Leveraging the future of diagnosis and management of diabetes: From old indexes to new technologies.

BACKGROUND: Diabetes is a heterogeneous and multifactorial disease. However, glycemia and glycated hemoglobin have been the focus of diabetes diagnosis and management for the last decades. As diabetes management goes far beyond glucose control, it has become clear that assessment of other biochemical parameters gives a much wider view of the metabolic state of each individual, enabling a precision medicine approach. METHODS: In this review, we summarize and discuss indexes that have been used in epidemiological studies and in the clinical practice. RESULTS: Indexes of insulin secretion, sensitivity/resistance and metabolism have been developed and validated over the years to account also with insulin, C-peptide, triglycerides or even anthropometric measures. Nevertheless, each one has their own objective and consequently, advantages and disadvantages for specific cases. Thus, we discuss how new technologies, namely new sensors but also new softwares/applications, can improve the diagnosis and management of diabetes, both for healthcare professionals but also for caretakers and, importantly, to promote the empowerment of people living with diabetes. CONCLUSIONS: In long-term, the solution for a better diabetes management would be a platform that allows to integrate all sorts of relevant information for the person with diabetes and for the healthcare practitioners, namely glucose, insulin and C-peptide or, in case of need, other parameters/indexes at home, sometimes more than once a day. This solution would allow a better and simpler disease management, more adequate therapeutics thereby improving patients' quality of life and reducing associated costs.

Prediabetes blunts DPP4 genetic control of postprandial glycaemia and insulin secretion.

AIMS/HYPOTHESIS: Imbalances in glucose metabolism are hallmarks of clinically silent prediabetes (defined as impaired fasting glucose and/or impaired glucose tolerance) representing dysmetabolism trajectories leading to type 2 diabetes. CD26/dipeptidyl peptidase 4 (DPP4) is a clinically proven molecular target of diabetes-controlling drugs but the DPP4 gene control of dysglycaemia is not proven. METHODS: We dissected the genetic control of post-OGTT and insulin release responses by the DPP4 gene in a Portuguese population-based cohort of mainly European ancestry that comprised individuals with normoglycaemia and prediabetes, and in mouse experimental models of Dpp4 deficiency and hyperenergetic diet. RESULTS: In individuals with normoglycaemia, DPP4 single-nucleotide variants governed glycaemic excursions (rs4664446, p=1.63x10-7) and C-peptide release responses (rs2300757, p=6.86x10-5) upon OGTT. Association with blood glucose levels was stronger at 30 min OGTT, but a higher association with the genetic control of insulin secretion was detected in later phases of the post-OGTT response, suggesting that the DPP4 gene directly senses glucose challenges. Accordingly, in mice fed a normal chow diet but not a high-fat diet, we found that, under OGTT, expression of Dpp4 is strongly downregulated at 30 min in the mouse liver. Strikingly, no genetic association was found in prediabetic individuals, indicating that post-OGTT control by DPP4 is abrogated in prediabetes. Furthermore, Dpp4 KO mice provided concordant evidence that Dpp4 modulates post-OGTT C-peptide release in normoglycaemic but not dysmetabolic states. CONCLUSIONS/INTERPRETATION: These results showed the DPP4 gene as a strong determinant of post-OGTT levels via glucose-sensing mechanisms that are abrogated in prediabetes. We propose that impairments in DPP4 control of post-OGTT insulin responses are part of molecular mechanisms underlying early metabolic disturbances associated with type 2 diabetes.

Increased Intake of Both Caffeine and Non-Caffeine Coffee Components Is Associated with Reduced NAFLD Severity in Subjects with Type 2 Diabetes.

