[Patients with a wearable cardioverter-defibrillator (WCD) : Prescription, function and rehabilitation support]. / Patienten mit einer tragbaren Defibrillatorweste ("wearable cardioverter-defibrillator", WCD) : Verschreibung, Wirkweise und Nachsorge.

Assessment of a permanent risk of life-threatening ventricular arrhythmia in patients with severely reduced left ventricular ejection fraction (LVEF <35%), e. g. after myocarditis, dilated cardiomyopathy, acute myocardial infarction, in patients with postpartum cardiomyopathy or implantable cardioverter-defibrillator (ICD) and cardiac resynchronization treatment plus defibrillator (CRT-D) infection with temporary explantation of the system is a medical challenge. This is time-consuming and unsafe because life-threatening ventricular arrhythmias may occur during the time of risk assessment. During this phase of risk stratification, a wearable cardioverter-defibrillator (WCD) is indicated. The WCD, which is usually worn by the patient for several months, combines continuous retrievable electrocardiogram (ECG) recordings with a reliable defibrillation capability. The prescription of a WCD guarantees safe rehabilitation procedures for patients following acute inpatient treatment. Rehabilitation measures in patients with a WCD are indicated because of the underlying systolic cardiac insufficiency due to severe myocardial disease. In almost half of the patients, who are potentially threatened by ventricular tachyarrhythmias or sudden cardiac death (SCD), the LVEF and heart failure symptoms improve under controlled medication within a few months. Thus, the risk of SCD is lowered so that in many cases a first line ICD implantation is no longer necessary. The purpose of this article is to provide recommendations for rehabilitation procedures of patients with a WCD. A review of the currently available data on WCD publications was carried out with special emphasis on the current national and international guidelines.

[Insulin therapy-new insulin analogues]. / Insulintherapie ­ neue Insulinanaloga.

A multitude of short-acting and long-acting insulin analogues are currently available for the treatment of diabetes mellitus, which mimic physiological insulin secretion better than normal insulins. By the use of ultrarapid insulin analogues postprandial glucose increases can be significantly reduced. Newer long-acting insulin analogues have a very stable action profile and reduce the rate of hypoglycemia, especially nocturnal hypoglycemia, even more than first generation long-acting insulin analogues. Future developments focus on a further acceleration of prandial insulin effects with a simultaneous shorter effect time and an even more prolonged action of long-acting insulin analogues.

The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes.

AIMS/HYPOTHESIS: The molecular mechanisms underlying insulin resistance in skeletal muscle are incompletely understood. Here, we aimed to obtain a global picture of changes in protein abundance in skeletal muscle in obesity and type 2 diabetes, and those associated with whole-body measures of insulin action. METHODS: Skeletal muscle biopsies were obtained from ten healthy lean (LE), 11 obese non-diabetic (OB), and ten obese type 2 diabetic participants before and after hyperinsulinaemic-euglycaemic clamps. Quantitative proteome analysis was performed by two-dimensional differential-gel electrophoresis and tandem-mass-spectrometry-based protein identification. RESULTS: Forty-four protein spots displayed significant (p < 0.05) changes in abundance by at least a factor of 1.5 between groups. Several proteins were identified in multiple spots, suggesting post-translational modifications. Multiple spots containing glycolytic and fast-muscle proteins showed increased abundance, whereas spots with mitochondrial and slow-muscle proteins were downregulated in the OB and obese type 2 diabetic groups compared with the LE group. No differences in basal levels of myosin heavy chains were observed. The abundance of multiple spots representing glycolytic and fast-muscle proteins correlated negatively with insulin action on glucose disposal, glucose oxidation and lipid oxidation, while several spots with proteins involved in oxidative metabolism and mitochondrial function correlated positively with these whole-body measures of insulin action. CONCLUSIONS/INTERPRETATION: Our data suggest that increased glycolytic and decreased mitochondrial protein abundance together with a shift in muscle properties towards a fast-twitch pattern in the absence of marked changes in fibre-type distribution contribute to insulin resistance in obesity with and without type 2 diabetes. The roles of several differentially expressed or post-translationally modified proteins remain to be elucidated.

Long-term graft function of cryostored alginate encapsulated rat islets.

