Andrzej Bartke, PhD - US grants

We are investigating the effects of different constitutents of marihuana (cannabinoids) on male reproductive functions. Treatment of male mice with the main psychoactive cannabinoid, Delta9-tetrahydrocannabinol (THC), or with non-psychoactive cannabinoids, cannabinol (CBN) or cannabidiol (CBD), can lead to impairment of sex hormone production, sexual behavior and fertility. We have shown that THC can both increase and reduce plasma testosterone (T) levels depending on the dose and interval between drug treatment and T measurement. In order to identify the methanisms responsible for these effects, we will examine content and turnover of dopamine, noradrenalin and serotonin in several brain areas; hypothalamic levels of LHRH; cytoplasmic androgen and estrogen receptors in the hypothalamus and the pituitary; pituitary responsiveness to LHRH and testicular responsiveness to LH in adult male mice given different doses of THC. We have demonstrated that cannabinoids can inhibit testicular T production in vitro. In order to evaluate the contributions of this direct gonadal effect to changes in plasma T levels observed in vivo in cannabinoid-treated animals, we will study the consequences of oral administration of THC to hypophysectomized, hCG-treated mice and of unilateral intratesticular THC injections in intact animals. In a separate series of experiments, we will examine the effects of chronic treatment with various doses of THC, CBN and CBD on fertility of treated mice and their progeny and the reversibility of these effects. We have preliminary evidence that cannabinoids may be mutagenic. Finally, we will compare the effects of combined chronic treatment with cannabinoids and alcohol to the consequences of chronic treatment with cannabinoids alone or alcohol alone. These studies will provide new information on the mechanisms of cannabinoid action on the pituitary and the testis, on the impact of chronic exposure to these compounds on fertility and on the interaction of marihuana and alcohol. This information is needed for evaluating the risks of marihuana use in human populations.

The long-term objectives of this research is to improve the present understanding of the hormonal control of male reproductive functions by elucidating the effects of prolactin (PRL) on the testes and the hypothalamic-pituitary system. Benefits from this project will include obtaining new information on the control of endocrine and reproductive functions in health and disease. One of the specific aims for the next five years will be to define hormonal control of testicular PRL receptors before and after sexual maturation and to relate the PRL-induced changes in the levels of LH and PRL receptors to the ability of the testis to respond to LH and PRL stimulation. Other aims of this project will be to elucidate the mechanisms responsible for the effects of PRL on the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary. The proposed studies will involve measurements of binding of radioactively labelled hormones to testicular membrane preparations, measurements of blood levels of pituitary and testicular hormones, assessment of noradrenergic, dopaminergic and serotonergic transmission in the hypothalamus by measurement of levels, turnover rates and metabolites of neurotransmitters in different brain regions, determinations of luteinizing hormone releasing hormone (LHR) stores in the hypothalamus and studying the control of its release in vivo and in vitro, as well as measurements of pituitary LHRH receptors and pituitary responsiveness to LHRH stimulation. Most of the proposed studies will be conducted in golden hamsters because in this species, PRL has already been shown to exert major and physiologically important effects on male reproductive functions, and PRL release can be readily manipulated by environmental lighting conditions without pharmacologic or surgical intervention. However, in order to more readily identify cause-effect relationships between the various effects of PRL and to determine whether our findings may apply to other mammalian species, we will conduct parallel comparative studies in the rat, the mouse and the Djungarian hamster.

A symposium on neuroendocrinology and two state-of-the-art lectures will be included in the program of the 19th annual meeting of the Society for the Study of Reproduction at Cornell University, Ithaca, NY, July 14-17, 1986. The symposium will focus on hypothalamic neurotransmitters and their role in the control of gonadotropins and prolactin release and sexual behavior. Hypothalamic control of reproductive function is a popular area of research among SSR members and there are many significant recent advances in this field, both in methodology and in conceptual grasp of the role of catecholaminergic neurons in the hypothalamus. The symposium will consist of lectures on hormones, neurotransmitters and sexual behavior, on dopaminergic control of pituitary function, and on the release of gonadotropin-releasing hormone in vivo. A lecture on the present state-of-the-art on experimental mammalian embryology, with particular emphasis on nuclear transfer, will be presented. Biology and experimental manipulation of early embryos are rapidly advancing areas of research, with prospect for numerous applications in animal breeding and medicine and are of immediate interest to a large segment of SSR membership. The second state-of-the-art lecture will be on the molecular mechanisms of gonadotropin-releasing hormone action. This area of research was chosen as a topic for invited lecture by a large proportion of SSR members responding to a recent questionnaire and the lecture will deal with some important new concepts in hormone action and in regulation of gonadotropin release.

