Clamp Period Research Articles

Novel PEGylated basal insulin LY2605541 has a preferential hepatic effect on glucose metabolism.

The impact of the novel basal insulin LY2605541 (LY) on hepatic and nonhepatic glucose uptake (non-HGU) was evaluated. Conscious dogs underwent euglycemic clamps with tracer and hepatic balance measurements. Clamp period infusions were peripheral venous regular insulin (0.1 nmol ⋅ kg−1 ⋅ h−1 [control], n = 6) or LY (bolus [nmol/kg], continuous [nmol ⋅ kg−1 ⋅ h−1]: 0.5, 0.5 [n = 6]; 0.375, 0.375 [n = 5]; 0.25, 0.25 [n = 4]), somatostatin, and glucose, as well as intraportal glucagon (basal). During the clamp, the dogs switched from net hepatic glucose output to uptake (rates reached 2.1 ± 1.2, 0.9 ± 2.1, 8.6 ± 2.3, and 6.0 ± 1.1 µmol ⋅ kg−1 ⋅ min−1 within 5 h in control, LY0.25, LY0.375, and LY0.5, respectively). Non-HGU in LY increased less than in control; the ratio of change from basal in non-HGU to change in net hepatic glucose balance, calculated when glucose infusion rates (GIRs) were ~20 µmol ⋅ kg-1 ⋅ min−1 in all groups, was higher in control (1.17 ± 0.38) versus LY0.25 (0.39 ± 0.33), LY0.375 (−0.01 ± 0.13), and LY0.5 (−0.09 ± 0.07). Likewise, the change from baseline in glucose Rd-to-Ra ratio was greatest in control (1.4 ± 0.3 vs. 0.6 ± 0.4, 0.5 ± 0.2, and 0.6 ± 0.2 in LY0.25, LY0.375, and LY0.5, respectively). In contrast to exogenously administered human insulin, LY demonstrated preferential hepatic effects, similar to endogenously secreted insulin. Therefore, the analog might reduce complications associated with current insulin therapy.

Apolipoprotein A-IV Reduces Hepatic Gluconeogenesis through Nuclear Receptor NR1D1

We showed recently that apoA-IV improves glucose homeostasis by enhancing pancreatic insulin secretion in the presence of elevated levels of glucose. Therefore, examined whether apolipoprotein A-IV (apoA-IV) also regulates glucose metabolism through the suppression of hepatic gluconeogenesis. The ability of apoA-IV to lower gluconeogenic gene expression and glucose production was measured in apoA-IV(-/-) and wild-type mice and primary mouse hepatocytes. The transcriptional regulation of Glc-6-Pase and phosphoenolpyruvate carboxykinase (PEPCK) by apoA-IV was determined by luciferase activity assay. Using bacterial two-hybrid library screening, NR1D1 was identified as a putative apoA-IV-binding protein. The colocalization and interaction between apoA-IV and NR1D1 were confirmed by immunofluorescence, in situ proximity ligation assay, and coimmunoprecipitation. Enhanced recruitment of NR1D1 and activity by apoA-IV to Glc-6-Pase promoter was verified with ChIP and a luciferase assay. Down-regulation of apoA-IV on gluconeogenic genes is mediated through NR1D1, as illustrated in cells with NR1D1 knockdown by siRNA. We found that apoA-IV suppresses the expression of PEPCK and Glc-6-Pase in hepatocytes; decreases hepatic glucose production; binds and activates nuclear receptor NR1D1 and stimulates NR1D1 expression; in cells lacking NR1D1, fails to inhibit PEPCK and Glc-6-Pase gene expression; and stimulates higher hepatic glucose production and higher gluconeogenic gene expression in apoA-IV(-/-) mice. We conclude that apoA-IV inhibits hepatic gluconeogenesis by decreasing Glc-6-Pase and PEPCK gene expression through NR1D1. This novel regulatory pathway connects an influx of energy as fat from the gut (and subsequent apoA-IV secretion) with inhibition of hepatic glucose production.

