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H H Klein

Publications and source records attributed to H H Klein.

At least 55 records · Page 3Linked to original sources
PubMed1997

Insulin stimulation of human adipocytes activates the kinase of only a fraction of the insulin receptors.

The degree of insulin receptor kinase activation by in situ stimulation was studied in isolated human adipocytes. Although maximal in situ stimulation increased the kinase activity approximately 10-fold, this activity could again be doubled by subsequent activation in a cell-free system. To investigate how in situ stimulation resulted in incomplete activation, receptors binding or not binding to anti-phosphotyrosine antibody (alpha-PY) were studied separately. Even after maximal insulin stimulation of the cells, approximately 50% of the receptors did not bind to alpha-PY and had low kinase activity. In the cell-free system, however, these receptors reached activity levels similar to the other receptors, suggesting that they were intact and that factors in their cellular environment had prevented their activation. The activity of the alpha-PY-binding receptors could only be slightly increased in the cell-free system, suggesting that almost complete activation had been attained in situ. In situ stimulation with increasing insulin concentrations increased the number of activated receptors rather than their individual activity. We conclude that factors in the in situ environment prevent insulin activation of approximately 50% of the insulin receptors in human adipocytes and might therefore be important regulators of insulin signaling.

Adipocytes↗
PubMed1996

Sustained signalling from the insulin receptor after stimulation with insulin analogues exhibiting increased mitogenic potency.

The metabolic and mitogenic potencies of six different insulin analogues were determined by measuring glucose transport in primary adipocytes and DNA synthesis in CHO cells respectively. Three analogues showed a disproportionately high mitogenic potency compared with their metabolic potency, and were up to 7 times more mitogenically than metabolically potent when compared with human insulin. The mitogenic/metabolic potency ratio of the analogues was found to be inversely correlated with the insulin receptor dissociation rate constant (Kd) in an exponential fashion (r=0.99), with a disproportionately greater increase in mitogenic potential compared with metabolic potential for analogues with Kd values of less than 40% of that of human insulin. To investigate the molecular mechanisms behind the correlation between the increased half-life of the receptor-ligand complex (low Kd) and mitogenicity, 3 h time-course experiments were performed. Slow ligand dissociation from the insulin receptor induced a parallel sustained activation of the insulin receptor tyrosine kinase. A similar pattern was observed for insulin receptor autophosphorylation and Shc phosphorylation, whereas the duration of insulin receptor substrate-1 phosphorylation with low-Kd analogues and with insulin was similar Thus the increased half-life of the ligand-receptor complex induces sustained activation of the insulin receptor tyrosine kinase and sustained phosphorylation of Shc, which may be the cause of the disproportionately high mitogenic potency seen for some insulin analogues.

Animals↗
PubMed1995

Myocardial protection by Na(+)-H+ exchange inhibition in ischemic, reperfused porcine hearts.

BACKGROUND: Studies in isolated myocytes and isolated heart preparations have suggested that Na(+)-H+ exchange is an important mechanism for myocardial ischemia-reperfusion injury. This study was undertaken to determine whether inhibition of Na(+)-H+ exchange limits infarct size and improves regional systolic shortening in regional ischemia and reperfusion in intact pigs. METHODS AND RESULTS: The left anterior descending coronary artery was occluded in 18 anesthetized and thoracotomized pigs for 45 minutes and then reperfused for 24 hours. As main end points of this study, regional systolic shortening (sonomicrometry) and infarct size (percentage of infarcted to ischemic myocardium) were determined at the end of the experiments. Infarcted myocardium was assessed by histochemistry (tetrazolium stain) and by quantitative histology of one heart slice. The Na(+)-H+ exchange inhibitor Hoe 694 was administered intravenously at a dose of 3 mg/kg in 6 pigs each either 10 minutes before ischemia (group A) or 10 minutes before the onset of reperfusion (group B). Six pigs served as controls (group C). Treatment with Hoe 694 before ischemia decreased histochemical infarct size from 65 +/- 18% (control group) to 13 +/- 8% (P < .01) and histological infarct size from 49 +/- 20% (control group) to 14 +/- 4% (P < .01). Histochemical (55 +/- 19%) and histological (42 +/- 15%) infarct sizes of group B were insignificantly reduced by 15%. Myocardial protection in group A was associated with an attenuated contracture after 10 minutes of reperfusion and an improved regional systolic shortening after 24 hours of reperfusion (group A, 25 +/- 12%; control group, 6 +/- 5%; P = .01). These parameters remained unaffected in group B. CONCLUSIONS: This study clearly demonstrates that Na(+)-H+ exchange is an important mechanism for cell death in myocardial ischemia and reperfusion in intact pigs; thus, inhibition of this exchange system may prove a promising new strategy in the clinical treatment of myocardial ischemia and reperfusion.

