Basal insulin therapy is associated with beneficial effects on postoperative infective complications, independently from circulating glucose levels in patients admitted for cardiac surgery.

BACKGROUND: Although hyperglycemia is a strong predictor of postoperative infective complications (PIC), little is known about the effect of basal insulin therapy (BIT) per se on PIC. AIM: To evaluate if there is an association between BIT, independent of glucose levels, and a possible improvement of PIC during the perioperative cardiosurgery period (PCP). METHODS: In 812 patients admitted for cardiac intervention and treated with a continuous intravenous insulin infusion (CIII) for hyperglycemic levels (>130 mg/dl), a retrospective analysis was performed during the PCP (January 2009-December 2011). Upon transfer to the cardiac surgery division, if fasting glucose was ≥130 mg/dl, a basal + premeal insulin therapy was initiated (121 patients, group 1); for <130 mg/dl, a premeal insulin alone was initiated (691 patients, group 2). FINDINGS: Compared with group 2, group 1 showed reductions in PIC (2.48% vs 7.96%, p < 0.049; odds ratio: 0.294; 95% CI: 0.110-0.780), C-Reactive Protein (p < 0.05) and white blood cell (p < 0.05) levels despite glucose levels and CIII that were higher during the first two days after surgery (179.8 ± 25.3 vs 169.5 ± 10.6 mg/dl, p < 0.01; 0.046 ± 0.008 vs 0.037 ± 0.015 U/kg/h, p < 0.05, respectively). Normal glucose levels were achieved in both groups from day 3 before the discharge. The mean length of hospital duration was 18% lower in group 1 than in group 2 (7.21 ± 05.08 vs 8.76 ± 9.08 days, p < 0.007), providing a significant impact on public health costs. CONCLUSIONS: Basal + preprandial insulin therapy was associated with a lower frequency of PIC than preprandial insulin therapy alone, suggesting a beneficial effect of basal insulin therapy on post-surgery outcome.

Lower endothelial progenitor cell number, family history of cardiovascular disease and reduced HDL-cholesterol levels are associated with shorter leukocyte telomere length in healthy young adults.

BACKGROUND AND AIMS: Leukocyte telomere length (LTL) is a novel marker of cardiovascular (CV) risk. The aim of the study was to investigate the major determinants of LTL in a healthy young population at very low CV risk. METHODS AND RESULTS: LTL was determined in 82 healthy subjects (49M/33F; age37 ± 9yrs), normotensive and not taking any medication with different family history of cardiovascular disease (CVD) (24yes/58no). Fasting blood samples were drawn in all subjects for the determination of lipid profile, high sensitive C-reactive protein, uric acid, Plasminogen Activator Inhibitor-1 (PAI-1), LTL and Endothelial Progenitor Cell (EPC) number. LTL was assessed with a specific real-time PCR reaction in leukocyte DNA samples. LTL resulted inversely correlated with family history of CVD (t = 2.70; p = 0.009), age (r = -0.238; p = 0.032), waist circumference (r = -0.256; p = 0.02), triglycerides (r = -0.218; p = 0.049), PAI-1 (r = -0.288; p = 0.009) and directly correlated with HDL-cholesterol (r = 0.316; p = 0.004) and EPC number (r = 0.358; p = 0.002). At a multivariate analysis, family history of CVD (p = 0.013), EPC count (p = 0.003), and HDL-cholesterol (p = 0.017) were independently associated with LTL (r = 0.62). CONCLUSION: LTL is independently associated to CV risk factors also in healthy young adults.

Effect of a long-term oral l-arginine supplementation on glucose metabolism: a randomized, double-blind, placebo-controlled trial.

