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D Brandenburg

Publications and source records attributed to D Brandenburg.

At least 73 records · Page 4Linked to original sources
PubMed1984

Effects of covalently linked insulin dimers on receptor kinase activity and receptor down regulation.

Certain covalently linked insulin dimers have previously been found to have a greater ability to bind to the insulin receptor than to stimulate lipogenesis in adipocytes. The present report presents data indicating that the same insulin dimers also have a greater ability to bind to the receptor than to stimulate the kinase activity of the insulin receptor. In particular, one such covalently linked insulin dimer had less than 1% the potency of native insulin in stimulating the receptor kinase although it could bind to the solubilized receptor with 30% the potency of native insulin. In contrast, this dimer could down regulate the insulin receptor with approximately 30% the potency of native insulin. These results suggest that stimulation of the receptor kinase may require more than simple occupancy of the receptor binding site whereas down regulation of the receptor may require only the binding of ligand to the receptor.

Dose-Response Relationship, Drug↗
PubMed1984

Biological potency of covalently linked insulin-receptor in rat adipocytes. Comparison with the potency of reversible complexes.

Irradiation of photoreactive insulin derivatives in the presence of isolated rat adipocytes produces a prolonged stimulation of lipogenesis in the cells even after exogenous and reversibly bound derivative has been removed by extensive washing. The quantitative nature of this response has now been studied using 125I-B2(2-nitro-4-azidophenylacetyl)des-PheB1-insulin. This derivative possesses nearly full biological potency and binding affinity prior to irradiation. After covalent linkage to adipocytes the efficacy of the derivative is reduced to 25 +/- 4% of the reversibly bound derivative, viz. 4-times as much needs to be covalently associated as reversibly bound to induce the same level of stimulation of lipogenesis. This reduced relative molar potency is due to a reduced ability of specific covalent insulin-receptor complexes to trigger a response.

Adipose Tissue↗
PubMed1984

Biological activity of covalently-linked insulin-receptor complexes in rat adipocytes. Effect of pH.

The relative molar potencies of covalently and reversibly-bound insulin-receptor complexes were studied as a function of pH. The insulin derivatives used were 125I-B2 (2-nitro-4-azidophenylacetyl)des-PheB1-insulin and 125I-B29(2-nitro-4-azidophenylacetyl)des-PheB1-insulin. The potencies of both types of reversible complexes were effectively identical and constant between pH 7 and 8. The relative potency of the covalent B2-complex increased from 25 to 75%, and of the covalent B29 complex from 30 to nearly 100%. This indicates that the covalently linked partners in the complex are able to flex about the cross-linkages. Variations in the potency are due to variations in the number of correctly associated, reversibly or covalently bound insulin-receptor complexes. The form of the pH dependance suggests that an ionizable group, possibly an amino group, must be deprotonated to allow effective interaction.

Adipose Tissue↗
PubMed1984

Assembly in vitro of a spanning membrane protein of the endoplasmic reticulum: the E1 glycoprotein of coronavirus mouse hepatitis virus A59.

The E1 glycoprotein of coronavirus mouse hepatitis virus A59 was synthesized in vitro by translation of viral mRNA in the presence of dog pancreatic microsomes. Its disposition in the membrane was investigated by digestion with proteases and by selective NH2-terminal labeling. The protein spans the membrane, but only small portions from the NH2 and COOH terminus are exposed respectively in the lumenal and cytoplasmic domains; the bulk of the molecule is apparently buried in the membrane. The protein lacks a cleavable leader sequence and does not acquire its characteristic O-linked oligosaccharides in rough microsomes. It may enter the membrane at any stage during synthesis of the first 150 amino acid residues. These unusual features of the protein might help to explain why it is not transported to the cell surface in vivo but remains in intracellular membranes, causing the virus to bud there.

Animals↗
PubMed1984

The biological potency of covalent insulin-receptor complexes. Dependence on site of cross-linkage.

A radioactive photosensitive insulin analogue, 125I-N epsilon B29-(4-azido-2-nitrophenyl-acetyl)insulin, was covalently bound to the receptors of isolated rat adipocytes by irradiation with UV light. This caused a stimulation of lipogenesis. The relative potency of the covalent complexes to that of normal reversible complexes was calculated by comparing the amounts of radioactivity required to be covalently or reversibly bound by adipocytes to cause the same levels of stimulation. For several different occupancies , this relative potency was constant at 50 +/- 3%. Previous studies had shown that the relative potency of covalently bound 125I-N alpha B2-(4-azido-2- nitrophenylacetyl )des- PheB1 -insulin was only 25 +/- 4% under identical conditions. This demonstrates that the sites of crosslinking have a marked effect on the potency of the covalent hormone-receptor complex. It appears that attachment through the C-terminus of the B-chain leads to a better stabilization of the biologically active form than linking through the more flexible N-terminus.

