Methyl donor deficiency impairs fatty acid oxidation through PGC-1α hypomethylation and decreased ER-α, ERR-α, and HNF-4α in the rat liver.

BACKGROUND & AIMS: Folate and cobalamin are methyl donors needed for the synthesis of methionine, which is the precursor of S-adenosylmethionine, the substrate of methylation in epigenetic, and epigenomic pathways. Methyl donor deficiency produces liver steatosis and predisposes to metabolic syndrome. Whether impaired fatty acid oxidation contributes to this steatosis remains unknown. METHODS: We evaluated the consequences of methyl donor deficient diet in liver of pups from dams subjected to deficiency during gestation and lactation. RESULTS: The deprived rats had microvesicular steatosis, with increased triglycerides, decreased methionine synthase activity, S-adenosylmethionine, and S-adenosylmethionine/S-adenosylhomocysteine ratio. We observed no change in apoptosis markers, oxidant and reticulum stresses, and carnityl-palmitoyl transferase 1 activity, and a decreased expression of SREBP-1c. Impaired beta-oxidation of fatty acids and carnitine deficit were the predominant changes, with decreased free and total carnitines, increased C14:1/C16 acylcarnitine ratio, decrease of oxidation rate of palmitoyl-CoA and palmitoyl-L-carnitine and decrease of expression of novel organic cation transporter 1, acylCoA-dehydrogenase and trifunctional enzyme subunit alpha and decreased activity of complexes I and II. These changes were related to lower protein expression of ER-α, ERR-α and HNF-4α, and hypomethylation of PGC-1α co-activator that reduced its binding with PPAR-α, ERR-α, and HNF-4α. CONCLUSIONS: The liver steatosis resulted predominantly from hypomethylation of PGC1-α, decreased binding with its partners and subsequent impaired mitochondrial fatty acid oxidation. This link between methyl donor deficiency and epigenomic deregulations of energy metabolism opens new insights into the pathogenesis of fatty liver disease, in particular, in relation to the fetal programming hypothesis.

Tissue kallikrein deficiency aggravates cardiac remodelling and decreases survival after myocardial infarction in mice.

BACKGROUND: Tissue kallikrein (TK) is a major kinin-releasing enzyme present in arteries. TK is involved in cardioprotection in the setting of acute myocardial ischaemia but its role in post-ischaemic heart failure (HF), a major cause of delayed mortality after myocardial infarction (MI), is unknown. AIM: To determine whether TK deficiency in the mouse influences survival and cardiac remodelling after MI. METHODS: MI was induced in 10 week-old male TK-deficient mice and wild-type littermates. Survival was assessed up to 14 months. Cardiac morphological and functional parameters were serially measured by echocardiography. In another experiment, myocardial capillary density and NOS content were evaluated at 3 months. RESULTS: Infarct size was similar in both genotypes. MI resulted in severe cardiac dysfunction. Up to 12 months after MI, TK(-/-) mice displayed an increased mortality rate (P<0.05, relative risk of death=3.41) and aggravation of left ventricular hypertrophy and dilatation by comparison with TK(+/+) (+18% and +27% respectively, both P<0.05). NOS1 and NOS3 were abnormally regulated in the heart of TK(-/-) mice after MI. CONCLUSIONS: TK exerts a protective role in HF in mice. Coronary effects are probably involved. As partial genetic deficiency in TK activity occurs in humans, TK-deficient subjects may be at increased risk of mortality in HF.

Methylglyoxal impairs the insulin signaling pathways independently of the formation of intracellular reactive oxygen species.