Microencapsulation of pancreatic islets before transplantation is a promising approach to enable graft function in an immunocompetent recipient without immunosuppression. However, the insufficient availability of allogenic islet tissue is a major problem. One concept to overcome these shortcomings is the cryopreservation of encapsulated allogenic islets. Recently, we reported a gentle cryopreservation protocol for rat islets encapsulated in an alginate-based microcapsule system. Here, we report for the first time long-term transplantation data of these cryopreserved microencapsulated islets. We detected a stable graft function for more than 12 month (experiments still continuing) after transplantation of 2500 cryopreserved microencapsulated CD rat islets in streptozotocin-diabetic Wistar rats. Moreover, the glucose clearance rate during an IPGTT was well preserved up to 56 weeks after transplantation. In addition, hyperglycemic blood glucose levels after removal of rat islet grafts 12 and 56 weeks after transplantation confirmed the efficacy of the encapsulated islets. Finally, the retrieved encapsulated rat islets responded well with a 7-fold increase of insulin secretion to a glucose stimulus (12 and 56 weeks). In conclusion, our study demonstrates for the first time that cryopreservation of encapsulated rat islets is possible without substantial losses on graft function for a very long time.

[New differential therapy of type 2 diabetes]. / Neue Möglichkeiten der Differenzialtherapie des Typ-2-Diabetes.

A variety of different drugs with distinct approaches are available for the treatment of type 2 diabetes. Different treatment options, as well as new developments, are needed, since an acceptable quality of glucose control often requires drug combinations. Furthermore, type 2 diabetes is known as a heterogeneous disease that is characterized by different phases and individual characteristics, such as risk profiles and contraindications. In particular the impact of antihyperglycemic therapies on cardiovascular risk, body weight and the risk for hypoglycemia is important, but also the consideration of other co-morbidities, occupational and social aspects plays a role. So far, each therapeutic step is based on lifestyle-interventions and, if possible, metformin. Dipeptidylpeptidase 4 (DPP 4) inhibitors und glucagon-like peptid 1 (GLP 1) mimetics represent a new important tool in the differential therapy of type 2 diabetes. The advantage of incretin-based concepts is an improvement of glucose quality without induction of hypoglycemia and additional weight gain. It is, however, still not known, whether these advantages will still be seen in end point studies, e. g. concerning the cardiovascular risk.

Fertility preservation and management of pregnancy in melanoma patients requiring systemic therapy.

Melanoma is one of the most common cancers in adolescents and adults at fertile age, especially in women. With novel and more effective systemic therapies that began to profoundly change the dismal outcome of melanoma by prolonging overall survival, the wish for fertility preservation or even parenthood has to be considered for a growing portion of melanoma patients-from the patients' as well as from the physicians' perspective. The dual blockade of the mitogen-activated protein kinase pathway by B-Raf proto-oncogene serine/threonine kinase and mitogen-activated protein kinase inhibitors and the immune checkpoint inhibition by anti-programmed cell death protein 1 and anti-cytotoxic T-lymphocyte-associated protein-4 monoclonal antibodies constitute the current standard systemic approaches to combat locally advanced or metastatic melanoma. Here, the preclinical data and clinical evidence of these systemic therapies are reviewed in terms of their potential gonadotoxicity, teratogenicity, embryotoxicity and fetotoxicity. Recommendations for routine fertility and contraception counseling of melanoma patients at fertile age are provided in line with interdisciplinary recommendations for the diagnostic work-up of these patients and for fertility-protective measures. Differentiated recommendations for the systemic therapy in both the adjuvant and the advanced, metastatic treatment situation are given. In addition, the challenges of pregnancy during systemic melanoma therapy are discussed.

In vitro phage display in a rat beta cell line: a simple approach for the generation of a single-chain antibody targeting a novel beta cell-specific epitope.

AIMS/HYPOTHESIS: The aim of the present study was to evaluate in vitro phage display in a beta cell line as a novel strategy for the isolation of beta cell-specific agents/biomarkers. METHODS: A single-chain antibody (SCA) library was pre-incubated with AR42J cells in order to eliminate SCAs with exocrine binding properties. It was then panned against INS-1 cells to select beta cell-targeted antibodies. RESULTS: By these means, we isolated a novel antibody, SCA B5, that binds rapidly (6.0 min) and with a 450-fold higher specificity to beta cells relative to exocrine cells. We estimated for SCA B5 a binding affinity in the low micromol/l range and 858 binding sites per beta cell. Confocal microscopy showed binding to the beta cell surface and confirmed subsequent internalisation. Moreover, staining of rat and human pancreatic tissue sections with SCA B5 suggests that the target epitope is presented in pancreatic beta cells of different origins. Infrared imaging revealed that labelling of beta cells with tracer SCA B5 is strictly dependent on beta cell mass. With competition assays we excluded insulin, glutamate decarboxylase, C-peptide and islet amyloid polypeptide as SCA B5 targets. In accordance with these predictions, SCA B5 homed in vivo highly selectively to normal beta cells and dysfunctional beta cells of diabetic rats. Moreover, accumulation of radioactively labelled SCA B5 in the pancreas was reduced by 80% after pre-injection with unlabelled SCA B5, thereby confirming the specific uptake in the pancreas. CONCLUSIONS/INTERPRETATION: We report a simple strategy for the generation of an SCA targeting a novel beta cell-specific epitope.

