Hyperglycemic Hyperinsulinemic Clamp Research Articles

A Cyclic Guanosine Monophosphate–Dependent Pathway Can Regulate Net Hepatic Glucose Uptake in Vivo

We previously showed that hepatic nitric oxide regulates net hepatic glucose uptake (NHGU), an effect that can be eliminated by inhibiting hepatic soluble guanylate cyclase (sGC), suggesting that the sGC pathway is involved in the regulation of NHGU. The aim of the current study was to determine whether hepatic cyclic guanosine monophosphate (cGMP) reduces NHGU. Studies were performed on conscious dogs with transhepatic catheters. A hyperglycemic-hyperinsulinemic clamp was established in the presence of portal vein glucose infusion. 8-Br-cGMP (50 µg/kg/min) was delivered intraportally, and either the glucose load to the liver (CGMP/GLC; n = 5) or the glucose concentration entering the liver (CGMP/GCC; n = 5) was clamped at 2× basal. In the control group, saline was given intraportally (SAL; n = 10), and the hepatic glucose concentration and load were doubled. 8-Br-cGMP increased portal blood flow, necessitating the two approaches to glucose clamping in the cGMP groups. NHGU (mg/kg/min) was 5.8 ± 0.5, 2.7 ± 0.5, and 4.8 ± 0.3, whereas the fractional extraction of glucose was 11.0 ± 1, 5.5 ± 1, and 8.5 ± 1% during the last hour of the study in SAL, CGMP/GLC, and CGMP/GCC, respectively. The reduction of NHGU in response to 8-Br-cGMP was associated with increased AMP-activated protein kinase phosphorylation. These data indicate that changes in liver cGMP can regulate NHGU under postprandial conditions.

In vivo effect of glucose-dependent insulinotropic peptide (GIP) on the gene expression of calcitonin peptides in human subcutaneous adipose tissue

BackgroundIncreased plasma levels of calcitonin gene-related peptide‐I (CGRP-I) and procalcitonin (Pro-CT) (both also named calcitonin peptides (CT peptides)) are associated with obesity and systemic inflammation. Glucose-dependent insulinotropic polypeptide (GIP), a nutrient-dependent incretin hormone, was recently found to induce CGRP-I and CT expression in human adipocytes in vitro. However, a physiological relevance of a possible interaction between GIP and CT peptides has not yet been studied. MethodsIn this study, we analyzed the effect of GIP on the expression of CGRP-I and CT mRNA in human subcutaneous adipose tissue within a randomized, controlled trial. Seventeen male obese subjects were infused with GIP [2.0pmolkg−1min−1 for 240min] or placebo, either in the fasting state, during euglycemic–hyperinsulinemic (EC) or hyperglycemic–hyperinsulinemic clamps (HC). ResultsThe CGRP-I gene expression was detected in all investigated adipose tissue samples, whereas very low CT expression was found in only 8 out of 116 analyzed samples. No significant influence of either GIP or glucose and insulin infusions on the CGRP-I and CT expression was observed in any of the individual experiments (GIP infusion, EC and HC) or in the combined analysis of all experiments with and without GIP. Furthermore, CGRP-I expression was not correlated with plasma GIP level before or after 240min of infusions or clamps. ConclusionIn contrast to in vitro data, an acute application of GIP has no effect on mRNA expression of CT peptides in subcutaneous adipose tissue of obese humans.

Insulin Up-Regulates Natriuretic Peptide Clearance Receptor Expression in the Subcutaneous Fat Depot in Obese Subjects: A Missing Link between CVD Risk and Obesity?

