transfection of cultured fat and muscle cell lines; luciferase and green fluorescent protein (GFP) reporter analysis; null and transgenic mouse analysis; histology and histochemistry; and voluntary cage wheel and treadmill running exercise training. In addition, we use an integrative approach spanning bioinformatics and sequence analysis, cell culture, animal models, and human clinical studies.

Personnel

• Director: David L. Allen, Ph.D.


• Professional Research Assistant: Sarah Lindsay, B.S.


• Undergraduate Students: Brianna Fowler, Lindsey Bird, Jennifer Lesh, Jake Lebin, Michelle Janer.


• Graduate Students: Ryan Mehan, Molly Madden.


• Collaborators: Dr. Virginia Ferguson and Dr. Louis Stodiek, BioServe Space Technologies, Aerospace Engineering, CU-Boulder.

Current Research Projects

• Transcriptional regulation of myostatin expression in 3T3-L1 adipocyctes and C₂C₁₂ myotubes. Myostatin is a member of the TGF-beta family of growth factors that is expressed exclusively in fat and muscle and which functions to inhibit muscle growth and development.  The factors regulating myostatin expression in skeletal muscle are currently not well defined and in fat are completely unknown; our goal is to identify the cis- and trans-regulatory elements and the signaling pathways involved in myostatin gene transcription in both muscle and fat, and furthermore to explore whether physiological changes such as exercise, obesity or aging affect myostatin expression in fat tissue.  We are using transfection of human and mouse myostatin promoter constructs into two well-defined in vitro models of adipocyte and myocyte cell culture, along with co-transfection with signaling and transcription factor expression vectors and mutagenesis, to address these issues.
  
  
• Regulation of myostatin expression in skeletal muscle and adipose during obesity and fasting.  Changes in food intake can result in dramatic changes in the growth of adipose and skeletal muscle, which in turn directly influences body composition and total metabolism.  The aim of this project is to determine the effects of obesity and fasting on the expression of myostatin and myostatin-associated genes in skeletal muscle and adipose, and to determine the role(s) of myostatin signaling in regulating proliferation, differentiation, survival, and cell growth during these states.



• Regulation of interleukin-6 (IL-6) expression in skeletal muscle in response to exercise.  IL-6 is a cytokine whose expression by and secretion from skeletal muscle is hugely increased following a single bout of endurance exercise.  IL-6 is thought to function as an “energy sensor”, linking an increase in muscle activity with the mobilization of glucose and lipid stores from liver and fat during prolonged exercise.  It is currently not known what the signal is for the increase in skeletal muscle IL-6 expression in response to exercise.  The goal of this project is to identify the transcriptional and/or post-transcriptional regulatory elements, and their associated signaling pathways, involved in activation of IL-6 expression in response to exercise.  For this study, the human and mouse IL-6 promoter and untranslated regions have been cloned into reporter constructs in order to determine the sequence(s) necessary for this systems level response.  Both cell culture and animal studies will be used to identify the mechanisms regulating IL-6 expression under basal and exercise-stimulated conditions.  In addition, human studies will be run in which subjects with specific IL-6 promoter and UTR genotypes are exercised to see whether naturally occurring polymorphisms in the human IL-6 regulatory regions influence the magnitude of the exercise IL-6 response. 
  
  
• Role of matrix metalloproteinases in exercise-induced muscle damage and repair.  A single bout of eccentric exercise often causes muscle damage and soreness for days afterward.  Currently the molecular etiology of this process is not well defined.  The goal of this project is to determine the role, if any, of connective tissue-degrading enzymes known as matrix metalloproteinases or MMPs in the exercise-induced damage/repair process.  MMPs are known to be secreted by many cell types involved in muscle damage and repair, including fibroblasts, immune cells, and the muscle fibers themselves.  We will explore the causal role of MMPs in exercise-induced damage and repair by inhibiting MMP activity pharmacologically and MMP expression genetically in eccentrically-exercised mice.  In addition, the factors regulating MMP transcription will be identified using MMP promoter constructs transfected into C2C12 myotubes in vitro and injected into mouse skeletal muscles in vivo.  Finally, human clinical studies will determine whether serum MMP levels mimic the serum levels of other indices of muscle damage in eccentrically exercised human subjects.
  
  

Opportunities for Undergraduates

• Undergraduate students who want to obtain a research experience in our Laboratory must meet the following requirements:


• Minimum 1 year commitment.
• Minimum hours - 6-8 hours per week in blocks of at least 2 hours.
• No prior experience required.
• Must be willing to work with rodents and occasionally work on weekends.


• For more information, contact Prof. David Allen (david.l.allen@colorado.edu)

Funding

• 2008-2011, NIH/NIAMS R03, "Regulation of Exercise-Induced IL-6 Expression in Skeletal Muscle".


• 2008-2009, University of Colorado Innovative Seed Grant, "Expression of Myostatin and its Receptor in Mouse and Human Adipose Tissue".


• 2004-2008, NIH KO1 AR050505-01, "Matrix Metalloproteinases, Exercise, and Muscle
  Damage".


• 2005-2006, Shih Chun Wang Young Investigator Award from the American
  Physiological Society, "Matrix Metalloproteinases, Exercise, and Muscle Damage".


• 2005-2006, Junior Faculty Development Award from the Council on Creative Research at the University of Colorado, Boulder, "Matrix Metalloproteinases, Exercise, and Muscle Damage".


• 2005-2006, Research Endowment Award from the American College of Sports Medicine, "Matrix Metalloproteinases, Exercise, and Muscle Damage".