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L Jarett

Publications and source records attributed to L Jarett.

At least 19 recordsLinked to original sources
PubMed1999

Angiotensin II inhibits insulin-induced egr-1 expression in mesangial cells.

The gene early growth response gene-1 (egr-1) encodes a zinc transcription factor involved in cell proliferation. Increased expression of egr-1 has been linked to heart and kidney disease. In mouse mesangial cells, insulin stimulated egr-1 expression more than angiotensin II, suggesting that insulin may play an important role in stimulating cell proliferation, leading to glomerulonephritis and diabetic nephropathy. Angiotensin II inhibited insulin-induced egr-1 expression but not c-fos expression, and the decrease in egr-1 expression was concurrent with a decrease in insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation. These results suggest that insulin-induced egr-1 expression in mouse mesangial cells is downstream of tyrosine phosphorylation of IRS-1 and activation of the MAP kinase pathway and that crosstalk between angiotensin II and insulin signaling pathways led to an inhibition of IRS-1 tyrosine phosphorylation and egr-1 expression.

Angiotensin II↗
PubMed1999

Mechanisms of nuclear translocation of insulin.

Insulin (Ins) and various other hormones and growth factors have been shown to be rapidly internalized and translocated to the cell nucleus. This review summarizes the mechanisms that are involved in the translocation of Ins to the nucleus, and discusses its possible role in Ins action, based on observations by the authors and others. Ins is internalized to endosomes by both receptor-mediated and fluid-phase endocytosis, the latter occurring only at high Ins concentrations. The authors recently demonstrated the caveolae are the primary cell membrane locations responsible for initiating the signal transduction cascade induced by Ins. Once Ins is internalized, Ins dissociates from the Ins receptor in the endosome, and is translocated to the cytoplasm, where most Ins is degraded by Ins-degrading enzyme (IDE), although how the polypeptides cross the lipid bilayer is unknown. Some Ins escapes the degradation and binds to cytosolic Ins-binding proteins (CIBPs), in addition to IDE. IDE and some CIBPs are known to be binding proteins for other hormones or their receptors, and are involved in gene regulation, suggesting physiological relevance of CIBPs in the signaling of Ins and other hormones. Ins is eventually translocated through the nuclear pore to the nucleus, where Ins tightly associates with nuclear matrix. The role of Ins internalization and translocation to the nucleus is still controversial, although there is substantial evidence to support its role in cellular responses caused by Ins. Many studies indicate that nuclear translocation of various growth factors and hormones plays an important role in cell proliferation or DNA synthesis. It would be reasonable to suggest that Ins internalization, its association with CIBPs, and its translocation to the nucleus may be essential for the regulation of nuclear events by Ins.

Animals↗
PubMed1999

Differential modulation of the tyrosine phosphorylation state of the insulin receptor by IRS (insulin receptor subunit) proteins.

In response to insulin, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins including insulin receptor subunit (IRS)-1, IRS-2, and Shc. Phosphorylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for the biological effects of insulin, and understanding regulation of insulin receptor phosphorylation and kinase activity is essential to understanding insulin action. Receptor tyrosine kinase activity may be altered by direct changes in tyrosine kinase activity, itself, or by dephosphorylation of the insulin receptor by protein-tyrosine phosphatases. After 1 min of insulin stimulation, the insulin receptor was tyrosine phosphorylated 8-fold more and Shc was phosphorylated 50% less in 32D cells containing both IRS-1 and insulin receptors (32D/IR+IRS-1) than in 32D cells containing only insulin receptors (32D/IR), insulin receptors and IRS-2 (32D/IR+IRS-2), or insulin receptors and a form of IRS-1 that cannot be phosphorylated on tyrosine residues (32D/IR+IRS-1F18). Therefore, IRS-1 and IRS-2 appeared to have different effects on insulin receptor phosphorylation and downstream signaling. Preincubation of cells with pervanadate greatly decreased protein-tyrosine phosphatase activity in all four cell lines. After pervanadate treatment, tyrosine phosphorylation of insulin receptors in insulin-treated 32D/IR, 32D/ IR+IRS-2, and 32D/IR+IRS-1F18 cells was markedly increased, but pervanadate had no effect on insulin receptor phosphorylation in 32D/IR+IRS-1 cells. The presence of tyrosine-phosphorylated IRS-1 appears to increase insulin receptor tyrosine phosphorylation and potentially tyrosine kinase activity via inhibition of protein-tyrosine phosphatase(s). This effect of IRS-1 on insulin receptor phosphorylation is unique to IRS-1, as IRS-2 had no effect on insulin receptor tyrosine phosphorylation. Therefore, IRS-1 and IRS-2 appear to function differently in their effects on signaling downstream of the insulin receptor. IRS-1 may play a major role in regulating insulin receptor phosphorylation and enhancing downstream signaling after insulin stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Insulin-induced protein tyrosine phosphorylation cascade and signalling molecules are localized in a caveolin-enriched cell membrane domain.