Natriuretic peptides (NP) regulate cardiovascular homeostasis and have multiple metabolic properties. Decreased levels of NP or "natriuretic handicap" are signs of insulin resistance such as central obesity. Increased expression of NP clearance receptor (NPRC) in sc adipose tissue (SAT) was observed in insulin-resistant subjects. We hypothesized that insulin acutely regulates NP receptor expression in adipose tissue. NPRA, NPRB, and NPRC mRNA expression was measured in paired samples of visceral adipose tissue (VAT) and SAT from 157 subjects (108 with type 2 diabetes). The effect of insulin on NPR gene expression in SAT was studied in euglycemic-hyperinsulinemic and hyperglycemic-hyperinsulinemic clamp experiments. Additionally, the effect of insulin and glucose on NPR expression in the culture of primary human monocytes and macrophages was tested. NPRA and NPRC gene expression was higher in VAT compared with SAT (P < 0.01), but only NPRC gene expression strongly correlated with fasting insulin levels (r = 0.65, P = 0.04 × 10(-3); and r = 0.54, P = 0.002, for VAT and SAT, respectively). NPRB expression was lower in VAT than in SAT in subjects with type 2 diabetes and was lower compared with nondiabetic subjects. NPRC gene expression was up-regulated in SAT during both euglycemic- and hyperglycemic-hyperinsulinemic clamps (P = 0.038 and P = 0.048, respectively), and was increased in high glucose and insulin treatment in monocytes (70.2%; P = 0.01), but not in mature macrophages. Insulin increased expression of NPRC in SAT independently of circulating glucose concentrations. Thus, insulin might suppress circulating NP via up-regulation of NPRC expression in obesity, providing a novel link between hyperinsulinemia and obesity.

Effects of raising muscle glycogen synthesis rate on skeletal muscle ATP turnover rate in type 2 diabetes

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. We hypothesized that any impairment in insulin-stimulated muscle ATP production could merely reflect the lower rates of muscle glucose uptake and glycogen synthesis, rather than cause it. If this is correct, muscle ATP turnover rates in type 2 diabetes could be increased if glycogen synthesis rates were normalized by the mass-action effect of hyperglycemia. Isoglycemic- and hyperglycemic-hyperinsulinemic clamps were performed on type 2 diabetic subjects and matched controls, with muscle ATP turnover and glycogen synthesis rates measured using 31P- and 13C-magnetic resonance spectroscopy, respectively. In diabetic subjects, hyperglycemia increased muscle glycogen synthesis rates to the level observed in controls at isoglycemia [from 19 ± 9 to 41 ± 12 μmol·l−1·min−1 (P = 0.012) vs. 40 ± 7 μmol·l−1·min−1 in controls]. This was accompanied by a modest increase in muscle ATP turnover rates (7.1 ± 0.5 vs. 8.6 ± 0.7 μmol·l−1·min−1, P = 0.04). In controls, hyperglycemia brought about a 2.5-fold increase in glycogen synthesis rates (100 ± 24 vs. 40 ± 7 μmol·l−1·min−1, P = 0.028) and a 23% increase in ATP turnover rates (8.1 ± 0.9 vs. 10.0 ± 0.9 μmol·l−1·min−1, P = 0.025) from basal state. Muscle ATP turnover rates correlated positively with glycogen synthesis rates (rs = 0.46, P = 0.005). Changing the rate of muscle glucose metabolism in type 2 diabetic subjects alters demand for ATP synthesis at rest. In type 2 diabetes, skeletal muscle ATP turnover rates reflect the rate of glucose uptake and glycogen synthesis, rather than any primary mitochondrial defect.

A Multiple Local Models Approach to Accuracy Improvement in Continuous Glucose Monitoring

Continuous glucose monitoring (CGM) devices estimate plasma glucose (PG) from measurements in compartments alternative to blood. The accuracy of currently available CGM is yet unsatisfactory and may depend on the implemented calibration algorithms, which do not compensate adequately for the differences of glucose dynamics between the compartments. Here we propose and validate an innovative calibration algorithm for the improvement of CGM performance. CGM data from GlucoDay(®) (A. Menarini, Florence, Italy) and paired reference PG have been obtained from eight subjects without diabetes during eu-, hypo-, and hyperglycemic hyperinsulinemic clamps. A calibration algorithm based on a dynamic global model (GM) of the relationship between PG and CGM in the interstitial space has been obtained. The GM is composed by independent local models (LMs) weighted and added. LMs are defined by a combination of inputs from the CGM and by a validity function, so that each LM represents to a variable extent a different metabolic condition and/or sensor-subject interaction. The inputs best suited for glucose estimation were the sensor current I and glucose estimations Ĝ, at different time instants [I(k), I(k)(-1), Ĝ(k)(-1)] (IIG). In addition to IIG, other inputs have been used to obtain the GM, achieving different configurations of the calibration algorithm. Even in its simplest configuration considering only IIG, the new calibration algorithm improved the accuracy of the estimations compared with the manufacturer's estimate: mean absolute relative difference (MARD)=10.8±1.5% versus 14.7±5.4%, respectively (P=0.012, by analysis of variance). When additional exogenous signals were considered, the MARD improved further (7.8±2.6%, P<0.05). The LM technique allows for the identification of intercompartmental glucose dynamics. Inclusion of these dynamics into the calibration algorithm improves the accuracy of PG estimations.

