Abstract
The neural circuits involved in energy homeostasis are complex and include multiple brain regions and neuropeptides. The many functions of the different neuropeptide systems in the hypothalamus have been described; however, the specific roles of the different neuropeptides in specific hypothalamic areas are not fully understood. The overall aim of
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this thesis was to modify leptin and melanocortin (MC) systems with viral vectors in order to further determine the role of leptin and MC signaling in food intake and energy expenditure. The first experiments were performed to determine the most optimal method to transduce neurons of the hypothalamus. The experiments in chapter 2 showed that adeno-associated viral vectors transduced more neurons in the lateral hypothalamus and amygdala than lentiviral vectors. Therefore, the transduction efficiency of AAV vectors in the hypothalamus was further optimized in chapter 3. This study implicated AAV vectors pseudotyped with AAV1 capsids are more efficient in transduction of the hypothalamus than AAV2, AAV8 and mosaic (1/2 and 2/8) capsids. In addition, 1x109 genomic copies of an AAV1 vector in a volume of 1 l is necessary to transduce almost all neurons of a certain nucleus in the hypothalamus, such as paraventricular nucleus and lateral hypothalamus. In chapter 4 the most optimal method to overexpress a gene was used to overexpress an antagonist for the brain melanocortin receptors 3 and 4 (MC3R, MC4R), namely Agouti related peptide (AgRP). This research showed that AgRP overexpression in specific hypothalamic nuclei (PVN, LH and VMH) increased body weight and food intake by an increase in meal size. However, there were brain nuclei specific differences in energy expenditure and leptin concentrations. In addition, comparison of AgRP overexpression data with NPY overexpression data showed that, although NPY and AgRP are co-released by neurons of the arcuate nucleus, they use different mechanisms to increase food intake. One disadvantage of neuropeptide overexpression with AAV vectors is that AAV vectors may transduce projection neurons and this may lead to secretion of the neuropeptide at distant projection site and not in the targeted area. In chapter 5 a novel method was developed and tested to establish local release of a neuropeptide minigene at the cell body instead of axonal release, through the use of a Von Willebrand factor signal peptide. Leptin signaling is upstream of MC and NPY signaling and is able to alter the mRNA levels of NPY, AgRP and POMC. In addition, obese animals and humans are leptin resistance. It is suggested that upregulation of suppressor of cytokine signaling 3 (Socs3)expression may be involved in the development of leptin resistance. Chapter 6 investigated the effects of knockdown of Socs3 in the mediobasal hypothalamus of rats which were fed a high fat high sucrose diet. In contrast to expectations, MBH-Socs3 knockdown were significant heavier and fatter than control rats at the end of the experiment. MBH-Socs3 knockdown increased NPY mRNA levels and this increase in NPY may explain the effects observed on energy balance such as decrease in locomotor activity and alterations in meal patterns.
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