PubMed Health⌕ Search

Biomedical subjects

J R Levy

Publications and source records attributed to J R Levy.

11 recordsLinked to original sources
PubMed1992

Regulation of insulin receptor gene expression. Cell cycle-mediated effects on insulin receptor mRNA stability.

Posttranscriptional mechanisms play important roles in insulin receptor gene regulation; variability in cellular insulin receptor number and the growth arrest-mediated increases in insulin receptor mRNA are secondary to changes in insulin receptor mRNA stability. Therefore, further characterization of the pathways and kinetics of insulin receptor mRNA degradation were investigated. The insulin receptor mRNA in the insulin receptor-rich Hep G2 cells is more stable compared with the insulin receptor-sparse MCF-7 cells. Growth arrest results in a significant rise in insulin receptor mRNA in both cell lines. The increase in mRNA is caused by changes in mRNA stability. The half-life of the insulin receptor mRNA in growth-arrested cells is approximately three times that of proliferating cells. The insulin receptor gene contains four polyadenylation sites that produce four species of mRNA of 5.4, 6.9, 8.0, and 9.4 kilobases (kb). The mRNA species are not coordinately regulated. The ratio of the most abundant species (9.4/6.9) is significantly larger in growth-arrested cells compared with proliferating cells. By utilizing a specific cDNA probe for the 9.4-kb mRNA species, it was determined that the diminished 9.4/6.9 ratio in proliferating cells was caused by a more rapid rate of the 9.4-kb mRNA degradation. The kinetics of insulin receptor mRNA degradation were investigated. Insulin receptor mRNA levels were reduced to 56% of their base line within 6 h when growth-arrested cells were stimulated to proliferate; protein inhibition with cycloheximide completely inhibited the decline in insulin receptor mRNA.

Actins↗
PubMed1992

Glucocorticoids decrease vitamin D receptor number and gene expression in human osteosarcoma cells.

The mechanisms by which glucocorticoids (GC) inhibit some actions of vitamin D [1,25-(OH)2D3] are not well understood, but there is growing evidence that GC alter vitamin D receptor (VDR) number. We studied the effects of dexamethasone (DEX) on VDR number and mRNA in the human osteosarcoma cell line, MG-63. The effects of DEX on 1,25-(OH)2D3 binding were examined by incubating confluent cells overnight in media without or with 10(-6) M DEX. DEX decreased VDR number (B max) by approximately 70% (110 versus 32 fmol/mg cellular protein, p less than 0.001) without significantly changing the apparent affinity (K'D) of 1,25-(OH)2D3 for its receptor (3.8 versus 2.2 x 10(-10) M, p greater than 0.05). Overnight incubation of MG-63 cells with DEX produced a time- and dose-responsive decrease in VDR mRNA compared to untreated controls (p less than 0.01). To determine the mechanism of the DEX-mediated decrease in VDR mRNA, the effect of DEX on VDR mRNA stability was studied. We found that the half-life for the VDR mRNA was approximately 5.7 h and was not significantly changed when the cells were incubated with DEX (approximately 6.3 h). We conclude that DEX decreases both VDR number and mRNA in MG-63 osteosarcoma cells. Since the half-life of VDR mRNA was not significantly modified by dexamethasone, glucocorticoids appear to decrease VDR mRNA by inhibiting VDR gene transcription or by affecting the processing of VDR mRNA.

Actins↗
PubMed1991

Effects of media conditions, insulin, and dexamethasone on insulin-receptor mRNA and promoter activity in HepG2 cells.

