Harald H. Klein

Simvastatin Acutely Reduces Myocardial Reperfusion Injury In Vivo by Activating the Phosphatidylinositide 3-kinase/akt Pathway

Long-term pretreatment with statins reduces myocardial injury after acute ischemia and reperfusion by increasing the expression of endothelial nitric oxide synthase (eNOS). We hypothesized that statins may act rapidly enough to protect the myocardium from ischemia/reperfusion injury when given right at the beginning of the reperfusion period and tried to delineate the role of PI 3-kinase/Akt pathway in early eNOS activation. Activated simvastatin was given intravenously 3 minutes before starting the reperfusion after temporary coronary artery occlusion (CAO) in anaesthetized rats. Simvastatin significantly increased myocardial PI 3-kinase activity, AktSer473, and eNOSSer1177 phosphorylation and reduced infarct size by 42%. Infarct size reduction as well as activation of PI 3-kinase/Akt/eNOS pathway were not observed in rats co-treated with the PI 3-kinase inhibitor wortmannin. Contribution of eNOS was further delineated using the NOS inhibitor l-NAME, which could completely block cardioprotection by the statin. In summary, simvastatin acutely reduces the extent of myocardial necrosis in normocholesterolemic rats in an NO- dependent manner by activating the PI 3-kinase/Akt pathway. This is the first study demonstrating short-term cardioprotective effects of simvastatin in an in vivo model of ischemia/reperfusion.

Association of Metformin's Effect to Increase Insulin-Stimulated Glucose Transport With Potentiation of Insulin-Induced Translocation of Glucose Transporters From Intracellular Pool to Plasma Membrane in Rat Adipocytes

To examine the cellular mechanism of the antihyperglycemic action of metformin, we studied its effect on various functional and molecular parameters involved in the pathogenesis of insulin resistance. Isolated rat adipocytes were incubated with or without metformin (1–100 μg/ml) for 2 h at 37°C followed by an incubation with or without insulin (1.72 nM). Metformin treatment had no significant effect on basal 3-O-methylglucose uptake. In contrast, metformin increased insulin-stimulated glucose transport in a dose-dependent manner up to 43 ± 7%. This effect was neither associated with a significant effect of metformin on trace insulin binding (1.74 ± 0.20% without metformin vs. 1.89 ± 0.30% with metformin; P > 0.05) nor with an effect of metformin on insulin-receptor kinase activity as measured by 32P incorporation into the 95,000-Mr β-subunit of the insulin receptor and an exogenous substrate, histone 2B. Determination of glucose-transporter numbers in subcellular membrane fractions, plasma membranes (PMs), and low-density microsomes (LDMs) with cytochalasin B binding revealed that metformins effect to increase insulin-stimulated glucose transport is associated with a potentiation (38 ± 5%) of insulininduced translocation of glucose transporter from the LDM to the PM, whereas the basal state was not significantly affected (basal LDM 61.3 ± 4.2 vs. basal metformin LDM 63.6 ± 7.1, P < 0.05; basal PM 6.4 ± 2.1 vs. basal metformin PM 7.2 ± 2.8, P > 0.05; insulin LDM 32.7 ± 5.4 vs. insulin-metformin LDM 15.8 ± 3.7, P < 0.01; insulin PM 28.6 ± 4.6 vs. insulin-metformin PM 45.3 ± 4.9 pmol/mg membrane protein, P < 0.01). Immunodetection of glucose-transporter isoforms HepG2 erythrocyte (GLUT1) and muscle adipose tissue (GLUT4) in adipocyte membrane fractions separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted to nitrocellulose revealed that insulin-induced translocation of GLUT1 and GLUT4 is potentiated by metformin. Determination of specific GLUT1 and GLUT4 mRNA levels revealed that this metformin effect is not associated with an increase in glucose-transporter gene expression. We conclude that 1) metformin increases insulinstimulated 3-O-methylglucose transport by potentiating insulin-induced translocation of GLUT1 and GLUT4 glucose transporter from LDM to the PM without affecting glucose-transporter gene expression, and 2) because neither insulin binding nor activation of insulin-receptor kinase in intact cells was significantly altered by metformin, these results suggest that metformin acts at the level of glucose transport.