[Hepatomegaly und elevated transaminases in a 24-year-old with type I diabetes mellitus]. / Ausgeprägte Hepatomegalie und Transaminasenerhöhung bei 24-Jährigem mit Typ-1-Diabetes.

Poorly controlled type I diabetes mellitus can lead to excessive hepatic glycogen storage, which is accompanied by elevated transaminases and hepatomegaly. Few cases have been reported and described as glycogenic hepatopathy. There is characteristically no progression to fibrosis and cirrhosis and the symptoms are reversible with improved glycemic control.

Effects of a short-term improvement in glycaemic control on skin microvascular dysfunction in Type 1 and Type 2 diabetic patients.

AIMS: To investigate whether Type 1 and Type 2 diabetic patients differ in the effects of short-term improvement in glycaemic control on skin microvascular dysfunction. METHODS: Fourteen Type 1 and 14 Type 2 diabetic patients admitted to hospital to improve glycaemic control were investigated. Two age- and sex-matched groups of non-diabetic subjects served as controls. Capillary blood cell velocity (CBV) was assessed at the dorsal middle phalangeal area of the ring finger at rest and after 3-min arterial occlusion using laser Doppler anemometry. RESULTS: Comparing the measurements before and after improvement in glycaemic control, there were no significant changes in peak CBV, time to peak CBV and vasomotion amplitudes in Type 1 and Type 2 diabetic patients. On admission to hospital, time to peak CBV was prolonged in Type 1 (20.9 +/- 2.9 vs. 12.3 +/- 1.6 s, P = 0.003) and Type 2 diabetic patients (20.6 +/- 2.6 vs. 11.9 +/- 1.3 s, P = 0.021) compared with control subjects. After improvement in glycaemic control, there was no significant difference in time to peak CBV between Type 1 diabetic patients and their control subjects (17.8 +/- 4.2 vs. 12.3 +/- 1.6 s, P = 0.535). In Type 2 diabetic patients, the time to peak CBV increased non-significantly. CONCLUSIONS: Short-term improvement in glycaemic control did not appear to reverse microcirculatory dysfunction in Type 1 and Type 2 diabetes. However, there was an improvement of the delayed reactive hyperaemia in Type 1 diabetic patients.

Regulation of glucose-6-phosphatase gene expression by insulin and metformin.

The biguanide derivative metformin is a potent anti-diabetic drug widely used in the treatment of type 2 diabetes mellitus. Its major effect on glucose metabolism consists in the inhibition of hepatic glucose production. Since the mechanisms of metformin action are only partially understood at the molecular level, we studied the regulation of the gene promoter activity of glucose-6-phosphatase (G6Pase), the central hepatic gluconeogenic enzyme, by this drug. We have found that both metformin and insulin inhibit the basal and dexamethasone/cAMP-stimulated G6Pase promoter activity in hepatoma cells. Since one of the pharmacological targets of metformin is AMP-activated protein kinase (AMPK) and activation of AMPK is known to inhibit hepatic glucose production by the suppression of G6Pase gene transcription, we studied the effect of AMPK in this context. Under nonstimulated conditions, the inhibitory effect of both insulin and metformin was partially counteracted to a similar extent by treatment with compound C, a specific inhibitor of AMPK. In contrast, under conditions of stimulation with dexamethasone and cAMP, treatment with compound C reversed the inhibitory effect of metformin on G6Pase promoter activity to a similar extent as compared to nonstimulated conditions, whereas the effect of insulin was almost resistant to treatment with the AMPK-antagonist. These data indicate a differential AMPK-dependent regulation of G6Pase gene expression by insulin and metformin under basal and dexamethasone/cAMP-stimulated conditions.

Influence of the duration of type 2 diabetes on early functional and morphological markers of atherosclerosis compared to the impact of coexisting classic cardiovascular risk factors.

