Insulin action in isolated fat cells. II. Effects of divalent cations on stimulation by insulin of protein synthesis, on inhibition of lipolysis by insulin, and on the binding of 125I-labelled insulin to isolated fat cells.

The effects of ommission of Ca2+ and Mg2+ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca2+ + Mg2+)-free incubation medium incorporation of L-[14C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[14C]leucine incorporation only in the presence of added CaCl2 or MgCl2. Incubation of the cells in the (Ca2+ + Mg2+)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of 125I-labelled insulin to the fat cells was reduced in the absence of Ca2+ and Mg2+ but was not abolished, even in the presence of EDTA. Ca2+ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg2+, Sr2+ and Ba2+. Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

Effect of glucose on beta-adrenergic induced downregulation of insulin receptor binding in human fat cells

Population genetics followed by purification suggested that "null" mutation in the mitochondrial E2 isozyme of human aldehyde dehydrogenase (EC 1.2.1.3) occurred in the Oriental individuals who are sensitive to alcohol. This report demonstrates that the Oriental E2, thought to be a "null" mutant, is catalytically active and except for maximal velocity and isoelectric point, identical with Caucasian E2 isozyme. The data presented are not inconsistent with mutation but preclude active site of the enzyme as the point at which alteration has occurred; they are, however, inconsistent with "null" mutation.     

Insulin action in isolated fat cells: II. Effects of divalent cations on stimulation by insulin of protein synthesis,on inhibition of lipolysis by insulin,and on the binding of 125I-labelled insulin to isolated fat cells

The effects of omission of Ca²⁺ and Mg²⁺ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca²⁺ + Mg²⁺)-free incubation medium incorporation of L-[¹⁴C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[¹⁴C]-leucine incorporation only in the presence of added CaCl₂ or MgCl₂. Incubation of the cells in the (Ca²⁺ + Mg²⁺)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of ¹²⁵I-labelled insulin to the fat cells was reduced in the absence of Ca²⁺ and Mg²⁺ but was not abolished, even in the presence of EDTA. Ca²⁺ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg²⁺, Sr²⁺ and Ba²⁺.Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

Ca binding to the human red cell membrane: characterization of membrane preparations and binding sites.

Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces...     

Lectin binding patterns in normal, metaplastic, and neoplastic gastric mucosa.

We investigated lectin binding patterns on tissue specimens of normal and metaplastic gastric surface mucosae, gastric adenomas, and intestinal and diffuse-type gastric carcinomas. Compared with normal gastric mucosa, metaplastic mucosa exhibited an increase of ConA binding and decreases of WGA, PNA, UEA-1, and DBA binding in the cytoplasm, and decreases of ConA, PNA, and UEA-1 binding at the luminal surface. Intestinal carcinomas were similar to metaplastic gastric surface mucosa in ConA, WGA, and UEA-1 binding in the cytoplasm, while diffuse-type carcinomas were similar to normal gastric mucosa in WGA and UEA-1 binding in the cytoplasm. Adenomas were similar to intestinal carcinomas in ConA and UEA-1 binding in the cytoplasm, but were different from intestinal carcinomas in Con A and UEA-1 binding at the luminal surface. For UEA-1, normal and metaplastic gastric surface mucosae did not show a significant difference between the blood type A, AB, B group and the O group. Intestinal and diffuse carcinomas and adenomas also did not show such a difference between the blood groups. For DBA, normal gastric surface mucosa showed a significant difference between the blood type B, O group and the A, AB group. Normal gastric mucosa of the blood type A, AB group was frequently positive for DBA binding in the cytoplasm and at the luminal surface. Metaplastic mucosa did not show a significant difference between the blood groups. Intestinal and diffuse-type carcinomas and adenomas also did not show a difference between the blood groups. DBA binding in the cytoplasm of intestinal carcinomas and adenomas was more frequently positive than that of normal and metaplastic mucosae, except for normal gastric mucosa of the blood type A, AB group. Compared with diffuse-type carcinomas, intestinal carcinomas were accompanied by a significant increase of ConA binding and decreases of WGA and PNA binding in the cytoplasm.     

