The Effect of Modifying the Combination of Insulin with Rat Adipose Cells on the Intracellular Levels of Cyclic Amp

1974 ◽  
Vol 46 (1) ◽  
pp. 75-87
Author(s):  
G. Jerums ◽  
D. J. Galton ◽  
C. Gilbert
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Insulin Receptor ◽  
Incubation Medium ◽  
Adrenergic Receptor ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Adipose Cells

1. Isoprenaline (1 μmol/l) and glucagon (1 μmol/l) raised the levels of cyclic AMP in isolated adipose cells of the rat to a maximum after incubation for approximately 15 min. 2. The effect of glucagon was impaired by pretreatment of adipose cells with trypsin (0.2 mg/ml and 2.0 mg/ml) and N-ethylmaleimide (5 mmol/l). The β-adrenergic receptor was insensitive to such forms of treatment. 3. Insulin (430 μunits/ml) lowered the intracellular levels of cyclic AMP in adipose cells stimulated with isoprenaline (1 μmol/l) and glucagon (1 μmol/l). This effect was observed after incubation for 5 min. 4. Pretreatment of cells with trypsin (0.2 mg/ml and 2.0 mg/ml) and N-ethylmaleimide (0.5 and 5 mmol/l) abolished the effect of insulin in decreasing the intracellular levels of cyclic AMP. At the higher concentration of trypsin a rise in intracellular levels of cyclic AMP was observed in the presence of insulin. 5. Similar concentrations of trypsin and N-ethylmaleimide decreased the disappearance of unlabelled insulin from the incubation medium and also decreased the binding of 125I-labelled insulin to isolated fat cells. 6. The effect of insulin on decreasing the intracellular levels of cyclic AMP in modified adipose cells significantly correlated with the disappearance of unlabelled insulin from the medium and with the percentage of total 125I-labelled insulin bound to cells. 7. The possibility is discussed of using the disappearance of insulin or binding of 125I-labelled insulin to adipose cells as a measure of insulin-receptor availability on the plasma membrane of fat-cells.

scholarly journals Insulin-Receptor Interaction in Isolated Fat Cells

1969 ◽  
Vol 244 (19) ◽  
pp. 5181-5188
Author(s):  
T. Minemura ◽  
Oscar B. Crofford
Keyword(s):  
Insulin Receptor ◽  
Receptor Interaction ◽  
Fat Cells ◽  
Isolated Fat Cells

scholarly journals Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor

1985 ◽  
Vol 232 (1) ◽  
pp. 71-78 ◽  
Author(s):  
J A Hedo ◽  
I A Simpson
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Insulin Receptor ◽  
Golgi Complex ◽  
Microsomal Fraction ◽  
Primary Cultures ◽  
Sodium Dodecyl ◽  
High Density ◽  
Low Density ◽  
Adipose Cells

We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.

Distribution of cyclic AMP phosphodiesterase in adipose tissue from trained rats

1986 ◽  
Vol 61 (4) ◽  
pp. 1546-1551 ◽  
Author(s):  
K. A. Kenno ◽  
J. L. Durstine ◽  
R. E. Shepherd
Keyword(s):  
Cyclic Amp ◽  
Specific Activity ◽  
Particulate Fraction ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Sprague Dawley ◽  
Enzyme Form ◽  
Cyclic Amp Phosphodiesterase ◽  
Sprague Dawley Rats ◽  
Michaelis Constants

Fat cells were isolated from sedentary and exercise trained female Sprague-Dawley rats and cyclic AMP phosphodiesterase (cyclic AMP-PDE) activities were determined from crude homogenates of the fat cells in the whole homogenate, P5, P48, and S48 fractions. Exercise training resulted in a significant increase in the mean specific activity of cyclic AMP-PDE (pmol X min-1 X mg-1) from the whole homogenate and S48 fraction at cyclic AMP concentrations of 4, 8, and 16 microM and in the P48 fraction at 8 and 16 microM cyclic AMP. Cyclic AMP-PDE kinetic plots according to Lineweaver-Burk for the calculation of Michaelis constants (Km) and maximum enzyme velocities (Vmax) were nonlinear, indicating both a low and high enzyme form. The Michaelis constants were significantly lower in trained rats than those of its control for the high Km form of cyclic AMP-PDE in the whole and soluble fractions and for the low Km form of the P5 particulate fraction. The Vmax of the high Km form of the P48 particulate fraction from trained animals was also significantly higher than that found in its control. Phosphodiesterase inhibition by methylxanthines in the various fractions was similar in both trained and sedentary animals. These changes in specific activity, Michaelis constants, and Vmax of cyclic AMP-PDE from crude homogenates of isolated fat cells from exercise trained animals may account for the decreased intracellular levels of cyclic AMP following catecholamine stimulation of isolated fat cells from trained rats.

