Euglycaemic glucose clamp: what it can and cannot do, and how to do it.

The hyperinsulinaemic-euglycaemic glucose clamp has always been regarded as the "gold standard" for the assessment of pharmacodynamic (PD) properties of insulin preparations; however, there has been controversy over a variety of methodogical details, such as study population, dosing time and the initial stabilization of blood glucose (BG) concentrations at the clamp target level, among clamp groups. As the impact of these details on PD results is unclear, the present review provides an overview of different methodological approaches for both the manual and the automated hyperinsulinaemic-euglycaemic glucose clamp. The advantages and limitations of several methodological details are discussed as well as the relevance of clamp results for the prediction of clinical outcomes. Overall, the best method strongly depends on the exact objective of the trial. If, for instance, duration of action is the primary objective, studies should be carried out in patients with type 1 diabetes to avoid any interference of endogenous insulin. This is less important for variables such as onset of action or early metabolic activity. The hyperinsulinaemic-euglycaemic glucose clamp has a high sensitivity to detect even minor differences between different insulin preparations. The practical relevance of potential differences, however, needs to be investigated in clinical studies. A major prerequisite for obtaining reliable glucose clamp results is the attainment of high clamp quality (i.e. keeping BG concentrations close to the clamp target throughout the experiments). Unfortunately, measures of clamp quality are often under-reported, as is the variability in PD profiles, although these might explain some unconfirmed extreme results obtained in a few clamp studies.

Twenty-four hour hyperinsulinemic-euglycemic clamp improves postoperative nitrogen balance only in low insulin sensitivity patients following cardiac surgery.

BACKGROUND: Critically ill patients often suffer from a protein catabolic state. The aim of this study was to demonstrate that nitrogen balance (NB) in cardiac patients admitted to the intensive care unit (ICU) is related to their insulin sensitivity level and that supraphysiologic doses of insulin can restore anabolism. MATERIALS AND METHODS: Twenty-eight patients that were admitted to ICU in enteral and/or parenteral nutrition have been enrolled in this study. All patients received a standard nutrition protocol for at least 3 days before starting the study. These patients received either enteral or parenteral nutrition based on 1.4 kcal/kg/h and 1.1 g/kg/24 h of proteins. Participants were studied for three 24 h periods (P1 , P2 , and P3 ). Twenty-four hour NB was calculated from urinary urea nitrogen excretion, fixed protein and energy intake during each of the three periods (P1 , P2 , and P3 ). Simultaneous to P2, a 24 h hyperinsulinemic-euglycemic clamp (HEC) was performed to determine patients' insulin sensitivity (IS) or insulin resistance (IR), as well as the impact of high doses of insulin on NB. RESULTS: Nitrogen balance remained consistently positive in the IS group regardless of the clamp. In IR patients, NB was negative before the clamp and became positive during P2 and P3 . Insulin sensitivity improved during the HEC in IR patients (P < 0.001). CONCLUSIONS: A negative NB was found only in insulin resistant patients admitted to the ICU for more than 7 days. A 24-h period HEC improved NB in these patients.

High prevalence of insulin resistance assessed by the glucose clamp technique in hormonal and non-hormonal contraceptive users.

OBJECTIVE: To assess the prevalence of insulin resistance (IR) and associated factors in contraceptive users. METHODS: A total of 47 women 18 to 40 years of age with a body mass index (kg/m(2)) < 30, fasting glucose levels < 100 mg/dl and 2-hour glucose level < 140 mg/dl after a 75-g oral glucose load were submitted to a hyperinsulinemic-euglycemic clamp. The women were distributed in tertiles regarding M-values. The analysed variables were use of combined hormonal/non-hormonal contraception, duration of use, body composition, lipid profile, glucose levels and blood pressure. RESULTS: IR was detected in 19% of the participants. The women with low M-values presented significantly higher body fat mass, waist-to-hip ratio, fasting insulin, HOMA-IR and were nulligravida, showed > 1 year of contraceptive use and higher triglyceride levels. IR was more frequent among combined oral contraceptive users, however no association was observed after regression analysis. CONCLUSIONS: The prevalence of IR was high among healthy women attending a family planning clinic independent of the contraceptive method used with possible long-term negative consequences regarding their metabolic and cardiovascular health. Although an association between hormonal contraception and IR could not be found this needs further research. Family planning professionals should be proactive counselling healthy women about the importance of healthy habits.

