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Caffeine ingestion decreases glucose disposal during a hyperinsulinemic-euglycemic clamp in sedentary humans.

The purpose of this investigation was to examine the effect of caffeine (an adenosine receptor antagonist) on whole-body insulin-mediated glucose disposal in resting humans. We hypothesized that glucose disposal would be lower after the administration of caffeine compared with placebo. Healthy, lean, sedentary (n = 9) men underwent two trial sessions, one after caffeine administration (5 mg/kg body wt) and one after placebo administration (dextrose) in a double-blind randomized design. Glucose disposal was assessed using a hyperinsulinemic-euglycemic clamp. Before the clamp, there were no differences in circulating levels of methylxanthines, catecholamines, or glucose. Euglycemia was maintained throughout the clamp with no difference in plasma glucose concentrations between trials. The insulin concentrations were also similar in the caffeine and placebo trials. After caffeine administration, glucose disposal was 6.38 +/- 0.76 mg/kg body wt compared with 8.42 +/- 0.63 mg/kg body wt after the placebo trial. This represents a significant (P < 0.05) decrease (24%) in glucose disposal after caffeine ingestion. In addition, carbohydrate storage was 35% lower (P < 0.05) in the caffeine trial than in the placebo trial. Furthermore, even when the difference in glucose disposal was normalized between the trials, there was a 23% difference in the amount of carbohydrate stored after caffeine administration compared with placebo administration. Caffeine ingestion also resulted in higher plasma epinephrine levels than placebo ingestion (P < 0.05). These data support our hypothesis that caffeine ingestion decreases glucose disposal and suggests that adenosine plays a role in regulating glucose disposal in resting humans.

Administration, Oral↗

The "patch-clamp" technique and its application in investigations of the properties of human T lymphocyte potassium channels.

The first part of this review presents a historical outline on the development of experimental methods in electrophysiology starting from the first experiments performed in the 1920s and ending with the "patch-clamp" technique. Recording configurations of the "patch-clamp" technique are briefly reviewed in the second section. The areas of application of the configurations are shown. The last section contains a short review on the available data of "patch-clamp" studies on the expression and properties of potassium channels in human T lymphocytes (TL). Problems that require further investigation are briefly presented.

Electrophysiology↗

[Assessment of insulin sensitivity during exercise training program in obese women. Comparison of simple indices with hyperinsulinemic euglycemic clamp technique].

Insulin resistance is a key element of metabolic syndrome, which includes disturbances of glucose tolerance, obesity, hypertension, coronary heart disease dyslipidemia and many other defects. An important problem in scientific research is precise measurement of insulin sensitivity. The method considered "the gold standard" is glucose clamp, however, it is difficult to apply this method in large studies. Therefore, simple indices of insulin resistance are proposed. It remains unclear whether those indices are able to reflect changes occurring during insulin-sensitizing intervention. The aim of the present study was to assess the use of indirect indices for the changes in insulin sensitivity during exercise training and to compare those indices with results derived from clamp. Fourteen obese normoglycemic women participated in 12-week exercise training program, which included exercise performed on a bicycle ergometer, 5 days a week for 30 minutes. Insulin sensitivity (M/FFM value) before and after training was measured with hyperinsulinemic euglycemic clamp technique. Simple indices of insulin resistance were also assessed: fasting plasma insulin (INS), logarithm INS (log [INS]), homeostasis model assessment (HOMA), logarithm HOMA (log [HOMA]) and quantitative insulin sensitivity check index (QUICKI). Before training, all those indices were markedly related to M/FFM. After training, an increase in M/FFM was observed. None of the examined indices markedly changed after training. There was no correlations between changes of evaluated indices and in M/FFM during training, and no relationships of those parameters after training. Our study indicates that simple indices are not able to reflect changes occurring during insulin-sensitizing intervention.

Adult↗

[Method for human peripheral blood eosinophil isolation for patch-clamp study].

