Eric P. Widmaier - US grants

The ability of an organism to respond lo challenges to its physiological homeostasis by increasing the activity of the brain- pituitary-adrenocortical hormonal axis is critical to survival in all mammals, including rats and man. In the absence of a functional adrenal cortex such challenges, or "stresses," are usually fatal. One of the most potent and reliable stimuli to secretion of adrenocorticotropic hormone (ACTH) from the pituitary is hypoglycemia, lypically induced experimentally by injections of insulin. The effect of hypoglycemia is now known to be mediated at least in part via glucose sensors in the hypothelomas. resulting in secretion of corticotropin-releasing factor (CRF) into the hypophysial-portal circulating. Conversely, in isolated rat hypothalami, hyperglycemia inhibits secretion of CRF and also of oxytocin, another hypothalamic peptide known to stimulate ACTH secretion. Failure to adequately regulate circulating products of metabolism (glucose, fatty acids) within normal limits is one of the most widespread endocrine associated disorders in human beings, and can result from numerous pathologies (diabetes, fasting). This 'rises the question of the capacity of individuals to respond to different physiological stresses, such as hemorrhage, during periods of abnormal metabolic status (hyperglycemia, hyperlipidemia). Fatty acids have been shown lo inhibit secretion of growth hormone in rats and man, at both the hypothalamic and pilutuary level. Since one of the major functions of adrenal glucoconlcoids is to catabolize triglycerides in order to provide usable energy substrates for the brain, it may well be that fatty acids, like glucose, inhibit the secretion of CRF and AGTH. Therefore, the long term goals of these projects will be to characterize in detail the effects of glucose and fatty acids on secretion of CRF and ACTH at the tissue or cellular level, and to relate these effects to the ability of rats to respond to stress in vivo under periods of experimentally altered intermediary metabolism. The role of other substances that themselves influence intermediary metabolism, such as catecholamines, in activating CRF release will also be examined. These studies will also determine the ontogeny of the ACTH and corticosterone secretory responses to insulin-induced hypoglycemia, since young rats typically show reduced sensitivity to certain stimuli that produce robust secretory responses in adults. Similarly, human newborns also undergo a period of abnormal responsivity to some, but not all, types of stress. Finally, the effects of hyperglycemia on suckling-induced secretion of oxytocin in rats will be examined, since glucose decreases oxytocin secretion in vitro, and since a relatively high proportion of pregnant women develop gestational deabetes in the last trimester of pregnancy, resulting in chronic hyperglycemia.

9513926 Widmaier Fatty acids serve numerous functions in living organisms. These lipids are building-blocks of cell membranes, and are a vital fuel source for most cells of the body. In addition, fatty acids appear to play an important role in cell:cell communication. In other words, fatty acids may have "regulatory" actions in the body that mediate hormonal and nervous signals. Certain fatty acids are more potent in this regard than others. The long-chain, unsaturated stearate are the least active. When blood levels of oleate are high, the function of several endocrine glands is affected, usually in a negative way (i.e., inhibited). One good model of fatty acid-induced changes in cell function is the adrenal gland, specifically the region of the gland (the cortex) that produces the steroid hormones known as glucocorticoids (cortisol in people, corticosterone in rats). In this grant, the mechanisms by which specific fatty acids induce changes in adrenal activity will be investigated by a variety of approaches. These include infusions of fatty acids into rats followed by analysis of hormone secretion, and the responses of cultured adrenal cells to additions of various fatty acids. The results of these experiments will significantly increase our understanding of how steroidogenic glands like the adrenal glands (and the gonads) are regulated under normal and high-fat conditions. In addition, basic information about cell signaling events associated with fatty acids will be obtained. This is important because the array of tissues that are sensitive to fatty acids is enormous, including the pancreas, ovary, testis, pituitary, liver, heart, muscle and others. Information gained from these studies may be expected to help neurobiologists understand how fatty acids induce changes in muscle membrane permeability to electrolytes, molecular biologists understand how fatty acids contribute to regulation of insulin secretion. An appreciation of the widespread and potent regulatory action s of these lipids is rapidly growing. This grant will begin the systematic exploration of the mechanisms by which these actions are produced.

