Malcolm J. Low, M.D., Ph.D - US grants

somatostatin; gene expression; peptide hormone biosynthesis; genetic manipulation; protein sequence; nucleic acid probes; median eminence; neuropeptides; gene therapy; hypothalamus; transfection; metallothionein; developmental neurobiology; tissue /cell culture; laboratory rabbit; laboratory mouse; radiotracer; microinjections; laboratory rat; aminoacid analyzer;

Cushing's disease is generally caused by benign corticotroph adenomas. Although a relatively uncommon condition, Cushing's diesease is accompanied by significant morbidity and mortality due to the excessive secretion fo ACTH and subsequenct hyperplasia of the adrenal glands. The long-term objectives of this proposal are to understand the regulation of proopiomelanocortin (POMC) gene expression in corticotrophs and define the pathogenesis of corticotroph adenoma formation. The first specific aim is to identify the cis-acting DNA elements of the rat POMC gene that are necessary and sufficient for tissue-specific expression in corticotrophs, melanotrophs, and particular neurons. This study will be accomplished by analyzing the cellular expression of deleted rat POMC genes in transgenic mice. To address the question of whether chronic corticotropin-releasing factor (CRF) secretion can cause hyperplasia and ultimately coricotroph adenomas, excess CRF will be produced in both transgenic mice and transkaryotic rats. Transkaryotic rats will harbor transplantable medullary thyroid carcinoma cells that have been stably transfected with a CRF-expression vector. The final specific aim is to determine whether constitutive expression of proto-oncogenes in corticotrophs can cause pituitary adenomas. The c-myc and c-fos proto-oncogene coding sequences will be fused to corticotroph-specific transcriptional regulatory elements from the POMC gene. These fusion genes will then be introduced into the germ line of transgenic mice and their pituitary glands examined histologically for the development of either corticotroph hyperplasia or neoplasia. Insights gained from these studies may be applicable to understanding the biology and pathogenesis of other benign endocrine tumors.

A genetic approach will be taken to investigate cell specification in the embryonic pituitary. A transgenic mouse will be made in which expression of the thymidine kinase gene will be directed to melanotropes and corticotropes using the promoter of the pro-opiomelanocortin gene. Cells that express the transgene will be ablated and the consequences of this ablation on the differentiation of other pituitary cell types, repopulation of the pituitary with pro-opiomelanocortin cells and re-differentiation of the hypothalamic-pituitary axis will be determined. These studies offer an innovative, non-invasive approach toward understanding the role of cell-cell interactions in neural development and specification.

The gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) are heterodimeric pituitary glycoprotein hormones that directly regulate gametogenesis and gonadal steroid hormone biosynthesis in the testis and ovary. Transcription of the gonadotropin subunit genes, and biosynthesis, assembly, and secretion of the biologically active hormones are precisely controlled by the hypothalamic neuropeptide gonadotropin releasing hormone (GnRH), and gonadal steroid hormones and peptides. Altered regulation of gonadotropin production is a major cause of infertility. Conversely, the selective control of gonadotropin production has implications for both the treatment of infertility disorders and the development of effective novel contraceptives. The major goal of this project is to understand at the molecular level the DNA components and transcription factors that are responsible for the gonadotroph-specific expression and hormonal regulation of the FSHbeta subunit gene. DNA elements will be coarsely mapped initially by a deletional and mutational analysis of the human and mouse FSHbeta genes expressed in the pituitary glands of transgenic mice. Gonadotroph-specific expression will be confirmed by histological techniques and the hormonal regulation of the transgenes will be studied by analysis of FSHbeta mRNA levels with species' specific probes and by radioimmunoassay of the expressed FSH. Standard reproductive endocrinological experiments will be performed using transgenic mice and hpg hypogonadal mice genetically crossed with the hpg mice. A comparison of functionally important sequences conserved between human and mouse will direct future fine scale mapping of the core DNA regulatory elements. The mechanism of androgen inhibition of the human FSHbeta subunit gene at the pituitary level will be studied by a combination of in vivo nuclear run on studies and in vitro DNA-protein binding reactions using purified androgen receptor and competition experiments with known positively regulated androgen response elements. Inhibin and activin regulation of hFSHbeta will be studied using perfused columns of primary pituitary cell cultures derived from transgenic mice. Finally, we will attempt to derive a continuous, well differentiated gonadotroph cell line from pituitary tumors induced in transgenic mice with a temperature sensitive simian virus 40 large T antigen oncogene. Alternatively we will attempt to immortalize gonadotrophs using a powerful papilloma virus oncogene carried in a retroviral vector or by the expression of other oncogenes in the gonadotrophs of transgenic mice. A beta-subunit expressing gonadotroph cell line would be invaluable for further functional analysis of regulatory elements in the gonadotropin subunit genes, to characterize gonadotroph-specific transcription factors, and to explore the physiological mechanisms underlying the coordinated regulation of gonadotropin gene expression and the cell biology of gonadotropin biosynthesis and secretion.

