Biomedical subjects

M Kellerer

Publications and source records attributed to M Kellerer.

49 records · Page 3Linked to original sources

PubMed1993

Inositol phospho-oligosaccharides from rat fibroblasts and adipocytes stimulate 3-O-methylglucose transport.

Inositol phospho-oligosaccharides (IPOs), which are released from liver membranes upon stimulation by insulin, mimic a wide spectrum of insulin effects in different cells, but not the stimulation of glucose transport. We investigated whether other insulin-sensitive tissues release glucose transport-stimulating IPOs and whether this is related to the human insulin receptor isoform-A or -B (HIR-A or HIR-B). Rat1 fibroblasts overexpressing HIR-A or -B (rat1-HIR cells) were labelled with [3H]glucosamine, [3H]mannose or myo-[3H]inositol. IPOs from the cell supernatant were partially purified by an AG1X2 anion-exchange column, and fractions were eluted at different pH values (pH 3, pH 2 and pH 1.3). The label from glucosamine, mannose and myo-inositol appeared predominantly in the pH 2 fraction. The biological activity of the fractions was determined by measuring 3-O-methylglucose transport and lipogenesis in fat cells. Using the pH 2 fraction from the supernatant of rat1-HIR fibroblasts, insulin increased the release of 3-O-methylglucose-transport-stimulating activity (HIR-A: without insulin, 22.4 +/- 5.4%; with insulin 54.0 +/- 8.4%; HIR-B: without insulin 21.6 +/- 7.5%, with insulin, 44.7 +/- 10.6%, given as a percentage of equilibrium glucose transport reached after 4 s) and lipogenesis-stimulating activity (HIR-A: without insulin, 1.24 +/- 0.17; with insulin, 4.69 +/- 0.2; HIR-B: without insulin, 1.34 +/- 0.18; with insulin, 4.98 +/- 0.31, given as nmol of [3H]glucose converted into lipids/min per 10(6) cells). Analogous experiments were performed with isolated rat fat cells expressing the physiological level of insulin receptors. Upon insulin stimulation of fat cells in the presence of 2.5 mM mannose, the release of 3-O-methylglucose-transport-stimulating activity was detected (for purified supernatant of adipocytes without insulin, 6.9 +/- 1.12%; with insulin, 41.0 +/- 3.6%) and lipogenesis-stimulating activity (without insulin, 0.93 +/- 0.17, with insulin 2.96 +/- 0.31 nmol/min per mg). These data suggest (1) that adipocytes and rat1-HIR fibroblasts release IPOs that are able to stimulate glucose transport, (2) that both insulin receptor isoforms (HIR-A and HIR-B) mediate the effect of insulin on IPO release, and (3) that overexpression of insulin receptors increases the basal release of IPOs.

3-O-Methylglucose↗

PubMed1993

Altered pattern of insulin receptor isotypes in skeletal muscle membranes of type 2 (non-insulin-dependent) diabetic subjects.

The human insulin receptor exists in two isoforms (HIR-A alpha-subunit 719 amino acids and HIR-B alpha-subunit 731 amino acids) which are generated by alternative splicing of a small exon and display distinct patterns of tissue-specific expression. Using the polymerase chain reaction we have recently shown that skeletal muscle of non-diabetic individuals contains predominantly mRNA encoding HIR-A while in skeletal muscle derived from subjects with Type 2 (non-insulin-dependent) diabetes mellitus similar amounts of each mRNA are expressed. We used a polyclonal antibody which discriminates between HIR-A and HIR-B to assess the isoform expression at the protein level. The antibody showed clearly distinct displacement of insulin binding in skeletal muscle membranes of non-diabetic subjects compared to Type 2 diabetic subjects (displacement of specific 125I-insulin binding: 13 non-diabetic subjects 70.0% +/- 14.34, 12 Type 2 diabetic subjects 32.6% +/- 17.45). A control antibody which does not discriminate between both isoforms showed similar displacement of 125I-insulin in membranes of non-diabetic and Type 2 diabetic subjects. These data suggest that the altered expression of receptor isotype mRNA in the skeletal muscle of Type 2 diabetic subjects leads to an altered receptor isoform pattern in the plasma membrane. While skeletal muscle membranes of non-diabetic subjects contain predominantly HIR-A, membranes of Type 2 diabetic subjects show an increased level of HIR-B in addition to HIR-A.