Effect of diacerein on insulin secretion and metabolic control in drug-naive patients with type 2 diabetes: a randomized clinical trial.

OBJECTIVETo assess the effect of diacerein on insulin secretion and metabolic control in drug-naïve patients with type 2 diabetes.RESEARCH DESIGN AND METHODSA randomized, double-blind, placebo-controlled clinical trial was carried out in 40 drug-naïve adult patients with type 2 diabetes. A metabolic profile including interleukin (IL)-1β, tumor necrosis factor-α, IL-6, and fasting insulin levels was carried out before the intervention and 2 months afterward. A hyperglycemic-hyperinsulinemic clamp technique was performed to assess the phases of insulin secretion and insulin sensitivity. After randomization, 20 patients received diacerein (50 mg once daily) for the first 15 days and twice daily for 45 additional days. The remaining patients received placebo. Intra- and intergroup differences were calculated by Wilcoxon signed rank and Mann-Whitney U tests.RESULTSThere were significant increases in first (102 ± 63 vs. 130 ± 75 pmol/L; P < 0.01), late (219 ± 111 vs. 280 ± 135 pmol/L; P < 0.01), and total insulin (178 ± 91 vs. 216 ± 99 pmol/L; P < 0.01) secretions without changes in insulin sensitivity after diacerein administration. There were significant decreases in fasting glucose (7.9 ± 1.4 vs. 6.8 ± 1.0 mmol/L; P < 0.01) and in A1C levels (8.3 ± 1.0 vs. 7.0 ± 0.8%; P < 0.001) after diacerein administration. There were no significant changes after placebo administration in the above-mentioned evaluations.CONCLUSIONSInsulin secretion increased and metabolic control improved after diacerein administration in drug-naïve patients with type 2 diabetes.

Metabolomic linkage reveals functional interaction between glucose-dependent insulinotropic polypeptide and ghrelin in humans

The gastric peptide ghrelin promotes energy storage, appetite, and food intake. Nutrient intake strongly suppresses circulating ghrelin via molecular mechanisms possibly involving insulin and gastrointestinal hormones. On the basis of the growing evidence that glucose-dependent insulinotropic polypeptide (GIP) is involved in the control of fuel metabolism, we hypothesized that GIP and/or insulin, directly or via changes in plasma metabolites, might affect circulating ghrelin. Fourteen obese subjects were infused with GIP (2.0 pmol·kg(-1)·min(-1)) or placebo in the fasting state during either euglycemic hyperinsulinemic (EC) or hyperglycemic hyperinsulinemic clamps (HC). Apart from analysis of plasma ghrelin and insulin levels, GC-TOF/MS analysis was applied to create a hormone-metabolite network for each experiment. The GIP and insulin effects on circulating ghrelin were analyzed within the framework of those networks. In the HC, ghrelin levels decreased in the absence (19.2% vs. baseline, P = 0.028) as well as in the presence of GIP (33.8%, P = 0.018). Ghrelin levels were significantly lower during HC with GIP than with placebo, despite insulin levels not differing significantly. In the GIP network combining data on GIP-infusion, EC+GIP and HC+GIP experiments, ghrelin was integrated into hormone-metabolite networks through a connection to a group of long-chain fatty acids. In contrast, ghrelin was excluded from the network of experiments without GIP. GIP decreased circulating ghrelin and might have affected the ghrelin system via modification of long-chain fatty acid pools. These observations were independent of insulin and offer potential mechanistic underpinnings for the involvement of GIP in systemic control of energy metabolism.

Impaired Leucine Oxidation During Hyperglycemia After Eccentric Exercise in Older Men

