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Objectives: The goal of this study was to assess mechanisms underlying atrial fibrillation (AF) promotion by exercise training in an animal model.Background: High-level exercise training promotes A...

Glucagon activates hepatic protein kinase A (PKA) to increase glucose production, but the gluco-stimulatory effect is transient even in the presence of continuous intravenous glucagon infusion. Continuous intravenous infusion of insulin, however, inhibits glucose production through its sustained actions in both the liver and the mediobasal hypothalamus (MBH). In a pancreatic clamp setting, MBH infusion with glucagon activated MBH PKA and inhibited hepatic glucose production (HGP) in rats, as did central glucagon infusion in mice. Inhibition of glucagon receptor-PKA signaling in the MBH and hepatic vagotomy each negated the effect of MBH glucagon in rats, whereas the central effect of glucagon was diminished in glucagon receptor knockout mice. A sustained rise in plasma glucagon concentrations transiently increased HGP, and this transiency was abolished in rats with negated MBH glucagon action. In a nonclamp setting, MBH glucagon infusion improved glucose tolerance, and inhibition of glucagon receptor-PKA signaling in the MBH enhanced the ability of intravenous glucagon injection to increase plasma glucose concentrations. We also detected a similar enhancement of glucose concentrations that was associated with a disruption in MBH glucagon signaling in rats fed a high-fat diet. We show that hypothalamic glucagon signaling inhibits HGP and suggest that hypothalamic glucagon resistance contributes to hyperglycemia in diabetes and obesity.

Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

Rationale: Dysregulation of hepatic triglyceride (TG)-rich very low-density lipoproteins (VLDL-TG) in obesity and type 2 diabetes contributes to the dyslipidemia that leads to cardiovascular morbidity. The central nervous system (CNS), particularly the hypothalamus, regulates hepatic lipid metabolism. Although the underlying neurocircuitry remains elusive, glycine has been documented to enhance CNS N -methyl- d -aspartate (NMDA) receptor-mediated transmission. Objective: We tested the hypothesis that glycine regulates hepatic VLDL-TG secretion by potentiating NMDA receptor-mediated transmission in the CNS. Methods and Results: Using 10-hour fasted male Sprague-Dawley rats implanted with stereotaxic cannulae into an extrahypothalamic region termed the dorsal vagal complex (DVC) and vascular catheters to enable direct DVC infusion and blood sampling, respectively, the rate of hepatic VLDL-TG secretion was measured following tyloxapol (an inhibitor of lipoprotein lipase) injection. Direct DVC infusion of glycine lowered VLDL-TG secretion, whereas NMDA receptor blocker MK-801 fully negated glycine's effect. NR1 subunit of NMDA receptor antagonist 7-chlorokynurenic acid, adenoviral injection of NR1 short hairpin RNA (shRNA), and hepatic vagotomy also nullified glycine's effect. Finally, DVC glycine normalized the hypersecretion of VLDL-TG induced by high-fat feeding. Conclusions: Molecular and pharmacological inhibition of the NR1-containing NMDA receptors in the DVC negated the ability of glycine to inhibit hepatic secretion of VLDL-TG in vivo. Importantly, the hypersecretion of VLDL-TG from the liver induced by a model of high-fat feeding was restored by the hepatic lipid control of CNS glycine sensing. These findings collectively suggest that glycine or glycine analogues may have therapeutic benefits in lowering plasma lipid levels in diabetes and obesity by triggering the CNS.

The upregulation of plasma inflammatory biomarkers in individuals with metabolic syndrome implies that activation of the innate immune response contributes to the pathogenesis of type 2 diabetes (1). Today, a large array of studies has demonstrated that activation of inflammatory pathways underlies obesity-associated insulin resistance at the liver and fat (2–4). However, little is known regarding whether obesity induces inflammation in the brain thereby disrupting the ability of insulin to control glucose homeostasis. Hotamisligil et al. (5) first established a link between obesity and the increased production of inflammatory molecules by demonstrating that tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is overexpressed in the adipose tissue of obese mice. This finding was confirmed in humans with obesity and insulin resistance (6,7). TNF-α induces peripheral insulin resistance in rodents (8,9) and alters insulin sensitivity and glucose homeostasis in humans (10,11). In fact, subjects with chronic inflammatory disease who are treated with TNF inhibitor show a 60% reduction in diabetes rates (12). Downstream of the inflammatory process lies the inhibitor of κB kinase (IKK-β) complex and its target, nuclear factor-ĸB (NF-ĸB), a transcription factor that regulates the expression of inflammatory genes (2–4) and mediates peripheral insulin resistance associated with overnutrition (2–4). In parallel, the c-Jun amino-terminal kinase (JNK), which can be activated in response to TNF-α or other stressors, is also implicated in insulin resistance of diabetic mice (2–4). NF-ĸB–mediated gene expression is regulated in part through the Toll-like receptors (TLRs), which serve to activate proinflammatory signaling cascades upon recognition of pathogen-associated molecular patterns (2–4 …