Animals↗
PubMed1995

Binding of human, porcine and bovine insulin to insulin receptors from human brain, muscle and adipocytes and to expressed recombinant alternatively spliced insulin receptor isoforms.

Previous studies have suggested that human and porcine insulin exert identical effects on blood glucose and counter-regulatory hormones but elicit different neurophysiological reactions. A major goal of the present study was to investigate whether this could be caused by different relative affinities of the insulins from different species to insulin receptors from the brain compared to other tissues. Insulin receptors isolated from human brain, muscle or adipocytes as well as from cultured cells over-expressing either of the human insulin receptor isoforms (exon 11- or exon 11 +) were immobilized to microwells coated with monoclonal anti-insulin receptor antibody. Subsequently the binding of human, porcine and bovine insulin was measured. While the receptors derived from the different tissues had different affinities for insulin, there were no tissue-specific differences in the relative binding of the insulins of the three species. The insulins of the three species were also not different with regard to their binding to the receptor isoforms. Finally, in human brain homogenates no differences in the degradation rates for human, porcine and bovine insulin were detected. Thus, our data do not support the hypothesis that different neurophysiological reactions during hypoglycaemia due to human or porcine insulin are caused by differences of the binding of the insulins to human brain insulin receptors or their degradation in the human brain.

Adipocytes↗
PubMed1995

Comparative study on the effects of intracoronary nicorandil and nitroglycerin in ischaemic, reperfused porcine hearts.

The direct cardioprotective properties of nitroglycerin and nicorandil were compared in regionally ischaemic (45 min), reperfused (24 h) porcine hearts. Intracoronary treatments, which were started 15 min prior to occlusion of the distal left anterior descending coronary artery (LAD), were continuously administered for 105 min. The following equi-hypotensive drug dosages were used in nine pigs each; nitroglycerin 6 micrograms.kg-1 x min before ischaemia and during 45 min of reperfusion, 0.6 microgram.kg-1 x min during ischaemia; nicorandil 5 micrograms.kg-1 x min before ischaemia and during 45 min of reperfusion, and 0.5 microgram.kg-1 x min during ischaemia. Nine control animals were treated with isotonic sodium hydrochloride solution (1 ml.min-1). Despite comparable effects on blood pressure, intracoronary nicorandil, in contrast to intracoronary nitroglycerin, did not increase heart rate. Although neither drug affected coronary blood flow significantly, nicorandil substantially reduced regional myocardial oxygen consumption before coronary artery occlusion (-37 +/- 22%, P = 0.003 vs control group, P = 0.01 vs nitroglycerin treatment). Infarct sizes (tetrazolium method) after 45 min of ischaemia and 24 h of reperfusion were significantly decreased by nicorandil (control group 76.9 +/- 19%, nicorandil group 49.3 +/- 24%, P = 0.012) whereas nitroglycerin exhibited a borderline effect (62.5 +/- 15%, P = 0.054). Both treatments resulted in improved regional systolic shortening of the reperfused segment at the end of the experiments but this was not significant. At these drug dosages the direct cardioprotective action of nicorandil is slightly superior to nitroglycerin. This may be ascribed to its K-channel opening property associated with reduced regional myocardial oxygen consumption before the onset of ischaemia.

Animals↗
PubMed1995

Effects of R56865, an Na(+)- and Ca(2+)-overload inhibitor, on myocardial injury in ischemic, reperfused porcine hearts.