We are studying the effects of marihuana on male endocrine, reproductive and sexual functions. Our objective is to define the effects of psychoactive and non-psychoactive constituents of marihuana on the pituitary, the testes and on male sexual behavior and to determine the mechanisms of these effects. During the next four years, we will address the mechanism of action of delta-9-tetrahydrocannabinol (THC) on male copulatory behavior and study the effects of perinatal or adult administration of THC, cannabinol (CBN) and cannabidiol (CBD) on hypothalamic and pituitary function. More specifically, we will test our hypothesis that the neurotransmitter-mediated alterations in the release of gonadotropin releasing hormone (GnRH) are responsible for the effects of THC on sexual behavior. We will also examine the effects of THC, CBN and CBD on the function of hypothalamic catecholaminergic neurons which are known to control the release of gonadotropins and prolactin from the anterior pituitary. These studies will be conducted in male rats and mice and will involve oral administration of different components of marihuana, measurements of levels and turnover rates of hypothalamic neurotransmitters, incubations of hypothalami and anterior pituitaries, determinations of GnRH receptors in the pituitary and radioimmunoassay measurements of blood levels of pituitary and testicular hormones. Copulatory behavior will be tested in the presence of ovariectomized females brought into sexual receptivity by estrogen and progesterone injections. Further studies will involve administration of blockers and precursors of hypothalamic neurotransmitters in an attempt to mimic or prevent cannabinoid effects on behavior and pituitary function as well as cuts, lesions and stimulations of specific sites in the brain aimed at localizing the sites of cannabinoid action and the anatomical pathways of their effects.

Most species have evolved reproductive strategies to insure that offspring are born during a time of year that provides maximum survival potential. Length of day supplemented by other environmental cues are used to predict these times. Although much progress has been made in understanding how photoperiodic information is perceived by animals, the tranduction of this information to hormonal signals that control reproductive function is still not well understood. The overall objectives of these studies is to identify brain mechanisms by which daylength influences the release of pituitary hormone that in turn regulate reproductive function. The golden hamster becomes reproductively active in the long days of summer and reproductively quiesent during winter. Natural changes in length of the light and dark portions of the day (less the 12.5h of light secretion rates of pituitary) hormones. Then, he will use drugs that affect specific chemical (neurotransmitter) systems in the brain to see if he can mimic or block the affects of daylength on hormone secretion and reproductive activity. The pineal gland, a small structure within the brain, is important in mediating the effects of daylength on reproductive function. The action of melatonin, the principal hormone of the pineal gland, on neurotransmitters and hormone secretion is not understood. He plans to study the effects of melatonin administration or removal of the pineal gland on the metabolism of brain neurotransmitters and hormone secretion. The results of these experiments should have important implications for understanding the control of reproductive function by both internal and external factors. Although he is specifically studying affects of daylength on reproductive function, the conclusions based on these studies are applicable to our understanding of how a variety of external stimuli such as food affect reproductive function.

Development of methods for mass production of recombinant growth hormone (GH) and for introduction of GH genes into mammalian embryos is certain to lead to widespread use of GH in medicine and agriculture. In this context it is important to understand the effects of chronic GH exposure on neuroendocrine function. Transgenic mice which express human or bovine GH genes exhibit various abnormalities in the function of catecholaminergic neurons in the hypothalamus, in the release of several hormones (including prolactin [PRL] and luteinizing hormone [LH]) from the pituitary and in female and male fertility. The broad objectives of the proposed studies are (i) to elucidate the mechanisms of GH action on the hypothalamic-adenohypophyseal function in transgenic animals, and (ii) to identify domains of the GH gene responsible for the various effects of chronic GH excess on neuroendocrine function and fertility. We will examine turnover rate, binding, uptake and release of dopamine (DA) and norepinephrine (NE) in various regions of the hypothalamus in transgenic mice expressing bovine GH (bGH) or human GH (hGH) genes and in normal mice treated with bGH. We will also examine hypothalamic secretion of LHRH and pituitary secretion of LH, FSH and PRL in these animals and relate the observed changes to gonadal function and fertility. The suspected cause:effect relationships will be probed with various adrenergic and dopaminergic agonists and antagonists in vivo and in vitro. Site-directed in vitro mutagenesis will be used to systematically convert designated segments of the bGH gene to the hGH gene and vice versa. These mutant genes will be combined with mouse metallothionein-I promoter and used to produce transgenic mice. Studies of neuroendocrine function in these animals will relate specific changes in the hypothalamic-hypophyseal function to defined domains of the GH gene. Results of these studies will produce new information on the mechanisms of GH action, on hypothalamic changes responsible for endocrine abnormalities of transgenic animals expressing various GH genes and on the functions of specific domains of the GH gene and by implication, the GH molecule. This information is needed for rational use of recombinant GH and of transgenic technology in medicine and agriculture.