Aged↗

PubMed1993

Tyrosine kinase activity of insulin-like growth factor I and insulin receptors in human endometrium during the menstrual cycle: cyclic variation of insulin receptor expression.

OBJECTIVE: To determine whether expression and binding or signaling characteristics of endometrial insulin-like growth factor I (IGF-I) and insulin receptors are modulated throughout the menstrual cycle. SETTING: Research laboratories of a university hospital and of the Institute for Diabetes Research. DESIGN: In vitro receptor binding and phosphorylation studies of human proliferative and secretory endometrial tissue and of cultured endometrial stromal cells. MAIN OUTCOME MEASURES: Binding and tyrosine kinase activation of IGF-I and insulin were studied in wheat germ agglutinin purified receptor protein. Binding data were analyzed by Scatchard plots. Autophosphorylation was measured by 32P incorporation into the 95 kd receptor beta-subunit; substrate phosphorylation was determined with poly(GluNa 4: Tyr 1). RESULTS: Binding studies revealed no differences of the affinities between cycle phases. Half-maximal displacement of both receptors was approximately 1 nM in both phases. Insulin-like growth factor I receptor number appeared to be unaltered in both phases, whereas insulin receptor numbers and tyrosine kinase activity in the secretory phase were significantly increased. The increase of tyrosine kinase activity was entirely because of the increased receptor number as calculated from the binding data. In cultured endometrial stromal cells the increase of expression of insulin receptors could be induced by sexual steroids. CONCLUSIONS: On the receptor level IGF-I signaling to human endometrium is not modulated during the menstrual cycle, whereas insulin binding and signaling are likely to be enhanced in the luteal phase. The increased insulin receptor level may be required for normal luteal function.

Cell Division↗

PubMed1992

Distinct alpha-subunit structures of human insulin receptor A and B variants determine differences in tyrosine kinase activities.

Human insulin receptor isoforms (HIR-A and -B) differ in their alpha-subunit structures which result from alternatively spliced precursor mRNAs. This structural difference causes distinct binding affinities for insulin. To determine the impact of the structural difference on receptor signaling, we characterized the tyrosine kinase activity of HIR-A and HIR-B in vitro and determined the insulin stimulated beta-subunit phosphorylation and tyrosine kinase activation in the intact cell. When 32P incorporation in beta-subunits of equal amounts of isolated HIR-A and HIR-B was measured, an increased 32P incorporation in tyrosine residues of the beta-subunit of HIR-B (2.5-fold) compared to that of HIR-A was found after in vitro insulin stimulation. This was paralleled by an increased rate of phosphorylation (2.0-fold) or poly(GluNa,Tyr 4:1). In vitro analysis of Km values for ATP were similar for HIR-A (Km = 14.3 microM +/- 3.8) and HIR-B (Km = 20.2 microM +/- 8.6), whereas the Vmax of HIR-B was significantly increased (HIR-A Vmax = 5.5 mumol/60 min micrograms-1 +/- 1.4, HIR-B Vmax = 42.5 mumol/60 min micrograms-1 +/- 19.2). HPLC analysis of tryptic beta-subunit phosphopeptides revealed identical patterns, suggesting that the difference in kinase activities is not due to an alteration of the phosphorylation-activation cascade within the beta-subunit. However, when cleavage of the alpha-subunit by short-time trypsinization was used to activate the tyrosine kinase, the differences in 32P incorporation between HIR-A and HIR-B were abolished.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

The nucleotide and partial amino acid sequences of rat fetuin. Identity with the natural tyrosine kinase inhibitor of the rat insulin receptor.