Coffee may protect against non-alcoholic fatty liver disease (NAFLD), but the roles of the caffeine and non-caffeine components are unclear. Coffee intake by 156 overweight subjects (87% with Type-2-Diabetes, T2D) was assessed via a questionnaire, with 98 subjects (all T2D) also providing a 24 h urine sample for quantification of coffee metabolites by LC-MS/MS. NAFLD was characterized by the fatty liver index (FLI) and by Fibroscan® assessment of fibrosis. No associations were found between self-reported coffee intake and NAFLD parameters; however, total urine caffeine metabolites, defined as Σcaffeine (caffeine + paraxanthine + theophylline), and adjusted for fat-free body mass, were significantly higher for subjects with no liver fibrosis than for those with fibrosis. Total non-caffeine metabolites, defined as Σncm (trigonelline + caffeic acid + p-coumaric acid), showed a significant negative association with the FLI. Multiple regression analyses for overweight/obese T2D subjects (n = 89) showed that both Σcaffeine and Σncm were negatively associated with the FLI, after adjusting for age, sex, HbA1c, ethanol intake and glomerular filtration rate. The theophylline fraction of Σcaffeine was significantly increased with both fibrosis and the FLI, possibly reflecting elevated CYP2E1 activity-a hallmark of NAFLD worsening. Thus, for overweight/obese T2D patients, higher intake of both caffeine and non-caffeine coffee components is associated with less severe NAFLD. Caffeine metabolites represent novel markers of NAFLD progression.

Loss of postprandial insulin clearance control by Insulin-degrading enzyme drives dysmetabolism traits.

Systemic insulin availability is determined by a balance between beta-cell secretion capacity and insulin clearance (IC). Insulin-degrading enzyme (IDE) is involved in the intracellular mechanisms underlying IC. The liver is a major player in IC control yet the role of hepatic IDE in glucose and lipid homeostasis remains unexplored. We hypothesized that IDE governs postprandial IC and hepatic IDE dysfunction amplifies dysmetabolic responses and prediabetes traits such as hepatic steatosis. In a European/Portuguese population-based cohort, IDE SNPs were strongly associated with postprandial IC in normoglycemic men but to a considerably lesser extent in women or in subjects with prediabetes. Liver-specific knockout-mice (LS-IDE KO) under normal chow diet (NCD), showed reduced postprandial IC with glucose intolerance and under high fat diet (HFD) were more susceptible to hepatic steatosis than control mice. This suggests that regulation of IC by IDE contributes to liver metabolic resilience. In agreement, LS-IDE KO hepatocytes revealed reduction of Glut2 expression levels with consequent impairment of glucose uptake and upregulation of CD36, a major hepatic free fatty acid transporter. Together these findings provide strong evidence that dysfunctional IC due to abnormal IDE regulation directly impairs postprandial hepatic glucose disposal and increases susceptibility to dysmetabolic conditions in the setting of Western diet/lifestyle.

S-Nitrosoglutathione Reverts Dietary Sucrose-Induced Insulin Resistance.

The liver is a fundamental organ to ensure whole-body homeostasis, allowing for a proper increase in insulin sensitivity from the fast to the postprandial status. Hepatic regulation of glucose metabolism is crucial and has been shown to be modulated by glutathione (GSH) and nitric oxide (NO). However, knowledge of the metabolic action of GSH and NO in glucose homeostasis remains incomplete. The current study was designed to test the hypothesis that treatment with S-nitrosoglutathione is sufficient to revert insulin resistance induced by a high-sucrose diet. Male Wistar rats were divided in a control or high-sucrose group. Insulin sensitivity was determined: (i) in the fast state; (ii) after a standardized test meal; (iii) after GSH + NO; and after (iv) S-nitrosoglutathione (GSNO) administration. The fasting glucose level was not different between the control and high-sucrose group. In the liver, the high-sucrose model shows increased NO and unchanged GSH levels. In control animals, insulin sensitivity increased after a meal or administration of GSH+NO/GSNO, but this was abrogated by sucrose feeding. GSNO was able to revert insulin resistance induced by sucrose feeding, in a dose-dependent manner, suggesting that they have an insulin-sensitizing effect in vivo. These effects are associated with an increased insulin receptor and Akt phosphorylation in muscle cells. Our findings demonstrate that GSNO promotes insulin sensitivity in a sucrose-induced insulin-resistant animal model and further implicates that this antioxidant molecule may act as a potential pharmacological tool for the treatment of insulin resistance in obesity and type 2 diabetes.