The long-term objective of this research is to elucidate multihormonal mechanisms which control male reproductive functions. During the next five years we will examine the role of growth hormone (GH) in this process and, specifically, the effects of endogenous GH on the release of gonadotropins and prolactin (PRL). We will neutralize endogenous GH by injection of specific high titer antisera or by administration of somatostatin and examine the effects of these treatments on plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and PRL, on the turnover of catecholamines in different regions of the hypothalamus, and on hypothalamic and pituitary function in vitro. Using similar approaches, we will evaluate the effects of GH treatment in genetically GH-deficient and in somatostatin-treated animals on the hypothalamus and the pituitary. The suspected cause:effect relationships between the changes in hypothalamic neurotransmitters and in pituitary hormonal release will be probed using various agonists, antagonists and precursors of dopamine and norepinephrine. Incubations and perifusions of hypothalamic and pituitary tissue with GH and insulin-like growth Factor-I (IGF-1) will be used to distinguish between direct and IGF-1 mediated actions of GH and to identify the site(s) of GH action. Additional experiments will address the suspected role of GH in the control of gonadotropin release by gonadal steroid feedback and in 'sexual maturation. Methods used for this research will include generation and characterization of antisera, radioimmunoassay measurements of pituitary and hypothalamic hormones, high performance liquid chromatography measurements of brain dopamine, norepinephrine and their metabolites, neuropharmacological studies, microdissection of brain tissues and breeding of mutant animals with hereditary GH deficiency. The results will answer whether physiological actions of GH include control of LH, FSH and PRL release and identify the mechanisms involved. This information is important for assessing the potential for undesirable side effects of GH treatment in medicine and in the animal industry.

The 1994 Gordon Research Conference on Prolactin will provide a forum for presentation and thorough critical discussion of the most recent developments in the study of prolactin (PRL) and other hormones of the PRL-growth hormone family. Invited lectures will cover topics including regulation of PRL gene expression, PRL and GH receptors, local synthesis and actions of PRL in the brain, novel effects of PRL and mechanisms of PRL action, placental lactogens, conditions of PRL excess and PRL deficiency, use of site directed in vitro mutagenesis to identify functional domains of PRL and GH molecules, and novel findings on regulation of PRL release. Data obtained in different species ranging from non-mammalian vertebrates to the human will be presented. In addition, all participants will have an opportunity to present their recent findings in poster sessions. In this application, we are requesting funds for partial coverage of travel and living expenses for 10 graduate students or postdoctoral fellows wishing to attend the Conference and present a poster. We are also requesting funds for covering the expenses of 4 invited speakers.

The overall objective of this pilot project is to determine whether transgenic mouse lines expressing various human and bovine growth hormone (GH) genes may serve as a model system for studies of the neuroendocrine determinants of susceptibility to ethanol dependence. The rationale for the proposed studies is based on the observation that transgenic mouse lines under investigation in this laboratory differ significantly in the metabolism of forebrain dopamine, a substrate which has been implicated in substance abuse in humans, including alcohol dependence. Of particular interest is to observation of opposite effects of different GH genes on hypothalamic dopamine transmission. The proposed research project encompasses three specific aims: (1) To determine whether transgenic mouse lines expressing various GH genes and differing in dopaminergic function differ in ethanol self-administration and in the plasma levels of ethanol they obtain in a two-bottle choice situation; (2) to determine whether these mouse lines also differ in dopamine agonist-induced locomotor activity; and (3) to assess the degree to which susceptibility to ethanol self-administration in these mouse lines is related to dopamine agonist-induced locomotor activity. If transgenic mouse lines differ in these behaviors as expected, this will constitute evidence supporting the hypothesis that genetically altered central dopaminergic function contributes to susceptibility to ethanol self- administration. Furthermore, it will provide the basis for future studies of the involvement of forebrain dopaminergic systems in ethanol dependence using transgenic mice as a model system. These studies will include neuropharmacological experiments to identify cause-effect relationships between changes in dopaminergic activity and behavior, as well as site-directed in vitro mutagenesis to identify specific features of the GH gene and GH molecule that are responsible for the observed effects.

In a recent study of life span in mice with hereditary dwarfism (Ames dwarf mice, df/df), we have discovered that dwarf animals live longer that their normal siblings. This difference is large (greater than 250 days for males and greater than 1 year for females) and highly significant (P less than 0.001). More than 1/2 of female dwarf animals survive to 3 1/2 years of age. The objective of this pilot project is to characterize of Ames dwarf mouse as a novel model for aging research. Specific aims include (i) verifying our initial observations in a large number of mice (100 dwarfs and 100 normals), (ii) pathological studies at gross and microscopic level directed at identifying the cause(s) of death in dwarf and normal animals, and (iii) studies of the effects of growth hormone (GH), thyroid hormone (thyroxine, T4) and prolactin (PRL) on life span of Ames dwarf mice. Studies of the effects of GH, T4, and PRL will test the hypothesis that greater life expectancy of Ames dwarf mice may be specifically related to known hormonal deficits of these animals and may provide some clues as to the mechanisms involved. The proposed studies will involve crossing dwarf mice to transgenic animals expressing GH, under control of the metallothionein promoter, to produce animals in which hereditary GH deficiency is compensated for by transgene expression. Other dwarf animals will receive transplants of pituitaries from normal donors to provide PRL replacement.