Nonenzymatic glycation is increased in diabetes and leads to elevated levels of advanced glycation end products (AGEs), which link hyperglycemia to the induction of insulin resistance. In hyperglycemic conditions, intracellularly formed alpha-ketoaldehydes, such as methylglyoxal, are an essential source of intracellular AGEs, and the abnormal accumulation of methylglyoxal is related to the development of diabetes complications in various tissues and organs. We have previously shown in skeletal muscle that AGEs induce insulin resistance at the level of metabolic responses. Therefore, it was important to extend our work to intermediates of the biosynthetic pathway leading to AGEs. Hence, we asked the question whether the reactive alpha-ketoaldehyde methylglyoxal has deleterious effects on insulin action similar to AGEs. We analyzed the impact of methylglyoxal on insulin-induced signaling in L6 muscle cells. We demonstrate that a short exposure to methylglyoxal induces an inhibition of insulin-stimulated phosphorylation of protein kinase B and extracellular-regulated kinase 1/2, without affecting insulin receptor tyrosine phosphorylation. Importantly, these deleterious effects of methylglyoxal are independent of reactive oxygen species produced by methylglyoxal but appear to be the direct consequence of an impairment of insulin-induced insulin receptor substrate-1 tyrosine phosphorylation subsequent to the binding of methylglyoxal to these proteins. Our data suggest that an increase in intracellular methylglyoxal content hampers a key molecule, thereby leading to inhibition of insulin-induced signaling. By such a mechanism, methylglyoxal may not only induce the debilitating complications of diabetes but may also contribute to the pathophysiology of diabetes in general.

Glucose intolerance and hypoadiponectinemia are already present in lean patients with chronic hepatitis C infected with genotype non-3 viruses.

OBJECTIVES: Steatosis and metabolic abnormalities seem to be frequent and deleterious in chronic hepatitis C. Changes in glucose homeostasis and in adiponectin levels, an adipokine with anti-inflammatory and insulin-sensitive properties, were evaluated in patients with chronic hepatitis C according to steatosis, liver fibrosis and body mass index. METHODS: Seventy-three patients with chronic hepatitis C (40 men, 33 women) infected with genotypes non-3 and 22 healthy controls (11 men and 11 women) were included in the study and all had a biochemical evaluation, including metabolic parameters, adiponectin measurement, and a liver biopsy. Insulin sensitivity was assessed with the HOMA 1-IR insulin resistance model. RESULTS: Steatosis was found in 65.7% of the patients and significant fibrosis (METAVIR F2-F4) was present in 28.7%. The presence of steatosis could only be predicted by fibrosis, whereas significant fibrosis could be predicted by steatosis and age. Adiponectin levels were significantly decreased (-32%) with the severity of the steatosis. Although overweight chronic hepatitis C patients (body mass index>or=25 kg/m2) had insulin resistance and hypoadiponectinemia, lean chronic hepatitis C patients (body mass index<25 kg/m2) had already significantly higher glycemia and lower adiponectin levels than in controls. CONCLUSIONS: This study confirms the high incidence of steatosis in patients infected by hepatitis C virus genotypes non-3, well linked to the development of fibrosis and metabolic abnormalities. Importantly, the present findings put emphasis on the early development of these metabolic abnormalities as they were already found in lean patients with chronic hepatitis C. The direct implication of hepatitis C virus is thus further stressed in the development of steatosis and insulin resistance, with or without involvement of host factors.

Effect of caspase inhibition on thymic apoptosis in hemorrhagic shock.

In hemorrhagic shock (HS) an increased thymic apoptosis (TA) was described. The aim of this study was to evaluate the effect of administration of the caspase inhibitor N-benzyloxy-carbonil-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) during the resuscitation phase on TA, organ dysfunctions, and tumor necrosis factor (TNF)-alpha release in HS. Forty rats were randomly assigned to four groups: no HS/resuscitation (sham); HS/resuscitation with shed blood and normal saline (control); HS/resuscitation with shed blood and phosphate-buffered solution (PBS) (vehicle); and HS/resuscitation with shed blood and Z-VAD-FMK (inhibitor). Rats were subjected to HS by blood removal to a MAP of 35-40 mmHg. After a 1-h shock period, the animals were resuscitated according to the protocol. At 1 and 3 h after resuscitation, transaminases, creatinine, urea, lipase, TNF-alpha, and TA were evaluated. Our study showed that a nonlethal HS is early able to induce organ dysfunctions and increased TA. Administration of Z-VAD-FMK did not significantly decrease organ dysfunctions, while it induced a significant TNF-alpha release. TA was significantly reduced by Z-VAD-FMK after 1 h, but not after 3 h. Our results suggest that postinjury caspase inhibition does not attenuate organ dysfunctions, and also does not permanently reduce TA induced by HS and resuscitation in rats.