The cellular localisation of time- and temperature-dependent 125I-insulin binding, insulin-sensitive signalling proteins and the insulin-induced protein tyrosine phosphorylation cascade were assessed in subcellular fractions isolated on Iodixanol gradients from control and insulin-treated H35 hepatoma cells. Western blot analysis demonstrated that the concentrations of IRS-1, Shc, GRB-2, SOS, Syp, PI 3-kinase, MAP kinase and Gi alpha were at least 10-fold higher in cell surface-derived, caveolin-enriched fraction than in a cell surface-derived, caveolin-poor fraction (i.e., the plasma membranes). Insulin treatment caused a 15-fold increase in tyrosine phosphorylation of IRS-1 in the caveolin-enriched fraction in 5 min at 37 degrees C compared with a 3-fold increase in plasma membranes and a 6-fold increases in the cytosol and endosomes. Insulin also increased tyrosine phosphorylation of both a 72-kDa protein and the 46-kDa Shc isoform only in the caveolin-enriched fraction. Insulin treatment did not change the concentrations of insulin receptors or Shc but increased IRS-1 in the caveolin-enriched fraction, possibly recruited from the cytosolic pool. Insulin also increased the concentrations of insulin receptors, IRS-1 and Shc in endosomes, suggesting insulin-induced internalization of the insulin receptors and proteins activated with them. Electron microscopic analysis, with the use of a combination of colloidal gold-labelled insulin to label the insulin receptor and immunolabelling to detect caveolin or IRS-1, demonstrated the co-localisation of insulin receptors in caveolin- and IRS-1 containing vesicular structures. Differences in the insulin-induced protein tyrosine phosphorylation and concentrations of these proximal signalling proteins in the caveolin-enriched fraction, plasma membranes, and cytosol suggest that insulin receptors in the caveolae play a major role in initiating insulin's signal transduction processes.

Binding Sites↗
PubMed1997

Receptor-mediated cellular entry of nuclear localizing anti-DNA antibodies via myosin 1.

A unique subset of anti-DNA antibodies enters living cells, interacts with DNase 1, and inhibits endonuclease activity, before their nuclear localization and subsequent attenuation of apoptosis. We now report that endocytosis of these immunoglobulins is mediated by cell surface binding to brush border myosin (myosin 1). Cellular entry and internalization via this unique receptor provides initial contact for entry and sorting these immunoglobulins to translocate to the nuclear pore and enter the nucleus, interact with DNase 1 within the cytoplasm, or recycle back to the cell surface. This internalization pathway provides clues to the translocation of large proteins across cell membranes and the functional effects of intracellular antibodies on cytopathology. This is the first demonstration that brush border myosin functions as a specific cell surface receptor for internalization of large proteins.

Amino Acid Sequence↗
PubMed1997

Insulin internalization and other signaling pathways in the pleiotropic effects of insulin.

Insulin is the major anabolic hormone in humans and affects multiple cellular processes. Insulin rapidly regulates short-term effects on carbohydrate, lipid, and protein metabolism and is also a potent growth factor controlling cell proliferation and differentiation. The metabolic and growth-related effects require insulin binding to its receptor and receptor phosphorylation. Evidence suggests these events result in subsequent substrate phosphorylation and activation of multiple signaling pathways involving Src homology domain-containing proteins and the internalization of the insulin:receptor complex. The role of insulin internalization in insulin action is largely speculative. For more than two decades, extensive investigation has been carried out by numerous laboratories of the mechanisms by which insulin causes its pleiotropic responses and the cellular processing of insulin receptors. This chapter reviews our current knowledge of the phosphorylation signaling pathways activated by insulin and presents evidence that substrates other than insulin receptor substrate-1 are involved in insulin's regulation of immediate-early gene expression. We also review the mechanisms involved in insulin internalization and present evidence that internalization may play a key role in insulin action through both signal transduction processes and translocation of insulin to the cell cytoplasm and nucleus.

