Search PubMed⌕ Search

Biomedical subjects

C Grunfeld

Publications and source records attributed to C Grunfeld.

At least 181 records · Page 10Linked to original sources
PubMed1983

The interaction of 125I-insulin with cultured 3T3-L1 adipocytes: quantitative analysis by the hypothetical grain method.

The murine 3T3-L1 fibroblast under appropriate incubation conditions differentiates into an adipocyte phenotype. This 3T3-L1 adipocyte exhibits many of the morphologic, biochemical, and insulin-responsive features of the normal rodent adipocyte. Using quantitative electron microscopic (EM) autoradiography we find that, when 125I-insulin is incubated with 3T3-L1 adipocytes, the ligand at early times of incubation localizes to the plasma membrane of the cell preferentially to microvilli and coated pits. When the incubation is continued at 37 degrees C, 125I-insulin is internalized by the cells and preferential binding to the villous surface is lost. With the internalization of the ligand, two intracellular structures become labeled, as determined by the method of hypothetical grain analysis. These include large clear, presumably endocytotic, vesicles and multivesicular bodies. Over the first hour of incubation the labeling of these structures increases in parallel, but in the second hour they diverge: the labeling of multivesicular bodies and other lysosomal forms continuing to increase and the labeling of large clear vesicles decreasing. At 3 hours limited but significant labeling occurs in small Golgi-related vesicles that have the typical distribution of GERL. The distinct morphologic features of this cell make it ideal for a quantitative morphologic analysis and allow for an unambiguous view of the sequence of events involved in receptor-mediated endocytosis of a polypeptide hormone. These events are likely to be representative of the processing of insulin by the mature rodent adipocyte.

Adipose Tissue↗
PubMed1983

Insulin-stimulated methylaminoisobutyric acid uptake in 3T3-L1 fibroblasts and fat cells.

3T3-L1 fibroblasts and fat cells have been extensively used to study the development of insulin-stimulated glucose and lipid metabolism during adipocyte differentiation in vitro. In this paper we explore the ability of insulin to stimulate amino acid uptake in 3T3-L1 cells using the nonmetabolizable amino acid analog methylaminoisobutyric acid (MAIB). In differentiated 3T3-L1 fat cells, a 12-h preincubation with insulin was required for maximal stimulation of MAIB uptake. In contrast, in the undifferentiated fibroblasts, insulin stimulation peaked between 6 and 8 h and then declined significantly. Maximal stimulation of MAIB uptake in the differentiated fat cell exceeded that in the fibroblast phenotype. This increased ability of insulin to stimulate MAIB uptake in fat cells appeared within the first day after removal of the differentiation medium. 3T3-L1 fat cells were 30 times more sensitive to the effects of insulin on MAIB transport than the undifferentiated fibroblasts. These findings are consistent with previous data on insulin-stimulated deoxyglucose uptake, in that the increased sensitivity to insulin with differentiation is more than can be accounted for by the increase in receptor number. The activity of porcine proinsulin indicates that this stimulation reflects the known characteristics of the insulin receptor. The stimulation of MAIB uptake by insulin in 3T3-L1 fat cells was blocked by inhibitors of protein synthesis (cycloheximide and puromycin) and mRNA synthesis (actinomycin D). Colchicine, an inhibitor of microtubule function, showed little inhibition of insulin-stimulated MAIB uptake. Insulin stimulation of MAIB uptake was greater when 3T3-L1 cells were preincubated with insulin in the absence of essential amino acids. Basal transport in 3T3-L1 cells was not influenced by the presence or absence of amino acids. Thus, amino acid deprivation appears specifically to enhance the ability of insulin to stimulate amino acid transport in cultured adipocytes.

Adipose Tissue↗
PubMed1983

Morphological changes of the 3T3-L1 fibroblast plasma membrane upon differentiation to the adipocyte form.

By quantitative evaluation carried out on freeze-fracture replicas, we have investigated the changes in plasma membrane organization as the 3T3-L1 fibroblast phenotype differentiates into the 3T3-L1 adipocyte form. As differentiation takes place there is a dramatic change in overall appearance of the cell as it acquires large lipid-laden vacuoles. On freeze-fracture replicas we find: (1) a ninefold increase in small invaginations as the cell differentiates from the fibroblast to the adipocyte phenotype; (2) virtually no quantitative change in the larger coated invaginations upon differentiation; (3) a greater density of intramembrane particles in the large invaginations as compared to the uninvaginated membrane in both the fibroblast and adipocyte form. This remains relatively constant with differentiation. By contrast, there is a marked increase of intramembrane particles in the undifferentiated membrane as the cell changes from fibroblast to adipocyte. The functional significance of these changes in plasma membrane organization is unknown, but they significantly correlate with the onset of lipogenesis by the cell.