AIM: This study assessed the efficacy of long-term l-arginine (l-arg) therapy in preventing or delaying type 2 diabetes mellitus. METHODS: A mono-centre, randomized, double-blind, parallel-group, placebo-controlled, phase III trial (l-arg trial) was conducted on 144 individuals affected by impaired glucose tolerance (IGT) and metabolic syndrome (MS). l-Arg/placebo was administered (6.4 g/day) on a background structured lifestyle intervention for 18 months plus a 12-month extended follow-up period after study drug termination. Fasting glucose levels and glucose tolerance after oral glucose tolerance test were evaluated throughout the study. RESULTS: After 18 months, l-arg as compared with placebo did not reduce the cumulative incidence of diabetes [21.4 and 20.8%, respectively, hazard ratio (HR), 1.04; 95% confidence interval (CI), 0.58-1.86] while the cumulative probability to become normal glucose tolerant (NGT) increased (42.4 and 22.1%, respectively, HR, 2.60; 95% CI, 1.51-4.46, p < 0.001). The higher cumulative probability to become of NGT was maintained during the extended period in subjects previously treated with l-arg (HR, 3.21; 95% CI, 1.87-5.51; p < 0.001). At the end of the extended period, the cumulative incidence of diabetes in subjects previously treated with l-arg was reduced as compared with placebo (27.2 and 47.1%, respectively, HR, 0.42; 95% CI, 0.24-0.75, p < 0.05). During both periods, l-arg significantly improved insulin sensitivity and ß-cell function. CONCLUSION: Among persons with IGT and MS, the supplementation of l-arg for 18 months does not significantly reduce the incidence of diabetes but does significantly increase regression to NGT.

Effects of chronic elevation of atrial natriuretic peptide and free fatty acid levels in the induction of type 2 diabetes mellitus and insulin resistance in patients with mitral valve disease.

BACKGROUND AND AIMS: The relationship between atrial natriuretic peptide (ANP), increased free fatty acid (FFA) and insulin resistance in patients with mitral valve disease (MVD), a group characterised by elevated atrial pressure and increased ANP levels, is not defined. The present study was performed to evaluate, in MVD patients, the relationship between increased ANP and FFA levels and insulin resistance and the role of mitral valve replacement/repair in ameliorating these metabolic alterations. Conversely, coronary heart disease (CHD) patients were evaluated before and after coronary artery bypass grafting (CABG), since they are known to be insulin resistant in the presence of chronic FFA increase. METHODS AND RESULTS: Fifty MVD patients and 55 CHD patients were studied before and 2 months after surgery and compared with 166 normal subjects. Before surgery, 56% of MVD patients had impaired glucose tolerance or newly diagnosed type 2 diabetes after a standard oral glucose load and this percentage decreased to 46% after surgery. In CHD, impaired glucose tolerance (IGT) or newly diagnosed type 2 diabetic patients were 67% of patients before and after CABG. In MVD, left atrial (LA) volume, ANP, FFA incremental area and insulin levels were higher and Insulin Sensitivity (IS) index significantly reduced while after surgery, LA volume, ANP and FFA significantly decreased and IS index significantly improved. In CHD, insulin resistance and hyperinsulinaemia were present both before and after surgery with increased tumour necrosis factor (TNF)-α and interleukin (IL)-6 levels. CONCLUSION: In MVD, a higher degree of abnormal glucose tolerance and insulin resistance are associated to increased levels of ANP and FFA, while these metabolic alterations are improved by mitral valve replacement/repair surgery. Clinical Trial.gov registration number NCT 00520962.

Beneficial role of L-arginine in cardiac matrix remodelling in insulin resistant rats.

