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In rats, cannulation of the jugular vein and the carotid artery precedes the use of the hyperinsulinemic euglycemic clamp to determine insulin sensitivity in vivo. Here, we present a vascular surgery protocol to allow the infusion of substances via the vein and the collection of blood samples from the artery on the day of the hyperinsulinemic euglycemic clamp. We describe steps for preparing for and performing catheterization surgery. We then detail procedures for clamp preparation and its use. For complete details on the use and execution of this protocol, please refer to Pereira et al. 1 , 2 , 3 ., Competing Interests: Declaration of interests M.K.H. received consultant fees from Alkermes, Inc. S.M.A. has served as a consultant for HLS Therapeutics and Boehringer Ingelheim Canada., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Accurate measurements of glycaemic control and the underpinning regulatory mechanisms are vital in human physiology research. Glycaemic control is the maintenance of blood glucose concentrations within optimal levels and is governed by physiological variables including insulin sensitivity, glucose tolerance and β-cell function. These can be measured with a plethora of methods, all with their own benefits and limitations. Deciding on the best method to use is challenging and depends on the specific research question(s). This review therefore discusses the theory and procedure, validity and reliability and any special considerations of a range common methods used to measure glycaemic control, insulin sensitivity, glucose tolerance and β-cell function. Methods reviewed include glycosylated haemoglobin, continuous glucose monitors, the oral glucose tolerance test, mixed meal tolerance test, hyperinsulinaemic euglycaemic clamp, hyperglycaemic clamp, intravenous glucose tolerance test and indices derived from both fasting concentrations and the oral glucose tolerance test. This review aims to help direct understanding, assessment and decisions regarding which method to use based on specific physiology-related research questions., (© 2024 The Author(s). Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The aim of the study was to compare the pharmacokinetics (PK) and pharmacodynamics (PD) of T-glu (GP40321, test drug), and reference insulin glulisine in a hyperinsulinemic-euglycemic clamp procedure. During this study, 34 healthy male volunteers underwent the hyperinsulinemic-euglycemic clamp procedure following subcutaneous 0.3 U/kg injection of T-glu or reference insulin glulisine in a randomized, double-blind, crossover study. Plasma glucose levels were monitored every 5 minutes for 8 hours. Glucose infusion rate adjustment was based on the blood glucose measurements. Evaluation of PD was performed using the glucose infusion rate values, while PK was calculated using insulin concentrations measured via enzyme-linked immunosorbent assay. The study results showed that the 90% CI for the geometric mean ratios of primary PK and PD of T-glu and reference insulin glulisine were within 80%-125% comparability limits, and that the safety profiles were comparable. PK, PD, and safety similarity of T-glu and reference insulin glulisine was demonstrated., (© 2024, The American College of Clinical Pharmacology.)

Background: Obstructive sleep apnea (OSA) is prevalent in people with obesity and is a major risk factor for type 2 diabetes (T2D). The effect of OSA on metabolic function and the precise mechanisms (insulin resistance, β-cell dysfunction, or both) responsible for the increased T2D risk in people with OSA are unknown., Design and Methods: We used a two-stage hyperinsulinemic-euglycemic clamp procedure in conjunction with stable isotopically labeled glucose and palmitate tracer infusions and 18F-fluorodeoxyglucose injection and positron emission tomography to quantify multi-organ insulin action and oral and intravenous tolerance tests to evaluate glucose-stimulated insulin secretion in fifteen people with obesity and OSA and thirteen people with obesity without OSA., Results: OSA was associated with marked insulin resistance of adipose tissue triglyceride lipolysis and glucose uptake into both skeletal muscles and adipose tissue, whereas there was no significant difference between the OSA and control groups in insulin action on endogenous glucose production, basal insulin secretion, and glucose-stimulated insulin secretion during both intravenous and oral glucose tolerance tests., Conclusions: These data demonstrate that OSA is a key determinant of insulin sensitivity in people with obesity and underscore the importance of taking OSA status into account when evaluating metabolic function in people with obesity. These findings may also have important clinical implications because disease progression and the risk of diabetes-related complications vary by T2D subtype (i.e. severe insulin resistance vs insulin deficiency). People with OSA may benefit most from the targeted treatment of peripheral insulin resistance and early screening for complications associated with peripheral insulin resistance.

Insulin aspart (IAsp) is one of the main therapies used to control blood glucose after a meal. This study aimed to compare the pharmacokinetics (PK) and pharmacodynamics (PD) of 2 rapid-acting IAsp products: a new IAsp biosimilar (RD10046) and NovoRapid. In a single-center, randomized, single-dose, 2-period, crossover, euglycemic clamp study (registry number: CTR20180517, registration date: 2018-05-30), healthy Chinese males were randomized to receive 0.2 U/kg of the IAsp biosimilar RD10046 and NovoRapid under fasted conditions on two separate occasions. PK and PD were assessed for up to 10 h. Of the 30 randomized subjects, all 30 completed both treatment periods. The PK (area under the curve [AUC] of total IAsp; maximum observed IAsp concentration [C max ]) and PD (maximum glucose infusion rate [GIR max ]; total glucose infusion during the clamp [AUC GIR,0-10h ]) were similar between the new IAsp biosimilar RD10046 and NovoRapid. In all cases, the 90% CIs for the ratios of the geometric means were completely contained in the prespecified acceptance limits of 0.80-1.25. No hypoglycemic events, allergic reactions, or local injection adverse reactions occurred in this trial. We concluded that the studied IAsp biosimilar (RD10046) was bioequivalent to NovoRapid.

Application of glucose clamp methodologies in multicenter studies brings challenges for standardization. The Restoring Insulin Secretion (RISE) Consortium implemented a hyperglycemic clamp protocol across seven centers using a combination of technical and management approaches to achieve standardization. Two-stage hyperglycemic clamps with glucose targets of 200 mg/dL and >450 mg/dL were performed utilizing a centralized spreadsheet-based algorithm that guided dextrose infusion rates using bedside plasma glucose measurements. Clamp operators received initial and repeated training with ongoing feedback based on surveillance of clamp performance. The precision and accuracy of the achieved stage-specific glucose targets were evaluated, including differences by study center. We also evaluated robustness of the method to baseline physiologic differences and on-study treatment effects. The RISE approach produced high overall precision (3%-9% variance in achieved plasma glucose from target at various times across the procedure) and accuracy (SD < 10% overall). Statistically significant but numerically small differences in achieved target glucose concentrations were observed across study centers, within the magnitude of the observed technical variability. Variation of the achieved target glucose over time in placebo-treated individuals was low (<3% variation), and the method was robust to differences in baseline physiology (youth vs. adult, IGT vs. diabetes status) and differences in physiology induced by study treatments. The RISE approach to standardization of the hyperglycemic clamp methodology across multiple study centers produced technically excellent standardization of achieved glucose concentrations. This approach provides a reliable method for implementing glucose clamp methodology across multiple study centers. NEW & NOTEWORTHY The Restoring Insulin Secretion (RISE) study centers undertook hyperglycemic clamps using a simplified methodology and a decision guidance algorithm implemented in an easy-to-use spreadsheet. This approach, combined with active management including ongoing central data surveillance and routine feedback to study centers, produced technically excellent standardization of achieved glucose concentrations on repeat studies within and across study centers.

