cyclic-gmp and Obesity

Investigations into the mixed muscle-secretory phenotype of cardiomyocytes from the atrial appendages of the heart led to the discovery that these cells produce, in a regulated manner, two polypeptide hormones - the natriuretic peptides - referred to as atrial natriuretic factor or atrial natriuretic peptide (ANP) and brain or B-type natriuretic peptide (BNP), thereby demonstrating an endocrine function for the heart. Studies on the gene encoding ANP (NPPA) initiated the field of modern research into gene regulation in the cardiovascular system. Additionally, ANP and BNP were found to be the natural ligands for cell membrane-bound guanylyl cyclase receptors that mediate the effects of natriuretic peptides through the generation of intracellular cGMP, which interacts with specific enzymes and ion channels. Natriuretic peptides have many physiological actions and participate in numerous pathophysiological processes. Important clinical entities associated with natriuretic peptide research include heart failure, obesity and systemic hypertension. Plasma levels of natriuretic peptides have proven to be powerful diagnostic and prognostic biomarkers of heart disease. Development of pharmacological agents that are based on natriuretic peptides is an area of active research, with vast potential benefits for the treatment of cardiovascular disease.

Topics: Animals; Atrial Appendage; Atrial Fibrillation; Atrial Natriuretic Factor; Atrial Remodeling; Biomarkers; Cyclic GMP; Diabetes Mellitus; Fibrosis; Gene Expression Regulation, Developmental; Heart Atria; Heart Failure; Humans; Hypertension; Lipid Metabolism; Metabolic Syndrome; Mice; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Obesity; Peptide Fragments; Prognosis; Protein Processing, Post-Translational; Pulmonary Arterial Hypertension; Receptors, Guanylate Cyclase-Coupled; Secretory Vesicles; Ventricular Remodeling; Water-Electrolyte Balance

Guanylate cyclase C (GC-C) receptor is a transmembrane receptor, predominantly expressed in intestinal epithelial cells, which is considered to play a main role in homeostasis and function of the digestive tract. The endogenous ligands for this receptor are the paracrine hormones uroguanylin and guanylin. Upon ligand binding, GC-C receptors increase cyclic guanosine monophosphate (cGMP) levels, regulating a variety of key cell-type specific processes such as chloride and bicarbonate secretion, epithelial cell growth, regulation of intestinal barrier integrity and visceral sensitivity. It has been suggested that GC-C acts as an intestinal tumor suppressor with the potential to prevent the initiation and progression of colorectal cancer. In fact, loss of ligand expression is a universal step in sporadic colorectal carcinogenesis. Interestingly, the role of GC-C is not limited to the digestive tract but it has been extended to several other systems such as the cardiovascular system, kidney, and the central nervous system, where it has been involved in a gut-hypothalamus endocrine axis regulating appetite. Objetive: In this review we summarize the physiology of the GC-C receptor and its ligands, focusing on newly developed drugs like linaclotide, and their suggested role to reverse/prevent the diseases in which the receptor is involved.. Available data points toward a relationship between uroguanylin and guanylin and their receptor and pathological processes like gastrointestinal and renal disorders, colorectal cancer, obesity, metabolic syndrome and mental disorders among others. Recent pharmacological developments in the regulation of GC-receptor may involve further improvements in the treatment of relevant diseases.

Topics: Animals; Colorectal Neoplasms; Cyclic GMP; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Kidney Diseases; Natriuretic Peptides; Obesity; Protein Binding; Protein Transport; Receptors, Peptide; Signal Transduction

The second messenger cyclic guanosine monophosphate (cGMP) is a key mediator in physiological processes such as vascular tone, and its essential involvement in pathways regulating metabolism has been recognized in recent years. Here, we focus on the fundamental role of cGMP in brown adipose tissue (BAT) differentiation and function. In contrast to white adipose tissue (WAT), which stores energy in the form of lipids, BAT consumes energy stored in lipids to generate heat. This so-called non-shivering thermogenesis takes place in BAT mitochondria, which express the specific uncoupling protein 1 (UCP1). The energy combusting properties of BAT render it a promising target in antiobesity strategies in which BAT could burn the surplus energy that has accumulated in obese and overweight individuals. cGMP is generated by guanylyl cyclases upon activation by nitric oxide or natriuretic peptides. It affects several downstream molecules including cGMP-receptor proteins such as cGMP-dependent protein kinase and is degraded by phosphodiesterases. The cGMP pathway contains several signaling molecules that can increase cGMP signaling, resulting in activation and recruitment of brown adipocytes, and hence can enhance the energy combusting features of BAT. In this review we highlight recent results showing the physiological significance of cGMP signaling in BAT, as well as pharmacological options targeting cGMP signaling that bear a high potential to become BAT-centered therapies for the treatment of obesity.

Topics: Adipocytes; Adipose Tissue, Brown; Animals; Cyclic GMP; Diabetes Mellitus; Humans; Mitochondria; Obesity; Signal Transduction

Natriuretic peptides (NPs) are a group of peptide-hormones mainly secreted from the heart, signaling via c-GMP coupled receptors. NP are well known for their renal and cardiovascular actions, reducing arterial blood pressure as well as sodium reabsorption. Novel physiological functions have been discovered in recent years, including activation of lipolysis, lipid oxidation, and mitochondrial respiration. Together, these responses promote white adipose tissue browning, increase muscular oxidative capacity, particularly during physical exercise, and protect against diet-induced obesity and insulin resistance. Exaggerated NP release is a common finding in congestive heart failure. In contrast, NP deficiency is observed in obesity and in type-2 diabetes, pointing to an involvement of NP in the pathophysiology of metabolic disease. Based upon these findings, the NP system holds the potential to be amenable to therapeutical intervention against pandemic diseases such as obesity, insulin resistance, and arterial hypertension. Various therapeutic approaches are currently under development. This paper reviews the current knowledge on the metabolic effects of the NP system and discusses potential therapeutic applications.

Topics: Animals; Cyclic GMP; Drug Design; Exercise; Humans; Hypertension; Insulin Resistance; Metabolic Syndrome; Natriuretic Peptides; Obesity

Obesity is a worldwide pandemic. Obesity-related health and economic costs are staggering. Existing strategies to combat obesity through lifestyle improvements and medical intervention have had limited success. Pharmacotherapy, in combination with lifestyle modification, may play a vital role in reversing the disease burden. However, past and current weight-loss medications have had serious safety risks, notably cardiovascular and psychiatric events.. The authors review the strategies for designing new anti-obesity drugs by describing those currently in development. They describe their target, mechanism of action and developmental or regulatory status. Furthermore, they discuss the problem of weight regain following weight loss, and its relevance to the long-term success of anti-obesity pharmacotherapy.. For weight management drugs to achieve the safety and efficacy required to be impactful, current studies are uncovering and characterizing new targets, including new signaling circuits and hormones regulating appetite and metabolism, and re-evaluating the role of pharmacotherapy in weight management. To avoid the safety failures of many past weight-loss drugs, the models and strategies covered in this article incorporate recent advances in knowledge and technology. We discuss the emergence of cGMP signaling as a potentially transformative target in weight management. Modulating cGMP signaling may represent an ideal goal for an anti-obesity pharmacotherapy, reflecting some of the major themes described in the present review: targeting pathways that are newly realized as relevant for weight management; promoting safety by re-purposing drugs that are safe, proven, and approved for clinical use; and having a synergistic effect on multiple, reinforcing pathways.

Topics: Anti-Obesity Agents; Appetite Depressants; Cyclic GMP; Drug Design; Humans; Life Style; Models, Theoretical; Obesity; Weight Gain; Weight Loss

Cardiac natriuretic peptides have emerged as potent metabolic hormones during the past decade. We here discuss recent work highlighting the potential importance of these hormones in metabolic physiology and diseases.. Natriuretic peptides signal through a cyclic guanosine monophosphate pathway to convey their biological effects at the cell level. Similarly to cyclic adenosine monophosphate, activation of cyclic guanosine monophosphate signaling induces a browning of white fat and thermogenesis. Natriuretic peptides also enhance oxidative capacity and fat oxidation in skeletal muscle of mice and humans. The molecular mechanism involves an upregulation of mitochondrial fat oxidative capacity and respiration. This may be particularly relevant to relay the physiological adaptations of chronic exercise. Population-based studies indicate that circulating natriuretic peptides are lowered in obesity and predict type 2 diabetes. Recent work also directly link natriuretic peptides with type 2 diabetes through a gut-heart axis.. Natriuretic peptides exhibit a wide range of biological actions to control metabolic homeostasis. Natriuretic peptides deficiency in obesity may trigger metabolic dysfunction and lead to type 2 diabetes. Increasing circulating natriuretic peptides level and tissue signaling may help to fight against metabolic complications of obesity.

Topics: Adipose Tissue; Animals; Cyclic GMP; Diabetes Mellitus, Type 2; Homeostasis; Humans; Lipid Metabolism; Mitochondria; Muscle, Skeletal; Natriuretic Peptides; Obesity; Oxidation-Reduction; Signal Transduction; Thermogenesis; Up-Regulation

Phosphodiesterase type 5 (PDE5) is expressed in many tissues (e.g. heart, lung, pancreas, penis) and plays a specific role in hydrolyzing cyclic guanosine monophosphate (cGMP). In adipocytes, cGMP regulates crucial functions by activating cGMP-dependent protein kinase (PKG). Interestingly, PDE5 was recently identified in adipose tissue, although its role remains unclear. Its inhibition, however, was recently shown to affect adipose differentiation and aromatase function. This review summarizes evidence supporting a role for the PDE5-regulated cGMP/PKG system in adipose tissue and its effects on adipocyte function. A better elucidation of the role of PDE5 in the adipocyte could reveal new therapeutic strategies for fighting obesity and metabolic syndrome.

Topics: Adipocytes; Adipogenesis; Adipose Tissue; Animals; Cell Differentiation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Activation; Humans; Insulin; Lipid Metabolism; Lipolysis; Obesity; Signal Transduction

The incidence of obesity in the developed world is increasing at an alarming rate. Concurrent with the increase in the incidence of obesity is an increase in the incidence of type 2 diabetes. Cyclic AMP (cAMP) and cGMP are key second messengers in all cells; for example, when it comes to processes of relevance for the regulation of energy metabolism, cAMP is a key mediator in the regulation of lipolysis, glycogenolysis, gluconeogenesis and pancreatic β cell insulin secretion. PDE3B, one of several enzymes which hydrolyze cAMP and cGMP, is expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, hypothalamic cells and β cells. It has been shown, using PDE3 inhibitors and gene targeting approaches in cells and animals, that altered levels of PDE3B result in a number of changes in the regulation of glucose and lipid metabolism and in overall energy homeostasis. This article highlights the complexity involved in the regulation of PDE3B by hormones, and in the regulation of downstream metabolic effects by PDE3B in several interacting tissues.

Topics: Adipocytes, White; Animals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Diabetes Mellitus, Type 2; Energy Intake; Energy Metabolism; Hepatocytes; Humans; Molecular Targeted Therapy; Obesity; Phosphodiesterase 3 Inhibitors; Phosphorylation; Protein Processing, Post-Translational; Second Messenger Systems

Studied for nearly 30 years for its ability to control many parameters, such as vascular smooth muscle cell relaxation, heart fibrosis, and kidney function, the natriuretic peptide (NP) system is now considered to be a key element in several other major metabolic pathways. After stimulation by NPs, natriuretic peptide receptors (NPR) convert GTP to the second messenger cGMP. In addition to its vasodilatory effects and natriuretic and diuretic functions, cGMP has been positively associated with fat cell function, apoptosis, and NPR expression/activity modulation. The NP system is also closely linked to metabolic syndrome (MetS) progression and obesity control. A new era is now on its way targeting the NP system to not only treat high blood pressure, but to also assist in the fight against the obesity pandemic. Here, we summarize recent data on the role of NPs in hypertension and MetS.

Topics: Cyclic GMP; Guanylate Cyclase; Humans; Hypertension; Metabolic Syndrome; Natriuretic Peptides; Obesity; Signal Transduction

Natriuretic peptides are a family of structurally related but genetically distinct hormones/paracrine factors that regulate blood volume, blood pressure, ventricular hypertrophy, pulmonary hypertension, fat metabolism, and long bone growth. The mammalian members are atrial natriuretic peptide, B-type natriuretic peptide, C-type natriuretic peptide, and possibly osteocrin/musclin. Three single membrane-spanning natriuretic peptide receptors (NPRs) have been identified. Two, NPR-A/GC-A/NPR1 and NPR-B/GC-B/NPR2, are transmembrane guanylyl cyclases, enzymes that catalyze the synthesis of cGMP. One, NPR-C/NPR3, lacks intrinsic enzymatic activity and controls the local concentrations of natriuretic peptides through constitutive receptor-mediated internalization and degradation. Single allele-inactivating mutations in the promoter of human NPR-A are associated with hypertension and heart failure, whereas homozygous inactivating mutations in human NPR-B cause a form of short-limbed dwarfism known as acromesomelic dysplasia type Maroteaux. The physiological effects of natriuretic peptides are elicited through three classes of cGMP binding proteins: cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels. In this comprehensive review, the structure, function, regulation, and biological consequences of natriuretic peptides and their associated signaling proteins are described.

Topics: Animals; Cyclic GMP; Growth Disorders; Heart Diseases; Humans; Hypertension; Natriuretic Peptides; Obesity; Protein Structure, Tertiary; Receptor Cross-Talk; Receptors, Peptide; Signal Transduction

We have demonstrated a potent and specific lipolytic effect of natriuretic peptides (NP) in human and primates' fat cells. The lipolytic effect of NP is mediated through intracellular production of cGMP and activation of the cGMP-dependent kinase 1alpha. Local infusion of atrial-NP (ANP), directly within the subcutaneous adipose tissue through a microdialysis catheter, increases lipolysis and stimulates blood flow through its vasodilating effect in lean healthy men. This effect is blunted in overweight men and can be recovered by endurance training. Intravenous infusion of physiological doses of ANP induces lipid mobilization. Higher concentrations of ANP that are encountered during heart failure also stimulate lipid oxidation. ANP activates lipolysis and free fatty acids release from adipose tissue during endurance exercise. This effect is paradoxically amplified when exercise is performed under beta-blockade treatment, because of an enhanced cardiac release of ANP. No gender differences in ANP-induced lipid mobilization during exercise have been found. Heart failure is associated with high circulating levels of NP that could participate to the progression toward cachexia. On contrary, a negative correlation between NP levels and body mass index is found in obese persons. The molecular basis of this inverse correlation is not yet demonstrated from a functional standpoint. Further studies are needed to clearly define the pathophysiological role of NP in obesity and heart failure.

