Search PubMed⌕ Search

Biomedical subjects

T Kolter

Publications and source records attributed to T Kolter.

29 records · Page 2Linked to original sources
PubMed1997

Growth promoting and metabolic activity of the human insulin analogue [GlyA21,ArgB31,ArgB32]insulin (HOE 901) in muscle cells.

[GlyA21,ArgB31,ArgB32]insulin (HOE 901) represents a biosynthetic human insulin analogue that, due to its isoelectric point, precipitates at neutral tissue pH leading to a retarded absorption rate and a corresponding longer duration of action. In the present investigation we have evaluated the growth promoting and metabolic activity of this analogue in muscle tissue using exponentially growing H9c2 cardiac myoblasts and adult rat ventricular cardiomyocytes. Equilibrium binding studies of 125I-labelled IGF-I (insulin-like growth factor I) to differentiating myoblasts revealed the presence of 7 x 10(3) IGF-I receptors per cell. In contrast, no specific binding of insulin could be detected. Competition binding experiments showed a slightly higher affinity of HOE 901 for the IGF-I receptor when compared to regular human insulin with IC50 (half-inhibitory concentration) values of 70 and 101 nM, respectively. However, the supermitogenic insulin analogue [AspB10]insulin competed significantly more efficiently for IGF-I binding (IC50: 44 nM). Maximum growth promoting activity of the peptides was then determined in serum-starved myoblasts by an incubation with the peptides (5 x 10(-7) M) for 16 h in the presence of [3H]thymidine. [Asp(B10)]Insulin produced a stimulation of DNA synthesis (about 3-fold) which was comparable to the effect of IGF-I and significantly (P < 0.005) higher than the effect of HOE 901 with the latter being essentially equipotent to native insulin. Comparable results were obtained at lower concentrations of the peptides (10(-9) to 10(-8) M). Metabolic activity of HOE 901 was determined by measuring the dose-dependent stimulation of 3-O-methylglucose transport in adult cardiomyocytes. Maximum transport stimulation was identical for insulin and HOE 901 with EC50 (half-effective concentration) values of 0.7 x 10(-10) and 1.9 x 10(-9) M, respectively. We concluded that the IGF-I receptor-mediated growth promoting activity of HOE 901 in muscle cells and the maximal metabolic activity of this analogue are not different from those of native human insulin. It is suggested that differential interaction with IGF-I receptors significantly contributes to the action profile of insulin analogues.

Animals↗
PubMed1997

Molecular analysis of insulin resistance in isolated ventricular cardiomyocytes of obese Zucker rats.

Isolated ventricular cardiomyocytes obtained from lean and genetically (fa/fa) obese Zucker rats were used to correlate alterations of insulin-induced glucose transport activation and GLUT-4 translocation to possible defects of the insulin signaling cascade. Maximal stimulation with insulin was found to produce an unaltered translocation of GLUT-4 to the plasma membrane (4.2- and 3.7-fold increase for lean and obese rats, respectively). However, a largely reduced sensitivity of 3-O-methylglucose transport could be detected in obese rats at physiological doses of insulin (completely unresponsive at 8 x 10(-11) M compared with 3-fold stimulation of glucose transport in lean controls). Tyrosine phosphorylation of the insulin receptor beta-subunit and the insulin receptor substrate 1 (IRS-1) was stimulated identically in cardiomyocytes from both lean and obese rats. Labeling of cells with [33P]orthophosphate revealed a marked increase in the serine and/or threonine phosphorylation of IRS-1 in the obese group (370% of lean controls), with a concomitant reduction in IRS-1 abundance (30-40%). The reduced sensitivity of glucose transport at 8 x 10(-11) M insulin was then found to correlate to a completely blunted response of IRS-1-associated phosphatidylinositol 3-kinase activity in cardiomyocytes from obese rats. Those data show that cardiac insulin resistance of obesity involves defective insulin signaling at low concentrations of the hormone, whereas GLUT-4 translocation is fully operative in the isolated cell. It is suggested that hyperphosphorylation of IRS-1 may significantly contribute to the pathogenesis of insulin resistance in the heart.

3-O-Methylglucose↗
PubMed1996

Only sphingolipid activator protein B (SAP-B or saposin B) stimulates the degradation of globotriaosylceramide by recombinant human lysosomal alpha-galactosidase in a detergent-free liposomal system.