Aging reduces insulin sensitivity in healthy sedentary adults, which may alter protein metabolism during periods of metabolic and physiologic stress. We determined the effects of age, and metabolic (hyperglycemia) and physiological [eccentric exercise (ECE)] stress, on whole-body amino acid turnover. [1-13C]-Leucine kinetics was measured in 8 healthy young (YM, 22±2 y; BMI 23±5 kg/m2) and 8 healthy older (OM, 66±6 y; BMI 25±3 kg/m2) men during fasting (F) and 3-hr hyperglycemic-hyperinsulinemic clamps (HG) that were performed before (CON) and 48 hours after ECE. Leucine oxidation (Lox) decreased (p<0.05) in YM during CON-HG, ECE-F and ECE-HG (CON-F 28.3±3.9; CON-HG 25.9±4.0; ECE-F 24.8±4.1; ECE-HG 21.4±3.2 μmol·kg FFM−1·h−1). In contrast, OM failed to suppress Lox from CON-F conditions (CON-F 26.0±3.9; CON-HG 26.8±3.7; ECE-F 25.1±5.7; ECE-HG 26.3±7.0 μmol·kg FFM−1·h−1; p>0.05). Insulin sensitivity was inversely correlated with Lox (r = −0.53; p<0.05) and was lower in OM (YM 7.1±0.6; OM 4.5±0.3 mg·kg FFM−1·min−1; p<0.05). Nonoxidative leucine disposal and leucine flux were not different between YM and OM (p>0.05). In conclusion, aging leads to an impaired capacity to adjust protein metabolism in response to metabolic or physiological stress, which is coupled with decreased insulin sensitivity. Supported by NIH grants T32DK007319 AG12834, AG15385, AG11811, RR000954, DK020579, DK056341

Regulation of hepatic fat and glucose oxidation in rats with lipid-induced hepatic insulin resistance

Pyruvate dehydrogenase plays a critical role in the regulation of hepatic glucose and fatty acid oxidation; however, surprisingly little is known about its regulation in vivo. In this study we examined the individual effects of insulin and substrate availability on the regulation of pyruvate dehydrogenase flux (V(PDH) ) to tricarboxylic acid flux (V(TCA) ) in livers of awake rats with lipid-induced hepatic insulin resistance. V(PDH) /V(TCA) flux was estimated from the [4-(13) C]glutamate/[3-(13) C]alanine enrichments in liver extracts and assessed under conditions of fasting and during a hyperinsulinemic-euglycemic clamp, whereas the effects of increased plasma glucose concentration on V(PDH) /V(TCA) flux was assessed during a hyperglycemic clamp in conjunction with infusions of somatostatin and insulin to maintain basal concentrations of insulin. The effects of increases in both glucose and insulin on V(PDH) /V(TCA) were examined during a hyperinsulinemic-hyperglycemic clamp. The effects of chronic lipid-induced hepatic insulin resistance on this flux were also examined by performing these measurements in rats fed a high-fat diet for 3 weeks. Using this approach we found that fasting V(PDH) /V(TCA) was reduced by 95% in rats with hepatic insulin resistance (from 17.2 ± 1.5% to 1.3 ± 0.7%, P < 0.00001). Surprisingly, neither hyperinsulinemia per se or hyperglycemia per se were sufficient to increase V(PDH) /V(TCA) flux. Only under conditions of combined hyperglycemia and hyperinsulinemia did V(PDH) /V(TCA) flux increase (44.6 ± 3.2%, P < 0.0001 versus basal) in low-fat fed animals but not in rats with chronic lipid-induced hepatic insulin resistance. These studies demonstrate that the combination of both hyperinsulinemia and hyperglycemia are required to increase V(PDH) /V(TCA) flux in vivo and that this flux is severely diminished in rats with chronic lipid-induced hepatic insulin resistance.

Effect of a 2-h hyperglycemic–hyperinsulinemic glucose clamp to promote glucose storage on endurance exercise performance

Carbohydrate stores within muscle are considered essential as a fuel for prolonged endurance exercise, and regimes for enhancing such stores have proved successful in aiding performance. This study explored the effects of a hyperglycaemic-hyperinsulinemic clamp performed 18 h previously on subsequent prolonged endurance performance in cycling. Seven male subjects, accustomed to prolonged endurance cycling, performed 90 min of cycling at ~65% VO(2max) followed by a 16-km time trial 18 h after a 2-h hyperglycemic-hyperinsulinemic clamp (HCC). Hyperglycemia (10 mM) with insulin infused at 300 mU/m(2)/min over a 2-h period resulted in a total glucose uptake of 275 g (assessed by the area under the curve) of which glucose storage accounted for about 73% (i.e. 198 g). Patterns of substrate oxidation during 90-min exercise at 65% VO(2max) were not altered by HCC. Blood glucose and plasma insulin concentrations were higher during exercise after HCC compared with control (p < 0.05) while plasma NEFA was similar. Exercise performance was improved by 49 s and power output was 10-11% higher during the time trial (p < 0.05) after HCC. These data suggest that carbohydrate loading 18 h previously by means of a 2-h HCC improves cycling performance by 3.3% without any change in pattern of substrate oxidation.