The development and coordinated function of the reproductive tract are dependent on the pituitary gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH). These two glycoprotein hormones are composed of a common alpha subunit and differing beta subunits that confer receptor, and therefore biological specificity. Transcriptional regulation of the individual subunit genes in response to a myriad of hypothalamic and gonadal factors plays a central role in the certain forms of human infertility, the design of new contraceptive methods, and the production of gonadotropins from the relatively common "null cell" pituitary adenomas. A detailed analysis of the cis-acting regulatory elements of the gonadotropin beta subunit genes and identification of their cognate nuclear binding proteins has been hampered by the unavailability of appropriate cell lines, however. The overall goal of this proposal is to define the mechanism of cell-specific and hormonally regulated expression of the gonadotropin genes. We will characterize initially the basal and hormonal regulation of human FSH beta gene expression in transgenic mice to test whether the human beta gene is expressed at levels comparable to the endogenous mouse gene and if it is regulated directly by gonadotropin releasing hormone and gonadal steroid hormones. A series of deletional mutants and FSH-reporter fusion genes will be introduced into transgenic mice to identify within 300 bp the FSH beta gene regulatory elements that are required for gonadotrope- specific expression and to determine their overlap with hormonal regulatory elements. We will establish lines of transgenic mice that develop well- differentiated gonadotropin-producing tumors to be used directly as a source of nuclear protein extracts and for the development of an immortalized glycoprotein beta subunit expressing cell line. Conditionally immortalized gonadotropes will be produced using either a temperature- sensitive SV40 T antigen or wild-type T antigen whose expression can be regulated in vitro and by the lac operator/repressor system. A cell line will be used for a more detailed analysis of the cis-acting regulatory elements of the hFSH beta gene and would provide a valuable resource for studying the cell biology of gonadotropin biosynthesis, assembly, and secretion. Finally, a multipronged approach of in vitro and in ovo transcription assays, and expression cloning techniques will be employed to characterize the gonadotrope-specific transactivator of the hFSH beta subunit gene and clone a cDNA encoding the protein. The new reagents resulting from this work will be useful for understanding the coordinated regulation of all the gonadotropin subunit genes in addition to FSH beta.

The mission of the transgenic core is to provide technical expertise, guidance in experimental design, and training opportunities in all aspects of the production of transgenic mice. A core laboratory will prevent the unnecessary and time consuming duplication of effort involved in the development of this technology by multiple investigators. The core will additionally provide better quality control, turn around time, and cost effectiveness than either commercially available services or multiple users within one institution. Projects 1 through 4 will use the core to produce transgenic mice that are essential to addressing a major theme of the Program Project: What are the physiological roles of specific molecular components of the hypothalamic-pituitary-adrenal axis? Project 1 will use transgenic mice that have altered naturietic peptide receptor expression to determine the role of these peptides in the control of ACTH secretion. Project 2 will generate mice with pituitary- specific expression of functional POMC minigenes to genetically "rescue" POMC gene "knock-out" mice produce by homologus recombination. Project 3 will explore the role of pituitary derived beta-endorphin in the stress response by blocking the posttranslational processing of POMC at particular dibasic residues with dominant negative or antisense type transgenes. Project 4 will use the technical expertise and facilities of the core to produce mutant mice carrying either null alleles of the neural-specific MSH receptors produced by homologous recombination in embryonic stem cells or constitutively activated receptors encoded by dominant transgenes. These aims will be accomplished through provision of the following services: 1. Embryo collection, nuclear microinjection, blastocyst injections, and oviduct and uterine transfer of manipulated embryos to generate transgenic mice. 2. Genotyping of transgenic mice by a combination of tail DNA dot-blot, Southern blot, or PCR analysis. 3. Supervision and maintenance of a mouse breeding colony providing inbred FVB mice for embryo donations and including stud and vasectomized males used for the initial production of transgenic embryos, and breeding and pedigree analysis of the transgenic mice. FVB mice from the colony will also be available to all investigators for use as controls in physiology experiments or for tissue harvesting. 4. Assistance in physiology experiments using mice including injections, anesthesia, cannulation, and surgical procedures. 5. Permanent storage of cryopreserved transgenic mouse embryos to protect valuable pedigrees from accidental loss, to minimize the continuous breeding of pedigrees that no longer are immediately needed for experiments, and to provide a potential resource for distribution of selected pedigrees to other qualified investigators outside of the institution.

Acute and chronic stress produce a characteristic array of metabolic, endocrine, and behavioral changes in the mammal. Among these changes are increased secretion of glucocorticoid hormones from the adrenal glands, an inhibition of reproductive function, and a form of endogenous opiate mediated analgesia. In the human numerous disorders including depression, anorexia, alcoholism and chronic diseases are associated with these stress responses. A family of neuropeptides including adrenocorticotrophic hormone (ACTH), alpha melanocyte stimulating hormone (alphaMSH), and beta-endorphin derived from the precursor protein proopiomelanocortin (POMC) are essential chemical mediators of the stress response. POMC is first produced in specialized neurons of the brain and in pituitary cells at critical points in development of the brain and endocrine glands suggesting that PMOC also plays an important role in regulating the growth and differentiation of these tissues. The goal of our project is to understand the physiological role of each of the peptides derived from POMC in the normal development of the neuroendocrine systems and in the regulation of the neuroendocrine responses to stress. To accomplish this goal, we will create loss of function mutations in the POMC gene by homologous recombination in embryonic stem cells and use these cells to subsequently generate chimeric transgenic mice by blastocyst injection. The resulting mice will carry mutant POMC alleles that either block the production of all functional POMC peptides or that result in a selective loss of beta- endorphin(1-31). Homozygous POMC gene mutated mice will be analyzed throughout prenatal development to characterize the effects on neuroendocrine cell proliferation and cytodifferentiation. These developmental studies will focus on the paraventricular and antero- ventral periventricular nuclei of the hypothalamus because they are nodal points of the central circuitry controlling the hypothalamic-pituitary- adrenal and hypothalamic-pituitary-gonadal axes, respectively. The beta- endorphin deficient mice are expected to survive into adulthood and will be used to determine the role of centrally produced beta-endorphin in the modulation of the hypothalamic-pituitary axis under basal and stress conditions and in the expression of stress-induced analgesia. In the future, our molecular genetic approach will also be applied to produce mice carrying POMC alleles mutated in such a way to prevent the production of alphaMSH or mutated alleles for specific POMC peptide receptor subtypes to prove the physiological function of each of these molecular components of the mammalian stress response.

