Distribution and connectivity of messenger molecules in the control of energy metabolism: focus on neuropeptides and calcium binding proteins

Feeding is an essential and complex behavior which aims to provide the energy required for maintaining physiological homeostasis. The drive to feed is a powerful stimulus arising from metabolic demands, and reinforced by evolutionary pressure. The current epidemic in obesity, and associated disorders such as diabetes, makes it clinically vital to understand the mechanisms behind the control of energy metabolism. Feeding is a process governed by the central nervous system (CNS); particularly through the interplay between different hypothalamic nuclei. At the heart of the feeding neuro-circuitry lies the arcuate nucleus (ARC) which acts as a metabolic sensor, taking stock of the supply and demands of energy in the body, and coordinating food intake and energy expenditure. The work in this thesis aimed to explore the neuro-anatomical substrate of metabolic control, and the mediators involved. The ARC contains two distinct sets of functionally antagonistic neurons. One group of neurons express the orexigenic peptides, neuropeptide Y (NPY) and agouti gene related peptide (AGRP); while the other set expresses the anorexigenic peptides, proopiomelanocortin (POMC) and cocaineand amphetamine-regulated transcript. In paper V, we describe the histochemistry of NPY/AGRP and POMC neurons with regard to their anatomical interrelationship at the cell body and terminal level. A common experimental problem is that the ARC NPY cell bodies are difficult to distinguish and visualize in electrophysiological experiments and for immunohistochemistry. Therefore, in paper III, a novel transgenic mouse which expresses bright Renilla green fluorescent protein in NPY neurons was generated. Using this model, a comprehensive map of NPY-expressing cells in the CNS was generated and the effects of the satiety-inducing gut-brain bombesin peptides on ARC neurons were explored. Bombesin was found to exert powerful depolarizing actions on NPY and POMC neurons alike. Calcium binding proteins (CaBPs) have been used extensively to delineate neuronal populations, but the ARC has not yet been subjected to such analysis. In Paper IV we show that three major CaBPs (calbindin D-28k, calretinin, and parvalbumin) are all expressed in the ARC, but displayed little co-localization with previously described cell groups. One exception was POMC neurons, of which distinct subpopulations stained for calbindin D-28k and calretinin, respectively. Another CaBP, nucleobindin 2 (NUCB2; also known as nesfatin), has recently been proposed as a central anorexigenic mediator. In Paper I, the CNS distribution of this protein was shown to include nuclei that participate in all three output channels of metabolic control, i.e. behavioral, endocrine and autonomic modulation. Our data also suggest that NUCB2 may not act as a cleaved and secreted messenger as proposed, but rather may play an intracellular role. The wide distribution of NUCB2 in the neuroendocrine system prompted us to explore this protein in the pancreas (Paper II). We show that NUCB2 is exclusively expressed in insulin-producing β cells, and that islet NUCB2 is dramatically decreased in the diabetic Goto-Kakizaki rat, an effect that is normalized by fasting. These data indicate that NUCB2 may play a role in metabolic control also outside of the CNS. LIST OF PUBLICATIONS The work in this thesis consists of the following communications. They will be referred to by their Roman numerals. I. Foo KS, Brismar H, Broberger C (2008). Distribution and neuropeptide coexistence of nucleobindin2 mRNA/nesfatin-like immunoreactivity in the rat central nervous system. Neuroscience; 156: 563-579. II. Foo KS*, Brauner H*, Ostenson C-G, Broberger C (2010). Nucleobindin2/Nesfatin in the endocrine pancreas: Distribution and relationship to glycaemic state. Journal of Endocrinology; 204 (3): 255-63. III. van den Pol, A. , Yao Y, Fu LY*, Foo K*, Huang H, Coppari R, Lowell BB, Broberger C (2009). Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons. Journal of Neuroscience; 29(14): 4622-39 IV. Foo KS and Broberger C (2010). Expression and co-localization patterns of calbindin-D28k, calretinin and parvalbumin in the rat hypothalamic arcuate nucleus. Manuscript V. Foo KS and Broberger C (2010). Projections and anatomical interactions between Neuropeptide Y/Agouti gene-related peptide and ProOpiomelanocortin neurons in the arcuate nucleus of the rat. Manuscript * Equal contribution.