Effect of Exenatide on Fat Deposition and a Metabolic Profile in Patients with Metabolic Syndrome

The aim of this study was to evaluate the effect of exenatide on fat deposition and a metabolic profile in patients with metabolic syndrome. An uncontrolled, open clinical study was carried out in 10 patients with metabolic syndrome and without pharmacological treatment. Patients received exenatide (5 μg) subcutaneously twice daily for 1 month. Before and after the intervention, metabolic profile and phases of insulin secretion and insulin sensitivity were estimated. To assess insulin secretion and sensitivity, the hyperglycemic-hyperinsulinemic clamp technique was performed. Computed tomography was performed to evaluate both subcutaneous and visceral fat. The Wilcoxon signed-rank test and Mann-Whitney U-test were used for statistical analyses. Weight, body mass index, waist circumference, and systolic blood pressure were decreased by exenatide. Subcutaneous fat deposition decreased by 4.4% compared to the basal value. There were significant decreases in total cholesterol and low-density lipoprotein cholesterol, as well as an increment in the first phase of insulin secretion after the intervention. One-month administration of exenatide significantly decreased subcutaneous fat deposition by 4.4%, improving the metabolic profile in patients with metabolic syndrome.

Insulin Resistance and Protein Energy Metabolism in Patients with Advanced Chronic Kidney Disease

Insulin resistance (IR), the reciprocal of insulin sensitivity is a known complication of advanced chronic kidney disease (CKD) and is associated with a number of metabolic derangements. The complex metabolic abnormalities observed in CKD such as vitamin D deficiency, obesity, metabolic acidosis, inflammation, and accumulation of "uremic toxins" are believed to contribute to the etiology of IR and acquired defects in the insulin-receptor signaling pathway in this patient population. Only a few investigations have explored the validity of commonly used assessment methods in comparison to gold standard hyperinsulinemic hyperglycemic clamp technique in CKD patients. An important consequence of insulin resistance is its role in the pathogenesis of protein energy wasting, a state of metabolic derangement characterized by loss of somatic and visceral protein stores not entirely accounted for by inadequate nutrient intake. In the general population, insulin resistance has been associated with accelerated protein catabolism. Among end-stage renal disease (ESRD) patients, enhanced muscle protein breakdown has been observed in patients with Type II diabetes compared to ESRD patients without diabetes. In the absence of diabetes mellitus (DM) or severe obesity, insulin resistance is detectable in dialysis patients and strongly associated with increased muscle protein breakdown, primarily mediated by the ubiquitin-proteasome pathway. Recent epidemiological data indicate a survival advantage and better nutritional status in insulin-free Type II DM patients treated with insulin sensitizer thiazolidinediones. Given the high prevalence of protein energy wasting in ESRD and its unequivocal association with adverse clinical outcomes, insulin resistance may represent an important modifiable target for intervention in the ESRD population.

Impaired-Inactivation of FoxO1 Contributes to Glucose-Mediated Increases in Serum Very Low-Density Lipoprotein

For patients with diabetes, insulin resistance and hyperglycemia both contribute to increased serum triglyceride in the form of very low-density lipoprotein (VLDL). Our objective was to define the insulin conditions in which hyperglycemia promotes increased serum VLDL in vivo. We performed hyperglycemic-hyperinsulinemic clamp studies and hyperglycemic-hypoinsulinemic clamp studies in rats, with metabolic tracers for glucose flux and de novo fatty acid synthesis. When blood glucose was clamped at hyperglycemia (17 mm) for 2 h under hyperinsulinemic conditions (4 mU/kg . min), serum VLDL levels were not increased compared with baseline. We speculated that hyperinsulinemia minimized glucose-mediated VLDL changes and performed hyperglycemic-hypoinsulinemic clamp studies in which insulin was clamped near fasting levels with somatostatin (17 mm blood glucose, 0.25 mU/kg . min insulin). Under low-insulin conditions, serum VLDL levels were increased 4.7-fold after hyperglycemia, and forkhead box O1 (FoxO1) was not excluded from the nucleus of liver cells. We tested the extent that impaired inactivation of FoxO1 by insulin was sufficient for glucose to promote increased serum VLDL. We found that, when the ability of insulin to inactivate FoxO1 is blocked after adenoviral delivery of constitutively active FoxO1, glucose increased serum VLDL triglyceride when given both by ip glucose tolerance testing (3.5-fold increase) and by a hyperglycemic clamp (4.6-fold). Under both experimental conditions in which insulin signaling to FoxO1 was impaired, we found increased activation of carbohydrate response element binding protein. These data suggest that glucose more potently promotes increased serum VLDL when insulin action is impaired, with either low insulin levels or disrupted downstream signaling to the transcription factor FoxO1.