Insulin secretion and sensitivity after single-dose amisulpride, olanzapine or placebo in young male subjects: double blind, cross-over glucose clamp study.

INTRODUCTION: Increased risks of weight gain and diabetes mellitus have been reported for schizophrenic patients under long-term treatment with several atypical antipsychotic drugs including olanzapine. Among other antipsychotic drugs, treatment with the selective dopamine D2 and D3 receptor antagonist amisulpride has been implicated with a lower risk for metabolic complications. PATIENTS AND METHODS: In this study we compared the acute, non-adiposity related effects of a single dose of olanzapine, amisulpride and placebo on insulin sensitivity and secretion in 10 healthy subjects in a randomised, double blind cross-over design. Subjects underwent euglycemic-hyperinsulinemic and hyperglycemic clamp tests using an automated clamp device. C-peptide and pro-insulin levels were determined using highly specific immuno-assays. RESULTS: Insulin sensitivity was not significantly different between both verum medications and placebo. However, C-peptide secretion during hyperglycemic clamp was significantly higher after administration of amisulpride than after olanzapine or placebo. This was true both for the early phase and for the second phase of insulin secretion (C-peptide at 0, 5,10 and 30 min: amisulpride 1.49±0.49; 4.22±1.45; 3.19±1.22; 5.33±1.85; olanzapine 1.35±0.47; 3.84±1.37; 2.72±0.91; 4.28±1.96; placebo 1.72±0.82; 3.59±1.19; 2.71±1.02; 4.54±1.42 ng/mL, mean±SD; ANOVA p=0.043). Pro-insulin levels did not differ significantly between groups. DISCUSSION: A low dose of the D2/D3 antagonist amisulpride, but not olanzapine appears to acutely increase pancreatic insulin secretion in healthy controls. Stimulation of ß-cells could be a protective factor against the development of diabetes mellitus.

Systemic hemodynamic function in humans with type 1 diabetes treated with protein kinase Cß inhibition and renin-angiotensin system blockade: a pilot study.

The protein kinase Cß (PKCß) system has been implicated in the deleterious vascular responses to hyperglycemia and angiotensin II (Ang II) in experimental models of diabetes (DM). Whether these interactions are important in humans is unknown. Flow-mediated vasodilatation (FMD) was measured during clamped euglycemia and hyperglycemia, before and after randomization to PKCß inhibition (ruboxistaurin; RBX, 32 mg daily, n = 13) or a placebo (n = 7) for 8 weeks in renin-angiotensin system (RAS) blockade-treated subjects with type 1 DM. Blood pressure responses to infused Ang II were measured before and after randomization to RBX or a placebo. The RBX and placebo groups displayed similar clinical characteristics. Before RBX, FMD declined in response to hyperglycemia (6.8% ± 2.8% to 4.9% ± 1.8%). This effect was reversed after treatment with RBX (5.6% ± 3.1% to 6.0% ± 1.6% (within-group change, p = 0.009 (ANOVA)). No changes were observed in the placebo group. Infused Ang II was associated with hypertensive responses in the RBX and placebo groups (p < 0.05 (ANOVA)), and RBX did not influence this effect. In conclusion, RBX blunted the effect of hyperglycemia on FMD, suggesting that PKCß may modulate endothelial function in type 1 DM. The lack of effect on Ang II responses suggests that PKCß inhibition may act through non-RAS pathways in humans with DM.

Hyperinsulinemic-Euglycemic Clamp Strengthens the Insulin Resistance in Nonclassical Congenital Adrenal Hyperplasia.

