In the central nervous system (CNS), the hypothalamus is a critical region of the brain that regulates appetite or energy balance by integrating metabolic signals such as nutrients or hormones. Although the action of hypothalamic neural circuits in control of feeding behavior or energy homeostasis widely has been elucidated, recent emerging evidence demonstrates that glial cells, non-neuronal cells, also have important functions to maintain energy balance. Especially, hypothalamic macroglial cells named tanycytes, which line the 3rd ventricle (3V), are components of the hypothalamic network that regulates a diverse array of metabolic functions for energy homeostasis. It has been suggested that tanycytes can sense nutrients such as glucose, amino acids, and lipids, and those nutritional signals may be transmitted to hypothalamic neurons participating in the appetite regulation. However, the mechanisms for the tanycytes-mediated regulation of energy metabolism remain poorly understood. The translocator protein (TSPO, 18kDa), previously discovered as a peripheral benzodiazepine receptor (PBR), is a protein mainly located in an outer-mitochondrial membrane. Recently, TSPO located at the periphery has emerged as a candidate regulator for cellular bioenergetics whereas the understanding of the role of TSPO in the brain related to energy metabolism remains elusive. In this study, I report that TSPO is highly enriched in tanycytes and regulates homeostatic responses to nutrient excess as a potential target for effective intervention in obesity. Administration of a TSPO ligand, PK11195, into the 3V, and tanycyte-specific deletion of Tspo reduced food intake and elevated energy expenditure, leading to negative energy balance in a high-fat diet challenge. Ablation of tanycytic Tspo elicited AMPK-dependent lipophagy, breaking down lipid droplets into free fatty acids, thereby elevating ATP in a lipid stimulus. Activated AMPK in inhibition of TSPO was due to elevation of cytosolic calcium caused by blocking the uptake of mitochondrial calcium. These findings suggest that tanycytic TSPO affects systemic energy balance through macroautophagy/autophagy regulated lipid metabolism, and highlight the physiological significance of TSPO in hypothalamic lipid sensing and bioenergetics in response to overnutrition.
Table Of Contents
Ⅰ. Introduction 1 1. Hypothalamus 1 1.1 Hypothalamic nuclei controlling feeding behavior and energy balance 1 2. Tanycytes 5 2.1 Morphology of tanycytes 5 2.2 The function of tanycytes on the regulation of energy metabolism 5 2.2.1 Metabolic sensing of tanycytes 6 2.2.2 Transporting hormones of tanycytes 7 3. TSPO 9 3.1 General information on TSPO 9 3.2 TSPO and metabolism 10 4. Autophagy and Lipophagy 11 5. Aims of the study 12
Ⅱ. Materials and methods 14 1. Mice 14 2. Cell culture 14 3. Antibodies 15 4. 3rd ventricle (3V) cannulation 16 5. Intracerebroventricular (ICV) injection 16 6. Genotyping 17 7. Stereotaxic injection of adeno-associated virus 17 8. Metabolic measurements 18 9. Glucose tolerance test and insulin tolerance test 19 10. In situ hybridization 19 11. siRNA and plasmid transfection 20 12. Lentivirus-mediated gene expression 20 13. Western blotting 21 14. ATP assay 22 15. Ca2+ imaging 23 16. Immunocytochemistry 23 17. Immunohistochemistry 24 18. BODIPY 493/503 and Lipid spot 610 staining 25 19. LysoTracker staining 25 20. Measurement of TG 26 21. Metabolomic analysis by GC-MS/MS 26 21.1 Free fatty acids analysis 26 21.2 Amino acids and cholesterol analysis 27 22. Assay of AMP:ATP ratio by UPLC 28 23. Real time-PCR 29 24. Statistical analysis 30 Ⅲ. Results 31 1. Hypothalamic TSPO is highly expressed in ependymal cells and tanycytes 31 2. PK11195, a TSPO-specific ligand, in the hypothalamus induces negative energy balance 33 3. A2/29 cells mimic tanycyte-like cells 37 4. Blockade of TSPO increases AMPK activity through modulating mitochondrial Ca2+ buffer-ing system in tanycyte-like cells 37 4.1 Pharmacological and genetic inhibition of TSPO activates AMPK in A2/29 cells 38 4.2 PK11195 activates AMPK through the CAMKK2 pathway in A2/29 cells 38 4.3 Inhibition of TSPO blocks mitochondrial uptake of Ca2+ in A2/29 cells 41 5. Inhibition of TSPO increases autophagy through AMPK activation in A2/29 cells 42 6. Tspo knockdown induces lipophagy and generates free fatty acids in A2/29 cells 48 7. Tanycyte-specific ablation of Tspo prevents obesity in diet-induced obese mice 55 8. Inhibition of TSPO induces lipophagy through AMPK activation in tanycytes in vivo 58 Ⅳ. Discussion 70 1. Lipophagy in tanycytes 70 2. Tanycytic TSPO is a regulator of lipophagy 71 3. Mechanism underlying lipophagy induced by TSPO inhibition: blockade of mitochondrial Ca2+ uptake and AMPK-dependent autophagy 72 4. Inhibition of tanycytic TSPO induces negative energy balance 74 Ⅴ. Conclusion 76 Ⅵ. References 77