Topics: Adipose Tissue; Cyclic GMP; Exercise; Humans; Lipid Mobilization; Lipolysis; Models, Biological; Natriuretic Peptides; Obesity; Receptors, Atrial Natriuretic Factor

Topics: Adenylyl Cyclases; Animals; Anti-Inflammatory Agents; Antiviral Agents; Asthma; Cardiovascular Diseases; Cricetinae; Cyclic AMP; Cyclic GMP; Disease; Enzyme Activation; Extracellular Space; Guanylate Cyclase; Humans; Hypoglycemic Agents; Isoenzymes; Kinetics; Mental Disorders; Mice; Neoplasms; Obesity; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Psoriasis; Rats

In view of the known vasodilatory effects of glucagon-like peptide-1 and exenatide, we investigated the effects of exenatide on vasoactive factors. We analysed blood samples and mononuclear cells (MNCs) from a previous study, collected after a single dose and 12 weeks of exenatide or placebo treatment in a series of 24 patients with type 2 diabetes mellitus. After exenatide treatment, plasma concentrations of atrial natriuretic peptide, cyclic guanyl monophosphate (cGMP) and cyclic adenyl monophosphate increased significantly at 12 weeks. Plasma cGMP and adenylate cyclase expression in MNCs increased significantly after a single dose. Angiotensinogen concentration fell significantly 2 hours after a single dose and at 12 weeks, while renin and angiotensin II levels fell significantly only after a single dose and not after 12 weeks of treatment. Exenatide also suppressed the plasma concentration of transforming growth factor-β and the expression of P311 in MNCs at 12 weeks. Thus, exenatide induces an increase in a series of vasodilators, while suppressing the renin-angiotensin system. These changes may contribute to the overall vasodilatory effect of exenatide.

Topics: Adenylyl Cyclases; Angiotensinogen; Anti-Obesity Agents; Antihypertensive Agents; Atrial Natriuretic Factor; Blood Pressure; Cyclic AMP; Cyclic GMP; Diabetes Mellitus, Type 2; Exenatide; Gene Expression Regulation; Glucagon-Like Peptide 1; Humans; Hypoglycemic Agents; Leukocytes, Mononuclear; Nerve Tissue Proteins; Obesity; Oncogene Proteins; Peptides; Renin-Angiotensin System; Reproducibility of Results; Single-Blind Method; Transforming Growth Factor beta; Venoms

The aim of this study was to evaluate the effects of a new L-arginine-enriched biscuit on endothelial function, insulin sensitivity/secretion and body composition.. The project was composed of two studies. The first study was an acute pilot postprandial study in 7 healthy subjects that evaluated bio-availability and vascular effects of L-arginine-enriched biscuits that contained 6.6 gL-arginine, 21.9 g carbohydrates, 3.6 g protein, 7.5 g fat and 4.3 g dietary fiber compared with placebo biscuits and 6.6 g powdered L-arginine. Subjects underwent the tests in random order, in at least 14-day intervals. The second study was a double-blind crossover study in 15 obese subjects with IGT and MS. These subjects consumed 6.6 g of L-arginine-enriched biscuits or placebo biscuits in a 1600 kcal diet. Each study period lasted 2 weeks with a 2-week washout in between. Endothelial function, glucose tolerance, insulin sensitivity and insulin secretion were evaluated at the end of each intervention period.. In the first study, the groups that received the L-arginine-enriched biscuits and the powdered L-arginine had similarly increased L-arginine, NOx and cGMP levels and post-ischemic blood flow (PI-BF). In both cases, these levels were significantly higher than those in the placebo biscuit recipient group. In the second study, the L-arginine-enriched biscuit recipient group displayed increased L-arginine, NOx, cGMP, PI-BF, and Matsuda index levels, whereas their circulating glucose, proinsulin/insulin ratio and fat mass were decreased compared with the placebo biscuit recipient group.. L-Arginine-enriched biscuits with low sugar and protein content enhance endothelial function and improve glucose metabolism, insulin sensitivity and insulin secretion in subjects with IGT and MS.

Topics: Arginine; Body Composition; Body Weight; Cholesterol; Cross-Over Studies; Cyclic GMP; Double-Blind Method; Endothelium, Vascular; Female; Glucose Intolerance; Humans; Insulin; Insulin Secretion; Male; Metabolic Syndrome; Middle Aged; Nitric Oxide Synthase Type II; Obesity; Pilot Projects; Snacks; Triglycerides

Intracellular levels of cyclic nucleotides can also be controlled by the action of multidrug resistance protein types 4 (MRP4) and 5 (MRP5). To date, no studies evaluated the role of their inhibition in an animal model of erectile dysfunction (ED).. To evaluate the effect of a 2-week treatment with MK571, an inhibitor of the efflux of cyclic nucleotides in the ED of obese mice.. Mice were divided in three groups: (i) lean, (ii) obese, and (iii) obese + MK571. The corpus cavernosum (CC) were isolated, and concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), and tadalafil in addition to electrical field stimulation (EFS) were carried out in phenylephrine pre-contracted tissues. Expression of ABCC4 and ABCC5, intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), the protein levels for pVASP. The MRPs inhibition by MK571 favored the accumulation of cGMP in the smooth muscle cells, thus improving the smooth muscle relaxation and the erectile function in obese mice.

Topics: Acetylcholine; Animals; ATP Binding Cassette Transporter, Subfamily B; Cyclic GMP; Erectile Dysfunction; Humans; Male; Mice; Mice, Obese; Nitroprusside; Obesity; Tadalafil

Chronic kidney disease (CKD) progression is associated with persisting oxidative stress, which impairs the NO-sGC-cGMP signaling cascade through the formation of oxidized and heme-free apo-sGC that cannot be activated by NO. Runcaciguat (BAY 1101042) is a novel, potent, and selective sGC activator that binds and activates oxidized and heme-free sGC and thereby restores NO-sGC-cGMP signaling under oxidative stress. Therefore, runcaciguat might represent a very effective treatment option for CKD/DKD. The potential kidney-protective effects of runcaciguat were investigated in ZSF1 rats as a model of CKD/DKD, characterized by hypertension, hyperglycemia, obesity, and insulin resistance. ZSF1 rats were treated daily orally for up to 12 weeks with runcaciguat (1, 3, 10 mg/kg/bid) or placebo. The study endpoints were proteinuria, kidney histopathology, plasma, urinary biomarkers of kidney damage, and gene expression profiling to gain information about relevant pathways affected by runcaciguat. Furthermore, oxidative stress was compared in the ZSF1 rat kidney with kidney samples from DKD patients. Within the duration of the 12-week treatment study, kidney function was significantly decreased in obese ZSF1 rats, indicated by a 20-fold increase in proteinuria, compared to lean ZSF1 rats. Runcaciguat dose-dependently and significantly attenuated the development of proteinuria in ZSF1 rats with reduced uPCR at the end of the study by -19%, -54%, and -70% at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo treatment. Additionally, average blood glucose levels measured as HbA1C, triglycerides, and cholesterol were increased by five times, twenty times, and four times, respectively, in obese ZSF1 compared to lean rats. In obese ZSF1 rats, runcaciguat reduced HbA1c levels by -8%, -34%, and -76%, triglycerides by -42%, -55%, and -71%, and cholesterol by -16%, -17%, and -34%, at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo. Concomitantly, runcaciguat also reduced kidney weights, morphological kidney damage, and urinary and plasma biomarkers of kidney damage. Beneficial effects were accompanied by changes in gene expression that indicate reduced fibrosis and inflammation and suggest improved endothelial stabilization. In summary, the sGC activator runcaciguat significantly prevented a decline in kidney function in a DKD rat model that mimics common comorbidities and conditions of oxidative stress of CKD patients. Thus, runcaciguat represents a promisin

Topics: Animals; Clinical Trials, Phase II as Topic; Cyclic GMP; Glycated Hemoglobin; Heme; Kidney; Obesity; Proteinuria; Rats; Renal Insufficiency, Chronic

Hydrogen sulfide (H

Topics: Adipose Tissue; Animals; Cyclic GMP; Fatty Acids, Nonesterified; Hydrogen Sulfide; Lipolysis; Male; Metabolic Syndrome; Obesity; Rats; Rats, Wistar; Receptors, Adrenergic, beta; Sulfides

Topics: Animals; Cyclic GMP; Diet, High-Fat; Hyperlipidemias; Inflammation; Male; Nitric Oxide; Nitric Oxide Synthase Type III; Nucleotides, Cyclic; Obesity; Orlistat; Propolis; Rats; Rats, Sprague-Dawley; Signal Transduction; Vasodilation

Robust associations between low plasma level of natriuretic peptides (NP) and increased risk of type 2 diabetes (T2D) have been recently reported in humans. Adipose tissue (AT) is a known target of NP. However it is unknown whether NP signalling in human AT relates to insulin sensitivity and modulates glucose metabolism. We here show in two European cohorts that the NP receptor guanylyl cyclase-A (GC-A) expression in subcutaneous AT was down-regulated as a function of obesity grade while adipose NP clearance receptor (NPRC) was up-regulated. Adipose GC-A mRNA level was down-regulated in prediabetes and T2D, and negatively correlated with HOMA-IR and fasting blood glucose. We show for the first time that NP promote glucose uptake in a dose-dependent manner. This effect is reduced in adipocytes of obese individuals. NP activate mammalian target of rapamycin complex 1/2 (mTORC1/2) and Akt signalling. These effects were totally abrogated by inhibition of cGMP-dependent protein kinase and mTORC1/2 by rapamycin. We further show that NP treatment favoured glucose oxidation and de novo lipogenesis independently of significant gene regulation. Collectively, our data support a role for NP in blood glucose control and insulin sensitivity by increasing glucose uptake in human adipocytes. This effect is partly blunted in obesity.

Topics: Adipocytes; Adipose Tissue; Biomarkers; Cyclic GMP; Diabetes Mellitus, Type 2; Gene Expression Regulation; Glucose; Humans; Insulin Resistance; Models, Biological; Natriuretic Peptides; Obesity; Proto-Oncogene Proteins c-akt; Receptors, Atrial Natriuretic Factor; Signal Transduction; TOR Serine-Threonine Kinases

Stimulation of thermogenic pathways appears to be a promising approach to find new ways of tackling metabolic diseases like obesity and diabetes mellitus type 2. Thermogenic, weight reducing and insulin sensitizing effects of phosphodiesterase 5 (PDE 5) inhibitors have recently been postulated, suggesting that modulators of endogenous cGMP signaling have the therapeutic potential to treat metabolic disorders. However, most studies have been performed in vitro or in animals that were not glucose intolerant. We, thus, aimed to test the metabolic effects of the PDE 5 inhibitor sildenafil by treating diet-induced obese (DIO) mice orally for 8 days. Surprisingly, our results revealed no changes in body temperature, brown adipose tissue (BAT) thermogenesis and gene expression in BAT and inguinal white adipose tissue (iWAT), thus excluding a thermogenic or 'browning' effect of sildenafil in preexisting obesity. In contrast, sildenafil-treated DIO mice displayed changes in liver metabolism and glucose homeostasis resulting in impaired glucose tolerance (P < 0.05), demonstrating for the first time an unfavorable metabolic effect of increased hepatic cGMP signaling in obesity. As sildenafil is commonly prescribed to treat pulmonary arterial hypertension and erectile dysfunction in diabetic and/or obese patients, follow up studies are urgently required to re-evaluate the drug safety.

Topics: Adipose Tissue; Adipose Tissue, Brown; Adipose Tissue, White; Animals; Blood Glucose; Cyclic GMP; Erectile Dysfunction; Glucose Intolerance; Homeostasis; Hypertension; Liver; Male; Mice, Inbred C57BL; Mice, Obese; Obesity; Phosphodiesterase 5 Inhibitors; Signal Transduction; Sildenafil Citrate; Thermogenesis

Obesity is assumed to be a major cause of human essential hypertension; however, the mechanisms responsible for weight-related increase in blood pressure (BP) are not fully understood. The prevalence of hypertension induced by obesity has grown over the years, and the role of the renin-angiotensin-aldosterone system (RAAS) in this process continues to be elucidated. In this scenario, the

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensins; Animals; Blood Pressure; Caloric Restriction; Cyclic GMP; Diet, High-Fat; Enzyme Inhibitors; Glutamyl Aminopeptidase; Kidney; Leptin; Male; Mice; Mice, Inbred C57BL; Obesity; Receptor, Angiotensin, Type 2; Sodium

Current worldwide figures suggest that obesity is pandemic. Understanding the underlying molecular mechanisms would help develop viable anti-obesity therapies. Here, we assess the influence of obesity-induced inflammation on white adipocyte cyclic guanosine monophosphate (cGMP) signaling, which is beneficial for adipocyte differentiation and thermogenesis. We find that murine gonadal and not inguinal fat is prone to obesity-induced inflammation. Correspondingly, the cGMP cascade is dysregulated in gonadal but not in inguinal fat of obese mice. Analysis of two independent human cohorts reveals a defective cGMP pathway only in visceral fat of obese subjects. Congruently, cGMP signaling is dysregulated in tumor necrosis factor α (TNF-α)-treated primary white adipocytes. TNF-α-mediated suppression of sGCβ

Topics: Adipogenesis; Adipose Tissue, White; Animals; Cell Differentiation; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Down-Regulation; Female; Gonads; Guanylate Cyclase; Humans; Inflammation; Inguinal Canal; Male; MAP Kinase Signaling System; Mice, Inbred C57BL; Mice, Obese; Models, Biological; NF-kappa B; Obesity; Phenotype; Signal Transduction; Solubility; Tumor Necrosis Factor-alpha

Childhood obesity is commonly associated with early signs of endothelial dysfunction, characterized by impairment of insulin signaling and vascular Nitric Oxide (NO) availability. However, the underlying mechanisms remain to be established. Hence, we tested the hypothesis that endothelial insulin-stimulated NO production and availability was impaired and related to Endoplasmic Reticulum (ER) in human umbilical vein endothelial cells (HUVECs) cultured with plasma obtained from pre-pubertal obese (OB) children. OB children (N = 28, age: 8.8 ± 2.2; BMI z-score: 2.15 ± 0.39) showed impaired fasting glucose, insulin and HOMA-IR than normal weight children (CTRL; N = 28, age: 8.8 ± 1.7; BMI z-score: 0.17 ± 0.96). The in vitro experiments showed that OB-plasma significantly impaired endothelial insulin-stimulated NO production and bioavailability compared to CTRL-plasma. In parallel, in HUVECs OB-plasma increased GRP78 and activated PERK, eIF2α, IkBα and ATF6 (all ER stress markers). Moreover, OB-plasma increased NF-κB activation and its nuclear translocation. Notably, all these effects proved to be significantly restored by using PBA and TUDCA, known ER stress inhibitors. Our study demonstrate for the first time that plasma from obese children is able to induce in vitro endothelial insulin resistance, which is characterized by reduced insulin-stimulated NO production and bioavailability, endothelial ER stress and increased NF-κB activation.