OBJECTIVE: Insulin sensitivity evaluation by hyperinsulinemic-euglycemic clamp in nonclassical congenital adrenal hyperplasia (NC-CAH) due to 21-hydroxilase deficiency. DESIGN AND SETTING: Cross-sectional study at university hospital outpatient clinics. PATIENTS AND METHODS: NC-CAH patients (25 females, 6 males; 24 ± 10 years) subdivided into C/NC (compound heterozygous for 1 classical and 1 nonclassical allele) and NC/NC (2 nonclassical alleles) genotypes were compared to controls. RESULTS: At diagnosis, C/NC patients presented higher basal and adrenocorticotropin-stimulated 17-hydroxyprogesterone and androstenedione levels than NC/NC genotype. Patients and controls presented similar weight, body mass index, abdominal circumference, and total fat body mass. NC-CAH patients showed higher waist-to-hip ratio, lower adiponectin and lower high-density lipoprotein cholesterol levels with no changes in fasting plasma glucose, glycated hemoglobin, homeostatic model assessment for insulin resistance, leptin, interleukin 6, tumor necrosis factor alpha, C-reactive protein, and carotid-intima-media thickness. All patients had used glucocorticoid (mean time of 73 months). Among the 22 patients with successful clamp, 13 were still receiving glucocorticoid-3 patients using cortisone acetate, 9 dexamethasone, and 1 prednisone (hydrocortisone equivalent dose of 5.5mg/m²/day), while 9 patients were off glucocorticoid but had previously used (hydrocortisone equivalent dose of 5.9mg/m2/day). The NC-CAH patients presented lower Mffm than controls (31 ± 20 vs 55 ± 23µmol/min-1/kg-1, P = 0.002). The Mffm values were inversely correlated with the duration of glucocorticoid treatment (r = -0.44, P = 0.04). There was association of insulin resistance and glucocorticoid type but not with androgen levels. CONCLUSION: Using the gold standard method, the hyperinsulinemic-euglycemic clamp, insulin resistance was present in NC-CAH patients and related to prolonged use and long-acting glucocorticoid treatment. Glucocorticoid replacement and cardiometabolic risks should be monitored regularly in NC-CAH.

Normalization of metabolic flux data during clamp studies in humans.

BACKGROUND: There is no consensus in the field regarding the optimal method for the expression of metabolic flux data, such as glucose disposal rates during hyperinsulinemic-euglycemic clamp experiments. Several normalization methods are in use today, but their impact on study outcomes is rarely discussed. METHODS: We illustrate this issue using clamp data from 92 lean and 66 obese subjects. Glucose kinetics and insulin sensitivity were determined during hyperinsulinemic-euglycemic clamp studies using [6,6-2H2]glucose. From this single dataset, we calculated 21 expression methods for the glucose disposal rate during hyperinsulinemic conditions. RESULTS AND DISCUSSION: With most normalization methods, the obese subjects demonstrated reduced insulin-stimulated glucose disposal as compared to the lean subjects. However, depending on the normalization method, glucose disposal rates in obese subjects ranged from 26 ±â€¯1% to 207 ±â€¯10% of glucose disposal rates in lean subjects. We conclude that data normalization methods greatly impacted metabolic flux outcomes in our dataset of lean and obese subjects. There is no compelling evidence to select one method over the other, but we encourage authors in the metabolic arena to think about, and provide a rationale for, the best normalization method for their specific research questions.

Comparison between euglycemic hyperinsulinemic clamp and surrogate indices of insulin sensitivity in children with growth hormone deficiency.