Metabolic Footprint, towards Understanding Type 2 Diabetes beyond Glycemia.

Type 2 diabetes (T2D) heterogeneity is a major determinant of complications risk and treatment response. Using cluster analysis, we aimed to stratify glycemia within metabolic multidimensionality and extract pathophysiological insights out of metabolic profiling. We performed a cluster analysis to stratify 974 subjects (PREVADIAB2 cohort) with normoglycemia, prediabetes, or non-treated diabetes. The algorithm was informed by age, anthropometry, and metabolic milieu (glucose, insulin, C-peptide, and free fatty acid (FFA) levels during the oral glucose tolerance test OGTT). For cluster profiling, we additionally used indexes of metabolism mechanisms (e.g., tissue-specific insulin resistance, insulin clearance, and insulin secretion), non-alcoholic fatty liver disease (NAFLD), and glomerular filtration rate (GFR). We found prominent heterogeneity within two optimal clusters, mainly representing normometabolism (Cluster-I) or insulin resistance and NAFLD (Cluster-II), at higher granularity. This was illustrated by sub-clusters showing similar NAFLD prevalence but differentiated by glycemia, FFA, and GFR (Cluster-II). Sub-clusters with similar glycemia and FFA showed dissimilar insulin clearance and secretion (Cluster-I). This work reveals that T2D heterogeneity can be captured by a thorough metabolic milieu and mechanisms profiling-metabolic footprint. It is expected that deeper phenotyping and increased pathophysiology knowledge will allow to identify subject's multidimensional profile, predict their progression, and treat them towards precision medicine.

Nutrition Education in Portuguese Medical Students: Impact on the Attitudes and Knowledge.

INTRODUCTION: Nutrition has been underrepresented in the curriculum of many medical schools and therefore physicians do not feel adequately prepared to provide dietary counselling. The aim of the present study is to determine the impact of a Nutrition and Metabolism curricular unit on nutrition attitudes, knowledge and confidence on future clinical practice of medical students. MATERIAL AND METHODS: All the students enrolled in the curricular unit (2017/2018) were invited to complete a questionnaire assessing their nutritional knowledge and eating habits at the beginning and at the end of the semester (n = 310). RESULTS: Initially, students reported good eating habits and nutrition knowledge. These aspects improved at the end of the study. Moreover, students reported that they felt more confident to do dietary counselling after intervention. DISCUSSION: Most medical students answered affirmatively to all questions related with good habits or eating behaviours, and the acquisition of knowledge had an impact in specific attitudes. After the Nutrition and Metabolism classes the students felt able to provide dietary counselling in different clinical settings, but none of the students felt extremely confident about their competencies for dietary counselling. This can be due to the fact that the students involved were in the first year of the integrated master's degree in medicine, which is a preclinical year, and thus distant from the medical reality and from contact with patients. CONCLUSION: Nutrition education can have a positive impact on attitudes and eating behaviours, knowledge and in the perception of competencies for dietary counselling.

Introdução: A nutrição não tem sido uma prioridade no programa curricular de muitas escolas médicas e, portanto, os médicos não se sentem devidamente preparados para realizar aconselhamento alimentar. O objetivo deste estudo consiste em determinar o impacto do ensino de Nutrição e Metabolismo nas atitudes e comportamentos, conhecimento e confiança na prática clínica futura dos alunos de medicina. Material e Métodos: Todos os estudantes (n = 310) inscritos na unidade curricular (2017/2018) foram convidados a preencher um questionário, no início e no final do semestre, para avaliar os seus hábitos e conhecimentos alimentares. Resultados: Inicialmente, os estudantes apresentavam bons hábitos alimentares e um bom conhecimento nutricional. Não obstante, no final do estudo verificou-se que estes parâmetros melhoraram. Mais ainda, após a unidade curricular os estudantes sentiam-se mais confiantes para realizar aconselhamento nutricional. Discussão: Após a unidade curricular, a maioria dos estudantes respondeu afirmativamente às questões referentes a atitudes e comportamentos alimentares, sendo que a aquisição de novos conhecimentos pode ser responsável pelo aumento significativo de respostas afirmativas a determinadas questões. Os estudantes sentiram-se capazes de realizar aconselhamento alimentar em diferentes contextos clínicos, mas foram poucos os que reportaram sentir-se fortemente confiantes. Tal pode dever-se ao facto de frequentarem o primeiro ano do mestrado integrado em medicina, ano pré-clínico, sem contacto com a prática médica e com o doente. Conclusão: Pode verificar-se que o ensino de Nutrição numa escola médica teve impacto positivo nas atitudes e comportamentos alimentares, conhecimento e perceção de competências para a realização de aconselhamento nutricional.