Aims: Sphingolipids(SPs) and their substrates and constituents, fatty acids (FAs), are implicated in the pathogenesis of various metabolic diseases associated. This study aimed to systematically investigate the associations between serum sphingolipids and insulin sensitivity as well as insulin secretion., Methods: We conducted a lipidomics evaluation of molecularly distinct SPs in the serum of 86 consecutive Chinese adults using LC/MS. The glucose infusion rate over 30 min (GIR 30 ) was measured under steady conditions to assess insulin sensitivity by the gold standard hyperinsulinemic-euglycemic clamp. We created the ROC curves to detect the serum SMs diagnostic value., Results: Total and subspecies of serum SMs and globotriaosyl ceramides (Gb3s) were positively related to GIR 30 , free FAs (FFA 16:1, FFA20:4), some long chain GM3 and complex ceramide GluCers showed strong negative correlations with GIR 30 . Notably, ROC curves showed that SM/Cer and SM d18:0/26:0 may be good serum lipid predictors of diagnostic indicators of insulin sensitivity close to conventional clinical indexes such as 1/HOMA-IR (areas under the curve > 0.80) based on GIR 30 as standard diagnostic criteria, and (SM/Cer)/(BMI*LDLc) areas under the curve = 0.93) is the best., Conclusions: These results provide novel associations between serum sphingolipid between insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp and identify two specific SPs that may represent prognostic biomarkers for insulin sensitivity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Aims: Hypoglycemia hinders optimal glycemic management in type 1 diabetes (T1D). Long diabetes duration and hypoglycemia impair hormonal counter-regulatory responses to hypoglycemia. Our study was designed to test whether (1) the metabolic responses and insulin sensitivity are impaired, and (2) whether they are affected by short-lived antecedent hypoglycemia in participants with T1D., Materials and Methods: In a randomized, crossover, 2x2 factorial design, 9 male participants with T1D and 9 comparable control participants underwent 30 minutes of hypoglycemia (p-glucose < 2.9 mmol/L) followed by a euglycemic clamp on 2 separate interventions: with and without 30 minutes of hypoglycemia the day before the study day., Results: During both interventions insulin sensitivity was consistently lower, while counter-regulatory hormones were reduced, with 75% lower glucagon and 50% lower epinephrine during hypoglycemia in participants with T1D, who also displayed 40% lower lactate and 5- to 10-fold increased ketone body concentrations following hypoglycemia, whereas palmitate and glucose turnover, forearm glucose uptake, and substrate oxidation did not differ between the groups. In participants with T1D, adipose tissue phosphatase and tensin homolog (PTEN) content, hormone-sensitive lipase (HSL) phosphorylation, and muscle glucose transporter type 4 (GLUT4) content were decreased compared with controls. And antecedent hypoglycemic episodes lasting 30 minutes did not affect counter-regulation or insulin sensitivity., Conclusions: Participants with T1D displayed insulin resistance and impaired hormonal counter-regulation during hypoglycemia, whereas glucose and fatty acid fluxes were intact and ketogenic responses were amplified. We observed subtle alterations of intracellular signaling and no effect of short-lived antecedent hypoglycemia on subsequent counter-regulation. This plausibly reflects the presence of insulin resistance and implies that T1D is a condition with defective hormonal but preserved metabolic responsiveness to short-lived hypoglycemia., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Metabolic pathways that are corrupted at early stages of insulin resistance (IR) remain elusive. This study investigates changes in body metabolism in clinically healthy and otherwise asymptomatic subjects that may become apparent already under compromised insulin sensitivity (IS) and prior to IR. 47 clinically healthy Arab male subjects with a broad range of IS, determined by hyperinsulinemic-euglycemic clamp (HIEC), were investigated. Untargeted metabolomics and complex lipidomics were conducted on serum samples collected under fasting and HIEC conditions. Linear models were used to identify associations between metabolites concentrations and IS levels. Among 1896 identified metabolites, 551 showed significant differences between fasting and HIEC, reflecting the metabolic switch in energy utilization. At fasting, 336 metabolites, predominantly di- and tri-acylglycerols, showed significant differences between subjects with low and high levels of IS. Changes in amino acid, carbohydrate and fatty acid metabolism in response to insulin were impaired in subjects with low IS. Association of altered mannose and amino acids with IS was also replicated in an independent cohort of T2D patients. We identified metabolic phenotypes that characterize clinically healthy Arab subjects with low levels of IS at their fasting state. Our study is providing further insights into the metabolic pathways that precede IR.

Gut hormones affect cardiac function and contractility. In this study, we examined whether insulin affects the cardiac atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression and release of proANP-derived peptides in pigs. Anaesthetized pigs were included in an experimental study comparing the effect of hyperinsulinemia in 15 pigs submitted to two different protocols versus 11 control pigs receiving saline infusion. Phosphorylation of Akt on Thr308 was determined by western blotting with a pAkt-Thr308 antibody. The mRNA contents of ANP and BNP were determined with real-time PCR; plasma and cardiac tissue proANP was measured with an immunoluminometric assay targeted against the mid-region of the propeptide and a processing-independent assay. Insulin stimulation increased phosphorylation of Akt Thr308 in both left atrium and left ventricle of porcine hearts (p < 0.005). No change was observed in ANP and BNP mRNA contents in the right or left atrium. BNP mRNA contents in the left ventricle, however, decreased 3-fold (p = 0.02) compared to control animals, whereas the BNP mRNA content in the right ventricle as well as ANP mRNA content in the right and left ventricle did not change following hyperinsulinemia. Moreover, the peptide contents did not change in the four cardiac chambers. Finally, proANP concentrations in plasma did not change during the insulin infusion compared to the control animals. These results suggest that insulin does not have direct effect on atrial natriuretic peptide expression but may have a role in the left ventricle., Competing Interests: Declaration of Competing Interest The authors have nothing to disclose., (Copyright © 2020. Published by Elsevier Inc.)

Aims: Insulin resistance in people with type 1 diabetes (T1D) is associated with increased risk of chronic complications and death. The gold standard to quantify insulin sensitivity, a euglycaemic hyperinsulinaemic clamp, is not applicable to clinical practice. We have employed clamp studies to develop a panel of formulae to estimate insulin sensitivity in adults with T1D for use in clinical practice and trials., Methods: Clamps were conducted in 28 adults with T1D, who were also characterised with 38 clinical and research biomarkers. Exhaustive search analysis was used to derive equations correlating with clamp-quantified glucose disposal rate (GDR), GDR/plasma insulin (M/I) and log 10 M/I., Results: Measured insulin sensitivity correlated with BMI, WHR, HDL-C, adipokines and inflammation markers on univariate analysis. Exhaustive search analysis derived three formulae correlating with clamp-derived GDR and logM/I (p < 0.0001), accounting for ≈62% of their variability. A formula using gender, age, HDL-C, pulse pressure and WHR performed as well as those containing inflammation and adipokine measures., Conclusions: The performance of formulae using routinely available parameters with/without research biomarkers in clinical studies and trials, particularly related to future complications, relevant lifestyle interventions, insulin delivery modes and insulin sensitisers is merited., Competing Interests: Declaration of Competing Interest AJJ, DNO and ASJ were investigators on the REMOVAL trial of metformin in Type 1 diabetes trial. No conflicts of interest from other authors., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Objective: The hyperinsulinemic euglycemic clamp (HEC) is the "gold standard" for measuring insulin sensitivity (Si-clamp). Here, we determined the reproducibility of serial HEC data in healthy subjects., Research Design and Methods: The Pathobiology of Prediabetes in A Biracial Cohort study assessed incident prediabetes in healthy African Americans (AA) and European Americans (EA) with parental type 2 diabetes mellitus during 5.5 years of follow-up. Assessments included anthropometry, OGTT, and HEC. Ninety subjects (44 AA, 46 EA) who underwent Year-1HEC consented to Year-3 HEC. We calculated coefficients of variation (CVs), 95% limits of agreement, and repeatability coefficients for Year-1 and Year-3 data, and assessed the association of change in Si-clamp with incident prediabetes., Results: The mean (SD) baseline age was 47.5 ± 8.13y, body mass index was 30.4 ± 9.16 kg/m 2 , fasting plasma glucose was 93.7 ± 7.82 mg/dL and 2-hrPG was 126 ± 26.8 mg/dL. Si-clamp (umol/kg/min·pmol/L -1 ) was 0.071 ± 0.04 in Year 1 and 0.067 ± 0.04 in Year 3 (P = 0.22). Year 1 and Year 3 values were strongly correlated (r = 0.81, P < 0.0001); the CV was 13.6% and repeatability coefficient was ±0.025. Intrasubject differences in serial Si-clamp were less than the repeatability coefficients and within the 95% limits of agreement. After 5.5 years of follow-up, 40 subjects progressed to prediabetes and 50 were nonprogressors. The change in Si-clamp was greater in progressors than nonprogressors (-10% vs. -2.5%, P = 0.02)., Conclusions: The HEC is reproducible over ~2 years in free-living individuals, with a temporal decline in Si-clamp that predicts prediabetes risk., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Impaired insulin secretion and action are important for development of type 2 diabetes (T2D) and metabolic syndrome (MetS). Despite recognized heterogeneity of these glucometabolic disorders, few data are available of biological variation in insulin secretion and action among individuals with T2D and MetS. The aim of this study was to explore the inter-individual variations using gold standard methods in a cross-sectional study of two independent cohorts of phenotypically well-characterized subjects. Cohort I included 486 subjects with MetS, and cohort II 62 subjects with established T2D. First phase insulin secretion was defined as the incremental area under the curve 0-8 min (iAUC 0-8 min ) during an intravenous glucose tolerance test (IVGTT). Insulin sensitivity was measured as the insulin sensitivity index (S I ) modelled from IVGTT in cohort I, and in II as total glucose disposal (TGD) estimated from a euglycaemic-hyperinsulinaemic clamp. Variation is given as total range and, fold-variation between 5%- and 95%-percentile. The iAUC 0-8 min ranged from -60 to 3397 mUL -1 min -1 among subjects with MetS and from -263 to 1194 mUL -1 min -1 in subjects with T2D, representing a more than 10-fold variation. Insulin sensitivity ranged from S I 0.19 to 15.29 (mU/L) -1 min -1 among subjects with MetS and TGD 12.9-101.6 μmolkgFFM -1 min -1 in subjects with T2D, representing a 6.8 and 5.5-fold variation, respectively. The other components of MetS; BMI, waist-hip ratio, HDL-cholesterol, triglycerides and blood pressure (BP), showed a 1.4-4.7-fold variation. In conclusion, our data demonstrated extensive inter-individual variations in insulin secretion and sensitivity. These variations may be essential to take into account when planning clinical research and treatment in subjects with T2D and MetS.