A comparison of the pharmacodynamic profiles of insulin detemir and insulin glargine: A single dose clamp study in people with type 2 diabetes

AimThe pharmacodynamic properties of a single dose of 0.5U/kg insulin detemir and insulin glargine were compared during two 24-h isoglycaemic clamps, one week apart. MethodsThe order of treatments was randomised. At approximately 0830h, persons with T2DM received subcutaneous administration of a 0.5U/kg dose of either insulin detemir or insulin glargine into the anterior abdominal wall. Plasma glucose was measured at 10-min intervals throughout the 24-h clamp period and isoglycaemia was maintained by variable infusion of 20% glucose. Glucose infusion rates (GIR) and plasma C-peptide were determined throughout each 24-h period. ResultsEleven persons with type 2 diabetes (8 male) with mean (SD) age 58.5years (8.5), BMI 30.8kg/m2 (2.8) and HbA1c 7.5% (0.6) were studied. Plasma glucose remained constant during the clamp (CV: insulin detemir 3.7%; insulin glargine 3.8%). Following injection of insulin detemir, GIR increased, reaching a mean peak of 2.29mg/kg/min (95% CI 1.64, 2.94) at 11.6h (range 8.9 to 14.3) compared to 1.71mg/kg/min (95% CI 1.4, 2.0) at 10.2h (8.1 to 12.3) for insulin glargine (P=0.025 for GIRmax). Plasma C-peptide decreased during the study period, remaining significantly lower than the fasting level at the study end after both analogues, insulin detemir (P=0.01) and insulin glargine (P=0.02). ConclusionIn persons with T2DM, no difference in duration of action following a single subcutaneous dose of insulin detemir and insulin glargine could be observed. Insulin detemir showed greater between subject variability and achieved a significantly higher maximum GIR than insulin glargine.

Intermittent cold-blood vs. warm-blood cardioplegia – a comparative study on myocardial acidosis during coronary bypass surgery

The aim of the study was to assess the impact of the intermittent cold-blood cardioplegia (ICBC) on myocardial acidosis in comparison to the warm-blood cardioplegic administration (IWBC) in patients undergoing coronary bypass surgery. In 15 patients undergoing elective bypass surgery with ICBC for myocardial protection, metabolic changes of global ischemia indicators with lactate and pH values, measured simultaneously in the arterial and coronary sinus blood, were analyzed and compared with those observed when warm-blood method was used. For comparison, the analysis of variance with repeated measurements including tests for a cross-over effect of group and time was used. In patients with ICBC compared to IWBC, the lactate production as well as degree of acidosis were generally lower with a significant group effect for both, lactate and pH values measured at the end of reperfusions. Our results suggest that ICBC has an inhibiting effect on potentially progressive myocardial acidosis during cross clamp period.

Evidence for a causal relationship between hyperkalaemia and axonal dysfunction in end-stage kidney disease

ObjectivePotassium (K+) has been implicated as a factor in the development of uraemic neuropathy. This study was undertaken to investigate whether hyperkalaemia plays a causal role in axonal dysfunction in end-stage kidney disease (ESKD). MethodsMedian motor nerve excitability studies were undertaken in four haemodialysis patients during a modified dialysis session. The serum K+ level was “clamped” (fixed) for the first 3h of dialysis, whilst allowing all other solutes to be removed, this was followed by dialysis against low dialysate K+ for a further 4h. Blood chemistry and nerve excitability studies were undertaken prior to, during and following dialysis. Results were compared to results from the same patients during routine dialysis sessions. ResultsAll patients demonstrated significant nerve excitability abnormalities reflective of nerve membrane depolarization in pre-dialysis recordings (p<0.01). After the 3h clamp period, serum K+ remained elevated (5.0mmol/L) and nerve excitability remained highly abnormal, despite the significant clearance of other uraemic toxins. In contrast, studies undertaken during routine dialysis sessions demonstrated significant improvement in both serum K+ and nerve function after 3h. ConclusionsThe current study has established a causal relationship between serum K+ and axonal membrane depolarization in haemodialysis patients. SignificanceFrom a clinical perspective, strict K+ control may help improve nerve function in ESKD.