Numerous physiological agents and conditions modulate cellular insulin sensitivity by downregulating or upregulating total cellular insulin receptors. In this study, we examined the effects of replacing complete medium in the absence or presence of insulin on the regulation of insulin-receptor gene expression in cultured human hepatoma cells (HepG2). Failure to replace complete medium resulted in growth arrest of HepG2 cells and a six- to sevenfold increase in insulin-receptor mRNA due to the prolongation of insulin-receptor mRNA half-life. Northern analysis revealed multiple insulin-receptor mRNA species; the largest species (11 kilobases) was disproportionately increased in growth-arrested cells. High concentrations of insulin (500 ng/ml) induced a 33.8% decrease in the abundance of insulin-receptor mRNA (n = 14). At lower concentrations, a trend of inhibition was observed but was not statistically significant. Insulin (500 ng/ml) did not affect insulin-receptor mRNA stability. The effect of conditioned media, insulin, and dexamethasone on insulin-receptor promoter activity was also examined. Various constructs of the 5'-flanking region of the insulin-receptor gene were attached immediately upstream to a chloramphenicol acetyltransferase (CAT) reporter gene and transiently transfected into HepG2 cells via a pBR322-derived plasmid (pCAT). In cells replaced with complete medium, 12 and 118% of the promoter activity was contained within 578 and 877 base pairs, respectively, from the major translational initiation site. Conditioned media from growth-arrested cells in culture for 7 days increased promoter activity approximately twofold in 48 h. However, this increase failed to localize to any specific region on the insulin-receptor promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1990

Down-regulated insulin receptors in HepG2 cells have an altered intracellular itinerary.

The delivery of insulin and the insulin receptor into an intracellular compartment may be important for eliciting some of the biologic responses of the cell to the hormone. Internalization of insulin-receptor complexes in cells from hyperinsulinemic type II diabetic patients is diminished, suggesting a possible role for this cellular process in insulin resistance. To examine whether hyperinsulinemia contributes to defective insulin-receptor processing in vitro, cultured hepatoma cells (HepG2) were incubated with high concentrations of (500 ng/ml) insulin from 1-3 days. Insulin induced a decrease in the number of total and surface insulin receptors within 24 hours; however, the hormone did not mediate a change in the number of intracellular receptors. The cellular itinerary of control and down-regulated receptors were then compared. Insulin mediated internalization of down-regulated receptors was impaired compared to control receptors; however, the down-regulated receptors that were internalized recycled back to the plasma membrane more efficiently. By covalently labeling the insulin receptor with the photoactive insulin derivative, 125I-NAPA-DP-insulin, it was demonstrated that the rates of receptor degradation of down-regulated and control receptors were similar. These results suggest that incubating HepG2 cells with high concentrations of insulin alters the cellular itinerary of the insulin receptor.

Carcinoma, Hepatocellular↗
PubMed1988

Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene.

The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblast insulin receptors. These cells bind and internalize insulin normally. Biochemical assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 degrees C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 degrees C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The total number of complexes reached a maximum by 5 min and decreased rapidly thereafter with a t 1/2 of approximately 10 min. There was a distinct delay in the appearance, rate of rise, and peak of intracellular free and degraded insulin. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored, based on the ability of dissociated insulin to rebind to receptor upon neutralization of acidic intracellular vesicles with monensin. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.

Animals↗
PubMed1988

Endocytotic uptake, processing, and retroendocytosis of human biosynthetic proinsulin by rat fibroblasts transfected with the human insulin receptor gene.

The cellular itinerary and processing of insulin and proinsulin were studied to elucidate possible mechanisms for the observed in vivo differences in the biologic half-lives of these two hormones. A rat fibroblast cell line transfected with a normal human insulin receptor gene was used. Due to gene amplification, the cells express large numbers of receptors and are ideal for studying a ligand, such as proinsulin, that has a low affinity for the insulin receptor. Competitive binding at 4 degrees C showed that the concentration of unlabeled insulin and proinsulin that is needed to displace 50% of tracer insulin or proinsulin was 0.85-0.95 nM and 140-150 nM, respectively. Binding to surface receptors and internalization occur at rates that are four to five times faster in cells incubated with insulin compared with proinsulin. Chloroquine led to an increase in cell-associated radioactivity of approximately 1.4-fold in cells incubated with insulin or proinsulin, but inhibited the appearance of degraded insulin by 54% and degraded proinsulin by only 10%. To study the fate of internalized ligand, cells were incubated with insulin and proinsulin until steady state binding occurred. Surface bound ligand was removed by an acid wash and the remaining cell-associated radioactivity represented internalized ligand. Cells were then reincubated in 37 degrees C buffer and the cell-associated radioactivity and radioactivity released into the medium were analyzed by TCA precipitation, Sephadex G-50, and HPLC. The results demonstrated that proinsulin more readily bypasses the intracellular degradative machinery and is therefore released intact from the cell via the retroendocytotic pathway. These results may help to explain the prolonged metabolic clearance rate and biologic responsiveness of proinsulin in vivo.