BACKGROUND: The study was performed to determine whether sucrose-induced insulin resistance could increase the expression of cardiac matrix metalloproteinases (MMPs), indices of matrix remodelling, and whether the addition of 1.25 g day(-1) of L-arginine (ARG) to a sucrose diet could prevent both the sucrose-induced metabolic abnormalities and elevated cardiac expression of matrix metalloproteinases in an insulin resistant stage that precedes frank type 2 diabetes. MATERIALS AND METHODS: Experiments were performed on 38 male Sprague-Dawley rats, 16 rats maintained a standard chow diet (ST), 12 rats were switched to a sucrose enriched diet (SU) and 10 rats to a sucrose plus L-arginine (1.25 g day(-1)) enriched diet (SU + ARG) for a period of 8 weeks. After 8 weeks of different diets, an intravenous glucose tolerance test (IVGTT) was performed and samples were drawn for the measurements of insulin, glucose, triglycerides, free fatty acids (FFA), plasma cyclic guanosine-monophosphate (c-GMP) and retroperitoneal, omental, epididymal fat pad and heart were dissected and weighed. RESULTS: At the end of the study, retroperitoneal fat, heart weight/body weight ratio, fasting plasma glucose, serum insulin, and serum triglyceride levels and integrated insulin area after IVGTT were significantly higher in SU than in SU + ARG and ST. All these parameters were comparable between SU + ARG and ST animals. FFA levels were significantly different among groups, with highest levels in SU and lowest levels in ST. Fasting plasma c-GMP levels and the integrated c-GMP area after IVGTT, an index of nitric oxide activity, were significantly lower in SU than in SU + ARG and ST, the result was similar in SU + ARG and in ST MMP-9 protein expression increased 10.5-fold, MMP-2 protein expression increased 2.4-fold and the expression of tissue inhibitors of metalloproteinase (TIMP-1) increased 1.7-fold in SU rats as compared to ST animals. This was accompanied with a significant increase of cardiac triglyceride concentrations. In contrast, cardiac MMP-9, MMP-2, and TIMP-1 protein expressions were not different between SU + ARG and ST animals. Cardiac triglyceride levels were not significantly different between SU + ARG and ST rats. CONCLUSIONS: SU rats developed insulin resistance and hyperlipidaemia, accompanied with increased fat deposition in the heart and enhanced MMP protein expression. Conversely, ARG supplementation prevents these metabolic abnormalities and restored MMP/TIMP-1 balance.

Triglycerides impair postischemic recovery in isolated hearts: roles of endothelin-1 and trimetazidine.

There is growing evidence that hypertriglyceridemia exacerbates ischemic injury. We tested the hypothesis that triglycerides impair myocardial recovery from low-flow ischemia in an ex vivo model and that such an effect is related to endothelin-1. Hyperglycemic (glucose concentration = 12 mmol/l) and hyperinsulinemic (insulin concentration = 1.2 micromol/l) isolated rat hearts were perfused with Krebs-Henseleit buffer (PO(2) = 670 mmHg, pH 7.4, 37 degrees C) added with increasing triglycerides (0, 1,000, 2,000, and 4,000 mg/dl, n = 6-9 rats/group). Hearts were exposed to 60 min of low-flow ischemia (10% of basal coronary flow), followed by 30 min of reperfusion. We found that increasing triglycerides impaired both the diastolic (P < 0.005) and systolic (P < 0.02) recovery. The release of endothelin-1 during reperfusion increased linearly with triglyceride concentration (P = 0.0009). Elevated triglycerides also increased the release of nitrite and nitrate (NO(x)), the end products of nitric oxide, up to 6 micromol/min. Trimetazidine (1 micromol) further increased NO(x) release, blunted endothelin-1 release, and protected myocardial function during recovery. We conclude that high triglyceride levels impair myocardial recovery after low-flow ischemia in association with endothelin-1 release. The endothelium-mediated effect of triglycerides on both contractile recovery and endothelin-1 release is prevented by 1 microM trimetazidine.

Long-term oral L-arginine administration improves peripheral and hepatic insulin sensitivity in type 2 diabetic patients.

