Elevated hypothalamic beacon gene expression in Psammomys obesus prone to develop obesity and type 2 diabetes

OBJECTIVE: To investigate hypothalamic beacon gene expression at various developmental stages in genetically selected diabetes-resistant and diabetes-prone Psammomys obesus. In addition, effects of dietary energy composition on beacon gene expression were investigated in diabetes-prone P. obesus. METHODS: Hypothalamic beacon gene expression was measured using Taqman fluorogenic PCR in 4-, 8- and 16-week-old animals from each genetically selected line. RESULTS: Expression of beacon was elevated in the diabetes-prone compared with diabetes-resistant P. obesus at 4 weeks of age despite no difference in body weight between the groups. At 8 weeks of age, hypothalamic beacon gene expression was elevated in diabetes-prone animals fed a high-energy diet, and was correlated with serum insulin concentration. CONCLUSION: P. obesus with a genetic predisposition for the development of obesity and type 2 diabetes have elevated hypothalamic beacon gene expression at an early age. Overexpression of beacon may contribute to the development of obesity and insulin resistance in these animals.     

Characterization of obesity phenotypes in Psammomys obesus (Israeli sand rats)

Psammomys obesus (the Israeli sand rat) has been well studied as an animal model of Type 2 diabetes. However, obesity phenotypes in these animals have not been fully characterized. We analyzed phenotypic data including body weight, percentage body fat, blood glucose and plasma insulin concentration for over 600 animals from the Psammomys obesus colony at Deakin University to investigate the relationships between body fat, body weight and Type 2 diabetes using regression analysis and general linear modelling. The body weight distribution in Psammomys obesus approximates a normal distribution and closely resembles that observed in human populations. Animals above the 75th percentile for body weight had increased body fat content and a greater risk of developing diabetes. Increased visceral fat content was also associated with elevated blood glucose and plasma insulin concentrations in these animals. A familial effect was also demonstrated in Psammomys obesus, and accounted for 51% of the variation in body weight, and 23-26% of the variation in blood glucose and plasma insulin concentrations in these animals. Psammomys obesus represents an excellent animal model of obesity and Type 2 diabetes that exhibits a phenotypic pattern closely resembling that observed in human population studies. The obesity described in these animals was familial in nature and was significantly associated with Type 2 diabetes.     

Impact of obesity and leptin treatment on adipocyte gene expression in Psammomys obesus

We examined the effects of leptin treatment on the expression of key genes in adipocyte metabolism in Psammomys obesus (P. obesus), a polygenic rodent model of obesity. Lean and obese P. obesus were given three daily intraperitoneal injections of either saline or leptin (total of 45 mg/kg per day) for 7 days. In lean animals, leptin treatment led to reductions in food intake, body weight and fat mass. Pair-fed animals matched for the reduction in food intake of the lean leptin-treated animals demonstrated similar reductions in body weight and fat mass. In obese P. obesus, leptin treatment failed to have any effect on body weight or body fat mass, indicating leptin resistance. Lipoprotein lipase, hormone-sensitive lipase and peroxisome proliferator activated receptor gamma 2 mRNA levels were significantly reduced in lean leptin-treated animals, whereas pair-fed animals were similar to lean controls. Uncoupling protein 2 and glycerol phosphate acyltransferase were also reduced in the lean leptin-treated animals, but not significantly so. Obese animals did not show any gene expression changes after leptin treatment. In conclusion, high circulating concentrations of leptin in lean P. obesus resulted in decreased gene expression of a number of key lipid enzymes, independent of changes in food intake, body weight and fat mass. These effects of leptin were not found in obese P. obesus.     

Leptin resistance in a polygenic, hyperleptinemic animal model of obesity and NIDDM: Psammomys obesus

OBJECTIVE: To investigate the effects of leptin administration to Psammomys obesus, a polygenic animal model of obesity and type 2 diabetes mellitus. DESIGN: Longitudinal intervention study utilising three separate leptin treatment protocols lasting 7-14 d. MEASUREMENTS: Body weight and food intake were measured daily, body fat and muscle content were estimated by carcass analysis on completion of the study. Blood glucose, plasma insulin, leptin, triglycerides and cholesterol were measured at baseline and twice each week during the study. RESULTS: Relatively high doses of leptin were required to significantly reduce food intake and body fat content in lean Psammomys obesus, but had no discernible effect on their obese littermates. CONCLUSION: As a species, Psammomys obesus appear to be relatively insensitive to the effects of leptin administration, compared with other rodents. Obese Psammomys obesus are leptin resistant relative to their lean littermates.     