Direct Effects of Locally Administered Lipopolysaccharide on Glucose, Lipid, and Protein Metabolism in the Placebo-Controlled, Bilaterally Infused Human Leg

Accumulating evidence suggests that chronic exposure to lipopolysaccharide (LPS, endotoxin) may create a constant low-grade inflammation, leading to insulin resistance and diabetes. All previous human studies assessing the metabolic actions of LPS have used systemic administration, making discrimination between direct and indirect effects impossible. We sought to define the direct, placebo-controlled effects of LPS on insulin resistance and protein and lipid metabolism in the infused human leg without systemic interference from cytokines and stress hormones. This was a randomized, placebo-controlled, single-blinded study. We studied 8 healthy volunteers with bilateral femoral vein and artery catheters during a 3-hour basal and 3-hour hyperinsulinemic-euglycemic clamp period with bilateral muscle biopsies in each period during infusion with saline and LPS. Overall, LPS perfusion significantly decreased leg glucose uptake, and during the clamp LPS decreased glucose arteriovenous differences (0.65 ± 0.07 mmol/L vs 0.73 ± 0.08 mmol/L). Net palmitate release was increased by LPS, and secondary post hoc testing indicated increased palmitate isotopic dilution, although primary ANOVA tests did not reveal significant dilution. Leg blood flows, phenylalanine, lactate kinetics, cytokines, and intramyocellular insulin signaling were not affected by LPS. LPS thus directly inhibits insulin-stimulated glucose uptake and increases palmitate release in the perfused human leg without detectable effects on amino acid metabolism. These data strongly suggest that the primary metabolic effect of LPS is increased lipolysis and muscle insulin resistance, which, together with secondary insulin resistance, caused by systemic cytokine and stress hormone release may lead to overt glucose intolerance and diabetes.

Hepatic glucose metabolism in late pregnancy: normal versus high-fat and -fructose diet.

Net hepatic glucose uptake (NHGU) is an important contributor to postprandial glycemic control. We hypothesized that NHGU is reduced during normal pregnancy and in a pregnant diet-induced model of impaired glucose intolerance/gestational diabetes mellitus (IGT/GDM). Dogs (n = 7 per group) that were nonpregnant (N), normal pregnant (P), or pregnant with IGT/GDM (pregnant dogs fed a high-fat and -fructose diet [P-HFF]) underwent a hyperinsulinemic-hyperglycemic clamp with intraportal glucose infusion. Clamp period insulin, glucagon, and glucose concentrations and hepatic glucose loads did not differ among groups. The N dogs reached near-maximal NHGU rates within 30 min; mean ± SEM NHGU was 105 ± 9 µmol⋅100 g liver−1⋅min−1. The P and P-HFF dogs reached maximal NHGU in 90–120 min; their NHGU was blunted (68 ± 9 and 16 ± 17 µmol⋅100 g liver−1⋅min−1, respectively). Hepatic glycogen synthesis was reduced 20% in P versus N and 40% in P-HFF versus P dogs. This was associated with a reduction (>70%) in glycogen synthase activity in P-HFF versus P and increased glycogen phosphorylase (GP) activity in both P (1.7-fold greater than N) and P-HFF (1.8-fold greater than P) dogs. Thus, NHGU under conditions mimicking the postprandial state is delayed and suppressed in normal pregnancy, with concomitant reduction in glycogen storage. NHGU is further blunted in IGT/GDM. This likely contributes to postprandial hyperglycemia during pregnancy, with potential adverse outcomes for the fetus and mother.