scholarly journals The properties and extracellular location of 5′-nucleotidase of the rat fat-cell plasma membrane

1975 ◽  
Vol 146 (3) ◽  
pp. 625-633 ◽  
Author(s):  
A C Newby ◽  
J P Luzio ◽  
C N Hales
Keyword(s):  
Plasma Membrane ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Fat Cell ◽  
Cell Plasma Membrane ◽  
Intact Cells ◽  
Low Concentrations ◽  
Cell Plasma ◽  
Atp Analogues ◽  
Extracellular Location

1. A phosphohydrolase specific for 5′-nucleotides was characterized by using a particulate fraction from isolated fat-cells. 2. The activity of intact cells towards 5′-AMP was studied. 3. The activity in either situation had the same KM for AMP (45 muM) and was inhibited by low concentrations of ATP (less than 50 muM), but less potently by the ATP analogues AMP-P(CH2)P(adenylyl (β γ-methylene)diphosphonate) and AMP-P)NH)P (adenylylimidodiphosphate). 4. Homogenization of intact fat-cells caused no increase in activity and at least 85% of the activity was recovered in the particulate preparation. 5. The preparation of fat-cells used in this work was not freely permeable to AMP. 6. The ability of intact fat-cells to hydrolyse AMP implies that 5′-nucleotidase is an ectoenzyme in fat-cells. 7. Concentrations of ATP 100 times lower than intracellular concentrations inhibit the enzyme when added extracellularly to intact fat-cells, implying that this effect is also medicated at the extracellular face of the membrane. 8. Antibodies raised to whole liver cells and whole fat-cells inhibit 5′-nucleotidase in intact cells. 9. Incubation of intact fat-cells with adrenaline (1 mug/ml) or insulin (50 mui.u./ml) failed to alter the KM or Vmax. of the enzyme.

scholarly journals PREPARATION AND CHARACTERIZATION OF A PLASMA MEMBRANE FRACTION FROM ISOLATED FAT CELLS

1970 ◽  
Vol 44 (2) ◽  
pp. 417-432 ◽  
Author(s):  
Daniel W. McKeel ◽  
Leonard Jarett
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Fat Cells ◽  
Isolated Fat Cells ◽  
Adenyl Cyclase Activity ◽  
Cytochrome C Reductase ◽  
Plasma Membrane Fraction

A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25–30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.

Effect of amitriptyline on lipolysis and cyclic AMP concentration in isolated fat cells

Metabolism ◽  
1972 ◽  
Vol 21 (3) ◽  
pp. 223-229 ◽  
Author(s):  
Fred C. Lovrien ◽  
Ann A. Steele ◽  
Joseph D. Brown ◽  
Daniel B. Stone
Keyword(s):  
Cyclic Amp ◽  
Fat Cells ◽  
Isolated Fat Cells

scholarly journals Multiple endosomal recycling pathways in rat adipose cells

1998 ◽  
Vol 331 (3) ◽  
pp. 829-835 ◽  
Author(s):  
Konstantin V. KANDROR ◽  
Paul F. PILCH
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Sedimentation Coefficient ◽  
Membrane Vesicles ◽  
Average Diameter ◽  
Subcellular Fractionation ◽  
Adipose Cells

Adipose and skeletal-muscle cells can translocate several membrane proteins from intracellular compartment(s) to the cell surface in an insulin-dependent fashion. Among these proteins is Glut4, a physiologically important glucose transporter which mediates insulin's effect on blood glucose clearance. Under basal conditions, Glut4 is localized in uniform, intracellular membrane vesicles with an average diameter of 50–70 nm and a sedimentation coefficient of 100–120 S. The nature of this compartment and its trafficking pathway to the plasma membrane is still unresolved. We show here that, in addition to Glut4, the aminopeptidase gp160 or insulin-responsive aminopeptidase (‘IRAP’), sortilin, and an acutely recycling population of the insulin-like growth factor-II/mannose 6-phosphate receptor, this compartment includes 60% of the intracellular population of the transferrin receptor. We used subcellular fractionation, cell-surface biotinylation, and radioactive-ligand (125I-transferrin) uptake to demonstrate that the transferrin receptor recycles between this compartment and the plasma membrane in response to insulin along with Glut4 and other protein components of these vesicles. The co-localization of Glut4 and several endosomal markers in the terminally differentiated fat-cells during several stages of their cycling pathways suggests that the ‘Glut4 pathway ’ may derive from the hormone-insensitive endosomes of undifferentiated preadipocytes. The insulin receptor is excluded from Glut4-containing vesicles in both insulin-stimulated and unstimulated adipocytes, and thus it is likely to traffic independently from Glut4 through different intracellular compartments. Our data show that, in adipose cells, the ligand-dependent recycling pathway of the insulin receptor is structurally separated from the ligand-independent pathway of the transferrin receptor, and that Glut4 is specifically targetted to the latter.

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