Hypothalamic neuronal circuits involved in the regulation of food intake and body weight : histochemical studies in lean rats and obese mutant mice

Abstract: Obesity is a major health problem because of its dramatic increase in prevalence and its association with type 2 diabetes, coronary heart diseases, hypertension and stroke. It is well known that signals generated in response to feeding behavior are integrated in the brain. In particular, neuronal circuits located in the hypothalamus exert powerful effects on food intake and body weight. Specific neuronal populations located in the ventromedial and lateral hypothalamus express mediators, which stimulate or inhibit food intake. Such neurons are targets for peripheral hormones that signal to the brain about the energy status of the organism. The aim of this thesis has been to study proteins and peptides that participate in the hypothalamic control of body weight. in order to better understand the neuronal circuits regulating feeding behavior, the cellular localization of transmitters, receptors and signal transduction molecules have been studied in normal rat hypothalamus. Special interest has been focused on possible alterations in neurochemical markers in animal models with obesity, thereby providing knowledge regarding the mechanisms of pathogenesis underlying the development of obesity. For this purpose obese ob/ob and tub/tub mice were used. The ob/ob mouse, which has a mutation in the ob gene, lacks functional leptin and exhibits a phenotype including severe obesity, insulin resistance and infertility. Leptin is an adipocyte tissue-derived hormone, which inhibits food intake and increases energy expenditure via an action on leptin receptors located in the hypothalamus. A mutation in the mouse tub gene causes maturityonset obesity, insulin resistance, retinal degeneration, and neurosensory hearing loss. Since the tub/tub mouse develops maturity-onset obesity, this obese mouse model is particularly interesting and relevant to human obesity. We have therefore investigated whether the neuronal circuits that participate in hypothalamic control of body weight are altered in the tub/tub mouse. Using immunohistochemistry, gamma-ammobutyric acid (GABA), an orexigenic neurotransmitter, was demonstrated to be present in leptin target neurons of the arcuate nucleus. The GABAergic leptin-target neurons belong to the orexigenic neuropeptide Y (NPY)/agouti-related protein (AGRP)-containing, but not the anotexigenic proopiomelanocortin (POMC)/cocaine- and amphetamme-regulated transcript (CART)-containing cell population of the arcuate nucleus. In order to find out whether leptin acts via GABA to regulate food intake, we compared mRNA levels for GABAergic markers in obese leptin-deficient o/lob mice with lean controls. There were no significant differences in the mRNA levels for glutamic acid decarboxylase (GAD) isoforms GAD65 and GAD67 or the vesicular GABA transporter (VGAT), which suggests that chronic leptin deficiency does not have a major regulatory role on transcription of these genes. However, a role for leptin on GABAergic neurotransmission other than transcriptional regulation cannot be excluded GABA binds to two types of receptors, ionotropic GABAA and metabotropic GABAB receptors. Immunoreactivities for both GABAA and GABAB receptors were demonstrated in several hypothalamic regions associated with regulation of body weight control and colocalized with peptides that are known mediators of ingestive behavior. Orexins, also called hypocretins, are produced exclusively in the lateral hypothalamic area and have important roles in regulation of feeding behavior and arousal. The orexins act via the two orexin receptors, OX-R1 and OX-R2. Immunohistochemical analysis was used to chemically characterize OX-R1 immunoreactive neurons in the hypothalamus. OX-R1-containing neurons were shown to contain several mediators that regulate feeding and drinking behavior. Behavioral and histochemical analysis revealed several abnormalities in obese tub/tub mice. It was concluded that tub/tub are obese as a results of increased eating behavior (hyperphagia). Immunohistochemistry showed presence of abnormally large nerve terminals surrounding blood vessels containing acetylcholine and GABA in the ventromedial part of the arcuate nucleus of obese tub/tub mice, but not in tub/+ mice. In situ hybridization and immunohistochemistry was used to demonstrate that tub/tub mice have a down-regulated expression of AGRP mRNA in the arcuate nucleus combined with lower fluorescence intensity and numbers of AGRP- and NPY-immunoreactive fibers and terminals in the hypothalamus. The results obtained in tub/tub mice may facilitate the understanding of the mechanism underlying the obese phenotype in mice with a mutation in the tub gene.