Manipulating the sequence of food ingestion improves glycemic control in type 2 diabetic patients under free-living conditions.

Lipid and protein ingested before carbohydrate reduce postprandial hyperglycemia. We tested feasibility, safety and clinical efficacy of manipulating the sequence of nutrient ingestion in patients with type 2 diabetes (T2D). After a 4-week run-in, 17 T2D patients were randomized to either a control diet (CD) or to an experimental diet (ED) allowing the consumption of high-carbohydrate foods only after high-protein and high-fat foods at each main meal (lunch+dinner). Both diets were accurately followed and neutral on arterial blood pressure, plasma lipids and indices of hepatic and kidney function. After 8 weeks, in spite of a similar reduction of body weight (ED -1.9 95% confidence interval (-3.4/-0.4)kg, P<0.03; CD -2.0 (-3.6/-0.5)kg, P<0.02) and waist circumference (ED -2.9 (-4.3/-1.5)cm, P<0.002; CD -3.3 (-5.9/-0.7)cm, P<0.02), the ED only was associated with significant reductions of HbA1c (-0.3 (-0.50/-0.02)%, P<0.04), fasting plasma glucose (-1.0 (-1.8/-0.3)mmol l(-1), P<0.01), postprandial glucose excursions (lunch -1.8 (-3.2/-0.4)mmol l(-1), P<0.01; dinner: -1.0 (-1.9/-0.1)mmol l(-1), P<0.04) and other indices of glucose variability (s.d.: -0.5 (-0.7/-0.2)mmol l(-1), P<0.02; Coefficient of variation: -6.6 (-10.4/-2.7)%, P<0.02). When compared with the CD, the ED was associated with lower post-lunch glucose excursions (P<0.02) and lower glucose coefficients of variation (P<0.05). Manipulating the sequence of nutrient ingestion might reveal a rapid, feasible, economic and safe strategy for optimizing glucose control in T2D.

Sustained effects of a protein and lipid preload on glucose tolerance in type 2 diabetes patients.

BACKGROUND: Small amounts of nutrients given as a 'preload' can reduce post-meal hyperglycaemic peaks in type 2 diabetes (T2D) patients by activating a number of mechanisms involved in glucose homoeostasis. This study was undertaken to ascertain whether this positive effect extends to the late absorptive phase and to identify the main mechanisms involved. MATERIAL AND METHODS: Eight well-controlled T2D patients, aged 40-70 years, were randomized to consume a 'preload' of either water or non-glucidic nutrients (50g of Parmesan cheese, one boiled egg) 30min before a 300-min oral glucose tolerance test. RESULTS: After the nutrient preload, significant reductions were observed in peak glucose (-49%; P<0.02), total plasma glucose (iAUC: -28%; P<0.03), exogenous glucose (iAUC: -30%; P<0.03) and insulin clearance (-28%; P<0.04), with enhancement of insulin secretion (iAUC: +22%; P<0.003). These effects were associated with higher plasma levels of GLP-1 (iAUC: +463%; P<0.002), GIP (iAUC: +152%; P<0.0003) and glucagon (iAUC: +144%; P<0.0002). CONCLUSION: In T2D patients, a protein and lipid preload improves glucose tolerance throughout the whole post-absorptive phase mainly by reducing the appearance of oral glucose, and improving both beta-cell function and insulin bioavailability.

The innovative "Bio-Oil Spread" prevents metabolic disorders and mediates preconditioning-like cardioprotection in rats.

BACKGROUND AND AIMS: Obesity is often associated with an increased cardiovascular risk. The food industry and the associated research activities focus on formulating products that are a perfect mix between an adequate fat content and health. We evaluated whether a diet enriched with Bio-Oil Spread (SD), an olive oil-based innovative food, is cardioprotective in the presence of high-fat diet (HFD)-dependent obesity. METHODS AND RESULTS: Rats were fed for 16 weeks with normolipidic diet (ND; fat: 6.2%), HFD (fat: 42%), and ND enriched with SD (6.2% of fat + 35.8% of SD). Metabolic and anthropometric parameters were measured. Heart and liver structures were analyzed by histochemical examination. Ischemic susceptibility was evaluated on isolated and Langendorff-perfused cardiac preparations. Signaling was assessed by Western blotting. Compared to ND rats, HFD rats showed increased body weight and abdominal obesity, dyslipidemia, and impaired glucose tolerance. Morphological analyses showed that HFD is associated with heart and liver modifications (hypertrophy and steatosis, respectively), lesser evident in the SD group, together with metabolic and anthropometric alterations. In particular, IGF-1R immunodetection revealed a reduction of hypertrophy in SD heart sections. Notably, SD diet significantly reduced myocardial susceptibility against ischemia/reperfusion (I/R) with respect to HFD through the activation of survival signals (Akt, ERK1/2, and Bcl2). Systolic and diastolic performance was preserved in the SD group. CONCLUSIONS: We suggest that SD may contribute to the prevention of metabolic disorders and cardiovascular alterations typical of severe obesity induced by an HFD, including the increased ischemic susceptibility of the myocardium. Our results pave the way to evaluate the introduction of SD in human alimentary guidelines as a strategy to reduce saturated fat intake.