The cardioprotective effect of R56865, an Na(+)- and Ca(2+)-overload inhibitor, was studied in 20 regionally ischemic reperfused (I/R) porcine hearts. Ten pigs were treated with a single intravenous (i.v.) injection of 0.4 mg/kg 15 min before occlusion of the distal left anterior descending coronary artery (LAD) for 45 min. Ten other animals served as controls. Infarct size (IS) was determined as percentage of infarcted (tetrazolium method) to ischemic (dye technique) myocardium after 24-h reperfusion. Regional systolic shortening (SS) was determined by sonomicrometry. Fifteen minutes after i.v. administration of R56865, a significant decrease in heart rate (HR) (from 83 +/- 15 to 74 +/- 16 beats/min), dP/dtmax (from 2,033 +/- 604 to 1,822 +/- 524 mm Hg/s), LAD blood flow (BF, from 17 +/- 7 to 14 +/- 7 ml/min), and calculated global myocardial O2 consumption (MVO2) (from 5.9 +/- 0.9 to 5.4 +/- 0.9 ml O2/min x 100 g) was observed. Although this investigational drug attenuated the increase in HR during early reperfusion, the incidence of ventricular fibrillation (VF) was not affected during either ischemia or reperfusion. R56865 reduced IS by 24% from 67.1 +/- 16% (control group) to 50.8 +/- 13%. In addition, this treatment improved systolic shortening after 24-h reperfusion from 6 +/- 8% (control group) to 15 +/- 9%. Our results support the concept that inhibition of intracellular Na(+)- and Ca(2+)-overload is a promising new principle in treatment of myocardial I/R.

Animals↗
PubMed1995

Severe insulin-resistant diabetes mellitus in patients with congenital muscle fiber type disproportion myopathy.

Congenital muscle fiber type disproportion myopathy (CFTDM) is a chronic, nonprogressive muscle disorder characterized by universal muscle hypotrophy and growth retardation. Histomorphometric examination of muscle shows a preponderance of smaller than normal type 1 fibers and overall fiber size heterogeneity. Concomitant endocrine dysfunctions have not been described. We report the findings of altered insulin secretion and insulin action in two brothers affected with CFTDM and glucose intolerance as well as in their nonconsanguineous glucose-tolerant parents. Results are compared with those of six normoglycemic control subjects. All study participants underwent an oral glucose tolerance test to estimate insulin secretion. The oldest boy and his parents volunteered for studies of whole-body insulin sensitivity consisting of a 4-h euglycemic hyperinsulinemic clamp in combination with indirect calorimetry. Insulin receptor function and glycogen synthase (GS) activity and expression were examined in biopsies of vastus lateralis muscle. Despite a 45-90-fold increase in both fasting and postprandial serum insulin levels, both CFTDM patients had diabetes mellitus. Clamp studies revealed that the oldest boy had severe insulin resistance of both liver and peripheral tissues. The impaired insulin-stimulated glucose disposal to peripheral tissues was primarily due to reduced nonoxidative glucose metabolism. These changes were paralleled by reduced basal values of muscle GS total activity, allosterical activation of GS by glucose-6-phosphate, GS protein, and GS mRNA. The father expressed a lesser degree of insulin resistance, and studies of muscle insulin receptor function showed a severe impairment of receptor kinase activity. In conclusion, CFTDM is a novel form of severe hyperinsulinemia and insulin resistance. Whether insulin resistance is causally related to the muscle disorder awaits to be clarified.

Adolescent↗
PubMed1995

Effect of in vivo thyroid hormone status on insulin signalling and GLUT1 and GLUT4 glucose transport systems in rat adipocytes.

