Fig 1: Effect of CB1 stimulation and the relative intramitochondrial pathway involved in isolated eWAT mitochondria. (A) Western blotting of isolated mitochondria extracts (mito) from rat eWAT compared to total cell lysate (TCL) and cytoplasmic (cyt) fraction. Membranes were incubated with antibodies against NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4, mitochondrial marker), glucose tansporter-1 (Glut1, plasma membrane marker), Ras-related protein Rab-7a (Rab7, endosomes marker), lysosomal-associated membrane protein-2 (LAMP, lysosomes marker), and tubulin (cytoplasm marker). (B) Western blotting of isolated mitochondria extracts (mito) from rat eWAT compared to total cell lysate (TCL) and cytoplasmic (cyt) fraction. Membranes were incubated with antibodies against CB1 receptor, Gi protein, sAC (soluble adenylyl cyclase), and cPKA (catalytic subunit of protein kinase A). (C) Oxygen consumption rate (OCR) in isolated mitochondria from rat eWAT. MPG, malate pyruvate glutamate; ADP, adenosine di-phosphate; Cyt.C, cytochrome C; Ama, antimycin A. The gray area indicates complex-I-dependent respiration (OXPHOS) used for testing cannabinoid agonist effects. (D) Dose-dependent effect of WIN (relative to vehicle, V) on complex-I-dependent OXPHOS respiration in isolated mitochondria from rat eWAT. (E,F) Effect of WIN 1 µM (E) or 2 µM (F) (mean of 3 time points, expressed as a percentage of V) on complex-I-dependent OXPHOS respiration in isolated mitochondria from rat eWAT pretreated with control vehicle, 2 µM CB1 antagonist JD5037, 5 µM sAC blocker KH7, and 500 µM PKA activator 8-br-cAMP. * p < 0.05; ** p < 0.01 from vehicle. # p < 0.05 from Veh pre-treatment.