Both growth hormone (GH) and the main mediator of its actions, insulin-like growth factor-I (IGF-I) can affect reproductive development and function. However, the multiple interactions between the GH-IGF-I axis and the hypothalamic-pituitary-gonadal (H-P-G) axis are poorly understood. In particular, the specific roles of direct actions of GH and IGF-I present in the systemic circulation versus the role of local IGF-I production in the control of puberty and adult reproductive functions remain to be delineated. Mice with GH resistance due to targeted disruption ("knock-out, KO) of the GH receptor gene (GH-R-KO mice) have delayed puberty and quantitative deficits in adult reproductive function, although most males and some females can reproduce. In contrast, IGF-I-KO mice with complete IGF-I deficiency have underdeveloped reproductive system and are sterile. This major difference between the effects of the disruption of the IGF-I gene and the GH-R gene implies differential role of GH dependent vs. GH-independent IGF-I production. We propose to utilize the GH-R-KO mice as a model system for identifying the role of GH-dependent IGF-I production in the control of sexual maturation, and for more clearly defining the role of GH-dependent (presumably brain and gonadal rather than hepatic) IGF-I in this process. We have already determined that administration of recombinant human IGF-I will advance the age of vaginal opening in GH-R-KO mice. In the proposed studies, we will determine whether exogenous IGF-I will advance the age of first ovulation in these GH-resistant mice, and will characterize the impact of GH resistance on the expression of IGF-I and IGF-I receptor (IGF-IR) in the brain, ovaries, and testes in pre-pubertal GH-R-KO mice. In addition, we will determine the impact of GH resistance on the time course of changes in the expression of IGF-I and IGF-IR and in the function of the H-P-G axis during spontaneous puberty and during acceleration of pubertal development by treatment with IGF-I. Results of these studies will indicate whether systemic IGF-I can influence development and function of the local IGF-I systems in the hypothalamus and in the gonads during sexual maturation.

Our long-term goals are to elucidate the role of hormonal (insulin/IGF-1) signaling in the control of aging and longevity in mammals and to determine how nutritional intake interacts with longevity genes. We have shown that caloric restriction (CR) increases longevity in the Ames dwarf mouse, a long-lived hypopituitary mutant, but surprisingly fails to extend longevity in another long-lived mutant, the growth hormone (GH) resistant, GH receptor knockout (GHRKO) mouse. Sensitivity to injected insulin was improved by CR in normal and in Ames dwarf mice but not in GHRKO animals. We hypothesize that failure of CR to prolong life in GHRKO mice is due to extremely low insulin levels and the inability of CR to further increase insulin sensitivity in these animals. We further hypothesize that stress resistance is related to insulin sensitivity and that CR will fail to improve stress resistance in GHRKO mice. These hypotheses will be tested by determining whether pancreatic islet-specific expression of IGF-1 that was shown to improve glucose tolerance and insulin secretion or replacement therapy with IGF-1 or insulin will normalize responses to short-term CR in GHRKO mice. Effects of these treatments will be assessed by measuring insulin and glucose tolerance, expression of insulin-related genes in the liver, skeletal muscle and heart (the "molecular signature" of responses to CR), resistance of cultured skin fibroblasts and intact mice to variousstressors, measurements of body core temperature (Tco) and study of other parameters related to insulin action and stress resistance. Subsequently, we will use therapy that most closely "normalizes" the responses to short- term CR in GHRKO mice to determine whether it will also result in GHRKO mice regaining the ability to respond to chronic CR by increasing longevity. We will also examine effects of CR in the recently developed Ghrh-KO mice with isolated GH deficiency and in transgenic mice expressing GH antagonist. Collectively, these studies will identify the effects of CR on insulin signaling and stress resistance that are consistently associated with prolonged longevity across a variety of genotypes. The results will provide novel insights into the mechanisms of anti-aging actions of CR and into the relationship of insulin level and actions to stress resistance, aging and longevity, an issue of major significance in public health in the context of the current "epidemic" of insulin resistance and metabolic syndrome.

DESCRIPTION (provided by applicant): The Seventh International Symposium on Neurobiology and Neuroendocrinology of Aging will be held in Bregenz, Austria July 18 -24, 2004. The purpose of this meeting is to bring together a group of investigators who made recent significant advances in the study of age-related alterations in the function of the central nervous system (CNS); neurodegenerative disease; neuroprotective mechanisms, endocrine changes in the aging individual, and endocrine replacement. We purposely include a wide range of topics and assemble a group of scientists that may not normally attend the same meeting. The format of the meeting is modeled after Gordon Research Conferences and encourages formal and informal discussions of the material presented. Proceedings will be published as a special issue of Experimental Gerontology within approximately six months after the Symposium. Participation of junior investigators, including graduate students and post-doctoral fellows is strongly encouraged and this proposal seeks funds for providing stipends to those US participants who are in training and who are planning to present their findings in the poster session. We are also seeking support for the presenters of the Special Lecture and the Keynote Lecture who will be traveling to Bregenz from the United States.