To examine the effect of thyroid hormone status on insulin action in isolated rat adipocytes, age- and weight-matched Sprague-Dawley rats were rendered hypothyroid (h) by i.p. injection of 2 mCi [131I]/kg. Another group of rats was made hyperthyroid (H) by i.p. injection of 500 micrograms L-thyroxine/kg/day for 7 days. The T4 levels in experimental groups were: controls, 33.5 +/- 0.95; h, 12.3 +/- 1.59; H, 133.2 +/- 8.8 micrograms/l. Adipocytes were isolated and 3-O-methylglucose transport (GT), insulin binding (IB) and insulin receptor kinase activity (IRKA) were determined. Subcellular membrane fractions (low-density microsomes, plasma membranes) were prepared and GLUT1 and GLUT4 glucose transporter immunodetected. Hyperthyroidism caused no significant effect on either IB or IRKA but increased insulin-stimulated GT by 43.6%. This increase of GT was associated with an increase of primarily GLUT4 glucose transporters. Hypothyroidism was associated with both increased insulin receptor affinity and enhanced IRKA. Despite a marked reduction of primarily GLUT4 glucose transporters, basal and insulin-stimulated GT was not reduced when compared with control. These results suggest that (1) in hyperthyroidism, increased insulin-stimulated glucose transport is associated with an increase of primarily GLUT4 glucose transporters, which may be responsible for the increment of peripheral glucose utilization in hyperthyroidism, and (2) the effect of hypothyroidism on insulin action in adipocytes is characterized by a state of increased insulin sensitivity, as indicated by the increase in insulin receptor affinity and tyrosine kinase activity. Despite the marked reduction of primarily GLUT4 glucose transporters, insulin-stimulated glucose transport is not diminished, which may suggest that functional activity of plasma membrane glucose transporters is enhanced in hypothyroidism.

Adipose Tissue↗
PubMed1995

Elevation of serum insulin concentration during euglycemic hyperinsulinemic clamp studies leads to similar activation of insulin receptor kinase in skeletal muscle of subjects with and without NIDDM.

The role of skeletal muscle insulin receptor kinase in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM) was investigated. Muscle biopsies from 13 patients with NIDDM and 10 control subjects at fasting serum insulin concentrations and approximately 1,000 pmol/l steady-state serum insulin during euglycemic hyperinsulinemic clamps were immediately frozen. The biopsies were then solubilized, and the receptors were immobilized to anti-insulin receptor antibody-coated microwells. Receptor kinase and binding activities were consecutively measured in these wells. The increase in serum insulin concentration (73 +/- 14 to 1,004 +/- 83 and 45 +/- 7 to 1,07 +/- 77 pmol/l in the NIDDM and control groups, respectively) had similar effects on receptor kinase activity in both study groups (12 +/- 1 to 42 +/- 5 and 12 +/- 2 to 47 +/- 5 amol P.fmol binding activity-1. min-1 in the NIDDM and control groups, respectively). Moreover, by selecting only the receptors that bound to anti-phosphotyrosine antibody, we found similar hyperinsulinemia-induced increases of this receptor fraction and its kinase activity in both study groups. In vitro activation of the immobilized receptors with 2 mmol/l ATP and insulin further increased their kinase activity to almost similar levels, independently of whether they had been previously stimulated in vivo or were from diabetic or nondiabetic subjects. Compared with this activity reached in vitro, the kinase activity obtained by in vivo stimulation at the clamp insulin concentration was only approximately 12%, because most receptors remained inactive and only a few reached almost the in vitro activation level.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Glucose↗
PubMed1994

Effect of epinephrine treatment during late ischemia and early reperfusion on regional myocardial function and infarct size in partially infarcted reperfused porcine hearts.

This study investigated whether epinephrine treatment during late ischemia and early reperfusion improves systolic shortening after 45 minutes of reperfusion at the cost of increased infarct size. A model consisting of both stunned and dead myocytes was used. The left anterior descending coronary arteries of 10 control and 10 treated pigs were occluded distally for 40 minutes and then reperfused for 3 days. Regional systolic shortening was determined by sonomicrometry, and infarct size was assessed as the percentage of infarcted (tetrazolium stain) to ischemic (dye technique) myocardium. Intravenous administration of epinephrine was started 10 minutes before the onset of reperfusion (5 micrograms/min) and continued until 45 minutes of reperfusion (mean 18 micrograms/min). Immediately before and during 45 minutes of reperfusion, left ventricular peak pressure, dp/dtmax, and heart rate were significantly increased in the treated animals. After 45 minutes of reperfusion, epinephrine treatment improved systolic shortening of the reperfused myocardium (treated group 9% +/- 8%; control group -1% +/- 6%; p < 0.01). Transient beta-adrenergic stimulation of the reperfused myocardium did not increase infarct size (treated group 57.2% +/- 19%; control group 55.4% +/- 17%). In conclusion, epinephrine treatment during late ischemia and early reperfusion improved systolic shortening after 45 minutes of reperfusion without affecting infarct size.