Animals↗
PubMed1987

The trafficking and processing of insulin and insulin receptors in cultured rat hepatocytes.

The processing and trafficking of insulin in cultured rat hepatocytes were studied. A time course of binding of radiolabeled insulin to hepatocytes at 37 C revealed a rapid rise in cell-associated radioactivity that reached a steady state by 20 min. Using an acid medium to extract insulin bound to surface receptors, the time courses of receptor binding and internalization of the ligand were characterized. The earliest event in insulin processing was the binding of insulin to surface receptors, reaching steady state by 20 min with a t1/2 of 4 min. The internalization rate of ligand was initially slower than the binding rate, with a t1/2 of 6 min. Similar internalization rates of the insulin receptor were found by measuring the trypsin sensitivity of hepatocyte insulin receptors covalently occupied with a photo-affinity-labeled derivative of insulin [( 125I]B2 (2-nitro-4-azido-phenylacetyl)Des-PheB1-insulin). At steady state, the internalized ligand and receptor comprised approximately 40-45% of the cell-associated radioactivity. The time course of intracellular degradation was assessed by trichloroacetic acid (TCA) precipitability and Sephadex G-50 gel chromatography of solubilized cells containing only internalized radioactivity. Intracellular TCA-soluble and low mol wt degradation products first appeared by 5 min and were released from the cell 3 min later. Chloroquine (100 microM) completely inhibited the formation of intracellular low mol wt degradation products as well as their appearance in the medium. The release of intracellular radioactivity was assessed by first removing surface-bound insulin with acid extraction. Eighty percent of the intracellular radioactivity was released in 45 min with a t1/2 of 8 min. The released radioactivity was assessed by TCA precipitability and gel chromatography. These results demonstrate that after 20 min, 43% of the released intracellular radioactivity is intact insulin. The percentage of intact insulin released increases in a dose-dependent fashion as the amount of insulin bound and internalized increases. In conclusion, the earliest event in insulin processing is binding to surface receptors. After a short delay, insulin and its receptor are internalized and trafficked into either a chloroquine-sensitive degradative pathway or a chloroquine-insensitive retroendocytotic pathway. The amount of insulin that traverses the nondegradative retroendocytotic pathway is proportional to the amount of insulin bound and internalized by the cell.

Animals↗
PubMed1986

Retroendocytosis of insulin in rat adipocytes.

A variety of ligands internalized by receptor-mediated endocytosis follow a short circuit pathway that does not lead to degradation but results in rapid exocytosis of intact ligand, a process termed retroendocytosis. We studied the time course of [125I]iodoinsulin processing and retroendocytosis after internalization in isolated rat adipocytes. After steady state binding and internalization, surface receptor-bound insulin was removed by exposing cells to a low pH at low temperatures. The cells containing internalized [125I]iodoinsulin were reincubated in fresh medium; subsequently, the radioactivity remaining within the cells and released into the medium were analyzed at various times by trichloroacetic acid (TCA) precipitation, Sephadex G-50 gel filtration, and reverse phase HPLC. Cell-associated radioactivity progressively decreased after reincubation in 37 C buffer, with 50% released in 9 min and 85% by 45 min. In the media, TCA-precipitable material appeared quickly, with a t1/2 of 2 min, and plateaued by 10 min. TCA-soluble material was released continually throughout the 45-min period. The release of both TCA-precipitable and TCA-soluble material was temperature and energy dependent. Sephadex G-50 chromatography demonstrated the loss of insulin from the intracellular pool and its appearance in the medium with a time course similar to that of TCA-precipitable material. Reverse phase HPLC demonstrated that the intracellular and medium radioactivity eluting in peak II (insulin peak) on Sephadex G-50 was composed of both intact insulin and intermediates. In conclusion, these studies demonstrated that after the internalization of insulin, rat adipocytes release not only small mol wt degradation products of insulin, but also insulin intermediates and intact insulin. The rate of retroendocytosis reported here is almost identical to the rate of insulin receptor recycling in rat adipocytes. Therefore, retroendocytosis may serve as an excellent in vitro reflection of the extent and rate of insulin receptor recycling.