Insulin at normal physiological levels does not prolong QTc interval in thorough QT studies performed in healthy volunteers

Food is known to shorten the QT(c) (QT(c)I and QT(c)F) interval and has been proposed as a non-pharmacological method of confirming assay sensitivity in thorough QT (TQT) studies and early phase studies in medicines research. Intake of food leads to a rise in insulin levels together with the release of C-peptide in equimolar amounts. However, it has been reported that euglycaemic hyperinsulinemia can prolong the QT(c) interval, whilst C-peptide has been reported to shorten the QT(c) interval. Currently there is limited information on the effects of insulin and C-peptide on the electrocardiogram (ECG). This study was performed to assess the effect of insulin, glucose and C-peptide on the QT(c) interval under the rigorous conditions of a TQT study. Thirty-two healthy male and female, Caucasian and Japanese subjects were randomized to receive six treatments: (1) placebo, (2) insulin euglycaemic clamp, (3) carbohydrate rich 'continental' breakfast, (4) calorie reduced 'American' FDA breakfast, (5) moxifloxacin without food, and (6) moxifloxacin with food. Measurements of ECG intervals were performed automatically with subsequent adjudication in accordance with the ICH E14 guideline and relevant amendments. No effect was observed on QT(c)F during the insulin euglycaemic clamp period (maximal shortening of QT(c) F by 2.6 ms, not significant). Following ingestion of a carbohydrate rich 'continental' breakfast or a calorie reduced 'American' FDA standard breakfast, a rapid increase in insulin and C-peptide concentrations were observed. Insulin concentrations showed a peak response after the 'continental' breakfast observed at the first measurement time point (0.25 h) followed by a rapid decline. Insulin concentrations observed with the 'American' breakfast were approximately half of those seen with the 'continental' breakfast and showed a similar pattern. C-peptide concentrations showed a peak response at the first measurement time point (0.25 h) with a steady return to baseline at the 6 h time point. The response to the 'continental' breakfast was approximately double that of the 'American' FDA breakfast. A rapid onset of the effect on QT(c) F was observed with the 'continental' breakfast with shortening by >5 ms in the time interval from 1 to 4 h. After the 'American' FDA breakfast, a similar but smaller effect was seen. The findings of this study demonstrate that there was no change in QT(c) during the euglycaemic clamp. Given that insulin was raised to physiological concentrations comparable with those seen after a meal, whilst the release of C-peptide was suppressed, insulin appears to have no effect on the QT(c) interval in either direction. The results suggest a relationship exists between the shortening of QT(c) and C-peptide concentrations and indicate that glucose may have a QT(c) prolonging effect, which will require further research.

Increased Hepatic Glucose Production in Fetal Sheep With Intrauterine Growth Restriction Is Not Suppressed by Insulin

Intrauterine growth restriction (IUGR) increases the risk for metabolic disease and diabetes, although the developmental origins of this remain unclear. We measured glucose metabolism during basal and insulin clamp periods in a fetal sheep model of placental insufficiency and IUGR. Compared with control fetuses (CON), fetuses with IUGR had increased basal glucose production rates and hepatic PEPCK and glucose-6-phosphatase expression, which were not suppressed by insulin. In contrast, insulin significantly increased peripheral glucose utilization rates in CON and IUGR fetuses. Insulin robustly activated AKT, GSK3β, and forkhead box class O (FOXO)1 in CON and IUGR fetal livers. IUGR livers, however, had increased basal FOXO1 phosphorylation, nuclear FOXO1 expression, and Jun NH2-terminal kinase activation during hyperinsulinemia. Expression of peroxisome proliferator–activated receptor γ coactivator 1α and hepatocyte nuclear factor-4α were increased in IUGR livers during basal and insulin periods. Cortisol and norepinephrine concentrations were positively correlated with glucose production rates. Isolated IUGR hepatocytes maintained increased glucose production in culture. In summary, fetal sheep with IUGR have increased hepatic glucose production, which is not suppressed by insulin despite insulin sensitivity for peripheral glucose utilization. These data are consistent with a novel mechanism involving persistent transcriptional activation in the liver that seems to be unique in the fetus with IUGR.