OBJECTIVE: To establish a rapid and economic method for isolating human peripheral blood eosinophils with high viability for patch-clamp studies and investigate the electrophysiological properties of Ca(2+)-activated K(+) channel of the isolated cells. METHODS: Peripheral blood eosinophils were isolated by modified discontinuous Percoll density gradient centrifugation, and the electric currents in the single Ca(2+)-activated K(+) channels of the cells were recorded using patch-clamp technique with cell-attached configuration. RESULTS: The purity of the eosinophils from healthy donors reached (90.5+/-1.6)%, with a viability rate over 99% and recovery rate of (48.2+/-6.9)%. The isolated cells were morphologically intact, from which Ca(2+)-activated K(+) channel activity could be detected. CONCLUSION: The peripheral blood eosinophils isolated using this rapid, simple and highly efficient method are characterized by high purity and viability without obvious cellular injuries, which are ideal for patch-clamp studies.

Cell Separation↗

[Serum adiponectin concentration in relation to body fat distribution and tissue insulin sensitivity in the glucose clamp study].

OBJECTIVE: To study serum adiponectin level in relation to body fat distribution and tissue insulin sensitivity by using the extended hyperinsulinemic euglycemic clamp technique. METHODS: Extended hyperinsulinemic euglycemic clamp was performed in 44 Chinese individuals with normal glucose tolerance from Shanghai area, including 12 subjects with normal weight [body mass index (BMI) < 25 kg/m(2)], 19 with overweight (25 kg/m(2) < or = BMI < 30 kg/m(2)), and 13 with obesity (BMI > or = 30 kg/m(2)). The rate of insulin-mediated glucose disposal (Rd) during the steady state of glucose clamp was used to assess the peripheral tissue insulin sensitivity. Total body fat depots [BMI, percentage of body fat (FAT%), and body fat mass (FM)], regional body fat depots [waist circumference (W), hip circumference (H), W and H ratio (WHR), abdominal subcutaneous fat area (SA), intra-abdominal fat area (VA), and femoral subcutaneous fat area (FA)] were measured on all subjects. Fasting serum adiponectin level was determined by radioimmunoassay. RESULTS: (1) After the adjustment for age and sex, the serum adiponectin concentrations in the overweight and obesity groups were (8.7 +/- 4.8) mg/L and (6.7 +/- 0.8) mg/L, both significantly lower than that in the normal weight group [(15.7 +/- 1.8) mg/L, both P < 0.01]. (2) Serum adiponectin concentration was negatively correlated with BMI, W, WHR, FAT%, FM and VA, especially with W and VA (r = -0.46, P < 0.01, and r = -0.53, P < 0.01). (3) Serum adiponectin concentration was positively correlated with Rd (r = 0.52, P < 0.01) and the rate of glycogen synthesis (r = 0.36, P < 0.05), and was negatively correlated with free fatty acid level (r = -0.41, P < 0.05). (4) Stepwise regression analysis indicated that in addition to sex, BMI, WHR or VA and SA, serum adiponectin level was also the independent contributing factor for Rd, which explains 3.52% and 4.84% of the variance respectively (P all < 0.05). CONCLUSION: (1) The decrease in serum adiponectin level is associated with the increase of total body fat depot, especially the increase of total body fat depot in intra-abdominal fat area. (2) Serum adiponectin level is an independent contributing factor for insulin sensitivity.

Adiponectin↗

Computer-aided formation of the whole-cell patch-clamp recording configuration.

The conventional patch-clamp technique requires well-trained experimenter. Few commercial automated patch-clamp systems, designed for drug development, are better suited for large-scale research then for standard electrophysiological experiments. Here we describe a state machine for automated recognition of recording states of the patch-clamp experiment. The principle of the state machine is based on evaluation of the charge carried by membrane current during specific time segments in responses to square wave voltage stimulation. The state machine may serve for generating various sound alerts, signals for automated control of other devices, assistance in micromanipulation, internal pipette pressure control, and holding potential adjustments. Algorithm of the state machine, designed to cover wide variety of cell types, was successfully tested on rat ventricular myocytes.