Adipose Tissue↗
PubMed1986

Demonstration of insulin receptors and modulation of alkaline phosphatase activity by insulin in rat osteoblastic cells.

Osteoporosis is a known complication of diabetes mellitus, suggesting a role for insulin in bone homeostasis. We studied insulin receptors and insulin action in the osteoblast-like rat osteogenic sarcoma cell line ROS 17/2.8. These cells share many common features with the osteoblast, such as 1,25-dihydroxyvitamin D3 receptors, PTH receptors, and 1,25-dihydroxyvitamin D3-induced modulation of alkaline phosphatase activity and osteocalcin. Competition binding studies revealed high affinity insulin receptors, with an ED50 for insulin of 1 nM. The receptors were highly specific for insulin, with 60% inhibition of insulin binding by an antireceptor antibody, no competition by epidermal growth factor, and an ED50 of 300 nM for proinsulin. Steady state maximal insulin binding was obtained by 40 min at 37 C, and insulin degradation, as measured by trichloroacetic acid solubility, was 1%/h at 37 C. ROS cells readily internalized insulin, and under steady state binding conditions at 37 C, 56% of the cell-associated radioactivity consisted of intracellular material. Chloroquine (100 microM) inhibited intracellular processing of insulin, leading to a 300% increase in cell-associated insulin by 2 h (37 C). Photoaffinity labeling of the insulin receptor with the photosensitive analog of insulin, B2 (2-nitro-4-azidophenyl-acetyl)des-pheB1-insulin, followed by solubilization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed specific bands of 125K and 430K mol wt under reducing and nonreducing conditions, respectively. Thus, the structure of insulin receptors in ROS cells appears comparable to that of insulin receptors of known target tissues. Insulin action was also examined. Insulin did not stimulate [2-3H]deoxyglucose uptake or [1-14C]leucine incorporation into protein. In contrast, physiological concentrations of insulin inhibited alkaline phosphatase activity in nonconfluent cells. After exposure to insulin for 24 h, alkaline phosphatase activity was decreased compared to basal by 39.5% and 50% with 5 and 50 ng/ml insulin, respectively. In conclusion, ROS cells bind insulin to specific receptors that are similar to insulin receptors on other target tissues; receptors internalize insulin, which is then processed through a chloroquine-sensitive pathway; insulin does not affect membrane substrate transport; and insulin does inhibit the activity of an enzyme that is important in bone metabolism. ROS cells represent a model for studying insulin effects on bone.

Affinity Labels↗
PubMed1975

Three variants of concealed bigeminy.

Long electrocardiographic strips were analyzed from five patients who exhibited periods of typical "concealed bigeminy," i. e., recurrent unifocal extrasystoles which were separated from one another by odd numbers of normally conducted sinus beats. However, in each of these patients, there were periods in which one of three different variants of concealed bigeminy was observed. Three patients displayed an "even number" variant; i. e., there were large numbers of consecutive extrasystoles which were separated exclusively or preponderantly by even rather than by odd numbers of sinus beats. One other patient exhibited an "interpolated extrasystole" variant: those interectopic intervals which were initiated by an interpolated extrasystole contained an even number of sinus beats, whereas all other interectopic intervals contained an odd number. In the fifth patient, the distribution of the numbers of sinus beats separating extrasystoles was such as to suggest a periodic fluctuation between the classical forms of concealed bigeminy and concealed trigeminy; i. e., a "combined bigeminy and trigeminy" variant.

Aged↗