Objective: This study aimed to determine the relationship between metabolic flexibility (MetFlex) measured during a euglycemic-hyperinsulinemic clamp and a prolonged fast. This study also analyzed the association between MetFlex and metabolic health., Methods: Eighteen healthy men (mean [SD]: 22 [2] years old; BMI: 22 [1] kg/m 2 ) performed two sessions: (1) euglycemic-hyperinsulinemic clamp (2 mIU/kg of insulin per minute) and (2) ~20-hour fast. Clamp MetFlex corresponded to the change in (Δ) respiratory quotient (RQ) (ΔRQ = postchallenge RQ - prechallenge RQ) adjusted for M value and prechallenge RQ. Prolonged fast MetFlex corresponded to the ΔRQ adjusted for the Δβ-hydroxybutyrate and prechallenge RQ., Results: MetFlex during the clamp related directly with MetFlex during prolonged fast (r = 0.59, P = 0.014). Using the median of MetFlex for each challenge, this study split participants into high or low MetFlex. Participants with high or low MetFlex to both challenges were identified. Participants with high MetFlex had 3% lower serum low-density lipoprotein cholesterol than participants with low MetFlex (P = 0.021)., Conclusions: Measuring MetFlex during a clamp or a prolonged fast produces similar results, despite challenging the oxidation of different substrates. An impaired MetFlex in response to these challenges may be an early event in the development of abnormal lipid metabolism., (© 2020 The Obesity Society.)

This work contributes to the improvement of glucose quantification using near-infrared (NIR), mid-infrared (MIR), and combination of NIR and MIR absorbance spectroscopy by classifying the spectral data prior to the application of regression models. Both manual and automated classification are presented based on three homogeneous classes defined following the clinical definition of the glycaemic ranges (hypoglycaemia, euglycaemia, and hyperglycaemia). For the manual classification, partial least squares and principal component regressions are applied to each class separately and shown to lead to improved quantification results compared to when applying the same regression models for the whole dataset. For the automatic classification, linear discriminant analysis coupled with principal component analysis is deployed, and regressions are applied to each class separately. The results obtained are shown to outperform those of regressions for the entire dataset., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aims: To investigate the effect of hypoglycaemia on platelet and coagulation activation in people with type 2 diabetes., Materials and Methods: This monocentric, open, single-arm, mechanistic trial included 14 people with established type 2 diabetes (four women, 10 men, age 55 ± 7 years, glycated haemoglobin concentration 51 ± 7 mmol/mol) receiving metformin monotherapy. A stepwise hyperinsulinaemic-hypoglycaemic clamp experiment (3.5 and 2.5 mmol/L, for 30 minutes respectively) was performed, aiming to investigate platelet and coagulation activity during predefined plateaus of hypoglycaemia, as well as 1 day and 7 days later., Results: While platelet activation assessed by light transmittance aggregometry did not significantly increase after the hypoglycaemic clamp procedure, the more sensitive flow cytometry-based measurement of platelet surface activation markers showed hypoglycaemia-induced activation 24 hours (PAC1 pos CD62P pos , PAC1 pos CD63P pos and PAC1 pos CD62P pos CD63 pos ; P < .01) and 7 days after the hypoglycaemic clamp (P < .001 for PAC1 pos CD63 pos ; P < .01 for PAC1 pos CD62P pos and PAC1 pos CD62P pos CD63 pos ) in comparison to baseline. Coagulation markers, such as fibrinogen, D-dimer, plasminogen activator inhibitor-1, von Willebrand factor activity and factor VIII, were also significantly increased, an effect that was most pronounced 24 hours after the hypoglycaemic clamp., Conclusion: A single event of insulin-induced hypoglycaemia led to an increase in markers of platelet activation and coagulation in people with early stages of type 2 diabetes on metformin therapy. However, the activation occurred with a delay and was evident 24 hours and 7 days after the actual hypoglycaemic episode., (© 2019 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Introduction: Insulin resistance plays a crucial role in the pathogenesis of type 2 diabetes and cardiovascular disease. The triglyceride to high-density lipoprotein cholesterol (TG/HDL-C) ratio, visceral adiposity index (VAI), lipid accumulation product (LAP) and triglycerides × fasting glucose (TyG) index are surrogate measures of insulin sensitivity based on anthropometric and/or biochemical parameters routinely collected in clinical practice. Herein, we compared the relationships of these four surrogate indexes with insulin sensitivity assessed by the gold standard euglycemic hyperinsulinemic clamp technique, and subclinical vascular damage., Research Design and Methods: 631 subjects with different degrees of glucose tolerance underwent euglycemic hyperinsulinemic clamp. The surrogate TG/HDL-C ratio, VAI, LAP and TyG indexes were computed. Pulse pressure and carotid intima-media thickness (IMT) were measured as indicators of subclinical vascular damage., Results: All the four surrogate indexes showed a significant correlation with insulin-stimulated glucose disposal in the whole study population. However, only LAP index had a significant association with insulin sensitivity across the different glucose tolerance groups. LAP index showed the highest area under the receiver operating characteristic curve (0.728) to detect individuals with insulin resistance defined as the bottom quartile of insulin-stimulated glucose disposal, followed by TG/HDL-C ratio (0.693), TyG index (0.688) and VAI (0.688). A significant association was found between the four indexes of insulin sensitivity and pulse pressure and IMT. All the four indexes have a similar ability to detect individuals with vascular atherosclerosis defined by IMT>0.9 mm. Conversely, LAP index had the greatest ability to recognize individuals with increased vascular stiffness defined by pulse pressure ≥60 mm Hg., Conclusion: Among the surrogate TG/HDL-C ratio, VAI, LAP and TyG indexes of insulin sensitivity, LAP index showed a significant association with insulin-stimulated glucose disposal across the different glucose tolerance categories and the highest ability to detect insulin resistance and subclinical vascular damage., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Applying the hyperinsulinemic-euglycemic clamp to estimate insulin resistance (IR) is accurate but time-consuming, so identifying a simple and effective index for IR is vitally important. The present study aimed to compare the lipid accumulation product (LAP), visceral adiposity index (VAI), body mass index (BMI), waist circumference (WC), homeostasis model assessment of insulin resistance (HOMA-IR), and Chinese visceral adiposity index (CVAI) using the hyperinsulinemic-euglycemic clamp as a reference and to screen a simple and effective indicator for IR in Chinese women of childbearing age. Methods: The present study included a cross-sectional study of 537 reproductive-aged women and an interventional study of 90 randomly chosen polycystic ovarian syndrome (PCOS) women. Physical, laboratory, and hyperinsulinemic-euglycemic clamp were completed, and the BMI, WC, LAP, VAI, CVAI, and HOMA-IR were calculated. A linear correlation and a receiver operating characteristic curve were performed. After intervention with metformin, the effects were estimated in the third month. Results: PCOS women had worse glycometabolism, serum lipid metabolism and IR, and higher prevalence rates of metabolic disorders than those without PCOS. The CVAI was strongly associated with the M value ( r  = -0.6953, P  < 0.0001) and outperformed other parameters with the largest area under the curve (0.903) and Youden index (71.07%) for IR diagnosis in Chinese reproductive-aged women, and the diagnostic point was >28.5. After 3 months of metformin therapy, IR improved with remarkable increases in M value and reductions in the CVAI. Conclusion: The CVAI can be used as an appropriate surrogate indicator for the hyperinsulinemic-euglycemic clamp to identify IR in Chinese women of childbearing age. The interventional trial part of this study has been registered as a clinical trial (no. ChiCTR-IIR-16007901).

Objective: Polycystic ovarian syndrome (PCOS) is a heterogeneous endocrine disorder associated with mitochondrial dysfunction and insulin resistance (IR). MOTS-c, a mitochondrial peptide, promotes insulin sensitivity (IS) through activating AKT and AMPK-dependent pathways. The current study was designed to examine the response of MOTS-c to lipids (intralipid) followed by insulin in PCOS and healthy subjects., Methods: All subjects underwent 5-hour intralipid/saline infusion with a hyperinsulinemic-euglycaemic clamp in the final 2 hours. Plasma samples were collected to measure circulating MOTS-c using a commercial ELISA kit. Subsequently, this was repeated following an eight-week exercise intervention., Results: Intralipid significantly increased plasma MOTS-c both in controls and PCOS subjects, whilst the insulin infusion blunted the intralipid-induced response seen for both lipids and MOT-c. Intralipid elevated plasma MOTS-c to 232 ± 124% of basal in control (P < 0.01) and to 349 ± 206% of basal in PCOS (P < 0.001) subjects. Administration of insulin suppressed intralipid-induced MOTS-c from 232 ± 124% to 165 ± 97% (NS) in control and from 349 ± 206% to 183 ± 177% (P < 0.05) in PCOS subjects, respectively. Following exercise, intralipid elevated plasma MOTS-c to 305 ± 153% of basal in control (P < 0.01) and to 215 ± 103% of basal in PCOS (P < 0.01) subjects; insulin suppressed intralipid-induced MOTS-c only in controls., Conclusions: In conclusion, this is the first study to show increased lipid enhanced circulating MOTS-c whilst insulin attenuated the MOTS-c response in human. Further, eight weeks of moderate exercise training did not show any changes in circulating MOTS-c levels in healthy controls and in women with PCOS., (© 2019 John Wiley & Sons Ltd.)