Proopiomelanocortin (POMC) is expressed abundantly in the pituitary gland and arcuate nucleus of the hypothalamus where it undergoes tissue- specific posttranslational processing into multiple biologically active peptides including adrenocorticotrophic hormone (ACTH), beta-endorphin, beta- and gamma lipotropins, and alpha, beta-, and gamma-melanocyte stimulating hormones (MSH). Inappropriate expression of the POMC gene and accompanying secretion of POMC-derived peptides from pituitary adenomas is a hallmark of Cushing's disease. POMC peptides are pleiotropic and regulate the mammalian stress response, modulate neuroendocrine homeostasis, and affect behavior. Importantly, the POMC gene is expressed early in development in both neurons and the pituitary gland suggesting additional roles for beta-endorphin and MSHs in the ontogeny of the nervous system and the hypophysis. The long term objectives of the parent grant and this FIRCA proposal are to understand the molecular mechanisms regulating cell-specific and developmental POMC gene transcription and to determine the physiological function of beta- endorphin in brain and pituitary development. These goals will be achieved by the following four specific aims already described in the parent grant together with two new ones belonging to the FIRCA grant. 1) Production of melanotrophic cell lines derived from transgenic mice expressing a POMC-SV40 T antigen fusion gene to study POMC gene regulation. 2) Identification of POMC specific transcription factors in the pituitary using DNA/protein interaction assays on AtT20 corticotroph cells, new melanotrophs cells and pituitaries of transgenic mice. 3) Structure and function characterization of a novel Sp1-like transcription factor mediating pituitary specific expression of the POMC gene by affinity purification or cloning. 4) A targeted mutation that blocks the production of beta-endorphin will be introduced in mice by homologous recombination in embryonic stem cells and we will analyze the effects of the lack of beta-endorphin during development. In close relation, the goals stated in the FIRCA grant are: 1) Identification of a distal cis- acting element responsible to confer appropriate neuronal expression of the POMC gene using transgenic mice as an expression system and 2) Localization of an additional corticotroph-specific enhancer(s) necessary to achieve adequate and integration position independent levels of expression of the POMC gene using a combined strategy of DNase I hypersentitive sites identification in AtT20 cells and a deletional analysis of POMC flanking sequences in transgenic mice. Together, these studies will provide complementary information important to understand the possible causes and consequences of the pathophysiological expression of the POMC gene.

9901278
Low

This Americas Program award will fund a three year cooperative research project between Dr. Malcolm Low, Vollum Institute, Oregon Health Sciences University, Portland, Oregon, and Dr. Marcello Rubinstein, Universidad de Buenos Aires, Argentina. They plan to utilize a new technology that will produce a conditional knockout of dopamine receptor subtypes in selective brain substrates of the adult mouse. The objective of these mutations in mammalian brain is to provide the experimental control of physiological processes. The generation of null alleles by homologous recombination in embryonic stem cells is a powerful technique to determine the physiological function of genes in vivo. However, drawbacks of this approach include the loss of multiple functions of a single gene and developmental compensation by changes in the expression of other genes processes in specific neurons. In this project the investigators will develop a new technology to overcome these limitations. If successful in the dopaminergic system, the same experimental strategy would be generally applicable to the functional analysis of many genes expressed in the brain.

The two investigators are experts in the techniques to be employed, therefore significant new mutational approaches, mouse strains and insights into dopamine receptor function will result from this work. The broader impact of this research includes, besides the development of new methods to produce localized, conditional mutations in central nervous systems genes, the understanding the functional significance of the five different dopamine receptor subtypes in localized brain regions
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Opiates and endogenous opioid peptides beta-endorphin, enkephalins and dynorphins primarily signal rewarding aspects of food consumption and are relatively unimportant in mediating feeding responses to metabolic demands. Classical pharmacological experiments have defined the contribution of various opioid receptor subtypes to the control of breeding but re limited in their power to distinguish among effects of the endogenous peptide ligands themselves. Moreover, beta-endorphin is synthesized together with melanocortin peptides from pro-opiomelanocortin (POMC) and probably is consecrated with melanocortins at many, if not all, POMC neuron terminals. There is abundant evidence for opposing and cooperative actions of beta-endorphin and melanocortins in several homeostatic brain functions, further complicating our understanding of the functional position of POMC neurons in hypothalamic circuits regulating the balance between caloric intake and energy expenditure. Our laboratory has initiated a genetic approach to investigate the physiological actions of individual endogenous opioid peptides, and the postulated interactions between opioids and melanocortins on feeding and metabolism. We will use beta-endorphin deficient mice previous generated in our lab, other existing spontaneous and induced mutants, and newly developed POMC mutants as physiological model systems to address the following specific hypotheses: 1.) The unusual growth pattern and resulting moderate obesity of beta-endorphin-deficient male mice, and wild-type male mice cross-fostered to beta-endorphin- deficient damns, is due to a combination of genetically determined factors contributed from both the mutant dams and the mutant offspring. Experimental approaches will include evaluations of relevant neuroendocrine circuits, maternal behavior, quantify and quality of mil from nursing dams, food intake, metabolic rates, and histology of adipocytes in white fat pads. 2.) Adult beta-endorphin- and enkephalin-deficient mice will exhibit altered food preferences and diminished motivation for eating because of decreased rewarding properties of food. These studies will use two existing strains of opioid-deficient mice and double homozygote mutant crosses between the strains for comparative analyses of food intake, food preference, macronutrient selection, and metabolic rate. Studies will be conducted in both food-deprived and sated mice to differentiate responses based on metabolic demands from rewarding properties of preferred reward. Two-bottle free choice drinking studies will be used to quantitate intake and preferences for sweet or salty liquid and aversion for quinine-laced water. 3.) POMC-derived beta-endorphin and melanocortins have coordinated actions on weight homeostasis. The major experimental models will be crosses between the beta-endorphin mutan6ts and melanocortin-receptor (MCR) knockout mice. We predict that the double homozygous beta- endorphin/MCR-4 KO mice will exhibit an additive or synergistic phenotype leading to more profound obesity in adults. Additionally, a new series of bigenic mouse mutants will be produce based on Cre-loxP recombination strategies that will carry spatially and/or temporally restricted null alleles for the POMC gene. Developmental, feeding and metabolic status of these various mouse models will be compared to each other and to the spontaneous Agamma yellow, obese mutant.