[unreadable] DESCRIPTION (provided by applicant): The 34th Annual Meeting of the American Aging Association, combined with the 19th Annual Meeting of the American College of Clinical Gerontology will be held in San Francisco, CA, June 3-6, 2005. The theme selected for the 2005 meeting of our organizations is "Aging, Mechanisms and Prevention". What we hope to accomplish is to bring together a group of scientists working in experimental gerontology, to review and discuss recent developments in this field with special emphasis on the regulation of aging and longevity in mammals and on studies aimed at delay on prevention of human aging and age-related disease, and to provide forum for presentation of new research data and for in-depth discussion of the presented material. The program will consist of invited lectures arranged into eight sessions, each consisting of three presentations and ample time for discussion, platform and poster presentations of material submitted by the registrants, and two special lectures. The purpose of the special lectures will be to provide a practical, clinical perspective and thus help the participants to view laboratory findings in the context of key issues in geriatric medicine. Topics selected for coverage by invited lectures include genetic and cellular mechanisms of aging, role of IGF-1 signaling, caloric restriction in the human, genetics of human longevity, brain aging and novel therapies for neurodegenerative disease, and use of genomics and proteomics in gerontological research. Financial assistance will be provided to those in training to encourage their participation in the meeting. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The Eighth and Ninth International Symposia on Neurobiology & Neuroendocrinology Aging will be held in Bregenz, Austria July 23-28, 2006 and in July 2008. The purpose of these meetings is to bring together a group of investigators who made recent significant advances in the study of mechanisms of aging, with particular emphasis on CNS aging, neuroprotection, detection and treatment of neurodegenerative disease. Specific topics to be covered in 2006 will include the role of damage of mitochondrial and chromosomal DNA, autophagy, insulin resistance, advanced glycation products, stress resistance, leptin, chaperones, and prions, neurogenesis and neuroprotective effects of TGF beta, IGF-1 isoforms, and estrogens, as well as [unreadable] novel approaches to early detection and treatment of Alzheimer's disease. In addition, new data on genetics of aging in C. elegans and on aging in an exceptionally short-lived vertebrate species will be presented. We purposely include a wide range of topics and assemble a group of scientists that may not normally attend the same meeting. The format of the meeting is modeled after Gordon Research Conferences and encourages formal and informal discussions of the material presented. Proceedings will be published as a special issue of Experimental Gerontology within approximately 6 months after the Symposium. Participation of junior investigators, including graduate students and post-doctoral fellows is strongly encouraged and this proposal seeks funds for providing stipends to those US participants who are in training or are in junior faculty positions and who are planning to present their findings in the poster session. We are also seeking partial support for the expenses of invited speakers from the United States. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to identify mechanisms responsible for the effects of growth hormone (GH) signaling on aging and longevity in mammals. Studies conducted by the applicants during the last 10 years, together with results obtained in other laboratories, have provided unequivocal evidence that GH deficiency and GH insensitivity in mice are associated with a marked increase in mean and maximal longevity. Studies of age-sensitive traits and analysis of survival characteristics in GH-deficient and GH-resistant mice indicate that increased longevity of these animals is coupled with and most likely due to a delay and/or deceleration of the process of biological aging. Our collaborative studies and work of other investigators defined a series of mechanisms that might, in principle, link GH signaling with longevity. In the proposed work we will use both well-characterized and novel mouse mutants to probe several of these specific mechanisms. Our aim is to develop a compelling model of the pathway or pathways that connect diminished GH or IGF-I signaling to extended longevity and to relate these pathways to maintenance of cognitive and physical function. The proposed collaborative studies will define the role of liver, adipose tissue and muscle in mediating the effects of GH and IGF-I on age-sensitive traits and longevity;and to elucidate the interactive effects of altered insulin signaling, adipocyte secretory profiles and stress resistance in mediating these effects. Project 1 will relate somatotropic and insulin signaling to stress resistance and aging. Project 2 will produce novel animals lacking the GHR/GHBP gene only in the liver, white adipose tissue or muscle. Project 3 will study mechanisms of stress resistance in fibroblasts and pre-adipocytes and evaluate longevity and age-sensitive traits in novel mutant mice developed in Project 2. Project 4 will characterize pre-adipocyte utilization, characteristics and senescence in mutants with altered GH signaling. Core A will coordinate research, information exchange and communication with Advisory Committee, develop shared data base and provide statistical support. Core B will assess gross and microscopic pathological changes with emphasis on novel mutants developed by Project 2. Improved understanding of the role of somatotropic and insulin signaling in the control of mammalian aging is needed for designing lifestyle and pharmacological interventions to maintain health and functionality during human aging. This information is also needed to develop rational basis for exploring or discouraging the use of human GH as an anti-aging agent. RELEVANCE: Improved understanding of the role of somatotropic and insulin signaling in the control of mammalian aging is needed for designing lifestyle and pharmacological interventions to maintain health and functionality during human aging. This information is also needed to develop rational basis for exploring or discouraging the use of human GH as an anti-aging agent. PRINCIPAL INVESTIGATOR: Dr. Bartke is Professor and Director of Geriatric Medicine in the Department of Internal Medicine at Southern Illinois University School of Medicine. His many years of experience studying the role of GH and IGF-I in mice brings a strong knowledge of endocrinology to this PPG. Dr. Bartke has developed a significant publication record in understanding animal models of aging and how manipulation of the growth hormone/IGF-I axis is related to extended longevity. His laboratory was the first to identify the link between the GH/IGF-1 signaling and longevity in the mouse. Dr. Barke has had substantial administrative experience and is very appropriate to be principal investigator of this PPG. CORE A - ADMINISTRATION;Dr. Andrzej Bartke, Core Leader (CL) DESCRIPTION (provided by applicant): A Program Project of this magnitude will require the coordination of personnel and technical expertise. The main functions of the Administrative Core will be to facilitate communication between the five laboratories involved in the proposed studies. This will entail development and maintenance of a shared database, statistical consultations, the solicitation of input from the Advisory Committee, and organization of an annual meeting of all participants in the Program Project. In addition, Core A will assist in issues related to the budget, distribution of animals and tissues, maintain regular contact with the Project Officer at NIA, and prepare annual progress reports/renewal applications. In Years 04 and 05, Core A will coordinate the planning of future directions of this Program Project and oversee preparation of the application for competing renewal. Five Specific Aims are proposed: 1. To facilitate communication between all participating investigators, all members of the participating laboratories including graduate students and postdoctoral fellows, and investigators and Advisory Committee. 2. To establish and maintain data management system that will facilitate exchange of samples and data between the participating laboratories and maximize utilization of every animal. 3. To provide statistical support and consultations for experimental design and data analysis, and to identify and recommend statistical approaches for combined analysis of data from different Projects/Cores. 4. To provide assistance and oversight of fiscal management of projects and Core B and of exchange of animals, tissues and other samples (plasma, tissue extracts, mRNA or cDNA preparations) between laboratories. 5. To prepare annual progress reports and coordinate preparation of a competing renewal application.

