Jonathan Fisher, Ph.D. - US grants

Although increase in size and number of mitochondria in skeleletal muscle is a well-known adaptation to endurance exercise, mechanisms for exercise-induced mitochondrial biogenesis remain unclear. Nuclear respiratory factor I (NRF-I) has been identified as an early-responding protein after exercise and has been suggested to coordinate transcription of mitochondrial enzymes. The goal of the proposed research is to examine potential pathways for exercise-induced mitochondrial biogenesis. First, a single bout of exercise known to increase expression of mitochondrial enzymes in rats will be used to determine the time course of NRF-1 expression. Second, possible exercise-related mechanisms for an increase in mitochondrial proteins, including increased intracellular cAMP, increased Ca2+, and reduced phosphorylation potential will be examined using cell culture models.

DESCRIPTION (provided by applicant): The immediate goal of this project is to train the candidate in molecular techniques (e.g. construction of vectors, transfection of mammalian cells, and protein transduction) to more closely examine the roles of individual proteins in the stimulation of glucose transport by insulin and other stimuli that the candidate studied during his postdoctoral training. The candidate's career aim is to apply diabetes research to a wide range of models, including cultured myotubes, isolated skeletal muscle, and muscle in vivo. The candidate's training at Saint Louis University will include mentored laboratory research, attendance of and participation in local research seminars and national conferences, and formal coursework in molecular biology and the responsible conduct of research. Diabetes researchers at a neighboring institution, Washington University, will provide additional technical training, career guidance, and research consultation. The environment offers numerous opportunities for the candidate to attend research seminars and interact with established researchers. The candidate has shown that stimulation of AMP-activated protein kinase (AMPK) activity is associated with increased insulin sensitivity in skeletal muscle, and he proposes to examine the role of ARK5, a newly-described Akt-activated AMPK family member, in the regulation of glucose transport. Since Akt is stimulated by muscle contractions (as recently reported in the literature and shown in preliminary data) and insulin, the hypothesis for this proposal is that ARK5 contributes to cross-talk between contraction- and insulin- signaling pathways in the stimulation of glucose transport. The candidate's preliminary data shows that skeletal muscle contains ARK5. The specific aims are 1) to determine whether ARK5 is activated by muscle contractions, insulin, and AMPK-activating stimuli, 2) to determine in myotubes (by means of overexpression of ARK5, expression of a constitutively active ARK5, and expression of ARK5 that cannot be regulated by Akt) the role of ARK5 in insulin-independent glucose transport and insulin sensitivity in the absence and presence of AMPK-activating stimuli, and 3) to determine, by transduction of ARK5 forms into myotubes, the acute role of ARK5 in the insulin sensitivity induced by AMPK-activating stimuli. The proposed training will develop the candidate in his transition to an independent researcher and is critical to the candidate's long-term ability to contribute to diabetes research