AIM: The increased incidence of atherosclerotic macrovascular disease in type 2 diabetic patients is associated both with diabetes specific factors and coexisting classic cardiovascular risk factors as components of the metabolic syndrome. The aim of this study was to investigate the association between the duration of diabetes and early functional and morphological markers of atherosclerosis compared to the impact of coexisting cardiovascular risk factors such as hypertension, dyslipoproteinemia and cigarette smoking. METHODS: 63 type 2 diabetic patients and 25 non-diabetic control subjects were investigated. Lumen diameter of the brachial artery was measured by high-resolution ultrasound at rest and after 5-min suprasystolic arterial compression. Endothelium-independent dilatation of the brachial artery was measured 4 min after sublingual administration of 400 mug of glycerol trinitrate (GTN). Percentage change of arterial lumen diameter during reactive hyperemia (FMD%) and after GTN administration (GTN%) relative to the baseline measurements were calculated. The intima-media thickness (IMT) of the common carotid artery was measured bilaterally and averages were calculated. RESULTS: FMD% (3.8+/-0.8% vs. 6.9+/-0.9%; p<0.01) and GTN% (5.6+/-0.7% vs. 14.9+/-1.7%; p<0.01) were reduced in the diabetic patients compared to their control subjects. IMT was increased in diabetic patients compared to their controls (0.82+/-0.02 mm vs. 0.62+/-0.02 mm; p<0.01). The age-adjusted diabetes duration was inversely related to FMD% (r=-0.27; p=0.016). On multiple regression analysis including packyears, hypertension, hypercholesterolemia, and hypertriglyceridemia, only diabetes duration remained a significant independent determinant of FMD. GTN% and IMT were not associated with diabetes duration, packyears, hypertension, hypercholesterolemia, and hypertriglyceridemia when all variables were taken into account. CONCLUSION: The present data lend support to the suggestion that diabetic specific factors compared to coexisting cardiovascular risk factors such as hypertension, hyperlipoproteinemia, and smoking are of major importance for the pathogenesis of endothelial dysfunction in type 2 diabetes, because only the diabetes duration was shown to be related to endothelium-dependent vasodilation when all variables were taken into account.

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.

Decreased kinase activity of insulin receptors from adipocytes of non-insulin-dependent diabetic subjects.

The tyrosine kinase activity of the insulin receptor was examined with partially-purified insulin receptors from adipocytes obtained from 13 lean nondiabetics, 14 obese nondiabetics, and 13 obese subjects with non-insulin-dependent diabetes (NIDDM). Incubation of receptors at 4 degrees C with [gamma-32P]ATP and insulin resulted in a maximal 10-12-fold increase in autophosphorylation of the 92-kDa beta-subunit of the receptor with a half maximal effect at 1-3 ng/ml free insulin. Insulin receptor kinase activity in the three experimental groups was measured by means of both autophosphorylation and phosphorylation of the exogenous substrate Glu4:Tyr1. In the absence of insulin, autophosphorylation and Glu4:Tyr1 phosphorylation activities, measured with equal numbers of insulin receptors, were comparable among the three groups. In contrast, insulin-stimulated kinase activity was comparable in the control and obese subjects, but was reduced by approximately 50% in the NIDDM group. These findings indicate that the decrease in kinase activity in NIDDM resulted from a reduction in coupling efficiency between insulin binding and activation of the receptor kinase. The insulin receptor kinase defects observed in NIDDM could be etiologically related to insulin resistance in NIDDM and the pathogenesis of the diabetic state.

Synthesis and evaluation of a glibenclamide glucose-conjugate: a potential new lead compound for substituted glibenclamide derivatives as islet imaging agents.

The search for novel SUR1-ligands originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity. Important application of novel conjugates might be their use as radioactively labelled tracer probes in the non-invasive investigation of the islet mass. It is known that the imaging quality of a tracer could be improved by increasing its hydrophilicity, which leads to a reduced plasma protein binding and diminished the unspecific uptake by various organs. In this study the glucose molecule was chosen as a substitute of glibenclamide to enhance hydrophilicity. As expected glucose conjugation leads to a 12-fold increase of the hydrophilicity. In vitro evaluation showed that the conjugate binds with high affinity to SUR1. Interestingly, in vivo the hypoglycaemic action of the conjugate was of significant shorter duration compared to glibenclamide. In accordance, the conjugate was cleared much faster from the blood stream, due to a significant lower plasma protein binding. In conclusion, glycosylation proved to be a powerful tool for the development of a high affinity glibenclamide ligand with completely different pharmacodynamics. Therefore, the glucose-conjugate could be a potential lead compound for the design of substituted glibenclamide derivatives as islet imaging ligands.

[The metabolic syndrome]. / Das metabolische Syndrom.