Our long-term objective is to elucidate the role of growth hormone (GH) in the control of aging. In mice, reduced somatotropic (GH / IGF-1) signaling leads to major increases in lifespan that are associated with various indices of delayed aging. We have proposed that reduced insulin release and enhanced insulin sensitivity are particularly important in mediating the effects of GH on aging. Our studies suggest that enhanced stress resistance is also involved. In the proposed studies, we will relate somatotropic signaling to insulin signaling in different tissues, stress resistance, physical function, healthspan and lifespan. We will use both well-characterized and novel models of whole animal or tissue-specific suppression of GH action, and pharmacological alterations of circulating GH levels or activity. The physiological role of GH at various stages of life history and its potential utility in geriatric medicine are poorly understood and controversial. To begin to address this issue, we will compare effects of GH in young, adult and aging animals. The following specific aims are proposed: 1: To determine the effects of GH replacement in hypopituitary mice on insulin signaling in different organs and cellular resistance to multiple forms of stress. 2: To determine the effects of liver-specific, adipose tissue-specific and muscle-specific deletion of GH receptor on insulin signaling, and expression of insulin-related genes in differentorgans. 3: To compare effects of adipocyte-specific GHR deletion to effects of surgical removal of visceral fat depots. 4: To compare the effects of replacement therapy with GH in young, middle-aged and old Ames dwarf mice. 5: To determine whether treatment of genetically normal mice with insulin sensitizers or a GH antagonist will improve stress resistance, promote maintenance of cognitive and physical function, health and vigor, and increase longevity. The results will identify alterations in the somatotropic axis and the importance of insulin signaling in the liver, muscle and adipose tissue that relate to enhanced stress resistance, improved cognition and increased healthspan and lifespan. RELEVANCE (See instructions): There is increasing evidence that the relationships of GH, IGF-1 and insulin to aging that were discovered in mice also apply to the human and, specifically, to the risk of age-related disease and to exceptional longevity. The results of the proposed research are expected to assist in development of interventions that may slow human aging and the associated functional decline and to provide factual basis for assessment of the potential utility of GH in geriatric medicine.

A Program Project of this magnitude will require the coordination of personnel and technical expertise. The main functions of the Administrative Core will be to facilitate communication between the five laboratories involved in the proposed studies. This will entail development and maintenance of a shared database, statistical consultations, the solicitation of input from the Advisory Committee, and organization of an annual meeting of all participants in the Program Project. In addition, Core A will assist in issues related to the budget, distribution of animals and tissues, maintain regular contact with the Project Officer at NIA, and prepare annual progress reports/renewal applications. In Years 04 and 05, Core A will coordinate the planning of future directions of this Program Project and oversee preparation of the application for competing renewal. Five Specific Aims are proposed: 1. To facilitate communication between all participating investigators, all members of the participating laboratories including graduate students and postdoctoral fellows, and investigators and Advisory Committee. 2. To establish and maintain data management system that will facilitate exchange of samples and data between the participating laboratories and maximize utilization of every animal. 3. To provide statistical support and consultations for experimental design and data analysis, and to identify and recommend statistical approaches for combined analysis of data from different Projects/Cores. 4. To provide assistance and oversight of fiscal management of projects and Core B and of exchange of animals, tissues and other samples (plasma, tissue extracts, mRNA or cDNA preparations) between laboratories. 5. To prepare annual progress reports and coordinate preparation of a competing renewal application. RELEVANCE (See instructions):