[unreadable] DESCRIPTION (provided by applicant): Humans and animals with deficient ataxia telangiectasia mutated (ATM) are insulin-resistant or hyperglycemic. ATM reportedly indirectly influences insulin signaling, including phosphorylation of Akt, in various cell types. There is also evidence that ATM is an activating kinase for the AMP-activated protein kinase (AMPK), a mediator of insulin-independent stimulation of glucose transport. However, the potential roles of ATM in AMPK- and insulin-stimulated glucose transport have not previously been addressed in skeletal muscle, the predominant tissue in insulin-stimulated glucose disposal. Preliminary data from cultured myotubes demonstrate that a specific ATM inhibitor prevents stimulation of glucose transport by insulin and the AMPK activator AICAR. The specific aims are: 1) to test the hypothesis that ATM plays a role in insulin-independent stimulation of glucose transport by AMPK-activating treatments (e.g. exercise and/or the AMPK activator AICAR) in skeletal muscle, 2) to test the hypothesis that ATM plays a role in insulin-stimulated glucose transport and insulin signaling in skeletal muscle, and 3) to test the hypothesis that ATM directly phosphorylates and/or alters activity of components of the insulin signaling pathway in skeletal muscle. Experimental tools will include ATM deficient transgenic mice, a specific inhibitor of ATM, and an antibody specific for proteins that have been phosphorylated by ATM. Elucidating the possible role of a factor like ATM that might play a role in regulation of glucose transport stimulated by either AMPK or insulin--or both--is potentially valuable to describing new approaches to prevention or treatment of diabetes. [unreadable] PUBLIC HEALTH RELEVANCE STATEMENT: The project will examine the potential role of a protein called ATM in regulation of sugar transport into muscle in response to exercise and insulin, a hormone that enters the bloodstream when blood sugar levels increase. Understanding the factors that cause muscles to clear sugar from the bloodstream is vital to developing strategies to treat or prevent the high blood sugar concentrations that are the hallmark of diabetes. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Regulation of GLUT1, the basal glucose transporter of skeletal muscle and the predominant dehydroascorbic acid (DHA) transporter, has not been fully-elucidated in terms of factors that influence GLUT1 cell surface abundance, GLUT1 trafficking, and GLUT1's activity toward its two substrates in skeletal muscle. Preliminary data suggest that ataxia telangiectasia mutated (ATM) may play a role in regulation of basal glucose transport and GLUT1 abundance in skeletal muscle. In addition, other investigators have reported a potential role for p38 in increasing intrinsic activity of GLUT1 toward glucose transport. There are three C-terminal phosphorylation sites in GLUT1;S490 is known target of ATM, and S473 and T478 match consensus target motifs for ATM and p38, respectively. The goal of the project is to determine the roles of these GLUT1 phosphorylation sites (or activation of the potential kinases for these sites) in regulation of GLUT1 abundance, localization, and activity in cultured skeletal muscle cells or mouse skeletal muscle. Specific Aim 1 of the project is to determine whether S473 and S490 of GLUT1 play roles in regulation of GLUT1 abundance or cell surface localization. The general hypotheses are that phosphorylation of these sites will preserve GLUT1 levels and cell surface localization, while GLUT1 that is not phosphorylated at these sites will be more prone toward internalization and degradation. Effects of S473 and S490 mutations will be determined for GLUT1 abundance, trafficking, and transport activity toward glucose and DHA, and effects of activation of ATM (a potential kinase for both sites) on GLUT1 will also be determined. Specific Aim 2 is to determine whether T478 plays a role in regulation of substrate specificity or intrinsic activity of GLUT1. The hypothesis is that T478 phosphorylation will stimulate increased GLUT1 activity toward glucose and decrease activity toward DHA. Effects of T478 mutations will be determined for intrinsic activity of GLUT1 toward glucose and DHA, though potential effects on GLUT1 abundance and trafficking will also be examined. Effects of activation of p38 (a potential kinase of T478) on GLUT1 activity and trafficking will be determined. Additionally, it will be determined whether factors that alter DHA transport also are associated with changes in levels of reactive oxygen species and/or can influence insulin action in muscle cells. Information provided by this project might be used to develop strategies to increase basal glucose transport in skeletal muscle or to increase DHA transport to support skeletal muscle antioxidant status. PUBLIC HEALTH RELEVANCE: This project will investigate factors that could regulate GLUT1, a protein that allows movement of sugar into skeletal muscle all day long (as opposed to another sugar-transporting protein that is responsible for moving sugar into muscle after meals or exercise). Additionally, the role of GLUT1 in providing the building block for muscle vitamin C, a key antioxidant that destroys free radicals, will be investigated. The goal of the project is to provide information that might be useful in improving blood sugar control or in maintaining antioxidant defenses in muscle.

? DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) have the capacity to cause insulin resistance and oxidative damage. A few specialized cell types have the capacity for extracellular ascorbate cycling. In this process, ascorbate is exported as an antioxidant, and after it is oxidized to dehydroascorbic acid (DHA), the DHA is taken up and reduced intracellularly to ascorbate for another round of antioxidant efflux. We have obtained preliminary data showing that mouse skeletal muscle has the ability to reduce extracellular electron acceptors in a process that is dependent on ascorbate release. Accordingly, the aims of the project are to elucidate the mechanism for, the regulation of, and the physiological consequences of extracellular ascorbate recycling by skeletal muscle. Aim 1 is to determine whether extracellular ascorbate recycling by skeletal muscle provides extracellular antioxidants. The hypotheses for Aim 1 are that muscle cells can reduce extracellular electron acceptors, that the electrons are carried by ascorbate, and that H2O2 plays a countervailing role in this process. Additional hypotheses are that GLUT1-mediated DHA transport supports ascorbate recycling and that anion channels are responsible for extracellular reduction activity and ascorbate efflux. Aim 2 is to determine whether extracellular reduction and ascorbate recycling by skeletal muscle are regulated by insulin and activation of the AMP-activated protein kinase (AMPK). In particular, the aim is to determine whether insulin produces a synergistic increase in DHA transport, glucose 6-phosphate dehydrogenase (G6PD) activity, and ascorbate efflux and to determine whether AMPK stimulates DHA transport but not G6PD activity or ascorbate efflux. Aim 3 is to determine functional roles of ascorbate recycling such as prevention of oxidative damage and maintenance of insulin action. Previously, ascorbate cycling was described for specialized tissues that are small relative to skeletal muscle, which contains 40% of whole-body ascorbate. This project will establish skeletal muscle as a primary generator of extracellular antioxidant and thus an important tissue in whole-body antioxidant status. Further, it could establish the importance of ascorbate cycling and in particular muscle DHA uptake in controlling ROS, thus maintaining normal insulin action. If successful, the project will introduce manipulation of skeletal muscle ascorbate cycling as a novel means for addressing insulin resistance.