The mechanistic target of rapamycin (mTOR) inhibitor everolimus is an antitumor agent known to cause hyperglycemia. However, the clinical course of everolimus-induced hyperglycemia, its pathophysiological basis, and the treatment strategy are not clear. In this case series report, we present the clinical course of everolimus-induced hyperglycemia in four patients. Hyperglycemia occurred 3-8 weeks after the administration of everolimus irrespective of the body mass index (range, 21.3-29.1 kg/m 2 ) or pre-existing diabetes. Insulin or insulin secretagogues were required for glycemic control in most of the patients. Of note, the hyperglycemia was reversible in all patients, and none of the patients required anti-diabetic agents to achieve adequate glycemic control after cessation of everolimus therapy. To investigate the underlying mechanism of everolimus-induced hyperglycemia, we assessed insulin secretion and sensitivity by 75 g oral glucose tolerance test, arginine challenge test, and/or hyperinsulinemic-euglycemic clamp study using stable isotope-labeled glucose tracer in two patients. Everolimus did not affect insulin sensitivity in the liver, skeletal muscle, or the adipose tissue. In contrast, everolimus impaired insulin secretion and thereby increased basal hepatic glucose production. These findings further our understanding of the role of mTOR in glucose homeostasis in humans and provide insights for treatment strategies against everolimus-induced hyperglycemia.

Aims: Simple surrogate indices of insulin sensitivity have been conceived to deal with costly and complicated approaches, such as the hyperinsulinemic-euglycemic clamp; however, their use has not been widespread given their variabilities in different populations. In this paper, we present two simple surrogate indices, one that uses fasting glucose and insulin values and the other based on the values from the oral glucose tolerance test., Materials and Methods: The proposed methods integrate easy-to-obtain anthropometric measures. Evolutionary algorithms were used to optimize the proposed methods by maximizing its correlation with the Stumvoll MCR method., Results and Conclusion: When the proposed indices were applied to three study groups (control subjects, metabolic syndrome, marathon runners), a reduction in the intergroup variability of the insulin sensitivity was obtained. Moreover, the proposed index based on the oral glucose tolerance test (OGTT), which considers the glucose metabolism process and the hepatic and peripheral insulin sensitivity, showed stronger correlations with the Stumvoll method and lower intergroup variability than the fasting one., (Copyright © 2019 Diabetes India. Published by Elsevier Ltd. All rights reserved.)

Insomnia-related sleep disruptions, such as short and disturbed sleep, have been tied to systemic insulin resistance in young adult populations. We therefore sought to confirm these findings in a cohort of elderly men. To this aim, we utilized variables from 980 men who participated in the investigation at age 70 of the Uppsala Longitudinal Study of Adult Men. Self-reported insomnia symptoms were assessed by questions about difficulty initiating sleep, early final awakening, and regular use of hypnotics. All participants also underwent the gold-standard hyperinsulinemic-euglycemic clamp technique to assess the insulin sensitivity index (M/I). Finally, fasting blood was collected to measure free fatty acids (FFAs) and adiponectin. Differences in blood parameters between men with and those without insomnia were determined by ANCOVA, and were adjusted for lifestyle and cardio-metabolic risk factors. Our analysis yielded no differences in M/I, FFAs, and adiponectin between men with and those without insomnia symptoms. Analyses in non-diabetic and diabetic subsamples confirmed these negative findings. Our cross-sectional results therefore suggest that insomnia symptoms may have a minimal effect, if any, on measures of insulin sensitivity in elderly men. Given the observational design of our study, future studies are needed to determine whether experimental sleep manipulations influence systemic insulin sensitivity in elderly humans, as has previously been shown in young adult populations., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Current reference methods for measuring glucose effectiveness (GE) are the somatostatin pancreatic glucose clamp and minimal model analysis of frequently sampled intravenous glucose tolerance test (FSIVGTT), both of which are laborious and not feasible in large epidemiological studies. Consequently, surrogate indices derived from an oral glucose tolerance test (OGTT) to measure GE (oGE) have been proposed and used in many studies. However, the predictive accuracy of these surrogates has not been formally validated. In this study, we used a calibration model analysis to evaluate the accuracy of surrogate indices to predict GE from the reference FSIVGTT (Sg MM )., Methods: Subjects (n = 123, mean age 48 ± 11 years; BMI 35.9 ± 7.3 kg/m 2 ) with varying glucose tolerance (NGT, n = 37; IFG/IGT, n = 78; and T2DM, n = 8) underwent FSIVGTT and OGTT on two separate days. Predictive accuracy was assessed by both root mean squared error (RMSE) of prediction and leave-one-out cross-validation-type RMSE of prediction (CVPE)., Results: As expected, insulin sensitivity, Sg MM , and oGE were reduced in subjects with T2DM and IFG/IGT when compared with NGT. Simple linear regression analyses revealed a modest but significant relationship between oGE and Sg MM (r = 0.25, p < 0.001). However, using calibration model, measured Sg MM and predicted Sg MM derived from oGE were modestly correlated (r = 0.21, p < 0.05) with the best fit line suggesting poor predictive accuracy. There were no significant differences in CVPE and RMSE among the surrogates, suggesting similar predictive ability., Conclusions: Although OGTT-derived surrogate indices of GE are convenient and feasible, they have limited ability to robustly predict GE.

This study attempted to elucidate the possible mechanism of magnesium sulfate (MgSO 4 ) administration on reducing insulin resistance in type 2 diabetic rats. Fifty Wistar rats were divided into five groups: NDC was fed the normal diet, CD received high-fat diet with 35 mg/kg of streptozotocin, CD-Mg animals received MgSO 4 via drinking water, CD-Ins1, and CD-Ins2 animals treated with low or high dose of insulin. Body weight and blood glucose levels were measured weekly. Intraperitoneal glucose tolerance test (IPGTT), insulin tolerance test, and metabolic cage assessment were performed monthly. After 12 weeks, the hyperinsulinemic-euglycemic clamp was performed for all animals and blood sample was taken to measure glycated hemoglobin (HbA1c), plasma insulin, glucagon, calcium, and magnesium levels. Liver and gastrocnemius muscle were isolated to measure glucagon receptor (GR), Glucose 6 phosphatase (G6Pase), Phosphoenolpyruvate carboxykinase (Pepck) and Glucose transporter 4 (Glut4) genes expression and GLUT4 protein translocation into the cell membrane. Consuming of high-fat diet generated insulin-resistant rats. Magnesium or insulin therapy altered insulin resistance, blood glucose, IPGTT, gluconeogenesis pathway, GR, body weight, the percentage of body fat, and HbA1C in diabetic rats. Administrations of MgSO 4 or insulin in Type 2 diabetes mellitus animals increase GLUT4 gene and protein expression. Mg could improve glucose tolerance via stimulation of Glut4 gene expression and translocation and also suppression of the gluconeogenesis pathway and GR gene expression. Mg also increased glucose infusion rate and displayed beneficial effects in the treatment of glucose metabolism and improved insulin resistance., (© 2018 Société Française de Pharmacologie et de Thérapeutique.)

We present a 62-year-old female who collapsed with a subarachnoid haemorrhage. This was complicated by profound shock secondary to neurogenic stunned myocardium. As the patient demonstrated life-threatening catecholamine-resistant shock that was unresponsive to conventional treatment measures, hyperinsulinaemic euglycaemic therapy was utilised as a rescue therapy. To our knowledge this has not previously been described in the literature. The patient proceeded to stabilise and made a good recovery.