Last year marked the ninetieth anniversary of the discovery of insulin by Nobel Laureates Frederick Banting and John Macleod, as well as Charles Best and James Bertram Collip. The initial success of insulin’s ability to lower glucose levels in type 1 diabetes is now shadowed by the urgent need to characterize insulin resistance and secretion defects in type 2 diabetes and obesity. Insulin triggers signaling pathways in liver, muscle, and fat that inhibit glucose production and increase glucose uptake (1). However, it has only been in recent years that the brain has received attention for being an insulin-sensitive organ that regulates food intake (2) and glucose (3) and lipid (4) homeostasis in animals (Fig. 1). FIG. 1. A working hypothesis for insulin action in the brain. Insulin triggers signaling cascades in the brain to regulate food intake and modulate the reward-related hedonic food response. In addition, brain insulin action alters glucose and lipid metabolism. The hypothalamus (blue) is the main effector of the metabolic changes induced by insulin and can coordinate the prefrontal cortex (green) to regulate the hedonic assessment of appetite. WAT, white adipose tissue. Circulating insulin crosses the blood-brain barrier and acts on its receptor in the hypothalamus to lower food intake and body weight (2). Infusion of insulin into the central nervous system (CNS) lowers food intake in rats (5), mice (6), and baboons (7), whereas hyperphagia is detected in neuronal insulin receptor knockout mice (8). Further, high-fat feeding induces hypothalamic insulin resistance in rodents (9). Although these animal studies expand the role of insulin to regulate appetite and suggest that characterizing insulin signaling pathways may reveal novel targets that reverse brain insulin resistance in obesity, a central question remains, Is …

Atrial fibrillation, NADPH oxidases, ryanodine receptor Background Gp91-containing NADPH oxidases (NOX2) are a significant source of reactive oxygen species (ROS) in the human atrial myocardium. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, whether an increase in atrial NOX2 activity is necessary to create a substrate for AF remains to be demonstrated. The purpose of this study is to determine whether an increase in NOX2-derived ROS directly contributes to the development of AF and to identify the molecular changes that occur downstream of NOX2 activation in atrial myocytes. Methods and Results Mice with myocardial NOX2 overexpression (NOX2-Tg) showed a 2-fold increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in both the left and right atria, without any other electrophysiological or structural abnormalities (assessed by surface ECG and echocardiography); however, AF susceptibility assessed in vivo by transesophageal pacing was significantly higher in NOX2-Tg mice compared with their wild-type (WT) littermate controls (AF probability: 16.9±1.8% vs. 10.2±1.6% respectively, n=25–26/genotype, p Conclusions Myocardial NOX2 overexpression and related increase in NADPH-stimulated ROS production are associated with increased AF susceptibility in vivo and reduced RyR phosphorylation at Ser2814. These findings suggest increased NOX2 ROS may promote arrhythmogenesis by modulating atrial calcium handling.

The brain emerges as a regulator of hepatic triglyceride-rich very-low-density lipoproteins (VLDL-TG). The neurocircuitry involved as well as the ability of fatty acids to trigger a neuronal network to regulate VLDL-TG remain unknown. Here we demonstrate that infusion of oleic acid into the mediobasal hypothalamus (MBH) activates a MBH PKC-δ→KATP-channel signalling axis to suppress VLDL-TG secretion in rats. Both NMDA receptor-mediated transmissions in the dorsal vagal complex (DVC) and hepatic innervation are required for lowering VLDL-TG, illustrating a MBH-DVC-hepatic vagal neurocircuitry that mediates MBH fatty acid sensing. High-fat diet (HFD)-feeding elevates plasma TG and VLDL-TG secretion and abolishes MBH oleic acid sensing to lower VLDL-TG. Importantly, HFD-induced dysregulation is restored with direct activation of either MBH PKC-δ or KATP-channels via the hepatic vagus. Thus, targeting a fatty acid sensing-dependent hypothalamic-DVC neurocircuitry may have therapeutic potential to lower hepatic VLDL-TG and restore lipid homeostasis in obesity and diabetes.

