Timothy J. Bartness - US grants

obesity; seasons;

The goal of this research is to determine the neural and peripheral tissue target sites and mechanisms of action of melatonin on body weight and energy metabolism. Melatonin is thought to be the neurochemical transducer of seasonal changes in reproduction, however, melatonin injections, like short day exposure, increase body weight and carcass lipin in Syrian hamsters (Mesocricetus auratus) as well. One set of experiments will examine CNS sites of action for the effects of melatonin on body weight and energy metabolism (including measures of brown adipose tissue thermogenic activity and white adipose tissue lipid deposition). Microknife cuts and radiofrequency lesions in combination with systemic melatonin injections or short day exposure will also be performed. Preliminary data suggest that the paraventricular nucleus may be a primary target site. A second set of experiments will examine peripheral targets of melatonin action such as brown and white adipose tissue and the thyroid gland using surgical denervation of brown fat and chemical throidectomy. A third set of experiments will examine the CNS site(s) and mechanism(s) behind the short day-induced pineal/melatonin independent increases in body weight and energy metabolism using many of the above techniques. Finally, the generality of the target sites for these effects of melatonin on body weight and energy metabolism will be examined in Siberian hamsters (Phodopus sungorus sungorus), a hamster species that shows short day- and melatonin-induced decreases in body weight and carcass lipid. These experiments represent an interdisciplinary approach to the study of naturally-occurring changes in adiposity by examining the interactions of neural and hormonal factors with environmental changes.

DESCRIPTION (provided by applicant): Obesity has reached epidemic proportions and its secondary health consequences are dependent on white adipose tissue (WAT) distribution with visceral fat increasing their incidence and severity. We study a naturally occurring photoperiodic obesity in Siberian hamsters because it shares the differential regulation of visceral vs subcutaneous WAT distribution with humans. This obesity is reversible by moving hamsters from long 'summer-like' to short 'winter-like' days (SDs) and is mediated by pineal melatonin (MEL). SDs trigger a greater increase in lipolysis by visceral vs subcutaneous WAT through the sympathetic nervous system (SNS) innervation of WAT. We continue and broaden our focus on this obesity to include other lipolytic stimuli and ask: How does the SNS control the differential mobilization of lipid from WAT? In Aim 1, we test for convergence of brain SNS outflow projections to different WAT pads, brown fat and adrenal medulla using multiple variants of a transneuronal viral tract tracer (pseudorabies virus [PRV]). We also test for colocalization of neurons activated by acute lipolytic stimuli (cold, glucoprivation) using c-fos, with PRV-labeled WAT SNS outflow neurons. We test for co-localization of neurotransmitter receptors involved in lipolysis, using in situ hybridization with PRV-labeled WAT SNS outflow neurons. We test for differences in SNS drive patterns across WAT pads by these acute lipolytic stimuli via norepinephrine turnover (NETO). In Aim 2, we test if implants of a MEL receptor antagonist at MEL1a receptor + PRV-labeled WAT SNS brain sites block SD-induced lipolysis. We test if SNS WAT denervation blocks SD-induced increases in WAT lipolysis/lipid utilization gene expression. We test which fat cell adrenoceptor subtypes underlie SD-induced SNS lipolysis by in vitro glyerol release assays in isolated adipocytes. In Aim 3, we test if WAT sensory denervation exaggerates acute- (cold, glucoprivation) and chronic (SDs)-induced WAT NETO. These studies will provide new information on the role of the WAT SNS innervation in lipid mobilization and obesity reversal.