Key Points: It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis after protein ingestion because leucine uniquely activates the mTOR-p70S6K signalling cascade. We compared the effects of ingesting protein or an amount of leucine equal to that in the protein during a hyperinsulinaemic-euglycaemic clamp (to eliminate potential confounding as a result of differences in the insulinogenic effect of protein and leucine ingestion) on muscle anabolic signalling and protein turnover in 28 women. We found that protein, but not leucine, ingestion increased muscle p-mTOR Ser2448 and p-p70S6K Thr389 , although only protein, and not leucine, ingestion decreased muscle p-eIF2α Ser51 and increased muscle protein synthesis., Abstract: It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis (MPS) after protein ingestion because leucine uniquely activates the mTOR-p70S6K signalling cascade. We tested this hypothesis by measuring muscle p-mTOR Ser2448 , p-p70S6K Thr389 and p-eIF2α Ser51 , as well as protein turnover (by stable isotope labelled amino acid tracer infusion in conjunction with leg arteriovenous blood and muscle tissue sampling), in 28 women who consumed either 0.45 g protein kg -1 fat-free mass (containing 0.0513 g leucine kg -1 fat-free mass) or a control drink (n = 14) or 0.0513 g leucine kg -1 fat-free mass or a control drink (n = 14) during a hyperinsulinaemic-euglycaemic clamp procedure (HECP). Compared to basal conditions, the HECP alone (without protein or leucine ingestion) suppressed muscle protein breakdown by ∼20% and increased p-mTOR Ser2448 and p-p70S6K Thr389 by >50% (all P < 0.05) but had no effect on p-eIF2α Ser51 and MPS. Both protein and leucine ingestion further increased p-mTOR Ser2448 and p-p70S6K Thr389 , although only protein, and not leucine, ingestion decreased (by ∼35%) p-eIF2α Ser51 and increased (by ∼100%) MPS (all P < 0.05). Accordingly, leg net protein balance changed from negative (loss) during basal conditions to equilibrium during the HECP alone and the HECP with concomitant leucine ingestion and to positive (gain) during the HECP with concomitant protein ingestion. These results provide new insights into the regulation of MPS by demonstrating that leucine and mTOR signalling alone are not responsible for the muscle anabolic effect of protein ingestion during physiological hyperinsulinaemia, most probably because they fail to signal to eIF2α to initiate translation and/or additional amino acids are needed to sustain translation., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Objective: To compare insulin sensitivity (M/I) and β-cell responses in youth versus adults with impaired glucose tolerance (IGT) or drug-naïve, recently diagnosed type 2 diabetes., Research Design and Methods: In 66 youth (80.3% with IGT) and 355 adults (70.7% IGT), hyperglycemic clamps were used to measure 1 ) M/I, 2 ) acute (0-10 min [first phase]) C-peptide (ACPR g ) and insulin (AIR g ) responses to glucose, 3 ) steady-state C-peptide and insulin concentrations at plasma glucose of 11.1 mmol/L, and 4 ) arginine-stimulated maximum C-peptide (ACPR max ) and insulin (AIR max ) responses at plasma glucose >25 mmol/L. The fasting C-peptide-to-insulin ratio was used as an estimate of insulin clearance., Results: Insulin sensitivity was 46% lower in youth compared with adults ( P < 0.001), and youth had greater acute and steady-state C-peptide (2.3- and 1.3-fold, respectively; each P < 0.001) and insulin responses to glucose (AIR g 3.0-fold and steady state 2.2-fold; each P < 0.001). Arginine-stimulated C-peptide and insulin responses were also greater in youth (1.6- and 1.7-fold, respectively; each P < 0.001). After adjustment for insulin sensitivity, all β-cell responses remained significantly greater in youth. Insulin clearance was reduced in youth ( P < 0.001). Participants with diabetes had greater insulin sensitivity ( P = 0.026), with lesser C-peptide and insulin responses than those with IGT (all P < 0.001) but similar insulin clearance ( P = 0.109)., Conclusions: In people with IGT or recently diagnosed diabetes, youth have lower insulin sensitivity, hyperresponsive β-cells, and reduced insulin clearance compared with adults. Whether these age-related differences contribute to declining β-cell function and/or impact responses to glucose-lowering interventions remains to be determined., (© 2018 by the American Diabetes Association.)

Cells respond to various extracellular stimuli through a limited number of signaling pathways. One strategy to process such stimuli is to code the information into the temporal patterns of molecules. Although we showed that insulin selectively regulated molecules depending on its temporal patterns using Fao cells, the in vivo mechanism remains unknown. Here, we show how the insulin-AKT pathway processes the information encoded into the temporal patterns of blood insulin. We performed hyperinsulinemic-euglycemic clamp experiments and found that, in the liver, all temporal patterns of insulin are encoded into the insulin receptor, and downstream molecules selectively decode them through AKT. S6K selectively decodes the additional secretion information. G6Pase interprets the basal secretion information through FoxO1, while GSK3β decodes all secretion pattern information. Mathematical modeling revealed the mechanism via differences in network structures and from sensitivity and time constants. Given that almost all hormones exhibit distinct temporal patterns, temporal coding may be a general principle of system homeostasis by hormones., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Impaired insulin sensitivity is a key abnormality underlying the development of type 2 diabetes. Measuring insulin sensitivity is therefore of importance in identifying individuals at risk of developing diabetes and for the evaluation of diabetes-focused interventions. A number of measures have been proposed for this purpose. Among these the hyperinsulinemic euglycemic clamp (HEC) is considered the gold standard. However, as the HEC is a costly, time consuming and invasive method requiring trained staff, there is a need for simpler so called surrogate measures., Main Message: A frequently used approach to evaluate surrogate measures is through correlation with the HEC. We discuss limitations with this method. We suggest other aspects to take into consideration, such as repeatability, reproducibility, systematic biases and discrimination ability. In addition, we focus on three frequently used surrogate measures. We argue that they are one-to-one transformations of each other, and therefore question the benefits of further comparison between them. They give the same results in all rank-based methods, for instance Spearman correlations, Mann-Whitney tests and receiver operating characteristic (ROC) analysis., Conclusions: We suggest investigating further aspects than correlation alone when evaluating a surrogate measure of insulin sensitivity. We recommend choosing one of the three surrogate measures HOMA-IR, QUICKI and FIRI for analysis of a clinical study.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) composition in skeletal muscle have been linked to insulin sensitivity. We evaluated the relationships between skeletal muscle PC:PE, physical exercise and insulin sensitivity. We performed lipidomics and measured PC and PE in m. vastus lateralis biopsies obtained from 13 normoglycemic normal weight men and 13 dysglycemic overweight men at rest, immediately after 45 min of cycling at 70% maximum oxygen uptake, and 2 h post-exercise, before as well as after 12 weeks of combined endurance- and strength-exercise intervention. Insulin sensitivity was monitored by euglycemic-hyperinsulinemic clamp. RNA-sequencing was performed on biopsies, and mitochondria and lipid droplets were quantified on electron microscopic images. Exercise intervention for 12 w enhanced insulin sensitivity by 33%, skeletal muscle levels of PC by 21%, PE by 42%, and reduced PC:PE by 16%. One bicycle session reduced PC:PE by 5%. PC:PE correlated negatively with insulin sensitivity (β = -1.6, P < 0.001), percent area of mitochondria (ρ = -0.52, P = 0.035), and lipid droplet area (ρ = 0.55, P = 0.017) on EM pictures, and negatively with oxidative phosphorylation and mTOR based on RNA-sequencing. In conclusion, PC and PE contents of skeletal muscle respond to exercise, and PC:PE is inversely related to insulin sensitivity.

Objective: To examine whether lowering plasma glucose concentration with the sodium-glucose transporter-2 inhibitor empagliflozin improves β-cell function in patients with type 2 diabetes mellitus (T2DM)., Methods: Patients with T2DM (N = 15) received empagliflozin (25 mg/d) for 2 weeks. β-Cell function was measured with a nine-step hyperglycemic clamp (each step, 40 mg/dL) before and at 48 hours and at 14 days after initiating empagliflozin., Results: Glucosuria was recorded on days 1 and 14 [mean ± standard error of the mean (SEM), 101 ± 10 g and 117 ± 11 g, respectively] after initiating empagliflozin, as were reductions in fasting plasma glucose levels (25 ± 6 mg/dL and 38 ± 8 mg/dL, respectively; both P < 0.05). After initiating empagliflozin and during the stepped hyperglycemic clamp, the incremental area under the plasma C-peptide concentration curve increased by 48% ± 12% at 48 hours and 61% ± 10% at 14 days (both P < 0.01); glucose infusion rate increased by 15% on day 3 and 16% on day 14, compared with baseline (both P < 0.05); and β-cell function, measured with the insulin secretion/insulin resistance index, increased by 73% ± 21% at 48 hours and 112% ± 20% at 14 days (both P < 0.01). β-cell glucose sensitivity during the hyperglycemic clamp was enhanced by 42% at 14 hours and 54% at 14 days after initiating empagliflozin (both P < 0.01)., Conclusion: Lowering the plasma glucose concentration with empagliflozin in patients with T2DM augmented β-cell glucose sensitivity and improved β-cell function.