Asystole to cross-clamp period predicts development of biliary complications in liver transplantation using donation after cardiac death donors

This study sought to determine the procurement factors that lead to development of intrahepatic bile duct strictures (ITBS) and overall biliary complications in recipients of donation after cardiac death (DCD) liver grafts. Detailed information for different time points during procurement (withdrawal of support; SBP < 50 mmHg; oxygen saturation <30%; mandatory wait period; asystole; incision; aortic cross clamp) and their association with the development of ITBS and overall biliary complications were examined using logistic regression. Two hundred and fifteen liver transplants using DCD donors were performed between 1998 and 2010 at Mayo Clinic Florida. Of all the time periods during procurement, only asystole-cross clamp period was significantly different between patients with ITBS versus no ITBS (P = 0.048) and between the patients who had overall biliary complications versus no biliary complications (P = 0.047). On multivariate analysis, only asystole-cross clamp period was significant predictor for development of ITBS (P = 0.015) and development of overall biliary complications (P = 0.029). Hemodynamic changes in the agonal period did not emerge as risk factors. The results of the study raise the possibility of utilizing asystole-cross-clamp period in place of or in conjunction with donor warm ischemia time in determining viability or quality of liver grafts.

Technical notes: a self-designed, simple, secure, and safe six-loop intracorporeal Pringle’s maneuver for laparoscopic liver resection

The aim of this study is to design a simple, secure, and safe technique of intracorporeal Pringle's maneuver to facilitate safer laparoscopic liver resection. A self-designed six-loop catheter was made using 20-French T-tube and 10-French nelaton urethral catheter. The cross head and stem of the T-tube were trimmed to 1 cm, respectively. The nelaton was shortened to 12-cm-long tube from the round tip, and the cut end was inserted and sutured to the stem of the T-tube. After establishment of pneumoperitoneum, the T-tube with nelaton was placed into the abdomen. The round end of the nelaton was inserted into the lesser sac and pulled through the foramen of Winslow, and the end of nelaton was then inserted into one end of the T-tube and pulled through the other end, forming a six-loop. The nelaton was pulled to occlude the hepatic inflow and temporarily fixed with 1-0 Vicryl on a curved round needle on the other end of the T-tube. The protocol of Pringle's maneuver was 15-min clamp and 5-min release periods. The liver parenchymal transection was performed using Harmonic scalpel. From November 2009 to August 2011, 20 patients received laparoscopic liver resection using the six-loop Pringle's maneuver. During operation, 17 patients were positioned supine, 2 patients in left decubitus, and 1 patient in supine followed by left decubitus position. There were 9 anatomical resections and 11 nonanatomical resections (18 patients for single lesion, 1 for two lesions, and 1 for three lesions). The average times of liver resection and Pringle's maneuver were 33.1 and 36.2 min, respectively. Mean blood loss was 102.5 ml. The postoperative course was uneventful, and average hospital stay was 4.4 days. Our self-designed six-loop intracorporeal Pringle's maneuver can facilitate safer laparoscopic liver resection.

Response of the human kidney to clamp ischemia.

365 Background: Structural changes in tubule cells during clamp ischemia are well characterized for animal models, but their timing and extent in the human kidney has not been established and may differ significantly. To better define the human response, we biopsied uninvolved areas of kidney in patients undergoing open partial nephrectomy for renal masses. Methods: Biopsies of 40 patients undergoing PN were obtained at specified time intervals: before renal artery clamping, then during periods ranging from 15 to 60 min. of warm and cold ischemia (80% &gt; 30 min.), and then after 5 minutes of reflow. These biopsies were assessed for ultrastructure (N=39) and for immunofluorescence and rhodamine phalloidin staining (N=22). Results: During the clamp period, apical membrane structure was remarkably well preserved with only patchy brush border clubbing, fragmentation, desquamation and blebbing and not in all patients. Mitochondria developed progressive swelling, which paradoxically was more prominent in distal than proximal tubule cells. This resolved during the 5 minutes of reflow in most cells in most patients, but persistence of swelling and development of matrix condensation occurred occasionally. Using a composite 0-5 scale covering the full spectrum of ultrastructural changes, average scores were: Preclamp 1.02±0.07, End clamp 2.18±0.07, Post clamp 1.86±0.09. Consistent with the ultrastructure, staining for F-actin with rhodamine phalloidin was well preserved. Immunsotaining for phosphotyrosine, which reflects cellular ATP content was decreased in 68.4% of the clamp biopsises and 52.6% of the postclamp biopsies with larger changes in proximal tubules, however B1 integrin was decreased in only one post clamp biopsy. ICAM-1 expression in peritubular capillaries was increased in 46.7% of the clamp biopsies and 66.7% of post clamp biopsies. None of the patients developed acute kidney injury. Conclusions: These data provide the first detailed analysis of the structural response of the human kidney to clamp ischemia and document many of the expected structural alterations based on prior animal work, but indicate a greater than expected resistance to injury in this commonly used clinical application of clamp ischemia.

