Hepatocyte-Specific Co-Delivery of Zinc Ions and Plasmid DNA by Lactosylated Poly(1-vinylimidazole) for Suppression of Insulin Receptor Internalization

The lactosylated poly(1-vinylimidazole) (PVIm-Lac) with various lactosylated degrees has been synthesized for the co-delivery of zinc ions (Zn) and plasmid DNA (pDNA). The Zn/DNA/PVIm-Lac complex formation has achieved the specific delivery of zinc ions to HepG2 cells. Especially, the resulting hepatocyte-specific delivery of zinc ions has increased the number of insulin receptors on the cell surface. Consequently, the Zn/DNA/PVIm-Lac complexes have suppressed insulin receptor internalization on the surface of the HepG2 cells, expecting to offer unique therapy to inhibit hepatic insulin clearance.

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Quantitative studies of the rate of insulin internalization in isolated rat hepatocytes

Biochemical Journal ◽

10.1042/bj2180307 ◽

1984 ◽

Vol 218 (2) ◽

pp. 307-312 ◽

Cited By ~ 28

Author(s):

B Draznin ◽

M Trowbridge ◽

L Ferguson

Keyword(s):

Cell Surface ◽

Insulin Receptor ◽

Insulin Receptors ◽

Rat Hepatocytes ◽

Isolated Hepatocytes ◽

Receptor Internalization ◽

Insulin Concentration ◽

Receptor Complex ◽

Isolated Rat Hepatocytes ◽

Isolated Rat

We studied internalization of 125I-labelled insulin in isolated rat hepatocytes. Using the acidification technique, we were able to dissociate the ligand from its cell-surface receptors, and thus to separate internalized from surface-bound insulin. Because during the first 5 min of incubation of 125I-labelled insulin with freshly isolated hepatocytes there is no loss of internalized label, the ratio of the amount of internalized ligand to the amount of cell-surface-bound ligand may serve as an index of insulin internalization. Within the first 10 min of insulin's interaction with hepatocytes, the plot of the above ratio as a function of time yields a straight line. The slope of this line is referred to as the endocytic rate constant (Ke) for insulin and denotes the probability with which the insulin-receptor complex is internalized in 1 min. At the insulin concentration of 0.295 ng/ml, the Ke is 0.049 min-1. It is independent of insulin concentration until the latter exceeds 1 ng/ml. At the insulin concentration of 3.2 ng/ml, the Ke accelerates to 0.131 min-1. With the Ke being the probability of insulin-receptor-complex internalization, 4.9% of occupied insulin receptors will be internalized in 1 min at an insulin concentration of 0.295 ng/ml, and 13.1% of occupied insulin receptors will be internalized in 1 min at 3.2 ng/ml. When the insulin concentration decreases from 3.2 to 0.3 ng/ml, the Ke decreases accordingly. The half-time of occupied receptor internalization is 15.4 min at the lower insulin concentration and 5.3 min at the higher insulin concentration.

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Insulin causes insulin-receptor internalization in human erythrocyte ghosts

Biochemical Journal ◽

10.1042/bj2410093 ◽

1987 ◽

Vol 241 (1) ◽

pp. 93-97 ◽

Cited By ~ 4

Author(s):

R S Kelleher ◽

E F Murray ◽

S W Peterson

Keyword(s):

Insulin Receptor ◽

Receptor Binding ◽

Human Erythrocyte ◽

Insulin Receptors ◽

Insulin Binding ◽

Erythrocyte Ghost ◽

Receptor Internalization ◽

Erythrocyte Ghosts ◽

And Control ◽

Vesicular Compartment

The effect of incubation with insulin on insulin-receptor internalization by erythrocyte ghosts was investigated. The number of surface insulin receptors decreased by 30-40% after incubation of ghosts with insulin. Total insulin-receptor binding to solubilized ghosts was the same in insulin-incubated and control ghosts, whereas insulin binding to an internal vesicular fraction was substantially increased in insulin-incubated ghosts. Our findings suggest that erythrocyte-ghost insulin receptors are internalized to a vesicular compartment in response to incubation with insulin.