The development of obesity, hyperinsulinemia, and hyperglycemia in ob/ob mice

The development of obesity, hyperglycemia, and hyperinsulinemia was examined in obese-hyperglycemic (ob/ob) mice, their lean littermates, and homozygous lean mice (+/+) between 17 days and 8 wk of age. By 4 wk of age ob/ob mice displayed many of the metabolic characteristics that are typical of the syndrome in adult animals, including elevated systemic insulin and glucose levels, increased body weight, obesity, reduced skeletal growth, and in vivo evidence of insulin resistance. In addition, 4-wk-old lean littermates of obese mice had greater body weights, increased per cent carcass lipid, and higher insulin levels than did +/+ mice of the same age that were raised under identical conditions. At 17 or 21 days of age ob/ob mice, defined by either (1) elevated carcass fat content when compared to littermates at time of killing or (2) by phenotypic expression of obesity at 6 wk of age, exhibited moderate hyperinsulinemia, hypoglycemia, reduced skeletal growth, and “obesity”, as expressed by the Lee index. The present results indicate that altered pancreatic β-cell function, obesity, and abnormalities of somatic growth all precede the onset of hyperglycemia and insulin “resistance” in ob/ob mice. Furthermore, the occurrence of these characteristics before 17 days of age suggests that the transition to laboratory diet is not essential for the expression of the ob mutation. The present data also support recent studies that have described a small but reliable effect of the ob gene on the metabolism of heterozygous lean mice.     

Physical exercise enhances hepatic insulin signaling and inhibits phosphoenolpyruvate carboxykinase activity in diabetes-prone Psammomys obesus

We have shown that physical exercise enhances insulin sensitivity of skeletal muscle in diabetes-prone Psammomys-obesus. In this study, we examined the effect of physical exercise on the liver of these animals. Three groups of animals were exposed to a 4-week protocol; high-energy diet (CH), high-energy diet and exercising (EH), and low-energy diet (CL). Different groups were studied either in a fed state or after an overnight fast, 30 minutes after intraperitoneal (IP) injection of 1 U insulin. Hepatic phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) activity was measured. Insulin signaling response was examined after insulin injection in the fast state by analyzing tyrosine phosphorylation of insulin receptor (IR) and the association between insulin receptor substrate-1 (IRS-1) and IRS-2 with phosphatidylinositol 3 kinase (PI3-K). After 4 weeks, none of the EH animals became diabetic, whereas all the CH animals became diabetic. PEPCK activity in the fed state was higher in the CH group compared with the CL and EH groups (480 +/- 28 nmol/min/mg protein, 280 +/- 30 nmol/min/mg protein, and 208 +/- 13 nmol/min/mg protein, respectively) (P < .02). G6Pase activity was higher in the CH and EH groups compared with the CL group (261 +/- 54 nmol/min/mg protein, 251 +/- 34 nmol/min/mg protein, and 75 +/- 32 nmol/min/mg protein, respectively) (P < .01). After insulin administration in the fast state, tyrosine phosphorylation of IR and association of IRS-2 with PI3-K were higher in the EH and CL groups than in the CH group. We conclude that exercise improves in vivo hepatic insulin sensitivity in diabetes-prone Psammomys-obesus.     

Cholesterol absorption efficiency and sterol metabolism in obesity

Role of enterohepatic cholesterol metabolism in obesity-induced increase of cholesterol synthesis was studied in healthy lean (BMI <24) and overweight (BMI >31) subjects by measuring serum lipids (including plant sterols, cholestanol and cholesterol precursors), cholesterol absorption % (double-label method), sterol balance and biliary lipids. New aspects of sterol metabolism in obesity were as follows: low efficiency of cholesterol absorption, reduced ratios to cholesterol of serum and biliary plant sterols and cholestanol (5alpha-derivative of cholesterol), and a marked increase of serum and biliary cholesterol precursor sterols. Percent of cholesterol absorption was positively related to serum cholestanol and plant sterols, and negatively to cholesterol synthesis, measured by the sterol balance technique or cholesterol precursor sterols in serum or bile. Total and endogenous cholesterol fluxes into the intestine were increased, but owing to low absorption percent, mass of cholesterol absorption was within control limits in the obese subjects. Thus, per gram of their large liver tissue the entry of intestinal cholesterol may even be subnormal. Percent of cholesterol absorption was insignificantly negatively (r=-0.256) related to intestinal cholesterol pool, but significantly to biliary concentrations of cholesterol (r=-0.581), bile acids (r=-0.513) and phospholipids (r=-0.469). Thus, dilution of labeled dietary cholesterol by expanded intestinal cholesterol pool could have contributed to subnormal efficiency of cholesterol absorption, or transfer of labeled dietary cholesterol from intestinal oil phase to micellar phase may be competitively inhibited by expanded biliary secretion, resulting in reduced absorption of dietary cholesterol. These mechanisms could have contributed to changes in metabolism of non-cholesterol sterols, especially of cholestanol and plant sterols.     