UNLABELLED: The aim of this study was to evaluate whether long-term administration of arginine acting through a normalization of NO/cyclic-guanosine-3' 5'-cyclic monophosphate (cGMP) pathway was able to ameliorate peripheral and hepatic insulin sensitivity in 12 lean type 2 diabetic patients. RESEARCH DESIGN AND METHODS: A double-blind study was performed for 3 months. In the first month, patients were treated with their usual diet. Then they were randomly allocated into to groups. In group 1, patients were treated with diet plus placebo (orally three times per day) for 2 months. In group 2 patients were treated for 1 month with diet plus placebo orally, three times per day) and then for 1 month with diet plus L-arginine (3 g three times per day). At the end of the first and the second month of therapy, patients underwent a euglycemic-hyperinsulinemic clamp combined with [6,6-2H2] glucose infusion. A total of 10 normal subjects underwent the same test as control subjects. RESULTS: In group 1, no changes in basal cGMP levels, systolic blood pressure, forearm blood flow, glucose disposal, and endogenous glucose production were observed throughout. In group 2, L-arginine normalized basal cGMP levels and significantly increased forearm blood flow by 36% and glucose disposal during the clamp by 34% whereas it decreased systolic blood pressure and endogenous glucose production by 14 and 29%, respectively. However, compared with normal subjects, L-arginine treatment was not able to completely overcome the defect in glucose disposal. CONCLUSIONS: L-Arginine treatment significantly improves but does not completely normalizc peripheral and hepatic insulin sensitivity in type 2 diabetic patients.

In middle-aged siblings of patients with type 2 diabetes mellitus normal glucose tolerance is associated with insulin resistance and with increased insulin secretion. The SPIDER study.

OBJECTIVES: To evaluate the frequency of impaired glucose tolerance (IGT) and of Type 2 diabetes mellitus (Type 2 DM) in siblings of patients with Type 2 DM, and to assess insulin release and insulin sensitivity in siblings with normal glucose tolerance (NGT), compared with NGT spouses of probands without family history of Type 2 DM. DESIGN AND METHODS: We evaluated 87 families including 103 Type 2 DM patients (87 probands), and we carried out an oral glucose tolerance test (OGTT) in 130 siblings and in 60 spouses. Among NGT subjects, 12 siblings and 16 spouses underwent a low-dose insulin-glucose infusion test (LDIGIT) to evaluate C-peptide release and insulin sensitivity. RESULTS: After the OGTT, 24 siblings were classified as having Type 2 DM, 31 as IGT, and only 14 spouses as IGT (P=0.0012 vs siblings). NGT siblings (n=75) showed higher insulin levels at 120 min than NGT spouses (n=46) at OGTT, in spite of identical blood glucose levels; at LDIGIT, NGT siblings secreted more C-peptide and showed a lower insulin sensitivity than NGT spouses. CONCLUSIONS: These data indicate that middle-aged siblings of probands with Type 2 DM have a high frequency of IGT and Type 2 DM, and that NGT siblings have increased insulin resistance and increased insulin secretion when compared with adequate controls.

Familial clustering of arterial blood pressure, HDL cholesterol, and pro-insulin but not of insulin resistance and microalbuminuria in siblings of patients with type 2 diabetes.

OBJECTIVE: To test the hypothesis that selected abnormalities cluster in type 2 diabetic families. Offspring of patients with type 2 diabetes have a 40-60% chance of developing type 2 diabetes and an increased frequency of impaired glucose tolerance (IGT) or unknown diabetes. These offspring also show metabolic abnormalities of type 2 diabetes, such as insulin resistance, high insulin and pro-insulin, low HDL cholesterol levels, arterial hypertension, and microalbuminuria. RESEARCH DESIGN AND METHODS: We studied 87 families including at least one type 2 diabetic patient, i.e., 87 probands and 146 siblings; 60 spouses of probands with no family history of diabetes were compared with siblings. Familial clustering was evaluated by 2 methods: concordance of siblings and probands for a given abnormality (method 1) and intraclass correlation coefficients of values within each family (method 2). RESULTS: At oral glucose tolerance testing, 24 siblings had type 2 diabetes, 31 siblings had IGT, and 14 spouses had IGT (P = 0.0012 vs. siblings). With method 1, familial clustering occurred for microalbuminuria, insulin resistance, arterial hypertension, HDL cholesterol and pro-insulin levels; with method 2, familial clustering was observed for the same variables except for microalbuminuria. With both method 1 and 2, familial clustering for insulin resistance disappeared, whereas familial clustering for arterial blood pressure, HDL cholesterol, and pro-insulin remained after correction for BMI; after further restriction of analysis to probands and to siblings with normal glucose tolerance, familial clustering for pro-insulin was observed only with method 2. CONCLUSIONS: These data indicate that siblings of diabetic patients are at high risk for selected features of type 2 diabetes.