The objectives of this application are to elucidate the role of the peripheral-type benzodiazepine receptor (PBR) in maturation of the adrenal glands, and to determine mechanisms of regulation of this receptor. PBR is expressed in all steroidogenic cells, including gonads, adrenals, placenta, and brain (glia), where it is believed to mediate transport of cholesterol to the inner mitochondrial membrane, the site of enzymes needed to convert cholesterol to pregnenolone. Inability to transfer cholesterol to the inner membrane is associated with decreased steroidogenesis. Since this is the rate-limiting step in steroidogenesis, it is critical that this process be fully understood if new and better therapeutic approaches to disorders of adrenal pathology (e.g., adrenal insufficiency or hypertrophy) are to be developed. Studies on regulation of this important receptor in have been hindered by the lack of a normal cell or animal model in which the receptor is expressed at low levels. The neonatal rat is a model of ACTH-insensitivity, since during this period adrenocortical cells respond weakly to ACTH (10 percent of adult levels). Insensitivity to ACTH is also observed in fetuses of larger mammals, and prevents neurotoxic effects of high levels of glucocorticoids on developing brain cells. Because adrenal insufficiency is the primary cause of morbidity and mortality in premature infants (glucocorticoids are required for induction of surfactant expression in fetal lungs), the process by which the developing adrenal gland matures is of great significance. Recent findings demonstrate that neonatal rat adrenals express PBR at 10 percent the level in adult adrenals. Thus, it is proposed to characterize the complete developmental profile of PBR and other factors suggested to be important in cholesterol transport, and correlate this with changes in adrenocortical sensitivity to ACTH in vivo and in vitro. PBR expression will be determined by immunoblot, Northern blot, in situ hybridization, and ligand binding assays from fetal life through weaning. The effects of chronic treatment with ACTH or cAMP on cholesterol transport, PBR expression, and steroidogenesis in vivo and in cultured neonatal adrenal cells will be examined. The ability of transfected PBR to restore ACTH sensitivity in cultured neonatal cells will also be examined. The ontogenic appearance of PBR ligands will be determined by radioimmunoassay. These studies, therefore, will simultaneously address two important questions: What are the factors that limit steroidogenesis in immature adrenal glands, and which factors regulate expression of PBR?

9875871
Widmaier
The existence of a circulating factor that regulates body weight in mammals has long been postulated. This hormone, now known as leptin, was identified in 1994, and found to be secreted from adipose (fat) tissue. Leptin signals the brain to reduce appetite and increase metabolic rate. Recently, it has become apparent that leptin levels in the blood are elevated during pregnancy in several mammalian species, including human beings. This, and other evidence, has led to the hypothesis that leptin may play previously unrecognized but important roles in the maintenance of pregnancy and the preparation for lactation. The work proposed in this application is designed to investigate this hypothesis, first by determining what tissues secrete leptin during pregnancy, then by determining what factors control its secretion, and ultimately determining what functions leptin performs in the pregnant animal. These goals will be facilitated by the ability to measure small amounts of leptin production from animal tissues in the culture dish. Dr. Widmaier proposes that the placenta becomes a significant source of circulating leptin during pregnancy and that reproductive hormones like estrogen and cortisol stimulate leptin production in placenta. Dr. Widmaier also proposes that the brain becomes less sensitive to leptin during pregnancy, permitting an increased appetite during pregnancy. These questions will be examined in free-ranging bats. A better appreciation of the hormonal correlates of reproduction will not only improve our ability to treat reproductive disorders in people, but will increase our ability to successfully maintain populations of ecologically important animal species. Finally, a better appreciation of the actions of leptin and the control of its secretion will improve our understanding of body weight pathologies in human and animal populations.