Algorithms↗

[A data interface based on USB bus technology for full auto patch-clamp system].

A USB bus based data interface technology for full auto Patch-Clamp system is discussed in the article. The main controller is CY2131QC (Cypress) and the logic controller is EPM3256A (Altera). Optocouplers are used to get rid of the noise from the interface. It makes the installation of the Patch-Clamp system easier by using the USB bus, and is suitable for the new generation of the Patch-Clamp system with a high speed of 1M bytes/s.

Computer-Aided Design↗

[Inhibition of pancreatic beta-cell secretion during a "glucose-clamp" in subjects with acanthosis nigricans].

It has long been known that acanthosis nigricans is accompanied by insulin resistance. In certain insulin-resistant states, including obesity, a more sluggish response to the insulin/insulin inhibition feedback normally present in pancreatic beta cells has been documented. Some have claimed a sort of beta-cell insulin resistance "parallel" to that of the peripheral tissues. The present study assesses the efficiency of the insulin/insulin feedback in acanthosis nigricans patients, measuring the inhibition of the production of C-peptide (the indicator of beta cell secretion) induced by the administration of exogenous insulin during glucose clamping. This was done in order to compare the roles of the peripheral tissues and the beta cells in producing the insulin resistance typical of acanthosis nigricans. The study using the glucose-insulin clamp technique was conducted on 4 Acanthosis Nigricans patients with normal glucose tolerance and 4 healthy controls, the drop in C-peptide levels after the administration of exogenous insulin being assessed in the course of both steady states. The results showed that the acanthosis nigricans patients retained a beta cell response to the exogenous insulin through their peripheral tissues presented a reduced sensitivity to insulin as revealed by the glucose-insulin clamp. It therefore seems reasonable to attribute the endocrine metabolic alteration found in Acanthosis Nigricans to a peripheral receptor and/or post receptor alteration rather than central alterations in the beta cells that have yet to be demonstrated. It is concluded that in acanthosis nigricans the peripheral insulin resistance is primarily independent phenomenon and not "parallel" to insulin/insulin feedback.

Acanthosis Nigricans↗

The effect of FK506 on glycemic response as assessed by the hyperglycemic clamp technique.

We studied seven nondiabetic subjects with the autoimmune diseases psoriasis, multiple sclerosis, and primary biliary sclerosis who were to receive FK506 as experimental immunotherapy. All subjects underwent two standard oral glucose tolerance tests and two 180-min hyperglycemic clamps immediately before and 10 weeks after starting FK506. There was no significant difference in weight or HbA1c pre- vs. post-FK506 treatment. FK506 levels were therapeutic and non-toxic (0.1-1 ng/ml) for all subjects studied. Repeated measures analysis of variance for interaction between time and treatment was performed on insulin (after outlier removed) and glucose values from the OGTT. There was neither time-by-treatment interaction, nor a treatment effect (P > 0.1). There were no significant differences in pre- vs. post-FK506 treatment values of plasma glucose during the hyperglycemic clamp mean acute insulin response to glucose (AIRG) 164 +/- 38 pmol/L vs. 148 +/- 46 pmol/L (P > 0.1); mean incremental area under the insulin curve (IAUC) during the first 10 min of the study, 473 +/- 109 pmol/L vs. 443 +/- 146 pmol/L (P > 0.1); total area under the insulin curve (TAUC) during the first 10 min of the study, 786 +/- 152 pmol/L vs. 781 +/- 18 pmol/L (P > 0.1); mean glucose infusion rate (GIR) 37.7 +/- 5.0 mumol/kg/min vs. 33.3 +/- 4.4 mumol/kg/min (P > 0.1); or mean insulin sensitivity index (ISI), 3.05 +/- 0.4 vs. 3.13 +/- 0.5 (P > 0.1). Mean steady-state insulin secretion (SSI) was significantly lower 244 +/- 43 pmol/L vs. 200 +/- 25.2 pmol/L (P = 0.03). Peak first-phase insulin secretion values of 321 +/- 62 pmol/L vs. 263 +/- 57 pmol/L approached significance (P = 0.07). No patient progressed to diabetes during the study. FK506 decreased steady-state insulin secretion during the last 60 min of the clamp, regardless of initial glucose tolerance. Insulin sensitivity and glucose infusion rate did not change in the group as a whole with FK506 treatment.