Portal infusion of escitalopram enhances hepatic glucose disposal in conscious dogs

To examine whether escitalopram enhances net hepatic glucose uptake during a hyperinsulinemic hyperglycemic clamp, studies were performed in conscious 42-h-fasted dogs. The experimental period was divided into P1 (0–90 min) and P2 (90–270 min). During P1 and P2 somatostatin (to inhibit insulin and glucagon secretion), 4× basal intraportal insulin, basal intraportal glucagon, and peripheral glucose (2× hepatic glucose load) were infused. Saline was infused intraportally during P1 in all groups. In one group saline infusion was continued in P2 (SAL, n = 11), while escitalopram was infused intraportally at 2 μg/kg/min (L-ESC, n = 6) or 8 μg/kg/min (H-ESC, n = 7) during P2 in two other groups. The arterial insulin concentrations rose ~four fold (to 123 ± 8, 146 ± 13 and 148 ± 15 pmol/L) while glucagon concentrations remained basal (41 ± 3, 44 ± 9 and 40 ± 3 ng/L) in all groups. The hepatic glucose load averaged 216 ± 13, 223 ± 19 and 202 ± 12 μmol/kg/min during the entire experimental period (P1 and P2) in the SAL, L-ESC and H-ESC groups, respectively. Net hepatic glucose uptake was 11.6 ± 1.4, 10.1 ± 0.9 and 10.4 ±2.3 μmol/kg/min in P1 and averaged 16.9 ± 1.5, 15.7 ± 1.3 and 22.6 ± 3.7 ( P < 0.05) in the SAL, L-ESC and H-ESC groups, respectively during the last hour of P2 (210–270 min). Net hepatic carbon retention (glycogen storage) was 15.4 ± 1.3, 14.9 ± 0.6 and 20.9 ± 2.6 ( P < 0.05) µmol/kg/min in SAL, L-ESC and H-ESC respectively during the last hour of P2. Escitalopram enhanced net hepatic glucose uptake and hepatic glycogen deposition, showing that it can improve hepatic glucose clearance under hyperinsulinemic hyperglycemic conditions. Its use in individuals with diabetes may, therefore, result in improved glycemic control.

Hyperglycemia prevents the suppressive effect of hyperinsulinemia on plasma adiponectin levels in healthy humans

Adiponectin is a fat-derived hormone with insulin-sensitizing properties. In patients with type 2 diabetes plasma adiponectin levels are decreased. Since these patients are characterized by high plasma insulin and glucose concentrations, hyperinsulinemia and hyperglycemia could be responsible for the downregulation of adiponectin. Insulin decreases adiponectin levels in humans. The effect of hyperglycemia is unknown. To determine the selective effects of insulin, glucose, or their combination on plasma adiponectin, clamps were performed in six healthy males on four occasions in a crossover design: 1) lower insulinemic-euglycemic clamp (100 pmol/l insulin, 5 mmol/l glucose) (reference clamp); 2) hyperinsulinemic-euglycemic clamp (400 pmol/l insulin, 5 mmol/l glucose); 3) lower insulinemic-hyperglycemic clamp (100 pmol/l insulin, 12 mmol/l glucose); and 4) hyperinsulinemic-hyperglycemic clamp (400 pmol/l insulin, 12 mmol/l glucose). Adiponectin concentrations and high-molecular-weight (HMW)-to-total adiponectin ratio were measured at the start and end of the 6-h clamps. After the 6-h study period, total plasma adiponectin levels were significantly (P = 0.045) decreased by 0.63 microg/ml in the lower insulinemic-euglycemic clamp (clamp 1). In both euglycemic groups (clamps 1 and 2) adiponectin concentrations significantly declined (P = 0.016) over time by 0.56 microg/ml, whereas there was no change in both hyperglycemic groups (clamps 3 and 4) (P = 0.420). In none of the clamps did the ratio of HMW to total adiponectin change. We conclude that insulin suppresses plasma adiponectin levels already at a plasma insulin concentration of 100 pmol/l. Hyperglycemia prevents the suppressive effect of insulin. This suggests that, in contrast to glucose, insulin could be involved in the downregulation of plasma adiponectin in insulin-resistant patients.