Topics: Activating Transcription Factor 6; Biological Availability; Biomarkers; Cell Nucleus; Child; Cyclic GMP; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Human Umbilical Vein Endothelial Cells; Humans; Insulin; Models, Biological; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type III; Obesity; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-akt; Puberty

Epidemiological evidence has shown that platelet activation markers are consistently elevated in obesity, contributing to its prothrombotic state. In order to improve the understanding of the regulation of platelet function in obesity, the aim of this study was to investigate the l-arginine-nitric oxide (NO) pathway in obese adults without other cardiovascular risk factor. Seventeen obese (body mass index [BMI] 35.9±1.0 kg/m(2) ) and eighteen age-matched normal weight subjects (BMI 22.0±0.6 kg/m(2) ) were included in this study. l-arginine influx was measured with incubation of l-[(3) H]-arginine. NO synthase (NOS) and arginase activities were determined by the citrulline assay and the conversion of l-[(14) C]-arginine to [(14) C]-urea, respectively. Cyclic guanosine monophosphate (cGMP) content was evaluated by enzyme-linked immunosorbent assay. In addition, the study analyzed: platelet aggregation; intraplatelet antioxidant enzymes, via superoxide dismutase (SOD) and catalase activities; and systemic levels of l-arginine, fibrinogen, and C-reactive protein (CRP). Obese patients presented a significant decrease of platelet l-arginine influx, NOS activity, and cGMP levels, along with platelet hyperaggregability. On the presence of NO donor, platelet aggregation was similar between the groups. The fibrinogen and CRP systemic levels were significantly higher and SOD activity was reduced in obesity. No significant differences were observed in plasma levels of l-arginine and intraplatelet arginase and catalase activities between groups. The diminished NO bioavailability associated with inflammatory status and impaired enzymatic antioxidant defence may contribute to future cardiovascular complications in obesity.

Topics: Adult; Arginine; Blood Platelets; Cross-Sectional Studies; Cyclic GMP; Female; Humans; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase; Obesity; Oxidative Stress; Platelet Aggregation

The aim of this study was to identify sex-dependent expression of renal transporter mRNA in lean and obese Zucker spontaneously hypertensive fatty (ZSF1) rats and to investigate the interaction of the most altered transporter, organic anion transporter 2 (Oat2), with diabetes-relevant metabolites and drugs. Higher incidence of glomerulosclerosis, tubulointerstitial fibrosis, and protein casts in Bowman's space and tubular lumen was detected by PAS staining in obese male compared to female ZSF1 rats. Real-time PCR on RNA isolated from kidney cortex revealed that Sglt1-2, Oat1-3, and Oct1 were higher expressed in kidneys of lean females. Oct2 and Mrp2 were higher expressed in obese males. Renal mRNA levels of transporters were reduced with diabetic nephropathy in females and the expression of transcription factors Hnf1β and Hnf4α in both sexes. The highest difference between lean and obese ZSF1 rats was found for Oat2. Therefore, we have tested the interaction of human OAT2 with various substances using tritium-labeled cGMP. Human OAT2 showed no interaction with diabetes-related metabolites, diabetic drugs, and ACE-inhibitors. However, OAT2-dependent uptake of cGMP was inhibited by furosemide. The strongly decreased expression of Oat2 and other transporters in female diabetic ZSF1 rats could possibly impair renal drug excretion, for example, of furosemide.

Topics: Animals; Biological Transport; Blood Pressure; Cyclic GMP; Diabetes Mellitus; Diabetic Nephropathies; Female; Furosemide; Gene Expression Profiling; Gene Expression Regulation; HEK293 Cells; Humans; Kidney; Male; Obesity; Organic Anion Transporters, Sodium-Independent; Rats; Rats, Inbred SHR; Rats, Zucker; RNA, Messenger; Sex Factors; Transcription Factors

Type 2 diabetes mellitus (DM2) and obesity are major risk factors for erectile dysfunction (ED). In diabetes, increased oxidative stress leads to decreased nitric oxide (NO) bioavailability, and diabetic patients appear to be less responsive to conventional therapy with phosphodiesterase type 5 inhibitors. We investigated whether the soluble guanylyl cyclase stimulator BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4ylamine) is effective in improving impaired corpus cavernosum (CC) relaxation in obese DM2 mice by reducing oxidative stress. Adult db/db(-/-) mice or their lean db(/+) littermates were used to assess vascular function, cGMP levels, antioxidant status, NADPH oxidase expression, and superoxide formation in the absence or presence of BAY 41-2272. Results showed that BAY 41-2272 (10(-8) to 10(-5) M) potently relaxed CC from db(/+) or db/db(-/-) mice in a similar manner. BAY 41-2272 significantly enhanced both endothelium-dependent and nitrergic relaxation induced by electrical field stimulation (EFS), and improved the impaired relaxation to acetylcholine and EFS in the diabetic animals in a concentration-dependent manner (10(-8) to 10(-7) M). BAY 41-2272 increased cGMP levels and potentiated relaxation responses to exogenous NO in CC. Total antioxidant status was reduced in plasma and urine whereas expression of vascular NADPH oxidase subunits (gp91phox, p22phox, and p47phox) was increased in the CC of db/db(-/-) mice, suggesting a state of oxidative stress. These effects were prevented by BAY 41-2272 in a concentration-dependent manner. These results suggest that BAY 41-2272 improves CC relaxation in db/db(-/-) mice by increasing cGMP and augmenting antioxidant status, making this drug is a potential novel candidate to treat ED.

Topics: Animals; Blood Glucose; Cyclic GMP; Diabetes Mellitus, Type 2; Drug Interactions; Epithelium; Gene Expression Regulation, Enzymologic; Guanylate Cyclase; Lipids; Male; Mice; Mice, Obese; Muscle Relaxation; NADPH Oxidases; Nitroprusside; Obesity; Oxidative Stress; Penile Erection; Penis; Pyrazoles; Pyridines; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Vasodilation

Guanylin (Gn), a bioactive peptide, and its receptor, guanylyl cyclase-C (GC-C), are primarily present in the intestine and maintain homeostasis in body fluids. Recently, rats whose macrophages overexpress Gn and GC-C were found to be resistant to diet-induced obesity. Considering that obesity is strongly related to a chronic inflammatory state in white adipose tissues, it is possible that Gn-GC-C macrophages contribute to the regulation of inflammation. In the present study, we investigated the inflammatory state of mesenteric fat in rats transgenic for both Gn and GC-C (double-transgenic [dTg] rats) by evaluating the levels of cyclic guanosine monophosphate (cGMP), a second messenger of Gn-GC-C, cGMP-dependent protein kinase (PKG), and phosphorylated vasodilator-stimulated phosphoprotein (VASP), a target protein of PKG. The levels of cGMP in dTg rats was higher than in WT rats fed the same diet. Although there were no significant differences in levels of PKG and phosphorylated VASP between WT and dTg rats fed a standard diet (STD), these levels in dTg rats fed a high fat diet (HFD) were markedly increased compared with levels in HFD WT rats. Furthermore, mRNA levels of proinflammatory factors in mesenteric fat were lower in HFD dTg rats than in HFD WT rats and were similar to levels in STD WT and dTg rats. These results indicate that the Gn-GC-C system in macrophages regulates the cGMP-PKG-VASP pathway and controls obesity through the downregulation of proinflammatory factors.

Topics: Animals; Cell Adhesion Molecules; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Diet, High-Fat; Gastrointestinal Hormones; Immunohistochemistry; Inflammation Mediators; Intra-Abdominal Fat; Macrophages, Peritoneal; Male; Microfilament Proteins; Natriuretic Peptides; Obesity; Panniculitis, Peritoneal; Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Random Allocation; Rats; Rats, Transgenic; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Second Messenger Systems

The adipokine chemerin has been implicated in cardiovascular complications associated with obesity and the metabolic syndrome. Chemerin has direct effects on the vasculature, augmenting vascular responses to contractile stimuli. As NO/cGMP signalling plays a role in vascular dysfunction associated with obesity and the metabolic syndrome, we hypothesized that chemerin induces vascular dysfunction by decreasing NO/cGMP signalling. Aortic rings from male Wistar rats (10-12 weeks of age) were incubated with chemerin (0.5 or 5 ng/ml for 1 h) or vehicle and isometric tension was recorded. Vasorelaxation in response to ACh (acetylcholine), SNP (sodium nitroprusside) and BAY 412272 [an sGC (soluble guanylate cyclase) stimulator] were decreased in chemerin-treated vessels. The NOS (NO synthase) cofactor BH4 (tetrahydrobiopterin), an O2- (superoxide anion) scavenger (tiron) and a SOD (superoxide dismutase) mimetic (tempol) abolished the effects of chemerin on ACh-induced vasodilation. eNOS (endothelial NOS) phosphorylation, determined by Western blotting, was increased in chemerin-treated vessels; however, the enzyme was mainly in the monomeric form, with decreased eNOS dimer/monomer ratio. Chemerin decreased the mRNA levels of the rate-limiting enzyme for BH4 biosynthesis GTP cyclohydrolase I. Chemerin-incubated vessels displayed decreased NO production, along with increased ROS (reactive oxygen species) generation. These effects were abrogated by BH4, tempol and L-NAME (NG-nitro-L-arginine methyl ester). sGC protein expression and cGMP levels were decreased in chemerin-incubated vessels. These results demonstrate that chemerin reduces NO production, enhances NO breakdown and also decreases NO-dependent cGMP signalling, thereby reducing vascular relaxation. Potential mechanisms mediating the effects of chemerin in the vasculature include eNOS uncoupling, increased O2- generation and reduced GC activity.

Topics: Acetylcholine; Adipokines; Animals; Aorta, Thoracic; Cyclic GMP; Endothelium, Vascular; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Obesity; Rats; Rats, Wistar; Signal Transduction; Superoxide Dismutase; Superoxides

Obesity is a major health problem. We investigated the effects of forskolin and rolipram in the diet of animals in which obesity had been induced. We used 50 female albino Wistar rats that were assigned randomly into five groups as follows: group 1, control; group 2, high fat diet; group 3, high fat diet + forskolin; group 4, high fat diet + rolipram; and group 5, high fat diet + rolipram + forskolin. The rats were fed for 10 weeks and rolipram and forskolin were administered during last two weeks. The animals were sacrificed and blood samples were obtained. Serum cAMP, cGMP and free fatty acids (FFA) levels were measured using ELISA assays. We also measured weight gain during the 10 week period. cAMP and FFA levels of groups 3, 4 and 5 were significantly higher than those of groups 1 and 2. We found no significant differences in serum cGMP levels among the groups. The weight gain in groups 3, 4 and 5 was significantly less than for group 2. We also found that the weight gain in group 5 was significantly less than in groups 3 and 4. We found that both forskolin and rolipram stimulated lipolysis and inhibited body weight increase by increasing cAMP levels. Also, combination therapy using the two agents may be more effective in preventing diet induced obesity than either agent alone. We found also that these agents did not effect cellular cGMP levels in diet induced obesity.

Topics: Animals; Colforsin; Cyclic AMP; Cyclic GMP; Diet, High-Fat; Fatty Acids, Nonesterified; Female; Lipolysis; Obesity; Random Allocation; Rats; Rats, Wistar; Rolipram; Weight Gain

Cardiovascular and endocrine-metabolic diseases associated with increased oxidative stress such as obesity lead to erectile dysfunction (ED). Activators of soluble guanylyl cyclase (sGC) such as BAY 60-2770 reactivate the heme-oxidized sGC in vascular diseases.. This study aimed to evaluate the effects of 2-week oral intake with BAY 60-2270 on a murine model of obesity-associated ED.. C57BL/6 male mice were fed for 12 weeks with standard chow or high-fat diet. Lean and obese mice were treated with BAY 60-2770 (1 mg/kg/day, 2 weeks).. Measurements of intracavernosal pressure (ICP), along with acetylcholine (10(-9) to 10(-5)  M) and electrical field stimulation (EFS; 4-10 Hz)-induced corpus cavernosum relaxations in vitro, were obtained. Levels of cyclic guanosine monophosphate (cGMP), reactive oxygen species (ROS), and sGC protein expressions in cavernosal tissues were measured.. Cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly lower in obese compared with lean mice (P < 0.05). Two-week therapy with BAY 60-2770 fully reversed the decreased ICP in obese group. Acetylcholine-induced cavernosal relaxations were 45% lower (P < 0.001) in obese mice, which were fully restored by BAY 60-2770 treatment. Likewise, the EFS-induced relaxations in obese mice were restored by BAY 60-2770. Basal cGMP content in erectile tissue was 68% lower (P < 0.05) in obese mice, an effect normalized by BAY 60-2770. Levels of ROS were 52% higher (P < 0.05) whereas protein expression of α1 sGC subunit was reduced in cavernosal tissue of obese mice, both of which were normalized by BAY 60-2770. In lean group, BAY 60-2770 did not significantly affect any functional, biochemical, or molecular parameter analyzed.. Two-week therapy with BAY 60-2770 restores the erectile function in obese mice that is associated with reduced ROS levels, up-regulation of α1 sGC subunit, and increased cGMP levels in the erectile tissue.

Topics: Animals; Benzoates; Biphenyl Compounds; Cyclic GMP; Diet, High-Fat; Enzyme Activators; Erectile Dysfunction; Guanylate Cyclase; Humans; Hydrocarbons, Fluorinated; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; Penile Erection; Penis; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Up-Regulation

With more than half a billion individuals affected worldwide, obesity has reached pandemic proportions. Development of "brown-like" or "brite" adipocytes within white adipose tissue (WAT) has potential antiobesity and insulin-sensitizing effects. We investigated the role of cyclic GMP (cGMP) signaling, focusing on cGMP-dependent protein kinase I (PKGI) in WAT. PKGI is expressed in murine WAT, primary adipocytes, and 3T3-L1. Treatment of adipocytes with cGMP resulted in increased adipogenesis, with a 54% increase in expression of peroxisome proliferator-activated receptor-γ. Lentiviral overexpression of PKGI further increased adipogenesis, whereas loss of PKGI significantly reduced adipogenic differentiation. In addition to adipogenic effects, PKGI had an antihypertrophic and anti-inflammatory effect via RhoA phosphorylation and reduction of proinflammatory adipokine expression. Moreover, PKGI induced a 4.3-fold increase in abundance of UCP-1 and the development of a brown-like thermogenic program in primary adipocytes. Notably, treatment of C57BL/6 mice with phosphodiesterase inhibitor sildenafil (12 mg/kg/d) for 7 d caused 4.6-fold increase in uncoupling protein-1 expression and promoted establishment of a brown fat cell-like phenotype ("browning") of WAT in vivo. Taken together, PKGI is a key regulator of cell size, adipokine secretion and browning of white fat depots and thus could be a valuable target in developing novel treatments for obesity.