Fetuins are among the major plasma proteins, yet their biological role has remained elusive. Here we report the molecular cloning of rat fetuin and the sequence analysis of a full-length clone, RF619 of 1456 bp with an open reading frame of 1056 bp encoding 352 amino acid residues. The coding part of RF619 was identical with the cDNA sequence of the natural inhibitor of the insulin receptor tyrosine kinase from rat (pp63) except for four substitutions and a single base insertion causing divergence of the predicted protein sequences. Partial amino acid sequences of rat plasma fetuin were in agreement with the predictions based on the RF619 cDNA. Purified rat fetuin inhibited the insulin receptor tyrosine kinase in vitro. Therefore, we conclude that RF619 and pp63 cDNA encode the same protein, i.e. authentic rat fetuin which is a functional tyrosine kinase inhibitor.

Amino Acid Sequence↗

PubMed1992

Normal insulin receptor tyrosine kinase activity and glucose transporter (GLUT 4) levels in the skeletal muscle of hyperinsulinaemic hypertensive rats.

The spontaneous hypertensive rat is an animal model characterized by a syndrome of hypertension, insulin resistance and hyperinsulinaemia. To elucidate whether in analogy to other insulin resistant animal models an inactivity of the insulin receptor kinase or an alteration of the glucose transporter (GLUT 4) level in the skeletal muscle might contribute to the pathogenesis of insulin resistance we determined insulin receptor kinase activity and GLUT 4 level in the hindlimbs of spontaneous hypertensive rats and normotensive control rats. Normotensive normoinsulinaemic Lewis and Wistar rats were used as insulin sensitive controls, obese Zucker rats were used as an insulin resistant control with known reduced skeletal muscle insulin receptor kinase activity. Binding of 125I-insulin, crosslinking of 125I-B26-insulin, autophosphorylation in vitro with 32P-ATP and phosphorylation of the synthetic substrate Poly (Glu 4: Tyr 1) were performed after partial purification of solubilized receptors on wheat germ agglutinin columns. GLUT 4 levels were determined by Western blotting of subcellular muscle membranes. Insulin receptors from spontaneous hypertensive rats compared to those from Lewis and Wistar rats showed no difference of the binding characteristics or the in vitro auto- and substrate phosphorylation activity of the receptor, while in the Zucker rats the earlier described insulin receptor kinase defect was clearly evident. Western blots of subcellular muscle membrane fractions with antibodies against GLUT 4 revealed no difference in transporter levels. These data suggest that insulin resistance in spontaneous hypertensive rats is caused neither by an insulin receptor inactivity nor by a decreased number of glucose transporters in the skeletal muscle.

Adenosine Triphosphate↗

PubMed1992

Insulin-like growth factor I-dependent tyrosine kinase activity in stromal cells of human endometrium in vitro.

The study was undertaken to identify and characterize insulin-like growth factor I (IGF-I) receptors in human endometrial stromal cells in culture and to examine whether these receptors are modulated by estradiol (E2) and/or progesterone (P). We found that partially purified plasma membrane proteins from these cells contain specific high-affinity binding sites for IGF-I (10 fmol/micrograms protein). Chemical cross-linking with 125I-labeled IGF-I and autophosphorylation with [32P]ATP-labeled proteins of relative molecular weight 135,000 and 95,000 correspond to the known Mr values of the alpha- and the beta-subunits of IGF-I receptors. Receptor autophosphorylation and phosphorylation of the substrate poly(Glu,Na4Tyr1) was stimulated in vitro by IGF-I (half-maximally at 1 nM, maximally at 100 nM). After stimulation of intact cells with IGF-I (5 nM) and subsequent partial purification of receptors in the presence of phosphatase inhibitors, a 2.5- to 3.6-fold stimulation of the kinase activity toward poly(Glu,Na4Tyr1) was found. Preincubation of the cells for 16 h with E2, P, and E2 + P did not modify the IGF-I binding characteristics nor the effect of IGF-I (5 nM) on tyrosine kinase stimulation in intact cells. This suggests that, in isolated humans, endometrial cell modulation of IGF-I receptor function by estrogen and P does not occur.

Adult↗

PubMed1992

The influence of pulmonary artery banding on outcome after the Fontan operation.