ObjectivesThe goal of this study was to assess mechanisms underlying atrial fibrillation (AF) promotion by exercise training in an animal model.BackgroundHigh-level exercise training promotes AF, but the underlying mechanisms are unclear.MethodsAF susceptibility was assessed by programmed stimulation in rats after 8 (Ex8) and 16 (Ex16) weeks of daily 1-h treadmill training, along with 4 and 8 weeks after exercise cessation and time-matched sedentary (Sed) controls. Structural remodeling was evaluated by using serial echocardiography and histopathology, autonomic nervous system with pharmacological tools, acetylcholine-regulated potassium current (IKACh) with patch clamp recording, messenger ribonucleic acid expression with quantitative polymerase chain reaction, and regulators of G protein–signaling (RGS) 4 function in knockout mice.ResultsAF inducibility increased after 16 weeks of training (e.g., AF >30 s in 64% of Ex16 rats vs 15% of Sed rats; p < 0.01) and rapidly returned to baseline levels with detraining. Atropine restored sinus rhythm in 5 of 5 Ex rats with AF sustained >15 min. Atrial dilation and fibrosis developed after 16 weeks of training and failed to fully recover with exercise cessation. Parasympathetic tone was increased in Ex16 rats and normalized within 4 weeks of detraining. Baroreflex heart rate responses to phenylephrine-induced blood pressure elevation and IKACh sensitivity to carbachol were enhanced in Ex16 rats, implicating both central and end-organ mechanisms in vagal enhancement. Ex rats showed unchanged cardiac adrenergic and cholinergic receptor and IKACh-subunit gene expression, but significant messenger ribonucleic acid downregulation of IKACh-inhibiting RGS proteins was present at 16 weeks. RGS4 knockout mice showed significantly enhanced sensitivity to AF induction in the presence of carbachol.ConclusionsChronic endurance exercise increased AF susceptibility in rats, with autonomic changes, atrial dilation, and fibrosis identified as potential mechanistic contributors. Vagal promotion is particularly important and occurs via augmented baroreflex responsiveness and increased cardiomyocyte sensitivity to cholinergic stimulation, possibly due to RGS protein downregulation.

The goal of this study was to assess mechanisms underlying atrial fibrillation (AF) promotion by exercise training in an animal model.High-level exercise training promotes AF, but the underlying mechanisms are unclear.AF susceptibility was assessed by programmed stimulation in rats after 8 (Ex8) and 16 (Ex16) weeks of daily 1-h treadmill training, along with 4 and 8 weeks after exercise cessation and time-matched sedentary (Sed) controls. Structural remodeling was evaluated by using serial echocardiography and histopathology, autonomic nervous system with pharmacological tools, acetylcholine-regulated potassium current (IKACh) with patch clamp recording, messenger ribonucleic acid expression with quantitative polymerase chain reaction, and regulators of G protein-signaling (RGS) 4 function in knockout mice.AF inducibility increased after 16 weeks of training (e.g., AF30 s in 64% of Ex16 rats vs 15% of Sed rats; p 0.01) and rapidly returned to baseline levels with detraining. Atropine restored sinus rhythm in 5 of 5 Ex rats with AF sustained15 min. Atrial dilation and fibrosis developed after 16 weeks of training and failed to fully recover with exercise cessation. Parasympathetic tone was increased in Ex16 rats and normalized within 4 weeks of detraining. Baroreflex heart rate responses to phenylephrine-induced blood pressure elevation and IKACh sensitivity to carbachol were enhanced in Ex16 rats, implicating both central and end-organ mechanisms in vagal enhancement. Ex rats showed unchanged cardiac adrenergic and cholinergic receptor and IKACh-subunit gene expression, but significant messenger ribonucleic acid downregulation of IKACh-inhibiting RGS proteins was present at 16 weeks. RGS4 knockout mice showed significantly enhanced sensitivity to AF induction in the presence of carbachol.Chronic endurance exercise increased AF susceptibility in rats, with autonomic changes, atrial dilation, and fibrosis identified as potential mechanistic contributors. Vagal promotion is particularly important and occurs via augmented baroreflex responsiveness and increased cardiomyocyte sensitivity to cholinergic stimulation, possibly due to RGS protein downregulation.