Plasma nitric oxide concentrations are elevated in insulin-resistant healthy subjects.

The goal of this study was to compare plasma nitric oxide (NO) concentrations in healthy subjects, defined as either insulin-resistant or insulin-sensitive on the basis of the plasma insulin response to a 75-g oral glucose challenge. For this purpose, 404 healthy subjects were divided into quartiles on the basis of the plasma insulin response to glucose, and 49 individuals were selected from the quartile with the lowest insulin response and 49 from the quartile with the highest insulin response. The two groups of 49 each were selected to be essentially identical in terms of age, gender distribution, body mass index (BMI), and waist to hip ratio (WHR). The quartile with the greatest insulin response also had a significantly higher plasma glucose response to oral glucose, faster heart rate, higher blood pressure, and the combination of higher triglyceride and lower high-density lipoprotein (HDL) cholesterol concentrations. In addition to the latter changes, previously shown to be associated with hyperinsulinemia, NO concentrations were also higher in the hyperinsulinemic group. It is speculated that this increase in the NO concentration in hyperinsulinemic and presumably insulin-resistant, subjects represents a compensatory effort to overcome the untoward effects of insulin resistance and/or hyperinsulinemia.

Detrimental effects of acute heparin administration on ischemic threshold in patients with coronary artery disease.

BACKGROUND: Recent studies have indicated that heparin administration might decrease endothelial nitric oxide production. The aim of this study was to investigate the effect of heparin on ischemic threshold. METHODS: Eighteen patients with a positive exercise test and proven coronary artery disease were submitted to a randomized, placebo-controlled trial using i.v. 0.9% NaCl as placebo and i.v. heparin (5,000 IU bolus + 1,000 IU/h). After both saline and heparin bolus, the infusion was started and, after 10 min, the exercise test was performed. Blood samples for nitric oxide metabolites and free fatty acid determinations were taken before, at peak exercise, and at ECG recovery. RESULTS: As compared to placebo, heparin significantly decreased time to 1 mm ST segment depression (241 +/- 160 vs 303 +/- 175 s, p = 0.003) and prolonged recovery (573 +/- 177 vs 441 +/- 195 s, p = 0.003), while exercise duration was similar. Accordingly, rate-pressure product at 1 mm ST segment depression was lower after heparin, while it was similar at peak exercise. No significant differences were found for plasma nitric oxide metabolite levels. Conversely, free fatty acid levels were higher after heparin throughout the study in all patients. The increase in free fatty acids was not correlated with the difference in rate-pressure product at 1 mm ST segment depression between placebo and heparin (r = 0.34, p = NS). CONCLUSIONS: In patients with stable coronary artery disease, heparin significantly decreased exercise ischemic threshold. The lower rate-pressure product at 1 mm ST segment depression during heparin, compared to placebo, suggests an impairment of coronary blood flow, which does not seem to be mediated by decreased nitric oxide production/release. The increased free fatty acid release, on the other hand, might contribute to the detrimental effect of heparin on exercise-induced ischemia, but the lack of a correlation with changes in ischemic threshold suggests that other, still unknown, factors are involved.

Alterations in nitric oxide/cyclic-GMP pathway in nondiabetic siblings of patients with type 2 diabetes.