OBJECTIVE: Data about the impact of growth hormone treatment (GHT) on insulin sensitivity in children are quite controversial, due to the different surrogate indices that have been used. DESIGN: We evaluated insulin sensitivity through the euglycemic hyperinsulinemic clamp, considered the gold standard technique, in 23 children affected by growth hormone deficiency (GHD) at baseline and after 12months of GHT and in 12 controls with short stature at baseline, and we compared the clamp-derived index (M-value) with the most commonly used surrogate index of insulin sensitivity, as ISI Matsuda, and with circulating plasma markers of insulin sensitivity, as adiponectin and resistin levels. RESULTS: At baseline, no significant difference in all metabolic parameters between GHD children and control subjects was found. After 12months of GHT, GHD children showed a significant increase in fasting insulin (p<0.001) and resistin (p=0.028) and a decrease in ISI Matsuda (p<0.001) and M-value (p<0.001), without significant change in fasting glucose, HbA1c and adiponectin. In GHD children, M-value showed a significant but weak correlation with ISI Matsuda (rho 0.418, p=0.047) at baseline, while no correlation with other parameters was found. After 12months of GHT, M-value did not show any significant correlation with any other metabolic parameter analyzed. CONCLUSIONS: This study highlights the limit of the evaluation of insulin sensitivity performed through surrogate indices or circulating markers, which may lead to controversial data and do not correlate with the gold standard technique to evaluate insulin sensitivity.

Effect of 2 weeks of theophylline on glucose counterregulation in patients with type 1 diabetes and unawareness of hypoglycemia.

BACKGROUND AND OBJECTIVE: A single dose of theophylline improves hypoglycemia unawareness in type 1 diabetic patients. Prolonged theophylline use is, however, associated with emergence of tolerance. This study investigated whether prolonged use of theophylline retains efficacy for counterregulatory defects in patients with type 1 diabetes and hypoglycemia unawareness. METHODS: Experiments were performed with 12 subjects with type 1 diabetes and hypoglycemia unawareness. All subjects participated in a crossover study of 2 randomly scheduled 15-day study periods during which 250 mg theophylline twice daily or matching placebo was used. On the final day of each period, hyperinsulinemic (360 pmol x m(-2) x min(-1)) hypoglycemic (5.0, 3.5, 2.5 mmol x L(-1)) glucose clamps were used to assess counterregulatory and cardiovascular responses. RESULTS: Under normoglycemic conditions, there were no differences between theophylline and placebo. Under hypoglycemic conditions, theophylline enhanced responses of growth hormone, symptoms, heart rate, and pulse pressure (all P <.05), induced sweating at higher plasma glucose levels (P =.039), and reduced exogenous glucose requirements (P =.018). Hypoglycemia-induced responses of epinephrine, norepinephrine, and cortisol were not enhanced by theophylline. CONCLUSIONS: Prolonged use of theophylline has a sustained effect on cardiovascular, metabolic, and symptom responses to hypoglycemia in patients with type 1 diabetes and hypoglycemia unawareness. Whether these results translate into clinical benefit remains to be determined.

Insulin sensitivity in type 2 diabetes: univariate and multivariate techniques to derive estimates of insulin sensitivity from the insulin modified intravenous glucose tolerance test (FSIGT).

The use of univariate and multivariate techniques to derive estimates of insulin sensitivity from the insulin modified FSIGT were investigated in 12 Type 2 diabetic subjects aged (mean+/-S.D.) 59+/-9.5 years and BMI 28.1+/-2.2 kg m(-2), who underwent both a FSIGT and an isoglycemic hyperinsulinemic clamp. Reproducibility of the FSIGT was tested in four patients on three separate occasions. FSIGT data were assessed by both univariate and multivariate techniques. The sensitivity index for the FSIGT ranged from 0.162 to 3.292 (mean 1.378) x 10(-4) x l min(-1) mU(-1) for the univariate approach and from 0.163 to 2.727 (mean 1.378) x 10(-4) x l min(-1) mU(-1) for the multivariate method. Mean S(Iclamp) was 44.41 x 10(-4) x l(-2) min(-1) x mU(-1) (range 22.0-77.92). The correlation of the insulin sensitivity indices between the clamp and the FSIGT was 0.51 (P=0.056) for the univariate and 0.67 (P=0.017) for the multivariate analyses. Repeated FSIGTs showed a lower variability for the multivariate than for the standard approach.