The NO stimulator, Catestatin, improves the Frank-Starling response in normotensive and hypertensive rat hearts.

The myocardial response to mechanical stretch (Frank-Starling law) is an important physiological cardiac determinant. Modulated by many endogenous substances, it is impaired in the presence of cardiovascular pathologies and during senescence. Catestatin (CST:hCgA352-372), a 21-amino-acid derivate of Chromogranin A (CgA), displays hypotensive/vasodilatory properties and counteracts excessive systemic and/or intra-cardiac excitatory stimuli (e.g., catecholamines and endothelin-1). CST, produced also by the myocardium, affects the heart by modulating inotropy, lusitropy and the coronary tone through a Nitric Oxide (NO)-dependent mechanism. This study evaluated the putative influence elicited by CST on the Frank-Starling response of normotensive Wistar-Kyoto (WKY) and hypertensive (SHR) hearts by using isolated and Langendorff perfused cardiac preparations. Functional changes were evaluated on aged (18-month-old) WKY rats and SHR which mimic human chronic heart failure (HF). Comparison to WKY rats, SHR showed a reduced Frank-Starling response. In both rat strains, CST administration improved myocardial mechanical response to increased end-diastolic pressures. This effect was mediated by EE/IP3K/NOS/NO/cGMP/PKG, as revealed by specific inhibitors. CST-dependent positive Frank-Starling response is paralleled by an increment in protein S-Nitrosylation. Our data suggested CST as a NO-dependent physiological modulator of the stretch-induced intrinsic regulation of the heart. This may be of particular importance in the aged hypertrophic heart, whose function is impaired because of a reduced systolic performance accompanied by delayed relaxation and increased diastolic stiffness.

Nesfatin-1 as a novel cardiac peptide: identification, functional characterization, and protection against ischemia/reperfusion injury.

Nesfatin-1 is an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide. It controls feeding behavior, water intake, and glucose homeostasis. If intracerebrally administered, it induces hypertension, thus suggesting a role in central cardiovascular control. However, it is not known whether it is able to directly control heart performance. We aimed to verify the hypothesis that, as in the case of other hypothalamic satiety peptides, Nesfatin-1 acts as a peripheral cardiac modulator. By western blotting and QT-PCR, we identified the presence of both Nesfatin-1 protein and NUCB2 mRNA in rat cardiac extracts. On isolated and Langendorff-perfused rat heart preparations, we found that exogenous Nesfatin-1 depresses contractility and relaxation without affecting coronary motility. These effects did not involve Nitric oxide, but recruited the particulate guanylate cyclase (pGC) known as natriuretic peptide receptor A (NPR-A), protein kinase G (PKG) and extracellular signal-regulated kinases1/2 (ERK1/2). Co-immunoprecipitation and bioinformatic analyses supported an interaction between Nesfatin-1 and NPR-A. Lastly, we preliminarily observed, through post-conditioning experiments, that Nesfatin-1 protects against ischemia/reperfusion (I/R) injury by reducing infarct size, lactate dehydrogenase release, and postischemic contracture. This protection involves multiple prosurvival kinases such as PKCε, ERK1/2, signal transducer and activator of transcription 3, and mitochondrial K(ATP) channels. It also ameliorates contractility recovery. Our data indicate that: (1) the heart expresses Nesfatin-1, (2) Nesfatin-1 directly affects myocardial performance, possibly involving pGC-linked NPR-A, the pGC/PKG pathway, and ERK1/2, (3) the peptide protects the heart against I/R injury. Results pave the way to include Nesfatin-1 in the neuroendocrine modulators of the cardiac function, also encouraging the clarification of its clinical potential in the presence of nutrition-dependent physio-pathologic cardiovascular diseases.

Receptor identification and physiological characterisation of glucagon-like peptide-2 in the rat heart.