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Insulin and secretagogues differentially regulate fluid-phase pinocytosis in insulin-secreting β-cells

Biochemical Journal ◽

10.1042/bj3180623 ◽

1996 ◽

Vol 318 (2) ◽

pp. 623-629 ◽

Cited By ~ 6

Author(s):

Gang XU ◽

Jennie HOWLAND ◽

Paul L ROTHENBERG

Keyword(s):

T Cells ◽

Cell Surface ◽

Insulin Receptor ◽

Cell Line ◽

Chinese Hamster Ovary Cells ◽

Insulin Receptors ◽

Fluid Phase ◽

Β Cells ◽

Β Cell ◽

Basal Rate

The physiological role of the β-cell insulin receptor is unknown. To evaluate a candidate function, the insulin regulation of fluid-phase pinocytosis was investigated in a clonal insulinoma cell line (βTC6-F7) and, for comparison, also in Chinese hamster ovary cells transfected with the human insulin receptor (CHO-T cells). In CHO-T cells, the net rate of fluid-phase pinocytosis was rapidly increased 3–4-fold over the basal rate by 100 nM insulin, with half-maximal stimulation at 2 nM insulin, as assayed by cellular uptake of horseradish peroxidase from the medium. Wortmannin, an inhibitor of phosphatidylinositol (PI)-3-kinase, blocked insulin-stimulated pinocytosis with an IC50 of 7.5 nM without affecting the basal rate of pinocytosis. In insulin-secreting βTC6-F7 cells, the secretagogues glucose and carbachol (at maximally effective concentrations of 15 mM and 0.5 mM respectively) augmented fluid-phase pinocytosis 1.65-fold over the basal rate. Wortmannin also inhibited secretagogue-stimulated pinocytosis in these β-cells with an IC50 of 7 nM but did not affect the basal rate of pinocytosis measured in the absence of secretagogues. Wortmannin did not influence either basal or secretagogue-induced insulin secretion. Although these βTC6-F7 cells have cell-surface insulin receptors, adding exogenous insulin or insulin-like growth factor 1 did not affect their rate of fluid-phase pinocytosis, either in the absence or presence of secretagogues. From these observations, we conclude that: (1) in both insulin-secreting β-cells and in conventional, insulin-responsive CHO-T cells, a common, wortmannin-sensitive reaction, which probably involves PI-3-kinase, regulates fluid-phase pinocytosis; (2) the insulin-receptor signal transduction pathway is dissociated from the regulation of fluid-phase pinocytosis in the insulin-secreting β-cell line we studied; and (3) the enhancement of fluid-phase pinocytosis associated with secretagogue-induced insulin release in βTC6-F7 cells is not attributable to autocrine activation of β-cell surface insulin receptors.

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Cell-surface insulin receptor cycling and its implication in the glycogenic response in cultured foetal hepatocytes

Biochemical Journal ◽

10.1042/bj2390609 ◽

1986 ◽

Vol 239 (3) ◽

pp. 609-615 ◽

Cited By ~ 12

Author(s):

P Soubigou ◽

E Pringault ◽

C Plas

Keyword(s):

Cell Surface ◽

Insulin Receptor ◽

Polyacrylamide Gel Electrophoresis ◽

Insulin Receptors ◽

Rat Hepatocytes ◽

Insulin Binding ◽

Surface Proteins ◽

Short Exposure ◽

Intermittent Exposure ◽

Initial Binding

The insulin-receptor cycle was investigated in cultured foetal rat hepatocytes by determining the variations in insulin-binding sites at the cell surface after short exposure to the hormone. Binding of 125I-insulin was measured at 4 degrees C after dissociation of prebound native insulin. Two protocols were used: exchange binding assay and binding after acid treatment; both gave the same results. Cell-surface 125I-insulin-receptor binding decreased sharply (by 40%) during the first 5 min of 10 nM-insulin exposure (t1/2 = 2 min) and remained practically constant thereafter; subsequent removal of the hormone restored the initial binding within 10 min. This fall-rise sequence corresponded to variations in the number of insulin receptors at the cell surface, with no detectable change in receptor affinity. The reversible translocation of insulin receptors from the cell surface to a compartment not accessible to insulin at 4 degrees C was hormone-concentration- and temperature-dependent. SDS/polyacrylamide-gel electrophoresis after cross-linking of bound 125I-insulin to cell-surface proteins with disuccinimidyl suberate showed that these variations were not associated with changes in Mr of binding components, in particular for the major labelled band of Mr 130,000. The insulin-receptor cycle could be repeated after intermittent exposure to insulin. Continuous or intermittent exposure to the hormone gave a similar glycogenic response, contrary to the partial effect of a unique short (5-20 min) exposure. A relationship could be established between the repetitive character of the rapid insulin-receptor cycle and the maximal expression of the biological effect in cultured foetal hepatocytes.