VGF ablation blocks the development of hyperinsulinemia and hyperglycemia in several mouse models of obesity

Targeted deletion of the gene encoding the neuronal and endocrine secreted peptide precursor called VGF (nonacronymic) produces a lean, hypermetabolic, hyperactive mouse. Because VGF mutant mice are resistant to specific forms of diet-, lesion-, and genetically induced obesity, we investigated the role that this polypeptide plays in glucose homeostasis. We report that VGF mutant mice have increased insulin sensitivity by hyperinsulinemic euglycemic clamp analysis, and by insulin and glucose tolerance testing. Blunted counterregulatory responses in VGF-deficient mice were likely influenced by their significantly lower liver glycogen levels. VGF deficiency lowered circulating glucose and insulin levels in several murine models of obesity that are also susceptible to adult onset diabetes mellitus, including A(y)/a agouti, ob/ob, and MC4R(-)/MC4R(-) mice. Interestingly, ablation of Vgf in ob/ob mice decreased circulating glucose and insulin levels but did not affect adiposity, whereas MC4R(-)/MC4R(-) mice that are additionally deficient in VGF have improved insulin responsiveness at 7-8 wk of age, when lean MC4R(-)/MC4R(-) mice already have impaired insulin tolerance but are not yet obese. VGF mutant mice also resisted developing obesity and hyperglycemia in response to a high-fat/high-carbohydrate diet, and after gold thioglucose treatment, which is toxic to hypothalamic glucose-sensitive neurons. Lastly, circulating adiponectin, an adipose-synthesized protein the levels of which are correlated with improved insulin sensitivity, increased in VGF mutant compared with wild-type mice. Modulation of VGF levels and/or VGF signaling may consequently represent an alternative means to regulate circulating glucose levels and insulin sensitivity.     

Time-dependent effect of hyperglycemia and hyperinsulinemia on oxidative and non-oxidative glucose metabolism in patients with NIDDM.

The present study was undertaken to compare the effect of hyperglycemia and euglycemia during identical hyperinsulinemic conditions on glucose metabolism in NIDDM subjects. Eight NIDDM subjects participated in a 4 h hyperglycemic (12.1 +/- 0.7 mmol/l), hyperinsulinemic (475 +/- 43 pmol/l) and in a 4 h euglycemic (5.5 +/- 0.5 mmol/l), hyperinsulinemic (468 +/- 36 pmol/l) insulin clamp in combination with indirect calorimetry and [3H]-3-glucose. Six non-diabetic subjects were studied during euglycemia (5.1 +/- 0.2 mmol/l) and hyperinsulinemia (474 +/- 35 pmol/l) and served as controls. In NIDDM patients the rate of insulin-stimulated glucose disposal was 57% greater during hyperglycemia compared with euglycemia throughout the 4 h clamp (p less than 0.01). The major part of the increase in glucose metabolism during hyperglycemia was due to an increase in the non-oxidative glucose metabolism (89%). Whereas glucose metabolism could not be normalized during the prolonged euglycemic hyperinsulinemic clamp in NIDDM patients (49.9 +/- 6.8 vs 57.5 +/- 5.4 mumol.(kgLBM)-1.min-1 in controls) the addition of hyperglycemia resulted in complete normalization of the glucose disposal rates (78.3 +/- 5.8 mumol.(kgLBM)-1.min-1). The effect of hyperglycemia was apparent already at 60 min of the clamp. The data thus suggest that glucose metabolism in NIDDM is insulin resistant, but that the defect in insulin-stimulated glucose uptake can be overcome by increasing the glucose concentration.     