Animals↗
PubMed1996

Insulin-induced egr-1 and c-fos expression in 32D cells requires insulin receptor, Shc, and mitogen-activated protein kinase, but not insulin receptor substrate-1 and phosphatidylinositol 3-kinase activation.

Many studies suggest that insulin utilizes multiple signal transduction pathways. Insulin's effects are initiated by insulin binding to the insulin receptor, resulting in tyrosine phosphorylation of insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1), IRS-2, or Shc. We recently demonstrated that immediate-early gene egr-1 transcription was fully induced without phosphorylation of IRS-1 in Chinese hamster ovary cells (Harada, S., Smith, R. M., Smith, J. A., Shah, N. , Hu, D.-Q. & Jarett, L. (1995) J. Biol. Chem. 270, 26632-26638). In the present study, we examined the effects of insulin on immediate-early gene egr-1 and c-fos expression in 32D cells overexpressing the insulin receptor (32D/IR), IRS-1 (32D/IRS), or both (32D/IR+IRS) and compared these effects with insulin-induced tyrosine phosphorylation. Insulin (17 nM) increased egr-1 and c-fos expression in 32D/IR and 32D/IR+IRS cells, but not in parental cells or 32D/IRS cells, as determined by Northern blot analysis. Insulin treatment (5 min at 37 degrees C) markedly increased tyrosine phosphorylation of several proteins, including the insulin receptor, IRS-1, and Shc, in 32D/IR+IRS cells as determined by immunoprecipitation and Western blot analysis with anti-phosphotyrosine antibody. In contrast, only two tyrosine-phosphorylated proteins, i.e. insulin receptor and Shc, were detected in 32D/IR cells. These data suggest that insulin receptor and Shc phosphorylation is necessary for insulin-induced egr-1 and c-fos expression, but IRS-1 phosphorylation is not necessary or sufficient for the expression of these genes. Furthermore, the effect of specific inhibitors on insulin-induced egr-1 expression was examined. Wortmannin (25 nM), a phosphatidylinositol 3-kinase inhibitor, had no effect on insulin-induced egr-1 expression. In contrast, PD 98059 (30 microM), a mitogen-activated protein kinase kinase inhibitor, totally blocked egr-1 expression induced by insulin. These data indicate that mitogen-activated protein kinase activation, but not phosphatidylinositol 3-kinase activation, is involved in insulin-induced egr-1 expression. Taken together, insulin receptor tyrosine phosphorylation, Shc tyrosine phosphorylation, and mitogen-activated protein kinase activation appear to be the signal transduction pathway responsible for insulin-induced egr-1 expression in 32D cells. These data demonstrate that insulin has multiple signal transduction pathways that vary from cell to cell.

Adaptor Proteins, Signal Transducing↗
PubMed1996

The expression of and insulin binding to cellular thyroid hormone binding protein, but not insulin degrading enzyme, is increased during 3T3-L1 adipocytes differentiation.

Specific insulin binding to several proteins (cytosolic insulin binding proteins; CIBPs) in the isolated cytoplasm of numerous cell types has been demonstrated. CIBPs include insulin degrading enzyme (IDE), CIBP p55 (identified as cellular thyroid hormone binding protein (CTHBP), which is also known as protein disulfide isomerase, or glutathione insulin transhydrogenase). To assess the possible role of CIBP p55/CTHBP in insulin action, we compared 125I-insulin binding to CIBP in cytosol isolated from 3T3-L1 cells at various time points during differentiation of the adipocytes. Insulin did not bind to CTHBP in fibroblasts, but the labeling was markedly increased during adipocyte differentiation. In contrast, insulin binding to IDE did not change during differentiation. Protein expression level of CTHBP in the cytosol fraction increased gradually during the differentiation of adipocytes, whereas that of IDE did not change throughout the period. These data indicate that CTHBP, but not IDE, was up-regulated during differentiation of the adipocytes, suggesting that CIBP p55/CTHBP may play a role in regulating some insulin action, especially the counter regulation between insulin and other hormones during adipocyte differentiation.