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The T-tubule is a cell-surface target for insulin-regulated recycling of membrane proteins in skeletal muscle

Biochemical Journal ◽

10.1042/bj3120393 ◽

1995 ◽

Vol 312 (2) ◽

pp. 393-400 ◽

Cited By ~ 28

Author(s):

P Muñoz ◽

M Rosemblatt ◽

X Testar ◽

M Palacín ◽

G Thoidis ◽

...

Keyword(s):

Skeletal Muscle ◽

Membrane Proteins ◽

Cell Surface ◽

Insulin Receptor ◽

Insulin Receptors ◽

Acute Administration ◽

Rat Skeletal Muscle ◽

Density Fractions ◽

A Cell ◽

T Tubules

(1) In this study we have determined the distribution of various membrane proteins involved in insulin-activated glucose transport in T-tubules and in sarcolemma from rat skeletal muscle. Two independent experimental approaches were used to determine the presence of membrane proteins in T-tubules: (i) the purification of T-tubules free from sarcolemmal membranes by lectin agglutination, and (ii) T-tubule vesicle immunoadsorption. These methods confirmed that T-tubules from rat skeletal muscle were enriched with dihydropyridine receptors and tt28 protein and did not contain the sarcolemmal markers dystrophin or beta 1-integrin. Both types of experiments revealed an abundant content of GLUT4 glucose carriers, insulin receptors and SCAMPs (secretory carrier membrane proteins) in T-tubule membranes. (2) Acute administration in vivo of insulin caused an increased abundance of GLUT4 in T-tubules and sarcolemma. On the contrary, insulin led to a 50% reduction in insulin receptors present in T-tubules and in sarcolemma, demonstrating that insulin-induced insulin receptor internalization affects T-tubules in the muscle fibre. The alteration in the content of GLUT4 and insulin receptors in T-tubules was a consequence of insulin-induced redistribution of these proteins. SCAMPs also redistributed in muscle membranes in response to insulin. They were recruited by insulin from intracellular high-density fractions to intracellular lighter-density fractions and to the cell surface, showing a pattern of insulin-induced cellular redistribution distinct from those of GLUT4 and the insulin receptor. (3) In conclusion, the T-tubule is a cell-surface target for membrane proteins involved in recycling such as SCAMPs or for membrane proteins that acutely redistribute in response to insulin such as GLUT4 or insulin receptors.

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Uncoupling between the insulin-receptor cycle and the cellular degradation of the hormone in cultured foetal hepatocytes. Effect of drugs and temperature that inhibit insulin degradation

Biochemical Journal ◽

10.1042/bj2460567 ◽

1987 ◽

Vol 246 (3) ◽

pp. 567-573 ◽

Cited By ~ 4

Author(s):

P Soubigou ◽

M Ali ◽

C Plas

Keyword(s):

Cell Surface ◽

Insulin Receptor ◽

Rat Hepatocytes ◽

Receptor Internalization ◽

Cell Surface Receptor ◽

Insulin Degradation ◽

Receptor Recycling ◽

Partial Restoration ◽

Receptor Sites ◽

Surface Receptor

Sequential changes in the numbers of cell-surface receptors induced by a transitory exposure to insulin in cultured 18-day foetal-rat hepatocytes were investigated in the presence of drugs and at a temperature of 22 degrees C, which inhibit cellular insulin degradation. Chloroquine (70 microM) and monensin (3 microM) did not greatly change the initial rate of internalization of cell-surface receptor sites after exposure to 10 nM-insulin, but led to a steady state after 20 min, which represented 40% of the initial binding, compared with 5 min and 60% in the absence of the drug. Moreover, these drugs strongly decreased the proportion of receptor sites recovered at the cell surface after subsequent removal of the hormone. They were ineffective when insulin was not present. The removal of monensin together with the hormone allowed partial restoration of cell-surface receptor sites and degradation of cell-associated insulin to start again at the initial speed, indicating a reversible effect of the drug. During this phase, the drug concentration-dependence for the two effects showed that receptor recycling was restored with concentrations of monensin not as low as for insulin degradation. The effect of vinblastine (50-100 microM) was similar to that of chloroquine and monensin, whereas no modification in the internalization and recovery processes was observed in the presence of bacitracin concentrations (1-3 mM) that inhibit insulin degradation by 70%. A temperature of 22 degrees C did not prevent the receptor internalization, but had a slowing effect on the recycling process, which appeared to vary in experiments where insulin degradation remained inhibited. The present study shows that the process of insulin degradation mediated by receptor endocytosis is not a prerequisite for insulin-receptor recycling in cultured foetal hepatocytes.

