Insulin and phorbol esters affect the maximum velocity rather than the half-saturation constant of 3-O-methylglucose transport in rat adipocytes.

EHD2 Interacts with the Insulin-Responsive Glucose Transporter (GLUT4) in Rat Adipocytes and May Participate in Insulin-Induced GLUT4 Recruitment

“…Adipocytes were isolated from epididymal fat pads of male Sprague-Dawley rats and stabilized as described (35). Subcellular membrane fractions enriched with plasma membranes (PM), nucleus/ mitochondria (NM), high-density microsome (HDM), and low-density microsomes (LDM) were prepared after homogenation as described (36). Subcellular fractions from hypotonically lysed adipocytes were prepared as previously described using hypotonic lysing medium (0.25 mM ATP, 2.5 mM MgCl 2 , 0.1 mM CaCl 2 , 0.1 mM NAD, 0.05 mM NADP, and 1 mM KHCO 3 , pH 7.4) (9).…”

Modulation of GLUT4 and GLUT1 Recycling by Insulin in Rat Adipocytes: Kinetic Analysis Based on the Involvement of Multiple Intracellular Compartments

“…Adipocytes were isolated from epididymal fat pads of male Sprague-Dawley rats and stabilized as described previously (12). Subcellular membrane fractions enriched with plasma membranes (PM), nuclear and mitochondria (NM), high-density microsomes (HDM), and low-density microsomes (LDM) were separated after homogenation as described previously (12). Subcellular fractions from hypotonically lysed adipocytes were prepared as previously described (11).…”

“…The 900g supernatant was centrifuged at 185000g for 2 h, and the pellet (185000g pellet) was resuspended in 600 µL of NaCl/HEPES buffer. A suspension of the 900g or 185000g pellet, without or after sonication, was then layered onto 9 mL of a 5 to 30% glycerol gradient prepared in NaCl/HEPES buffer over 400 µL of a 50% (w/ v) sucrose pad (height of 10 cm), and centrifuged in a 40.2SW rotor (Beckman) at 60000g for 1 h, and 13 fractions were collected from the bottom to the top (P and [1][2][3][4][5][6][7][8][9][10][11][12]. This typically separated GLUT4 into two peaks, a sharp, heavy peak (fractions P, 1, and 2) and a broad, light peak (fractions 3-12).…”

“…Rat epididymal adipocyte intracellular compartments were separated into three fractions (see Materials and Methods), namely, a fraction trapped inside plasma membrane sheets (T) and two plasma membrane-free fractions, H and L. Fraction T was further separated after sonication into fractions T H and T L on glycerol gradient centrifugation. A plasma membrane fraction (PM) was prepared by the conventional homogenization-subcellular fractionation method (12). We have previously shown that the intracellular GLUT4 pools separated in T, H, and L are distinct and may represent the compartments for GLUT4 sorting (SR), storage (ST), and exocytic vesicles (EV), respectively (11).…”

“…Using hypotonic lysis instead of mechanical homogenization, we (11) have recently separated the rat adipocyte intracellular GLUT4 compartments into three distinct fractions, T, H, and L, where T contained all of the plasma membrane in addition to intracellular organelles trapped in it while H and L were exclusively of intracellular organelles with no plasma membrane contamination. In this study, we isolated a plasma membrane fraction (PM) separately by the conventional homogenization and subcellular fractionation method (12) and measured its GLUT4 content, which also allowed us to assess the size of the intracellular GLUT4 compartment trapped in T by a simple subtraction. Distribution of various recycling proteins and endosomal markers further suggested that the intracellular GLUT4 in T, H, and L (G4T, G4H, and G4L, respectively) are putative sorting, storage, and exocytotic vesicle compartments, respectively, in the GLUT4 recycling itinerary.…”