Thirty-eight patients were selected from a total of 120 patients who underwent the Fontan operation between 1974 and 1988. They were classified into two groups. Group 1 consisted of 18 patients with previous pulmonary artery banding at a mean age of 7 months (2 days to 59 months), and group 2 comprised 20 patients with native pulmonary stenosis. In group 1, 10 children had tricuspid atresia (seven with normally connected and three with transposed great arteries), six had double-inlet ventricle, and two had complex heart malformations. Group 2 consisted of 12 patients with tricuspid atresia and normally connected great arteries, six with double-inlet ventricle, and two with complex malformations. The following clinical and hemodynamic parameters at cardiac catheterization and cineangiocardiography were determined in both groups before the Fontan operation: age and body surface area, hemoglobin concentration and hematocrit value, atrial and pulmonary artery pressures, end-diastolic pressure of the systemic ventricle, arterial oxygen saturation, pulmonary/systemic flow ratio, end-diastolic volume, ejection fraction and mass of the systemic ventricle, cardiac index, and Nakata index. After the Fontan operation in all patients, the presence or absence of pericardial and pleural effusions, ascites, protein-losing enteropathy, and liver and kidney dysfunction was assessed and the clinical status was classified according to New York Heart Association criteria. All preoperative and postoperative parameters were tested for differences between the two groups, and they were compared with normal values. Hematocrit value was higher in group 2 than in group 1 (57.8% versus 53.1%; p less than 0.05). Ventricular mass index was increased in group 1 when compared with group 2 (125.8 gm/m2 versus 87 gm/m2; p less than 0.05). Severe pericardial effusions in the early postoperative period were significantly more frequent in group 1 and were particularly prevalent in the subgroup with long-standing pulmonary artery banding (p less than 0.01). Subaortic stenosis was observed more frequently in group 1. The remaining parameters were not statistically different between the two groups. We conclude that the significant increment in ventricular mass after pulmonary artery banding may represent a risk for unfavorable outcome after the Fontan operation, which increases with time. Therefore, long-standing pulmonary artery banding as a palliative procedure for candidates for the Fontan operation should be avoided.

Adolescent↗

PubMed1991

Stimulation of phospholipase C activity by insulin is mediated by both isotypes of the human insulin receptor.

The human insulin receptor exists in two isoforms, HIR-A and HIR-B. We studied whether both insulin receptor isotypes are able to mediate an insulin signal to phospholipase C. Plasma membranes were prepared from rat-1 fibroblasts transfected either with HIR-A or HIR-B and insulin stimulated PIP-hydrolysis was determined. We found that insulin stimulates PIP-hydrolysis in a similar dose dependent manner and to a similar extent in plasma membranes expressing HIR-A and HIR-B. These data suggest that both receptor isoforms are equally able to activate phospholipase-C.

Adenosine Triphosphate↗

PubMed1991

Insulin activates GTP binding to a 40 kDa protein in fat cells.

The first steps in insulin action are binding of insulin to its receptor and activation of the insulin receptor kinase. As there is indirect evidence that further signal transduction might involve a guanine-nucleotide-binding protein (G-protein), we studied whether insulin modulates GTP binding to plasma membrane proteins of fat cells and skeletal muscle. We found that insulin rapidly increased (30 s) binding of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in a dose dependent manner (0.03-2.0 nM). This effect was not altered by pertussis toxin, but it was abolished by cholera toxin treatment of fat cells. Scatchard analysis of the binding data showed that the increased GTP[S] binding is due to a decrease in the Kd for GTP from 100 nM to 50 nM. Furthermore, binding of GTP to these plasma membranes inhibited both the binding of 125I-insulin to the insulin receptor and the stimulation of the insulin receptor kinase, suggesting a feedback interaction between the insulin-stimulated GTP-binding site and the insulin receptor. In order to identify this insulin-stimulated GTP-binding site, plasma membranes were labelled with the photoreactive GTP analogue [alpha-32P]GTP gamma-azidoanilide. We found that insulin selectively stimulated GTP binding to a 40 kDa protein. In conclusion, in plasma membranes of fat cells and skeletal muscle, the insulin receptor interacts with a 40 kDa GTP-binding site. We speculate that this 40 kDa GTP-binding site might be a G-protein which is involved in insulin signal transmission.

Adenosine Diphosphate Ribose↗

PubMed1991

Prevention by protein kinase C inhibitors of glucose-induced insulin-receptor tyrosine kinase resistance in rat fat cells.