Adipose Tissue↗
PubMed1983

Evidence concerning the mechanism of insulin-receptor interaction and the structure of the insulin receptor from biological properties of covalently linked insulin dimers.

Covalently linked insulin dimers have been prepared by cross-linking two insulin monomers with a flexible suberoyl chain at either the B1 phenylalanine or the B29 lysine residue. Binding potencies of dimers determined by inhibition of binding of 125I-insulin to isolated rat liver plasma membranes or adipocytes were 2.5-7-fold greater than their abilities to stimulate lipogenesis in adipocytes. Rates of liver plasma-membrane-associated degradation of labelled insulin and dimers, measured by gel filtration, were similar at 37 degrees C. Binding and lipogenesis potencies of dimers prepared by substitution of each monomeric half of an asymmetrical dimer with desoctapeptide insulin, an almost inactive derivative, implicated the B1-cross-linked monomeric half as predominantly interacting with the insulin receptor. These results suggest that (1) dimers bind univalently to a bivalent insulin-receptor complex, in which the two individual binding subunits are arranged with anti-parallel symmetry and (2) the mechanism by which insulin binds and initiates its biological responses requires a conformational change within the insulin-receptor complex and/or in the insulin molecule for full biological expression.

Adipose Tissue↗
PubMed1983

Structural differences between insulin receptors in the brain and peripheral target tissues.

Insulin receptors in various brain regions (olfactory tubercle, hippocampus, and hypothalamus) were photoaffinity labeled using the photoreactive analogue of insulin B2(2-nitro,4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). A protein with an apparent Mr of 400,000 was specifically labeled with 125I-NAPA-DP-insulin in all three brain regions. When radiolabeled proteins were reduced with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a protein with an apparent Mr of 115,000 and to a much lesser extent in a protein with an apparent Mr of 83,000. The size of these receptor proteins, based on their electrophoretic mobilities, was consistently smaller than insulin receptor proteins in adipocytes. The covalent labeling of insulin receptors in brain by 125I-NAPA-DP-insulin was not blocked by anti-insulin receptor antiserum. Additionally, in contrast to effects observed in peripheral target tissues, this antisera did not inhibit the binding of 125I-insulin to brain membranes. Neuraminidase treatment resulted in an increase in the electrophoretic mobilities of insulin receptor subunits in adipocytes, but, had no effect on receptor subunits in brain. Solubilized insulin receptors from adipocytes were retained by wheat germ agglutinin columns and specifically eluted with N-acetylglucosamine. In contrast, solubilized insulin receptors from brain did not bind to these columns. The results from this study indicate that structural differences, including molecular weight, antigenicity, and carbohydrate composition exist between insulin receptors in brain and peripheral target tissues.

Adipose Tissue↗
PubMed1983

Insulin receptors in isolated human adipocytes. Characterization by photoaffinity labeling and evidence for internalization and cellular processing.

