Pilot and Feasibility


P & F STUDIES

Function of mitochondrial NT-PGC-1a in mitochondrial DNA transcription

PI:  JS Chang, Ph.D.
Professional Career Status at time awarded: Postdoctoral Researcher
Current Professional Career Status: Assistant Professor-Research
P&F Title: Function of mitochondrial NT-PGC-1a in mitochondrial DNA transcription

Overview of project including significance and salient results:
Brown adipose tissue (BAT) dissipates energy in the form of heat, defending against hypothermia and obesity. An increase in mitochondrial electron transport chain (ETC) system within the mitochondrium is important for BAT thermogenesis since mitochondrial respiration through the ETC facilitates fuel oxidation and provides the energy needed for heat production. Nevertheless, the mechanisms by which mitochondrial genome encoding 13 essential subunits of ETC is transcriptionally regulated during thermogenesis remain undetermined.  Our data show that the short isoform of PGC-1α transcriptional coactivator (NT-PGC-1α) localizes to the mitochondria and binds to the mitochondrial genome for transcriptional induction of mitochondrial ETC genes. This study provides novel insight into the transcriptional control of mitochondrial ETC respiratory function in brown adipocytes.  

List of Resulting Publications:

  • Chang JS, Fernand V, Zhang Y, Shin J, Jun HJ, Joshi Y, Gettys TW. (2012). NT-PGC-1α is sufficient to link β3-adrenergic receptor activation to the transcriptional and physiological components of adaptive thermogenesis.  J Biol Chem, 287(12):9100-9111. PMCID: PMC3308807

  • Chang JS, Gettys TW. (2013). Analyzing phosphorylation-dependent regulation of subcellular localization and transcriptional activity of transcriptional coactivator NT-PGC-1alpha. Methods Mol Biol, 952:163-173. PMCID: PMC4035304.

  • Jun HJ, Gettys TW, Chang JS. (2012). Transcriptional activity of PGC-1α and NT-PGC-1α is differentially regulated by Twist-1 in brown fat metabolism. PPAR Res, 2012:320454. PMCID: PMC3474972

  • Jun HJ, Joshi Y, Patil Y, Noland RC, Chang JS. (2014). NT-PGC-1α activation attenuates high-fat diet-induced obesity by enhancing brown fat thermogenesis and adipose tissue oxidative metabolism. Diabetes, PMC Journal - In Progress.

Any peer-reviewed subsequent funding in the same or related area:

  • R01DK104748, Pending for funding (6th % as new investigator) The short isoform of PGC-1α in the regulation of adaptive thermogenesis


Defining the importance of skeletal muscle peroxisomal function in regulating insulin resistance

PI:  Robert Noland, PhD
Professional Career Status at time awarded: Assistant Professor
Current Professional Career Status: Assistant Professor
P&F Title: Defining the importance of skeletal muscle peroxisomal function in regulating insulin resistance

Overview of project including significance and salient results:
Peroxisomes have a primary role in modulating the cellular lipid environment. We predict peroxisomes provide an alternative route for lipid catabolism that can improve insulin sensitivity by limiting mitochondrial lipid overload and reducing intracellular lipid accrual in skeletal muscle. The purpose of this project was two-fold. First, we set forth to determine if peroxisomal function is linked to ceramide buildup. Results support a model where peroxisomal content and ceramide levels are inversely correlated. These findings led us to examine potential mechanisms responsible for this apparent link, which are ongoing studies. Second, to test our primary hypothesis that peroxisomes help maintain glucose homeostasis we began development of a muscle-specific, peroxisome-deficient mouse by targeting deletion of Pex5 (Pex5SkM-/-). Pex5 is a protein that is vital to entry of enzymes into the peroxisomal matrix that are responsible for lipid breakdown, thus deletion of Pex5 fundamentally results in peroxisome deficiency. We have successfully generated, and are currently characterizing this mouse model. Initial results indicate Pex5SkM-/- mice exhibit significant reductions in lipid oxidation. Moreover, while Pex5SkM-/- mice do not exhibit differences in body weight or composition (lean mass or fat mass) compared to control mice, they do exhibit impaired glucose clearance. Globally, findings from these studies support the concept that peroxisomes in skeletal muscle function to maintain glucose tolerance and ongoing studies are aimed at defining the mechanism/s through which this occurs. Lastly, the animal models developed and results obtained from studies supported by this NORC pilot & feasibility grant have been included in an R01 application which was scored in 20th percentile and we are currently waiting to receive final notification on whether it is funded.

List of Resulting Publications: (none stemming directly P&F to date but others are listed through collaborations)

  • Jun HJ, Joshi Y, Patil Y, Noland RC, Chang JS. NT-PGC-1alpha activation attenuates high-fat diet-induced obesity by enhancing brown fat thermogenesis and adipose tissue oxidative metabolism. Diabetes 2014.

