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Item Cilia Associated Signaling in Adult Energy Homeostasis(2022-05) Bansal, Ruchi; Berbari, Nicolas F.; Perrin, Benjamin J.; Mastracci, Teresa L.; Baucum, Anthony J.; Dunn, Kenneth W.Primary cilia are solitary cellular appendages that function as signaling centers for cells in adult energy homeostasis. Here in chapter 1, I introduce cilia and how dysfunction of these conserved organelles results in ciliopathies, such as Bardet-Biedl Syndrome (BBS), which present with childhood obesity. Furthermore, conditional loss of primary cilia from neurons in the hypothalamus leads to hyperphagia and obesity in mouse models of ciliopathies. Classically, cilia coordinate signaling often through specific G-protein coupled receptors (GPCRs) as is the case in both vision and olfaction. In addition, neurons throughout the brain including hypothalamic neurons possess primary cilia whose dysfunction contributes to ciliopathy-associated obesity. How neuronal cilia regulate the signaling of GPCRs remains unclear and many fundamental cell biology questions remain about cilia mediated signaling. For example, how cilia coordinate signaling to influence neuronal activity is unknown. To begin to address some of these cell biology questions around neuronal cilia, chapter 2, describes the development and use of a system for primary neuronal cultures from the hypothalamus. Using this system, we found that activation of the cilia regulated hedgehog pathway, which is critical in development, influenced the ability of neurons to respond to GPCR ligands. This result highlights the role of the developmentally critical hedgehog pathway on terminally differentiated hypothalamic neurons. One challenge facing the cilia field is our ability to assess cilia in large numbers without potential bias. This is especially true in tissues like the brain, where cilia appear to have region-specific characteristics. Work included in Chapter 3 describes the use of a computer-assisted artificial intelligence (Ai) approach to analyze cilia composition and morphology in a less biased and high throughput manner. Cilia length and intensities are important parameters for evaluation of cilia signaling. Evidence suggests that activation of some ciliary GPCRs results in shortening of cilia whereas deviations from normal cilia length in mutant phenotypes affects normal physiological processes such as decreased mucociliary clearance. Therefore, to analyze a large number of cilia, we describe the use of the Ai module from in vitro and in vivo samples in a reproducible manner that minimizes user bias. Using this approach, we identified that Mchr1 expression is significantly stronger in the cilia of paraventricular nucleus than that in the arcuate nucleus of adult mice. Work in Chapter 4 continues to explore the integration between hedgehog pathway and ciliary GPCR signaling in the central nervous system, and its relevance with energy homeostasis. We evaluated the hedgehog ligand in the plasma of mice in acute and long-term metabolic changes and identified that the activity of the ligand changed under altered metabolic conditions. We also developed a genetic mouse model where hedgehog signaling was constitutively active in neuronal cilia. These mice become hyperphagic and obese. These results further emphasize the potential role of the hedgehog signaling pathway in regulation of feeding behavior in adult vertebrates. Overall, results from this work will provide a better understanding of the defects not only underlying ciliopathy-associated obesity but may also reveal more common mechanisms of centrally mediated obesity. In addition, the tools I have developed will help in understanding how neuronal cilia are used for intercellular communications and ultimately how they regulate behaviors like feeding.Item Enhancing hepatic mitochondrial fatty acid oxidation stimulates eating in food-deprived mice(APS, 2015-01) Mansouri, Abdelhak; Pacheco-López, Gustavo; Ramachandran, Deepti; Arnold, Myrtha; Leitner, Claudia; Prip-Buus, Carina; Langhans, Wolfgang; Morral, Núria; Department of Medical & Molecular Genetics, IU School of MedicineHepatic fatty acid oxidation (FAO) has long been implicated in the control of eating. Nevertheless, direct evidence for a causal relationship between changes in hepatic FAO and changes in food intake is still missing. Here we tested whether increasing hepatic FAO via adenovirus-mediated expression of a mutated form of the key regulatory enzyme of mitochondrial FAO carnitine palmitoyltransferase 1A (CPT1mt), which is active but insensitive to inhibition by malonyl-CoA, affects eating and metabolism in mice. CPT1mt expression increased hepatocellular CPT1 protein levels. This resulted in an increase in circulating ketone body levels in fasted CPT1mt-expressing mice, suggesting an increase in hepatic FAO. These mice did not show any significant changes in cumulative food intake, energy expenditure, or respiratory quotient after 4-h food deprivation. After 24-h food deprivation, however, the CPT1mt-expressing mice displayed increased food intake. Thus expression of CPT1mt in the liver increases hepatic FAO capacity, but does not inhibit eating. Rather, it may even stimulate eating after prolonged food deprivation. These data do not support the hypothesis that an increase in hepatic FAO decreases food intake.