Thyrotropin interactions with human fat cell membrane preparations and the finding of soluble thyrotropin binding component.

Three criteria are presented that permit a distinction to be made between the conventional rapid equilibrium Random Bi Bi mechanism and a unique Ordered Bi Bi mechanism in which the rapid equilibrium assumption is made[C. Frieden (1976) Biochem. Biophys. Res. Commun., 68, 914–917]. A choice between these mechanisms cannot be made using kinetic procedures now in vogue for differentiating between Ordered and Random Bi Bi mechanisms. However, transition state analogs, pulse-chase experiments, and pre-equilibrium isotope exchange procedures can be employed to make this distinction.     

Equilibrium binding of insulin to rat white fat cells at 15 degrees C

Equilibrium binding of insulin to isolated rat epididymal fat cells was investigated. A temperature of 15 degrees C was chosen for the study to minimize lysosomal degradation of insulin. Indeed, medium insulin lost only 1% of its precipitability in trichloroacetic acid during the 4-h incubation required to approach equilibrium. Binding was measured by a method that did not perturb the equilibrium of the system. A new formalism for analyzing binding data in general was introduced. A correction for trapping of insulin in the interstitial space of cell pellets was both necessary and sufficient to derive specific binding data from raw observations. Thus, so-called "nonspecific binding" was unmasked as a misnomer, and the expression "correction for trapping" was proposed as a substitute. Equations for one and two independent classes of binding sites were fit to the data by the method of maximum likelihood, and the best fit was selected based on Akaike's information criterion, as adapted for a constant fractional error. More than 99.7% of the binding sites were found to be describable by a simple binding isotherm with Kd,app = 8.8 multiplied by over divided by 1.3 nM. Less than 0.3% sites had a higher affinity (Kd approximately equal to 8 multiplied by over divided by 3 pM). There were 99,000 x/divided by 1.6 binding sites/cell. These equilibrium parameters are in agreement with values derived from a kinetic analysis, presented in the subsequent paper (Lipkin, E. W., Teller, D. C., and de Ha?n, C. (1986) J. Biol. Chem. 260, 1702-1711).     

Kinetics of insulin binding to rat white fat cells at 15 degrees C

The kinetics of insulin binding to isolated rat epididymal fat cells was investigated at 15 degrees C, at which temperature the system was simplified by the absence of lysosomal insulin degradation. The data were fit by maximum likelihood criteria with differential equations describing a number of models for the interaction of insulin and cells. Among those models that yielded a fit, the selection criteria were minimization of the Akaike information criterion and compatibility of the overall equilibrium constant for the system calculated from rate constants with the previously obtained experimental value. The results of the analysis indicated that insulin, I, first reversibly bound to cell surface receptors, R, whereupon this initial insulin-receptor complex, RI, reversibly altered its state or cellular location to R'I, according to the following equation. (Formula: see text) No evidence was found that insulin could either associate or dissociate from R'I directly. The association rate constant was kappa 12 = 1.6 x/divided by 1.4 X 10(5) liter mol-1 s-1, a value shown to be incompatible with diffusion control. The other rate constants were: kappa 21 = 3.4 x/divided by 1.6 X 10(-3) s-1, kappa 23 = 3.2 x/divided by 1.5 X 10(-4) s-1, and kappa 32 = 2.0 x/divided by 1.5 X 10(-4) s-1. From these rate constants, an equilibrium constant of 8.4 x/divided by 1.5 nM was calculated, in excellent agreement with the previously measured value of 8.8 x/divided by 1.3 nM (Lipkin, E. W., Teller, D. C., and de Ha?n, C. (1986) J. Biol. Chem. 260, 1694-1701). The kinetic analysis also yielded receptor numbers similar to those obtained by equilibrium binding studies. The nature of the R'I state is discussed in terms of an internalized state, in terms of insulin receptor complex in caveolae, in terms of receptor aggregates, and in terms of being a Michaelis complex between insulin bound to the receptor and cell surface-bound insulin protease.     