Animals↗
PubMed1994

[Diagnosis of Ebstein's anomaly with transesophageal echocardiography].

We describe a 63-year old female patient presenting with increasing dyspnea on exertion. The chest X-ray was suggestive but not compelling for Ebstein's anomaly. Although Ebstein's anomaly is most often discovered first in childhood, rare cases, such as ours, are described in late adulthood, however. Obesity may be one reason why it was not possible in our patient to detect the characteristic features in transthoracic echocardiography. The diagnosis of Ebstein's anomaly could be established only by transesophageal echocardiography: There the right atrium was grossly enlarged due to the distal insertion of the septal tricuspid valve leaflet. Colour flow echocardiography clearly demonstrated severe tricuspid valve incompetence. No additional congenital or acquired cardiac disorder could be detected. Cardiac catheterization confirmed the echocardiographical findings, whereas magnetic resonance tomography did not show the abnormal insertion of the tricuspid valve leaflet. If Ebstein's anomaly is suspected and cannot be ascertained by transthoracic echocardiography, transesophageal echocardiography can be employed to definitely diagnose this fairly rare form of congenital heart disease in an adult population.

Angina Pectoris↗
PubMed1994

[Eosinophilic endomyocarditis post partum or pregnancy-related cardiomyopathy].

We report the case of a 28-year old asthmatic female patient, who developed an acute heart failure beginning with diarrhea, fever, and dyspnea 5 weeks after delivery. After improvement of all vital functions and dismissal from hospital care unit a marked blood hypereosinophilia, left ventricular congestive heart failure, pericardium effusion and fever up to 40 degrees C followed. Endomycardial, bone marrow and skeletal muscle biopsies and the pericardial fluid showed a marked eosinophilic infiltration or polymyositis, respectively, which could be treated successfully with steroids and azathioprin. During steroid medication cytomegalovirus-associated myocarditis developed and was diagnosed by in situ hybridization. CMV hyperimmunoglobulin treatment (Cytotect, Biotest) was started (2 ml/kg bw on day 1 and 3, and 1 ml/kg on days 5, 7 and 9), which led to the eradication of the residual infiltrate and CMV-DNA in the myocardium. After discontinuation of all medication, eosinophilia and asthma recurred so that immunosuppressive treatment was continued.

Adult↗
PubMed1993

Comparison of death rates from acute myocardial infarction in a single hospital in two different periods (1977-1978 versus 1988-1989).

The in-hospital prognosis of patients with definite acute myocardial infarction (AMI) treated at the University Medical Center in Marburg, Germany, was retrospectively evaluated for the years 1977 to 1978 and 1988 to 1989. Diagnosis of AMI was established in 357 patients (251 men, 106 women) in 1977 to 1978 and in 338 patients (240 men, 98 women) in 1988 to 1989. Overall in-hospital death rate increased from 19.6% (1977 to 1978) to 28.7% (1988 to 1989) (p < 0.01). Median ages of both groups were comparable. Patients treated in 1988 to 1989 had a higher prevalence of arterial hypertension (p < 0.001), hypercholesterolemia (p < 0.0001), reinfarction (p < 0.01), and successful resuscitation before hospital arrival (p < 0.0002). Univariate risk factor evaluation suggested the following unfavorable prognostic variables: age, successful resuscitation before hospital arrival, diabetes mellitus, reinfarction, and female sex. A favorable prognosis was associated with a history of smoking, higher serum cholesterol concentration and thrombolysis. Logistic regression analysis identified age, smoking, serum cholesterol concentration, and the combination of treatment period with either successful resuscitation before hospital arrival, or diabetes mellitus, as independent variables of in-hospital prognosis. In conclusion, the apparent increase of in-hospital death rate between 1977 to 1978 and 1988 to 1989 could mainly be attributed to differences in the 2 study groups.