How Inflammation Impinges on NAFLD: A Role for Kupffer Cells.

Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming the most prevalent cause of liver disease worldwide and afflicts adults and children as currently associated with obesity and insulin resistance. Even though lately some advances have been made to elucidate the mechanism and causes of the disease much remains unknown about NAFLD. The aim of this paper is to discuss the present knowledge regarding the pathogenesis of the disease aiming at the initial steps of NAFLD development, when inflammation impinges on fat liver deposition. At this stage, the Kupffer cells attain a prominent role. This knowledge becomes subsequently relevant for the development of future diagnostic, prevention, and therapeutic options for the management of NAFLD.

Acute glucagon induces postprandial peripheral insulin resistance.

Glucagon levels are often moderately elevated in diabetes. It is known that glucagon leads to a decrease in hepatic glutathione (GSH) synthesis that in turn is associated with decreased postprandial insulin sensitivity. Given that cAMP pathway controls GSH levels we tested whether insulin sensitivity decreases after intraportal (ipv) administration of a cAMP analog (DBcAMP), and investigated whether glucagon promotes insulin resistance through decreasing hepatic GSH levels.Insulin sensitivity was determined in fed male Sprague-Dawley rats using a modified euglycemic hyperinsulinemic clamp in the postprandial state upon ipv administration of DBcAMP as well as glucagon infusion. Glucagon effects on insulin sensitivity was assessed in the presence or absence of postprandial insulin sensitivity inhibition by administration of L-NMMA. Hepatic GSH and NO content and plasma levels of NO were measured after acute ipv glucagon infusion. Insulin sensitivity was assessed in the fed state and after ipv glucagon infusion in the presence of GSH-E. We founf that DBcAMP and glucagon produce a decrease of insulin sensitivity, in a dose-dependent manner. Glucagon-induced decrease of postprandial insulin sensitivity correlated with decreased hepatic GSH content and was restored by administration of GSH-E. Furthermore, inhibition of postprandial decrease of insulin sensitivity L-NMMA was not overcome by glucagon, but glucagon did not affect hepatic and plasma levels of NO. These results show that glucagon decreases postprandial insulin sensitivity through reducing hepatic GSH levels, an effect that is mimicked by increasing cAMP hepatic levels and requires physiological NO levels. These observations support the hypothesis that glucagon acts via adenylate cyclase to decrease hepatic GSH levels and induce insulin resistance. We suggest that the glucagon-cAMP-GSH axis is a potential therapeutic target to address insulin resistance in pathological conditions.

Risk of postprandial insulin resistance: the liver/vagus rapport.

Ingestion of a meal is the greatest challenge faced by glucose homeostasis. The surge of nutrients has to be disposed quickly, as high concentrations in the bloodstream may have pathophysiological effects, and also properly, as misplaced reserves may induce problems in affected tissues. Thus, loss of the ability to adequately dispose of ingested nutrients can be expected to lead to glucose intolerance, and favor the development of pathologies. Achieving interplay of several organs is of upmost importance to maintain effectively postprandial glucose clearance, with the liver being responsible of orchestrating global glycemic control. This dogmatic role of the liver in postprandial insulin sensitivity is tightly associated with the vagus nerve. Herein, we uncover the behaviour of metabolic pathways determined by hepatic parasympathetic function status, in physiology and in pathophysiology. Likewise, the inquiry expands to address the impact of a modern lifestyle, especially one's feeding habits, on the hepatic parasympathetic nerve control of glucose metabolism.