Validation of a simple index (SIisOGTT) of insulin sensitivity in a population of sedentary men.

AIM: The ongoing obesity epidemic is associated with numerous health problems related to altered metabolic function. Among these is type 2 diabetes, characterized by lowered insulin sensitivity (IS). Consequently, the development of simple indices to assess IS has research and clinical importance. The SI(is)OGTT, a new index of IS, was recently described by Bastard et al. (Diabetes & Metabolism 2007;33:261-8), and validated in sedentary, non-diabetic, overweight and obese postmenopausal women. The aim of the present study was to validate the index in men. METHODS: The data used in this project came from sedentary men (n=36), aged 34-53 years, all of whom underwent a hyperinsulinaemic-euglycaemic clamp and 2-hour oral glucose tolerance test (OGTT). Correlations with M/I (glucose infusion rate [GIR] divided by insulin concentration), GIR and GIR divided by fat-free mass (FFM) were obtained by four well-known indices (HOMA, QUICKI, Cederholm and Matsuda) as well as with the new SI(is)OGTT index. Pearson correlations and Bland-Altman analyses were obtained for every index versus clamp value. RESULTS: The best correlate of IS in the present study was the SI(is)OGTT (r=0.84, P<0.0001). The agreement of this method with the hyperinsulinaemic-euglycaemic clamp, as assessed by Bland-Altman plots, was similar to those of the other indices and to those previously described in postmenopausal women. CONCLUSION: The new index proposed by Bastard et al. is as good a predictor of IS in sedentary men as the other commonly used indices, and appears to be as reliable in this population as it was in the original study of postmenopausal women.

Whole-body skeletal muscle mass is not related to glucose tolerance or insulin sensitivity in overweight and obese men and women.

The relationship between skeletal muscle mass, visceral adipose tissue, insulin sensitivity, and glucose tolerance was examined in 214 overweight or obese, but otherwise healthy, men (n = 98) and women (n = 116) who participated in various exercise and (or) weight-loss intervention studies. Subjects had a 75 g oral glucose tolerance test and (or) insulin sensitivity measures by a 3 h hyperinsulinemic-euglycemic clamp technique. Whole-body skeletal muscle mass and visceral adipose tissue were measured using a multi-slice magnetic resonance imaging protocol. Total body skeletal muscle mass was not associated with any measure of glucose metabolism in men or women (p > 0.10). These observations remained independent of age and total adiposity. Conversely, visceral adipose tissue was a significant predictor of various measures of glucose metabolism in both men and women with or without control for age and (or) total body fat (p < 0.05). Although skeletal muscle is a primary site for glucose uptake and deposition, these findings suggest that unlike visceral adipose tissue, whole-body skeletal muscle mass per se is not associated with either glucose tolerance or insulin sensitivity in overweight and obese men and women.

Sex differences in insulin action and body fat distribution in overweight and obese middle-aged and older men and women.

Controversy exists as to whether there are differences in insulin action between older men and women, and what factors contribute to these differences. This study tests the hypothesis that sex differences in regional fat distribution contribute to a disparity in insulin sensitivity in older men vs. older women. Healthy, older (50-71 years), sedentary men (n = 28) and women (n = 29) were recruited to participate in the study. Body fat, fat-free mass (FFM), and visceral (VAT) and subcutaneous abdominal (SAT) adipose tissue areas were measured by DXA and computed tomography (CT). For measurements of insulin-stimulated glucose disposal (M), insulin was infused at a constant rate of 240 pmol.m(-2).min(-1), and M was calculated between the 90th and 120th min of the hyperinsulinemic-euglycemic clamp. The men weighed 16% more and had 16% higher waist and 4% lower hip circumferences than women (p < 0.05 for all). Total fat mass and SAT were 21% and 33% lower and FFM was 49% higher in men than in women, whereas waist-to-hip ratio (WHR) and VAT:SAT ratio were 21% and 56% higher in men than in women (p < 0.05 for all). Although insulin concentrations during the glucose clamp were higher in men, M was 47% lower in men vs. women (21.7 +/- 1.1 vs. 46.7 +/- 3.1 micromol.L(-1).kg FFM(-1).min(-1), p < 0.05). The sex-related differences in M persisted after controlling for insulin concentrations during the glucose clamp, for waist, WHR, and VAT:SAT. Older men are more insulin resistant than women, despite lower body fat and subcutaneous abdominal fat. This difference in insulin sensitivity is not explained by abdominal fat distribution, therefore other metabolic factors contribute to the sex differences in insulin sensitivity.