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Adipose Tissue, Brown; Adipose Tissue, White; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Ion Channels; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Proteins; Obesity; Signal Transduction; Uncoupling Protein 1

Prominent features of myocardial remodeling in heart failure with preserved ejection fraction (HFPEF) are high cardiomyocyte resting tension (F(passive)) and cardiomyocyte hypertrophy. In experimental models, both reacted favorably to raised protein kinase G (PKG) activity. The present study assessed myocardial PKG activity, its downstream effects on cardiomyocyte F(passive) and cardiomyocyte diameter, and its upstream control by cyclic guanosine monophosphate (cGMP), nitrosative/oxidative stress, and brain natriuretic peptide (BNP). To discern altered control of myocardial remodeling by PKG, HFPEF was compared with aortic stenosis and HF with reduced EF (HFREF).. Patients with HFPEF (n=36), AS (n=67), and HFREF (n=43) were free of coronary artery disease. More HFPEF patients were obese (P<0.05) or had diabetes mellitus (P<0.05). Left ventricular myocardial biopsies were procured transvascularly in HFPEF and HFREF and perioperatively in aortic stenosis. F(passive) was measured in cardiomyocytes before and after PKG administration. Myocardial homogenates were used for assessment of PKG activity, cGMP concentration, proBNP-108 expression, and nitrotyrosine expression, a measure of nitrosative/oxidative stress. Additional quantitative immunohistochemical analysis was performed for PKG activity and nitrotyrosine expression. Lower PKG activity in HFPEF than in aortic stenosis (P<0.01) or HFREF (P<0.001) was associated with higher cardiomyocyte F(passive) (P<0.001) and related to lower cGMP concentration (P<0.001) and higher nitrosative/oxidative stress (P<0.05). Higher F(passive) in HFPEF was corrected by in vitro PKG administration.. Low myocardial PKG activity in HFPEF was associated with raised cardiomyocyte F(passive) and was related to increased myocardial nitrosative/oxidative stress. The latter was probably induced by the high prevalence in HFPEF of metabolic comorbidities. Correction of myocardial PKG activity could be a target for specific HFPEF treatment.

Topics: Aortic Valve Stenosis; Biopsy; Cohort Studies; Comorbidity; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Diabetes Mellitus; Female; Heart; Heart Failure; Humans; Male; Middle Aged; Myocardium; Natriuretic Peptide, Brain; Obesity; Oxidative Stress; Stroke Volume; Tyrosine

• Obesity induced by high-fat diet (HFD) is one of the most important risk factor for the development of erectile dysfunction (ED) in man. This study aimed to characterize the ED resulting from obesity associated with HFD in mice.. • C57BL/6 mice fed for 10 weeks with either HFD to induce obesity or a standard-chow diet (SD) were used. Corpus cavernosum was surgically dissected free, and strips were mounted in 10-mL organ baths containing Krebs solution. • Functional responses to endothelium-dependent and -independent agents, as well as to electrical-field stimulation were measured in the cavernosal tissue. Levels of cGMP in erectile tissue were detected by enzyme immunoassay assay.. • The potency (pEC(50)) and maximal response (E(max)) to acetylcholine were significantly lower in the HFD group compared with the SD group. A marked decrease in the non-adrenergic non-cholinergic (nitrergic) cavernosal relaxations in the HFD group was also detected. There were no significant differences between the SD and HFD groups for the cavernosal relaxations in response to sodium nitroprusside. • The contractile responses elicited by the α(1) -adrenoceptor agonist phenylephrine were significantly greater in the HFD group compared with the SD group. • Similarly, the electrical-field stimulation (2-8 Hz)-induced adrenergic contractions were markedly greater in HFD mice. The pEC(50) for endothelin-1 was about 6.9-fold higher in the HFD compared with SD group. • The basal cGMP content was 47% lower in HFD strips compared with SD group. There were no morphological alterations in erectile tissue of HFD group compared with SD mice.. • Obesity associated with HFD favours ED as result of impaired endothelial and nitrergic cavernosal relaxations along with increased contractile responses to adrenergic stimulation and endothelin-1 receptor activation.

Topics: Animals; Cyclic GMP; Dietary Fats; Impotence, Vasculogenic; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Obesity; Penile Erection; Penis; Receptor, Endothelin A

The purposes of these studies were to quantify the concentrations of total nitrate and nitrite (NO(x)(-)) cyclic guanosine monophosphate (cGMP), and nitrotyrosine over skin surface in normal weight healthy volunteers (n = 64) compared to overweight/obese subjects (n = 54). A semi-circular plastic tube was taped to the skin along acupuncture points (acupoints), meridian line without acupoint (MWOP), and nonmeridian control and filled with a 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl solution for 20 min. The concentrations of NO(x)(-), cGMP, and nitrotyrosine in the samples were quantified in a blinded fashion using chemiluminescence and enzyme-linked immunosorbent assay, respectively. In normal weight healthy volunteers, NO(x)(-) and cGMP concentrations were consistently increased over the pericardium meridian (PC) 4-7 compared with nonmeridian areas. NO(x)(-) concentration is enhanced over the bladder meridian (BL) 56-57, but cGMP level is similar between the regions. In overweight/obese subjects, NO(x)(-) contents were increased or tended to be elevated over PC and BL regions. cGMP is paradoxically decreased over PC acupoints and nonmeridian control on the forearm but the decreases were blunted along BL regions on the leg. Nitrotyrosine concentrations are markedly elevated (five- to sixfold) over both PC and BL in all areas of overweight/obese subjects. This is the first evidence showing that nitrotyrosine level is tremendously elevated over skin accompanied by paradoxical changes in nitric oxide (NO)-cGMP concentrations over PC skin region in overweight/obese subject. The results suggest that NO-related oxidant inflammation is systemically enhanced while cGMP generation is impaired over PC skin region but not over BL region in obesity.

Topics: Acupuncture Points; Adult; Cyclic GMP; Cyclic N-Oxides; Female; Humans; Imidazoles; Male; Nitrates; Nitric Oxide; Nitrites; Obesity; Skin; Tyrosine

8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), formed nitric oxide (NO)-dependently, is a physiological second messenger, yet little is known about its role in the pathophysiology of vascular diseases. To study the pharmacological activity of 8-nitro-cGMP in diabetic mice, we compared its effects on vascular reactivity of aortas from non-diabetic and diabetic mice.. Vascular tension recording was performed in thoracic aortic rings from wild-type (C57BL/6), non-diabetic db/+ and obese/diabetic db/db mice. Endothelial NO synthase (eNOS) uncoupling and superoxide were tested by Western blot and dihydroethidium fluorescence respectively.. 8-Nitro-cGMP, at concentrations up to 10 µM, enhanced phenylephrine-induced contractions in aortas from C57BL/6 and db/+ mice, but not from db/db mice. This enhancement was not observed with 8-bromo-cGMP. Pretreatment of aortas from C57BL/6 and db/+ mice with l-NAME (100 µM), superoxide dismutase (100 U·mL(-1) ) or tiron (1 mM), abolished 8-nitro-cGMP-induced enhancement of the phenylephrine contraction. In 8-nitro-cGMP (10 µM)-treated C57BL/6 aortas, eNOS dimer/monomer ratio was significantly decreased and vascular superoxide production increased, suggesting that 8-nitro-cGMP-induced superoxide production via eNOS uncoupling may mediate the enhancement of the phenylephrine contraction. At higher concentrations (>10 µM), 8-nitro-cGMP produced relaxation of the phenylephrine-contracted aortas from C57BL/6, db/+ and db/db mice. The 8-nitro-cGMP-induced relaxation in db/db mouse aortas was found to be resistant to a phosphodiesterase 5 inhibitor, zaprinast (1 µM).. The vasodilator effect of 8-nitro-cGMP may contribute to amelioration of the vascular endothelial dysfunction in diabetic mice, representing a novel pharmacological approach to prevent the complications associated with diabetes.

Topics: Animals; Aorta, Thoracic; Blotting, Western; Cyclic GMP; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Endothelium, Vascular; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type III; Obesity; Phenylephrine; Vasoconstriction; Vasodilation

Obesity is characterized by chronic inflammation of adipose tissue, which contributes to insulin resistance and diabetes. Although nitric oxide (NO) signaling has antiinflammatory effects in the vasculature, whether reduced NO contributes to adipose tissue inflammation is unknown. We sought to determine whether (1) obesity induced by high-fat (HF) diet reduces endothelial nitric oxide signaling in adipose tissue, (2) reduced endothelial nitric oxide synthase (eNOS) signaling is sufficient to induce adipose tissue inflammation independent of diet, and (3) increased cGMP signaling can block adipose tissue inflammation induced by HF feeding.. Relative to mice fed a low-fat diet, an HF diet markedly reduced phospho-eNOS and phospho-vasodilator-stimulated phosphoprotein (phospho-VASP), markers of vascular NO signaling. Expression of proinflammatory cytokines was increased in adipose tissue of eNOS-/- mice. Conversely, enhancement of signaling downstream of NO by phosphodiesterase-5 inhibition using sildenafil attenuated HF-induced proinflammatory cytokine expression and the recruitment of macrophages into adipose tissue. Finally, we implicate a role for VASP, a downstream mediator of NO-cGMP signaling in mediating eNOS-induced antiinflammatory effects because VASP-/- mice recapitulated the proinflammatory phenotype displayed by eNOS-/- mice.. These results imply a physiological role for endothelial NO to limit obesity-associated inflammation in adipose tissue and hence identify the NO-cGMP-VASP pathway as a potential therapeutic target in the treatment of diabetes.

Topics: Adipose Tissue; Animals; Cell Adhesion Molecules; Cyclic GMP; Dietary Fats; Disease Models, Animal; Endothelium, Vascular; Inflammation; Insulin Resistance; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Nitric Oxide; Nitric Oxide Synthase Type III; Obesity; Phosphodiesterase 5 Inhibitors; Phosphoproteins; Phosphorylation; Piperazines; Purines; Signal Transduction; Sildenafil Citrate; Sulfones

Proinflammatory activation of Kupffer cells is implicated in the effect of high-fat feeding to cause liver insulin resistance. We sought to determine whether reduced endothelial nitric oxide (NO) signaling contributes to the effect of high-fat feeding to increase hepatic inflammatory signaling and if so, whether this effect 1) involves activation of Kupffer cells and 2) is ameliorated by increased NO signaling.. Effect of NO/cGMP signaling on hepatic inflammation and on isolated Kupffer cells was examined in C57BL/6 mice, eNos(-/-) mice, and Vasp(-/-) mice fed a low-fat or high-fat diet.. We show that high-fat feeding induces proinflammatory activation of Kupffer cells in wild-type mice coincident with reduced liver endothelial nitric oxide synthase activity and NO content while, conversely, enhancement of signaling downstream of endogenous NO by phosphodiesterase-5 inhibition protects against high fat-induced inflammation in Kupffer cells. Furthermore, proinflammatory activation of Kupffer cells is evident in eNos(-/-) mice even on a low-fat diet. Targeted deletion of vasodilator-stimulated phosphoprotein (VASP), a key downstream target of endothelially derived NO, similarly predisposes to hepatic and Kupffer cell inflammation and abrogates the protective effect of NO signaling in both macrophages and hepatocytes studied in a cell culture model.. These results collectively imply a physiological role for endothelial NO to limit obesity-associated inflammation and insulin resistance in hepatocytes and support a model in which Kupffer cell activation during high-fat feeding is dependent on reduced NO signaling. Our findings also identify the NO/VASP pathway as a novel potential target for the treatment of obesity-associated liver insulin resistance.

Topics: Animals; Cell Adhesion Molecules; Cells, Cultured; Cyclic GMP; Cytokines; Dietary Fats; Endothelial Cells; Gene Expression Regulation; Hepatitis; Insulin Resistance; Kupffer Cells; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Molecular Targeted Therapy; Nitric Oxide; Nitric Oxide Synthase Type III; Obesity; Phosphodiesterase 5 Inhibitors; Phosphoproteins; Signal Transduction

Apart from controlling energy balance, leptin, secreted by adipose tissue, is also involved in the regulation of cardiovascular function. Previous studies have demonstrated that acutely administered leptin stimulates natriuresis and vascular nitric oxide (NO) production and that these effects are impaired in obese animals. However, the mechanism of resistance to leptin is not clear. Because obesity is associated with chronically elevated leptin, we examined if long-term hyperleptinemia impairs acute effects of leptin on sodium excretion and NO production in the absence of obesity. Hyperleptinemia was induced in lean rats by administration of exogenous leptin at a dose of 0.5mg/kg/day for 7 days, and then acute effect of leptin (1mg/kg i.v.) was studied under general anesthesia. Leptin increased fractional sodium excretion and decreased Na(+),K(+)-ATPase activity in the renal medulla. In addition, leptin increased the level of NO metabolites and cyclic GMP in plasma and aortic wall. These acute effects of leptin were impaired in hyperleptinemic animals. In both control and hyperleptinemic groups the effect of leptin on Na(+) excretion and renal Na(+),K(+)-ATPase was abolished by phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, but not by protein kinase B/Akt inhibitor, triciribine,. In contrast, acute effect of leptin on NO metabolites and cGMP was abolished by triciribine but not by wortmannin. Leptin stimulated Akt phosphorylation at Ser(473) in aortic tissue but not in the kidney, and this effect was comparable in control and hyperleptinemic groups. These results suggest that hyperleptinemia may mediate "renal" and "vascular" leptin resistance observed in obesity.

Topics: Androstadienes; Animals; Aorta; Blood Pressure; Cyclic GMP; Humans; Insulin; Kidney Medulla; Leptin; Male; Natriuresis; Nitric Oxide; Obesity; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Ribonucleosides; Sodium; Sodium-Potassium-Exchanging ATPase; Substance P; Wortmannin

Nitric oxide (NO) is a short-lived gaseous messenger with multiple physiological functions including regulation of blood flow, platelet adhesion and aggregation inhibition. NO synthases (NOS) catalyze the conversion of cationic amino acid L-arginine in L-citrulline and NO. Despite an increasing prevalence of obesity and metabolic syndrome (MetS) in the last decades, the exact mechanisms involved in the pathogenesis and cardiovascular complications are not fully understood. We have examined the effects of obesity and MetS on the L-arginine-NO-cGMP pathway in platelets from a population of adolescents.. A total of twenty six adolescent patients (13 with obesity and 13 with MetS) and healthy volunteers (n=14) participated in this study. Transport of L-arginine, NO synthase (NOS) activity and cGMP content in platelets were analyzed. Moreover, platelet function, plasma levels of L-arginine, metabolic and clinical markers were investigated in these patients and controls.. L-arginine transport (pmol/10(9) cells/min) in platelets via system y(+)L was diminished in obese subjects (20.8±4.7, n=10) and MetS patients (18.4±3.8, n=10) compared to controls (52.3±14.8, n=10). The y(+)L transport system correlated negatively to insulin levels and Homeostasis Model Assessment of Insulin Resistance (HOMA IR) index. No differences in NOS activity and cGMP content were found among the groups. Moreover, plasma levels of L-arginine were not affected by obesity or MetS.. Our study provides the first evidence that obesity and MetS lead to a dysfunction of L-arginine influx, which negatively correlates to insulin resistance. These findings could be a premature marker of future cardiovascular complications during adulthood.