Fasting, But Not Exercise, Increases Adipose Triglyceride Lipase (ATGL) Protein and Reduces G(0)/G(1) Switch Gene 2 (G0S2) Protein and mRNA Content in Human Adipose Tissue

Fasting and exercise are characterized by increased lipolysis, but the underlying mechanisms are not fully understood. The study was designed to test whether fasting and exercise affect mRNA and protein levels of adipose triglyceride lipase (ATGL) and G(0)/G(1) switch gene 2 (G0S2), a recently discovered ATGL inhibitor, in humans. We studied eight healthy men (age, 25.5 ± 4.3 yr) for 6 h (a 4-h basal and a 2-h clamp period) on three occasions in a randomized crossover design: 1) in the basal state and after; 2) 72-h fasting; and 3) 1-h exercise (65% VO(2max)). Subcutaneous abdominal adipose tissue (AT) biopsies were taken at t = 30 and 270 min. The study was conducted at a university hospital research unit. Circulating free fatty acids and GH were increased, and C-peptide was decreased by both fasting and exercise. During fasting, insulin failed to suppress free fatty acid levels, suggesting AT insulin resistance. ATGL protein was increased 44% (P < 0.001), and G0S2 mRNA and protein were decreased 56% (P = 0.02) and 54% (P = 0.01), respectively, after fasting, but both ATGL and G0S2 were unaffected by exercise. Protein levels of hormone-sensitive lipase and comparative gene identification-58 were unaffected throughout. We found increased AT content of ATGL and decreased protein and mRNA content of the ATGL inhibitor G0S2, suggesting increased ATGL activity during fasting, but not after short-term exercise. These findings are compatible with the notion that the ATGL-G0S2 complex is an important long-term regulator of lipolysis under physiological conditions such as fasting in humans.

Whole Body Deletion of AMP-activated Protein Kinase β2 Reduces Muscle AMPK Activity and Exercise Capacity

AMP-activated protein kinase (AMPK) β subunits (β1 and β2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK β2 (β2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that β2 KO mice are viable and breed normally. β2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the β1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in β2 KO mice. During treadmill running β2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of β2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet β2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK β2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.

Similarity of pharmacodynamic effects of a single injection of insulin glargine, insulin detemir and NPH insulin on glucose metabolism assessed by 24‐h euglycaemic clamp studies in healthy humans

Two long-acting insulin analogues, insulin glargine and insulin detemir, have been developed as alternatives to neutral protamine Hagedorn (NPH) insulin, which has been the preferred basal insulin preparation for decades. The aim was to directly compare the pharmacodynamic properties of the long-acting insulin analogues and NPH insulin after a single subcutaneous injection. The study was conducted as a double-blind, controlled, three-arm, crossover study including 10 healthy lean male volunteers. On three different occasions, each subject was challenged with 0.4 U kg(-1) of either insulin glargine, insulin detemir or NPH insulin. Plasma glucose was maintained at 0.3 mmol l(-1) below fasting level by glucose clamping for 24 h. C-peptide, insulin, free fatty acids (FFAs) and counter regulatory hormones were measured throughout the clamp period, whereas endogenous glucose release (EGR) was assessed by isotope dilution technique (3-(3)H-glucose). The mean glucose infusion rate (GIR)-time profiles revealed no significant differences between the three preparations in the primary endpoints: Maximal GIR of approximately 3.4 mg kg(-1) min(-1) (P = 0.68), time to maximal GIR of approximately 10 h (TR(max)) (P = 0.35) and area under the GIR curve (GIR(AUC)) (P = 0.81). Compared with the other insulin preparations, EGR (see above)was lower for insulin detemir at the beginning of the clamp period (330-360 min) (P = 0.007) while GIR was lower (P = 0.005) and FFA concentrations were higher (P = 0.005) during the last 4 h of the clamp. In this experimental design, only minor pharmacodynamic differences were demonstrated between insulin detemir, insulin glargine and NPH insulin.