Sitagliptin, a dipeptidyl peptidase-IV inhibitor (DPP-4), sustains activity of the incretin hormones GLP-1 and GIP and improves hyperglycemia in Type 2 diabetes mellitus (T2DM). It has however proven challenging to quantify the effect of sitagliptin on rates of insulin secretion (ISR) during a prandial challenge. The tight feedback governance of ISR by plasma glucose means that in the face of treatment-related lowering of postprandial glycemia, corresponding stimulation of ISR is lessened. We postulated that sustaining a stable level of moderate hyperglycemia before and during a meal challenge (MC) would be a platform that enables greater clarity to assess the effect of sitagliptin on ISR and an approach that could be valuable to evaluate novel targets that increase insulin secretion directly and by augmenting incretins. A hyperglycemic clamp (HGC) at 160 mg/dl was conducted in 12 healthy volunteers (without diabetes) for 6 h; 3 h into the HGC, MC was administered while maintaining stable hyperglycemia of the HGC for an additional 3 h. Modeling of C-peptide response was used to calculate ISR. In crossover design of three periods (sitagliptin twice and placebo once), the effect of sitagliptin vs. placebo on ISR and the reproducibility of the response to sitagliptin were assessed. Sitagliptin increased ISR compared with placebo by 50% and 20% during the HGC alone and the HGC-MC phases, respectively ( P < 0.001 for both). There was an associated significant treatment-based increase in circulating insulin, as well as active levels of GLP-1. Robust reproducibility of the sitagliptin-mediated ISR response was observed; the intraclass correlation value was 0.94. The findings delineate the effect of sitagliptin to stimulate insulin secretion, and these benchmark data also demonstrate that an HGC-MC can be a useful platform for interrogating therapeutic targets that could potentially modulate ISR via direct action on beta-cells as well as by augmenting release or action of incretins.

Objective: Lactate is an intermediate of glucose metabolism that has been implicated in the pathogenesis of insulin resistance. This study evaluated the relationship between glucose kinetics and plasma lactate concentration ([LAC]) before and after manipulating insulin sensitivity by progressive weight loss., Methods: Forty people with obesity (BMI = 37.9 ± 4.3 kg/m 2 ) were randomized to weight maintenance (n = 14) or weight loss (n = 19). Subjects were studied before and after 6 months of weight maintenance and before and after 5%, 11%, and 16% weight loss. A hyperinsulinemic-euglycemic clamp procedure in conjunction with [6,6- 2 H 2 ]glucose tracer infusion was used to assess glucose kinetics., Results: At baseline, fasting [LAC] correlated positively with endogenous glucose production rate (r = 0.532; P = 0.001) and negatively with insulin sensitivity, assessed as the insulin-stimulated glucose disposal (r = -0.361; P = 0.04). Progressive (5% through 16%) weight loss caused a progressive decrease in fasting [LAC], and the decrease in fasting [LAC] after 5% weight loss was correlated with the decrease in endogenous glucose production (r = 0.654; P = 0.002) and the increase in insulin sensitivity (r = -0.595; P = 0.007)., Conclusions: This study demonstrates the interrelationships among weight loss, hepatic and muscle glucose kinetics, insulin sensitivity, and [LAC], and it suggests that [LAC] can serve as an additional biomarker of glucose-related insulin resistance., (© 2018 The Obesity Society.)

Context: Complete loss of β-cell function in patients with type 1 diabetes mellitus (T1DM) may lead to an increased risk of severe hypoglycemia., Objective: We aimed to determine the impact of C-peptide status on glucagon response and endogenous glucose production (EGP) during hypoglycemia in patients with T1DM., Design and Setting: We conducted an open, comparative trial., Patients: Ten C-peptide positive (C-pos) and 11 matched C-peptide negative (C-neg) patients with T1DM were enrolled., Intervention: Plasma glucose was normalized over the night fast, and after a steady-state (baseline) plateau all patients underwent a hyperinsulinemic, stepwise hypoglycemic clamp with glucose plateaus of 5.5, 3.5, and 2.5 mmol/L and a recovery phase of 4.0 mmol/L. Blood glucagon was measured with a specific and highly sensitive glucagon assay. EGP was determined with a stable isotope tracer technique., Main Outcome Measure: Impact of C-peptide status on glucagon response and EGP during hypoglycemia., Results: Glucagon concentrations were significantly lower in C-pos and C-neg patients than previously reported. At baseline, C-pos patients had higher glucagon concentrations than C-neg patients (8.39 ± 4.6 vs 4.19 ± 2.4 pmol/L, P = 0.016, mean ± standard deviation) but comparable EGP rates (2.13 ± 0.2 vs 2.04 ± 0.3 mg/kg/min, P < 0.391). In both groups, insulin suppressed glucagon levels, but hypoglycemia revealed significantly higher glucagon concentrations in C-pos than in C-neg patients. EGP was significantly higher in C-pos patients at hypoglycemia (2.5 mmol/L) compared with C-neg patients., Conclusions: Glucagon concentrations and EGP during hypoglycemia were more pronounced in C-pos than in C-neg patients, which indicates that preserved β-cell function may contribute to counterregulation during hypoglycemia in patients with T1DM.

Objective: Insulin resistance is a major risk factor for type 2 diabetes. ApolipoproteinJ (ApoJ) has been implicated in altered pathophysiologic states including cardiovascular and Alzheimer's disease. However, the function of ApoJ in regulation of glucose homeostasis remains unclear. This study sought to determine whether serum ApoJ levels are associated with insulin resistance in human subjects and if they change after interventions that improve insulin sensitivity., Methods: Serum ApoJ levels and insulin resistance status were assessed in nondiabetic (ND) and type 2 diabetic (T2D) subjects. The impacts of rosiglitazone or metformin therapy on serum ApoJ levels and glucose disposal rate (GDR) during a hyperinsulinemic/euglycemic clamp were evaluated in a separate cohort of T2D subjects. Total ApoJ protein or that associated with the HDL and LDL fractions was measured by immunoblotting or ELISA., Results: Fasting serum ApoJ levels were greatly elevated in T2D subjects (ND vs T2D; 100±8.3 vs. 150.6±8.5AU, P<0.0001). Circulating ApoJ levels strongly correlated with fasting glucose, fasting insulin, HOMA-IR, and BMI. ApoJ levels were significantly and independently associated with HOMA-IR, even after adjustment for age, sex, and BMI. Rosiglitazone treatment in T2D subjects resulted in a reduction in serum ApoJ levels (before vs. after treatment; 100±13.9 vs. 77±15.2AU, P=0.015), whereas metformin had no effect on ApoJ levels. The change in ApoJ levels during treatment was inversely associated with the change in GDR. Interestingly, ApoJ content in the LDL fraction was inversely associated with HOMA-IR., Conclusion: Serum ApoJ levels are closely correlated with the magnitude of insulin resistance regardless of obesity, and decrease along with improvement of insulin resistance in response only to rosiglitazone in type 2 diabetes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: A glucose clamp procedure is the most reliable way to quantify insulin pharmacokinetics and pharmacodynamics, but skilled and trained research personnel are required to frequently adjust the glucose infusion rate. A computer environment that simulates glucose clamp experiments can be used for efficient personnel training and development and testing of algorithms for automated glucose clamps., Methods: We built 17 virtual healthy subjects (mean age, 25±6 years; mean body mass index, 22.2±3 kg/m 2 ), each comprising a mathematical model of glucose regulation and a unique set of parameters. Each virtual subject simulates plasma glucose and insulin concentrations in response to intravenous insulin and glucose infusions. Each virtual subject provides a unique response, and its parameters were estimated from combined intravenous glucose tolerance test-hyperinsulinemic-euglycemic clamp data using the Bayesian approach. The virtual subjects were validated by comparing their simulated predictions against data from 12 healthy individuals who underwent a hyperglycemic glucose clamp procedure., Results: Plasma glucose and insulin concentrations were predicted by the virtual subjects in response to glucose infusions determined by a trained research staff performing a simulated hyperglycemic clamp experiment. The total amount of glucose infusion was indifferent between the simulated and the real subjects (85±18 g vs. 83±23 g; p=NS) as well as plasma insulin levels (63±20 mU/L vs. 58±16 mU/L; p=NS)., Conclusions: The virtual subjects can reliably predict glucose needs and plasma insulin profiles during hyperglycemic glucose clamp conditions. These virtual subjects can be used to train personnel to make glucose infusion adjustments during clamp experiments., (Copyright © 2017 Diabetes Canada. Published by Elsevier Inc. All rights reserved.)