Effects of CPAP on nitrate and norepinephrine levels in severe and mild-moderate sleep apnea.

BACKGROUND: Reduced plasma nitrate (NO(x)) levels and increased urinary norepinephrine (U-NE) levels have been described in severe obstructive sleep apnea (OSA), and are reverted by continuous positive airway pressure (CPAP). The effect of CPAP on these biomarkers in mild-moderate OSA is not well understood. The aim of this study was to compare NO(x) and U-NE levels and blood pressure (BP) between male patients with mild-moderate and severe OSA and determine the impact of 1 month of CPAP therapy on these parameters. METHODS: We undertook a prospective study of 67 consecutive OSA patients (36 mild-moderate, 31 severe). Measurements of plasma NO(x) at 11 pm, 4 am and 7 am, 24-h U-NE and ambulatory BP were obtained at baseline and after 1 month of CPAP. RESULTS: At baseline, NO(x) levels showed a significant decrease during the night in both groups (p < 0.001). U-NE level and BP were significantly higher in the severe OSA group. After 1 month of CPAP, there was a significant increase in NO(x) levels and a reduction in U-NE level and BP only in patients with severe OSA. CONCLUSIONS: One month of CPAP results in significant improvements in NO(x) levels, 24-h U-NE level and BP in patients with severe OSA, but not in patients with mild-moderate OSA. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01769807.

Postprandial but not fasting insulin resistance is an early identifier of dysmetabolism in overweight subjects.

The dynamic response to insulin is highly potentiated after meal ingestion, and this meal-induced insulin sensitization (MIS) in healthy subjects is dependent on cholinergic mechanisms. The main objective of this study was to test the hypothesis that the reduced response to insulin observed in moderately overweight subjects, in comparison with control lean subjects, is due to MIS impairment and not to a reduction in the direct hypoglycemic action of insulin. Both lean and overweight male subjects were recruited. Insulin sensitivity (IS) was assessed by the rapid insulin sensitivity test (RIST) performed after a 24 h fast, as well as after a standardized meal. Fasting glucose disposal was similar between lean and overweight subjects. Following the meal, glucose disposal increased more extensively in lean than overweight subjects. The insulin profiles, in both fasted and fed states, were superimposable, suggesting that the absence of a factor other than insulin is responsible for the decreased postprandial insulin sensitivity observed in overweight subjects. Our data suggest that in overweight subjects, MIS contribution is decreased, which is responsible for the postprandial impaired IS observed and is suggested to be the cause, not effect, of mild adiposity.

Meal-induced insulin sensitization and its parasympathetic regulation in humans.

In animal studies, the whole-body glucose disposal effect of insulin is low in the fasted state or after atropine infusion, but doubles after a meal, consistent with the hepatic insulin-sensitizing substance (HISS) hypothesis. We tested how a standardized test meal and atropine affected the dynamic response to insulin in humans. Insulin sensitivity was assessed in healthy male subjects (aged 28.9 +/- 1.9 years, body mass index 23.3 +/- 0.8 kg.m-2) by using the rapid insulin sensitivity test (RIST), which is a transient euglycemic clamp. After a 24-hour fasting period, dynamic insulin sensitivity was assessed and then repeated 100 min after the test meal. In a second protocol, the volunteers were fed the standardized test meal and intravenous atropine (0.5 mg) or saline (control group) was administered 50 min before insulin sensitivity assessment. Insulin sensitivity increased in the fed state (232.1% +/- 46.3%, n = 7) in comparison with the 24-hour fasted state. In the atropine protocol, the drug partially blocked (56.5% +/- 11.6%, n = 6) insulin sensitivity. In humans, feeding resulted in increased insulin sensitivity. The low dose of atropine in humans lead to a partial HISS-dependent decrease in insulin sensitivity. Meal-induced insulin sensitization occured in humans by a similar mechanism as that reported in other species. The sensitization process was regulated by a cholinergic 'feeding signal.'

