In vitro metabolism by human skin and fibroblasts of retinol, retinal and retinoic acid.

The metabolism of radio-labelled retinol, retinal and retinoic acid by fresh human skin as well as by human dermal fibroblasts have been investigated in vitro. Surgically removed human skin biopsies were placed at the air liquid interface, and treated topically for 24 h with retinoids. At the end of the treatment period, epidermis and dermis were separated by heat.

A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects.

Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RAR alpha, RAR beta, and RAR gamma. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes.

Ligand specificities of recombinant retinoic acid receptors RAR alpha and RAR beta.

Binding of retinoic acid (RA) to specific RA receptors alpha and beta (RAR alpha and RAR beta) was studied. Receptors were obtained in two ways: (1) full-length receptors were produced by transient expression of the respective human cDNAs in COS 1 cells; and (2) the ligand-binding domains of RAR alpha and RAR beta were produced in Escherichia coli. RA binding to the wild-type and truncated forms of the receptor was identical for both RAR alpha and RAR beta, indicating that the ligand-binding domains have retained the binding characteristics of the intact receptors.

Acute and chronic regulation of phosphoenolpyruvate carboxykinase mRNA by insulin and glucose.

Using the well differentiated rat hepatoma Fao we have studied the regulation of phosphoenolpyruvate carboxykinase (PEPCK) mRNA by insulin and glucose and compared these results to glucose production as estimated by glucose release into the medium. Fao cells possess an active gluconeogenic pathway and, when grown in glucose-free medium, release glucose for over 8 h. Addition of the cAMP analog, 8-(4-chlorophenyl-thio) cAMP (8-CTP-cAMP) or increasing the concentration of dihydroxyacetone and oxaloacetate results in an increase in glucose release which can be suppressed by insulin at concentrations between 1 and 100 nM.

Transcriptional and posttranscriptional regulation of tyrosine aminotransferase by insulin in rat hepatoma cells.

The molecular mechanisms of induction of tyrosine aminotransferase (TAT) by insulin were studied in the well-differentiated rat hepatoma cell line Fao. Incubation of Fao cells with insulin resulted in a 2-fold increase in TAT activity and TAT mRNA measured by Northern blot analysis with an oligonucleotide probe to the 5' end of the gene. The effect of insulin on TAT activity had a lag period of 30-60 min and was maximal within 4-5 h.

Characterization of the receptors for insulin and the insulin-like growth factors on micro- and macrovascular tissues.

Insulin and insulin-like growth factors (IGFs) have been implicated in the pathogenesis of diabetic retinopathy and peripheral vascular complications. Previously, we have shown that retinal capillary endothelial cells responded to insulin and IGFs for metabolic and growth effects, whereas aortic endothelial cells were not responsive. In contrast, vascular supporting cells from both retinal capillaries (i.

Structure, phosphorylation and desensitization of the insulin receptor in hepatoma cells.

Long-term exposure of rat hepatoma cells to insulin results in a total desensitization of the cells to the action of the hormone. This is characterized by changes in binding and post-binding steps of insulin action, including a decrease in the number and phosphorylation of receptors, and a major modification in receptor oligomerization.

Cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors.

Madin-Darby canine kidney (MDCK) cells were previously shown to have few or no plasma membrane insulin binding sites (Hofmann et al: J Biol Chem 258:11774, 1983]. Accordingly, neither insulin-stimulated incorporation of [14C]glucose into glycogen, nor insulin-induced uptake of radiolabeled alpha-aminoisobutyrate ([3H]AIB) could be demonstrated. To probe for receptors, MDCK cultures were surface-labeled with Na125I or were labeled with [35S]methionine.

Insulin receptor regulation and desensitization in rat hepatoma cells. The loss of the oligomeric forms of the receptor correlates with the change in receptor affinity.

We have previously reported that prolonged incubations of Fao cells, a cell line derived from the well-differentiated Reuber H35 rat hepatoma, with 10(-6) M insulin, induced a decrease in receptor number (down-regulation), an increase in receptor affinity for insulin, and a loss of insulin's biological effect (desensitization). In the present study, we have investigated the relationship between these changes in insulin binding and action and changes in the structure of the insulin receptor. Intact cells were surface labeled with Na125I and lactoperoxidase, and the 125I-labeled insulin receptor was immunoprecipitated using specific antibodies and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Processing of insulin by bovine endothelial cells in culture. Internalization without degradation.