Blood Glucose↗

Insulin responses to intravenous glucose, intravenous arginine and a hyperglycaemic clamp in ICA-positive subjects with different degrees of glucose tolerance.

The relationship between altered insulin secretion and impaired glucose tolerance was studied in 32 non-obese subjects aged 14-49 years with islet-cell antibodies (ICA) and fasting blood glucose below 7.9 mmol/l, using oral (OGTT) and intravenous (IVGTT) glucose tolerance tests. Glucose tolerance was normal in 19 subjects, impaired (IGT) in 4 and satisfied diabetic criteria in 9. Fifteen of these subjects and 8 ICA-negative controls also underwent a hyperglycaemic clamp (10 mmol/l) and a glucose-potentiated IV arginine bolus. Acute insulin response to IVGTT and insulin and C-peptide responses to the hyperglycaemic clamp and the arginine bolus were dramatically lower (p < 0.001) in diabetic and IGT subjects than in ICA-positive patients with normal glucose tolerance and control subjects. Insulin responses to the three tests were inversely correlated with plasma glucose levels and the area under the curve of OGTT. The correlations between the degree of glucose tolerance and insulin responses to IVGTT, the hyperglycaemic clamp and the arginine bolus were virtually identical. It is concluded that insulin responses to the three stimuli were severely altered in ICA-positive patients with impaired glucose tolerance or asymptomatic diabetes, normal in normotolerant ICA-positive subjects, and correlated with glucose tolerance.

Administration, Oral↗

[Study of electrophysiological properties of large conductance K(+)-channels in outer hair cells from the guinea pig with patch-clamp technique].

The basic electrophysiological characteristics of large conductance K(+)-channels in the basolateral cell membrane of outer hair cells (OHC) from the guinea pig cochlea were studied with patch-clamp technique. Acutely isolated OHC preparations were obtained by enzymatic digestion and excised patches were made in cell-attached configuration to record single-channel currents led by patch-clamp micropipette and fed to patch-clamp amplifier. Data were sampled and analyzed on-line with a computer. The result showed that the typical properties of single channels of large conductance K(+)-channels were: 1. The channel was activated by depolarization of membrane and it showed a marked voltage-dependence. The channel had a unitary conductance of about 133 pS and the channel current reversed at about -30mV under asymmetrical K+ concentration gradient. 2. Tetraethylammonium (TEA, > 10 mmol/L), a potassium channel blocker, dose-dependently abolished the outward current. 3. The open probability (Po) and mean open time of this channel was markedly increased with elevation of cytoplasmic Ca2+ concentration. These results suggest that a population of voltage-dependent and Ca(2+)-activated K+ channel exists in the basolateral membrane of OHC of the guinea pig; it is characterized by a very high unitary conductance and high sensitivity to Ca2+ and is blocked by K+ channel blocker. This work provided valuable basic materials for further study on the regulation and drug influence of the type of single channels.

Animals↗

[Analysis of molecular basis of neuronal properties using the patch-clamp RT-PCR method].

The AMPA-type glutamate receptor mediates fast neurotransmission in most of the excitatory synapses in the brain. Expression studies of the AMPA receptor subunits, GluR1-GluR4, have shown that the functional properties of AMPA receptors, such as rectification and Ca2+ permeability, are determined by their subunit composition. To analyze the molecular basis of functional properties of AMPA receptors at the single neuron level, we have combined the whole-cell patch clamp recording with the reverse transcription-polymerase chain reaction (patch-clamp RT-PCR method). Using this method, we have demonstrated that expression of the GluR2 subunit determines rectification properties and Ca2+ permeability of AMPA receptors in different types of neurons. The patch-clamp RT-PCR technique will be useful for a wide range of physiological studies to elucidate the molecular basis of functional properties at the single cell level.

