FATP2 is a hepatic fatty acid transporter and peroxisomal very long-chain acyl-CoA synthetase

저자: Falcon A, Doege H, Fluitt A, Tsang B, Watson N, Kay MA, Stahl A.

저널: Am J Physiol Endocrinol Metab. 2010 Sep;299(3):E384-93

사용한 제품:  Dynabeads Streptavidin MyOne T1 (Cat# 65601, 65602)

Abstract

Fatty acid transport protein (FATP)2, a member of the FATP family of fatty acid uptake mediators, has independently been identified as a hepatic peroxisomal very long-chain acyl-CoA synthetase (VLACS). Here we address whether FATP2 is 1) a peroxisomal enzyme, 2) a plasma membrane-associated long-chain fatty acid (LCFA) transporter, or 3) a multifunctional protein. We found that, in mouse livers, only a minor fraction of FATP2 localizes to peroxisomes, where it contributes to approximately half of the peroxisomal VLACS activity. However, total hepatic (V)LACS activity was not significantly affected by loss of FATP2, while LCFA uptake was reduced by 40%, indicating a more prominent role in hepatic LCFA uptake. This suggests FATP2 as a potential target for a therapeutic intervention of hepatosteatosis. Adeno-associated virus 8-based short hairpin RNA expression vectors were used to achieve liver-specific FATP2 knockdown, which significantly reduced hepatosteatosis in the face of continued high-fat feeding, concomitant with improvements in liver physiology, fasting glucose, and insulin levels. Based on our findings, we propose a model in which FATP2 is a multifunctional protein that shows subcellular localization-dependent activity and is a major contributor to peroxisomal (V)LACS activity and hepatic fatty acid uptake, suggesting FATP2 as a potential novel target for the treatment of nonalcoholic fatty liver disease.

제품 사용법

Peroxisome purification.

Peroxisomes were isolated according to a modification of a previously described protocol (21). Briefly, 1 ml of ice-cold 250 mM sucrose, 10 mM MOPS (pH 7.4), 0.1 mM EDTA, 0.2 mM DTT, and protease inhibitor cocktail was added to 500 mg of liver. After 60 stokes of a Dounce homogenizer, the liver lysates were centrifuged at 1,000 g for 10 min. The supernatant was centrifuged at 5,000 g for 10 min. The resulting supernatant was mixed with prepared streptavidin magnetic beads (Dynabeads MyOne streptavidin T1 magnetic beads were purchased from Invitrogen (Carlsbad, CA)). The beads were prepared as follows: 100  l (1 mg) of beads were mixed with 500㎕ of PBS; then the beads were washed three times for 30 min with PBS on a magnetic sorter, and the supernatant was removed. The beads were mixed with 10  g of biotinylated anti-PMP70 and 500㎕ of PBS with gentle rotation for 30 min at room temperature, washed three times with PBS, and mixed with 20 nmol of biotin and 500㎕ of PBS with rotation for 30 min at room temperature. Finally, the beads were washed three times with PBS. Prepared beads in 500㎕ of PBS and 500㎕ of the supernatant from the liver homogenates were mixed with gentle rotation at 4°C for 1 h. Peroxisomes were isolated bound to magnetic beads after four washes with PBS.