Age Factors↗
PubMed1993

Failure of chronic, high-dose, oral vitamin E treatment to protect the ischemic, reperfused porcine heart.

Lipid peroxidation, presumably the result of free radical-mediated injury, has been shown to occur during myocardial ischemia and reperfusion. Since vitamin E is a very effective, naturally occurring, chain-breaking antioxidant, it was investigated whether a vitamin E-supplemented diet increased myocardial tolerance towards ischemia and reperfusion in pigs. In addition to a standard diet which contained 30 mg vitamin E/kg (approximately daily vitamin E intake 30 mg), ten pigs were fed with 10 g vitamin E (all-rac-alpha-tocopherol acetate, Merck AG, Darmstadt, Germany) daily for at least 4 weeks. Ten control pigs remained on the standard diet. In an open chest preparation, the left anterior descending coronary artery was distally ligated for 45 min followed by 3 d of reperfusion. Infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic myocardium (dye technique). Regional systolic shortening was evaluated by sonomicrometry. Vitamin E concentrations in plasma and myocardium were measured by high-performance liquid chromatography. Global hemodynamic characteristics did not differ between the two groups. Oral pretreatment with vitamin E raised the plasma concentration of this vitamin from 1.1 +/- 0.3 to 5.0 +/- 1.0 mg/l and the myocardial content from 4.2 +/- 0.7 to 18.6 +/- 2.7 ng/mg fresh weight. Vitamin E treatment did not reduce infarct size, which amounted to 71.3 +/- 5% in the control group and to 71.7 +/- 8.2% in the treated animals. Furthermore, recovery of regional systolic shortening of the reperfused segment did not significantly differ in the two groups after 3 d of reperfusion; it measured 2 +/- 4% in the controls and 6 +/- 6% (p = 0.16) in the treated animals. Therefore, chronic, oral treatment with vitamin E which raised myocardial and plasma concentrations of this vitamin 4- to 5-fold did not increase myocardial tolerance towards ischemia and reperfusion in this animal model.

Administration, Oral↗
PubMed1993

In vivo metformin treatment ameliorates insulin resistance: evidence for potentiation of insulin-induced translocation and increased functional activity of glucose transporters in obese (fa/fa) Zucker rat adipocytes.

To examine the cellular mechanism of the antihyperglycemic action of in vivo metformin (M) we used an animal model of severe insulin resistance, the genetically obese (fa/fa) Zucker rat. The animals were treated with or without M (250 mg/kg.day) which was supplied with the drinking water. Three weeks of in vivo M-treatment had no effect on body weight and several blood lipid parameters, but markedly reduced plasma insulin levels by 45% (-M: 2932 +/- 166 vs. +M: 1614 +/- 85 pmol/liter, P < 0.01); plasma glucose was slightly but significantly decreased by 8.3% (-M: 7.2 +/- 0.2 vs. +M: 6.6 +/- 0.16 mmol/liter, P < 0.05). Adipocytes were isolated and incubated with or without insulin. In vivo M-treatment had no effect on basal 3-O-methylglucose uptake. In contrast, in vivo M-treatment increased insulin-stimulated glucose transport by 2.6 +/- 0.6-fold (P < 0.01). Measurement of cell surface insulin receptors revealed no effect of M on neither specific [125I]insulin binding nor on insulin receptor kinase activity. Insulin-mediated translocation of both GLUT1 and GLUT4 glucose transporters was enhanced by in vivo M-treatment, GLUT1 by 26.1%, GLUT4 by 30.5%. To fully account for the M-induced increment of insulin-stimulated glucose transport (2.6-fold), these data suggest that M increased the functional activity of glucose transporters. We conclude that amelioration of insulin resistance in (fa/fa) Zucker rats after 3 weeks of in vivo M-treatment is associated with 1) a marked reduction of in vivo hyperinsulinemia, 2) an increase of insulin-stimulated glucose transport in adipocytes; 3) this increase of insulin-stimulated glucose transport is accompanied with both a potentiation of insulin-induced translocation of GLUT1 and GLUT4 glucose transporters from an intracellular pool to the plasma membrane as well as increased functional activity of plasma membrane glucose transporters. 4) This M-effect seems to be independent of de novo glucose transporter synthesis, since total cellular GLUT1 and GLUT4 glucose transporter number were uneffected by M. 5) These results strongly suggest a direct action of M at the level of glucose transport, since neither tracer insulin binding nor insulin receptor kinase activity were significantly altered by M.