This Fogarty International Collaboration Award between the U.S. and Argentina is in support of the scientific goals outlined in its parent grant P01DK55819, "Neuroendocrine Control of Feeding and Metabolism" subproject 4, "Modulation of Feeding and Metabolism by Opioid Peptides." Obesity and its metabolic sequelae are major health problems world-wide. The opioid peptide beta-endorphin and a family of melanocortin peptides exhibit (POMC), that is expressed in specific populations of neurons within the arcuate nucleus of the hypothalamus and the nucleus tractus solitarius in the brain stem. Both of these brain nuclei are key areas involved in the complex integration of neuroendocrine and autonomic control of a peptide and feeding. Recent genetic studies in human populations have strongly implicated the POMC gene as a quantitative trait locus for obesity and null mutations in molecular basis for neural-specific expression of the POMC gene, although we hypothesize that disordered POMC expression in the hypothalamus or brain stem may be a contributing factor to disorders of energy homeostasis. The goals of this project are to elucidate the transcriptional machinery responsible for POMC gene expression in the brain and to develop novel molecular tools for the further physiological characterization of POMC peptides and co-transmitters produced in POMC neurons in the control of appetite and feeding. These goals will be addressed by the following specific aims: 1) Identify a neural-specific enhancer(s) in the mouse POMC gene utilizing a deletional and mutational analysis in transgenic mice; 2) characterize putative transcriptional factors that interact with the neuronal in cancer; and 3) Develop an immortalized hypothalamic neuronal cell lines that expresses the POMC gene under regulation of known physiological mediators such as leptin.

DESCRIPTION (provided by applicant): A widely held view of the mechanism underlying the reinforcing properties of opiates, thereby contributing to drug craving, is that drugs such as morphine and heroin usurp existing neural circuits that mediate the incentive value of natural reinforcers. If this is true, then one would expect that positive stimuli may be identical at the motivational level while differing at the perceptual or discriminative level. Pharmacology has suggested this to be the case, in that endogenous opioids modulate neural circuits involved in motivation to differing classes of stimuli, namely food and drugs of abuse. There remain many questions concerning the function of specific endogenous opioid peptides in the modulation of reward to positive stimuli. Our laboratory and others have generated mutant mice lacking specific opioid peptides or receptors to characterize the physiological function of these molecules. The proposed experiments are designed to test our hypothesis that the endogenous opioid peptides fulfill specific functions in modulating the incentive value of different classes of positive stimuli. The animal models we will use are fully congenic strains of C57BL/6J and DBA/2J mice harboring single mutations that result in the total absence of either Beta-endorphin, all forms of enkephalin, or a combination of both opioid peptide families. The primary behavioral task to assess reward is operant responding for food, saccharin, or morphine. Since the incentive value of positive stimuli vary with motivational states, we will study operant responding in the mice under both deprived and non-deprived conditions. Additional behavioral tasks that will be assessed in all groups of mice to further test the specificity of differences in operant responding include response extinction, measurements of taste preference, and locomotor activity.

DESCRIPTION (provided by applicant) This competitive renewal of a Fogarty International Cooperative Award between the U.S.A. and Argentina is in continued support of the scientific goals outlined in its parent grant P01 DK55819, "Neuroendocrine Control of Feeding and Metabolism," subproject 4, "Modulation of Feeding and Metabolism by Opioid Peptides." Obesity and its metabolic sequelae are major health problems world-wide. The opioid peptide beta-endorphin and a family of melanocortin peptides are both important modulators of weight homeostasis and products of the same prohormone, proopiomelanocortin (POMC), that is expressed in specific populations of neurons within the arcuate nucleus of the hypothalamus and the nucleus tractus solitarius in the brain stem. Both of these brain nuclei are key areas involved in the complex integration of neuroendocrine and autonomic control of appetite and feeding. Null mutations of the POMC gene have rarely been associated with early onset morbid obesity and hyperphagia in children. However, genetic studies in several human populations have strongly implicated the POMC gene as a quantitative trait locus for obesity and leptin levels. Mutations in the coding region of the POMC gene that would alter peptide production or function do not account for this linkage, suggesting the alternative possibility of mutations in POMC regulatory sequences. Little is known about the molecular basis for neural-specific expression of the POMC gene, although we hypothesize that dysregulated neuronal POMC expression may be a contributing factor to disorders of energy homeostasis. The overall goal of this FIRCA project is to elucidate the transcriptional machinery responsible for neuronal-specific and leptin-regulated POMC gene expression in the brain and to develop novel molecular tools for the further physiological characterization of POMC peptides in the control of appetite and feeding. This goal will be addressed by the following specific aims: 1) Identify a hypothalamic neural-specific enhancer(s) in the POMC gene utilizing a deletional and mutational analysis in transgenic mice; 2) Characterize the leptin-responsive element in the POMC gene; and 3) Determine a phylogenetic transcriptional code for the POMC gene by sequence comparisons of genomic elements from divergent orders of vertebrate species and the analysis of transgenic mice carrying POMC regulatory sequences of Fugu rubripes, a teleost fish that diverged from placental mammals 450 million years ago.