Neurotransmitters released from sympathetic and parasympathetic nerve terminals in the SAN exert their effects via G-protein-coupled receptors. Integration of these different G-protein signals within pacemaker cells of the sinoatrial node (SAN) is critical for proper regulation of heart rate and function. For example, excessive parasympathetic signaling can be associated with sinus node dysfunction and supraventricular arrhythmias. Our previous work has shown that one member of the regulator of G-protein signaling (RGS) protein family, RGS4, is highly and selectively expressed in pacemaker cells of the SAN. Consistent with its role as an inhibitor of parasympathetic signaling, RGS4-knockout mice have reduced basal heart rates and enhanced negative chronotropic responses to parasympathetic agonists. Moreover, RGS4 appears to be an important part of SA nodal myocyte signaling pathways that mediate G protein-coupled inwardly-rectifying potassium channel (GIRK) channel activation/deactivation and desensitization. Since RGS4 acts immediately downstream of M2 muscarinic receptors, it is tempting to speculate that RGS4 functions as a master regulator of parasympathetic signaling upstream of GIRKs, HCNs and L-type Ca2+ channels in the SAN. Thus, loss of RGS4 function may lead to increased susceptibility to conditions associated with increased parasympathetic signaling, including bradyarrhythmia, sinus node dysfunction, and atrial fibrillation.

OBJECTIVE Circulating glucose inhibits glucose production in normal rodents and humans, but this glucose effectiveness is disrupted in diabetes due partly to sustained hyperglycemia. We hypothesize that hyperglycemia in diabetes impairs hypothalamic glucose sensing to lower glucose production, and changes of glucose transporter-1 (GLUT1) in the hypothalamic glial cells are responsible for the deleterious effects of hyperglycemia in vivo. RESEARCH DESIGN AND METHODS We tested hypothalamic glucose effectiveness to increase hypothalamic glucose concentration and lower glucose production in rats induced with streptozotocin (STZ) uncontrolled diabetes, STZ and phlorizin, and whole-body and hypothalamic sustained hyperglycemia. We next assessed the content of glial GLUT1 in the hypothalamus, generated an adenovirus expressing GLUT1 driven by a glial fibrillary acidic protein (GFAP) promoter (Ad-GFAP-GLUT1), and injected Ad-GFAP-GLUT1 into the hypothalamus of rats induced with hyperglycemia. Pancreatic euglycemic clamp and tracer-dilution methodologies were used to assess changes in glucose kinetics in vivo. RESULTS Sustained hyperglycemia, as seen in the early onset of STZ-induced diabetes, disrupted hypothalamic glucose sensing to increase hypothalamic glucose concentration and lower glucose production in association with reduced GLUT1 levels in the hypothalamic glial cells of rats in vivo. Overexpression of hypothalamic glial GLUT1 in STZ-induced rats with reduced GLUT1 acutely normalized plasma glucose levels and in rats with selectively induced hypothalamic hyperglycemia restored hypothalamic glucose effectiveness. CONCLUSIONS Sustained hyperglycemia impairs hypothalamic glucose sensing to lower glucose production through changes in hypothalamic glial GLUT1, and these data highlight the critical role of hypothalamic glial GLUT1 in mediating glucose sensing to regulate glucose production.

International audience; Over the last 20 years, melatonin, a pineal hormone synthesized from serotonin, has been implicated in various studies on the autism spectrum disorder (ASD) and altered melatonin levels were detected in subgroups of subjects with ASD. Its effect on sleep disturbances got the attention of clinicians and several investigations were carried out to determine the usefulness and safety of melatonin administration in this disorder. Hypotheses were also raised regarding the possibility that the dysfunctional synthesis and secretion of melatonin detected in subgroups of subjects with ASD may increase the risk as well the severity of ASD. The purpose of this paper is to review our pharmacokinetic knowledge on melatonin and present results from recent studies on sleep disorders in autism, their treatment with melatonin and the impact of melatonin prescription in children with ASD evaluated in a Diagnostic Center for Autism Spectrum Disorder in Paris, France.