A cross-sectional and short-term longitudinal characterisation of NIDDM in Psammomys obesus

Summary The present study was undertaken to examine the cross-sectional and short-term longitudinal changes in glucose and insulin concentrations as well as measure the enzymatic activity of PEPCK and glycogen synthase in our Psammomys obesus colony. In the cross-sectional study, blood samples were taken from one group of animals at 19 weeks of age (n=37) in the fed state and following a 4-h fast. In a separate group of 19-week-old animals (n = 69), samples were taken l h following an OGTT (1 g/kg body weight) in Psammomys subjected to a 16-h fast. In the longitudinal study, blood samples were taken from one group of animals in the fed state at 7, 11, 15 and 19 weeks of age. All of the cross-sectional data have described the classic inverted U-shaped curve (Starling's curve of the pancreas) in the relationship between glucose and insulin levels. This trend was also reflected by Psammomys subjected to the OGTT; a mild impairment in glucose tolerance was associated with an increase in the insulin response and a further impairment in glucose tolerance was associated with a reduction in the insulin response. Similar results were obtained following a 4-h fast. The short-term longitudinal glucose and insulin data revealed that of the 37 animals examined over the 12-week period, 16 progressed along the inverted U-shaped curve described by the cross-sectional data. Of the other animals, 8 remained unchanged, 7 were unclassifiable and 6 hyperglycaemic Psammomys developed normoglycaemia at the expense of elevated insulin levels. Psammomys were divided into four separate groups based on their glucose and insulin levels in the fed state at age 19 weeks: group A were normoglycaemic (4.5 ± 0.2 mmol/l) and normoinsulinaemic (81±15 mU/l); group B were normoglycaemic (5.3±0.3 mmol/l) and hyperinsulinaemic (267±19 mU/l); group C were hyperglycaemic (16.9±1.4 mmol/l) and hyperinsulinaemic (356±29 mU/l); group D were hyperglycaemic (20.4±1.4 mmol/l) and normoinsulinaemic (92±56 mU/l). Analysis of liver and soleus muscle tissue revealed that active glycogen synthase activity was reduced in the hyperglycaemic Psammomys (C and D). Total glycogen synthase activity (in liver and muscle) and PEPCK activity (in liver) were not different between groups A-D. These findings add to those already published which suggest Psammomys is an ideal animal model for the study of NIDDM.Abbreviations NIDDM Non-insulin-dependent diabetes mellitus - OGTT oral glucose tolerance test - PEPCK phosphoenolpyruvate carboxykinase     

A sustained, non-insulin related, hypoglycaemic effect of electroacupuncture in diabetic Psammomys obesus

Aim/hypothesis. Electroacupuncture has been shown to induce a short-term hypoglycaemic effect in streptozotocin diabetic rats. We designed an experiment to investigate the effect of electroacupuncture in Psammomys obesus, a model of insulin resistance and non-insulin-dependent diabetes mellitus.¶Methods. We divided 29 diabetic Psammomys randomly into three groups: abdominal electroacupuncture (real, n = 11), back electroacupuncture (placebo, n = 9) and control (anaesthesia, n = 9). Electroacupuncture was carried out on days 1, 3 and 5 of the experiment. During the first week of the experiment, blood glucose was tested three times on treatment days and once on the following days. Over the next 2 weeks, blood glucose was tested every other day. Animals were weighed at the same time of blood sampling. After 3 weeks, at the end of the experiment, blood was drawn for measurement of insulin, fructosamine, cholesterol and triglycerides.¶Results. At day 5 (end of intervention), blood glucose (as per cent of primary concentrations, means ± SE) was 57 ± 10, 93 ± 13 and 89 ± 11 for the real, placebo and control groups respectively (p = 0.02). At day 8, blood glucose 68 ± 14, 86 ± 16 and 97 ± 9 for the real, placebo and control groups respectively (p = 0.04). At day 22, blood glucose was 79 ± 11, 85 ± 15 and 131 ± 2 for the real, placebo and control groups (p = 0.04). Comparison of the decline in blood glucose, throughout the 3 weeks, between the real and placebo groups by ANOVA was highly significant (p < 0.0001), the difference between the placebo and control groups at the same time was not significant (p > 0.05). Animal weight gain, serum insulin, fructosamine, cholesterol and triglycerides were not significantly different between real and placebo groups.¶Conclusion/interpretation. Electroacupuncture at special abdominal acupoints induces a sustained hypoglycaemic effect in diabetic Psammomys compared with electroacupuncture at non-specific points, without weight loss. No hypoinsulinaemic effect was shown in the real and placebo groups. [Diabetologia (2000) 43: 809–813]     