Heritability and genetic correlations of insulin sensitivity measured by the euglycaemic clamp.

AIMS: Studies investigating genetic factors influencing insulin sensitivity/insulin resistance have measured this phenotype using a variety of methods. In this study, genetic correlations and heritability of insulin sensitivity measured using the euglycaemic hyperinsulinaemic clamp and related phenotypes were examined. METHODS: The study population included 818 non-diabetic individuals from 297 nuclear families. Genetic correlations and heritability estimates were calculated using variance components methods. RESULTS: Homeostasis model of insulin resistance (HOMA-IR) and fasting insulin were very highly phenotypically and genetically correlated (r = 0.99 and r = 0.99). HOMA-IR and insulin sensitivity measured with the euglycaemic clamp were only moderately genetically correlated (r = -0.53), suggesting that the two traits may be influenced, at least in part, by different genes. Heritabilities for fasting insulin (h2 = 0.36) and HOMA-IR (h2 = 0.38) were consistent with the published literature, but heritability for insulin sensitivity measured by the euglycaemic clamp was slightly lower than other published estimates (h(2) = 0.24). CONCLUSIONS: Because HOMA-IR (or fasting insulin) and insulin sensitivity measured with the euglycaemic clamp are not highly genetically correlated, they should not be used interchangeably in genetic studies. Given the very high correlations between fasting insulin and HOMA-IR, HOMA-IR does not offer any advantage over fasting insulin in analyses of insulin sensitivity in this population.

Correlation of calculated indices of insulin resistance (QUICKI and HOMA) with the euglycaemic hyperinsulinaemic clamp technique for evaluating insulin resistance in critically ill patients.

BACKGROUND AND OBJECTIVE: Insulin resistance is frequently observed in critical illness. It can be quantified by the expensive and time-consuming euglycaemic hyperinsulinaemic clamp technique (M-value) and calculated indices of insulin resistance (Quantitative Insulin Sensitivity Check Index; QUICKI and Homeostasis Model Assessment; HOMA) with lower costs and efforts. We performed an observational study to assess the reliability of QUICKI and HOMA to evaluate insulin resistance in critically ill patients compared with the current gold standard method, the euglycaemic hyperinsulinaemic clamp technique. METHODS: Insulin resistance was measured in 30 critically ill medical patients by the euglycaemic hyperinsulinaemic clamp technique (M-value) as well as calculated using QUICKI and HOMA. Correlations between the M-values as well as QUICKI and HOMA were assessed by means of the Pearson's correlation coefficient. RESULTS: M-value, QUICKI and HOMA indicated insulin resistance in all 30 patients. However, both indices QUICKI and HOMA did not correlate with the M-values in our patients (r2 = 0.008 and 0.0005, respectively). A significant negative correlation was found between the M-value and the severity of illness assessed by the APACHE (Acute Physiology and Chronic Health Evaluation) III score (r2 = 0.16; P < 0.05). In contrast, neither HOMA nor QUICKI correlated with the APACHE III score (r2 = 0.034 and 0.033, respectively). CONCLUSIONS: Although QUICKI and HOMA indicated insulin resistance in the critically ill medical patients, both indices did not correlate with the M-value. Therefore, the euglycaemic hyperinsulinaemic clamp technique remains the gold standard for estimating insulin resistance in critically ill patients.