PI: Fisher, Jonathan
Proposal Number:1541612

This proposal, whose driving goal is the need for a portable device capable of quantifying biomarkers of traumatic brain injury (TBI), focuses on determining the utility of Electroencephalography (EEG) and Diffuse Correlation Spectrography (DCS) for detecting TBI biomarkers in an animal model.

TBI is a very significant issue in both military and civilian medicine. Diagnosis and assessment of TBI patients is generally performed using clinical imaging modalities (MRI, CT). However, the future availability of portable devices for assessing TBI would fundamentally improve the outlook for TBI patients. The diagnosis and treatment of TBI is impeded by gaps in regulatory science due to a lack of quantifiable biomarkers. Two complementary measurement parameters have potential to fill this gap. (1) measurement of somatosensory evoked electrophysiological potentials (SSEPs); and (2) diffuse correlation spectroscopy (DCS). This proposal seeks to evaluate the utility of these parameters for detecting TBI in an animal model without the need for a baseline, pre-injury measurement.

PI: Fisher, Jonathan
Proposal Number: 1641133

Existing monitoring methods have technological limitations, such as the inability to detect flow changes in small blood vessels, as well as practical ones, such as portability and safety issues involved in bedside monitoring. Cost is an additional limitation for current clinical diagnostics; small hospitals often do not have such capabilities. Transcranial optical techniques such as diffuse correlation spectroscopy (DCS) can provide rapid, real-time monitoring of brain activity for a fraction of the cost of other methods. The PI will evaluate diffuse correlation spectroscopy (DCS) as a noninvasive optical methods for monitoring real-time changes in cerebral blood flow. The proposed research will partner researchers, faculty and postdocs at the New York Medical College, the University of Colorado, the University of Pennsylvania, and the FDA. The proposal includes a well-designed mentoring plan to be implemented by the PI. The plan includes tracking progress through Individual Development Plans; informal discussions to assess satisfaction with the mentoring program' and tracking progress toward career goals.

This project will provide information on the types of changes that can be interrogated using the low-resolution diffuse correlation spectroscopy (DCS) technique. Direct comparison to a high resolution technique will provide necessary information on sensitivity and specificity for DCS. The use of stimulated changes during monitoring will provide the types of necessary detail on the populations of blood vessels that can be assessed. Cerebral blood flow can be used to inform medical interventions in cases of neurological injury or disease.

This proposal will work on identifying non-invasive markers for traumatic brain injury (TBI). The proposal lays out a plan to verify quantitative biomarkers for mild (m)TBI that persist in the weeks following an injury. The proposed research will partner with faculty and postdocs at the New York Medical College, the University of Colorado, the University of Pennsylvania, and the FDA. The proposal includes a well-designed mentoring plan to be implemented by the PI. The plan includes tracking progress through Individual Development Plans; informal discussions to assess satisfaction with the mentoring program' and tracking progress toward career goals.

The principal investigator will evaluate diffuse correlation spectroscopy as a noninvasive optical method for monitoring real-time changes in cerebral blood flow, in order to better inform medical interventions in the event of neurological injury or disease. This project will provide information on the types of changes that can be interrogated using the low-resolution diffuse correlation spectroscopy (DCS) technique. Direct comparison to a high resolution technique will provide necessary information on sensitivity and specificity for DCS. The use of stimulated changes during monitoring will provide the types of necessary detail on the populations of blood vessels that can be assessed. Cerebral blood flow can be used to inform medical interventions in cases of neurological injury or disease.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.