DESCRIPTION (provided by applicant): Obesity and its complications stemming from type 2 diabetes mellitus are now leading health concerns worldwide. Research in the past decade has established a critical role for the arcuate nucleus of the hypothalamus and the nucleus tractus solitarius (NTS) in the CNS control of appetite and energy expenditure. We and others have shown that peptidergic neurons expressing proopiomelanocortin (POMC), located within both of these nuclei, are key cellular elements of the neural circuits that mediate energy homeostasis. The melanocortin peptides, including alpha, beta, and gamma-melanocyte stimulating hormones and adrenocorticotrophic hormone inhibit feeding and increase metabolic rate. Mutations in either of the neural melanocortin receptors (MC4-R and MC3-4) or POMC cause obesity in humans and rodent models, emphasizing the importance of this class of neuropeptide transmitter. In contrast, POMC derived beta-endorphin stimulates feeding and mediates the behavioral reinforcing properties of palatable food. We have recently discovered that a substantial proportion of POMC neurons also synthesize the classical, fast inhibitory neurotransmitter gamma-amino butyric acid (GABA), a finding that challenges the widely held assumption that POMC neurons would coexpress the excitatory amino acid transmitter glutamate. GABA was previously co-localized in an adjacent population of orexigenic, neuropeptide Y/agouti gene-related transcript neurons in the arcuate nucleus, which generally oppose the function of POMC neurons. Based on these data and other studies showing qualitative differences among POMC neurons in their individual responses to hormonal and synaptic signals, we hypothesize that POMC neurons are not a synchronized population of homogenous cells, but instead composed of subpopulations with distinct roles in energy balance. Furthermore, we postulate that the coordinated release of GABA and peptide modulators from POMC neurons may be a key aspect of their mechanism of action at autoreceptors, and pre- and postsynaptic sites on other neurons. Therefore, the overall goal of this project is to further delineate the neurochemical basis of subpopulations of POMC neurons within the arcuate nucleus and NTS and to match these subpopulations with their specific actions in the regulation of metabolic rate and food intake. The specific aims are to: 1) Utilize a newly developed mutant mouse model with selective neuronal deficiency of POMC to determine the specific physiological functions of the different melanocortin receptor ligands in the central nervous system control of energy homeostasis; 2) Map the co-localization of GABA and POMC peptides within the arcuate nucleus and hypothalamic projections of POMC neurons and test how GABA release from POMC neurons is integrated with the release and physiological activity of melanocortins and beta-endorphin on energy homeostasis; and 3) Probe the functional organization of the arcuate nucleus and NTS using the genetic techniques of: a) Cell-specific ablation of selected groups of POMC neurons by transgene-directed toxin delivery; and b) Aggregation chimera analysis to juxtapose multiple different combinations of wild-type and POMC-deficient neurons within the brain. Results from these studies are expected to significantly increase our basic understanding of the brain areas and mechanisms involved in the regulation of body weight and lead to new therapeutic approaches for the prevention and treatment of obesity.

[unreadable] DESCRIPTION (provided by applicant): Obesity and associated metabolic disorders including diabetes have become epidemic worldwide and threaten the health of millions. A recent confluence of data from human population genetics studies, mouse transgenic experiments, and neuropeptide pharmacology has implicated the proopiomelanocortin (POMC) gene, its encoded peptides, and their cognate G-protein coupled receptors as essential components of the neural circuits that regulate food intake and energy expenditure. Evolution has resulted in functionally and topographically distinct DNA regulatory elements that control POMC transcription in either neurons or pituitary endocrine cells, and which are highly conserved across mammalian species. These distinctions in cell-type specific POMC promoter and enhancer elements are mirrored by unique complements of transcription factors that differentially regulate POMC gene expression in the brain versus pituitary gland. POMC expression in hypothalamic neurons is quantitatively associated with adipose mass over the lifetime of a mouse and we hypothesize that in humans the analogous control of neuronal POMC gene expression is a key regulatory switch determining the ultimate balance of fat storage or utilization. The first aim of this project is to further characterize the neuronal-specific enhancer elements of the POMC gene using a combination of in vivo transgenic and in vitro primary neuronal culture expression systems, guided by additional cross-species comparative sequencing. A second aim will specifically determine the interaction of POMC gene regulatory elements with components of the leptin receptor-signaling pathway to result in activation of neuronal POMC gene transcription by the adipocyte-derived hormone. Third, the quantitative and qualitative effects of selective neural enhancer mutations in the context of the endogenous mouse POMC gene will be rigorously tested by genetic knock-in strategies. Fourth, the mRNA "transcriptome" of POMC neurons isolated by lasercapture microscopy will be defined, and the cognate DNA-binding transcription factors responsible for neuron-specific POMC gene expression will be identified and cloned using both computational genomics and screening of yeast one-hybrid cDNA libraries constructed from amplified POMC neuron-specific RNA. Throughout these studies we will collaborate with our clinical colleagues, who are involved in human genome-wide screens and index case-based gene sequencing projects, in a translational effort to identify and functionally characterize additional polymorphic alleles of either the POMC gene itself or candidate neuronal POMC gene transcription factors underlying altered weight regulation. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Obesity and its complications stemming from type 2 diabetes mellitus are now leading health concerns worldwide. Research in the past decade has established a critical role for the arcuate nucleus of the hypothalamus and the nucleus tractus solitarius (NTS) in the CNS control of appetite and energy expenditure. We and others have shown that peptidergic neurons expressing proopiomelanocortin (POMC), located within both of these nuclei, are key cellular elements of the neural circuits that mediate energy homeostasis. The melanocortin peptides, including alpha, beta, and gamma-melanocyte stimulating hormones and adrenocorticotrophic hormone inhibit feeding and increase metabolic rate. Mutations in either of the neural melanocortin receptors (MC4-R and MC3-4) or POMC cause obesity in humans and rodent models, emphasizing the importance of this class of neuropeptide transmitter. In contrast, POMC derived beta-endorphin stimulates feeding and mediates the behavioral reinforcing properties of palatable food. We have recently discovered that a substantial proportion of POMC neurons also synthesize the classical, fast inhibitory neurotransmitter gamma-amino butyric acid (GABA), a finding that challenges the widely held assumption that POMC neurons would coexpress the excitatory amino acid transmitter glutamate. GABA was previously co-localized in an adjacent population of orexigenic, neuropeptide Y/agouti gene-related transcript neurons in the arcuate nucleus, which generally oppose the function of POMC neurons. Based on these data and other studies showing qualitative differences among POMC neurons in their individual responses to hormonal and synaptic signals, we hypothesize that POMC neurons are not a synchronized population of homogenous cells, but instead composed of subpopulations with distinct roles in energy balance. Furthermore, we postulate that the coordinated release of GABA and peptide modulators from POMC neurons may be a key aspect of their mechanism of action at autoreceptors, and pre- and postsynaptic sites on other neurons. Therefore, the overall goal of this project is to further delineate the neurochemical basis of subpopulations of POMC neurons within the arcuate nucleus and NTS and to match these subpopulations with their specific actions in the regulation of metabolic rate and food intake. The specific aims are to: 1) Utilize a newly developed mutant mouse model with selective neuronal deficiency of POMC to determine the specific physiological functions of the different melanocortin receptor ligands in the central nervous system control of energy homeostasis; 2) Map the co-localization of GABA and POMC peptides within the arcuate nucleus and hypothalamic projections of POMC neurons and test how GABA release from POMC neurons is integrated with the release and physiological activity of melanocortins and beta-endorphin on energy homeostasis; and 3) Probe the functional organization of the arcuate nucleus and NTS using the genetic techniques of: a) Cell-specific ablation of selected groups of POMC neurons by transgene-directed toxin delivery; and b) Aggregation chimera analysis to juxtapose multiple different combinations of wild-type and POMC-deficient neurons within the brain. Results from these studies are expected to significantly increase our basic understanding of the brain areas and mechanisms involved in the regulation of body weight and lead to new therapeutic approaches for the prevention and treatment of obesity.