Prolactin binding to dissociated cells from rat mammary tumors and mammary gland.

Prolactin binding activity was studied in suspensions of cells which had been enzymatically dissociated from R3230AC mammary tumors, 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors and lactating rat mammary glands. Prolactin bound specifically with high affinity (apparent binding affinity = 4.0 X 10(9) M-1) to R3230AC tumor cells. Hormone binding at room temperature was proportional to cell number and increased with time of incubation up to 120-180 min. Prolactin binding to R3230AC tumor cells from diabetic animals was reduced by about 50%. Specific prolactin binding activity was also demonstrated in preparations of cells from DMBA-induced tumors and lactating mammary gland. The levels of hormone binding in both dissociated cells and subcellular particles prepared from these tissues varied as follows: DMBA-induced tumors > lactating mammary gland > R3230AC mammary adenocarcinoma.     

Characterization of insulin binding to bovine liver and mammary microsomes

Bovine liver and mammary gland (MG) appear metabolically independent of insulin, yet the specificity and kinetics of 125I-insulin (125I-INS) binding to bovine liver and MG microsomes (MIC) indicate the presence of insulin receptors in MIC from both tissues. The insulin receptors from bovine liver (Kd = 7.6 X 10(-10) M) and MG (Kd = 9.6 X 10(-11) M) were similar to each other and to other insulin receptors in their binding affinities and pH optima. Perturbation of rat liver and bovine MG MIC by phospholipase or NaCl treatment increased 125I-INS binding to the membranes, suggesting exposure of cryptic insulin receptors. Different responses in 125I-INS binding to membrane perturbation suggest differences between rat and bovine membranes.     

Autocrine and juxtacrine effects of amphiregulin on the proliferative, invasive, and migratory properties of normal and neoplastic human mammary epithelial cells

Amphiregulin (AR) autocrine loops have been associated with several types of cancer. We demonstrate that SUM149 breast cancer cells have a self-sustaining AR autocrine loop. SUM149 cells are epidermal growth factor (EGF)-independent for growth, and they overexpress AR mRNA, AR membrane precursor protein, and secreted AR relative to the EGF-dependent human mammary epithelial cell line MCF10A. MCF10A cells made to overexpress AR (MCF10A AR) are also EGF-independent for growth. Treatment with the pan-ErbB inhibitor CI1033 and the anti-EGF receptor (EGFR) antibody C225 demonstrated that ligand-mediated activation of EGFR is required for SUM149 cell proliferation. AR-neutralizing antibody significantly reduced both SUM149 EGFR activity and cell proliferation, confirming that an AR autocrine loop is required for mitogenesis in SUM149 cells. EGFR tyrosine phosphorylation was dramatically decreased in both SUM149 and MCF10A AR cells after inhibition of AR cleavage with the broad spectrum metalloprotease inhibitor GM6001, indicating that an AR autocrine loop is strictly dependent on AR cleavage in culture. However, a juxtacrine assay where fixed SUM149 cells and MCF10A AR cells were overlaid on top of EGF-deprived MCF10A cells showed that the AR membrane precursor can activate EGFR. SUM149 cells, MCF10A AR cells, and MCF10A cells growing in exogenous AR were all considerably more invasive and motile than MCF10A cells grown in EGF. Moreover, AR up-regulates a number of genes involved in cell motility and invasion in MCF10A cells, suggesting that an AR autocrine loop contributes to the aggressive breast cancer phenotype.     

Cell cycle control in normal and neoplastic cells.

Recent studies of cell cycle control suggest that cyclin-dependent protein kinases play a central role in the cell's commitment to a new division cycle in late G1. The regulation of these kinases in normal and neoplastic growth is becoming clear.