This grant proposal is a request for an ADAMHA RSDA (Level II). Humans and other animals display seasonal changes in body weight. The environmental signal for this response and for seasonal reproduction cycles in some species is daylength change, the neuroendocrine transducer of which is the pineal gland and its hormone, melatonin (MEL). Furthermore, the duration is the critical feature of the nocturnal MEL secretion profile. Siberian hamsters will be studied, a species that exhibit dramatic seasonal fluctuations in body fat following exposure to short 'winter-like' photoperiods. The goal of this research is to identify the CNS target sites of MEL that trigger seasonal changes in body fat. The target sites of MEL will be identified by determining: 1) the localization of MEL binding in brain using autoradiography and examining environmental and hormonal influences on MEL binding (e.g., time-of-day, photoperiod, pinealectomy [PINX]), 2) whether lesions of MEL binding sites will eliminate short day-induced changes in body weight and fat, food intake and reproductive status, 3) whether lesions of MEL binding sites block short day responses by eliminating the reception of the short day MEL signal by giving PINX hamsters bearing lesions programmed subcutaneous (s.c.) MEL infusions that mimic the MEL signal associated with transfer to short days, 4) the neural input and output pathways of the target sites using new, highly sensitive anterograde and retrograde tracers, 5) whether short day responses elicited by programmed s.c. MEL infusions can be blocked by selective damage of fibers of passage or cell bodies with microknife cuts and neurotoxins, respectively, in PINX hamsters, 6) whether short day responses can be elicited by directly microinfusing MEL into the target sites in PINX hamsters, and 7) the neurochemical substrate that receives the MEL signal by immunocytochemical methods. These studies should identify the CNS site(s) responsible for the photoperiodic (MEL) control of several seasonal cycles and add to our understanding of how naturally-occurring changes in the environment can have dramatic effects on body fat. The acquisition of the neuroanatomical techniques will compliment the interdisciplinary research program of the Principal Investigator and create a unique research environment for examining brain/behavior/energy metabolism.

Animals in nature must forage for food to help pay their energy debts. When their current energy bills are paid, excess energy can be stored internally as body fat, or externally as a food hoard. Little is known about the distribution of energy among these alternatives and less is known about energy distribution with the addition of other energy consumers - offspring - whether as fetuses during pregnancy, or as young during lactation. The first goal is to determine the energy distribution strategies during the energetically demanding conditions of pregnancy or lactation. A second goal is to determine if a proposed signal of body fat stores, leptin, affects energy distribution strategies. This will accomplished using a species that naturally hoards food, Siberian hamsters. Foraging effort will be controlled by varying the required number of running wheel revolutions to earn food pellets from outside a semi-natural burrow. We will measure the amount of food foraged, eaten, hoarded, stored as fat, and deposit in their offspring during pregnancy, lactation, or with leptin treatment. Collectively, these results should help define the energy budget strategies of animals raising their young. This is a fundamental problem in the regulatory biology of all species.

DESCRIPTION: Obesity is a disease of literally and figuratively enormous proportions. We have chosen a model of naturally occurring obesity-- photoperiod-induced seasonal obesity in Siberian hamsters (Phodopus sungorus sungorus). Siberian hamsters are naturally obese when housed in long "summer- like" days (LDs) and lose body mass, nearly exclusively as body fat, when exposed to short, "winter-like" days. After prolonged SD exposure and/or with increasing daylengths, body and lipid mass increase to their previous obese LD levels. Thus, the obesity is reversible. Three Specific Aims (SA) are proposed, each represented by a set of experiments. 1: What is the functional role of the sympathetic nervous system (SNS) in the photoperiod-induced differential decrease in white adipose tissue (WAT) mass and what is its CNS neuroanatomical substrate? We have demonstrated previously that a relatively separate direct SNS innervation exists for WAT pads. Experiments are designed to test further the separate neurologies associated with the SNS innervation of WAT and the function of this innervation. Therefore, we will answer the questions: 1) Can the SD-induced decreases in body fat occur independently of the SNS innervation of WAT? and 2) Does the relatively separate SNS innervation of WAT converge in the CNS? SA 2: What is the extent of the sensory innervation of WAT and what is its function in the photoperiod-induced changes in body fat? We have extended the initial observation of sensory innervation of WAT in laboratory rats to Siberian hamsters. However, in both studies, only subcutaneous WAT was tested for sensory innervation. In addition, we do not know the neurotransmitter phenotypes of these neurons, nor their function. Therefore, we will answer the questions: 1) What is the extent of the sensory innervation of WAT and what are the neurotransmitter phenotypes of the sensory neurons innervating this tissue? 2) What is the functional role of the sensory innervation of WAT in the SD-induced decreases in body fat? and 3) Is the SNS drive on the tissue increased following sensory denervation? SA 3: What are the neural contributions to total body fat regulation? We have shown that total body fat appears to be regulated based on the results of lipectomy experiments in Siberian hamsters; however, we do not know the mechanism underlying this regulation. Therefore, we will answer the following questions: 1) Is the SNS drive on the fat pads that show compensatory increases in fat pad mass decreased following lipectomy? 2) What is the role of the SNS innervation of WAT in the regulation of total body fat following lipectomy? and 3) What is the role of the sensory innervation of WAT in the regulation of total body fat by lipectomized hamsters? The results of these experiments should provide new information about the importance of the neural innervation of WAT, the regulation of total body fat and in the photoperiodic control of seasonal body fat cycles. In addition, insight into the fundamental processes involved in the development, maintenance and reversal of obesity also should occur.