Project Summary: Obesity and its associated metabolic diseases are major health problems around the world. Although the reasons for the rapid increase in rates of obesity and overweight, particularly in children, are multifactorial, it is clear that neural circuits in the brain play a major role in sensing energy stores and regulating energy balance. These neural pathways are candidate targets for the development of new pharmacotherapies aimed at reversing the obesity epidemic and therefore it is essential that we understand their function in detail, including the complete map of interconnections with other neural systems and their utilization of multiple neurotransmitters and intracellular signalling pathways. This project focuses on a key component of the brain[unreadable]s energy balance circuitry, the proopiomelanocortin (POMC) neurons located in the hypothalamus and brainstem. Genetic deletion of POMC function from the brain results in an obesity and metabolic syndrome characterized by extreme hyperphagia and reduced basal metabolic rate. However, POMC neurons are heterogeneous in many aspects and accumulating evidence suggests that different subpopulations of the neurons regulate separate neurological processes that together result in normal or pathological control of caloric balance. The overall goals of this project are to identify specific functions of these neuronal subpopulations and the neuroanatomic and molecular pathways that they utilize. Specific aim 1 includes a series of behavioral and neuropharmacological studies to probe the underlying component processes and neural substrates contributing to hyperphagia in POMC-deficient mouse models. These experiments utilize a newly developed method for meal pattern analysis and will test the hypothesis that melanocortin signaling coordinately modulates stereotyped motor, reward, and hedonic aspects of feeding behavior; processes which are particularly relevant to human issues surrounding food choice and meal size in the clinical pathogenesis of obesity. Specific aim 2 continues our ongoing work to define the neurochemical phenotype of POMC neurons and their collateral axonal projections to multiple brain sites. A novel retrograde tracing method involving site-specific microinjections of a canine adenoviral vector expressing Cre recombinase into mutant mice with a reversibly silenced POMC gene allele will be used to map axon collaterals to specific combinations of target sites. We will also use multilabel immunohistochemical and genetic techniques to study the unexpectedly complex dendrites of these neurons that receive synaptic inputs from distal sites. Finally, in Specific aim 3 we will use complementary genetic approaches to study the unique functional role of spatially distinct POMC neuron subpopulations or developmentally altered POMC gene expression in the prevention or mitigation of obesity. The techniques involved are aggregation chimera formation and Cre recombinase mediated reactivation of POMC expression from a neuron-specific and reversibly silenced POMC allele.

DESCRIPTION (provided by applicant): This revised application requests continuing support for a training program which provides graduate students studying for the PhD degree with broad state-of-the-art training in Systems and Integrative Biology. Students will learn and apply tools and approaches of genomics, bioinformatics, proteomics as well as cell and molecular biology to the study of integrated systems and organismic biology. The program is centered in the Department of Molecular & Integrative Physiology because of its longstanding commitment to integrative biology but students will enter from an initial year in a combined gateway Program in Biomedical Sciences (PIBS) and can be enrolled in multiple PhD programs including Physiology, Pharmacology, Neuroscience, Bioinformatics and Human Genetics. Trainees will usually be supported for two years. The potential applicant pool is all students applying to PIBS and includes both students who come to Michigan to study integrative biology and those who enter undecided and are then drawn to integrative biology. The training includes formal coursework and seminars, teaching, and research experiences with faculty in a variety of departments at the University of Michigan. All students will take graduate level courses in Integrative Biology, Cell and Molecular function, Molecular Biology and Genetics, and Computational Biology. Additional courses are selected by the student in accordance with his/her interests. Students also participate in weekly student research seminars in their PhD program which they are expected to critically evaluate the presentations of their peers. This broad training is further reinforced by each student taking a written preliminary examination at the end of the second year of study that requires and understanding of function, from the cellular and molecular level to that of the organism including interactions between the environment and the organism. A unique aspect of the proposed training is that all students will have two dissertation co-chairs, rather than the more traditional single chair, at least one of whom will be schooled in the most recent techniques in cellular, molecular, or computational biology and one being an integrative biologist. The training faculty consists of 58 outstanding scientists all of whom are expert in either cellular and molecular or systems and integrative biology and committed to training individuals to answer questions of integrative physiological relevance.