Context: Pheochromocytoma and paraganglioma are catecholamine-producing tumors that often impair glucose tolerance. The effects of these tumors on insulin sensitivity and insulin secretion in patients have remained unclear, however., Objective: To characterize the influence of pheochromocytoma or paraganglioma on glucose tolerance, we comprehensively analyzed various parameters related to insulin secretion or insulin sensitivity in patients with these tumors., Design: Hyperglycemic and hyperinsulinemic-euglycemic clamps, as well as an oral glucose tolerance test (OGTT), were performed in patients before and after tumor excision., Setting: Patients underwent metabolic analyses on admission to Kobe University Hospital., Patients: Eleven patients with pheochromocytoma and two with paraganglioma were examined., Intervention: None., Main Outcome Measures: We evaluated various parameters related to insulin secretion or insulin sensitivity as determined by an OGTT and by hyperglycemic and hyperinsulinemic-euglycemic clamp analyses., Results: Surgical treatment of the tumor reduced urinary catecholamine excretion and improved glucose tolerance. The insulinogenic index, but not total insulin secretion, measured during the OGTT as well as the first phase, but not the second phase, of insulin secretion during the hyperglycemic clamp were improved after surgery. The insulin sensitivity index determined during the hyperinsulinemic-euglycemic clamp remained unchanged after surgery., Conclusion: These results suggest pheochromocytoma and paraganglioma impair glucose tolerance primarily through impairment of insulin secretion-in particular, that of the early phase of the insulin secretory response. A prospective study with more patients is warranted to further confirm these results., (Copyright © 2017 Endocrine Society)

Background/objective: Impairments in metabolic flexibility (MF) and substrate handling are associated with metabolic syndrome. However, it is unknown whether metabolic inflexibility causes insulin resistance. We therefore measured MF and substrate handling before and after 8 weeks of overfeeding in initially healthy adults as a model of the early stages of insulin resistance., Subjects/methods: Twenty-nine healthy men (27±5 years old; body mass index 25.5±2.3 kg m -2 ) were overfed by 40% above baseline energy requirements for 8 weeks and gained 7.6±2.1 kg of weight. Before and after overfeeding, energy expenditure, substrate oxidation and MF were measured in two ways: (a) during 1 day of eucaloric feeding in a whole-room indirect calorimeter and (b) during a two-step hyperinsulinemic-euglycemic clamp., Results: Eight weeks of overfeeding decreased insulin sensitivity at low and high doses of insulin (P=0.001 and P=0.06, respectively). This was accompanied by decreases in the respiratory quotient (RQ) while sleeping (from 0.877±0.020 to 0.864±0.026; P=0.05) and at low insulin levels during the clamp (from 0.927±0.047 to 0.907±0.032; P=0.01). Overfeeding did not affect MF as measured during a clamp (P⩾0.17), but it tended to increase 24-h MF (awake RQ-sleep RQ) as measured by chamber by 0.010±0.028 (P=0.08). In terms of substrate oxidation, overfeeding increased protein oxidation by 13±23 g day -1 (P=0.003) and tended to increase fat oxidation by 6±16 g day -1 (P=0.07) but did not affect carbohydrate oxidation (P=0.64). Individuals with greater metabolic adaptation to overfeeding had higher carbohydrate oxidation rates (r=0.66, P=8 × 10 -5 ) but not fat oxidation rates (P=0.09)., Conclusions: The early stages of insulin resistance are accompanied by modest declines in the RQs during sleep and during a clamp, with no changes in fasting RQ or signs of metabolic inflexibility. Our data therefore suggest that metabolic inflexibility does not cause insulin resistance.

Roux-en-Y gastric bypass (RYGB) induces weight loss and improves insulin sensitivity when evaluated by the hyperinsulinemic-euglycemic clamp (HEC). Surrogate indices of insulin sensitivity calculated from insulin and glucose concentrations at fasting or after an oral glucose tolerance test (OGTT) are frequently used, but have not been validated after RYGB. Our aim was to evaluate whether surrogate indices reliably estimate changes in insulin sensitivity after RYGB. Four fasting surrogates (inverse-HOMA-IR, HOMA2-%S, QUICKI, revised-QUICKI) and three OGTT-derived surrogates (Matsuda, Gutt, OGIS) were compared with HEC-estimated peripheral insulin sensitivity ( R d or R d /I, depending on how the index was originally validated) and the tracer-determined hepatic insulin sensitivity index (HISI) in patients with preoperative type 2 diabetes ( n = 10) and normal glucose tolerance ( n = 10) 1 wk, 3 mo, and 1 yr postoperatively. Post-RYGB changes in inverse-HOMA-IR and HOMA2-%S did not correlate with changes in R d at any visit, but were comparable to changes in HISI at 1 wk. Changes in QUICKI and revised-QUICKI correlated with R d /I after surgery. Changes in the Matsuda and Gutt indices did not correlate with changes in R d /I and R d , respectively, whereas OGIS changes correlated with R d changes at 1 yr post-RYGB. In conclusion, surrogate measures of insulin sensitivity may not reflect results obtained with gold standard methodology after RYGB, underscoring the importance of critical reflection when surrogate endpoints are used. Fasting surrogate indices may be particularly affected by post-RYGB changes in insulin clearance, whereas the validity of OGTT-derived surrogates may be compromised by surgical rearrangements of the gut., (Copyright © 2017 the American Physiological Society.)

The cut-off point of intrahepatic triglyceride (IHTG) content to define nonalcoholic fatty liver disease (NAFLD) by proton magnetic resonance spectroscopy ( 1 H-MRS) was established based on the 95th percentile in a group of healthy individuals (i.e., ≥5.56%). Whether this threshold correlates with metabolic and histological changes and whether a further accumulation of IHTG is associated with worsening of these parameters has not been properly assessed in a large cohort of patients. In this cross-sectional study, 352 subjects were carefully characterized with the following studies: liver 1 H-MRS; euglycemic insulin clamp with measurement of glucose turnover; oral glucose tolerance test; and a liver biopsy. Hepatic insulin sensitivity (suppression of endogenous glucose production by insulin) was affected early on after IHTG content was ∼1.5% and remained uniformly impaired (∼40%-45%), regardless of further IHTG accumulation. Skeletal muscle insulin sensitivity showed a gradual impairment at low degrees of IHTG accumulation, but remained unchanged after IHTG content reached the ∼6 ± 2% threshold. A similar pattern was observed for metabolic changes typically associated with NAFLD, such as hypertriglyceridemia and low high-density lipoprotein cholesterol (HDL-C). In contrast, adipose tissue insulin sensitivity (suppression of free fatty acids by insulin) showed a continuous worsening across the spectrum of IHTG accumulation in NAFLD (r = -0.38; P < 0.001). Histological severity of liver disease (inflammation, ballooning, and fibrosis) was not associated with the amount of IHTG content., Conclusion: IHTG accumulation is strongly associated with adipose tissue insulin resistance (IR), supporting the current theory of lipotoxicity as a driver of IHTG accumulation. Once IHTG accumulation reaches ∼6 ± 2%, skeletal muscle IR, hypertriglyceridemia, and low HDL-C become fully established. Histological activity appears to have an early threshold and is not significantly influenced by increasing amounts of IHTG accumulation. (Hepatology 2017;65:1132-1144)., (© 2016 by the American Association for the Study of Liver Diseases.)

Context and Objective: Adipose tissue insulin resistance may cause hepatic and skeletal muscle insulin resistance by releasing excess free fatty acids (FFAs). Because no consensus exists on how to quantify adipose tissue insulin sensitivity we compared three methods for measuring adipose tissue insulin sensitivity: the single step insulin clamp, the multistep pancreatic clamp, and the adipose tissue insulin resistance index (Adipo-IR)., Design and Participants: We studied insulin sensitivity in 25 adults by measuring the insulin concentration resulting in 50% suppression of palmitate flux (IC50) using both a multistep pancreatic clamp and a one-step hyperinsulinemic-euglycemic clamp. Palmitate kinetics were measured using a continuous infusion of [U-13C]palmitate. Adipo-IR was calculated from fasting insulin and fasting FFA concentrations., Results: Adipo-IR was reproducible (sample coefficient of variability, 10.0%) and correlated with the IC50 measured by the multistep pancreatic clamp technique (r, 0.86; P < 0.001). Age and physical fitness were significant predictors of the residual variation between Adipo-IR and IC50, with a positive relationship with age (r, 0.47; P = 0.02) and a negative association with VO2 peak (r, -0.46; P = 0.02). Likewise, IC50 measured by the multistep pancreatic clamp technique correlated with IC50 measured using the one-step hyperinsulinemic-euglycemic clamp technique (r, 0.73; P < 0.001)., Conclusion: Adipo-IR and the one-step hyperinsulinemic-euglycemic clamp technique using a palmitate tracer are good predictors of a gold standard measure of adipose tissue insulin sensitivity. However, age and physical fitness systematically affect the predictive values. Although Adipo-IR is suitable for larger population studies, the multistep pancreatic clamp technique is probably needed for mechanistic studies of adipose tissue insulin action., (Copyright © 2017 by the Endocrine Society)