BACKGROUND AND AIMS: The anorexigenic glucagon-like peptide (GLP)-2 is produced by intestinal L cells and released in response to food intake. It affects intestinal function involving G-protein-coupled receptors. To verify whether GLP-2 acts as a cardiac modulator in mammals, we analysed, in the rat heart, the expression of GLP-2 receptors and the myocardial and coronary responses to GLP-2. METHODS AND RESULTS: GLP-2 receptors were detected on ventricular extracts by quantitative real-time polymerase chain reaction (Q-RT-PCR) and Western blotting. Cardiac GLP-2 effects were analysed on Langendorff perfused hearts. Intracellular GLP-2 signalling was investigated on Langendorff perfused hearts and by Western blotting and enzyme-linked immunosorbent assay (ELISA) on ventricular extracts. By immunoblotting and Q-RT-PCR, we revealed the expression of ventricular GLP-2 receptors. Perfusion analyses showed that GLP-2 induces positive inotropism at low concentration (10-12 mol l(-1)), and negative inotropism and lusitropism from 10 to 10 mol l(-1). It dose-dependently constricts coronaries. The negative effects of GLP-2 were independent from GLP-1 receptors, being unaffected by exendin-3 (9-39) amide. GLP-2-dependent negative action involves Gi/o proteins, associates with a reduction of intracellular cyclic adenosine monophosphate (cAMP), an increase in extracellular signal regulated kinases 1 and 2 (ERK1/2) and a decrease in phospholamban phosphorylation, but is independent from endothelial nitric oxide synthase (eNOS) and protein kinase G (PKG). Finally, GLP-2 competitively antagonised ß-adrenergic stimulation. CONCLUSIONS: For the first time, to our knowledge, we found that: (1) the rat heart expresses functional GLP-2 receptors; (2) GLP-2 acts on both myocardium and coronaries, negatively modulating both basal and ß-adrenergic stimulated cardiac performance; and (3) GLP-2 effects are mediated by G-proteins and involve ERK1/2.

Distinct signalling mechanisms are involved in the dissimilar myocardial and coronary effects elicited by quercetin and myricetin, two red wine flavonols.

BACKGROUND AND AIMS: Moderate red wine consumption associates with lower incidence of cardiovascular diseases. Attention to the source of this cardioprotection was focused on flavonoids, the non-alcoholic component of the red wine, whose intake inversely correlates with adverse cardiovascular events. We analysed whether two red wine flavonoids, quercetin and myricetin, affect mammalian basal myocardial and coronary function. METHODS AND RESULTS: Quercetin and myricetin effects were evaluated on isolated and Langendorff perfused rat hearts under both basal conditions and α- and ß-adrenergic stimulation. The intracellular signalling involved in the effects of these flavonoids was analysed on perfused hearts and by western blotting on cardiac and HUVEC extracts. Quercetin induced biphasic inotropic and lusitropic effects, positive at lower concentrations and negative at higher concentrations. Contrarily, Myricetin elicits coronary dilation, without affecting contractility and relaxation. Simultaneous administration of the two flavonoids only induced vasodilation. Quercetin-elicited positive inotropism and lusitropism depend on ß1/ß2-adrenergic receptors and associate with increased intracellular cAMP, while the negative inotropism and lusitropism observed at higher concentrations were α-adrenergic-dependent. NOS inhibition abolished Myricetin-elicited vasodilation, also inducing Akt, ERK1/2 and eNOS phosphorylation in both ventricles and HUVEC. Myricetin-dependent vasodilation increases intracellular cGMP and is abolished by triton X-100. CONCLUSIONS: The cardiomodulation elicited on basal mechanical performance by quercetin and the selective vasodilation induced by myricetin point to these flavonoids as potent cardioactive principles, able to protect the heart in the presence of cardiovascular diseases.

A new membrane G protein-coupled receptor (GPR30) is involved in the cardiac effects of 17beta-estradiol in the male rat.

In the present study, we evaluated the transduction pathways involved in the cardiac effects elicited by 17beta-estradiol (E2) on the isolated, Langendorff perfused male Wistar rat heart. E2 and selective agonists for ERalpha and ERbeta induced a dose-dependent reduction of contractility which was blocked by the ER inhibitor ICI 182,780. Moreover, the potential involvement of the novel membrane estrogen receptor GPR30 in mediating estrogen activity was determined using the selective GPR30 ligand G-1. Notably, specific inhibitors of ERK, PI3K, PKA, and eNOS transduction pathways abolished the cardiac responses to E(2). Taken together, our data suggest that ERalpha and ERbeta along with several signaling cascades are involved in the action of E(2) on the male rat heart. Our results also point to a potential role of GPR30, however further evaluation is required in order to fully understand the contribution of the different estrogen receptors in mediating estrogen activity on cardiac performance.

The homologous rat chromogranin A1-64 (rCGA1-64) modulates myocardial and coronary function in rat heart to counteract adrenergic stimulation indirectly via endothelium-derived nitric oxide.