Collectively, rodents and bats comprise roughly 75% of all known mammalian species. Within the Chiroptera, temperate bats such as the little brown bat (Myotis lucifugus) play critical ecological and economic roles in the United States and elsewhere. For example, these species are important in controlling insect populations, which would otherwise flourish in the absence of insectivorous bats. Understanding the regulation of basic reproductive processes in these species is therefore of vital concern, but surprisingly many fundamental aspects of reproductive control are still unclear in bats. The use of a comparative approach will provide information on the ubiquity in nature of these regulatory processes. Recently, the so-called obesity hormone, leptin, has been suggested to serve a role in mammalian reproduction. This hypothesis originally arose from the observation that levels of leptin in blood of pregnant animals increase considerably. Leptin's established functions are to suppress appetite and stimulate metabolic rate when body weight increases. As fat mass in the body increases, adipose cells secrete leptin into the blood, which signals the brain that sufficient fat reserves are available. Whether leptin exerts regulatory effects during reproduction is still uncertain. Also uncertain is the mechanism(s) by which plasma leptin levels are elevated during pregnancy. Recent evidence suggests that both the placenta and adipose tissue are important in secreting leptin during pregnancy, and that leptin may not only be secreted by the placenta, but may act on it as well (e.g., to promote its growth and development). In this project the regulatory factors that influence leptin secretion from both tissue sources during this energetically-demanding period of a mammal's life history will be identified. Further proposed experiments will permit determination of whether leptin is actively synthesized from placentas of rodents and bats. Finally, experiments will provide tests of the hypothesis that changes in expression of brain leptin receptors account in part for observed leptin-insensitivity during pregnancy (i.e., the ability of pregnant females to continue eating above a normal amount despite elevated levels of this appetite-suppressing hormone). Such experiments may help explain why some people are apparently insensitive to the appetite-suppressing effects of leptin. These studies will extend our knowledge of reproductive processes in Chiroptera, specifically, of the placenta as a mammalian endocrine organ, and will also be applicable to other mammalian orders, since the control of leptin secretion during pregnancy appears similar in bats and humans, for example. Elucidation of the sequence of leptin and its receptor in bats will provide useful probes for detecting the presence of receptor in brain and placenta, and aid in future phylogenetic and structure/function analyses of the protein. This project will also contribute to science education by including numerous undergraduate students who will participate in various phases of the project, plus several graduate students or post-doctoral fellows. These students will gain valuable dual experience in field and laboratory biology, while engaged in an extremely topical project with wide-ranging ramifications and benefits to society at large. The PI and the co-PI have a long and successful history of promoting science through research and teaching at all levels, from K-12 through college, and continuing education of high school science teachers. It is anticipated that this trend will continue with this new project.

Dissertation Research: Hormonal Correlates of Prehibernatory Fattening in Bats
Eric P. Widmaier and Kristy Townsend
Boston University

Obesity is associated with adverse health consequences in people and other animals. However, increased fat accumulation is also an important survival adaptation during certain stages of the life histories of many animals. For example, fat deposition is beneficial to successful pregnancies. Another example of adaptive fattening occurs in hibernating mammals. How seasonal animals deposit adequate fat stores to survive hibernation is currently unresolved, however; clarifying the mechanism of prehibernatory fattening is a key long-term goal of this research.
Leptin is an important hormone through which adipose tissue communicates with the brain in mammals. Leptin is produced by adipose cells in proportion to body fat. It acts within the brain by activating cell signaling molecules that result in increased metabolic rate and decreased appetite. During the prehibernatory period, when fat must be accumulated, the brain must somehow ignore the appetite-suppressing action of leptin. One way in which this could happen is if the cells of the brain which normally respond to leptin, fail to do because of reduced signaling molecules or leptin receptors. A second way in which the brain can be released from leptin inhibition is to reduce the amount of leptin secreted by adipose tissue. This project will test both possibilities by examining adipose tissue secretion of leptin in vitro in little brown bats before or during the prehibernatory fattening period. The activational state of the leptin gene will be determined using a sophisticated procedure called MALDI-TOF-mass spectrometry, which is capable of distinguishing the ratio of modified bases in DNA to non-modified bases (a marker of one key way in which genes are activated). In addition, the brains of the animals will be examined to test the hypothesis that active forms of the leptin receptor and the signaling molecules generated by the receptors are decreased in the prehibernatory period. Little brown bats are chosen as experimental subject animal for many reasons. They are extremely abundant and exist in very large, thriving maternity colonies and prehibernatory colonies, are of great importance ecologically, and are among the most well-understood hibernators in terms of their basic physiology and reproduction, providing substantial information on which to base this study.
Broader impacts of this research include: integrated training of one graduate student and several undergraduate students in field biology and molecular endocrinology; providing critical information about survival strategies in an ecologically important mammalian order; and possible relation of mechanisms of seasonal changes in leptin biology to abnormal leptin biology in obesity.