We photolabeled and characterized insulin receptors in isolated adipocytes from normal human subjects and then studied the cellular fate of the labeled insulin-receptor complexes at physiologic temperatures. The biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)des-PheB1-insulin (NAPA-DP-insulin) was used to photoaffinity label the insulin receptors, and the specifically labeled cellular proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. At saturating concentrations, the binding of 125I-NAPA-DP-insulin to the isolated adipocytes at 16 degrees C was rapid (half-maximal in approximately 1 min and maximal in approximately 10 min) and approximately 25% of the specifically bound ligand was covalently linked to the cells by a 3-min exposure to long-wave (366 nm) ultraviolet light. Analysis of the photolabeled cellular proteins by PAGE in the absence of disulfide reductants revealed the specific labeling of a major protein band of Mr 330,000 and two less intense bands of Mr 295,000 and 260,000. Upon reduction of disulfide bonds with dithiothreitol, all three unreduced forms of the insulin receptor were converted into a major labeled Mr-125,000 band and a less intensely labeled Mr-90,000 band. The labeling of the Mr-125,000 receptor subunit was saturable and native porcine insulin effectively inhibited (half-maximal inhibition at 12 ng/ml) the photolabeling of this binding subunit by NAPA-DP insulin. When intact adipocytes photolabeled at 16 degrees C (a temperature that inhibits endocytosis) were immediately trypsinized, all of the labeled receptor bands were converted into small molecular weight tryptic fragments, indicating that at 16 degrees C all of the labeled insulin-receptor complexes remained on the cell surface. However, when the photolabeled cells were further incubated at 37 degrees C and then trypsinized, a proportion of the labeled receptors became trypsin insensitive, indicating that this fraction has been translocated to the cell interior and thus was inaccessible to the trypsin in the incubation medium. The intracellular translocation of the labeled receptors was observed within 2 min, became half-maximal by 10 min, and maximal by approximately 30 min of incubation at 37 degrees C. Cellular processing of the internalized insulin-receptor complexes also occurred, since incubation at 37 degrees C (but not 16 degrees C) resulted in the generation of a Mr-115,000 component from the labeled receptors. Inclusion of chloroquine, a drug with lysosomotropic properties, in the incubation media caused a time-dependent increase (maximal increase of 50% above control by 2 h at 37 degrees C) in the intracellular pool of labeled receptors. In contrast to these findings in human adipocytes, no appreciable internalization of insulin-receptor complexes and no chloroquine effect was observed in cultures human IM-9 lymphocytes during a 1-h incubation at 37 degrees C. We concluded that in isolated human adipocytes: (a) the subunit structure of insulin receptors is the same as that reported for several other tissues, (b) insulin-receptor complexes are rapidly internalized and processed at physiologic temperatures, and (c) the cellular processing of insulin-receptor complexes occurs at one or more chloroquine-sensitive intracellular site(s).

Adipose Tissue↗
PubMed1983

Degradation of insulin receptors in rat adipocytes.

Insulin receptors on viable rat adipocytes were affinity-labeled using a biologically active and photosensitive analogue of insulin, 125I-B2(2-nitro, 4 azidophenylacetyl)-des-PheB1-insulin (125I-NAPA-DP-insulin). The radiolabeled proteins were identified by SDS polyacrylamide gel electrophoresis and autoradiography. Binding of 125I-NAPA-DP-insulin (40 ng/ml) to rat adipocytes at 16 degrees C, followed by photolysis, resulted in the specific labeling of essentially one protein with an apparent molecular weight of 430-450,000 daltons. When this radiolabeled protein was treated with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a 125,000-dalton protein and to a lesser extent in a 90,000-dalton protein. In addition, there was a minimal amount of specific labeling of a 115,000-dalton protein. Under certain experimental conditions, the nonreduced form of the photoaffinity-labeled receptor appeared as a heterogeneous population of proteins having apparent molecular weights of 430,000, 350,000, and 270,000 daltons. Subsequent to photoaffinity labeling of insulin receptors at 16 degrees C, adipocytes were incubated at 37 degrees C for various periods of time to allow for internalization. This resulted in an initial rapid loss of radioactivity in the 430,000- and 125,000-dalton bands. At 60 min the amount of radioactivity in each of these bands was approximately 50% of that present before incubation at 37 degrees C and stayed constant for 120 min. A first-order plot of the decline in receptor-associated radioactivity was biphasic with the initial phase having a half-life of 1.4 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1982

Photoaffinity labelling of the insulin receptor in intact rat hepatocytes, mouse soleus muscle, and cultured human lymphocytes.

Using the photoreactive, biologically active insulin analogue, B2-(2 nitro, 4-azidophenylacetyl)des-PheB1 insulin, which can be covalently bound to receptor molecules upon photolysis, the insulin receptor has been studied in three different types of cells or tissues: isolated rat hepatocytes, intact murine soleus muscle and cultured human lymphocytes. When compared with native insulin, this analogue displayed a slightly reduced binding affinity. Accordingly, the biological potency of the photoreactive analogue was decreased by approximately 30% compared with native insulin when tested for its ability to stimulate amino acid transport in hepatocytes, and deoxyglucose uptake in soleus muscles. It was as effective as insulin, however, at maximally stimulating concentrations and therefore is a full insulin agonist. This photoprobe was used to specifically label the insulin receptor in the three tissues: after ultra-violet irradiation, sodium dodecyl sulphate-polyacrylamide gel analysis of extracts under reducing conditions revealed that most of the radioactivity was associated with a 130,000 dalton band. In isolated hepatocytes, two bands at 125,000 and 23,000 daltons were also specifically labelled. In three different cell types from three different animal species, the 130,000 dalton band appeared to be the major subunit of the insulin receptor.

Affinity Labels↗
PubMed1982

Xid-defective male (CBA/N X C57BL/6)F1 accessory cells present bovine insulin to long-term cultured F1-restricted T-cells.

