Brain control of energy balance : Localization and regulation of proteins involved in the central control of food intake and body weight

Abstract: Obesity has become a worldwide threat to human well-being and is now so common that it is beginning to replace undernutrition and infectious disease as the most significant contributor to ill health. Obesity increases the risk for aquiring serious diseases such as diabetes type 2, hypertension, coronary heart disease and certain forms of cancer. The brain, in particular the hypothalamus, but also the brainstem and neuronal circuits associated with reward, play critical roles in the regulation of body weight. Peripheral signals from adipose tissue, liver and the gastrointestinal tract are delivered to the brain via the circulation and afferent neurons that control food intake and energy balance. The target neurons in hypothalamic nuclei express mediators that either stimulate or inhibit food intake and which affect other neuronal circuits in the brain. The aim of this thesis was to study neuronal circuits involved in regulation of energy balance in relevant areas of the brain. Areas investigated were the hypothalamus, the brainstem raphe nucleus and the diencephalic habenular complex. Special interest has been focussed on leptin, an adipose tissuederived cytokine hormone, which inhibits food intake and increases energy expenditure and thereby reduces body weight. Leptin receptor immunoreactivity was demonstrated in neurons in the hypothalamic arcuate neurons, which expressed STAT3, a leptin-induced signal transduction molecule. Using in situ hybridization and immunohistochemistry, it was shown that STAT3 mRNA and protein levels are down-regulated in vivo in the a rcuate nucleus of obese leptin-deficient ob/ob mice as compared to lean control mice. Together these results support the view that STAT3 in arcuate neurons is essential for leptin action in the hypothalamus. Leptin receptors were also demonstrated in serotonin (5-hydroxytryptamine; 5-HT)-containing neurons of the brainstem dorsal raphe nucleus. Obese leptin-deficient ob/ob mice exhibited significantly down-regulated levels of serotonin transporter mRNA levels in the dorsal raphe nucleus and they displayed behavioural depression, indicating that leptin may act to regulate the brain serotonergic system. The effect of serotonin on food intake is believed to be partly mediated via postsynaptic 5-HT1A receptors. Presence of 5-HT1Areceptor immunoreactivity and mRNA were shown in several hypothalamic neuronal populations producing peptides that are known to affect food intake, e.g. in neuropeptide Y (NPY)/agouti-related peptide (AGRP) and in proopiomelanocortin (POMC)/cocaine- and amphetamine -regulated transcript (CART) containing neurons of the arcuate nucleus as well as in melanin-concentrating hormone (MCH) and orexin-containing neurons in the lateral hypothalamic area (LHA). Efforts were also made to identify classical neurotransmitters in neurons located in areas known to affect feeding. Glutamate is the most abundant excitatory transmitter in the mammalian brain. After synaptic release, glutamate is taken up by the nerve terminal via a plasma membrane-bound protein EAAT3. In the nerve terminal, glutamate is pumped into synaptic vesicles by different vesicular glutamate transporter proteins (VGLUT1-3), which represent unique markers for glutamatergic neurons. Using in situ hybridization and immunohistochemistry, the presence of EAAT3, glutaminase and VGLUT1-3 mRNA and protein was demonstrated in hypothalamic neurons known to be involved in the regulation of energy balance, indicating that several key neurons involved in regulation of energy balance are glutamatergic and/or densely innervated by glutamatergic nerve terminals. Whereas orexigenic NPY/AGRP neurons situated in the ventromedial part of the arcuate nucleus contain EAAT3, glutaminase and VGAT but lack VGLUT 1-3 and therefore should be considered as GABAergic, it is shown for the first time that anotexigenic POMC/CART neurons of the ventromedial arcuate nucleus contain EAAT3, glutamate and VGLUT2, and therefore should be considered to be glutamatergic. The family of G protein-coupled receptors (GPCRs) is one of the largest protein families in the mammalian genome. A novel and previously not identified orphan GPCR was cloned and identified. In rat brain, in situ hybridization showed that GPCR-2037mRNA expression was exclusively detected in neurons of the habenular complex, indicating a unique function for this orphan GPCR. A role for the habenular complex in feeding behaviour may be related to reward mechanisms.