DESCRIPTION (provided by applicant): It is well established that developmental events can act as determinants of adult disease. For example, maternal undernutrition, as well as overnutrition, represent risk factors for development of various components of metabolic syndrome in the offspring. Yet it is unknown whether (or to what extent) early growth and development influence mammalian aging. On the basis of preliminary data derived from hormonal replacement therapy in long-lived mutants, we propose that the period of rapid pre- and post-weaning growth may represent a critical "time window" for the effects of early hormonal milieu on healthspan and lifespan. To test the validity of this novel concept and to begin identifying the underlying mechanisms, we will determine whether a defined (six week) period of hormonal therapy, started at different stages of postnatal development, can influence phenotypic characteristics associated with longevity and whether the hormone-induced alterations in these characteristics will persist after the treatment is stopped. To test the hypothesis that the period of rapid postnatal growth represents a critical time window for development of metabolic characteristics that influence (and likely predict) aging, the following specific aims are proposed: 1. To determine the effects of treating long-lived hypopituitary Prop1df (Ames dwarf) mice with growth hormone (GH) or with a combination of hormones starting at one week, two weeks or two months of age on oxygen consumption (VO2), respiratory quotient (RQ), body temperature and expression of genes related to insulin action, fat and carbohydrate metabolism. 2. To determine whether hormone-induced changes in VO2, RQ, body temperature, insulin signaling and gene expression persist after the treatment is stopped. 3. To determine the effects of early treatment with a combination of GH, prolactin and thyroxine as compared to treatment with GH alone on adipokine levels, insulin and glucose tolerance and other characteristics associated with extended longevity of Ames dwarf mice. The results will begin to fill the gap in the present understanding of the developmental influences on aging and set the stage for addressing broader questions of major public health significance, e.g.: How does nutrition and nutrition-related endocrine signaling during different stages of development affect growth and ultimately healthspan and lifespan? What is the relationship of key metabolic parameters in young adults to aging and longevity? And what early lifestyle and/or pharmacological interventions could effectively increase healthspan and life expectancy? PUBLIC HEALTH RELEVANCE: There is considerable evidence that growth hormone (GH) and other hormones that stimulate growth are also importantly involved in the control of aging and longevity. Our recent findings indicate that the actions of GH early in life (starting before weaning and continuing to the age of puberty) influence life expectancy. In the proposed studies we will use normal as well as long- lived mutant mice, and therapy with GH or with a combination of hormones to elucidate this novel and somewhat unexpected effect. We will determine at which stages of early postnatal life hormone levels influence metabolic characteristics that differ in normal and long-lived mice and thus may predict longevity. We will also determine which of these hormone-induced changes persist after hormone treatment is stopped. Because nutrition has major effects on the level of various hormones, understanding the relationships between early hormone action and aging is important for discovering how aging can be postponed and life expectancy improved by judicious modifications of the diet during rapid pre-pubertal growth.

DESCRIPTION (provided by applicant): Prenatal developmental events can influence adult phenotype and the risk of adult disease. However, it is unknown whether (or to what extent) the actions of hormones and nutrients on early postnatal growth can influence mammalian aging, adult health and longevity. We have recently shown that: (i) the remarkable extension of longevity in hypopituitary Ames dwarf mice can be completely reversed by a six-week course of growth hormone (GH) injections started at the age of 2 weeks; and (ii) limiting nutrient availability during the first three weeks of postnatal life by increasing the numbers of pups in a litter increases both median and maximal lifespan in genetically normal mice. Based on these novel and largely unexpected findings, we hypothesize that the period of rapid postnatal growth represents a critical time window for development of lifelong metabolic and other phenotypic characteristics that influence-and likely predict-longevity and functionality during old age. To test the validity of this hypothesis, we will examine the impact of modestly reducing the amount of available nutrients during suckling (or suckling and the immediate post-weaning period) in normal mice, and the effects of early (starting at one or two weeks) as compared to late (starting at eight weeks) GH replacement therapy in Ames dwarfs and in GHRH-/- mice with isolated GH deficiency. Endpoints will include both longevity and a series of age-sensitive traits related to health span, including indices of immune function, locomotion, stress resistance, and cataract development. We will pay special attention to endpoints related to fuel metabolism, with tests of somatotropic and insulin signaling, pancreatic beta cell function, metabolic rate and respiratory quotient, and expression of GH and insulin-related genes in the liver, skeletal muscle, and both subcutaneous and intra-abdominal (visceral) fat. Lastly, we will in parallel studies see whether early life nutrient limitation, before or before and after weaning, will benefit GHRKO (Laron dwarf) mice, which unlike Ames dwarf mice fail to benefit from calorie restriction (CR) when CR is started at the age of 2 months. Results of the proposed studies will be used to accept or reject specific hypotheses concerning mechanisms by which early hormonal or nutritional interventions can influence aging and longevity. In sum, we will take our studies of the interactions of longevity genes and CR in a novel direction to address the impact of transient changes in nutritional and hormonal signals during early postnatal life on mammalian aging, health span and longevity. Results of these studies will address critical gaps in the present understanding of developmental influences on aging and set a stage for addressing the relationships between early nutrition, growth and adult health which are of major public health significance.