Topics: Adolescent; Arginine; Biological Transport; Blood Platelets; Cardiovascular Diseases; Case-Control Studies; Cyclic GMP; Humans; Insulin Resistance; Metabolic Syndrome; Nitric Oxide; Obesity

Emerging evidence indicates that aldosterone causes oxidative stress by stimulating proinflammatory/oxidative mediators, including nuclear factor-kappaB, activating protein (AP-1), and c-Jun N-terminal kinase. Thus, in insulin-resistant type 2 diabetes (T2D), oxidative stress generated by hyperglycemia and aldosterone would potentiate the oxidative destruction of tissue and important regulators of glucose metabolism like adiponectin and insulin. Although heme oxygenase (HO)-1 is cytoprotective, its effects on T2D have not been fully characterized. Here we report an enduring antidiabetic effect of the HO inducer, hemin, on Zucker diabetic-fatty rat (ZDF), a model of insulin-resistant T2D. Chronically applied hemin to ZDF reduced and maintained significantly low fasting and postprandial hyperglycemia for 4 months after therapy. The antidiabetic effect was accompanied by enhanced HO activity, catalase, cyclic GMP, bilirubin, ferritin, total antioxidant capacity, and insulin. In contrast, reduced aldosterone alongside markers/mediators of oxidative stress, including 8-isoprostane, c-Jun N-terminal kinase, nuclear factor-kappaB, AP-1, and AP-2 were observed. Interestingly, in hemin-treated ZDF, inhibitory proteins of insulin-signaling, such as glycogen synthase kinase-3 and protein-tyrosine phosphatase-1B were reduced, whereas agents that promote insulin signaling including adiponectin, cAMP, AMP-activated protein kinase, aldolase-B, and glucose transporter-4 (GLUT4), were robustly increased. Correspondingly, hemin improved ip glucose tolerance, reduced insulin intolerance, and lowered insulin resistance (homeostasis model assessment of insulin resistance), and the inability of insulin to enhance GLUT4 was overturned. These results suggest that the suppression of hyperglycemia and aldosterone-induced oxidative stress alongside the potentiation of insulin-sensitizing pathways may account for the 4-month enduring antidiabetic effect. The synergistic interaction between the HO system, aldolase-B, adiponectin, AMP-activated protein kinase, and GLUT4 may be explored for novel strategies against postprandial/fasting hyperglycemia and insulin-resistant T2D.

Topics: Aldosterone; Animals; Cyclic GMP; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Drug Evaluation, Preclinical; Fasting; Heme Oxygenase (Decyclizing); Hemin; Hyperglycemia; Insulin Resistance; Male; Muscle, Skeletal; Obesity; Oxidative Stress; Rats; Rats, Sprague-Dawley; Rats, Zucker; Signal Transduction

Obesity and diabetes are major risk factors for the development of vascular disease in the lower limbs. Previous studies have demonstrated reduced nitric oxide (NO)-mediated vasodilation, increased adrenergic constriction, and inward, atrophic remodeling in the limb circulation of obese Zucker rats, but the component of the "metabolic syndrome" driving these changes is unclear. Because insulin resistance precedes the state of frank diabetes, the current study hypothesized that insulin resistance independent of obesity induced by fructose feeding would impair microvascular function in the skeletal muscle circulation in lean Zucker rats (LZR). A 66% fructose diet impaired glucose tolerance and induced moderate insulin resistance with no changes in whole-body hemodynamics of anesthetized rats (FF-LZR), compared to control LZR. NO-mediated vasodilation of isolated gracilis arteries, assessed in vitro with acetylcholine and sodium nitroprusside, was reduced approximately 20% in FF-LZR vs. LZR. NO-independent cGMP-mediated vasodilation was unimpaired. Pretreatment of isolated vessels with the superoxide scavenger, tempol, improved responses to both vasodilators. Reactivity to adrenergic stimulation was unaltered in FF-LZR vs. LZR, although constriction to endothelin was increased. Structural and passive mechanical characteristics of isolated gracilis arteries were similar in both LZR and FF-LZR. Taken together, these findings indicate that moderate insulin resistance is sufficient to impair endothelial function in an oxidant-dependent manner in the rat hindlimb circulation. Other aspects of skeletal muscle vascular function documented in obese models, specifically adrenergic tone and inward remodeling, must reflect either severe insulin resistance or other aspects of obesity. The factors accounting for nonendothelial vasculopathies remain unknown.

Topics: Acetylcholine; Animals; Arteries; Cyclic GMP; Diet; Endothelium, Vascular; Fructose; Hindlimb; Insulin Resistance; Male; Muscle, Skeletal; Nitric Oxide; Nitroprusside; Obesity; Oxidants; Rats; Rats, Zucker; Sweetening Agents; Vasoconstriction; Vasodilation; Vasodilator Agents

We examined mechanisms leading to the impairment of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-dependent vasorelaxation in response to acutely administered leptin in rats with the metabolic syndrome.. Effects of leptin on blood pressure and NO and cGMP in the aortic wall were studied in four groups of rats: (1) lean control, (2) obese, fed "cafeteria diet" for 3months (hyperleptinemia and hyperinsulinemia), (3) hyperleptinemia induced by administration of exogenous leptin for 8days, and (4) fructose-fed, receiving 20% fructose in the drinking water for 8weeks (hyperinsulinemia with slightly elevated leptin).. Stimulatory effect of leptin on NO and cGMP production in the aortic wall was impaired in obese and hyperleptinemic groups but not in the fructose group. In contrast, EDHF-mimetic effect of leptin was impaired in obese and fructose-fed but not in the hyperleptinemic group. Leptin increased tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) in the aortic wall, and this effect was impaired in obese and fructose-fed animals. The EDHF-mimetic effect of leptin was abolished by phosphoinositide 3-kinase inhibitor, wortmannin, whereas its effect on NO was not. In addition, IRS-1 phosphorylation at Ser(307) and Ser(612) was enhanced in obese and fructose-fed but not in hyperleptinemic rats.. These results indicate that: (1) long-term hyperleptinemia induces resistance to acute vascular NO-mimetic effect of leptin in obesity/metabolic syndrome, (2) leptin stimulates EDHF in IRS-1 and PI3K-dependent manner, and this effect is impaired in obesity due to excessive serine phosphorylation of IRS-1.

Topics: Animals; Aorta; Biological Factors; Blood Pressure; Cyclic GMP; Disease Models, Animal; Endothelium, Vascular; Insulin; Leptin; Male; Metabolic Syndrome; Nitric Oxide; Obesity; Phosphorylation; Rats; Rats, Wistar; Recombinant Proteins; Vasodilation

Natriuretic peptides (NPs) have been characterized as vascular hormones that regulate vascular tone via guanylyl cyclase (GC), cyclic GMP (cGMP), and cGMP-dependent protein kinase (cGK). Recent clinical studies have shown that plasma NP levels were lower in subjects with the metabolic syndrome. The present study was conducted to elucidate the roles for NP/cGK cascades in energy metabolism.. We used three types of genetically engineered mice: brain NP (BNP) transgenic (BNP-Tg), cGK-Tg, and guanylyl cyclase-A (GCA) heterozygous knockout (GCA(+/-)) mice and analyzed the metabolic consequences of chronic activation of NP/cGK cascades in vivo. We also examined the effect of NPs in cultured myocytes.. BNP-Tg mice fed on high-fat diet were protected against diet-induced obesity and insulin resistance, and cGK-Tg mice had reduced body weight even on standard diet; surprisingly, giant mitochondria were densely packed in the skeletal muscle. Both mice showed an increase in muscle mitochondrial content and fat oxidation through upregulation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1alpha and PPARdelta. The functional NP receptors, GCA and guanylyl cyclase-B, were downregulated by feeding a high-fat diet, while GCA(+/-) mice showed increases in body weight and glucose intolerance when fed a high-fat diet. NPs directly increased the expression of PGC-1alpha and PPARdelta and mitochondrial content in cultured myocytes.. The findings together suggest that NP/cGK cascades can promote muscle mitochondrial biogenesis and fat oxidation, as to prevent obesity and glucose intolerance. The vascular hormone, NP, would contribute to coordinated regulation of oxygen supply and consumption.

Topics: Animals; Blood Glucose; Cells, Cultured; Cyclic GMP; Dietary Fats; Down-Regulation; Genetic Engineering; Glucose Intolerance; Insulin Resistance; Lipid Peroxidation; Mice; Mice, Knockout; Mitochondria; Molecular Sequence Data; Muscle Cells; Muscle, Skeletal; Natriuretic Peptide, Brain; Natriuretic Peptides; Obesity; Oxygen Consumption; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; PPAR delta; PPAR gamma; Protein Kinases; Receptors, Atrial Natriuretic Factor; Trans-Activators; Transcription Factors; Up-Regulation

To study the role of endogenous proinflammatory cytokines in endothelial dysfunction in diabetes, we administered semapimod, an inhibitor of proinflammatory cytokine production, to obese Zucker (OZ) rats, and examined its effect on endothelium-dependent vasorelaxation. Endothelium-dependent vasorelaxation induced by acetylcholine and adrenomedullin (AM) was significantly reduced in OZ rats compared to a control group of lean Zucker rats. Semapimod significantly restored endothelium-dependent vasorelaxation in OZ rats. This effect of semapimod was well correlated with the reduction in the serum concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-6, and C-reactive protein, as well as with the recovery of AM-induced Akt phosphorylation and cGMP production. Furthermore, acute administration of TNF-alpha significantly suppressed endothelium-dependent vasorelaxation and AM-induced cGMP production. These results implicate endogenous proinflammatory cytokines, especially TNF-alpha, in endothelial dysfunction in diabetes, and indicate that blockade of these cytokines will be a promising strategy for inhibiting the progression of vascular inflammation.

Topics: Adrenomedullin; Animals; Biomarkers; C-Reactive Protein; Cyclic GMP; Endothelium, Vascular; Hydrazones; Hypertension; Immunosuppressive Agents; Interleukin-6; Male; Obesity; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Zucker; Tumor Necrosis Factor-alpha; Vasculitis; Vasodilation

The impact of obesity on nitric oxide (NO)-mediated coronary microvascular responses is poorly understood. Thus NO-mediated vasomotor responses were investigated in pressurized coronary arterioles ( approximately 100 microm) isolated from lean (on normal diet) and obese (fed with 60% of saturated fat) rats. We found that dilations to acetylcholine (ACh) were not significantly different in obese and lean rats (lean, 83 +/- 4%; and obese, 85 +/- 3% at 1 microM), yet the inhibition of NO synthesis with N(omega)-nitro-l-arginine methyl ester reduced ACh-induced dilations only in vessels of lean controls. The presence of the soluble guanylate cyclase (sGC) inhibitor oxadiazolo-quinoxaline (ODQ) elicited a similar reduction in ACh-induced dilations in the two groups of vessels (lean, 60 +/- 11%; and obese, 57 +/- 3%). Dilations to NO donors, sodium nitroprusside (SNP), and diethylenetriamine (DETA)-NONOate were enhanced in coronary arterioles of obese compared with lean control rats (lean, 63 +/- 6% and 51 +/- 5%; and obese, 78 +/- 5% and 70 +/- 5%, respectively, at 1 microM), whereas dilations to 8-bromo-cGMP were not different in the two groups. In the presence of ODQ, both SNP and DETA-NONOate-induced dilations were reduced to a similar level in lean and obese rats. Moreover, SNP-stimulated cGMP immunoreactivity in coronary arterioles and also cGMP levels in carotid arteries were enhanced in obese rats, whereas the protein expression of endothelial NOS and the sGC beta1-subunit were not different in the two groups. Collectively, these findings suggest that in coronary arterioles of obese rats, the increased activity of sGC leads to an enhanced sensitivity to NO, which may contribute to the maintenance of NO-mediated dilations and coronary perfusion in obesity.

Topics: Acetylcholine; Adaptation, Physiological; Animals; Arterioles; Blotting, Western; Coronary Vessels; Cyclic GMP; Dietary Fats; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Guanylate Cyclase; Immunohistochemistry; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroprusside; Nitroso Compounds; Obesity; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Vasodilation; Vasodilator Agents

Impairment of platelet response to antiaggregatory agents is seen in individuals with central obesity and may play a role in the increased cardiovascular risk associated with obesity. In this study we evaluated whether this impairment involves the antiaggregatory pathways regulated by cAMP and cGMP.. We obtained platelet-rich plasma from 12 obese individuals and 12 controls. We investigated the effects of the cyclic nucleotide analogs 8-pCPT-cAMP (10-500 micromol/L) and 8-pCPT-cGMP (10-500 micromol/L) on ADP-induced platelet aggregation as assessed by decreased light scattering. We assessed the activation of cAMP- and cGMP-dependent protein kinases by measuring phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) at Ser157 and Ser239.. The antiaggregatory effect of both cyclic nucleotide analogs was impaired in obese individuals compared to controls, with mean (SE) half-maximal inhibitory concentrations (IC(50)) (after 20-min incubation) of 123 (33) micromol/L vs 5 (1) micromol/L, respectively, for 8-pCPT-cAMP (P <0.01) and of 172 (43) micromol/L vs 17 (8) micromol/L, respectively, for 8-pCPT-cGMP (P <0.01). The Homeostasis Model Assessment Index of Insulin Resistance was independently correlated with cyclic nucleotide analog IC(50). In obese individuals, VASP phosphorylation at Ser157 and Ser239 in response to cyclic nucleotides was significantly lower than in controls.. In central obesity the reduced ability of cyclic nucleotides to inhibit platelet aggregation is associated with reduced activation of their specific kinases. Because cyclic nucleotides help regulate platelet antiaggregation, alteration of this ability is consistent with platelet hyperactivity in obesity.