  • Muoio DM, Noland RC, Kovalik JP, Seiler SE, Davies MN, DeBalsi KL, Ilkayeva OR, Stevens RD, Kheterpal I, Zhang J, Covington JD, Bajpeyi S, Ravussin E, Kraus W, Koves TR, Mynatt RL. Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility. Cell Metab 2012, 15:764-77. **PMCID: PMC3348515

  • Ruggiero C, Elks CM, Kruger C, Cleland E, Addison K, Noland RC, Stadler K. Albumin-bound fatty acids but not albumin itself alter redox balance in tubular epithelial cells and induce a peroxide-mediated redox-sensitive apoptosis. Am J Physiol Renal Physiol 2014, 306:F896-906. **PMCID: 3989633

  • Jun HJ, Joshi Y, Patil Y, Noland RC, Chang JS. NT-PGC-1alpha activation attenuates high-fat diet-induced obesity by enhancing brown fat thermogenesis and adipose tissue oxidative metabolism. Diabetes 2014.

  • Muoio DM, Noland RC, Kovalik JP, Seiler SE, Davies MN, DeBalsi KL, Ilkayeva OR, Stevens RD, Kheterpal I, Zhang J, Covington JD, Bajpeyi S, Ravussin E, Kraus W, Koves TR, Mynatt RL. Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility. Cell Metab 2012, 15:764-77. **PMCID: PMC3348515

  • Ruggiero C, Elks CM, Kruger C, Cleland E, Addison K, Noland RC, Stadler K. Albumin-bound fatty acids but not albumin itself alter redox balance in tubular epithelial cells and induce a peroxide-mediated redox-sensitive apoptosis. Am J Physiol Renal Physiol 2014, 306:F896-906. **PMCID: 3989633

Any peer-reviewed subsequent funding in the same or related area:

  • R01DK103860 (2015-2019) Defining the role of skeletal muscle peroxisomes in glucose homeostasis.


Nutrient Detection in the Hindbrain

PI:  David McDougal, BS, BA, PhD
Professional Career Status at time awarded: Instructor      
Current Professional Career Status: Assistant Professor - Research
P&F Title: Nutrient Detection in the Hindbrain

Overview of project including significance and salient results:
The principle aim of our P&F project is to utilize the Cre-LoxP transgenic technology to generate cell specific and/or tissue specific knockout of the type 2 glucose transporter (GLUT2) in the in vivo mouse model.  These transgenic mouse models will then be utilized to investigate the relative role of astrocytes and neurons in the CNS detection and response to severe hypoglycemia. We proposed to accomplish the aims of this study through the completion of the following 3 projects:

  • Project 1: Generation of a GLUT2-loxP mouse.
  • Project 2: Optimization of cell type/brain region specific Cre mice.
  • Project 3: Generation of tissue/cell specific GLUT2 null mice.

During our first year of funding we made significant progress towards completion of our study.  Project 1 and 2 were nearly completed.


Details of progress towards completion of Project 1:
The initial steps of project 1, i.e. blastocyst injections, generation of chimeric mouse, and germline transmission, were completed.  These steps represented the largest technical and biological impediments to realization of the goals of this project. To date, 8 germline pups were generated [genotype verified via PCR] and the final two breeding steps necessary for completion of this project were initiated.
           
Details of progress towards completion of Project 2:
We have also made significant progress in validation of 3 separate cell specific Cre lines [synapsin-cre, GFAP-cre, and GLAST-cre].  We have successfully induced cell specific red fluorescent protein [RFP] expression in all 3 Cre lines via crosses with a tdTomato-loxP strain.  2 of the 3 Cre lines required tamoxifen administration in order to induce Cre expression.  We have optimized the induction process in these 2 lines and are currently producing control and experimental animals in all 3 Cre lines.  Using immunohistochemical methods, we have begun to evaluate the efficiency and specificity of astrocyte and neuron specific cre expression in the hypothalamus and hindbrain of these lines.  In addition, all three of these Cre-loxP combinations represent novel transgenic lines, previously unreported in the literature.

List of Resulting Publications:

  • McDougal DH, Hermann GE, Rogers RC. Astrocytes in the nucleus of the solitary tract are activated by low glucose or glucoprivation: evidence for glial involvement in glucose homeostasis. Front Neurosci 2013, 7:249. **PMCID: 3868892
  • McDougal DH, Hermann GE, Rogers RC. Vagal afferent stimulation activates astrocytes in the nucleus of the solitary tract via AMPA receptors: evidence of an atypical neural-glial interaction in the brainstem. J Neurosci 2011, 31:14037-45. **PMCID: PMC3445261

Any peer-reviewed subsequent funding in the same or related area:

  • American Diabetes Association Junior Faculty Award:

Grant Reference #7-14-JF-37- CNS Mechanisms of Glucose Detection: Role of the Type 2 Glucose Transporter [GLUT2] in Hypoglycemic Counterregulation
Recommended for funding
Start Date: January 2015-2017