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

Klein, Harald; Kowalewski, B.; Drenckhan, Maren; Neugebauer, S.; Matthaei, S.; Kotzke, G.

doi:10.2337/diabetes.42.6.883

Cited by 13 publications

(19 citation statements)

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“…Skeletal muscle is the most important site of peripheral insulin resistance [25] but this tissue is relatively inaccessible for routine evaluation of insulin action in humans. Mononuclear cells are not a typical insulin target tissue but they do have the advantage of being the only easily accessible human cell system available to study insulin action at the cellular level [26]. 2-DG uptake represents hexose transport and glucose uptake in isolated monocytes has been previously shown to respond to insulin in a dose-dependent manner [15].…”

Section: Discussionmentioning

confidence: 99%

A Mediterranean and a high-carbohydrate diet improve glucose metabolism in healthy young persons

et al. 2001

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Insulin resistance, or the relative inability of insulin to facilitate the disposal of glucose in tissues, is considered to be a risk factor for both diabetes and coronary heart disease (CHD). In 1988 ªSyndrome Xº was [1] first described as a cluster of pathophysiologic phenomena including insulin resistance, glucose intolerance, dyslipidemia, and abdominal obesity.The relation between diet and chronic disease is well established, although there is debate over the details of this relation. In general, high intakes of dietary fat have been associated with obesity and its comorbid conditions, including heart disease and diabetes [2]. All these factors are related to the globalization of the ªWesternº lifestyle and dietary habits. Insulin resistance usually precedes the diagnosis of Type II (non-insulin-dependent) diabetes mellitus by Diabetologia (2001) Abstract Aims/hypothesis. Insulin resistance usually precedes the diagnosis of Type II (non-insulin-dependent) diabetes mellitus. However, in most patients, the clinical expression of the disease could be prevented by dietary and lifestyle changes. We investigated the effects of a diet enriched in monounsaturated fatty acids (Mediterranean diet) and a low fat, high-carbohydrate diet on in vivo and in vitro glucose metabolism in 59 young subjects (30 men and 29 women). Methods. We carried out an intervention dietary study with a saturated fat phase and two randomized-crossover dietary periods: a high-carbohydrate diet and a Mediterranean diet for 28 days each. We analysed the plasma lipoproteins fractions, free fatty acids, insulin sensitivity and glucose uptake in isolated monocytes at the end of the three dietary periods.Results. In comparison to the saturated fat diet, the CHO and Mediterranean diets induced a decrease of LDL-cholesterol (p < 0.001) and HDL-cholesterol (p < 0.001). Steady-state plasma glucose decreased (p = 0.023) and basal and insulin-stimulated 2-deoxiglucose uptake in peripheral monocytes increased in both diets (CHO and Mediterranean), (p = 0.007) indicating an improvement in insulin sensitivity. Fasting free fatty acids plasma values were correlated positively with steady state plasma glucose (r = 0.45; p < 0.0001). In addition, there was an inverse correlation between the mean glucose of the steady state plasma glucose period and logarithmic values of basal (r = ±0.34; p = 0.003) and insulin stimulated glucose uptake in monocytes (r = ±0.32; p = 0.006). Conclusion/interpretation. Isocaloric substitution of carbohydrates and monounsaturated fatty acids for saturated fatty acids improved insulin sensitivity in vivo and in vitro, with an increase in glucose disposal. Both diets are an adequate alternatives for improving glucose metabolism in healthy young men and women. [Diabetologia (2001

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Section: Discussionmentioning

confidence: 99%

A Mediterranean and a high-carbohydrate diet improve glucose metabolism in healthy young persons

et al. 2001

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“…Insulin receptor kinase and binding activities were measured essentially as described [33,34]. Briefly, 40 µl of solubilized muscle sample were added to microwells coated with anti-insulin receptor antibody for 16 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…The wells were washed and receptor-mediated 32 P-incorporation into recombinant IRS-1 measured at a 32 P-ATP concentration of 120 nmol/l and 2.3 µg/ml recombinant IRS-1 (Upstate Biotechnology Incorporated, New York, USA). [ 125 I-Tyr-A 14 ]-monoiodoinsulin (Amersham-Pharmacia, Freiburg, Germany) binding to immobilized insulin receptors was also measured in the wells as described [33]. Insulin binding capacity was defined as the amount of specifically bound insulin at a concentration of 8.7 nmol/l [18].…”

Section: Methodsmentioning

confidence: 99%

Insulin signalling in skeletal muscle of subjects with or without Type II-diabetes and first degree relatives of patients with the disease