3T3 Cells↗
PubMed1996

Dexamethasone inhibits insulin binding to insulin-degrading enzyme and cytosolic insulin-binding protein p82.

We recently demonstrated that insulin specifically binds to several cytosolic insulin-binding proteins (CIBPs) including insulin-degrading enzyme (IDE) and CIBP p82 in cytosol isolated from H35 rat hepatoma cells. Insulin binding to these CIBPs was regulated by culture conditions, such as serum or insulin. In the present study, we examined the effect of dexamethasone on insulin binding to CIBPs in H35 cells. When the cells were treated with 100 nM dexamethasone for 24 hrs, insulin binding to IDE and CIBP p82 was decreased by about 50% without decreasing the expression level of IDE. Insulin added with the dexamethasone prevented the steroid's effect. Furthermore, dexamethasone directly blocked insulin binding to CIBPs in isolated cytosol. These results suggest that dexamethasone, directly or as a complex with other proteins, binds to IDE and CIBP p82 and changes their ability to bind insulin, possibly by inducing a conformational change or by blocking insulin binding sites. IDE was recently identified as a receptor accessory factor for androgen and glucocorticoid receptors and plays an important role in the regulation of gene transcriptional responses. Combined with previous reports, our findings suggest IDE and other CIBPs such as CIBP p82 may play a role in the cross-talk between insulin and the signal transduction pathways of steroid hormones.

Animals↗
PubMed1996

Report of the American Diabetes Association's Task Force on standardization of the insulin assay.

Recent large-scale epidemiological studies demonstrate that blood concentrations of immunoreactive insulin predict the development of NIDDM and IDDM and are associated with the risk of several degenerative diseases, such as coronary and peripheral vessel atherosclerosis, hypertension, and dyslipidemia. The reliability of these measurements is dependent on a biological assay that has not been well standardized between laboratories. Recognizing this, the American Diabetes Association organized a task force to assess comparability of blood insulin measurements between laboratories and to suggest techniques to improve comparability. The task force found that identical serum and plasma samples measured in different laboratories produced widely disparate values that were unacceptable for population comparisons. Use of a single reference standard did little to improve comparability. Assay characteristics such as linearity, recovery, accuracy, and cross-reactivity to proinsulin and its primary conversion intermediates varied among the laboratories, and they did not readily explain differences in the measurements made from assay to assay. Use of the same assay kit in different laboratories did not always ensure comparable measurements. Linear regression of assay results from one laboratory to an arbitrarily chosen reference assay greatly improved comparability and demonstrated the potential value in comparing each assay to a reference method. The task force report defines acceptable assay characteristics and proposes a three-step process of insulin assay proficiency and comparability. A central reference assay and ongoing sample exchange will be needed to allow reliable comparisons of insulin measurements made in different laboratories. Rigorous quality control and continuous quality improvement are needed to maintain reliability of the insulin measurement.

Diabetes Mellitus↗
PubMed1995

The effects of brefeldin A on the glucose transport system in rat adipocytes. Implications regarding the intracellular locus of insulin-sensitive Glut4.

Insulin activates glucose transport by recruiting Glut4 glucose transporters from an intracellular pool to plasma membrane (PM). To localize intracellular translocating Glut4, we studied the effects of brefeldin A (BFA), which disassembles Golgi and prevents trans-Golgi vesicular budding, on the glucose transport system. Isolated rat adipocytes were treated with and without both BFA (10 micrograms/ml) and insulin. BFA did not affect maximal rates of either 2-deoxyglucose or 3-O-methyl-glucose transport or the insulin:glucose transport dose-response curve but did increase basal transport by approximately 2-fold (p < 0.05). We also measured Glut4 in PM, low (LDM) and high density microsome subfractions. In basal cells, BFA increased PM Glut4 by 58% concomitant with a 18% decrease in LDM (p < 0.05). Insulin alone increased PM Glut4 by 3-fold concomitant with a 56% decrease in LDM. BFA did not affect insulin-induced changes in Glut4 levels in PM or LDM. Most intracellular Glut4 was localized to sub-PM vesicles by immunoelectron microscopy in basal cells, and BFA did not affect insulin-mediated recruitment of immunogold-labeled Glut4 to PM. In summary, 1) in basal cells, BFA led to a small increase in glucose transport activity and redistribution of a limited number of transporters from LDM to PM; 2) BFA did not affect insulin's ability to stimulate glucose transport or recruit normal numbers of LDM Glut4 to PM; and 3) insulin action is predominantly mediated by a BFA-insensitive pool of intracellular Glut4, which localizes to sub-PM vesicles. Thus, the major translocating pool of Glut4 in rat adipocytes does not involve trans-Golgi.