The primary goals and function of the MNORC Animal Phenotyping Core are to provide expert consultation, state-of-the art equipment and technical services that are critical for the detailed metabolic phenotyping of rodent models of diabetes and obesity. A thorough understanding of the physiological responses to nutrients and environmental factors and of the pathophysiological mechanisms that contribute to diabetes and related metabolic diseases is required if we are to effectively combat these conditions. However, the resources and technology necessary to phenotypically probe whole animal models of altered glucose homeostasis and metabolism at a level that reveals basic underlying mechanisms of control are not available in most investigators' laboratories. The Animal Phenotyping Core meets these needs through a comprehensive, convenient and cost-effective menu of platforms that includes: a) Glucose homeostasis and metabolic clamps. The Core performs hyperinsulinemic/ euglycemic clamp studies including specialized analysis of metabolite storage and release in rats and mice. b) Whole animal metabolic assessment. The CLAMS apparatus and other systems are used to examine metabolic rate, respiratory quotient, food consumption, and locomotor activity in rodent models. c) Body composition measurement by NMR. d) Radiotelemetric monitoring. Systems are in place for remote, chronic monitoring of cardiovascular parameters and core body temperature in rats and diurnal running wheel behavior in mice. e) Ingestive behavior. Meal microstructure and reinforcing properties of dietary constituents are measured in either home-cage or operant-conditioning paradigms. f) Automated blood/body fluids sampling and infusion utilizing a Culex/Empis system to remotely collect serial samples and infuse substances to freely behaving, unstressed rodents. g) In vivo optogenetics combined with the analysis of a wide range of behavioral outputs. Altogether, the Animal Phenotyping Core provides consultation and advice on experimental design, reliable data from a range of validated assays and essential data analysis relevant to the needs of multiple investigators in the MNORC.

Project Summary / Abstract ? Overall Metabolic disorders and their vascular complications are among the most pressing healthcare issues worldwide. The University of Michigan Medical School has strategically invested in both human and physical resources over the past 15 years under the umbrella of the Michigan Comprehensive Diabetes Center in a concerted attack on this problem. One component of the strategy has been to foster the development of core laboratories within the university to provide key expertise and specialized phenotyping services at reasonable cost to researchers throughout the institution. We now propose to coalesce a quartet of these phenotyping cores into the Michigan MMPC to continue providing comprehensive and state of the art services internally, while expanding their availability to researchers on a national level, and continuing the development of more sensitive and more specific tools to probe the pathogenesis and biochemical consequences of diabetes and obesity in mouse models. The Michigan MMPC will provide services in four major areas. The Metabolism, Bariatric Surgery and Behavior Core will perform a variety of in vivo physiological assessments encompassing glucose homeostasis (glucose tolerance, insulin tolerance, hyperinsulinemic/euglycemic clamps), energy homeostasis (indirect calorimetry by CLAMS, dietary challenge), ultradian hormone secretion (Culex platform for serial biological fluid sampling from unrestrained mice), behavioral measurements (locomotor activity, meal pattern analysis, operant conditioning) and generation of bariatric surgery models. The Cardiovascular Pathophysiology Core will assess cardiovascular function (exercise tolerance, blood pressure, ECG, telemetry, echocardiogram), offer mouse surgical models of cardiac ischemia and pressure overload and imaging using ultrasound. The Microvascular Complications Core will provide basic and advanced assessment of diabetes complications including neuropathy, nephropathy, and retinopathy. The Microbiome Core will provide anaerobic microbial culture, microbiome genomic and transcriptomic libraries, 16S ribosomal gene and RNA sequencing with associated bioinformatic analysis. Mouse importation, housing, veterinary care, clinical chemistry and histopathology services will be provided by the Animal Care Core. In addition, the latter core hosts a germ-free mouse facility and will interact with the Microbiome core to produce and distribute germ-free and gnotobiotic mouse models. The Administrative Core will manage all Center activities, oversee financial operations, provide training opportunities, and collaborate with the other phenotyping centers and the Central Coordinating and Bioinformatics Unit of the MMPC Consortium by providing all collected phenotyping data and protocols and participating in the Pilot and Feasibility award and MICROMouse funding programs. Together, our Core resources will provide in a single Center access to a broad range of advanced phenotyping capabilities, including our unique focus and expertise on microvascular complications, microbiome analysis and germ-free mice and bariatric surgery models.

Project Summary / Abstract ? Core C. Metabolism, Bariatric Surgery and Behavior Core The primary goals and function of the Metabolism, Bariatric Surgery and Behavior Core are to provide expert consultation, state-of-the art equipment and technical services that are critical for the detailed metabolic and behavioral phenotyping of mouse models of diabetes, obesity and associated disorders. A thorough understanding of the physiological responses to nutrients and environmental factors and of the pathophysiological mechanisms that contribute to diabetes and related metabolic diseases is required if we are to effectively combat these conditions. However, the resources and technology necessary to phenotypically probe whole animal models of altered glucose homeostasis and metabolism at a level that reveals basic underlying mechanisms of control are not available in most investigators' laboratories. The Metabolism, Bariatric Surgery and Behavior Core meets these needs through a comprehensive, convenient and cost- effective menu of platforms that includes: a) Glucose homeostasis and metabolic clamps. The Core performs hyperinsulinemic clamp studies including specialized analysis of metabolite storage and release in mice. b) Whole animal metabolic assessment: The CLAMS and TSE indirect calorimetry systems are used to examine metabolic rate, respiratory quotient, food consumption, and locomotor activity in mouse models. c) Body composition measurement by NMR. d) Radiotelemetric monitoring of diurnal running wheel behavior in mice. e) Ingestive behavior: Meal microstructure and reinforcing properties of dietary constituents are measured in either home-cage or operant-conditioning paradigms. f) Automated blood/body fluids sampling and infusion utilizing a Culex/Empis platform to remotely collect serial samples and infuse substances to freely behaving, unstressed mice. g) Vertical sleeve gastrectomy model of bariatric surgery is performed in mice and subsequent phenotyping can be performed with the combined resources of all the Cores in the Michigan MMPC. Altogether, the Metabolism, Bariatric Surgery and Behavior Core provides consultation and advice on experimental design, reliable data from a range of validated assays, essential data analysis relevant to the needs of multiple investigators and training opportunities in the established methodologies of the Core to researchers both at the University of Michigan and throughout the country.