Topics: Adult; Blood Platelets; Cell Adhesion Molecules; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Drug Resistance; Enzyme Activation; Female; Humans; Light; Male; Microfilament Proteins; Nucleotides, Cyclic; Obesity; Phosphoproteins; Phosphorylation; Platelet Aggregation Inhibitors; Scattering, Radiation; Thionucleotides

SHR/NDmcr-cp (SHR-cp) rats display typical symptoms and features of the metabolic syndrome. We previously reported that endothelium-dependent relaxation decreases in the thoracic aortas of SHR-cp rats, despite increased nitric oxide (NO) production from the endothelium. In the present study, to search for the reasons for this contradiction, we investigated whether vascular abnormality could be reduced by treatment of SHR-cp rats with antihypertensive drugs; a calcium channel blocker (amlodipine), an alpha 2 and imidazoline receptor agonist (moxonidine), and an angiotensin II type 1 (AT1) receptor antagonist (telmisartan). Telmisartan but not amlodipine and moxonidine ameliorated the impairment of relaxation in response to acetylcholine and the increased protein expression of endothelium NO synthase in thoracic aortas. All three drugs significantly lowered the blood pressure. Telmisartan decreased the serum levels of lipid peroxide and 8-hydroxy-2'-deoxyguanosine, oxidative stress markers, and also the aortic levels of the protein expression of gp91, a component of NADPH oxidase, and 3-nitrotyrosine, a biomarker of peroxynitrite. These findings suggest that NADPH oxidase-derived superoxide, probably produced due to stimulation of AT1 receptors, reacts with NO to form peroxynitrite, and consequently decreases active NO, leading to attenuation of endothelium-dependent relaxation. Angiotensin receptor antagonists may be effective for preventing endothelial dysfunction in metabolic syndrome.

Topics: Acetylcholine; Amlodipine; Animals; Aorta, Thoracic; Blood Glucose; Blood Pressure; Blotting, Western; Cholesterol; Cyclic GMP; Dose-Response Relationship, Drug; Heart Rate; Hypertension; Imidazoles; In Vitro Techniques; Insulin; Male; Nitric Oxide Synthase Type III; Nitroprusside; Obesity; Peroxynitrous Acid; Rats; Rats, Inbred SHR; Rats, Mutant Strains; Triglycerides; Vasodilation

We investigated the role of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in hemodynamic action of leptin. The effect of leptin (1 mg/kg i.p.) on systolic blood pressure (SBP) was examined in lean rats and in rats made obese by feeding highly palatable diet for either 1 or 3 months. Separate groups received NO synthase inhibitor, L-NAME, or EDHF inhibitors, the mixture of apamin+charybdotoxin or sulfaphenazole, before leptin administration. Leptin increased NO production, as evidenced by increase in plasma and urinary NO metabolites and cyclic GMP. This effect was impaired in both obese groups. In lean rats either leptin or EDHF inhibitors had no effect on blood pressure. L-NAME increased blood pressure in lean animals and this effect was prevented by leptin. However, when leptin was administered to animals pretreated with both L-NAME and EDHF inhibitors, blood pressure increased even more than after L-NAME alone. In the 1-month obese group leptin had no effect on SBP, however, pressor effect of leptin was observed in animals pretreated with EDHF inhibitors. In the 3-month obese group leptin alone increased SBP, and EDHF inhibitors did not augment its pressor effect. The results suggest that leptin may stimulate EDHF when NO becomes deficient, e.g. after NOS blockade or in short-term obesity. Although the effect of leptin on NO production is impaired in the 1-month obese group, BP does not increase, probably because EDHF compensates for NO deficiency. In contrast, leptin increases BP in 3-month obesity because its effect on EDHF is also attenuated.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Biological Factors; Blood Pressure; Body Weight; Cyclic GMP; Diet; Indicators and Reagents; Isoprostanes; Leptin; Male; Nitric Oxide; Obesity; Rats; Rats, Wistar

Activation of protein kinase C (PKC) in vascular tissue is associated with endothelial dysfunction and insulin resistance. However, the effect of vascular PKC activation on insulin-stimulated endothelial nitric oxide (NO) synthase (eNOS) regulation has not been characterized in obesity-associated insulin resistance. Diacylglycerol (DAG) concentration and PKC activity were increased in the aorta of Zucker fatty compared with Zucker lean rats. Insulin-stimulated increases in Akt phosphorylation and cGMP concentration (a measure of NO bioavailability) after euglycemic-hyperinsulinemic clamp were blunted in the aorta of fatty compared with lean rats but were partly normalized after 2 weeks of treatment with the PKCbeta inhibitor ruboxistaurin (LY333531). In endothelial cell culture, overexpression of PKCbeta1 and -beta2, but not PKCalpha, -delta, or -zeta, decreased insulin-stimulated Akt phosphorylation and eNOS expression. Overexpression of PKCbeta1 and -beta2, but not PKCalpha or -delta, also decreased Akt phosphorylation stimulated by vascular endothelial growth factor (VEGF). In microvessels isolated from transgenic mice overexpressing PKCbeta2 only in vascular cells, Akt phosphorylation stimulated by insulin was decreased compared with wild-type mice. Thus, activation of PKCbeta in endothelial cells and vascular tissue inhibits Akt activation by insulin and VEGF, inhibits Akt-dependent eNOS regulation by insulin, and causes endothelial dysfunction in obesity-associated insulin resistance.

Topics: Animals; Atherosclerosis; Blood Vessels; Cattle; Cells, Cultured; Cyclic GMP; Diglycerides; Enzyme Activation; Insulin Resistance; Male; Mice; Mice, Transgenic; Nitric Oxide Synthase Type III; Obesity; Phosphorylation; Protein Kinase C; Protein Kinase C beta; Proto-Oncogene Proteins c-akt; Rats; Rats, Zucker; Vascular Endothelial Growth Factor A

Abnormal neurohormonal regulation of renal sodium handling plays an important role in obesity-associated hypertension. We investigated the effect of experimental obesity on renal response to atrial natriuretic peptide (ANP).. The effect of ANP was studied in three groups of rats: (1) lean controls, (2) animals made obese by a highly palatable diet, (3) rats treated with adipose tissue hormone, leptin, for 7 days to reproduce hyperleptinemia observed in obesity.. ANP administered at a dose of 50 pmol/kg min(-1) induced about a 3-fold lower increase in Na+ and cGMP excretion in obese and leptin-treated rats than in the control group. ANP decreased Na+,K+-ATPase activity in the renal medulla only in the control group. Natriuretic effect of exogenous cGMP was also impaired in obese and leptin-treated rats. In contrast, hydrolysis-resistant cGMP derivative, 8-bromo-cGMP exerted comparable natriuretic effects in all groups. Neutral endopeptidase inhibitor, phosphoramidon, and ANP clearance receptor antagonist, C-ANP, increased urinary ANP excretion in all groups to a similar level, but their natriuretic effect was impaired in obese and leptin-treated groups. A specific inhibitor of cGMP-degrading phosphodiesterase, zaprinast, had comparable natriuretic and Na+,K+-ATPase-lowering effects in all groups and restored normal sensitivity to ANP.. (1) Dietary-induced obesity is accompanied by impaired natriuretic effect of ANP, (2) ANP resistance in obesity may be accounted for by increased leptin level, (3) accelerated degradation of cGMP may contribute to ANP resistance associated with obesity and hyperleptinemia, suggesting that inhibiting cGMP-specific phosphodiesterases may be useful in the treatment of obesity-associated hypertension.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Atrial Natriuretic Factor; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Drug Resistance; Hyperlipidemias; Kidney; Male; Obesity; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Rats; Rats, Wistar; Sodium

We have reported recently that the renal angiotensin II type 2 (AT2) receptors are upregulated and involved in promoting natriuresis/diuresis in obese but not in lean Zucker rats. In the present study, we tested the hypothesis that there is an enhanced AT2 receptor signaling via NO/cGMP pathway leading to greater inhibition of the Na(+), K(+)-ATPase (NKA) activity in the proximal tubules (PT) of obese rather than lean Zucker rats. The AT2 agonist CGP42112 (0.1 to 100 nmol/L) inhibited (33% at 100 nmol/L) the NKA activity in the PTs of obese but not in lean Zucker rats. The AT2 antagonist PD123319 (1 micromol/L), not the angiotensin II type 1 antagonist losartan (1 micromol/L), significantly diminished the CGP42112-induced inhibition of the NKA activity in obese rats. The AT2 agonist (10 nmol/L)-induced NKA inhibition was abolished by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10 micromol/L), the NO synthase inhibitor NG-nitro-L-arginine methyl ester (100 micromol/L), and the protein kinase G inhibitor K1388 (2 micromole/L). CGP42112 (10 nmol/L) caused an increase in serine phosphorylation of NKA alpha1-subunit in PT of obese rats. Measurement of cGMP and NO revealed that CGP42112 (0.1 to 100 nmol/L) increased cGMP and NO accumulation in the PTs of obese but not lean rats. The CGP42112-induced stimulation of NO and cGMP was blocked by PD123319 (1 micromol/L), NG-nitro-L-arginine methyl ester (100 micromol/L), and 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10 micromol/L) but not by losartan (1 micromol/L). The data suggest that the AT2 receptor activation via stimulation of the NO/cGMP/protein kinase G pathway directly inhibits the tubular NKA activity that provides as a mechanism responsible for the AT2 receptor-mediated natriuresis in obese but not in lean Zucker rats.

Topics: Animals; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Imidazoles; Kidney Tubules, Proximal; Male; Nitrates; Nitrites; Obesity; Oligopeptides; Phosphorylation; Pyridines; Rats; Rats, Zucker; Receptor, Angiotensin, Type 2; Sodium-Potassium-Exchanging ATPase; Thinness

Leptin resistance leads to obesity and may affect responses to the second messenger cGMP. We tested the hypothesis that the myocardial negative metabolic response to cGMP would be enhanced in leptin-resistant animals. This hypothesis was tested in anesthetized open-chest Zucker obese (n = 16) and age-matched control rats (n = 13). Coronary blood flow (microspheres) and O2 extraction (microspectrophotometry) measurements were used to determine myocardial O2 consumption (VO2). Protein phosphorylation by cGMP protein kinase and cAMP phosphodiesterase activity were also determined. Either vehicle (saline) or 8-Br-cGMP (10(-3) M) was topically applied to the left ventricular surface. Body weight was significantly greater in the obese rats (523 +/- 17 versus 322 +/- 12 g). There were no hemodynamic differences between groups. There was no difference in VO2 between lean (52 +/- 13 mL O2/min/100 g) and obese (54 +/- 9) vehicle-treated rats. 8-Br-cGMP significantly lowered VO2 in obese (35 +/- 6) but not lean (45 +/- 7) rats. This was not related to altered protein phosphorylation by the cGMP protein kinase. Cyclic GMP inhibited cAMP phosphodiesterase activity in lean but not obese hearts. Thus, the high myocardial oxygen consumption of lean rats was not significantly affected by cGMP but was reduced in obese hearts. This appeared to be related to a reduced inhibition of cAMP phosphodiesterase activity by cGMP in the Zucker obese rat.

Topics: Animals; Cyclic GMP; Heart; In Vitro Techniques; Male; Obesity; Oxygen Consumption; Rats; Rats, Zucker

Subjects with central obesity exhibit platelet hyperactivity, which is involved in the atherosclerotic process and therefore can account for the increased risk of cardiovascular morbidity and mortality. The aim of the study was to evaluate whether alterations of platelet function in obesity involve synthesis and/or action of the two antiaggregating cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP).. In platelets from 16 obese and 15 control subjects we investigated the influence on platelet responses to the Adenosine-5-diphosphate sodium salt (ADP) exerted by (i) prostacyclin analogue Iloprost (0.31-5 nmol L(-1)) and the cAMP analogue 8-bromo-cAMP (10-500 micro mol L(-1)); and by (ii) nitric oxide (NO) donor sodium nitroprusside (SNP) (5-100 micro mol L(-1)) and the cGMP analogue 8-bromo-cGMP (10-500 micro mol L(-1)). IC(50) (minimal concentration of each inhibitor necessary to reduce platelet response to ADP by half) was determined. Iloprost and SNP ability to increase cyclic nucleotides was also measured.. Significantly greater IC(50) were observed in obese subjects than in healthy controls (1.59 +/- 0.16 vs. 0.80 +/- 0.08 nmol L(-1), P = 0.0001 for Iloprost, and 27.6 +/- 6.5 vs. 7.0 +/- 1.7 micro mol L(-1), P = 0.006, for SNP); when data from control and obese subjects were pooled together, IC(50) of Iloprost and SNP correlated with the homeostasis model assessment (HOMA IR), which is a parameter used to measure the insulin resistance (r = 0.588, P = 0.029 and r = 0.640, P = 0.006, respectively). Also the antiaggregating effect of 8-Br-cAMP and 8-Br-cGMP was smaller in the obese subjects. Finally, the ability of Iloprost to increase platelet cAMP and the ability of SNP to increase both cGMP and cAMP were reduced in obese subjects.. Platelet resistance to the antiaggregating effects of prostacyclin and NO in obesity is attributable to impairment of cyclic nucleotide synthesis and action. As cyclic nucleotides are the main effectors of platelet antiaggregation, the resistance to them can account for platelet hyperactivity in obesity.

Topics: Adult; Body Mass Index; Cyclic AMP; Cyclic GMP; Female; Humans; Iloprost; Male; Obesity; Platelet Aggregation; Platelet Aggregation Inhibitors

Leptin is a regulator of body weight and affects nitric oxide (NO) production. This study was designed to determine whether the myocardial NO-cGMP signal transduction system was altered in leptin-deficient obese mice. Contractile function, guanylyl cyclase activity, and cGMP-dependent protein phosphorylation were assessed in ventricular myocytes isolated from genetically obese (B6.V-Lepob) and age-matched lean (C57BL/6J) mice. There were no differences in baseline contraction between the lean and obese groups. After stimulation with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10-6 and 10-5 M) or a membrane-permeable cGMP analog 8-bromo-cGMP (8-Br-cGMP, 10(-6) and 10(-5) M), cell contractility was depressed. However, 8-Br-cGMP had significantly greater effects in obese mice than in lean controls with percent shortening reduced by 47 vs. 39% and maximal rate of shortening decreased by 46 vs. 36%. The negative effects of SNAP were similar between the two groups. Soluble guanylyl cyclase activity was not attenuated. This suggests that the activity of the cGMP-independent NO pathway may be enhanced in obesity. The phosphorylated protein profile of cGMP-dependent protein kinase showed that four proteins were more intensively phosphorylated in obese mice, which suggests an explanation for the enhanced effect of cGMP. These results indicate that the NO-cGMP signaling pathway was significantly altered in ventricular myocytes from the leptin-deficient obese mouse model.