Irreversibility of nutritionally induced NIDDM in Psammomys obesus is related to beta-cell apoptosis

Psammomys lapses into fully fledged diabetes when maintained on a high-energy diet. Progression to diabetes has been classified into stage A of normoglycemia and normoinsulinemia (<120 mg/ml and 100 mU/L, respectively); stage B of hyperinsulinemia (100-300 mU/L) with marked insulin resistance in the face of normoglycemia; stage C of pronounced hyperinsulinemia with hyperglycemia < or =500 mg/ml; stage D at 6-10 weeks after stage C, featuring further hyperglycemia and loss of insulin. Insulin resistance expressed in Psammomys at stages B and C was demonstrated by nonsuppression of the hepatic gluconeogenesis enzyme phosphoenolpyruvate carboxykinase by the endogenous hyperinsulinemia and by the reduced capacity of insulin to activate muscle and liver tyrosine kinase of the insulin receptor. Diabetes at stage C, but not at stage D, was fully reversed to stage A by restricting the food ration of animals by half (from 14 to 7 g/day) for 10-14 days. We examined islet beta cells of Psammomys in the four stages of progression to diabetes by staining for insulin as well as for apoptosis by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and visualizing the biotin-labeled cleavage sites. Psammomys in stage A had insulin-laden beta cells. In stage B, a hypertrophy and partial insulin depletion of beta cells was evident with negative TUNEL staining. In stage C, beta cells were markedly depleted of insulin, and their number within the islets decreased, but the TUNEL staining was virtually negative. In stage D, beta cells were markedly diminished within the islets, almost void of insulin, showing distinct TUNEL staining of beta cells. These results indicate that prolonged exposure of islets to in vivo hyperglycemia with beta-cell overtaxation induces nuclear disintegration with irreversible damage to the insulin-secretion apparatus. This precludes the return to normalcy by restricting the food intake of Psammomys. The appearance of cells with TUNEL-positive staining may serve as a marker of impending irreversibility of nutritionally induced diabetes.     

The relationship of sex hormones to hyperinsulinemia and hyperglycemia

Mexican-Americans, a high-risk population for non-insulin-dependent diabetes mellitus (NIDDM), have been previously reported to have decreased levels of sex-hormone-binding globulin (SHBG). We measured total testosterone, total estradiol and SHBG, glucose and insulin in premenopausal women (58 Mexican-Americans and 38 non-Hispanic whites) as part of the San Antonio Heart Study, a population-based study of cardiovascular risk factors. Although total estradiol and total testosterone were, in general, not correlated with metabolic variables, SHBG was negatively correlated with glucose and insulin. After adjustment for body mass index (BMI), ratio of waist-to-hip circumference (WHR) and ratio of subscapular-to-triceps skinfold (Centrality Index), SHBG was still significantly correlated with insulin concentrations (P less than .001). Since Mexican-Americans were previously reported to be more hyperinsulinemic than non-Hispanic whites, we examined the effect of adjusting for SHBG on insulin levels in this small population. While unadjusted insulin concentrations in Mexican-Americans were higher than in non-Hispanic whites (354 microU/mL v 236 microU/mL, respectively, P = .009), adjustment for BMI, WHR, and centrality index reduced the ethnic difference in insulin levels considerably (P = .014). However, only after adjusting for SHBG as well, did the ethnic difference in insulin levels became nonsignificant. Our data suggest that alterations in sex hormones and SHBG in particular may be related to the hyperinsulinemia and the high rates of NIDDM in Mexican-Americans.     

