Pascal Bousquet

Murine models for pharmacological studies of the metabolic syndrome

Metabolic syndrome has been described as the association of insulin resistance, hypertension, hyperlipidemia and obesity. Its prevalence increased dramatically, mainly in developed countries. Animal models are essential to understand the pathophysiology of this syndrome. This review presents the murine models of metabolic syndrome the most often used in pharmacological studies. The most common metabolic syndrome models exhibit a non-functional leptin pathway, or metabolic disorders induced by high fat diets. In a first part, and after a short introduction on leptin, its receptor and mechanism of action, we provide a detailed description of each model: SHROB, SHHF, JCR:LA-cp, Zucker, ZDF, Wistar Ottawa Karlsburg W, and Otsuka Long-Evans Tokushima Fatty rats, ob/ob, db/db, agouti yellow and Mc4R KO mice. The second part of this review is dedicated to metabolic syndrome models obtained by high fat feeding.

Contribution of catecholaminergic neurons of the dorsomedial and ventrolateral medulla oblongata to the hypotensive effect of clonidine in spontaneously hypertensive rats : in vivo voltammetric studies

OBJECTIVE Our previous electrochemical studies in the normotensive Wistar-Kyoto (WKY) rat showed a positive correlation between the hypotensive effect of low doses of clonidine (2-10 micrograms/kg intravenously) and inhibition of the activity of catecholaminergic neurons within the brainstem, and that this action was mediated by imidazoline-preferring receptors. In the present study the possibility of a relationship between the centrally mediated hypotensive effect of clonidine and the metabolic activity of catecholaminergic neurons of the ventrolateral and dorsomedial medulla oblongata was investigated in pentobarbital-anaesthetized spontaneously hypertensive rats (SHR). DESIGN AND METHODS Neuronal metabolic activity was monitored by in vivo electrochemistry in the nucleus reticularis lateralis region of the ventrolateral medulla and in the nucleus tractus solitarii region of the dorsomedial medulla oblongata. RESULTS Hypotensive doses of intravenously administered clonidine (10 micrograms/kg) failed to inhibit the neuronal metabolic activity of the nucleus reticularis lateralis region; fivefold higher doses were required to inhibit these neurons. The low dose of clonidine (10 micrograms/kg) decreased the metabolic activity of the nucleus tractus solitarii region. Nevertheless, this effect did not appear to be correlated with a reduction in blood pressure and it was not attenuated by a prior central injection of idazoxan (5 nmol/kg intracisternally), which almost completely prevented the hypotensive and the neuronal inhibitory effects of clonidine in the nucleus reticularis lateralis region of the normotensive rat. CONCLUSION Both the site and the mechanism of the central hypotensive action of clonidine in the SHR appear to be different from those in normotensive control WKY rats.

Cardiac Muscarinic Receptor Overexpression in Sudden Infant Death Syndrome

Background Sudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated. Methodology/Principal Findings Left ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [3H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled Bmax value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls). Conclusions In the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism.

Methylation of imidazoline related compounds leads to loss of α2-adrenoceptor affinity. Synthesis and biological evaluation of selective I1 imidazoline receptor ligands

Methylated analogues of imidazoline related compounds (IRC) were prepared; their abilities to bind I(1) imidazoline receptors (I(1)Rs), I(2) imidazoline binding sites (I(2)BS) and α(2)-adrenoceptor subtypes (α(2)ARs) were assessed. Methylation of the heterocyclic moiety of IRC resulted in a significant loss of α(2)AR affinity. Amongst the selective ligands obtained, LNP 630 (4) constitutes the first highly selective I(1)R agent showing hypotensive activity after intravenous administration.

A New Pyrroline Compound Selective for I1-Imidazoline Receptors Improves Metabolic Syndrome in Rats

Symptoms of the metabolic syndrome (MetS), such as insulin resistance, obesity, and hypertension, have been associated with sympathetic hyperactivity. In addition, the adiponectin pathway has interesting therapeutic potentials in MetS. Our purpose was to investigate how targeting both the sympathetic nervous system and the adipose tissue (adiponectin secretion) with a drug selective for nonadrenergic I1-imidazoline receptors (I1Rs) may represent a new concept in MetS pharmacotherapy. LNP599 [3-chloro-2-methyl-phenyl)-(4-methyl-4,5-dihydro-3H-pyrrol-2-yl)-amine hydrochloride], a new pyrroline derivative, displaced the specific [125I]para-iodoclonidine binding to I1R with nanomolar affinity and had no significant affinity for a large set of receptors, transporters, and enzymes. In addition, it can cross the blood-brain barrier and has good intestinal absorption, permitting oral as well as intravenous delivery. The presence of I1Rs was demonstrated in 3T3-L1 adipocytes; LNP599 had a specific stimulatory action on adiponectin secretion in adipocytes. Short-term administration of LNP599 (10 mg/kg i.v.) in anesthetized Sprague-Dawley rats markedly decreased sympathetic activity, causing hypotension and bradycardia. Long-term treatment of spontaneously hypertensive heart failure rats with LNP599 (20 mg/kg PO) had favorable effects on blood pressure, body weight, insulin resistance, glucose tolerance, and lipid profile, and it increased plasma adiponectin. The pyrroline derivative, which inhibits sympathetic activity and stimulates adiponectin secretion, has beneficial effects on all the MetS abnormalities. The use of one single drug with both actions may constitute an innovative strategy for the management of MetS.