β-cell development and turnover during prenatal life in humans

INTRODUCTION beta-cell regeneration is an area under active investigation for the future treatment of diabetes, but little is known about the patterns and dynamics of prenatal beta-cell development in humans. In particular, the quantitative changes in beta-cell mass in the developing pancreas have not been elucidated in detail. We addressed the following questions in prenatal humans: i) what is the timing of beta-cell occurrence and islet growth? ii) What are the dynamics of beta-cell replication and apoptosis? METHODS Pancreatic tissue was obtained from 65 human embryos and foetuses aged between 8 weeks post conception (p.c.) and birth. Sections were stained for insulin, glucagon, Ki67 (proliferation marker), TUNEL (apoptosis marker) and CD31 (blood vessel marker), and morphometric analyses were performed. RESULTS beta-cells were detected from gestational week 9 onward, whereas glucagon expression was detected already at week 8. The fractional beta-cell area of the pancreas increased in a linear fashion until birth (r=0.60, P<0.001). The first endocrine cells were found within or adjacent to the primitive ductal epithelium. beta-cell replication was readily detected in the newly forming islets already starting at week 9 p.c. (average frequency 2.8+/-0.4%). A small percentage of cells co-expressed insulin and glucagon during the early foetal period. There was a close relationship between the development of endocrine islets and blood vessels during all stages of prenatal pancreas development suggesting a possible interaction between both cell types. The frequency of beta-cell apoptosis was relatively high throughout all ages (1.5+/-0.3%). CONCLUSIONS beta-cell differentiation in humans occurs from week 9 p.c. onward. The first endocrine cells are closely associated with the ductal epithelium suggesting differentiation from precursor cells. High rates of beta-cell replication suggest that this mechanism plays an important role in the prenatal expansion of beta-cell mass.

Generation of Novel Single-Chain Antibodies by Phage-Display Technology to Direct Imaging Agents Highly Selective to Pancreatic β- or α-Cells In Vivo

OBJECTIVE Noninvasive determination of pancreatic β-cell mass in vivo has been hampered by the lack of suitable β-cell–specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet cells in vivo. RESEARCH DESIGN AND METHODS To generate agents specifically binding to pancreatic islets, a phage library was screened for single-chain antibodies (SCAs) on rat islets using two different approaches. 1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 min. 2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo. RESULTS We report the generation of SCAs that bind highly selective to either β- or α-cells. These SCAs are internalized by target cells, disappear rapidly from the vasculature, and exert no toxicity in vivo. Specific binding to β- or α-cells was detected in cell lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [125I]-labeled SCAs after intravenous administration in rats strongly predicted the β-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test. CONCLUSIONS Our data provide strong evidence that the presented SCAs are highly specific for pancreatic β-cells and enable imaging and quantification in vivo.

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 ∼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 1,070 ± 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 ∼12%, because most receptors remained inactive and only a few reached almost the in vitro activation level. Our data suggest 1) that 1,000 pmol/1 serum insulin activates only a small fraction of insulin receptors in intact muscle and 2) that NIDDM is not associated with impaired insulin activation of insulin receptor kinase in skeletal muscle, at least not when euglycemia is maintained.

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

Aims/hypothesisThe 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.MethodsSkeletal 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.ResultsForty-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/interpretationOur 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.

Salivary cortisol and psychological mechanisms in patients with acute versus chronic low back pain

This study was designed to explore whether the basal adrenocortical activity is related with pain-related coping, nonverbal pain behavior, depressive mood, and fatigue in patients with acute and chronic nonspecific low back pain. 19 patients with acute low back pain (ALBP) and 24 with chronic low back pain (CLBP) participated in the study. The adrenocortical activity was assessed through the cortisol awakening response. All participants provided five saliva samples (0, 15, 30, 45, and 60min after waking) on two consecutive days off work. Pain-related coping [fear-avoidance coping (FAC) and endurance coping (EC)], nonverbal pain behavior (NPB), depressive mood, and fatigue were assessed through questionnaires. Among ALPB patients, EC was negatively associated with the cortisol release, whereas fatigue was positively associated with it. Among CLBP patients, FAC, NPB, depressive mood, and fatigue were negatively associated with the cortisol awakening response, whereas EC tended to be positively associated with it. The results indicate that pain-related coping strategies which are expected to be successful appear to lower the adrenocortical activity among ALBP patients, whereas affective distress may enhance the level of cortisol in this group. Among CLBP patients, long-term maladaptive coping strategies might contribute to hypocortisolism.