Hepatic-dependent and -independent insulin actions are impaired in the obese Zucker rat model.

OBJECTIVE: Whole-body insulin sensitivity (IS) depends on a hepatic pathway, involving parasympathetic activation and hepatic nitric oxide (NO) production. Both atropine and N-monomethyl-L-arginine (L-NMMA, NO synthase inhibitor) induce insulin resistance (IR). IR is associated with obesity. Because NO action was shown to be impaired in animal models of obesity, such as the obese Zucker rat (OZR), we tested the hypothesis that the hepatic-dependent pathway is diminished in OZR, resulting in IR. RESEARCH METHODS AND PROCEDURES: Lean Zucker rats (LZRs) were used as OZR controls. IS was evaluated in terms of glucose disposal [milligrams of glucose per kilogram of body weight (bw)]. Two groups were submitted to two protocols. First, a control clamp was followed by a post-atropine (3 mg/kg intravenously) clamp. Second, after the control clamp, L-NMMA (0.73 mg/kg intraportally) was given, and a second clamp was performed. Hepatic-dependent IS was assessed by subtracting the response after atropine or L-NMMA from the basal response. RESULTS: In the first protocol, basal IS was lower in OZR than in LZR (OZR, 73.7 +/- 14.2; LZR, 289.2 +/- 24.7 mg glucose/kg bw; p < 0.001), and atropine decreased IS in the same proportion for both groups (OZR, 41.3 +/- 8.0%; LZR, 40.1 +/- 6.5%). Equally, in the second protocol, OZR presented lower IS (OZR, 79.3 +/- 1.6; LZR, 287.4 +/- 22.7 mg glucose/kg bw; p < 0.001). L-NMMA induced IS inhibition in both groups (OZR, 48.3 +/- 6.6%; LZR, 46.4 +/- 4.1%), similar to that after atropine. DISCUSSION: We show that the IR in OZR is due to similar impairment of both hepatic-dependent and -independent components of insulin action, suggesting the existence of a defect common to both pathways.

A new technique to assess insulin sensitivity in humans: the rapid insulin sensitivity test (RIST).

The objective of this study was to develop a Rapid Insulin Sensitivity Test (RIST) in humans, a test already used in animal studies. Insulin sensitivity was assessed using a rapid modified euglycemic clamp, the RIST. In this test, glucose disposition was determined after an intravenous (i.v.) bolus (50mU/kg bw administered over 30 seconds) of insulin, before and after feeding a standardized test meal, in healthy male subjects (aged 27.8 +/- 2.4 years, BMI 23.5 +/- 1.2 kg/m2). The RIST uses as the index of insulin sensitivity, the total amount of glucose required to be infused to maintain euglycemia during insulin action following an i.v. bolus of insulin. During the RIST, glucose levels are determined at 2-min intervals in order to clamp the glycemia at baseline values. Following a 24 hr fasting period, the RIST index was 225.6 +/- 25.1 mg glucose/kg bw. The volunteers were then fed a standardized test meal, a new stable glucose level was obtained 100 min after the meal, and a second RIST was performed. The glucose requirement (RIST index) increased to 647.9 +/- 73.5 mg glucose/kg bw following the standardized test meal (n = 5, p < 0.001). This report describes a new technique to evaluate insulin sensitivity in healthy humans. The RIST is a powerful research tool to assess the glucose utilization action of an insulin bolus in fasted and fed states both evaluated in the same day.

Carvedilol's actions are largely mediated by endogenous nitric oxide.