Adipose Tissue↗
PubMed1983

Surface interactions and intracellular localization of cationic ferritin: similarity to 125I-insulin in 3T3-L1 adipocytes.

We previously reported that in 3T3-L1 adipocytes 125I-insulin associates preferentially with microvilli and coated pits at low temperatures and early times of incubation. At higher temperatures it is internalized through a series of membrane limited intracellular compartments. In the present study, we used a high resolution probe, cationic ferritin (CF), to track adsorptive endocytosis in the 3T3-L1 adipocyte. We find that CF initially associates with coated pits at 2 min of incubation at 37 degrees C. With further incubation at 37 degrees C CF is internalized and after 2 to 10 min of incubation is predominantly localized to coated and non-coated clear vesicles. Approximately 50% of the apparent coated vesicles seen near the plasma membrane on single thin sections are shown by serial sectioning to be true vesicles (i.e., without a surface connection). At later time points CF is localized predominantly to lysosomal structures and, to a much smaller extent, Golgi-related structures. The remarkable similarity between 125I-insulin and CF with respect to post-binding processing suggests that while the membrane receptor confers the initial specificity, post-binding events are common for different types of ligands after they bind to cell surfaces and are subject to adsorptive endocytosis.

Adipose Tissue↗
PubMed1982

Autoantibodies to insulin receptors in man: immunological determinants and mechanism of action.

The insulin receptor is a membrane glycoprotein of high Mr which binds insulin with high affinity and specificity and transmits some intracellular signal(s) that initiate(s) insulin action. Antibodies to the receptor have been identified in patients with a syndrome characterized by severe resistance to endogenous and exogenous insulin, varying degrees of glucose intolerance, and the skin lesion acanthosis nigricans. The syndrome is most common in non-Caucasian, middle-aged women, but occurs in patients of all races, both sexes, and spanning the ages of 12-62. Most patients have evidence of other autoimmune disease with increased erythrocyte sedimentation rate and gamma globulins, anti-DNA and anti-nuclear antibodies, leucopenia, and other signs and symptoms of autoimmune disease. Antibodies to the insulin receptor are detected by their ability to inhibit 125I-insulin binding or to immunoprecipitate solubilized insulin receptors. In vitro these antibodies acutely mimic most of insulin's metabolic effects. This insulin-like activity depends on antibody bivalence; monovalent Fab fragments block insulin binding and action but lack intrinsic activity. With prolonged exposure of cells to anti-receptor antibody the insulin-like effect is lost and a state of insulin resistance ensues. This is due to both a blockage of insulin binding and a form of post-receptor desensitization. The possible causation of anti-receptor antibodies in this condition is discussed.

Antibody Specificity↗
PubMed1982

Receptor-mediated endocytosis of insulin: role of microvilli, coated pits, and coated vesicles.

When 125I-labeled insulin (125I-insulin) is incubated with 3T3-L1 adipocytes and cells processed for electron microscopic autoradiography, the ligand initially localizes preferentially to microvilli and coated pits. As a function of time and temperature, this initial preferential localization to microvilli is lost, and the ligand is internalized by the cell. Serial sections of apparent coated vesicles near the cell surface indicate that about half of these structures are true vesicles and, therefore, intermediates in this receptor-mediated endocytotic process. With time, 125I-insulin localizes to larger intracellular membrane-bounded structures. When cells are incubated with another ligand, cationic ferritin, that is taken up by adsorptive endocytosis, essentially the same structures are involved as for the endocytosis of 125I-insulin. The data suggest that specificity for receptor-mediated endocytosis is conferred by the specific ligand receptor and possibly by ligand-induced receptor mobility in the plane of the plasma membrane. Other structures such as coated pits, coated vesicles, larger vesicles, and secondary lysosomes are common for different ligands.

Animals↗
PubMed1982

Autoantibodies to the insulin receptor and insulin-resistant diabetes.