In this study, we have compared resistance to insulin-mediated glucose disposal and plasma concentrations of nitric oxide (NO) and cyclic-GMP in healthy volunteers with (n = 35) or without (n = 27) at least one sibling and one parent with type 2 diabetes. The 62 volunteers were further divided into groups of those with normal glucose tolerance or impaired glucose tolerance. Insulin-mediated glucose disposal was quantified by determining the insulin sensitivity index (ISI) in response to a low-dose, constant infusion of insulin (25 mU/kg x h) and glucose (4 mg/kg x min) for 150 min. The mean (+/-SEM) ISI [(mL kg(-1) min(-1)/pmol/L) x 10(3)] was significantly greater in those without a family history (30.3 +/- 2.3) as compared with nondiabetic volunteers with a family history of type 2 diabetes, whether they had normal glucose tolerance (17.0 +/- 7.2) or impaired glucose tolerance (9.5 +/- 1.4). In addition, basal NO levels, evaluated by the measurement of its stable end products [i.e. nitrite and nitrate levels (NO2-/ NO3-)], were significantly higher, and cyclic-GMP levels, its effector messenger, were significantly lower in those with a family history, irrespective of their degree of glucose tolerance, when compared with healthy volunteers without a family history of type 2 diabetes. Furthermore, when the 62 volunteers were analyzed as one group, there was a negative correlation between ISI and NO2-/NO3- levels (r = -0.35; P < 0.005) and a positive correlation between ISI and cyclic-GMP levels (r = 0.30; P < 0.02). These results have shown that alterations of the NO/cyclic-GMP pathway seem to be an early event in nondiabetic individuals with a family history of type 2 diabetes and these changes are correlated with the degree of insulin resistance.

Relationship between endothelin-1 concentration and metabolic alterations typical of the insulin resistance syndrome.

The purpose of the study was to examine the relationship between the endothelin-1 (ET-1) concentration and the metabolic variables characteristic of the insulin resistance syndrome ([IRS] hyperinsulinemia, insulin resistance, hypertriglyceridemia, low high-density lipoprotein [HDL] cholesterol, visceral obesity, and glycemic abnormalities). The measurement of circulating ET-1 is a well-recognized marker of endothelial atherosclerotic and cardiovascular disease. Two hundred subjects were divided into 3 groups. Group 1 included 50 subjects with impaired glucose tolerance (IGT) or non-insulin-dependent diabetes mellitus (NIDDM) with IRS. Group 2 included 50 subjects with IGT or NIDDM without IRS. Group 3 included 100 normal subjects as controls. ET-1 levels were higher in group 1 versus groups 2 and 3 in women (11.2 +/- 0.7 v 7.9 +/- 0.5 and 6.6 +/- 0.4 pg/mL, P < .01) and men (10.1 +/- 0.6 v 6.5 +/- 0.8 and 7.2 +/- 0.3 pg/mL, P < .01). No differences were found between groups 2 and 3. With simple regression analysis, ET-1 levels significantly correlated with insulin, glycosylated hemoglobin, body weight, waist to hip ratio, and triglyceride values. However, with multiple regression analysis, only triglycerides (P < .009) and glycosylated hemoglobin (P < .001) remained independently correlated with ET-1. In conclusion, this cross-sectional study indicates that glycosylated hemoglobin and triglycerides are independently correlated with ET-1 levels in patients with IRS.

Effects of endothelin-1 and nitric oxide on glucokinase activity in isolated rat hepatocytes.

To test the hypothesis that endothelin-1 (ET-1) and nitric oxide (NO) influence glucokinase (GK) activity in an opposite manner, we evaluated the effects of ET-1, L-NAME, an inhibitor of NO synthase, and L-arginine, a substrate for NO synthase, on GK activity and glycogen content in isolated rat hepatocytes. Moreover, to understand the receptor involved in the process, the effects of BQ 788, a specific antagonist of ETB receptor, and PD 142893, an antagonist of ETA-ETB receptors, were also evaluated. GK activity, cyclic guanosine monophosphate (cGMP), and glycogen intracellular content were measured on isolated hepatocytes, while glucose levels and NO as NO2-/NO3- were determined in the medium. High ET-1 levels induced a 20% decrease of NO2-/NO3- levels and cGMP intracellular content, followed by a 49% reduction of GK activity and a 15% decrease of glycogen. In parallel, a 10% increase of glucose in the medium was observed. In the presence of L-NAME, GK activity and glycogen levels showed analogous decrements as observed with ET-1. Also in this case, a significant decrease of the intracellular content of cGMP was observed. No synergistic effects of ET-1 and L-NAME were observed. L-Arginine was able to counteract the inhibitory effect of ET-1 on cGMP and GK activity. Glycogen content was slightly but not significantly reduced, and under those conditions, a significant decrease of glucose in the medium was observed. When hepatocytes were incubated with ET-1 plus BQ 788 or ET-1 plus PD 142893, GK activity was unchanged. Interestingly, no changes were observed in NO2-/NO3- levels and the intracellular content of cGMP was not modified when the antagonists of ET-1 receptors were added to the medium. In conclusion, the present study shows that the NO pathway seems to be an important regulator of GK activity and glycogen content through cGMP activity. In addition, ET-1 seems to be not active per se, but its activity seems mediated by a simultaneous decrease of NO levels.