Selective Downregulation of the High–Molecular Weight Form of Adiponectin in Hyperinsulinemia and in Type 2 Diabetes

Adiponectin is an adipocyte-specific secretory protein found in circulation in several different forms and is present at significantly lower levels in the plasma of diabetic patients compared with that of insulin-sensitive individuals. We wanted to test whether insulin per se is a contributing factor toward lower plasma adiponectin concentrations and, if so, whether the splanchnic bed contributes to this phenomenon. We sampled femoral artery and hepatic venous samples and measured the high-molecular weight (HMW) and low-molecular weight (LMW) fractions of adiponectin in 11 type 2 diabetic and 7 nondiabetic subjects matched for age, sex, and BMI during basal conditions and during a hyperglycemic (approximately 9.5 mmol/l) hyperinsulinemic (approximately 700 pmol/l) clamp. Under these conditions, total arterial adiponectin, HMW, and the ratio of HMW to total adiponectin all were lower (P < 0.01) in the diabetic versus nondiabetic subjects, whereas the LMW form did not significantly differ. Under hyperinsulinemic conditions, total adiponectin levels dropped, primarily due to a reduction of the HMW form, whereas LMW forms were not significantly affected. HMW adiponectin and the ratio of HMW to total adiponectin are lower in individuals with diabetes than in nondiabetic subjects. We conclude that HMW adiponectin is downregulated in hyperinsulinemia and type 2 diabetes.

Adiponutrin gene is regulated by insulin and glucose in human adipose tissue

Adiponutrin is a new transmembrane protein specifically expressed in adipose tissue. In obese subjects, short- or long-term calorie restriction diets were associated with a reduction in adiponutrin gene expression. Adiponut.rin mRNA level was previously shown to be negatively correlated with fasting glucose plasma levels and associated with insulin sensitivity of non-diabetic obese and non-obese subjects. The purpose of the present work was to get more insight into the regulation of adiponutrin gene expression by insulin and/or glucose using clamp studies and to examine its potential dysregulation in subjects with a deterioration of glucose homeostasis. Adiponutrin gene expression was quantified by reverse transcriptase-quantitative PCR in s.c. adipose tissue of healthy lean subjects after an euglycemic hyperinsulinemic clamp (EGHI), a hyperglycemic euinsulinemic clamp, and a hyperglycemic hyperinsulinemic (HGHI) clamp. Adiponutrin gene expression was also analyzed in patients with different levels of insulin resistance. During EGHI, insulin infusion induced adiponutrin gene expression 8.4-fold (P = 0.008). Its expression was also induced by glucose infusion, although to a lesser extend (2.2-fold, P = 0.03). Infusion of both insulin and glucose (HGHI) had an additive effect on the adiponutrin expression (tenfold, P = 0.008). In a pathological context, adiponutrin gene was highly expressed in the adipose tissue of type-1 diabetic patients with chronic hyperglycemia compared with healthy subjects. Conversely, adiponutrin gene expression was significantly reduced in type-2 diabetics (P = 0.01), but remained moderately regulated in these patients after the EGHI clamp (2.5-fold increased). These results suggest a strong relationship between adiponutrin expression, insulin sensitivity, and glucose metabolism in human adipose tissue.

Effect of pioglitazone on insulin secretion in patients with both impaired fasting glucose and impaired glucose tolerance

Aim To evaluate the effect of a thiazolidinedione, pioglitazone, on insulin secretion in patients with both impaired fasting glucose and impaired glucose tolerance. Methods A randomized, double-blind, placebo-controlled clinical trial was carried out in 18 overweight or obese patients with both impaired fasting glucose and impaired glucose tolerance. Pharmacological intervention consisted of an oral morning administration of pioglitazone (30 mg) or a placebo with a similar presentation for 30 days. Before and after the intervention, glucose, creatinine, lipid profile and uric acid concentrations were measured. To evaluate insulin secretion (early, late and total phases) and insulin sensitivity, a hyperglycemic-hyperinsulinemic clamp was also performed. Results There were significant reductions ( p = 0.008) in fasting insulin concentration (121 versus 45 pmol/l), late (565 versus 307 pmol/l) and total insulin secretion (474 versus 254 pmol/l), as well as, in 2 h postload glucose levels (9.7 versus 6.9 mmol/l, p = 0.028), with an increment in insulin sensitivity after pioglitazone administration (7.5 versus 9.9). Conclusion Pioglitazone administration during a period of 4 weeks decreased late and total insulin secretion phases, fasting insulin and 2 h postload glucose levels, and improved insulin sensitivity in patients with both impaired fasting glucose and impaired glucose tolerance.