Autoantibodies to the insulin receptor are a rare cause of insulin-resistant diabetes, but when they occur they produce a profound clinical syndrome. These antibodies block insulin binding, immunoprecipitate solubilized insulin receptors, and their acute effect is to mimic the biological effects of insulin. However, prolonged exposure of cells to these antibodies produces a state of insulin resistance. Since the antigen to which the antibody is directed is relatively well-characterized, many of the observations in this syndrome can serve as a model for elucidating molecular mechanisms in other diseases with antibodies against membrane components. The autoantibodies to the insulin receptor have also provided valuable probes in the study of insulin receptor structure and insulin action.

Adipose Tissue↗
PubMed1980

Antibody-induced desensitization of the insulin receptor. Studies of the mechanism of desensitization in 3T3-L1 fatty fibroblasts.

Antibodies against the insulin receptor (Anti-R), which are found in the serum of type B patients with the syndrome of insulin resistance and acanthosis nigricans, inhibit the binding of insulin to its receptor and mimic the actions of insulin when studied acutely in vitro. After prolonged exposure of 3T3-L1 cells to Anti-R, the insulinomimetic activity is lost, and the cells show a marked decrease in their maximal response to insulin (antibody-induced desensitization), thus providing a model for the insulin resistance seen in vivo. This study explores in detail the mechanism and specificity of desensitization in 3T3-L1 cells.Desensitization, like the insulinomimetic activity of Anti-R, requires bivalence. Monovalent preparations of Anti-R inhibit insulin binding and shift the insulin biological dose-response curve to the right, but do not decrease the maximal insulin response. The affinity of monovalent Anti-R is less than that of the native antibody. Cross-linking of monovalent Anti-R reconstitutes its insulinomimetic activity and partially reconstitutes desensitization. Desensitized cells are resistant to the insulinomimetic actions of concanavalin A, which interacts with the insulin receptor, but are not desensitized to spermine and vitamin K(5), insulinomimetic agents that are thought to act independently of the insulin receptor. Glucose, pyruvate, or certain hexoses are required in the incubation media for desensitization to occur. Although Anti-R is taken up into cells and degraded by lysosomes, chloroquine, cycloheximide, colchicine, and cytochalasin E have little influence on the induction of or recovery from antibody-induced desensitization. These data suggest that desensitization is not merely due to the inhibition of insulin binding, but is a complex process involving a decreased ability of the receptor to generate a biological response.

Animals↗
PubMed1979

Primary defect of insulin receptors in skin fibroblasts cultured from an infant with leprechaunism and insulin resistance.

Insulin binding to insulin receptors, on skin fibroblasts established in culture from an infant with insulin resistance and clinical features of leprechaunism was markedly decreased in comparison with cultures from an age-matched control. By contrast, the binding of epidermal growth factor, a polypeptide growth factor chemically unrelated to insulin, to patient's and control fibroblasts was indistinguishable. The selective defect in insulin binding to patient's fibroblasts was reflected in an impaired ability of insulin to stimulate 2-deoxyglucose uptake. These results most likely indicate a primary genetic defect of insulin receptors.

Biological Transport↗
PubMed1979

Desensitization of the insulin receptor at an early postreceptor step by prolonged exposure to antireceptor antibody.

We have used an adipocyte-like cell line, the 3T3-L1 fatty fibroblasts, to compare acute and chronic effects of autoantibodies directed against the insulin receptor. Acute exposure of the cells in tissue culture to the antibodies resulted in a blockade of insulin binding and stimulation of 2-deoxy-glucose transport and glucose oxidation. Maximal acute effects were reached within 30-120 min. Subsequently, the stimulatory response decayed and, after 6 hr in the continuous presence of the antibodies, basal glucose metabolism had returned to the level observed with unexposed cells and a state of severe insulin resistance prevailed. In contrast to the decay of bioresponse, no change in insulin binding was detectable over the same time period. The mechanism of desensitization seemed to involve events early after insulin binding to receptor because cells exposed to antibody for prolonged periods of time, although unresponsive to insulin and antireceptor antibodies, responded normally to both spermine and vitamin K(5), agents that stimulate glucose metabolism independently of the insulin receptor. These data suggest that prolonged or continuous occupancy of the insulin receptor by a ligand, in this case antireceptor antibodies, does not produce a continuous biological response. Instead, there is desensitization at some early step in the pathway for insulin action. These observations have important implications with respect to the mechanism of insulin action and to other situations in which there is long-term exposure of cells to antibodies that react with membrane components.

Adipose Tissue↗