LAY ABSTRACT
BARTNESS, TIMOTHY

Animals in nature must forage for food to help pay their energy debts. Once they find a food source, the food found has three fates. First, it can be eaten and the energy used immediately if they have an immediate energy debt to pay. Alternatively, if no immediate need exists, the energy can be stored as body fat. Finally, it can be brought to their homes and stored as a food hoard. Little is know about what drives the animals to forage for food, in terms of underlying brain or hormonal factors, or what causes them to eat and burn the calories, eat and store the calories as fat, or hoard the food for later consumption. The purpose of the first experiments is to test some brain chemicals that control these fates of the foraged food, as well as foraging itself by applying them to the brain. The purpose of the second experiments is to test whether changes in body fat affect foraging, food hoarding and food eaten by experimentally removing or adding body fat and measuring these responses. How animals acquire, use and store food energy is a fundamental problem in the regulatory biology of all species.

DESCRIPTION (provided by applicant): Obesity is a life threatening disease with economically devastating consequences. Food intake (FI) reduction is the cornerstone for obesity treatment and has largely been unsuccessful. We offer an alternative approach- reducing food hoarding (FH) that thereby decreases stored foods thus decreasing the opportunities and increasing the efforts required to overeat. Most animals, including humans, hoard food. FH has morphed from a strategy to combat food scarcity into excessive external energy storage due to abundant, inexpensive, calorically-dense food, larger food storage units and improved food shelf lives - factors we believe significantly contribute to the human obesity increases. The most potent stimulator of FH is hunger exacerbating 'normal' food purchases by humans or foraged/hoarded food by rodents. Moreover, because ~85% of FI occurs at home and because obese store more calories per person than their lean counterparts, it is even easier for the obese to overeat. It's not surprising that a common recommendation for weight loss is reducing food stored at home. The innovation of this proposal lies with: 1) study of an unappreciated behavior --FH, 2) the novel animal model of human FH -- Siberian hamsters, a laboratory model that mimics FH in nature, 3) our unique housing system where hamsters earn food pellets to eat and hoard by wheel running, and 4) several approaches: fluorescent in situ hybridization, an unique inhibitor of ghrelin activation (GO-CoA-Tat) and tests of the role of brai peroxisome proliferator- activated receptor gamma (PPAR?) in FH. We hypothesize that a FH network is stimulated by FD causing increases in ghrelin secretion. Ghrelin stimulates neurons bearing ghrelin receptors [growth hormone secretagogue (GHSR)]. These include arcuate nucleus (Arc) AgRP/gamma-amino butyric acid (GABA) neurons and ventral tegmental area (VTA) dopamine (DA) neurons. Stimulation of their GHSRs increases PPAR? found therein. Finally, Arc AgRP neurons release AgRP at several terminal sites and GABA from their projections to the lateral parabrachial nucleus (LPBN) collectively stimulating FH. Our overarching hypothesis is that FD engages a distributed FH network with the hunger-released stomach peptide ghrelin a key initiator that interacts with several central sites and neurochemicals traditionally designated as controllers of FI and reward, as well as a virtually unexplored central factor, PPAR?. We will test this hypothesis in two Specific Aims 1: How does ghrelin initiate the persisting stimulation of FH? and What is role of AgRP neurons and brain PPAR? in the persisting increases in FH? Collectively, we will identify key components driving the prolonged FH increases deepening our understanding of this fundamental ingestive behavior of humans and rodents that we almost exclusively study. This will greatly impact behavioral or drug therapies for obesity and for the genetic disorder Prader-Willi-Syndrome characterized by obesity and uncontrollable FH.