3-O-Methylglucose↗
PubMed1995

Insulin-induced egr-1 expression in Chinese hamster ovary cells is insulin receptor and insulin receptor substrate-1 phosphorylation-independent. Evidence of an alternative signal transduction pathway.

Insulin's effects primarily are initiated by insulin binding to its plasma membrane receptor and the sequential tyrosine phosphorylation of the insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1). However, studies suggest some insulin effects, including those at the nucleus, may not be regulated by this pathway. The present study compared the levels of insulin binding, insulin receptor and IRS-1 tyrosine phosphorylation, and phosphatidylinositol 3'-kinase activity to immediate early gene c-fos and egr-1 mRNA expression in Chinese hamster ovary (CHO) cells expressing only neomycin-resistant plasmid (CHONEO), overexpressing wild type human insulin receptor (CHOHIRc) or ATP binding site-mutated insulin receptors (CHOA1018K). Insulin binding in CHONEO cells was markedly lower than that in other cell types. 10 nM insulin significantly increased tyrosine phosphorylation of insulin receptor and IRS-1 in CHOHIRc cells. Phosphorylation of insulin receptor and IRS-1 in CHONEO and CHOA1018K cells was not detected in the presence or absence of insulin. Similarly, insulin increased phosphatidylinositol 3-kinase activity only in CHOHIRc cells. As determined by Northern blot, nuclear run-on analysis, and in situ hybridization, insulin induced c-fos mRNA expression, through transcription, in CHOHIRc cells but not in CHONEO and CHOA1018K cells, consistent with previous reports. In contrast, all three cell types showed a similar insulin dose-dependent increase of egr-1 mRNA expression through transcription. These data indicated that insulin-induced egr-1 mRNA expression did not correlate with the levels of insulin binding to insulin receptor or phosphorylation of insulin receptor and IRS-1. These results suggest that different mechanisms are involved in induction of c-fos and egr-1 mRNA expression by insulin, the former by the more classic insulin receptor tyrosine kinase pathway and the latter by a yet to be determined alternative signal transduction pathway.

Adenosine Triphosphate↗
PubMed1995

The heterotrimeric G-protein Gi is localized to the insulin secretory granules of beta-cells and is involved in insulin exocytosis.

Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from beta-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting beta-cell line beta-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized beta-TC3 cells. Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion. Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized beta-TC3 cells. Mastoparan-induced insulin secretion from permeabilized beta-TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin-sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western immunoblotting performed on beta-TC3 homogenate and ISG demonstrated that G alpha i was dramatically enriched in ISG. Levels of G alpha o and G alpha q were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of G alpha i to ISG was confirmed with electron microscopic immunocytochemistry in beta-TC3 cells and beta-cells from rat pancreas. In contrast to G alpha o and G alpha q, G alpha was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein G alpha i located in the ISG of beta-cells to stimulate insulin secretion.

Animals↗
PubMed1995

Demonstration of specific insulin binding to cytosolic proteins in H35 hepatoma cells, rat liver and skeletal muscle.