Increased endothelin-1 (ET-1) and reduced endothelial nitric oxide phosphorylation (peNOS) are hypothesized to reduce insulin-stimulated blood flow in type 2 diabetes (T2D), but studies examining these links in humans are limited. We sought to assess basal and insulin-stimulated endothelial signaling proteins (ET-1 and peNOS) in skeletal muscle from T2D patients. Ten obese T2D [glucose disposal rate (GDR): 6.6 ± 1.6 mg·kg lean body mass (LBM) -1 ·min -1 ] and 11 lean insulin-sensitive subjects (Lean GDR: 12.9 ± 1.2 mg·kg LBM -1 ·min -1 ) underwent a hyperinsulinemic-euglycemic clamp with vastus lateralis biopsies taken before and 60 min into the clamp. Basal biopsies were also taken in 11 medication-naïve, obese, non-T2D subjects. ET-1, peNOS (Ser1177), and eNOS protein and mRNA were measured from skeletal muscle samples containing native microvessels. Femoral artery blood flow was assessed by duplex Doppler ultrasound. Insulin-stimulated blood flow was reduced in obese T2D (Lean: +50.7 ± 6.5% baseline, T2D: +20.8 ± 5.2% baseline, P < 0.05). peNOS/eNOS content was higher in Lean under basal conditions and, although not increased by insulin, remained higher in Lean during the insulin clamp than in obese T2D (P < 0.05). ET-1 mRNA and peptide were 2.25 ± 0.50- and 1.52 ± 0.11-fold higher in obese T2D compared with Lean at baseline, and ET-1 peptide remained 2.02 ± 1.9-fold elevated in obese T2D after insulin infusion (P < 0.05) but did not increase with insulin in either group (P > 0.05). Obese non-T2D subjects tended to also display elevated basal ET-1 (P = 0.06). In summary, higher basal skeletal muscle expression of ET-1 and reduced peNOS/eNOS may contribute to a reduced insulin-stimulated leg blood flow response in obese T2D patients., New & Noteworthy: Although impairments in endothelial signaling are hypothesized to reduce insulin-stimulated blood flow in type 2 diabetes (T2D), human studies examining these links are limited. We provide the first measures of nitric oxide synthase and endothelin-1 expression from skeletal muscle tissue containing native microvessels in individuals with and without T2D before and during insulin stimulation. Higher basal skeletal muscle expression of endothelin-1 and reduced endothelial nitric oxide phosphorylation (peNOS)/eNOS may contribute to reduced insulin-stimulated blood flow in obese T2D patients., (Copyright © 2017 the American Physiological Society.)

Alzheimer's disease (AD) may have heterogeneous pathophysiological underpinnings, with risk factors including apolipoprotein rmvarep4 (APOE4) genotype and insulin resistance. We hypothesized that distinct phenotypes exist within AD. We examined APOE4 and metabolic biomarkers in 338 subjects (n = 213 nondemented (ND), n = 125 AD). We further characterized steady state free fatty acid (FFA) levels in a subset of 45 participants who had also participated in a hyperinsulinemic-euglycemic clamp. Insulin resistance (HOMA-IR) was elevated in AD versus ND (p = 0.04) and in APOE4 noncarriers versus carriers (p < 0.01). This was driven by increased fasting insulin in AD versus ND (p < 0.01) and in APOE4 non-carriers versus carriers (p = 0.01). Fasting glucose was not different. In subjects who underwent a clamp, there was a group x genotype interaction on FFA levels during hyperinsulinemia (p = 0.03). APOE4 non-carriers with AD had higher FFA levels, while APOE4 carriers with AD exhibited lower FFA levels. Metabolic dysfunction is overrepresented in individuals with AD dementia who do not carry the APOE4 allele. This suggests that important subsets of AD phenotypes may exist that diverge metabolically.

Aims: We compare spectral analysis of photoplethysmography (PTG) with insulin resistance measured by the hyperinsulinemic euglycemic clamp (HEC) technique., Material and Method: A total of 100 nondiabetic subjects, 43 men and 57 women aged 20-63years, 30 lean, 42 overweight and 28 obese were enrolled in the study. These patients underwent an examination with HEC, and an examination with the PTG spectral analysis and calculation of the PTG Total Power (PTG-TP). Receiver-operating characteristic (ROC) curves were constructed to determine the specificity and sensitivity of PTG-TP in the assessment of insulin resistance., Results: There is a moderate correlation between insulin sensitivity (M-value) and PTG-TP (r=- 0.64, p<0.0001). The ROC curves showed that the most relevant cutoff to the whole study group was a PTG-TP>406.2. This cut-off had a sensitivity=95.7%, specificity =84,4% and the area under the ROC curve (AUC)=0.929 for identifying insulin resistance. All AUC ROC curve analysis were significant (p<0.0001)., Conclusion: The use of the PTG-TP marker measured from the PTG spectral analysis is a useful tool in screening and follow up of IR, especially in large-scale studies., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: In vitro, beta cells immediately secrete stored but readily releasable insulin in response to a rise of glucose. During a prolonged insulin response, this is followed by newly synthesized insulin. Our aim was to develop an in vivo test to determine the ratio between readily available and newly synthesized insulin after a stimulus in humans by labelling newly synthesized insulin., Methods: A stable isotope tracer of 1.0 g 13 C leucine with C-peptide as target peptide was administered 45 min prior to 75 g glucose load of a frequently blood sampled 210-min oral glucose tolerance test (OGTT). Our OGTT also encompassed collection of urine, which has a high content of C-peptide. Prior, the optimal conditions under which the tracer 13 C leucine was administered for enrichment of (pre) proinsulin were established. Also, techniques to obtain urinary C-peptide under highly purified circumstances were set up. Our main outcome measure was the stable isotope enrichment of de novo C-peptide, which we related to early plasma insulin and glucose AUC. Twelve healthy Caucasian individuals (M4F8, age 41.8 ± 2.3, BMI 28.3 ± 1.7) with normal glucose tolerance underwent our OGTT., Results: We found that during a 75-g OGTT, newly synthesized insulin contributed approximately 20 % of total insulin secretion. The pattern of isotope enrichment obtained by collecting multiple urine voids was suggestive that the newly synthesized insulin contributes to the late phase of insulin secretion. De novo C-peptide correlated negatively with both early plasma insulin AUC (r = -0.629, P = 0.028) and early plasma glucose AUC (r = -0.605, P = 0.037)., Conclusions: With stable isotope technique added to OGTT, we were able to measure newly synthesized insulin in healthy individuals. This new technique holds the promise that it is feasible to develop a direct in vivo beta cell function test., Competing Interests: The authors declare that they have no conflict of interest. Ethical standard The authors declare that no ethical issues apply to the present study. Human and animal rights disclosure All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent disclosure Informed consent was obtained from all patients for being included in the study.

Aims: Insulin resistance underlies the etiology of both type 2 diabetes and gestational diabetes. In pregnancy, insulin resistance is also associated with an unfavorable metabolic programming of the fetus, potentially contributing to a higher risk of obesity and type 2 diabetes in the offspring. To assess insulin sensitivity, several methods based on glucose and insulin levels during a 75-g oral glucose tolerance test (OGTT) exist. It is unclear how they perform during pregnancy, where physiologically altered metabolism could introduce a bias., Methods: In a cohort comprising 476 non-diabetic subjects undergoing OGTT and hyperinsulinemic-euglycemic clamp (HEC), we used cross-validation to develop an insulin sensitivity index also based on non-esterified fatty acids (NEFA) that could be more robust during pregnancy (NEFA-index). We tested commonly used OGTT-based indexes and the NEFA-index in a different cohort of 42 women during pregnancy and 1 year after delivery., Results: The Matsuda and OGIS index failed to detect lower insulin sensitivity during pregnancy as compared to the follow-up OGTT 1 year after delivery (p > 0.09). The new NEFA-index incorporating BMI, plasma insulin and NEFA, but not glucose, clearly indicated lower insulin sensitivity during pregnancy (p < 0.0001). In the non-pregnant cohort, this NEFA-index correlated well with the gold-standard HEC-based insulin sensitivity index, and outperformed other tested indexes for the prediction of HEC-measured insulin resistance., Conclusions: This insulin/NEFA-based approach is feasible, robust, and could be consistently used to estimate insulin sensitivity also during pregnancy.

Introduction: Anesthesia induces insulin resistance, which may contribute to elevated blood glucose and adverse post-operative outcomes in critically ill patients, and impair glycemic control in surgical patients with diabetes. However, little is known about the mechanisms by which anesthesia impairs insulin sensitivity. Here we investigate the effects of anesthesia on insulin sensitivity in metabolic tissues., Methods: Hyperinsulinemic-euglycemic clamps were performed in 32 lean (control diet; n = 16 conscious versus n = 16 anesthetized) and 24 fat-fed (6 weeks fat-feeding; n = 16 conscious versus n = 8 anesthetized) adult male mongrel dogs in conjunction with tracer methodology to differentiate hepatic versus peripheral insulin sensitivity. Propofol was administered as an intravenous bolus (3mg/kg) to initiate anesthesia, which was then maintained with inhaled sevoflurane or isoflurane (2-3%) for the duration of the procedure., Results: Anesthesia reduced peripheral insulin sensitivity by approximately 50% in both lean and fat-fed animals as compared to conscious animals, and insulin action at the liver was almost completely suppressed during anesthesia such that hepatic insulin sensitivity was decreased by 75.5% and; 116.2% in lean and fat-fed groups, respectively., Conclusion: Inhaled anesthesia induces severe hepatic insulin resistance in a canine model. Countermeasures that preserve hepatic insulin sensitivity may represent a therapeutic target that could improve surgical outcomes in both diabetic and healthy patients., Competing Interests: The authors have declared that no competing interests exist.