The degradation of globotriaosylceramide (GbO-se3Cer) by insect-cell derived recombinant human alpha-galactosidase (EC 3.2.1.22) was carried out in a detergent-free liposomal system in order to mimic intralysosomal conditions. GbOse3Cer incorporated into unilamellar liposomes was used as the substrate, and naturally occurring sphingolipid activator proteins, rather than detergents, were used to stimulate the enzyme reaction. The degradation of GbOse3Cer was dependent on the presence of both alpha-galactosidase and sphingolipid activator protein B (SAP-B or saposin B). It proceeded optimally at pH 4.6, and was enhanced by increasing amounts of both alpha-galactosidase (0.24-24 mU/50 microliters assay) and SAP-B (0-5 micrograms/50 microliters assay). The enzyme reaction was not affected by SAP-A, SAP-C, or SAP-D. Therefore, our results indicate that only SAP-B is essential for the degradation of GbOse3Cer by alpha-galactosidase.

Animals↗
PubMed1996

Topology of glycosphingolipid degradation.

Glycosphingolipids (GSLs) form cell-type-specific patterns on the surface of eukaryotic cells. Degradation of plasma-membrane-derived GSLs in the lysosomes after internalization through the endocytic pathway is achieved through the concerted actions of hydrolysing enzymes and sphingolipid activator proteins. The latter are proteins necessary for the degradation of GSLs possessing short oligosaccharide chains. Some activator proteins bind to GSLs and form water-soluble complexes, which lift out of the membrane and give the water-soluble hydrolysing enzymes access to the regions of the GSL that would otherwise be obscured by the membrane. The inherited deficiency of both lysosomal hydrolases and sphingolipid activator proteins gives rise to sphingolipid storage diseases. An analysis of these diseases suggests a new model for the topology of endocytosis and lysosomal digestion, which is discussed in this article.

Journal Article↗
PubMed1995

Failure of insulin-regulated recruitment of the glucose transporter GLUT4 in cardiac muscle of obese Zucker rats is associated with alterations of small-molecular-mass GTP-binding proteins.

Cardiac ventricular tissue of lean and genetically obese (fa/fa) Zucker rats was used to study the expression, subcellular distribution and insulin-induced recruitment of the glucose transporter GLUT4 and to elucidate possible molecular alterations of the translocation process. Hearts were removed from basal and insulin-treated (20 min) lean and obese Zucker rats, and processed for subcellular fractionation and Western blotting of proteins. In obese rats, the total GLUT4 content in a crude membrane fraction was reduced to 75 +/- 8% (P = 0.019) of lean controls. In contrast, GLUT4 abundance in plasma membranes was not significantly different between lean and obese rats with a concomitant decrease (47 +/- 3%) in the microsomal fraction of obese animals. In plasma membranes of lean animals insulin was found to increase the GLUT4 abundance to 294 +/- 43% of control with a significantly (P = 0.009) reduced effect in the obese group (139 +/- 10% of control). In these animals insulin failed to recruit GLUT4 from the microsomal fraction, whereas the hormone induced a significant decrease (41 +/- 4%) of microsomal GLUT4 in lean controls. In GLUT4-enriched membrane vesicles, obtained from cardiac microsomes of lean rats, a 24 kDa GTP-binding protein could be detected, whereas no significant labelling of this species was observed in GLUT4 vesicles prepared from obese animals. In addition to the translocation of GLUT4, insulin was found to promote the movement of the small GTP-binding protein rab4A from the cytosol (decrease to 61 +/- 13% of control) to the plasma membrane (increase to 177 +/- 19% of control) in lean rats with no effect of the hormone on rab4A redistribution in the obese group. In conclusion, cardiac glucose uptake of insulin-resistant obese Zucker rats is subject to multiple cellular abnormalities involving a reduced expression, altered redistribution and defective recruitment of GLUT4. We show here an association of the latter defect with alterations at the level of small GTP-binding proteins possibly leading to an impaired trafficking of GLUT4 in the insulin-resistant state.

Animals↗
PubMed1995

[Glycolipids of the cell surface--biochemistry of their decomposition].

Glycosphingolipids (GSL) form cell-type-specific patterns on the surface of eukaryotic cells. For the maintenance of these patterns, biosynthesis, intracellular transport, and degradation of GSL are thought to proceed in a coordinated fashion. After transport from the plasma membrane to the lysosomes the degradation of GSL requires protein cofactors to solubilize the membrane-bound substrates of the degradative enzymes. Inborn errors of metabolism leading to defective hydrolases or activator proteins give rise to sphingolipid storage diseases; in some cases the residual enzyme activity can be correlated with the clinical manifestations.