We previously demonstrated that internalized insulin enters the cytoplasm before accumulating in nuclei of H35 rat hepatoma cells. This finding raises the possibility that insulin may interact with cytosolic proteins in addition to insulin-degrading enzyme (IDE). In the present study, cytosol from H35 hepatoma cells, rat liver or muscle was incubated with A14- or B26-125I-insulin at 4 degrees C for 5-120 min in the absence or presence of 25 micrograms/ml unlabelled insulin. 125I-insulin was cross-linked to cytosolic proteins by disuccinimidyl suberate and analysed by reducing or non-reducing SDS/PAGE and autoradiography. Our results demonstrate the presence of both tissue-specific and common cytosolic proteins which specifically bind insulin. In muscle cytosol, only two proteins of 27 and 110 kDa were specifically labelled with B26-125I-insulin. Seven major bands, of 27, 45, 55, 60, 76, 82 and 110 kDa, were labelled in rat liver cytosol. Detection of cytosolic insulin-binding proteins in H35-cell cytosol was dependent on cell-culture conditions. Labelling in cytosol from serum-deprived cells was decreased or absent compared with cytosol prepared from serum-fed or serum-deprived cells treated with 100 ng/ml insulin for 1 h before preparation of the cytosol, in which six bands, of 32, 41, 45, 55, 82 and 110 kDa, were specifically labelled with B26-125I-insulin. This result suggests that the concentration or binding activity of some cytosolic insulin-binding proteins is rapidly regulated. Labelling of both rat liver and H35 cytosolic insulin-binding proteins was time-dependent, and decreased or disappeared at 120 min in parallel with the degradation of labelled insulin. Fewer bands were specifically labelled with A14-125I-insulin than with B26-125I-insulin. The number of labelled bands observed under reducing and non-reducing conditions was not different in any of the cytosols. The 110 kDa band in all cytosols was identified as IDE by Western-blot analysis; the other proteins did not react with anti-IDE antibody and remain unidentified. 1,10-Phenanthroline (2 mM) increased IDE labelling, but decreased the labelling of 82 and 27 kDa bands. The marked difference in the number of cytosolic insulin-binding proteins in muscle and either H35 cells or liver suggests both that the labelling is specific and that these proteins serve a function and may be involved in some heretofore unknown mechanism of the signalling pathway by which insulin regulates cell growth or differentiation.

Animals↗
PubMed1995

Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides.

An enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques. Chemical cross-linking studies with a biotin-labeled P-ODN (bP-ODN), revealed the existence of five major cell surface P-ODN binding protein groups ranging in size from approximately 20-143 kD. Binding to these proteins was competitively inhibited with unlabeled P-ODN, but not free biotin, suggesting specificity of the interactions. Additional experiments suggested that binding proteins likely exist as single chain structures, and that carbohydrate moieties may play a role in P-ODN binding. Uptake studies with 35S-labeled P-ODN revealed that endocytosis, mediated by a receptor-like mechanism, predominated at P-ODN concentrations < 1 microM, whereas fluid-phase endocytosis prevailed at higher concentrations. Cell fractionation and ultrastructural analysis demonstrated the presence of ODN in clathrin coated pits, and in vesicular structures consistent with endosomes and lysosomes. Labeled ODN were also found in significant amounts in the nucleus, while none was associated with ribosomes, or ribosomes associated with rough endoplasmic reticulum (ER). Since nuclear uptake was not blocked by wheat germ agglutinin or concanavalin A, a nucleoporin independent, perhaps diffusion driven, import process is suggested. These data imply that antisense DNA may exert their effect in the nucleus. They also suggest rational ways to design ODN which might increase their efficiency.

Biological Transport↗
PubMed1995

Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin.

Insulin affects numerous metabolic processes as well as nuclear events such as gene transcription. Our previous ultrastructural and biochemical studies demonstrated insulin accumulation in nuclei of cultured and rapidly proliferating cells, and biochemical evidence suggested that insulin entered the cell cytoplasm before accumulating in the nucleus. The present study was undertaken to develop a covalently linked electron-dense insulin complex that could be used to visualize the intracellular translocation of insulin and confirm that insulin enters the cytoplasm of cells. Insulin was cross-linked to 1.4-nm diameter Nanogold particles. The complex binds to the plasma membrane insulin receptor, is biologically active, and is degraded by cellular insulin-degradative enzymes. Ultrastructural analysis after silver intensification of the gold particles confirmed that insulin internalization culminates in the translocation of some internalized insulin to the cytoplasm and nuclei. When cytoplasmic insulin-degrading enzyme (IDE) activity was inhibited with 1,10-phenanthroline, an increase in the number of cytoplasmic and nuclear Nanogold-insulin particles was observed. The results of this and previous studies suggest that 1) the translocation of insulin to the cytoplasm, 2) the regulation of insulin degradation in the cytoplasm by IDE, 3) the possible interaction of insulin with cytoplasmic proteins other than IDE, and 4) the subsequent accumulation of intact insulin or insulin complexed with cytoplasmic proteins in nuclei may play a role in insulin's regulation of gene transcription and cell proliferation.

Adipose Tissue↗