Central Sympathetic Modulation Reverses Microvascular Alterations in a Rat Model of High-Fat Diet-Induced Metabolic Syndrome.

The objective of this study was to investigate the role of the SNS on hemodynamic, metabolic, and microvascular alterations in a rat model of HFD‐induced MS with salt supplementation.

Expression of Circulating Muscarinic Receptors in Infants With Severe Idiopathic Life-Threatening Events.

Expression of Circulating Muscarinic Receptors in Infants With Severe Idiopathic Life-Threatening Events About half of the apparent life-threatening events (ALTEs) that occur among infants remain unexplained and are called idiopathic.1 Yet there is no biomarker associated with idiopathic ALTEs. Although the connection between ALTEs and sudden infant death syndrome (SIDS) remains controversial, the cholinergic system has been investigated in the pathogenesis of both.2-4 We investigate the M2 muscarinic receptors’ expression in the blood samples of infants who experienced severe ALTEs.

Imidazoline-like drugs improve insulin sensitivity through peripheral stimulation of adiponectin and AMPK pathways in a rat model of glucose intolerance

Altered adiponectin signaling and chronic sympathetic hyperactivity have both been proposed as key factors in the pathogenesis of metabolic syndrome. We recently reported that activation of I1 imidazoline receptors (I1R) improves several symptoms of the metabolic syndrome through sympathoinhibition and increases adiponectin plasma levels in a rat model of metabolic syndrome (Fellmann L, Regnault V, Greney H, et al. J Pharmacol Exp Ther 346: 370-380, 2013). The present study was designed to explore the peripheral component of the beneficial actions of I1R ligands (i.e., sympathoinhibitory independent effects). Aged rats displaying insulin resistance and glucose intolerance were treated with LNP509, a peripherally acting I1R agonist. Glucose tolerance, insulin sensitivity, and adiponectin signaling were assessed at the end of the treatment. Direct actions of the ligand on hepatocyte and adipocyte signaling were also studied. LNP509 reduced the area under the curve of the intravenous glucose tolerance test and enhanced insulin hypoglycemic action and intracellular signaling (Akt phosphorylation), indicating improved glucose tolerance and insulin sensitivity. LNP509 stimulated adiponectin secretion acting at I1R on adipocytes, resulting in increased plasma levels of adiponectin; it also enhanced AMPK phosphorylation in hepatic tissues. Additionally, I1R activation on hepatocytes directly enhanced AMPK phosphorylation. To conclude, I1R ligands can improve insulin sensitivity acting peripherally, independently of sympathoinhibition; stimulation of adiponectin and AMPK pathways at insulin target tissues may account for this effect. This may open a promising new way for the treatment of the metabolic syndrome.

[PP.12.01] SYMPATHETIC INHIBITION IMPROVES INFLAMMATION AND ENDOTHELIAL DYSFUNCTION IN BRAIN MICROCIRCULATION OF RATS WITH METABOLIC SYNDROME

Objective: We aimed to investigate the effects of a chronic oral treatment using centrally-acting sympatho-inhibitory drugs, clonidine (CLO) or a selective I1 imidazoline agonist LNP599 (LNP) on the brain microcirculation of rats under long-term high-fat diet (HFD). Design and method: Male Wistar rats were maintained under normal diet (CON, n = 10) or high-fat diet (HFD, n = 30) during 20 weeks. Thereafter, the HFD group received oral CLO (0.1 mg/kg), LNP (20 mg/kg) or vehicle. Systolic blood pressure (SBP) was evaluated by photo-plethysmography during the long-term treatment, cerebral microcirculation flow was evaluated by Laser Speckle contrast Imaging and the brain functional capillary density, endothelial function and endothelial-leukocyte interactions were evaluated by intravital microscopy. Brain oxidative stress was evaluated by gene expressions of endothelial NOS (e-NOS) and NADPH oxidase using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique in brain samples of HFD animals compared to CON group. All experimental procedures were conducted in accordance with the internationally accepted principles for the Care and Use of Laboratory Animals and were approved by the Oswaldo Cruz Foundation Animal Welfare Committee (CEUA license #LW31/11) compared to CON group. Results: HFD group presented a decreased blood perfusion in the brain (163 ± 25, arbitrary perfusion units, APU) compared to CON group (239 ± 14 APU). Chronic sympathetic inhibition increased cerebral blood flow (CLO:211 ± 33APU and LNP:226 ± 22APU vs HFD group; p < 0.05) and the vasodilation response to Ach. CLO and LNP treatment reversed the brain capillary rarefaction (326 ± 51 and 378 ± 8 vs HFD:117 ± 11 capillaries/mm2) and decreased 3 fold the cerebral rolling leukocytes observed in pial venules. Decreases in gene expressions of NADPH oxidase and increases of eNOS in the brain of HFD treated groups suggest a reduction in oxidative stress and improvement of endothelial function induced by sympathetic inhibition. Conclusions: These results suggest that the modulation of sympathetic activity results in an improvement of brain perfusion, capillary rarefaction and microvascular endothelial dysfunction and oxidative stress in a diet-induced rat model of metabolic syndrome.