Insulin binding and processing was studied in monolayer cultures of bovine aortic endothelial cells. Specific 125I-insulin binding was both time and temperature dependent. Maximum binding at 37 degrees C occurred at 90 min, and was 3.

Phosphorylation and dephosphorylation of the insulin receptor: evidence against an intrinsic phosphatase activity.

We have studied the reversibility of insulin receptor phosphorylation to establish the relation between this autophosphorylation reaction and the initiation of insulin action and between dephosphorylation and the termination of insulin effects in cells. In cultured Fao hepatoma cells labeled with 32PO4(3-), insulin increased 5-fold the phosphorylation of the beta-subunit of the insulin receptor at serine, threonine, and tyrosine residues. Addition of anti-insulin antiserum to cells incubated with insulin caused dissociation of insulin from the receptor and concurrent dephosphorylation of the beta-subunit.

Insulin receptor regulation and desensitization in rat hepatoma cells. Concomitant changes in receptor number and in binding affinity.

We have studied the effects of chronic exposure to insulin on the binding and the biologic activity of the hormone using a well-differentiated cell line (Fao) derived from the Reuber H35 rat hepatoma. Prolonged incubation (24 h) with 10(-6) M insulin produced a 20-25% decrease in binding of tracer concentrations (2 X 10(-11) M) of 125I-insulin, and a leftward shift of the curve for inhibition by unlabeled insulin. Scatchard analysis of the binding data revealed that a 75-80% decrease in the number of binding sites had occurred in the insulin-treated cells, but was accompanied by an increase in apparent receptor affinity.

Dysregulation of glucose transport in hearts of genetically obese (fa/fa) rats.

Overall D-glucose metabolism and 3-0-methylglucose transport were measured in the perfused heart preparation of lean and genetically obese (fa/fa) rats. Absolute values of basal and insulin-stimulated glucose metabolism were decreased in hearts of 15-week-old obese rats when compared to lean age-matched controls. Basal and maximally stimulated (i.

Analysis of insulin action using differentiated and dedifferentiated hepatoma cells.

Hepatoma cells in culture exhibit a range of differentiated functions. Well differentiated hepatoma cells retain most of the functions of the adult liver, whereas dedifferentiated cells have lost most of them. In the present study, insulin binding and insulin effects were studied in four differentiated and two dedifferentiated cell lines derived from the Reuber hepatoma and in a partially differentiated cell line, HTC, derived from the Morris 7288C hepatoma.

Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.

Expression of insulin receptors and insulin action in cell hybrids and cybrids.

The expression of insulin receptors and insulin action was studied in cell hybrids and cybrids produced by fusion of the BWIJ mouse hepatoma cell line with nucleated and enucleated mouse L-cells (LEA-2A) respectively. The BWIJ parent and the cybrids expressed high numbers of insulin receptors, whereas the hybrids resembled the L-cell parent with low numbers of receptors. Likewise, the hybrids resembled the LEA-2A cells with high levels of glycogen synthase, whereas the BWIJ cells and cybrids had much lower levels.

Glucagon regulation of amino acid transport in hepatocytes: effect of cell enucleation.

Glucagon and cAMP analogs stimulate amino acid transport in freshly isolated hepatocytes by inducing the synthesis of new transport proteins. The role of the cell nucleus in the glucagon regulation of amino acid transport has been studied in rat hepatocytes enucleated by centrifugation through a discontinuous Ficoll gradient in the presence of cytochalasin B. Enucleated hepatocytes take up alpha-aminoisobutyric acid (AIB) through a Na+-dependent transport component with kinetic properties similar to those found in intact hepatocytes.

Physical training of lean and genetically obese Zucker rats: effect on fat cell metabolism.

The effects of 6-wk treadmill training program on the metabolism of isolated adipose cells from obese (fa/fa) and lean (Fa/?) Zucker rats were studied. Glucose metabolism and transport, insulin binding, and lipolysis were measured in adipose cells prepared from sedentary control and exercise-trained (ET) lean and/or obese rats. Two- to threefold increases in glucose metabolism were observed in cells from lean and obese ET rats compared with their respective controls.

Role of microtubules in insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes.

The effects of the microtubule inhibitor, colchicine, on insulin or glucagon stimulation of alpha-amino[1-14C]-isobutyric acid (AIB) transport were investigated in isolated hepatocytes from normal fed rats. Under all conditions tested, AIB uptake appeared to occur through two components of transport: a low affinity (Km approximately 50 mM) component and a high affinity (Km approximately 1 mM) component. Within 2 h of incubation, insulin and glucagon, at maximal concentrations, increase AIB (0.