Proinsulin in girls: relationship to obesity,hyperinsulinemia, and puberty

In adults with impaired glucose tolerance (IGT) and obesity (OB), an elevated proinsulin (PI) is predictive of type 2 diabetes mellitus (DM) and precedes the diagnosis by 5-20 yr. In type 2 DM, the PI is disproportionately elevated, i.e. increased PI/insulin ratio (PI/I). Few studies have evaluated PI in children at risk for type 2 DM. In the face of the current epidemic, we evaluated the relationship of PI and PI/I to IGT, insulin resistance (IR) defined by homeostasis model of assessment (HOMA), degree of OB, and stage of puberty in 70 girls (mean age 10.8 yr; body mass index z-score 3.5; ethnicity 64% Hispanic, 19% white, 16% African-American, and 1% other). Family history of DM was reported in 83%, and acanthosis nigricans was present in 80%. Subjects underwent a 2-h oral glucose tolerance test with glucose, insulin, and PI determinations every 30 min. All had normal hemoglobin A1c and fasting glucose. Five had IGT. With higher HOMA-IR, PI increased (P < 0.05), yet the ratio of fasting PI/I was lower (P < 0.05). Girls with body mass index z-score greater than 4 (n = 29) had higher PI than nonobese girls (n = 19, P < 0.05), but PI/I ratios were not different. PI-0 was increased in late puberty (n = 29), compared with prepuberty (n = 26, P < 0.05), but PI/I ratios showed no statistical difference. We found PI increased with increasing IR and OB in girls. Overall, PI/I was not different, suggesting the elevated PI reflects increased beta-cell output proportional to the elevated insulin in these groups and not a defect in PI processing or secretion. In fact, the lower fasting PI/I of the highest HOMA-IR quartile vs. the lowest HOMA quartile indicates more efficient conversion of PI to I in the presence of increasing IR in these girls.     

Insulin resistance in the NIDDM model Psammomys obesus in the normoglycaemic,normoinsulinaemic state

Summary The desert gerbil Psammomys obesus (“sand rat”), a model of nutritionally induced insulin resistance and non-insulin-dependent diabetes mellitus, was treated after weaning with exogenous insulin implants in the normoglycaemic, normoinsulinaemic state. Albino rats matched for weight and age served as high energy diet adjusted reference animals. Insulin administration, elevating the serum insulin to 6000 pmol/l resulted in only a mild reduction in blood glucose levels in Psammomys, but caused a severe, often fatal hypoglycaemia in the albino rats. The hepatic response to insulin-induced hypoglycaemia in rats involved a significant loss in glycogen and suppression of phosphoenolpyruvate carboxykinase (PEPCK) activity. In Psammomys under similar hyperinsulinaemia no appreciable changes in liver glycogen and PEPCK activity were evident, indicating that blood glucose was replenished by continuing gluconeogenesis. Euglycaemic, hyperinsulinaemic clamp caused a complete shut-down of hepatic glucose production in albino rats. However, in both diabetes-prone and diabetes-resistant Psammomys lines, mean hepatic glucose production was reduced by only 62 to 53 % respectively, despite longer lasting and higher levels of hyperinsulinaemia. These results indicate that Psammomys is characterized by muscle and liver insulin resistance prior to diet-induced hyperglycaemia and hyperinsulinaemia. This is assumed to be a species feature of Psammomys, exemplifying a metabolic adjustment to survival in conditions of food scarcity of both animal and human populations. It may reflect a propensity to insulin resistance and hyperglycaemia in population groups exposed to affluent nutrition. [Diabetologia (1996) 39: 1269–1275] Received: 11 April 1996 and in revised form: 4 July 1996     

Circulating vascular endothelial growth factor is unaffected by acute hyperglycemia and hyperinsulinemia in type 1 diabetes mellitus

BACKGROUND: Circulating levels of vascular endothelial growth factor (VEGF) may predict microvascular complications in type 1 diabetes mellitus and are elevated when metabolic control is poor. We tested whether serum VEGF is influenced by prevailing glucose and insulin levels. METHODS: In 15 type 1 diabetic patients, serum VEGF, plasma von Willebrand factor antigen (vWF-Ag), and serum soluble intercellular adhesion molecule-1 (s-ICAM) levels were measured after 210 min of hyperglycemia (blood glucose target 12.0 mmol/l) and hyperinsulinemia (insulin infused at 120 mU/kg/h), alone and in combination. These were then compared with the levels obtained at the end of a 210-min normoglycemic (blood glucose target 5.0 mmol/l) standard insulin clamp (insulin infused at 30 mU/kg/h). RESULTS: VEGF (p>0.60) as well as vWF-AG (p>0.80) and s-ICAM (p>0.20) remained unchanged at the end of the three intervention periods. CONCLUSION: These findings suggest that no special precautions, in terms of concurrent measurement of glucose or timing of insulin administration, are necessary when interpreting circulating VEGF in this patient category.