et al. 2002

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Aims/hypothesis. Alterations in insulin signalling could contribute to insulin resistance in Type II (non-insulindependent) diabetes mellitus. Some of these alterations could be secondary to the diabetic state, ie. the hyperglycaemia or increased NEFA concentrations. We sought to exclude such secondary factors and to investigate whether Type II diabetes in itself is associated with altered insulin signalling in skeletal muscle. Methods. Hyperinsulinaemic-euglycaemic clamps were performed in 10 obese Type II diabetic patients whose glucose concentrations had been normalised for 8 h by plasma glucose-adapted insulin infusion, 10 BMImatched first-degree relatives of Type II diabetic patients, and 10 BMI-matched non-diabetic subjects. Muscle biopsies were obtained before and at the end of the clamps, and insulin receptor kinase activity, phosphatidylinositol-3′-kinase activity, Akt-Thr 308 -phosphorylation, and glycogen synthase activity determined. Results. At similar steady-state clamp insulin concentrations (~400 pmol/l) similar receptor kinase activities, phosphatidylinositol-3′-kinase activities, AktThr 308 -phosphorylation, and glycogen synthase activities were found in all subject groups although glucose disposal was reduced in the diabetic subjects and relatives. Pre-clamp signalling levels were different between subject groups, most likely due to different preclamp insulin concentrations. Conclusion/interpretation. Our results in subjects at risk for the development of diabetes and Type II diabetic patients with normalized glucose concentrations suggest that Type II diabetes in itself is not associated with reduced signalling intensity at the studied signalling molecules, at least not at the chosen clamp insulin concentration and under the chosen conditions. Alterations responsible for the reduced glucose disposal could be located downstream of the investigated steps or in alternative insulin signalling pathways. A different spatial organisation of the investigated signalling molecules can also not be excluded. [Diabetologia (2002) Type II (non-insulin-dependent) diabetes mellitus is associated with a reduced insulin-stimulated glucose disposal in skeletal muscle and other tissues [1]. Because insulin resistance is also observed in non-diabetic first-degree relatives of diabetic subjects [2,3,4,5], and because it seems to precede the development of frank diabetes [6], an inherited basis of at least a part of the diabetes-associated insulin resistance has been proposed [7]. On the other hand metabolic abnormalities in the diabetic state itself such as hyper-

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“…IRK and binding capacities. These were measured as previously described (34,36,37). Briefly, 40 l muscle sample lysates were added to microwells coated with anti-insulin receptor antibody for 16 h at 4°C.…”

Section: Subjectsmentioning

confidence: 99%

“…The wells were washed, and receptor-mediated 32 P incorporation into recombinant IRS-1 (17 nmol/l) (Upstate Biotechnology, New York, NY) was measured at 120 nmol/l 32 P-ATP. [ 125 I-Tyr-A 14 ]monoiodoinsulin (Amersham-Pharmacia, Freiburg, Germany) binding to immobilized insulin receptors was also measured in the wells (36). Insulin binding capacity was defined as the amount of specifically bound insulin at a concentration of 8.7 nmol/l (34,37).…”

Section: Subjectsmentioning

confidence: 99%

Troglitazone Treatment Increases Protein Kinase B Phosphorylation in Skeletal Muscle of Normoglycemic Subjects at Risk for the Development of Type 2 Diabetes

et al. 2002

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We investigated whether the effect of troglitazone on glucose disposal is associated with altered insulin signaling. Nondiabetic first-degree relatives of type 2 diabetic patients (age 30 ؎ 2 years, BMI 30 ؎ 1 kg/m 2 ; n ‫؍‬ 20) were randomized in a double-blind manner to 3 months of troglitazone (200 mg/day) or placebo treatment. Before and after treatment, 3-h euglycemic-hyperinsulinemic glucose clamps (40 mU ⅐ m ؊2 ⅐ min ؊1 ) were performed, and muscle biopsies were obtained immediately before and after the clamps. In the biopsies, insulin receptor kinase (IRK) activity, insulin receptor substrate (IRS)-1-associated phosphatidylinositol 3-kinase (PI3K) activity, Ser 473 and Thr 308 phosphorylation of protein kinase B (PKB), and protein expression of IRS-1, IRS-2, phosphoinositol-dependent kinase-1 (PDK-1), PKB, and GLUT-4 were determined. After troglitazone treatment, insulin-stimulated glucose disposal was increased compared with pretreatment and placebo (279 ؎ 37 vs. 211 ؎ 26 and 200 ؎ 25 mg ⅐ m ؊2 ⅐ min ؊1 ; both P < 0.05). IRK and PI3K activities were not altered by troglitazone, but PKB Ser 473 phosphorylation was enhanced compared with pretreatment and placebo at the clamp insulin level (138 ؎ 36 vs. 77 ؎ 16 and 55 ؎ 13 internal standard units; both P < 0.05) and with pretreatment at the basal level (31 ؎ 9 vs. 14 ؎ 4 internal standard units; P < 0.05). PKB Thr 308 phosphorylation also tended to be higher, but this was not statistically significant. Troglitazone did not alter insulin receptor number or IRS-1, IRS-2, PKB, PDK-1, or GLUT-4 protein expression. We conclude that increased PKB phosphorylation may contribute to the insulinsensitizing effects of thiazolidinediones in human skeletal muscle.

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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

Cited by 13 publications

References 0 publications

A Mediterranean and a high-carbohydrate diet improve glucose metabolism in healthy young persons

A Mediterranean and a high-carbohydrate diet improve glucose metabolism in healthy young persons

Insulin signalling in skeletal muscle of subjects with or without Type II-diabetes and first degree relatives of patients with the disease

Troglitazone Treatment Increases Protein Kinase B Phosphorylation in Skeletal Muscle of Normoglycemic Subjects at Risk for the Development of Type 2 Diabetes

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