Chromogranin A (CGA), produced by human and rat myocardium, generates several biologically active peptides processed at specific proteolytic cleavage sites. A highly conserved cleavage N-terminal site is the bond 64-65 that reproduces the native rat CGA sequence (rCGA1-64), corresponding to human N-terminal CGA-derived vasostatin-1. rCGA1-64 cardiotropic activity has been explored in rat cardiac preparations. In Langendorff perfused rat heart, rCGA1-64 (from 33 nM) induced negative inotropism and lusitropism as well as coronary dilation, counteracting isoproterenol (Iso) - and endothelin-1 (ET-1) -induced positive inotropic effects and ET-1-dependent coronary constriction. rCGA1-64 also depressed basal and Iso-induced contractility on rat papillary muscles, without affecting calcium transients on isolated ventricular cells. Structure-function analysis using three modified peptides on both rat heart and papillary muscles revealed the disulfide bridge requirement for the cardiotropic action. A decline in Iso intrinsic activity in the presence of the peptides indicates a noncompetitive antagonistic action. Experiments on rat isolated cardiomyocytes and bovine aortic endothelial cells indicate that the negative inotropism observed in rat papillary muscle is probably due to an endothelial phosphatidylinositol 3-kinase-dependent nitric oxide release, rather than to a direct action on cardiomyocytes. Taken together, our data strongly suggest that in the rat heart the homologous rCGA1-64 fragment exerts an autocrine/paracrine modulation of myocardial and coronary performance acting as stabilizer against intense excitatory stimuli.

Beta3-adrenoceptors modulate left ventricular relaxation in the rat heart via the NO-cGMP-PKG pathway.

AIMS: Using a model of isolated and Langendorff-perfused rat heart we analysed whether activation of beta3-adrenergic receptors (beta3-ARs) influences ventricular lusitropic performance. We also focused on the NOS/NO/cGMP/PKG cascade as the signal transduction mechanism. METHODS: Hearts were treated with increasing concentrations (from 10(-12) to 10(-6) m) of BRL(37344), a selective beta3-AR agonist, and cardiac performance was evaluated by analysing both lusitropic parameters and coronary motility. Cardiac preparations were also perfused with BRL(37344) in the presence of either isoproterenol (ISO) or nadolol, or pertussis toxin (PTx), or selective inhibitors of the NOS/NO/cGMP/PKG pathway. RESULTS: BRL(37344) caused a significant concentration-dependent reduction in (LVdP/dt)(min), a decrease in half time relaxation significant starting from 10(-12) m, and an increase in (LVdP/dt)(max)/(LVdP/dt)(min) ratio (T/-t). BRL(37344) abolished the ISO-mediated positive lusitropism. beta3-AR-dependent effects on relaxation were insensitive to beta(1)/beta2-AR inhibition by nadolol (100 nm), and were abolished by G(i/o) protein inhibition by PTx (0.01 nm). NO scavenging by haemoglobin (10 microm), and nitric oxide synthase (NOS) inhibition by NG-monomethyl-l-arginine (10 microm) revealed the involvement of NO signalling in BRL(37344) response. Pre-treatment with inhibitors of either soluble guanylate cyclase (ODQ; 10 microm) or PKG (KT(5823); 100 nm) abolished beta3-AR-dependent negative lusitropism. In contrast, anantin (10 nm), an inhibitor of particulate guanylate cyclase, did not modify the effect of BRL(37344) on relaxation. CONCLUSION: Taken together, our findings provide functional evidence for beta3-AR modulation of ventricular relaxation in the rat heart which involves PTx-sensitive inhibitory Gi protein and occurs via an NO-cGMP-PKG cascade. Whether the effects of beta3-AR stimulation on lusitropism are beneficial or detrimental remains to be established.

[Diltiazem in paroxysmal supraventricular tachycardia: electrocardiographic findings at termination]. / Diltiazem nella tachicardia parossistica sopraventricolare: quadri elettrocardiografici alla interruzione.

The effects of diltiazem hydrochloride (0.3 mg/kg i.v. over 2 min.) was studied by continuous electrocardiographic monitoring in 60 patients. Conversion to sinus rhythm was achieved in 55 patients (91%). Electrocardiographic findings were: undisturbed sinus rhythm in 20 patients; A-V junctional rhythm in 4 patients; complex ventricular arrhythmias (ventricular tachycardia or complex VPCs) in 19 patients. Patients with complex ventricular arrhythmias were matched against patients with normal sinus rhythm, with respect to the following parameters: age, sex, heart disease, tachycardia duration, tachycardia cycle length, sinus cycle length, pre- and post-infusion blood pressure. No differences between the two groups of patients were found. Ventricular arrhythmias occurring at the termination of supraventricular tachycardia are difficult to explain. Nevertheless, these arrhythmias are not associated with organic heart disease. They could be the expression of triggered activity.