Kevin Kelley, Ph.D. - US grants

Thy-1 is a member of the immunoglobulin supergene family that codes for a cell surface recognition molecule that is expressed only in the immune and nervous systems. The function of Thy-1 is unknown. Transgenic mice will be developed to study the function and cellular expression of Thy-1 in the developing nervous system. Genetic techniques to make mutant embryonic stem cells lacking the Thy-1.2 allele will be used to make transgenic mice lacking expression of Thy-1. These mice will be used to study the role of this protein in neural development. In addition, transgenic mice containing a marker protein whose expression is directed by the Thy-1 promoter will be used to study the lineages of cells that express Thy-1 in the neuroepithelium.

Dopamine is an important neurotransmitter in the central nervous system and its various actions are mediated by several dopamine receptor subtypes. One of these subtypes, designated as the D3 receptor, is expressed in phylogenetically older regions of the brain, including the limbic system. This suggest that D3 receptors play a role in cognition and in the expression of emotive behavioural phenotypes. However, conventional animal studies on the particular function of D3 receptors proved to be difficult because of the high homology and the similar pharmacological characteristics of this receptor subtype with other brain dopamine receptors. To circumvent these difficulties, this proposal lays the groundwork for studies on the specific function of D3 receptors in the whole animal by generating mutant mice that lack functional D3 receptors. This will be achieved by introducing a mutant (non-functional) D3 gene into embryonic stem (ES) cells of mice. Such ES cells are then used to generate chimeric mice that have the ability to transmit the mutant D3 gene to their offspring. Mutant offspring will have a decreased (heterozygous mutants) expression of the D3-receptor protein and such mice can be interbred to generate homozygous mutant mice in which the D3 receptor expression is abolished. Initial studies on such mutant animals aim at testing whether a decreased or abolished D3-receptor protein expression affects the development of brain regions in which D3 receptors are normally expressed. At present, nothing is known about the importance of D3 receptor expression for the normal development of the brain. Thus, comparative anatomical studies of such brain sections obtained at defined developmental stages from mutant and normal (wildtype) mice are proposed. Furthermore, because of the significant anatomical overlap in the expression of D3 receptors and another (very similar) dopamine receptor subtype (named D2), and the potential functional complementarity bet ween these two receptor subtypes, one might expect changes in the expression of D2 receptors to occur in response to a decreased or abolished expression of D3 receptors. Such changes could mask the functional consequences of a "loss of D3 receptor expression" in our mutant animals. Therefore, experiments are proposed that will test whether the expression of D2 receptors differs in wildtype and D3- receptor-mutant animals. The work proposed here will provide a powerful mouse model for future studies on the importance of D3 receptor expression for the expression of certain behavioural phenotypes.

One of the most fundamental biological roles of the pancreatic hormone insulin is to promote cellular uptake of glucose and other fuels from the blood and extracellular spaces. The development of an insulin-regulated glucose transporter system is arguably a key physiological adaptation that supports the heightened energetic demands of cells and tissues in endothermic ("warm-blooded") animals, such as mammals. Conversely, in ectothermic vertebrates, this and other aspects of insulin-regulated metabolism are hypothetically less developed. Current understanding of insulin-regulated cellular glucose transport has been dependent almost entirely on studies of the insulin-regulated GLUT-4 glucose transporter of mammals. By contrast, the status of this system "before" the evolution of an endothermic physiology (e.g., in fishes) is not understood, due in part to the lack of available models of insulin deficiency in ectothermic vertebrates to facilitate experimental analysis of the system.
The objectives of the proposed studies are (1) to characterize a teleost fish insulin-regulated glucose transporter system at the molecular and cellular levels, (2) to elucidate its physiological role in the regulation of glucose homeostasis in this vertebrate, and (3) to identify the likely evolutionary differences relative to that which exist in mammals. The gobiid fish, Gillichthys mirabilis, is a particularly useful model for such studies, since it can be experimentally manipulated into a state of insulin-deficiency reminiscent of type-1 diabetes mellitus. Previous work on this model indicates that insulin-deficient gobies exhibit high blood glucose levels and a pronounced impairment of insulin-regulated glucose transport activity in skeletal muscle, while insulin replacement therapy corrects these metabolic deficiencies. In addition, insulin treatment stimulates glucose clearance in vivo and muscle glucose transport in vitro. It is therefore hypothesized that an insulin-regulated glucose transporter, related to mammalian GLUT-4, exists in muscle of this teleost fish and plays an important role in glucoregulation, albeit with differences likely to reflect the different metabolic strategies represented by the ectothermic fish versus endotherms. In support of this hypothesis, recent molecular cloning data in the goby and some other teleost fish species have identified at least two different cDNAs related to mammalian GLUTs 1 & 4. The following three specific aims are proposed: 1) to isolate, clone, and characterize GLUT cDNAs from goby muscle, principally putative GLUT4 and GLUT 1, and to develop specific molecular probes (cDNA, antisera) for use in subsequent biochemical analyses; 2) to define insulin's specific regulatory actions on the muscle glucose transporter system in the goby, both at the level of gene expression and the level of the cell-physiological response to insulin; and 3) to characterize the interaction of insulin and the hormones that antagonize its actions (so-called counterregulatory hormones) in regulating the muscle GLUT system and hepatic metabolism, in order to advance a more integrative understanding of glucoregulatory physiology in this fish. These studies should provide a novel biological perspective and understanding of a fundamental action of the hormone insulin.