3-O-Methylglucose↗
PubMed1993

Muscle-specific expression of human insulin receptor in transgenic mice.

Variations in skeletal muscle insulin signaling are thought to have important effects on in vivo glucose homeostasis. To address the role of the insulin receptor in insulin action in muscle, we overexpressed human insulin receptors in the skeletal muscle of transgenic mice. A muscle-specific transgene (TMPE/HIR) was constructed by using promotor and enhancer elements derived from the rat MLC1/3 locus coupled to the intact protein-coding region of the human insulin-receptor cDNA. After testing the transgene for expression in cultured C2C12 myotubes, six founder mice transgenic for TMPE/HIR were generated. We determined that one line of mice had significant expression of human insulin-receptor mRNA in skeletal muscle. The analysis of several tissues from these mice by immunoprecipitation of labeled insulin receptors with a human-specific antireceptor antibody, revealed exclusive expression of human insulin receptors in skeletal muscle. Using both human-specific and non--species-specific anti-insulin receptor antibodies, we developed two immunoassays capable of quantitating the relative amounts of human and total insulin receptors in muscle. Compared with nontransgenic littermate controls, the total number of insulin receptors was increased by 30% in heterozygous transgenics and 68% in homozygotes. Human insulin-receptor protein contributed substantially to the total insulin-receptor pool present in transgenic muscle (42% for heterozygotes, 61% for homozygotes). Intraperitoneal glucose and insulin tolerance tests were performed with homozygous transgenic and nontransgenic littermate mice. Results with both approaches were significantly different for the two groups of mice, suggesting that the modest increase in insulin receptors in the muscle of transgenic mice causes a direct increase in insulin responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A microtiter well assay system to measure insulin activation of insulin receptor kinase in intact human mononuclear cells. Decreased insulin effect in cells from patients with NIDDM.

A sensitive microtiter well-based assay for the measurement of insulin activation of insulin receptor kinase in intact human circulating mononuclear cells has been developed and characterized. Mononuclear cells from 100-150 ml blood were incubated with various insulin concentrations to activate the receptor kinase. The cells were then solubilized in the presence of phosphatase and kinase inhibitors and the receptors immobilized to microwells coated with anti-insulin receptor antibody (efficiency of receptor immobilization > 85%). Receptor kinase activity and binding activity were then consecutively measured in the same wells. Insulin incubation of the cells increased the kinase activity three- to fourfold with a half-maximal effect at 5 nM and a maximal effect at 87 nM. In mononuclear cells from 16 subjects with NIDDM, the insulin effect on receptor kinase activation was significantly reduced compared with 16 nondiabetic control subjects (0.135 +/- 0.016 vs. 0.195 +/- 0.024 fmol P.fmol binding activity-1 x min-1, respectively; P < 0.05). We conclude that; 1) it is possible to determine insulin activation of receptor kinase in intact cells in this easily accessible human tissue; 2) insulin activation of insulin receptor kinase is impaired in intact mononuclear cells from patients with NIDDM; and 3) the finding that kinase activation in NIDDM is reduced in a tissue that, according to the literature, contains only the A isoform of the insulin receptor, suggests that mechanisms other than a different abundance of the A and B insulin receptor isoforms must exist that contribute to the decreased kinase activity in NIDDM.

Aged↗