Endothelial and metabolic characteristics of patients with angina and angiographically normal coronary arteries: comparison with subjects with insulin resistance syndrome and normal controls.

OBJECTIVES: This study was performed to characterize the endothelial and metabolic alterations of patients with angina and angiographically normal coronary arteries ("cardiac" syndrome X [CSX]) compared with subjects with insulin resistance syndrome ("metabolic" syndrome X [MSX]) and normal controls. BACKGROUND: Previous studies have found high endothelin-1 levels, impaired endothelium-dependent vasodilation and insulin resistance in patients with angina pectoris and angiographically normal coronary arteries. On the other hand, subjects with insulin resistance syndrome have shown high endothelin-1 levels. METHODS: Thirty-five subjects were studied: 13 patients with angina pectoris and angiographically normal coronary arteries (CSX group); 9 subjects with insulin resistance syndrome (MSX group) and 13 normal controls. All subjects received an acute intravenous bolus of insulin (0.1 U/kg) combined with a euglycemic clamp and forearm indirect calorimetry. Endothelin-1 levels, nitrite/nitrate (NOx) levels, end products of nitric oxide metabolism, glucose infusion rates (index of insulin sensitivity) and their incremental areas (deltaAUCs [area under curves]) were measured during this period. RESULTS: Basal endothelin-1 levels were higher in CSX and MSX groups than in normal controls (8.19 +/- 0.46 and 6.97 +/- 0.88 vs. 3.67 +/- 0.99 pg/ml; p < 0.01), while basal NOx levels were significantly higher in MSX group than in CSX and normal controls (36.5 +/- 4.0 vs. 24.2 +/- 3.3 and 26.8 +/- 3.2 mol/liter, p < 0.05). After insulin administration, the deltaAUCs of NOx (p < 0.05) were lower in CSX group than in MSX and normal controls, and the deltaAUCs of endothelin-1 were lower in group CSX than in normal controls. Glucose infusion rate was significantly lower in CSX and MSx groups than in normal controls (p < 0.01), suggesting that in both CSX and MSX groups insulin resistance is present. A positive correlation was found between the deltaAUCs of nitric oxide and the AUCs of glucose infusion rate. CONCLUSIONS: Blunted nitric oxide and endothelin responsiveness to intravenously infused insulin is a typical feature of patients with angina pectoris and angiographically normal coronary arteries and may contribute to the microvascular dysfunction observed in these subjects.

Reversal of diabetes in mice by implantation of human fibroblasts genetically engineered to release mature human insulin.

Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

Mediation of the hepatic effects of growth hormone by its lipolytic activity.