Animals↗

Clenbuterol-induced insulin resistance in calves measured by hyperinsulinemic, euglycemic clamp technique.

Hyperinsulinemic, euglycemic clamp tests were performed on calves before and after clenbuterol treatment. Clenbuterol was given as 2 intramuscular injections with an interval of about 12 h. The dose used was 1 microgram/kg b.w. The treatment resulted in increased plasma levels of insulin and glucose. The results of the clamp tests showed that clenbuterol induced a transient decrease in insulin sensitivity. Both insulin mediated glucose disposal (M), expressed as mumol/kg live b.w./min. and the M/I-index (M divided by the average insulin concentration at steady state) were significantly reduced after treatment. The effect of clenbuterol on carbohydrate metabolism seemed to be rather short-lived, since significant changes occurred only in animals treated 5-6 h prior to the test. According to the literature, the metabolic effects of clenbuterol have been studied only after the high doses used for growth promoting purposes. The results from the present study showed that similar changes occur also after doses at the therapeutic level. The hyperinsulinemic, euglycemic clamp test was considered to be a valuable tool for the study of insulin sensitivity in cattle.

Animals↗

Myocardial preservation with intermittent aortic cross-clamping technique during coronary artery bypass grafting.

OBJECTIVE: To evaluate the safety and efficacy of intermittent aortic cross-clamping technique for myocardial preservation during coronary artery bypass grafting and to compare the results with other published series using the same technique. DESIGN: An institution-based analytical study. PLACE AND DURATION OF STUDY: Study was carried out from July 1995 to December 1997 at Punjab Institute of Cardiology, Lahore. PATIENTS AND METHODS: The data of 100 consecutive patients were analyzed who had coronary artery bypass grafting without cardioplegia using intermittent aortic cross-clamping. All the patients who had emergency or concomitant major surgery, reoperations, and aortic disease with calcification were excluded from this study. RESULTS: Despite minor complications there was no mortality, no rhythm disturbance or perioperative infarction. Inotropic support was needed in 8% of patients. CONCLUSION: Our results compared favourably with other published series using the same technique. These results provide re-assurance that intermittent aortic cross-clamping provides excellent myocardial protection and operative conditions for primary coronary artery bypass. It also provides a simple and safe option for myocardial protection during coronary artery bypass grafting.

Aorta↗

Effect of clamp versus anastomotic-induced ischemia on critical ischemic time and survival of rat epigastric fasciocutaneous flap.

BACKGROUND: There are many models used to explore ischemic-related phenomena. The rat epigastric fasciocutaneous flap model is the one most commonly used. Critical ischemic time is the maximum ischemic insult that tissue can undergo and still remain viable. Experimentally, ischemia is induced either by clamping the vascular pedicle or by dividing the pedicle then performing microvascular arterial and venous anastomosis. We sought to determine what effect the different methods of inducing ischemia have on the critical primary ischemic time and, thus, flap survival. METHODS: A right 3 cm x 6 cm groin flap based on the inferior epigastric vessels was raised in each rat. Ischemic times of 4, 6, 8, or 10 hours were induced either by placing temporary occlusion clamps on each vessel of the vascular pedicle (island pedicle group) or by ligation and division of the pedicle with subsequent microvascular anastomosis (free flap group). Survival was assessed at 7 days. RESULTS: The primary ischemic time at which one half of free flaps are predicted to die was calculated to be 7.60 hours, compared with 6.09 hours for the island pedicle flaps (p<.05). CONCLUSIONS: Fasciocutaneous flaps undergoing ligation and anastomosis are more resistant to ischemia than are those undergoing clamping of the pedicle. Possible etiologic factors responsible for this experimental finding are discussed.

Anastomosis, Surgical↗

Intermittent vascular exclusion of the liver (without vena cava clamping) during major hepatectomy.