The reactivity of H-2b-restricted murine T cells towards bovine insulin was reported to depend on the expression of Ia. W39, a private specificity of I-Ab, on antigen-presenting cells. Cells of male (CBA/N X B6)F1 mice carrying the mutation xid on the X chromosome lack IA. W39 on the cell surface. These cells are unable to present bovine insulin to primed T cells derived from female (CBA/N X B6)F1 mice. We show here that spleen cells of male (CBA/N X B6)F1 hybrids served perfectly as accessory cells for the insulin-dependent induction of a proliferative response of long-term cultured T cells with (B10 X B10.BR)F1 genotype, restricted to recognizing insulin in the context of F1-unique I-A determinants.l The epitope on the insulin molecule essential for stimulation was determined to depend on the glutamic acid residue in position 4 of the A chain of insulin. This contracts with the H-2b-restricted response of B6 mice to bovine insulin, which appears to be directed at the A chain loop determinant (amino acids A8 and A10). These data suggest that distinct I-Ab-encoded structures, the expression of which is regulated independently, may serve as components of restriction elements for H-2b and (H-2b X H-2k)F1 restricted T cells, which are specific for different epitopes of bovine insulin.

Animals↗
PubMed1982

Internalization and molecular processing of insulin receptors in isolated rat adipocytes.

The cellular fate of insulin receptors in isolated rat adipocytes was studied by using a biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin), to photoaffinity label the insulin receptors. Insulin receptors specifically labeled with 125I-labeled NAPA-DP-insulin were identified by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Under nonreducing conditions, specific bands of Mr 330,000, 295,000, and 260,000 were identified; under disulfide reducing conditions, these were converted into Mr 125,000 and 90,000 subunits. When cells labeled at 16 degrees C were immediately trypsinized, all of the receptor bands were degraded into lower molecular weight fragments, indicating that the labeled receptors were all on the cell surface. However, when the labeled cells were incubated at 37 degrees C for 1 hr prior to trypsin exposure, approximately equal to 30% of the receptors were found to be trypsin insensitive, indicating that this fraction was translocated intracellularly. Processing of the insulin receptors appeared to occur; incubation at 37 degrees C (but not at 16 degrees C) resulted in generation of a Mr 115,000 component from the Mr 125,000 subunit as well as in the disappearance of the Mr 330,000 and 295,000 species. Inclusion of chloroquine during photoaffinity labeling at 16 degrees C and during the subsequent incubation at 37 degrees C showed that this agent (i) increased the trypsin-insensitive (intracellular) receptor pool, (ii) blocked conversion of the Mr 125,000 subunit into the Mr 115,000 component, and (iii) prevented the disappearance of the Mr 330,000 and 295,000 species. These studies show that insulin-receptor complexes are internalized and processed intracellularly at a chloroquine-sensitive site(s).

Adipose Tissue↗
PubMed1982

Internalized insulin receptors are recycled to the cell surface in rat hepatocytes.

We have followed the fate of cell surface insulin receptors in isolated rat hepatocytes by both a biochemical and a morphological approach. Hepatocytes were labeled with the photoreactive and biologically active 125I-labeled insulin analogue, [2-nitro-4-azidophenylacetylB2]des-PheB1-insulin, under conditions that allow for minimal internalization (2 hr at 15 degrees C). Analysis of the cell-associated radioactivity by NaDodSO4/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography revealed the specific labeling of a major insulin receptor subunit with Mr 130,000 and a minor degradation product with Mr 125,000. When the cells were exposed at 15 degrees C to trypsin at the end of the association period, these two bands were no longer observed, indicating that the labeled receptors were at the cell surface. This trypsin sensitivity of the receptor disappeared within 30-60 min of incubation of the cells at 37 degrees C, reflecting the internalization of the hormone-receptor complexes. Over the subsequent 4 hr of incubation, this was followed by a progressive reappearance of the receptor complexes at the cell surface, as indicated by the recovery of trypsin sensitivity of the labeled insulin receptors. An identical (both chronologically and quantitatively) journey of the insulin receptors was observed when the labeled material was studied by quantitative electron microscopic autoradiography. Thus, when the cells were incubated at 37 degrees C there was a rapid decrease (30-60 min) in the percentage of autoradiographic grains associated with the plasma membrane, followed by a progressive increase in this percentage over the subsequent 4 hr of incubation. In conclusion, using a biochemical and morphological approach to trace the photoaffinity-labeled insulin receptor, we have shown that the internalized hormone-receptor complex is recycled back to the cell surface.

Affinity Labels↗
PubMed1982

Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes.

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.

Animals↗