Central to an understanding of successful pregnancies is determining how the placenta
attaches itself to the uterine wall of the mother. The outer lining of the placenta contains a layer
of cells (trophoblasts), which have the property of being able to invade the uterine lining, thus
establishing the placenta. How the invasion process is initiated and regulated is uncertain. In
this project, the hypothesis that the reproductive hormone leptin stimulates invasiveness of
trophoblast cells will be investigated in trophoblast cells grown on an artificial, protein-coated
membrane. Invasive cells secrete enzymes that degrade the protein coating, and permit the cells
to move through tiny pores in the membrane; the number of cells penetrating the membrane are
counted under a microscope. The ability of leptin to stimulate invasion of trophoblasts from
placentas at early and late gestation will be examined; we predict that leptin will promote
invasion only in trophoblasts from early-stage placentas. The molecular mechanisms by which
leptin stimulates invasiveness will be determined by examining changes in intracellular signaling
molecules and gene expression induced by leptin. Placentas will be studied in species
representing the two most abundant mammalian orders. The mouse placenta does not produce
leptin, but expresses receptors for leptin and is therefore sensitive to leptin actions. The little
brown bat placenta produces and secretes leptin (as does the human placenta), and like the mouse
placenta is a target for leptin. The effects of leptin on mouse and bat placental function will be
compared. Broader impacts of this project include: 1) documentation of the importance of leptin
in establishing the placenta in diverse mammals; 2) understanding the role of leptin as a general
signal for cell migration and invasion (i.e., in other cell types where leptin is known to act and
where cells are invasive); 3) understanding gestational stage-dependent actions of hormones on
trophoblast cells; and 4) education of young scientists-in-training and outreach to include
participation of underrepresented minorities (one postdoctoral fellow, one to two graduate
students and four undergraduates students will be mentored per year on this project).

White-Nose Syndrome has been linked to unprecedented morbidity and mortality among five of the nine species of bats in the northeastern United States, first observed in six hibernacula in New York State in the winter of 2006-2007, and subsequently reported from at least 33 caves and mines in four states (New York, Vermont, Massachusetts, and Connecticut) in the winter of 2007-2008, with mortality reported as high as 95% at some sites. The hypothesis being tested is that a reduction in the quantity and quality fat deposited by bats during the pre-hibernation period in autumn may compromise successful hibernation, and their ultimate reproductive success. White adipose tissue (WAT) is the primary source of energy that sustains bats (and other hibernators) throughout the winter when they have no access to food. Over-winter survival and subsequent reproductive success requires a sufficient quantity and quality of WAT deposited during the pre-hibernation period, and sufficient fat reserves to sustain deep torpor and periodic arousals throughout the winter, and a final arousal in spring. Because insectivorous bats cannot synthesize polyunsaturated fatty acids (PUFAs), deficiencies in dietary PUFAs during the pre-hibernation period in autumn may reduce the duration and depth of torpor during hibernation. Frequent arousals during hibernation may result in premature depletion of WAT before the end of the hibernation period. Moreover, depleted WAT at this time may contribute to a decrease in leptin production (necessary for ovulation and successful reproduction by females), or, in the worst cases, the inability to arouse from torpor or inability to mount an immune response to possible pathogens.

Analyses of body composition (including PUFAs) of bats of little brown myotis (Myotis lucifugus) during the pre-hibernation period will be conducted at sites affected by and unaffected by WNS to test three hypotheses that may help reveal or rule out causes of premature deaths or compromise reproductive success in these hibernating bats. Body composition analysis provides the most accurate and reliable estimates of total body water, WAT, and lean dry mass, and analyses of PUFAs in WAT collected from bats during the pre-hibernation fattening period. These data promise to provide valuable insight for testing proposed hypotheses to help explain why hibernating bats are dying prematurely at hibernacula in the northeastern US, and in turn suggest directions for future study to better understand WNS.

The proposed research will facilitate cross-disciplinary research between an ecologist, biochemical ecologist, and an endocrinologist to address an urgent environmental problem: unexpected mortality in hibernating insectivorous bats in the northeastern US. The proposed research will provide new insight into the mechanisms that influence winter survival and reproductive success in bats. Moreover, this research will provide an excellent opportunity to highlight the ecological and economic importance of bats in temperate ecosystems through print media, radio, TV, and web-based Internet blogs.