13C nuclear magnetic resonance spectra—VI: Stereochemical dependence of γanti, heterosubstituent effects on 13C chemical shifts of bridgehead substituted molecules1

Abstract The 13C NMR data of some mono- and disubstituted adamantanes, homoadamantenes and homonoradamantenes are presented. A model of dependencies of the γanti substituent induced shifts on torsion angles and internuclear distances between substituents and the γ carbons is proposed. Furthermore, it is shown that for the α substituent induced shifts strain within the molecular frameworks of these compounds plays no significant part.

Differential modulation of insulin actions by dexamethasone: studies in primary cultures of adult rat hepatocytes

BACKGROUND/AIMS Steroid diabetes is associated with hepatic insulin resistance; in hepatic cell models, however, mainly insulin-permissive effects have been described. Here we investigate modulation by dexamethasone of a larger number of insulin actions. METHODS Adult rat hepatocytes were cultured+/-dexamethasone for 48 h; insulin actions were studied subsequently. RESULTS Stimulation of glycolysis by insulin but not by glucose required culture with dexamethasone. Activation of glycogen synthesis by insulin or glucose was strongly enhanced by dexamethasone, the insulin effects on glycogenolysis and amino acid uptake were not modulated. When dexamethasone was omitted from the culture, insulin was incapable to activate glycogen synthase, inactivate glycogen phosphorylase or elevate the level of fructose 2,6-bisphosphate. Dexamethasone did not alter insulin binding, insulin receptor number or kinase activity, insulin receptor substrate-1 and Akt protein expression/phosphorylation. Insulin-stimulated association of phosphatidylinositol 3-kinase with insulin receptor substrates-1 and -2 was increased with dexamethasone, the increased association with IRS-2 may, at least partially, be explained by higher IRS-2 protein expression. CONCLUSIONS The steroid does not cause hepatic resistance in vitro. The differential attenuation under steroid deprivation points to defects in branches of the insulin signal chain and/or loss of hormonal regulation at the level of target enzymes.

Nonthyroidal Illness Syndrome in Cardiac Illness Involves Elevated Concentrations of 3,5-Diiodothyronine and Correlates with Atrial Remodeling

Background: Although hyperthyroidism predisposes to atrial fibrillation, previous trials have suggested decreased triiodothyronine (T3) concentrations to be associated with postoperative atrial fibrillation (POAF). Therapy with thyroid hormones (TH), however, did not reduce the risk of POAF. This study reevaluates the relation between thyroid hormone status, atrial electromechanical function and POAF. Methods: Thirty-nine patients with sinus rhythm and no history of atrial fibrillation or thyroid disease undergoing cardiac surgery were prospectively enrolled. Serum concentrations of thyrotropin, free (F) and total (T) thyroxine (T4) and T3, reverse (r)T3, 3-iodothyronamine (3-T1AM) and 3,5-diiodothyronine (3,5-T2) were measured preoperatively, complemented by evaluation of echocardiographic and electrophysiological parameters of cardiac function. Holter-ECG and telemetry were used to screen for POAF for 10 days following cardiac surgery. Results: Seven of 17 patients who developed POAF demonstrated nonthyroidal illness syndrome (NTIS; defined as low T3 and/or low T4 syndrome), compared to 2 of 22 (p < 0.05) patients who maintained sinus rhythm. In patients with POAF, serum FT3 concentrations were significantly decreased, but still within their reference ranges. 3,5-T2 concentrations directly correlated with rT3 concentrations and inversely correlated with FT3 concentrations. Furthermore, 3,5-T2 concentrations were significantly elevated in patients with NTIS and in subjects who eventually developed POAF. In multivariable logistic regression FT3, 3,5-T2, total atrial conduction time, left atrial volume index and Fas ligand were independent predictors of POAF. Conclusion: This study confirms reduced FT3 concentrations in patients with POAF and is the first to report on elevated 3,5-T2 concentrations in cardiac NTIS. The pathogenesis of NTIS therefore seems to involve more differentiated allostatic mechanisms.