? DESCRIPTION (provided by applicant): This competitive renewal application requests continuing support for a training program that provides predoctoral graduate students with broad state-of-the-art training in Systems and Integrative Biology. Students will learn and apply tools and approaches of genomics, bioinformatics, proteomics as well as cell and molecular biology to the study of integrated systems and organismic biology. The program is centered in the Department of Molecular & Integrative Physiology because of the Department's longstanding commitment to integrative biology, but students will enter after their initial year in a combined gateway Program in Biomedical Sciences (PIBS) and can be enrolled in multiple PhD programs including Physiology, Pharmacology, Neuroscience, Cell and Molecular Biology, Molecular and Cellular Pathology, Cell and Developmental Biology, Human Genetics, Biochemistry, Bioinformatics and the Medical Scientist Training Program. Trainees will usually be supported for their second and third years. Therefore, the six available slots are usually assigned to three new trainees and three reappointed trainees each year. The potential applicant pool is all students applying to PIBS and includes both students who come to Michigan to study integrative biology and those who enter undecided and are then drawn to integrative biology. The training includes formal coursework and seminars, teaching, and research experiences with faculty in a variety of departments at the University of Michigan. All students will take graduate level courses in Integrative Biology, Cell & Molecular function, Genetics & Molecular Biology, and Computational Biology. Additional courses are selected by the student in accordance with his/her interests. Students also participate in a course on Responsible Conduct of Research and weekly student research seminars in their respective PhD programs in which they are expected to critically evaluate the presentations of their peers. Rather than offering a standard journal club, our program has a highly interactive monthly workshop where the trainees work together with faculty to apply the principles of mathematical modeling and computational biology to a real set of data from one of the participating faculty's research programs. This broad training is further reinforced by each student taking a written and oral preliminary or candidacy examination mid-way or at the end of the second year of study that requires an understanding of biological function, from the cellular and molecular level to that of the whole animal including interactions between the environment and the organism. A unique aspect of this training program is that all students have two dissertation co-chairs, rather than the more traditional single chair, at least one of whom will be schooled in the most recent techniques in cellular, molecular, or computational biology and one being an integrative biologist. The training faculty consists of 52 outstanding scientists all of whom are experts either in cellular and molecular or systems and integrative biology and committed to training individuals to answer questions of integrative physiological relevance.

Project Summary / Abstract ? Core A. Administrative Core The Administrative Core will provide overall administrative and scientific oversight for the Michigan Mouse Metabolic Phenotyping Center. The Center director will meet monthly with his associate director, Core directors and the Internal Steering Committee to review all activities of the Center and provide input to the Core directors. The Core director will actively participate in the monthly phone conferences and annual meeting with the MMPC Consortium Executive Steering Committee and NIDDK participants. The director will also participate in special focus committees and be responsive to periodic reviews and recommendations by the Expert Scientific Panel. A Center website will be maintained and updated in consultation with the MMPC Coordinating and Bioinformatics Unit (CBU) and the Administrative Core will assure that all Material Transfer Agreements, contracts, service fees, and data records are handled efficiently and recorded in the CBU database in a timely manner. The Administrative Core will oversee core services' pricing determination in conjunction with the University of Michigan Office of Financial Analysis, oversee the expenditures and revenue from each core, provide for rebudgeting across cores if appropriate based on service demands and scientific need and prepare annual progress reports for NIDDK. The Administrative Core will also oversee research development and training resources for the Michigan MMPC. Finally, the Core will solicit and participate in the review of applications for the MMPC Pilot and Feasibility Award and MICROMouse funding Programs from the Opportunity Pool in cooperation with the national CBU.

Abstract Naturally occurring and genetically modified mouse models are powerful preclinical tools to study the pathogenesis of human disease. Specifically, they are commonly used to study obesity, the metabolic syndrome and the mechanistic connections among obesity, diabetes, the gut microbiome, cardiovascular disease and many forms of cancer. In order to understand the physiology of such models and the alterations in metabolism that underlie their phenotypes, equipment capable of specialized and precise measurements must be employed to examine energy balance and metabolic variables. The equipment requested is a Promethion Core for simultaneous metabolic and behavioral measurements of 16 mice. It includes two Sable-modified Darwin temperature-controlled cabinets that each hold 8 mouse cages. Sable Systems, Inc. HD High Definition Technology is built into each Promethion Core system to capture high-resolution respirometry (indirect calorimetry) data including oxygen consumption and carbon dioxide production. These data are used to calculate energy expenditure and respiratory quotient. The entire system is controlled by Ethoscan software for additional, automated behavioral analyses relevant to studies of energy balance. Each cage will include the following modular monitoring devices: an access control module for ad libitum, paired or yoked food access from a food intake module, a water intake module, a body weight module, a running wheel activity module, a position/movement module and a rearing activity module. The calorimetry system will also include a monitor that detects the concentration of the naturally occurring stable isotopes of carbon, [13C] and [12C]. The ratio of these two nonradioactive isotopes in the exhaled carbon dioxide of mice housed within the calorimetry chambers is the most sensitive measurement of the relative oxidation of dietary lipids and carbohydrates. These ?breath tests? are also used for the analysis of gut absorption, gastric emptying time and other metabolic parameters whose detection can be amplified by the prior administration of artificially enriched [13C]-labeled precursor molecules. With the addition of 8 modified cages designed to contain germ-free mice in a sterile condition and specialized gas monitoring devices for hydrogen and volatile organic chemicals, the system will also be used to study the interactions of the gut microbiome, genotype and diet on the regulation of body weight and glucose homeostasis. The Michigan Mouse Metabolic Phenotyping Center (MMPC) currently has one 16-cage and one 8-cage Comprehensive Laboratory Animal Monitoring Systems for indirect calorimetry (CLAMS) that are in constant use. One of these systems is 11 years old and frequently in need of costly repairs. It will be replaced by the state-of-the-art Promethion Core to continue meeting current and future demand in a timely fashion and provide an array of unique experimental measurements not possible with our existing CLAMS instruments.