The aim of the study was to investigate the acute effect of GH per se, independent from its lipolytic activity, on glucose and lipid oxidation and glucose turnover in seven healthy subjects. Five tests lasting 360 min were performed. Each test consisted of a 4-h equilibration period followed by a euglycemic hyperinsulinemic (25 mU/kg x h) clamp lasting 2 h. In test 1 (control experiment) saline was infused, leaving GH and FFA at basal levels. In tests 2, 3, and 4, GH was infused (80 ng/kg x min) to increase GH levels. Whereas in test 2 FFA levels were free to increase due to GH lipolytic activity, in test 3 FFA elevation was prevented by using an antilipolytic compound (Acipimox) that allowed evaluation of the effect of GH at low FFA levels. In test 4 (GH+Acipimox+heparin) GH infusion was associated with the administration of Acipimox and heparin to maintain FFA at the basal level to evaluate the effect of GH per se independent from GH lipolytic activity. In test 5 Acipimox and a variable heparin infusion were given to evaluate possible effects of Acipimox other than the inhibition of lipolysis. During the euglycemic hyperinsulinemic clamp in the presence of high GH and FFA levels (test 2), glucose oxidation was significantly lower and lipid oxidation was significantly higher than in tests 1, 3, 4, and 5. During the same period, hepatic glucose production was completely suppressed in the control study (test 1; 94%) and in test 5 (99.6%), whereas it was significantly less inhibited (65%, 74%, and 73%) when GH was administered in tests 2, 3, and 4. In conclusion, these results suggest that GH directly mediates the reduction of insulin's effect on the liver. In addition, the effect of GH on glucose and lipid oxidation is not direct, but is mediated by its lipolytic activity.

Long-term in vitro exposure to high glucose increases proinsulin-like-molecules release by isolated human islets.

The aim of this study was to determine the effect of long-term in vitro exposure to high glucose on the release and content of proinsulin and insulin in human islets. After 48 h culture in CMRL medium at 5.5 mM (control islets) and 16.7 mM glucose (experimental islets), islets were perifused and acutely stimulated with 16.7 mM glucose, followed by 3.3 mM glucose. Compared with control islets, experimental islets showed a higher basal release of true insulin and proinsulin-like-molecules (PLM), with no increase of true insulin and PLM release in response to 16.7 mM glucose, and a paradoxical true insulin release in response to 3.3 mM glucose; the PLM/total insulin ratio increased significantly after 16.7 mM glucose. Moreover these islets showed a decreased true insulin content and an increased PLM/total insulin ratio. Quantitative ultrastructural analysis of granules, supported by double gold immunostaining with monoclonal antibodies against proinsulin and insulin, showed an increased proinsulin to insulin ratio in beta-cells from experimental islets. These data support in vitro what was recently shown in vivo, and further confirm that culture in high glucose is a useful tool to mimic the effect of in vivo chronic hyperglycemia on human beta-cell function.

Gluconeogenesis in isolated rat hepatocytes evaluated by gas chromatography/mass spectrometry using deuterated water.

Tritiated water and radioactive tracers have been used to monitor glucose production by primary cultures of hepatocytes. More recently, 3H2O has been replaced for by 2H2O in 'in vivo' studies addressed at the evaluation of the relative contribution of gluconeogenesis to total glucose production. In this work, the possibility of using 2H2O to determine the ratio between the glucogenic flux and the overall flux through glucose 6-phosphate in isolated liver cells in vitro was evaluated. For this purpose, hepatocytes from either fasted or fed rats were incubated with a medium containing 6, 12 and 25% of 2H2O in the presence of either 2 or 20 mM pyruvate. Isotopomer analysis of six different mass clusters (m/z 328, 314, 242, 212, 187 and 145) was carried out by gas chromatography/mass spectrometry (GC/MS) of glucose aldonitrile pentaacetate. For each cluster, ions at m/z +1, +2, +3 and +4 were monitored. From the combination of different clusters the enrichment at C-6 and C-2 of glucose was computed and the C-6/C-2 ratio was considered to represent the contribution of gluconeogenesis to total glucose production, as suggested previously. Based on the results obtained, conditions selected to be optimum for the use of the method in studies on the modulation of gluconeogenesis were as follows: incubation of hepatocytes with 20 mM pyruvate in 12% 2H2O followed GC/electron ionization MS analysis of the clusters of ions at m/z 328, 314 and 187 of the glucose derivative to calculate enrichment at the C-2 and C-6 positions of glucose.