Hyperglycemia causes insulin-receptor kinase (IRK) resistance in fat cells. We characterized the mechanism of IRK inhibition and studied whether it is the consequence of a glucose-induced stimulation of protein kinase C (PKC). Fat cells were incubated for 1 or 12 h in culture medium containing either a low-(5-mM) or high- (25-mM) glucose concentration. IRK was isolated, insulin binding was determined, and autophosphorylation was studied in vitro with [gamma-32P]ATP or was determined by Western blotting with anti-phosphotyrosine antibodies. Substrate phosphorylation was investigated with the artificial substrate poly(Glu80-Tyr20). Partially purified insulin receptor from rat fat cells, which were cultured under high-glucose conditions for 1 or 12 h, showed no alteration of insulin binding but a reduced insulin effect on autophosphorylation (30 +/- 7% of control) and poly(Glu80-Tyr20) phosphorylation (55.5 +/- 9% of control). Lineweaver-Burk plots of the enzyme kinetics revealed, beside a reduced Vmax, and increased KM (from 30 microM to 80 microM) for ATP of IRK from high-glucose-treated cells. Because a similar inhibition pattern was earlier found for IRK from fat cells after acute phorbol ester stimulation, we investigated whether activation of PKC might be the cause of the reduced IRK activity. We isolated PKC from the cytosol and the membrane fraction of high- and low-glucose fat cells and determined the diacylglycerol- and phospholipid-stimulated PKC activity toward the substrate histone. There was no significant change of cytosolic PKC; however, membrane-associated PKC activity was increased in high-glucose-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

TPA inhibits insulin stimulated PIP hydrolysis in fat cell membranes: evidence for modulation of insulin dependent phospholipase C by proteinkinase C.

Proteinkinase-C (PKC) stimulating phorbolesters induce in vitro insulin resistance of isolated adipocytes. This effect might be explained by an inhibition of insulin signal transduction at the level of the insulin receptor kinase. There is now some evidence that a phospholipase C is a potential candidate as a signal transducer at the postreceptor level. In order to determine whether phorbol esters might inhibit insulin signalling also at the level of a phospholipase C, we studied the insulin dependent [3H] phosphatidylinositol 4-monophosphate (PIP) hydrolysis of fat cell membranes. PIP hydrolysis was measured after in vitro stimulation with and without insulin. Insulin stimulated PIP hydrolysis in a dose dependent way. When plasma membranes from phorbolester (TPA) treated fat cells were used, this insulin stimulated phospholipase C activity was suppressed, provided, membranes have been prepared in a buffer containing serine phosphatase inhibitors. These data suggest that fat cell membranes contain an insulin dependent phospholipase C which is inhibited by TPA most likely via serine phosphorylation through proteinkinase C.

Adipose Tissue↗

PubMed1990

An altered IGF-I receptor is present in human leukemic cells.

We have characterized and analyzed IGF-I- and insulin-stimulated cell growth, receptor binding, and autophosphorylation in the human leukemic cell line HL-60. IGF-I-stimulated cell growth occurred at low (5 ng/ml) and insulin stimulated only at high (500 ng/ml) concentrations. Binding of 125I-IGF-I to partially purified plasma membrane proteins followed the characteristics of IGF-I receptor binding. 125I-IGF-I binding, as determined by chemical cross-linking, occurred to a 145-kDa protein. IGF-I, as well as insulin, stimulated the autophosphorylation of a 105-kDa band (pp105), but we could not detect a 95-kDa band corresponding to the known molecular mass of the IGF-I and insulin receptor beta-subunits. Phosphorylation of pp105 followed the dose-response characteristics of the IGF-I receptor. The phosphorylation of pp105 occurred at tyrosine and threonine, and the pattern of HPLC tryptic peptide maps showed marked differences when compared with that of a phosphorylated insulin receptor beta-subunit. Enzymatic deglycosylation of pp105 resulted only in a slight reduction of the molecular weight. These data suggest that pp105 is the beta-subunit of an IGF-I receptor variant with a higher molecular weight, similar to that found in fetal tissue. The HL-60 cell may acquire, at least in part, malignant growth characteristics through reexpression of the fetal version of the IGF-I receptor.

Cell Division↗