Topics: Animals; Cyclic GMP; Heart Ventricles; Leptin; Mice; Mice, Inbred C57BL; Mice, Obese; Myocardial Contraction; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Donors; Obesity; Penicillamine

We investigated whether the platelets from obese subjects are sensitive as those from controls to the antiaggregating effects of N-acetyl-L-cysteine (NAC)-an antioxidant thiol that increases availability of endogenous nitric oxide (NO)-and of superoxide dismutase (SOD) and amifostine which act as scavengers of superoxide anion.. In platelets from obese subjects (n=20, body mass index [BMI]=34.2+/-1.9 kg/m(2), homeostasis model assessment [HOMA] index=5.5+/-1.1) and controls (n=20, BMI=21.4+/-0.6 kg/m(2), HOMA index=1.4+/-0.2), we investigated the effects of NAC on aggregation and on 3',5'-cyclic guanosine monophosphate (cGMP) synthesis and the interplay between NAC and the organic nitrates glyceryl trinitrate (GTN) and sodium nitroprusside (SNP). Similar experiments were carried out with SOD and amifostine.. We found that a 3-min platelet exposure to NAC decreased aggregation and increased cGMP in controls, but not in obese subjects. Only more prolonged incubations exerted a small effect also in obese subjects. GTN and SNP increased platelet cGMP in both groups, but their effect was much lower in obese subjects. NAC (3 mmol/l), SOD (150 U/ml), and amifostine (50 micromol/l) enhanced the increase of cGMP elicited by NO donors, but again, the effect was much lower in obese subjects.. Since antioxidants do not restore the effects of NO in platelets from obese subjects, we hypothesize that oxidative stress is not the unique cause of platelet resistance to NO in obesity and suggest that a resistance to the NO action at the guanylate cyclase level could play a role in this phenomenon, potentially involved in the increased atherothrombotic risk linked to obesity.

Topics: Acetylcysteine; Adenosine Diphosphate; Adult; Amifostine; Antioxidants; Collagen; Cyclic GMP; Drug Resistance; Female; Free Radical Scavengers; Humans; Insulin Resistance; Male; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Nitroprusside; Obesity; Platelet Aggregation; Platelet Aggregation Inhibitors; Superoxide Dismutase

A key question for understanding the mechanisms of pulsatile insulin release is how the underlying beta-cell oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) are synchronized within and among the islets in the pancreas. Nitric oxide has been proposed to coordinate the activity of the beta-cells by precipitating transients of [Ca2+]i. Comparing ob/ob mice and lean controls, we have now studied the action of carbon monoxide (CO), another neurotransmitter with stimulatory effects on cGMP production. A strong immunoreactivity for the CO-producing constitutive heme oxygenase (HO-2) was found in ganglionic cells located in the periphery of the islets and in almost all islet endocrine cells. Islets from ob/ob mice had sixfold higher generation of CO (1 nmol.min-1.mg protein-1) than the lean controls. This is 100-fold the rate for their constitutive production of NO. Moreover, islets from ob/ob mice showed a threefold increase in HO-2 expression and expressed inducible HO (HO-1). The presence of an excessive islet production of CO in the ob/ob mouse had its counterpart in a pronounced suppression of the glucose-stimulated insulin release from islets exposed to the HO inhibitor Zn-protoporhyrin (10 microM) and in a 16 times higher frequency of [Ca2+]i transients in their beta-cells. Hemin (0.1 and 1.0 microM), the natural substrate for HO, promoted the appearance of [Ca2+]i transients, and 10 microM of the HO inhibitors Zn-protoporphyrin and Cr-mesoporphyrin had a suppressive action both on the firing of transients and their synchronization. It is concluded that the increased islet production of CO contributes to the hyperinsulinemia in ob/ob mice. In addition to serving as a positive modulator of glucose-stimulated insulin release, CO acts as a messenger propagating Ca2+ signals with coordinating effects on the beta-cell rhythmicity.

Topics: Animals; Blood Glucose; Body Weight; Calcium; Carbon Monoxide; Cyclic GMP; Cytoplasm; Enzyme Inhibitors; Female; Glucose; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hemin; Insulin; Insulin Secretion; Islets of Langerhans; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Obese; Nitric Oxide; Obesity; Protoporphyrins; Signal Transduction

We investigated the effect of leptin on nitric oxide production in lean and rats made obese by a high-calorie diet.. The animals were placed in metabolic cages, and urine was collected in 2-hour periods after leptin (1 mg/kg intraperintoneally) or vehicle administration. Blood was obtained 0.5, 1, 2, 4, or 6 hours after injection.. Leptin had no effect on systolic blood pressure in either lean or obese animals. Plasma concentration of NO metabolites (nitrites + nitrates, NOx) increased in lean rats by 31.5%, 58.0%, and 27.9% at 1, 2, and 4 hours after leptin injection, respectively. In the obese group, plasma NOx increased only at 2 hours (+36.5%). Leptin increased urinary NOx excretion by 31.8% in the first 2-hour period after injection in lean but not in obese rats. In lean animals, leptin elevated plasma cyclic 3',5'-guanosine monophosphate (cGMP) at 1, 2, and 4 hours by 35.3%, 96.3%, and 57.3%, respectively. In the obese group, plasma cGMP was higher only at 2 and 4 hours (+44.6% and +32.1%, respectively). Urinary excretion of cGMP increased in lean animals by 67.1% in the first period and by 50.4% in the second period. In the obese group, leptin induced a 53.9% increase in urinary cGMP excretion only in the first 2-hour period.. The stimulatory effect of leptin on NO production is impaired in dietary-induced obesity; however, leptin does not increase blood pressure in obese animals, suggesting that other NO-independent depressor mechanisms are stimulated.

Topics: Animals; Arginine; Blood Pressure; Cyclic GMP; Leptin; Male; Nitric Oxide; Nitric Oxide Donors; Obesity; Random Allocation; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine

Insulin resistance is associated with an increased risk of atherothrombotic vascular disease, but the mechanisms are poorly understood. We determined how insulin in vivo regulates platelet activation in nonobese and obese subjects by using methods mimicking thrombus formation. Twelve nonobese (aged 42+/-2 years, body mass index 24.0+/-0.4 kg/m(2)) and 14 obese (aged 43+/-1 years, body mass index 37.2+/-1.5 kg/m(2)) subjects were studied under euglycemic hyperinsulinemic (3-hour insulin infusion of 1 mU. kg(-1). min(-1)) conditions. Before and at the end of hyperinsulinemia, the following were determined: (1) platelet-related early hemostasis (shear rate of approximately 4000 s(-1)) by platelet function analysis; (2) platelet deposition to collagen during whole-blood perfusion (shear rate of 1600 s(-1)); (3) aggregation responses to collagen, thrombin receptor-activating peptide, ADP, and epinephrine; and (4) platelet cGMP concentrations. Insulin action on glucose metabolism was 69% lower in the obese subjects (1.6+/-0.2 mg. kg(-1). min(-1)) than in the nonobese subjects (5.4+/-0.4 mg. kg(-1). min(-1), P<0.0001). The in vivo insulin infusion inhibited platelet deposition to collagen from 4.3+/-0.6x10(6) to 3.5+/-0.4x10(6) per square centimeter in the nonobese subjects (P<0.05) but failed to do so in the obese subjects (5.2+/-0.8x10(6) versus 5.5+/-0.7x10(6) per square centimeter, P=NS; P<0.01 versus nonobese subjects). Epinephrine- and ADP-primed closure times by platelet function analysis were prolonged by insulin in the nonobese but not the obese subjects (P<0.05 for between-group difference). In the nonobese subjects, insulin decreased aggregation to all agonists and significantly increased platelet cGMP concentrations (2.5+/-0.3 versus 3.2+/-0.5 pmol/10(9) for before versus after insulin, respectively; P<0.01). In the obese subjects, insulin did not alter collagen-induced aggregation or cGMP concentrations (1.9+/-0.2 versus 1.8+/-0.1 pmol/10(9) for before versus the end of in vivo hyperinsulinemia, respectively; P=NS). These data demonstrate that normal in vivo insulin action inhibits platelet interaction with collagen under conditions mimicking thrombus formation and reduces aggregation to several agonists. These platelet-inhibitory actions of insulin are blunted or absent in obese subjects and could provide 1 mechanism linking insulin resistance to atherothrombotic disease.

Topics: Adenosine Diphosphate; Adult; Analysis of Variance; Blood Platelets; Body Mass Index; Collagen; Cyclic GMP; Enzyme-Linked Immunosorbent Assay; Epinephrine; Glucose; Glucose Clamp Technique; Hemostasis; Humans; Insulin; Insulin Resistance; Obesity; Peptide Fragments; Platelet Aggregation

We recently demonstrated that natriuretic peptides (NP) are involved in a pathway inducing lipolysis in human adipose tissue. Atrial NP (ANP) and brain NP (BNP) operate via a cGMP-dependent pathway which does not involve phosphodiesterase-3B inhibition or cAMP. The study was performed to evaluate the effect of ANP on lipid mobilization in obese women and secondly to examine the possible effect of a low-calorie diet (LCD) on the lipolytic response of subcutaneous abdominal fat cells to NP and on the lipid mobilization induced by ANP infusion (1 microg/m(2) min for 60 min).. Ten obese women from 40.5+/-3.4 y old were selected for this study. Their body weight was 96.4+/-5.7 kg and their BMI was 35.3+/-1.7 kg/m(2). They received a 2.5-2.9 MJ/day formula diet for 28 days.. Before and during the LCD, an adipose tissue biospy was performed for in vitro studies and, moreover, ANP was perfused i.v. to evaluate its lipid mobilizing action in toto and in situ in subcutaneous abdominal adipose tissue (SCAAT) using microdialysis.. The lipolytic effects of isoproterenol, ANP, BNP and bromo-cGMP (an analogue of cGMP) on fat cells increased by about 80-100% during LCD. The lipid mobilization during i.v. ANP infusion, assessed by plasma non-esterified fatty acids (NEFA) increase was enhanced during the LCD. However, during LCD, ANP infusion induced a biphasic effect on glycerol concentration in plasma and interstitial fluid of SCAAT; a significant increase was observed in glycerol levels during the first 30 min infusion period, followed by a steady decrease. The concentration of glycerol was lower during the post-infusion period than during the baseline period. This effect was stronger in obese subjects submitted to the LCD with a low-carbohydrate composition. Other plasma parameters were weakly increased (noradrenaline) or not modified (insulin, glucose) by ANP infusion and no difference was found before and during LCD treatment.. The present study shows that NP are powerful lipolytic agents in subcutaneous fat cells and that both isoproterenol- and NP-induced lipolysis increase during LCD, in obese women. These changes seem to be associated with an improvement of the lipolytic pathway at a post-receptor level. Moreover, i.v. administration of ANP induced a lipid mobilizing effect which was enhanced by a LCD in these objects.

Topics: Adipocytes; Adipose Tissue; Adult; Cyclic GMP; Diet, Reducing; Female; Humans; Infusions, Intravenous; Isoproterenol; Lipid Metabolism; Lipolysis; Middle Aged; Natriuretic Agents; Natriuretic Peptide, Brain; Obesity; Women's Health

The biochemical properties of an in vivo hormonally regulated low Km cAMP phosphodiesterase (PDE) activity associated with a liver Golgi-endosomal (GE) fraction have been characterized. DEAE-Sephacel chromatography of a GE fraction solubilized by a lysosomal extract resulted in the sequential elution of three peaks of activity (numbered I, II, and III), while ion-exchange HPLC resolved five peaks of activity (numbered 1, 2, 3, 4, and 5). Based on the sensitivity of the eluted activity to cGMP and selected phosphodiesterase inhibitors, two phosphodiesterase isoforms were resolved: a cGMP-stimulated and EHNA-inhibited PDE2, eluted in DEAE-Sephacel peak I and HPLC peak 2 and a cGMP-, a cilostamide-, and ICI 118233-inhibited PDE3, eluted in DEAE-Sephacel peak III and HPLC peaks 3, 4, and 5. GE fractions isolated after acute treatments with insulin, tetraiodoglucagon, and growth hormone displayed an increase in phosphodiesterase activity relative to saline-injected controls, as did GE fractions from genetically obese and hyperinsulinemic rats relative to lean littermates. In all experimental rats, an increase in PDE3 activity associated with DEAE-Sephacel peak III and HPLC peaks 4 and 5 was observed relative to control animals. Furthermore, in genetically obese Zucker rats, an increase in the sensitivity of PDE activity to cilostamide and in the amount of PDE activity immunoprecipitated by an antibody to adipose tissue PDE3 was observed relative to lean littermates. These results extend earlier studies on isolated hepatocytes and show that liver PDE3 is the main if not sole PDE isoform activated by insulin, glucagon, and growth hormone in vivo.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Endosomes; Gene Expression Regulation, Enzymologic; Glucagon; Golgi Apparatus; Growth Hormone; Hormones; Insulin; Isoenzymes; Liver; Male; Obesity; Phosphodiesterase Inhibitors; Pyridazines; Quinolones; Rats; Rats, Zucker; Subcellular Fractions

We have recently made the novel observation that a pro-oxidant challenge with hydroquinone in combination with buthionine sulfoximine (each at 50 mg/kg i.p. daily for 7 days) provokes the onset of type II diabetes mellitus in a model of insulin resistance, the obese Zucker rat. Since endothelial dysfunction in oxidant stress may aggravate in vivo insulin resistance, we have now investigated endothelium-dependent and nitric oxide (NO)-mediated vascular responses in the obese Zucker rat in vivo following this pro-oxidant insult. Pro-oxidant-treated animals exhibited defective vasodepression to the endothelium-dependent agent acetylcholine and to a lesser extent, the NO donor glyceryl trinitrate, together with a reduction in circulating levels of cGMP. Our data therefore suggest that the progression to type II diabetes mellitus in the obese Zucker rat mediated by a pro-oxidant insult is associated with impairments in agonist-stimulated, endothelium-dependent vasodilation and vascular NO signalling.

Topics: Acetylcholine; Animals; Antioxidants; Area Under Curve; Body Weight; Buthionine Sulfoximine; Cyclic GMP; Diabetes Mellitus, Type 2; Endothelium, Vascular; Enzyme Inhibitors; Hemodynamics; Hydroquinones; Insulin Resistance; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Obesity; Oxidants; Rats; Rats, Zucker

The present study was undertaken to characterise the phosphodiesterases (PDEs) present in brown adipose tissue (BAT) of Zucker rat pups and to determine whether the capacity for degradation of cyclic nucleotides was affected by the fatty genotype. Regardless of the genotype, PDE2-4 contributed to total PDE activity, the PDE3 activity equalling the sum of PDE2 and 4 activities. In fa/fa compared to Fa/fa rats, (a) PDE2 activity was significantly increased, (b) Western blot analysis of PDE2 revealed two signals at 71 and 105 kDa, with changes in protein being in good parallelism with changes in activity, (c) the PDE2 mRNA concentration was also significantly increased. In good agreement, the cGMP concentration was decreased in BAT from fa/fa pups.

