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  • Tanycytic TSPO inhibition induces lipophagy to regulate lipid metabolism and improve energy balance.

Tanycytic TSPO inhibition induces lipophagy to regulate lipid metabolism and improve energy balance.

Autophagy (2019-08-31)
Seolsong Kim, Nayoun Kim, Seokjae Park, Yoonjeong Jeon, Jaemeun Lee, Seung-Jun Yoo, Ji-Won Lee, Cheil Moon, Seong-Woon Yu, Eun-Kyoung Kim
ABSTRACT

Hypothalamic glial 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. Herein, we report that TSPO (translocator protein), an outer mitochondrial protein, is highly enriched in tanycytes and regulates homeostatic responses to nutrient excess as a potential target for an 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. Our 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. 3V: 3rd ventricle; ACAC: acetyl-Coenzyme A carboxylase; AGRP: agouti related neuropeptide; AIF1/IBA1: allograft inflammatory factor 1; AMPK: AMP-activated protein kinase; ARC: arcuate nucleus; Atg: autophagy related; Bafilo: bafilomycin A1; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CNS: central nervous system; COX4I1: cytochrome c oxidase subunit 4I1; FFA: free fatty acid; GFAP: glial fibrillary acidic protein; HFD: high-fat diet; ICV: intracerebroventricular; LAMP2: lysosomal-associated membrane protein 2; LD: lipid droplet; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MBH: mediobasal hypothalamus; ME: median eminence; MEF: mouse embryonic fibroblast; NCD: normal chow diet; NEFM/NFM: neurofilament medium; NPY: neuropeptide Y; OL: oleic acid; POMC: pro-opiomelanocortin-alpha; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; Rax: retina and anterior neural fold homeobox; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RER: respiratory exchange ratio; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TG: triglyceride; TSPO: translocator protein; ULK1: unc-51 like kinase 1; VCO2: carbon dioxide production; VMH: ventromedial hypothalamus; VO2: oxygen consumption.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Insulin solution human, sterile-filtered, BioXtra, suitable for cell culture
Sigma-Aldrich
PK 11195
Sigma-Aldrich
Hexadimethrine bromide, ≥94% (titration)
Sigma-Aldrich
1,2-Dilinoleoyl-3-palmitoyl-rac-glycerol, ≥95% (TLC), liquid
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
TWEEN® 20, viscous liquid
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially fatty acid free, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose, L-glutamine, and sodium bicarbonate, without sodium pyruvate, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
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2-Isopropylmalic acid, 98%
Sigma-Aldrich
Benzamidine, ≥95.0%
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Dorsomorphin, ≥98% (HPLC)
Sigma-Aldrich
STO-609-acetic acid, ≥95% (HPLC), solid
Millipore
Immobilon®-P PVDF Membrane, 1 roll, 27 cm x 3.75 m, 0.45 µm pore size, Hydrophobic PVDF Transfer Membrane for western blotting.
Sigma-Aldrich
Benzamidine hydrochloride hydrate, ≥99%
Sigma-Aldrich
Sodium oleate, ≥99%
Sigma-Aldrich
Carbonyl cyanide 3-chlorophenylhydrazone, ≥97% (TLC), powder
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Triton X-100, for molecular biology
Sigma-Aldrich
Poly-L-lysine solution, 0.01%, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Bafilomycin A1 from Streptomyces griseus, ≥90% (HPLC)
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Fluo-3, suitable for fluorescence, ~70%
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MISSION® TRC2 pLKO.5-puro Non-Target shRNA Control Plasmid DNA, Targets no known genes from any species
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Anti-NeuN Antibody, clone A60, clone A60, Chemicon®, from mouse
Sigma-Aldrich
ECO TWEEN® 20, viscous liquid