Animals↗
PubMed1994

Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle: studies on the role of small-molecular-mass GTP-binding proteins.

Subcellular fractions obtained from rat cardiac ventricular tissue were used to elucidate a possible functional relationship between small-molecular-mass G-proteins and the insulin-responsive glucose transporter GLUT4. Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Incubation with [alpha-32P]GTP revealed the presence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsomal membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriched membrane vesicles. This material was found to contain a much decreased amount of small G-proteins, with the exclusive presence of the 24 kDa species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decreased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enriched vesicles from insulin-treated animals. Western-blot analysis of microsomal membranes with a panel of antisera against rab GTP-binding proteins indicated the presence of rab4A, with a molecular mass of 24 kDa, whereas rab1A, rab2 and rab6 were not observed. rab4A was barely detectable in GLUT4-enriched vesicles; however, insulin produced an extensive shift of rab4A from the cytosol and the microsomal fraction to the plasma membrane with a parallel increase in GLUT4. These data show that a small GTP-binding protein is co-localized with GLUT4 in an insulin-responsive intracellular compartment, and strongly suggest that this protein is involved in the exocytosis of GLUT4 in cardiac muscle. Furthermore, the observed translocation of rab4A is compatible with insulin-induced endosome recycling processes, possibly including the glucose transporters.

Animals↗
PubMed1994

Photoaffinity labelling of cardiac membrane GTP-binding proteins in response to insulin.

Plasma membranes from rat cardiac ventricular tissue and insulin receptors partially purified by wheat-germ-agglutinin chromatography were subjected to direct photoaffinity labelling with [alpha-32P]GTP in order to elucidate the presence of insulin-receptor-coupled GTP-binding proteins. In plasma membranes three proteins have been identified that exhibit an enhanced photolabelling with the nucleotide in response to insulin. The apparent molecular masses of these proteins were found to be 56, 60 and 74 kDa. Photolabelling of partially purified insulin receptors showed the copurification of the 60-kDa species, whereas the 56-kDa and 74-kDa proteins could not be detected. Furthermore, the 60-kDa G-protein was found to be specifically co-immunoprecipitated with the insulin receptor. Incubation of insulin receptors with insulin increased the labelling of the 60-kDa band to 205 +/- 27% (n = 5) of control. Immuno- and ligand-blotting experiments revealed the additional presence of a 39-kDa G(o)-like protein and two G-proteins with molecular masses of 24 and 26 kDa in the receptor preparation. Under basal conditions the insulin receptor and the 60-kDa G-protein exhibited an apparent inverse distribution between plasma and microsomal membranes with the G-protein being extensively labelled in the microsomal fraction. In conclusion, our data show that, in its native environment, the cardiac insulin receptor couples to at least three GTP-binding proteins. Out of these, a 60-kDa species of microsomal origin, copurifies with the insulin receptor. It is suggested that this G-protein is associated with the insulin receptor and may be involved in insulin receptor signalling in target cells.

Affinity Labels↗
PubMed1992

Contraction-induced translocation of the glucose transporter Glut4 in isolated ventricular cardiomyocytes.

Field stimulation of isolated adult ventricular cardiomyocytes was used to study the effect of contractile activity on 3-O-methylglucose transport and the subcellular distribution of Glut4. Cells contracting at a frequency of 1 Hz for 30 min exhibited unaltered basal and insulin-stimulated rates of glucose transport when compared to resting cells. However, at 5 Hz 3-O-methylglucose transport increased to 224% of control after 5 min. Under these conditions insulin was unable to produce a significant additional stimulation of glucose transport. Immunoblotting with an anti-Glut4 polyclonal antibody showed that both insulin and contraction (5 Hz) increased the amount of Glut4 in a plasma membrane fraction by about 8-fold with a parallel decrease in an intracellular membrane fraction by 60-65%. These data suggest the existence of an identical insulin- and contraction-recruitable Glut4 transporter pool in cardiomyocytes.

3-O-Methylglucose↗
PubMedDate not supplied

The glycosphingolipidoses-from disease to basic principles of metabolism.

The glycosphingolipidoses are a set of diseases that are caused by defects in the lysosomal degradation of glycolipids derived from the plasma membrane. By investigating the molecular bases of the diseases, basic principles of storage disease pathology and of membrane digestion were discovered. The generation of mouse models has facilitated the development of new and promising therapeutic strategies for these diseases, most of which are not treatable at present. Lately, the discovery of the importance of glycosphingolipid metabolism for skin development has opened a new and interesting field.

Animals↗