Exercise and Fasting Activate Growth Hormone-Dependent Myocellular Signal Transducer and Activator of Transcription-5b Phosphorylation and Insulin-Like Growth Factor-I Messenger Ribonucleic Acid Expression in Humans

GH secretion is increased by stress, such as fasting and exercise. Our study was designed to test the hypothesis that fasting and exercise increase GH levels, GH-dependent signal transducer and activator of transcription (STAT)-5b phosphorylation, and IGF-I mRNA levels in human skeletal muscle. Eight healthy men (25.5 +/- 4.3 yr) were studied for 6 h (a 4 h basal and a 2 h clamp period) in the basal state and after 72 h fasting and a 1-h ergometer cycling at 65% maximal oxygen uptake. Muscle biopsies were taken at t = 30 and 270 min. The study was conducted at a university clinical research unit. During exercise, GH concentrations rapidly increased to greater than 10 ng/ml (P < 0.001). Thirty minutes after exercise, we observed a 4-fold increase in signal transducer and activator of transcription (STAT)-5 phosphorylation (P < 0.001) followed by an increase in IGF-I mRNA after 270 min (P = 0.026). During fasting, more sporadic GH bursts occurred, leading to an overall 3-fold increase in GH area under the curve(t=0-270) (P < 0.001). Similarly, STAT5 patterns were less consistent, with a tendency toward increased phosphorylation (P = 0.067, area under the curve(t=0-270)), whereas IGF-I mRNAs were persistently increased (P < 0.01). Our data show that myocellular GH signaling is stimulated after exercise and fasting in terms of increased STAT5 phosphorylation and/or IGF-I gene expression. This suggests that exercise with brief, well-defined GH peaks leads to distinct STAT5 phosphorylation and subsequent IGF-I gene expression, whereas fasting induces more sporadic GH bursts and less distinct but more persistent activation of the GH signal.

Salt sensitivity and insulin resistance in normotensives.

Among hypertensive patients salt sensitivity and insulin resistance are commonly observed together. We investigated if a causal relationship already exists in young normotensive adults. With a standardized dietary regimen we determined salt sensitivity in 35 male volunteers by measuring diastolic blood pressure (24-hours-RR-recording). Insulin resistance was tested using hyperinsulinaemic-euglycaemic-clamp-technique by de Fronzo after a freely chosen diet, after 7 days of salt loading (260 mmol/d) and after 7 days of salt restriction (60 mmol/d). Data from euglycaemic-clamp technique were available from 27 subjects. 18 of them (67%) could be characterized as salt resistant; 9 persons (33%) were salt sensitive. Glucose infusion rate, mean glucose and insulin concentrations were measured in plasma, metabolic clearance rate (MCR) and indices of insulin sensitivity (ISI) were calculated. The results of MCR and ISI show large interindividual variances. There were no differences between the salt sensitive and salt resistant group regarding the mean insulin concentrations and also the mean glucose uptake in the steady state clamp period and also the calculated MCR and ISI. Comparing the periods of different salt intake, there were no differences between salt loading and salt restriction. Whereas salt sensitivity can already be shown in the normotensive state, with this experimental design a changed insulin sensitivity is not detectable. This supports the idea, that insulin resistance is not causally linked with salt sensitivity. It may be a secondary phenomenon of salt induced hypertension.

