Leptin targets in the brain regulating body weight : receptors and down-stream mediators of leptin in neurons of the hypothalamus and brainstem

Abstract - Obesity is a disease which has become a global epidemic that is beginning to replace undernutrition and infectious diseases as the most significant contributor to ill health in the world. In particular, obesity is associated with diabetes mellitus, hypertension and coronary heart disease. The brain, primarily the hypothalamus, plays a fundamental role in regulating body weight. During the last decade our knowledge on the specific mediators and neuronal networks that regulate food intake and body weight has increased dramatically. An important contribution to the understanding of hypothalamic control of food intake has been the characterization of the ob gene product (leptin) via positional cloning. Leptin (from the Greek word leptos meaning thin) is a cytokine hormone produced and secreted by adipose tissue, which acts via neuronal circuits in the brain to reduce food intake and body weight. Absence of leptin as seen in ob/ob mice results in obesity, diabetes and infertility. The hormone leptin binds to and activates specific leptin receptors (Ob-R) and regulates gene transcription via activation of the JAK-STAT signal transduction pathway (JAK = Janus kinase; STAT = signal transducers and activators of transcription). There are at least five different isoforms of Ob-R, one of which has a long intracellular domain (Ob-Rb) and is considered the most important for JAK-STAT signaling. The aim of this thesis was to identify cellular targets for leptin in the brain and thereby clarifying the central mechanisms by which leptin regulates body weight. In situ hybridization showed widespread expression of Ob-R mRNA in the brain. Particularly strong expression was demonstrated in the choroid plexus, representing short Ob-R isoform mRNA, and the hypothalamus, reflecting mainly Ob-Rb mRNA. Using direct immunofluorescence histochemistry, Ob-R immunoreactivity was demonstrated in ventromedial arcuate neurons containing neuropeptide Y (NPY), agouti-related protein (AGRP) and [gamma]-aminobutyric acid (GABA), and in ventrolateral arcuate neurons containing proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART), in corticotropin-releasing hormone (CRH)-containing neurons of the parvicellular part of the paraventricular nucleus, and in melanin-concentrating hormone (MCH)- and hypocretin/orexin-containing neurons of the lateral hypothalamic area, indicating that these neurons are targets for circulating leptin. Transcription factor STAT3, an intracellular leptin mediator in the hypothalamus, was demonstrated in leptin receptor-containing NPY/AGRP/GABA- and POMC/CART-containing neurons of the arcuate nucleus and in MCH and hypocretin/orexin neurons of the lateral hypothalamic area, suggesting that leptin may exert direct effects on these neurons. STAT3 mRNA and protein levels were down-regulated in the arcuate nucleus of obese leptin-deficient ob/ob mice as compared to lean control mice, indicating that STAT3 is an important signal transducer for leptin's action in the hypothalamic arcuate nucleus. The mRNA levels for the glutamic acid decarboxylase (GAD) isoforms GAD65 and GAD67, and mRNA levels for the vesicular GABA transporter (VGAT) were not altered in ob/ob mice, suggesting that chronic leptin-deficiency does not result in altered transcription of these genes. Serotonergic neurons of the dorsal raphe nucleus contain Ob-R and Ob-Rb. Obese ob/ob mice have significantly lower levels of serotonin transporter mRNA and display behavioral depression, suggesting an altered serotonin neurotransmission in the absence of leptin and a role for leptin in the regulation of serotonindependent brain functions. Cholinergic neurons of the dorsal motor nucleus of the vagus nerve (DMX), which innervate the stomach, contain Ob-R/Ob-Rb and STAT3. Leptin inhibits gastric emptying when administered into the fourth ventricle, indicating that leptin also acts via the brainstem to regulate food intake and body weight