Fat Cell Plasma Membranes Research Articles

The effect of insulin on fat cells. An insulin degrading system extracted from plasma membranes of insulin responsive cells.

The major effects of insulin on glucose, fatty acid and amino acid metabolism and on ion flux are initiated by the interaction of insulin with certain unidentified components of the cell membrane. These early events in the action of insulin were studied further with purified plasma membranes prepared from rat epididymal adipose cells. The binding of 125I-labeled insulin to fat cell plasma membranes gives results quantitatively similar to those reported previously with unlabeled insulin. In addition, intact fat cells, plasma membranes from fat cells and a soluble extract from the plasma membranes contain a highly active insulin degrading system. Both the capacity of the plasma membranes to bind insulin and the insulin degrading activity of the membrane extract can be inhibited by the addition of unlabeled insulin to the incubation medium even though albumin is present in excess. The degraded products have not been fully identified but are soluble in 10 per cent trichloroacetic acid, nonreactive with anti-insulin guinea pig serum, nonadsorbable to a 125I adsorbing resin and, therefore, probably represent fragments of the insulin molecule. When isolated fat cells are treated with trypsin they become insulin unresponsive. Plasma membranes derived from such cells have a reduced capacity to bind insulin, and the insulin degrading system of the membranes or in the soluble extract from the membranes is inactive. This suggests that both the insulin receptor and the insulin degrading system are located on the external surface of the plasma membranes of the fat cells. It is postulated that an insulin degrading system of the plasma membrane may be one of the physiological mechanisms by which the response of fat cells to insulin is terminated.

Loss of adenyl cyclase hormone receptors during purification of fat cell plasma membranes.

The response of adenyl cyclase to glucagon, ACTH and epinephrine was determined during the various steps of a fractionation scheme yielding purified fat cell plasma membrane. The adenyl cyclase activity of the homogenate was stimulated about 5X by 10.0 HIM NaF, 7X by 5.0 μg of epinephrine/ml, 3.5X by 500 munits of ACTH/ml and 5.5X by 10.0 pig of glucagon/ml and a combination of the three hormones was non-additive, producing only a 7X response. During the fractionation process the hormonal responsiveness was lost in distinctive amounts and patterns so that the final plasma membrane adenyl cyclase had none of the original glucagon, 20% of the original ACTH and 30% of the original epinephrine responses. During the purification steps 70% of the homogenate adenyl cyclase activity was lost in a pattern distinct from the loss of hormone receptors and the loss could not be restored or prevented. The ACTH and epinephrine stimulation of the plasma membrane adenyl cyclase was blocked by EGTA and propranolol, respect...

Spectroscopic evidence for β structure in rat adipocyte plasma membrane protein

Infrared spectra in the Amide I region of fat cell plasma membrane indicates the presence of a significant proportion of β conformation in the membrane proteins. This is noted both in spectra of dried membrane films and membrane suspensions in 2H 2O. The effect of various solvents is described. This finding illustrates the diversity in protein conformation in plasma membranes obtained from various mammalian cell types.

Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells

1. 1. Plasma membrane and endoplasmic reticulum of free fat cells were isolated in high yield as a mixed microsomal fraction by sucrose gradient centrifugation. 2. 2. The semipermeable behavior of these particles was explored by observing shifts in isopycnic density in continuous polymer (Dextran) gradients over a range of pH and ionic conditions. 3. 3. Using a discontinuous Dextran gradient, a plasma membrane fraction was partially purified. The plasma membrane fraction showed a 12–16-fold enrichment of 5′-nucleotidase over the homogenate. Na +-K +-stimulated ATPase was recovered in highest specific activity in this fraction. Mitochondrial contamination was 3% where endoplasmic reticulum contamination was 27%. 4. 4. A sensitive radioassay for 5′-nucleotidase is described.

Adenyl Cyclase in Fat Cells: III. STIMULATION BY SECRETIN AND THE EFFECTS OF TRYPSIN ON THE RECEPTORS FOR LIPOLYTIC HORMONES

Abstract The similarity in the chemical structures of secretin and glucagon prompted an investigation of the comparative effects of these hormones on adenyl cyclase activity in ghosts of rat fat cells and in a preparation of purified plasma membranes from rat liver. Secretin did not activate adenyl cyclase or inhibit the stimulatory effects of glucagon on an adenyl cyclase system in plasma membranes of rat liver. Secretin and glucagon stimulated adenyl cyclase activity in ghosts of fat cells. Secretin was a more potent stimulator of the enzyme; concentrations of secretin and glucagon giving half-maximal activities were, respectively, 0.1 and 0.3 µg per ml, and maximal activity given by secretin was about twice that given by glucagon. Secretin was also more effective than glucagon in stimulating lipolysis in isolated fat cells. Combination of secretin and glucagon at maximal concentrations (20 µg per ml) did not give additive adenyl cyclase activities in ghosts, indicating that the hormones activate the same enzyme system in fat cells. Ethylene glycol bis(β-aminoethyl ether)-N,N'-tetraacetic acid, a chelator of calcium, abolished the effects of adrenocorticotropin (ACTH) on adenyl cyclase in ghosts but did not inhibit the effects of secretin, glucagon, thyroid-stimulating hormone, and luteinizing hormones. 1-(2,4-Dichlorophenyl)-1-hydroxy-2-t-butylamino)ethane hydrochloride, a β-adrenergic agent, stimulated adenyl cyclase and inhibited the effects of epinephrine. DCB did not inhibit the effects of secretin, glucagon, and ACTH. Pretreatment of adipose tissue with trypsin decreased by 44% the lipolytic response of isolated fat cells to glucagon, but it had no effect on their response to secretin and ACTH. Glucagon did not stimulate adenyl cyclase activity in ghosts prepared from trypsin-treated fat cells; the effects of secretin and ACTH on the enzyme were reduced by 60 and 40%, respectively. Trypsin treatment had no effect on the activation of adenyl cyclase by fluoride ion and epinephrine. It is concluded that secretin activates adenyl cyclase in fat cells via a receptor that is distinct from the receptors for glucagon and the other lipolytic hormones. It is suggested that the receptors for the peptide hormones are protein in character and are localized on the outer surface of the plasma membrane of fat cells.

Metabolism of Isolated Fat Cells: V. PREPARATION OF “GHOSTS” AND THEIR PROPERTIES; ADENYL CYCLASE AND OTHER ENZYMES

Abstract A procedure is described for preparing sacs of plasma membrane from free fat cells. The resultant spherical were essentially devoid of fat and contained 25% of the mitochondria, 2% of the soluble glycolytic enzymes measured (glucose 6-phosphate dehydrogenase, fructose 1,6-diphosphate aldolase, lactic dehydrogenase), and 15% of the hexokinase activity originally present in fat cells. A large fraction of the hexokinase activity was associated with particles. Some ghosts were nucleated, but all contained undefined particles and vesicles. The plasma membrane of ghosts was semipermeable to ions and sucrose and thus retained some of the characteristics of permeability shown by the plasma membrane of intact fat cells. Adenyl cyclase activity was found in ghosts and in a particulate fraction containing mitochondria released during lysis of fat cells. Ghosts contained 62% of the adenyl cyclase activity, with specific activities that averaged 2.5 times that found in the mitochondrial fraction. The enzyme was not dissociated from membranes by sonic oscillation. Adrenocorticotropin stimulated adenyl cyclase activity in both ghosts and mitochondria fractions, the effect of the hormone being greater on ghosts.