Blocking NMDA receptor signaling does not decrease hormonal counterregulation to hypoglycemia in humans

In animals, blocking of glutamate signaling at the N-methyl-D-aspartate (NMDA) receptor reduces the neuroendocrine counterregulation to hypoglycemia. Hence, it has been proposed that increased excitatory glutamatergic input to the hypothalamus signals enforced central nervous energy demand under conditions of reduced supply. We examined the effect of the NMDA receptor antagonist memantine on hypoglycemia counterregulation in healthy humans. Hypoglycemic clamp experiments were performed in 10 healthy men after oral administration of 20 mg memantine and placebo. Counterregulatory hormones were measured during baseline and a clamp period of 120 min with hypoglycemia of 2.4 mmol/l lasting for 50 min. In addition, symptoms related to glycemic changes were assessed. Unexpectedly, the counterregulatory responses to hypoglycemia of adrenocorticotropin, cortisol and epinephrine were not decreased but tended to be increased by memantine, while norepinephrine and growth hormone were not affected. Glucagon levels were increased by memantine treatment during baseline and throughout the hypoglycemic period. After memantine administration, subjects also experienced more neuroglycopenic symptoms during hypoglycemia, whereas differences in autonomic symptoms did not reach significance. Contrasting with findings in animals, blocking the NMDA receptor does not decrease the counterregulatory responses to hypoglycemia in humans. Our data do not support the view that in humans, enhanced glutamate signaling during hypoglycemia supports the satisfaction of increased central nervous energy demands by enforcing hormonal counterregulation.

Reduction of food intake by insulin detemir in comparison to regular human insulin

Systemic insulin is considered to serve as a major negative feedback signal in the central nervous regulation of food intake. Accordingly, euglycemic infusion of regular human insulin (RI) has been shown to reduce hunger in a dose dependent fashion. Due to its increased lipophility, the long-acting insulin analogue insulin detemir (DI) might cross the blood-brain barrier faster and in higher quantities than RI and exceed stronger anorexigenic effects. To test this hypothesis, we measured food intake after DI and RI administration. 15 healthy, normal-weight men were examined in two hyperinsulinemic euglycemic clamp experiments taking place in the morning after an overnight fast. In both conditions, an insulin bolus injection (DI, 90 mU/kg body weight; RI, 17.75 mU/kg) was followed by a steady 90min insulin infusion (2.0 vs. 1.0 mU/kg/min). To compensate for the relatively slower onset of the action of DI, a higher dosage was chosen to induce comparable peripheral effects of both compounds. Mean blood glucose levels during the clamp period were likewise comparable between conditions (p<0.384). Twenty minutes after infusion, subjects were allowed to eat ad libitum from a standardized breakfast buffet. ID reduced total food intake in comparison to RI (1257±82 vs. 1560±139kcal, p<0.04). This effect was also observed for the intake of protein (p=0.004), tended to be observed for the intake of carbohydrates (p=0.063) but not of fat (p=0.201). Although systemic contributions to the observed effects cannot be ruled out, our data suggest that compared to regular human insulin, insulin detemir induces stronger effects on brain networks that control food intake. They further support the notion of insulin as an anorexigenic feedback signal in the central nervous regulation of energy homeostasis.

Combined PET‐Stable Isotope Measurements on Muscle and Whole Body Glucose Metabolism with Insulin Clamp

Positron Emission Tomography (PET) identifies the altered metabolism in tumor versus normal tissues. We applied this technique to non-invasively quantify substrate metabolism in muscle tissues (MT) and whole body (WB) by combing PET-stable isotope technique. Four healthy volunteers were studied for glucose metabolism (GM). Each subject underwent two experiments: that under fasting state and that including euglycemic insulin clamp (EGIC) period. PET studies were conducted using 18F-deoxyglucose (18FDG) tracer in these conditions on thigh MT. [6,6, 2H2]glucose was infused or WB GM. Results and Conclusion: PET provided a unique quantitative information on GM in different groups of MT on the same thigh (Table), MT contributes to 63 ±9 % of WB GM in vivo; EGIC caused increment of GM to 9.6 ± 2.7 times in MT but 4.1 ± 0.3 times in WB, hence GM in MT is more sensitively regulated by insulin than other tissues.