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Hyperinsulinemia Induces Insulin Receptor Dysfunction in Brain Microvascular Endothelial Cells

10.20944/preprints202111.0479.v1 ◽

2021 ◽

Author(s):

Luke S. Watson ◽

Brynna Wilken-Resman ◽

Alexus Williams ◽

Guadalupe Sanchez ◽

Taylor Lowry McLeod ◽

...

Keyword(s):

Endothelial Cells ◽

Insulin Receptor ◽

High Fat Diet ◽

Insulin Receptors ◽

Receptor Internalization ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Receptor Function ◽

High Fat ◽

Microvascular Endothelial

Insulin receptors are internalized by endothelial cells; however, the impact of hyperinsulinemia on this process is not known. Thus, the aim of this study is to determine the role of hyperinsulinemia on insulin receptor function and internalization, as well as the potential impact of protein tyrosine phosphatase 1B (PTP1B). To this end, hippocampal microvessels were isolated from male C57Bl/6J mice on either a control or high-fat diet and assessed for insulin receptor signaling. Cell surface insulin receptors in brain microvascular endothelial cells were labelled with biotin to assess the role hyperinsulinemia plays on receptor internalization in response to stimulation, with and without Claramine treatment, a potent PTP1B antagonist. Our results indicated that insulin receptor levels increased in tandem with insulin receptor dysfunction in the high-fat diet mouse hippocampal microvessels. Hyperinsulinemic cell-receptors demonstrate a shift in splice variation towards decreased IR-A/IR-B ratios and demonstrate a higher membrane-localized proportion. This corresponded with decreased autophosphorylation at sites critical for receptor internalization and signaling, however, Claramine restored signaling and receptor internalization in hyperinsulinemic cells. In conclusion, hyperinsulinemia negatively impacts brain microvascular endothelial cell insulin receptor function and internalization, likely through both alternative splicing and increased negative feedback from PTP1B.

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Glucocorticoids increase insulin binding and the amount of insulin-receptor mRNA in human cultured lymphocytes

Biochemical Journal ◽

10.1042/bj2490715 ◽

1988 ◽

Vol 249 (3) ◽

pp. 715-719 ◽

Cited By ~ 11

Author(s):

Y Shibasaki ◽

H Sakura ◽

M Odawara ◽

M Shibuya ◽

Y Kanazawa ◽

...

Keyword(s):

Cell Surface ◽

Insulin Receptor ◽

De Novo ◽

Insulin Receptors ◽

Fold Increase ◽

Insulin Binding ◽

Dependent Manner ◽

Receptor Mrna ◽

Dose Dependent ◽

Receptor Mrnas

The effect of steroid hormones on insulin binding and the amount of insulin-receptor mRNA was examined in IM-9 lymphocytes. Cortisol and cortexolone, but not oestrogen, increased both the binding of insulin and the amount of insulin-receptor mRNA in a time- and dose-dependent manner. Cortisol was most potent, and induced a 2-fold increase in insulin binding and a 4-fold increase in mRNA. The elevation in binding was due to an increased number of insulin receptors at the cell surface. The increase in mRNA involved all four of the insulin-receptor mRNAs and could not be inhibited by cycloheximide. The cortisol-induced increase in mRNA was associated with a 3-4-fold increase in the synthesis of pro-receptor. The relative potency of the three steroids indicated that these effects were mediated by an interaction with the glucocorticoid receptor. The results of this study suggest that cortisol can increase the number of insulin receptors at the cell surface by increasing the amounts of insulin-receptor mRNA and the synthesis de novo of insulin receptors.

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