Topics: Adipose Tissue, Brown; Animals; Animals, Newborn; Blotting, Northern; Cyclic AMP; Cyclic GMP; Cytosol; Female; Gene Expression Regulation; Immunoblotting; Isoenzymes; Leptin; Male; Obesity; Phosphoric Diester Hydrolases; Proteins; Rats; Rats, Zucker

Nitric oxide activates guanylate cyclase to form cGMP, comprising a signalling system that is believed to be a distinct mechanism for increasing glucose transport and metabolism in skeletal muscle. The effects of a selective cGMP phosphodiesterase inhibitor, zaprinast, on basal glucose utilization was investigated in incubated rat soleus muscle preparations isolated from both insulin-sensitive (lean Zucker; Fa/?) and insulin-resistant (obese Zucker; fa/fa) rats. Zaprinast at 27 microM significantly increased cGMP levels in incubated soleus muscle isolated from lean, but not obese, Zucker rats. Muscles were incubated with 14C-labelled glucose and various concentrations of zaprinast (3, 27 and 243 microM). Zaprinast (at 27 and 243 microM) significantly increased rates of net and 14C-labelled lactate release and of glycogen synthesis in lean Zucker rat soleus muscle; glucose oxidation was also increased by 27 microM zaprinast. In addition, regardless of concentration, the phosphodiesterase inhibitor failed to increase any aspect of 14C-labelled glucose utilization in soleus muscles isolated from obese Zucker rats. The maximal activity of nitric oxide synthase (NOS) was significantly decreased in insulin-resistant obese Zucker muscles. Thus the lack of effect of zaprinast in insulin-resistant skeletal muscle is consistent with decreased NOS activity. To test whether there is a defect in insulin-resistant skeletal muscle for endogenous activation of guanylate cyclase, soleus muscles were isolated from both insulin-sensitive and insulin-resistant Zucker rats and incubated with various concentrations of the NO donor sodium nitroprusside (SNP; 0.1, 1, 5 and 15 mM). SNP significantly increased rates of net and 14C-labelled lactate release, as well as glucose oxidation in muscles isolated from both insulin-sensitive and insulin-resistant rats. A decreased response to SNP was observed in the dose-dependent generation of cGMP within isolated soleus muscles from insulin-resistant rats. A possible link between impaired NO/cGMP signalling and abnormal glucose utilization by skeletal muscle is discussed.

Topics: Animals; Cell Fractionation; Cyclic GMP; Female; Glucose; Glycogen; Insulin; Insulin Resistance; Lactic Acid; Muscle, Skeletal; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Obesity; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Zucker; Signal Transduction

Previous studies in our laboratory showed that the platelet anti-aggregating effect exerted by insulin, mediated by a nitric oxide (NO)-induced increase of guanosine-3',5'-cyclic monophosphate (cGMP), is lost in the insulin-resistant of obesity and obese NIDDM. It is not clear 1) whether the alterations observed in obese NIDDM patients are attributable to the obesity-related insulin resistance or to diabetes per se and 2) whether insulin-resistant states present a normal or a blunted response to NO. This study has been conducted to investigate 1) the platelet sensitivity to insulin in lean NIDDM and 2) the platelet sensitivity to an NO donor, glyceryl trinitrate (GTN), in obesity and in both lean and obese NIDDM.. We determined 1) ADP-induced platelet aggregation and platelet cGMP content in platelet-rich plasma (PRP) obtained from 11 lean NIDDM patients, after a 3-min incubation with insulin (0, 240, 480, 960, 1,920 pmol/l) and 2) ADP-induced platelet aggregation and platelet cGMP content in PRP obtained from 9 obese subjects, 11 lean and 8 obese NIDDM patients, and 18 control subjects, after a 3-min incubation with 0, 20, 40, and 100 mumol/l GTN.. Insulin dose-dependently decreased platelet aggregation in lean NIDDM patients (P = 0.0001): with 1,920 pmol/l of insulin, ADP ED50 was 141.5 +/- 6.4% of basal values (P = 0.0001). Furthermore, insulin increased platelet cGMP (P = 0.0001) from 7.5 +/- 0.2 to 21.1 +/- 3.7 pmol/10(9) platelets. These results were similar to those previously described in healthy subjects. GTN reduced platelet aggregation in all the groups (P = 0.0001) at all the concentrations tested (P = 0.0001), but GTN IC50 values were much higher in insulin-resistant patients: 36.3 +/- 5.0 mumol/l in healthy control subjects, 26.0 +/- 6.0 mumol/l in lean NIDDM patients (NS vs. control subjects), 123.6 +/- 24.0 mumol/l in obese subjects (P = 0.0001 vs. control subjects), and 110.1 +/- 19.2 mumol/l in obese NIDDM patients (P = 0.0001 vs. control subjects). GTN dose-dependently increased platelet cGMP in all the groups (P = 0.0001 in control subjects, lean NIDDM patients, and obese subjects; P = 0.04 in obese NIDDM patients). Values reached by obese subjects and obese NIDDM patients, however, were lower than those reached by control subjects (with 100 mumol/l of GTN, P = 0.001 and P = 0.0001, respectively). In healthy control subjects and in obese subjects, the insulin:glucose ratio, used as an indirect measure of insulin sensitivity, was positively correlated to GTN IC50 (r = 0.530, P = 0.008), further suggesting that the sensitivity to NO is reduced in the presence of insulin resistance.. The insulin anti-aggregating effect is preserved in lean NIDDM; platelet sensitivity to GTN in preserved in lean NIDDM but is reduced in the insulin-resistant states of obesity and obese NIDDM. Resistance to nitrates, therefore, could be considered another feature of the insulin-resistance syndrome.

Topics: Adenosine Diphosphate; Adult; Analysis of Variance; Blood Glucose; Blood Platelets; Blood Pressure; Body Mass Index; Cholesterol; Cholesterol, HDL; Cyclic GMP; Diabetes Mellitus; Diabetes Mellitus, Type 2; Female; Glycated Hemoglobin; Humans; Insulin; Insulin Resistance; Male; Nitroglycerin; Obesity; Platelet Aggregation; Platelet Aggregation Inhibitors; Reference Values; Thinness; Triglycerides

1. The effects of nitric oxide (NO) on vascular reactivity and platelet function in the obese (cp/cp) and lean (+/?) JCR:LA-cp rats were investigated. 2. Phenylephrine (PE; 0.1 nM-10 microM) induced contraction of isolated aortic rings in both genotypes (cp/cp and +/?) of JCR:LA-cp rats. The sensitivity to contraction with PE was enhanced in cp/cp compared with +/? rings. Rings from both genotypes showed an increased contraction upon removal of the endothelium. 3. Acetylcholine (ACh; 0.1 nM-10 microM)-induced endothelium-dependent relaxation of rings was not significantly different in the two genotypes. Both were inhibited to a similar extent by NG-nitro-L-arginine methyl ester (L-NAME; 0.01-1 mM) when administered in vitro. 4. The nitric oxide synthase (NOS) inhibitor (L-NAME; 0.3, 1 or 3 mg ml(-1), p.o.) when administered in vivo increased blood pressure in cp/cp rats but not in +/? rats. 5. L-NAME resulted in greater inhibition of ACh-induced relaxation in cp/cp rings compared with +/? rings. 6. L-NAME treatment in vivo caused a decrease in cyclic GMP and NOS activity in rings from cp/cp but not +/? rats. 7. The NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 0.1 nM-10 microM)-induced relaxation of rings from +/? rats, an effect enhanced by the treatment with L-NAME in vivo. 8. Oral administration of L-NAME did not enhance the vasorelaxant effect of SNAP on rings of aorta from cp/cp animals. 9. Platelet aggregation and NOS activity were similar in both genotypes and were not modified by oral administration of L-NAME. 10. These results show that unimpaired generation of NO is crucial for maintenance of vascular tone particularly under conditions of vascular insult exemplified by insulin resistance, obesity and dyslipidemia detected in cp/cp rats.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Blood Platelets; Blood Pressure; Cyclic GMP; Diabetes Mellitus; Enzyme Inhibitors; Genotype; Insulin Resistance; Muscle Contraction; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Obesity; Penicillamine; Phenylephrine; Rats; S-Nitroso-N-Acetylpenicillamine; Vasoconstrictor Agents; Vasodilator Agents

We have investigated the effects of early phases of chronic stress on generation and actions of nitric oxide (NO) in JCR:LA-cp rats both lean (+/+) and obese (cp/cp). Restraint stress was carried out for a 15-min single exposure or for 1 hr every day during 4, 9 or 14 days. The stress reaction was evidenced by significant increase in plasma cortisol. The exposure to stress for 14 days led to a neuronal damage in lean rats as evidenced by a decrease in glutamate uptake and an increase in the release of lactate in synaptosomes. This effect was not observed in obese rats. Concomitantly, the levels of glutamate increased in the hippocampus at 14 days in lean, but not obese rats, that showed higher basal levels of glutamate than lean rats. The activity of NO synthase (NOS) and guanosine cyclic monophosphate levels increased in the hippocampus preceding the neuronal damage. The neuronal lesions were prevented by inhibition of NOS without affecting cortisol levels. In the cardiovascular system, chronic stress exerted no significant effect on blood pressure, aortic contractility or platelet aggregation. However, there were significant changes in plasma nitrite/nitrate that reached maximum at 4 to 9 days. It is concluded that the generation of NO contributes to the systemic response to the organism to stress. In the brain, NO appears to be detrimental as this molecule mediates glutamate-dependent hippocampal damage, this effect being cortisol-independent. In contrast, in the vascular system, increased generation of NO may attenuate the vasoconstrictor and platelet aggregatory effects of catecholamines and other mediators of stress.

Topics: Animals; Cyclic GMP; Glutamic Acid; Hippocampus; Homeostasis; Hydrocortisone; Male; Mice; Mice, Inbred ICR; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Obesity; Platelet Aggregation; Rats; Stress, Psychological

1. The obese fa/fa Zucker rat is a genetic model of obesity and insulin resistance which develops a number of metabolic and endocrine features of non-insulin-dependent diabetes, including hypertension, proteinuria and glomerular sclerosis. 2. We have investigated the urinary excretion of the metabolites of thromboxane (thromboxane B2) and prostacyclin (6-keto prostaglandin F1 alpha), and of endothelin and cyclic GMP as markers for changes in the balance of renal haemodynamic factors in the obese Zucker rat. 3. Obese fa/fa Zucker rats were hypertensive compared with their lean counterparts (161 +/- 3 and 138 +/- 3 mmHg respectively, P < 0.01); obese animals were also markedly proteinuric (16.7 +/- 6.7 versus 1.1 +/- 0.1 mg/ml) and albuminuric (8.3 +/- 2.9 versus 0.4 +/- 0.25 mg/ml) and excreted less creatinine than lean animals (all P < 0.01). Urinary excretion of endothelin was greater in obese rats (123 +/- 24 versus 62 +/- 10 pg/15 h, P < 0.05) as was the level of pre-proendothelin mRNA, but excretion of cyclic GMP was depressed (12.5 +/- 1.6 versus 27.2 +/- 3.1 nmol/ 15 h, P < 0.01). Histological examination of kidneys from obese animals showed evidence of focal glomerulosclerosis and cortical tubular damage. 4. These results show that increased urinary endothelin is associated with proteinuria and early stage nephropathy in this animal model of non-insulin-dependent diabetes mellitus. This finding, coupled with a decreased excretion of cyclic GMP, suggests that these increased renal vasoconstrictor/vasodilator forces might contribute to the renal functional changes in non-insulin-dependent diabetes mellitus.

Topics: 6-Ketoprostaglandin F1 alpha; Albuminuria; Animals; Cyclic GMP; Diabetes Mellitus; Diabetes Mellitus, Type 2; Endothelin-1; Immunohistochemistry; Kidney; Male; Obesity; Rats; Rats, Zucker; RNA, Messenger; Statistics, Nonparametric; Thromboxane B2

Hypertension is commonly associated with diabetes mellitus. The aim of the present study was to explore the pathophysiological significance of the natriuretic peptide (NP) system in hypertension associated with genetically obese/hyperglycemic Wistar fatty rats. The messenger RNA (mRNA) levels of the two biologically active NP receptors, NP-A receptor [more specific for atrial natriuretic peptide (ANP)] and NP-B receptor [more specific for C-type natriuretic peptide (CNP)], and CNP mRNA levels were determined in the aorta and kidney by ribonuclease protection assay. Plasma ANP levels were determined by RIA. Both NP-A and NP-B receptor mRNA levels in the aortae of Wistar fatty rats were double those in Wistar lean rats. Plasma ANP levels and CNP mRNA levels in the aorta of Wistar fatty rats were also significantly higher than those in Wistar lean rats. In contrast, there was no significant difference in renal levels of the mRNA for both NP receptors and CNP between the two strains. Administration of a NP-A and -B receptor antagonist, HS-142-1, to Wistar fatty rats resulted in a significant increase in systolic blood pressure and a larger decrease in plasma cGMP level than that in Wistar lean rats, with no difference in the extents of decrease in urine volume and urinary sodium excretion between the two strains. These results suggest that both the ANP/NP-A system and the CNP/NP-B system in vessels are up-regulated at the level of gene expression and may, thus, play an important role in counteracting the hypertension associated with diabetes mellitus.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Blood Vessels; Cyclic GMP; Diuresis; Guanylate Cyclase; Hyperglycemia; Hypertension; Kidney; Male; Natriuresis; Natriuretic Peptide, C-Type; Obesity; Polysaccharides; Proteins; Rats; Rats, Wistar; Receptors, Atrial Natriuretic Factor; RNA, Messenger

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adipose Tissue; Animals; Cyclic AMP; Cyclic GMP; Diabetes Mellitus; Diabetes Mellitus, Experimental; Epinephrine; Glucagon; Humans; Insulin; Isoproterenol; Liver; Male; Mice; Mice, Obese; Obesity; Rats

Topics: Animals; Cyclic AMP; Cyclic GMP; Diabetes Mellitus; Energy Metabolism; Enzyme Activation; Humans; Obesity; Protein Kinases

Topics: Adrenergic alpha-Agonists; Adrenergic beta-Agonists; Amino Acids; Animals; Binding Sites; Blood Glucose; Calcium; Carbohydrate Metabolism; Cyclic AMP; Cyclic GMP; Diabetes Mellitus; Gastrointestinal Hormones; Gluconeogenesis; Growth Hormone; Hormones; Humans; Hypoglycemia; Hypoglycemic Agents; Insulin; Insulin Resistance; Insulin Secretion; Islets of Langerhans; Lipid Metabolism; Obesity; Proteins; Receptors, Cell Surface; Sulfonylurea Compounds

Topics: Adenosine Triphosphate; Animals; Bucladesine; Butyrates; Cyclic AMP; Cyclic GMP; Cytosine Nucleotides; Drug Synergism; Female; Glucose; Guanosine Triphosphate; Hyperglycemia; In Vitro Techniques; Inosine Nucleotides; Insulin; Insulin Antibodies; Insulin Secretion; Iodine Radioisotopes; Islets of Langerhans; Mice; Nucleotides, Cyclic; Obesity; Uracil Nucleotides