The "metabolic syndrome" consists of some common risk factors for cardiovascular diseases: central obesity, diabetes mellitus, hyperlipidaemia and hypertension. The metabolic syndrome (MTS) leads to increased morbidity and mortality and to higher direct and indirect healthcare costs. The MTS can be diagnosed using the NCEP/ATP III criteria. The prevalence of the MTS in Germany is estimated at 23.8% and is expected to rise further, due to increasing obesity among children and adolescents. Studies have shown that the MTS leads to increased cardiovascular and total mortality and thus to a decreased life expectancy. Studies estimating the total costs of MTS are missing, but after addition of the costs for all the risk factors it is assumed that MTS costs amount to 5% of total healthcare costs. This is the result of the more frequent demand of health services and longer hospitalisation of patients with MTS. Even moderate weight loss can decrease the rates of morbidity and healthcare costs.

Na(+)/H(+) exchange inhibitor cariporide attenuates cell injury predominantly during ischemia and not at onset of reperfusion in porcine hearts with low residual blood flow.

BACKGROUND: This study investigated whether myocardial protection by inhibition of Na(+)/H(+) exchange (NHE) occurs during ischemia and/or during reperfusion. METHODS AND RESULTS: The left anterior descending coronary artery was occluded in 32 pigs for 60 minutes and then reperfused for 24 hours. Infarct sizes (nitroblue tetrazolium [NBT] stain, histology) were determined at the end of the experiments. An extracorporeal bypass was used to achieve a constant residual blood flow of 3 mL/min in the myocardium at risk during ischemia. The NHE-1 inhibitor cariporide or distilled water was infused into the extracorporeal bypass system. In group 1, active treatment was administered from the onset of ischemia until 10 minutes of reperfusion (n=8). In group 2, active treatment was infused during the first 30 minutes of ischemia only (n=8). The group 3 animals (n=8) received intracoronary cariporide after 45 minutes of ischemia until 10 minutes of reperfusion. The control animals (group 4, n=7) were treated similarly to group 1 animals, with the cariporide solution being replaced by distilled water. Infarct sizes of group 1 (NBT stain, 41.5+/-20%; histology, 44. 6+/-12%) and group 2 (NBT stain, 33.5+/-14%; histology 34.9+/-15%) differed significantly (at least P:=0.012) from infarct sizes of group 3 (NBT stain, 71.6+/-15%; histology, 69.2+/-12%) and the control group (NBT stain, 76+/-9%; histology 72.4+/-12%). Cariporide treatment in group 1 and group 2 significantly improved functional recovery after 24 hours of reperfusion. CONCLUSIONS: Myocardial protection by cariporide is predominantly achieved by NHE inhibition during ischemia and not during early reperfusion.

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)

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.

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)

Insulin signaling and action in cultured skeletal muscle cells from lean healthy humans with high and low insulin sensitivity.

The aim of these studies was to investigate whether insulin resistance is primary to skeletal muscle. Myoblasts were isolated from muscle biopsies of 8 lean insulin-resistant and 8 carefully matched insulin-sensitive subjects (metabolic clearance rates as determined by euglycemic-hyperinsulinemic clamp: 5.8 +/- 0.5 vs. 12.3 +/- 1.7 ml x kg(-1) x min(-1), respectively; P < or = 0.05) and differentiated to myotubes. In these cells, insulin stimulation of glucose uptake, glycogen synthesis, insulin receptor (IR) kinase activity, and insulin receptor substrate 1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity were measured. Furthermore, insulin activation of protein kinase B (PKB) was compared with immunoblotting of serine residues at position 473. Basal glucose uptake (1.05 +/- 0.07 vs. 0.95 +/- 0.07 relative units, respectively; P = 0.49) and basal glycogen synthesis (1.02 +/- 0.11 vs. 0.98 +/- 0.11 relative units, respectively; P = 0.89) were not different in myotubes from insulin-resistant and insulin-sensitive subjects. Maximal insulin responsiveness of glucose uptake (1.35 +/- 0.03-fold vs. 1.41 +/- 0.05-fold over basal for insulin-resistant and insulin-sensitive subjects, respectively; P = 0.43) and glycogen synthesis (2.00 +/- 0.13-fold vs. 2.10 +/- 0.16-fold over basal for insulin-resistant and insulin-sensitive subjects, respectively; P = 0.66) were also not different. Insulin stimulation (1 nmol/l) of IR kinase and PI 3-kinase were maximal within 5 min (approximately 8- and 5-fold over basal, respectively), and insulin activation of PKB was maximal within 15 min (approximately 3.5-fold over basal). These time kinetics were not significantly different between groups. In summary, our data show that insulin action and signaling in cultured skeletal muscle cells from normoglycemic lean insulin-resistant subjects is not different from that in cells from insulin-sensitive subjects. This suggests an important role of environmental factors in the development of insulin resistance in skeletal muscle.