Intermittent vascular exclusion of the liver (IVEL) combines clamping of the hepatic pedicle with clamping of the main hepatic veins without interruption of caval flow. In this retrospective study, eight cases of total IVEL and eight of partial IVEL were analysed (involving only the middle and left hepatic veins) during major hepatectomy for malignant tumours. Liver parenchyma was pathological in nine cases. IVEL was feasible in 16 of the 18 attempts and was efficient in reducing bleeding during hepatectomy in 15 cases. Mean duration of IVEL was 60.2 (range 37-140) min, mean blood loss was 1230 (range 300-2800) ml and there were no postoperative complications related to the procedure. The major advantages of this technique of liver vascular exclusion (good tolerance and possibility of long duration) merit its inclusion in the list of different clamping techniques available for use during hepatectomy.

Adult↗

LigaSure versus clamp-and-tie thyroidectomy for benign nodular disease.

BACKGROUND: LigaSure is an alternative bipolar diathermy system (BDS) combining vessel sealing with reduced thermal spread, which has been successfully used in many abdominal operations; however, there is a little experience in open thyroidectomy. This study compares the efficacy and the advantages of the LigaSure BDS when used for total thyroidectomy for benign thyroid disease with the conventional clamp-and-tie technique. METHODS: Between May 1998 and October 2002, 517 patients underwent total thyroidectomy for benign thyroid multinodular goiter. Among them, from May 1998 until May 2000, 247 patients (group I) were operated on with the conventional clamp-and-tie technique, whereas from May 2000 until October 2003, 270 patients (group II) underwent total thyroidectomy for benign multinodular goiter with LigaSure BDS through a 4-cm transverse suprasternal incision. Demographics, pathologic characteristics, gland mass, operative time, blood loss, and complications were assessed. RESULTS: There were no intraoperative complications. Thyroid mass was similar in both groups, but the operative time was shorter in group II than in group I (mean +/- standard deviation, 71 +/- 14 minutes vs 86 +/- 22 minutes; p < .01). Intraoperative total blood loss was similar between the two groups, but postoperative drain volume was less in group II than in group I (21 +/- 15 mL; p < .01). Major post-thyroidectomy complications (ie, laryngeal nerve palsy, hematoma, and hypocalcemia) occurred less frequently in the LigaSure group than in the clamp-and-tie group (0.7%, 0.4%, 1.1% vs 4%, 2%, 4.8%, respectively; p < .05). The mean +/- standard deviation postoperative hospital stay was significantly less for the patients in group II than for those in group I (2.3 +/- 1.7 days vs 2.8 +/- 1.3 days; p < .05). CONCLUSIONS: The use of the LigaSure vessel sealer for thyroid surgery is an effective and safe alternative that reduces the overall operative time and could be successfully applied through a narrow surgical incision.

Adult↗

Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells.

Changes in the cytoplasmic free calcium concentration ([Ca2+]i) in pancreatic B-cells play an important role in the regulation of insulin secretion. We have recorded [Ca2+]i transients evoked by single action potentials and voltage-clamp Ca2+ currents in isolated B-cells by the combination of dual wavelength emission spectrofluorimetry and the patch-clamp technique. A 500-1000 ms depolarization of the B-cell from -70 to -10 mV evoked a transient rise in [Ca2+]i from a resting value of approximately 100 nM to a peak concentration of 550 nM. Similar [Ca2+]i changes were associated with individual action potentials. The depolarization-induced [Ca2+]i transients were abolished by application of nifedipine, a blocker of L-type Ca2+ channels, indicating their dependence on influx of extracellular Ca2+. Following the voltage-clamp step, [Ca2+]i decayed with a time constant of approximately 2.5 s and summation of [Ca2+]i occurred whenever depolarizations were applied with an interval of less than 2 s. The importance of the Na(+)-Ca2+ exchange for B-cell [Ca2+]i maintenance was evidenced by the demonstration that basal [Ca2+]i rose to 200 nM and the magnitude of the depolarization-evoked [Ca2+]i transients was markedly increased after omission of extracellular Na+. However, the rate by which [Ca2+]i returned to basal was not affected, suggesting the existence of additional [Ca2+]i buffering processes.

Animals↗