Hyperglycemia Stimulates Coagulation, Whereas Hyperinsulinemia Impairs Fibrinolysis in Healthy Humans

Type 2 diabetes and insulin resistance syndromes are associated with an increased risk for cardiovascular and thrombotic complications. A disturbed balance between coagulation and fibrinolysis has been implicated in the pathogenesis hereof. To determine the selective effects of hyperglycemia and hyperinsulinemia on coagulation and fibrinolysis, six healthy humans were studied on four occasions for 6 h: 1) lower insulinemic-euglycemic clamp, 2) lower insulinemic-hyperglycemic clamp, 3) hyperinsulinemic-euglycemic clamp, and 4) hyperinsulinemic-hyperglycemic clamp. In the hyperglycemic clamps, target levels of plasma glucose were 12 versus 5 mmol/l in the normoglycemic clamps. In the hyperinsulinemic clamps, target plasma insulin levels were 400 versus 100 pmol/l in the lower insulinemic clamps. Hyperglycemia exerted a procoagulant effect irrespective of insulin levels, as reflected by mean twofold rises in thrombin-antithrombin complexes and soluble tissue factor, whereas hyperinsulinemia inhibited fibrinolysis irrespective of glucose levels, as reflected by a decrease in plasminogen activator activity levels due to a mean 2.5-fold rise in plasminogen activator inhibitor type 1. The differential effects of hyperglycemia and hyperinsulinemia suggest that patients with hyperglycemia due to insulin resistance are especially susceptible to thrombotic events by a concurrent insulin-driven impairment of fibrinolysis and a glucose-driven activation of coagulation.

Effect of acute hyperglycemia and/or hyperinsulinemia on polymorphonuclear functions in healthy subjects

Abnormal immune functions of polymorphonuclear (PMN) cells occur in a variety of pathophysiological conditions. There exists a close link between glucose metabolism and PMN functions. The aim of this study was to assess the effect of short-term hyperglycemia and/or hyperinsulinemia on phagocytosis and respiratory burst of PMN cells in healthy subjects in vivo. The study was performed on 12 healthy subjects (mean age, 26.9 ± 1.6 years; body mass index, 24.4 ± 0.84 kg/m 2). Acute hyperglycemia and/or hyperinsulinemia was induced by three 4-hour-long clamp studies—hyperglycemic hyperinsulinemic clamp (HHC), hyperinsulinemic euglycemic clamp (HEC), and isolated hyperglycemic clamp with insulin secretion blockade (HGC). Polymorphonuclear cell phagocytosis and PMN cell respiratory burst (mean percentage and mean fluorescent intensity of phagocyting/activated PMN cells, phagocytic, and respiratory burst indexes) were evaluated by flow cytometry under basal and stimulated conditions. Results detected during clamp studies were compared with those found during a control study with saline infusion. Significant reductions in the mean percentage of phagocyting cells measured under basal conditions after the HHC (6.7% ± 1.3% vs 12.1% ± 4.3%; P < .05) and HGC (4.5% ± 1.8% vs 9.9% ± 2.1%; P < .05) were found in comparison with the preclamp study period; however, these results did not differ significantly from those detected during the control clamp (CC) study. Significantly higher phagocytic (115.1 ± 65 vs 35.8 ± 18.6; P < .05) and respiratory burst indexes (16.5 ± 3 vs 10.1 ± 1.4; P < .05) measured under basal conditions were found after HEC in comparison with the preclamp data. However, these data did not differ significantly from those found after the CC study. No significant differences in other parameters of detected PMN cell immune functions were found after HHC, HEC, and HGC. In conclusion, immune functions of PMN cells were not significantly influenced by short-lasting hyperglycemia and/or hyperinsulinemia induced in vivo by clamp techniques in healthy subjects compared to changes induced by the CC study. Further studies on the short-term effect of glucose metabolism on PMN functions in diabetic patients should be considered necessary.