Carvedilol's adrenergic antagonism does not fully explain its therapeutic actions. We therefore tested the hypothesis that its action is associated with an increase in NO synthesis. Wistar rats (male, 9 weeks, n = 10) anesthetized with sodium pentobarbital were used. Arterial NO concentration ([NO]), determined by chemiluminescence, and mean arterial pressure (MAP) were monitored throughout the experiment. In protocol 1), the effects of carvedilol (1 mg/kg, iv) were studied over a eriod of 90 min. In protocol 2), carvedilol was p administered, followed by the NO synthase (NOS) inhibitor L-NAME (5 mg/kg, iv) and by a second carvedilol administration. In protocol 1), carvedilol induced a significant fall in MAP (from 125,0 +/- 4,5 mmHg to 78.2 +/- 2.6 mmHg; p <0.001), reaching a minimum at t = 11.7 +/- 2.1 min and recovering 60 min afterwards (105.7 +/- 5.9 mmHg). Plasma [NO] varied in response to carvedilol in inverse proportion to MAP: baseline, 19.8 +/- 0.9 microM; t = 11.7 +/- 2.1 min, 32,3 +/- 2,3 microM; t = 60 min, 17.3 +/- 1.9 microM. In protocol 2), L-NAME administration blocked the effects of carvedilol (L-NAME: MAP, 129.9 +/- 5.0 mmHg; [NO], 13,1 +/- 2,3 microM. Post-L-NAME carvedilol administration resulted in MAP of 108.3 +/- 8.0 mmHg, NS, and [NO], 21.3 +/- 1.3 microM, NS. These results suggest that carvedilol increases plasma [NO], which is associated with a fall in MAP. Furthermore, carvedilol's hemodynamic action was blocked by NOS inhibition, suggesting that it depends on endogenous NO production, thus possibly explaining carvedilol's effects in hypertension and in cardiac failure.

Carvedilol action is dependent on endogenous production of nitric oxide.

BACKGROUND: Carvedilol is known to be an adrenoreceptor blocker and free radical scavenger, used in hypertension and cardiac failure. However, its therapeutic actions cannot be fully explained by these mechanisms. In these studies, we tested the hypothesis that carvedilol action is associated with the synthesis/release of nitric oxide (NO). METHODS: Male Wistar rats (n = 22), 9 weeks old, were anesthetized with an intraperitoneal injection of sodium pentobarbital. Mean arterial pressure and arterial NO levels were monitored throughout the experiments. Carvedilol (1 mg/kg, intravenously [iv]) effects were evaluated before and after NO synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 5 mg/kg, iv). RESULTS: Carvedilol induced a significant decrease in basal arterial pressure (from 126.6 +/- 4.3 mm Hg to 75.9 +/- 3.0 mm Hg, P < .001) and significant increase in NO levels (from 17.9 +/- 1.7 micromol/L to 32.2 +/- 2.5 micromol/L, P < .001). After administration of L-NAME the arterial pressure increased (129.9 +/- 5.0 mm Hg, P < .001) with concomitant decrease in NO levels (13.4 +/- 1.6 micromol/L, P < .01). The second carvedilol administration (post-L-NAME) did not affect either arterial pressure (108.3 +/- 8.0 mm Hg) or NO levels (22.1 +/- 1.3 micromol/L). CONCLUSIONS: Our results suggest that the carvedilol-induced decrease of blood pressure is associated with an increase of plasma NO levels. Furthermore, NOS inhibition results in impairment of carvedilol hemodynamic effects and plasma NO levels. Therefore, these results are consistent with the hypothesis that the hemodynamic effect of carvedilol is in part dependent on endogenous NO production.

In vitro nitrosation of insulin A- and B-chains.

The physiological roles of insulin and nitric oxide (NO) have been recently recognized by several studies. A diversity of chemical modifications of insulin is reported both in vivo and in vitro. S-nitrosation, the covalent linkage of NO to cysteine free thiol is recognized as an important post-translational regulation in many proteins. Here we report the in vitro synthesis of an S-nitrosothiol of bovine insulin A- and B-chains. These compounds were characterized by their HPLC chromatographic behavior, monitored by UV visible spectroscopy and electron spray ionization mass spectrometry. The experimental results indicate that each A- and B-chain were S- nitrosated with only one NO group. Stability and solubility of these synthesized derivatives is described for physiological purposes. In this work, nitroso A- and B-chains of insulin were synthesized in vitro in order to better understand the possible interactions between insulin and NO that may be involved in the etiology of insulin resistance.