Abstract Although the influence of metabolic rate on longevity has been studied and debated for decades, the cause:effect relationship between different parameters of energy metabolism and the process of aging is poorly understood and controversial. Hypopituitary Ames dwarf (Prop1df) mice and growth hormone receptor deleted (GHRKO) mice are remarkably long-lived and exhibit many features of delayed, slower and healthy aging. Many phenotypic characteristics shared by these mutants are believed to represent mechanisms of extended longevity. These characteristics include increased oxygen consumption (VO2) per gram of total or lean body mass and reduced respiratory quotient (RQ = respiratory exchange ratio, RER), indicative of increased reliance on lipids as metabolic fuel. Intriguingly, the increase in VO2 and the reduction in RQ in Ames dwarf and GHRKO mice disappear or are severely attenuated after 24 hours of acclimation to thermoneutral temperature. From these novel findings, we suspect that increased heat loss and the consequent increase in energy demand for thermogenesis in these diminutive mutants induce alterations in energy metabolism (increased VO2 and fatty acids ? oxidation; reduced RQ) that promote delayed, healthy aging and extended longevity. We hypothesize that chronic exposure of Ames dwarf and GHRKO mice to thermoneutral temperature will accelerate aging and reduce or eliminate their longevity advantage. We further hypothesize that chronic activation of thermogenesis in genetically normal (?wild type?) animals by exposure to reduced environmental temperature will induce metabolic characteristics that are associated with the delayed aging and extended longevity seen in GH-related mutants and thus lead to a slower rate of aging. As the first step in testing these hypotheses, we will determine whether characteristics associated with delayed aging are normalized by extended (weeks to months) exposure of long-lived mutant animals to thermoneutral environment and whether they can be induced by extended exposure of normal animals to reduced environmental temperature. Two Specific Aims are proposed. Specific Aim 1. To determine whether chronic exposure of adult Ames dwarf and GHRKO mice to thermoneutral ambient temperature (30°C) will rescue (normalize) energy metabolism (as assessed by measurements of V02, RQ), glucose homeostasis, circulating adiponectin, IL-6 and lipid levels and expression of genes related to mitochondrial uncoupling in brown and white adipose tissue, metabolism, inflammation, insulin and mTOR signaling. Specific Aim 2. To determine whether chronic exposure of adult normal (?wild type?) mice from the same strains to reduced environmental temperature (17°C) will induce alterations in thermogenesis, energy metabolism, glucose homeostasis, adipokine levels and expression of selected genes resembling those observed in long-lived GH-related mutants exposed to standard animal room temperature (23°C).

Abstract It is well known that the risk of adult disease can be altered by factors acting during development, both pre- and postnatally. Recent studies in our laboratory have shown that hormonal intervention limited to six weeks during early postnatal life can alter adult metabolic characteristics and determine how long an animal will live. However, the potential of early life interventions to slow the rate of aging and reduce the risk of age- related chronic diseases has not been adequately explored. The proposed HYPOTHESIS is that interventions during rapid pre- and peri-pubertal growth can produce permanent beneficial changes in carbohydrate, lipid, and energy metabolism, thus promoting healthy aging. This hypothesis will be tested using two pharmacological and one environmental intervention which have already been shown to improve glucose homeostasis in adults and to be well tolerated by juveniles. In three SPECIFIC AIMS, juvenile mice will be treated with metformin, a drug with multiple beneficial effects in adults with type 2 diabetes or with MSI-1436, a novel experimental anti-obesity and anti-diabetic drug, or exposed to a modest reduction of environmental temperature, a safe, cost free and readily translatable intervention. The effects of these three interventions will be tested on glucose homeostasis, energy metabolism, body composition, physical endurance, responses to nutritional stress (high fat diet), and gene expression. Using RT-PCR, multiple tissues will be analyzed for expression of genes mechanistically related to healthspan and longevity, including those related to insulin, GH, IGF-1, mTORC1 and mTORC2 signaling, glucose and lipid metabolism, and inflammation. Unbiased RNA-Seq studies of adipose tissue will also be conducted to search for novel mechanisms linking early life interventions and adult phenotype and aging. Separate cohort of animals from each treatment group will be used for studies of glucose homeostasis, energy metabolism, body composition, and physical endurance at the ages of 24 and 30 months and for determination of longevity. The results will determine whether simple and safe lifestyle interventions during childhood and adolescence can promote extension of healthspan and lifespan and begin to identify mechanisms of developmental programing of mammalian aging and potential targets for pharmacological interventions. Three Specific Aims are proposed: Specific Aim 1: To determine the impact of early life treatment with metformin on adult metabolic characteristics causally related to aging. Specific Aim 2: To determine the impact of early life treatment with MSI-1436 (an experimental anti- obesity drug) on adult metabolic characteristics causally related to aging. Specific Aim 3: To determine the impact of a mild reduction of environmental temperature during early life on adult metabolic characteristics causally related to aging.