DESCRIPTION: (provided by applicant) Skeletal muscle function declines with age. With the increase in man?s average expected life span and the increasing elderly population worldwide, scientific research is attempting to preserve the aging population?s quality of life. One theory at the forefront of explanations for the effects of aging has been the cumulative negative effects of free radical production over time. Evidence suggests that aged organisms grow increasingly vulnerable to damage from reactive oxygen species (ROS). In addition to the progressive atrophy and functional declines observed in aging skeletal muscle, common medical conditions in the elderly such as respiratory and circulatory insufficiencies can compromise the body?s ability to deliver oxygen to tissues throughout the body. These combined effects of aging and hypoxia on skeletal muscle car severely compromise functional capacity in these individuals and further hasten the loss of physical independence. I propose to use the single skeletal muscle fiber model in which experimental conditions can be tightly controlled and several intracellular factors can be examined in an intact, contracting fiber. By I studying the alterations that occur in the aged muscle?s oxidative stress and its ability to respond to exercise and hypoxia, a better understanding of how exercise prescription and drug therapy can prevent or improve muscle function with age may be achieved.

The goal for this symposium is to advance the recognition of hormone-binding proteins (HBPs) as centrally-positioned regulators in endocrine systems, that have significant influences both in the integration of cell and physiological function as well as in the evolution of endocrine systems. In this symposium, HBPs will be defined as proteins in the extracellular environment (mostly) that bind hormones or similar factors, usually with a high affinity, and that therefore maintain a central regulatory position between a hormone and its ability to transduce its signal through its cell receptor. The emphasis of the symposium will be to look beyond the role of HBPs as 'carrier proteins' and instead to bring in the emerging concepts of HBPs as multifunctional regulatory proteins that can play dynamic --even predominant-- roles in integrating endocrine systems. Of particular interest will be where HBPs have developed regulatory properties beyond that of primary hormone-binding, including those that broadcast into other endocrine systems. With the diverse properties of HBPs being increasingly defined, it is also becoming clear that many HBPs have interesting evolutionary histories, not only in the development of their properties through different taxa, but also in their influences on the evolution of the hormone and receptor systems with which they interact. Such data are providing an enhanced understanding, and biological perspective, of the roles of HBPs in endocrine systems.
In this symposium, there will be 13 plenary lectures by speakers who will serve as "ambassadors" for their respective HBP(s) of interest. The speakers are encouraged to present their own work in the context of general and underlying regulatory mechanisms ("integrative context") and, where possible, in the context of the evolution of the respective endocrine systems ("evolutionary context"). It is expected that several of the speakers will also draw connections between different HBP/endocrine systems, as well as to diverse physiological systems. Given the involvement of HBP/endocrine systems in the integration and evolution of diverse physiological systems, including reproduction, development, neural systems, immune systems, and others, this type of symposium will afford an opportunity for a wide spectrum of biologists to learn about a forefront area in endocrinology with relevance to them. Twelve speakers will consider more specific aspects of certain HBPs, with a thirteenth lecture to be more general and given as the first annual Howard A. Bern Lecture in Comparative Endocrinology and presented by the namesake himself this year. On the following day, there will be an associated HBP Mini-Symposium at which students, postdocs, and others will have an opportunity to participate. The internationally recognized Journal of Endocrinology will publish the symposium proceedings.
To date, general recognition of the importance of HBPs to endocrine regulation and to the evolution of endocrine systems has been limited, despite increasingly compelling evidence for their central, key roles in many systems. As far as we are aware, there has not yet been a broad-reaching symposium of this type, which orients itself on a "cross-cutting" theoretical basis. Particularly unique are the emphases on the multifunctional nature of HBPs and on their role in endocrine system evolution.

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce of the United States. The program recognizes and supports outstanding graduate students who are pursuing research-based master's and doctoral degrees in science, technology, engineering, and mathematics (STEM) and in STEM education. The GRFP provides three years of financial support for the graduate education of individuals who have demonstrated their potential for significant research achievements in STEM and STEM education. This award supports the NSF Graduate Fellows pursuing graduate education at this GRFP institution.

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.