Rat liver injury after normothermic ischemia is prevented by a phosphinic matrix metalloproteinase inhibitor.

Hepatic ischemia occurs in liver transplantation, hemodynamic or cardiogenic shock, and liver resection associated with trauma or tumor. Ischemia/reperfusion (I/R) injury results in microcirculation failure followed by apoptosis and necrosis. Matrix metalloproteinases (MMPs) are involved in many physiological and pathological processes, but their expression and function during liver I/R remains poorly documented. In this study, we evaluated the expression of nine MMPs and their natural inhibitors, tissue inhibitors of MMPs (TIMPs), in a rat model of liver I/R. Analysis of MMP and TIMP expression show that although most of these genes are not constitutively expressed in the normal liver, they are induced in a specific time-dependent manner following I/R. Stromelysin-1, gelatinase B, and collagenase-3 are induced during the early phase of acute liver injury associated with inflammation and increased necrosis/apoptosis, whereas gelatinase A, membrane type-MMP, stromelysin-3, metalloelastase, TIMP-1, and TIMP-2 are essentially detectable during the recovery phase of liver injury corresponding to hepatocyte regeneration. This observation suggested that MMPs and TIMPs could play both deleterious and beneficial roles following I/R. We thus tested the effect of a specific phosphinic MMP inhibitor on acute liver I/R injury. Inhibition of MMP activity was shown to significantly decrease liver injury in ischemic/reperfused liver tissue as assessed by histological studies and serum hepatic enzyme levels. We therefore propose that MMP inhibitors may be of clinical relevance in liver-associated ischemic diseases or after liver transplantation.

Fluvastatin prevents renal dysfunction and vascular NO deficit in apolipoprotein E-deficient mice.

OBJECTIVE: The objective of this study was to investigate the effects of fluvastatin on atherosclerosis, systemic and regional hemodynamics, and vascular reactivity in apolipoprotein E-deficient (ApoE(-/-)) mice. METHODS AND RESULTS: Hemodynamics (fluospheres) and vasomotor responses of thoracic aorta and carotid artery were evaluated in male wild-type (WT) and untreated (ApoE(-/-) Control) or fluvastatin-treated (50 mg/kg per day for 20 weeks) ApoE(-/-) mice, all fed a Western-type diet. Plasma cholesterol and aortic root atherosclerotic lesions (ALs) were greater in ApoE(-/-) Control mice (19+/-1 mmol/L and 63,0176+/-38,785 micro m(2), respectively) than in WT mice (2+/-1 mmol/L and 1+/-1 micro m(2), respectively, P<0.01). Fluvastatin significantly decreased plasma cholesterol (-53%) but failed to limit ALs. Renal blood flow was significantly reduced in ApoE(-/-) Control versus WT (-25%, P<0.05) mice. This reduction was prevented by fluvastatin. Aortic and carotid endothelium-dependent relaxations to acetylcholine were not altered in ApoE(-/-) Control versus WT mice. In carotid arteries from WT mice, these responses were abolished after nitro-L-arginine (L-NA), whereas those from ApoE(-/-) Control were only partially inhibited after L-NA but fully abolished after L-NA+diclofenac. Thus, in carotid arteries from ApoE(-/-) mice, vasodilating prostanoids compensate the deficit in NO availability. Fluvastatin prevented this carotid NO deficit. CONCLUSIONS: In ApoE(-/-) mice, chronic fluvastatin treatment preserved renal perfusion and vascular NO availability independently from atherosclerotic lesion prevention.

Role of microvascular rarefaction in the increased arterial pressure in mice lacking for the endothelial nitric oxide synthase gene (eNOS3pt-/- ).

OBJECTIVES: Mechanisms involved in hypertension in homozygous mice for the defective endothelial nitric oxide synthase gene (eNOS-/-) have not been fully elucidated. As NO is a potent vasodilator agent and possibly promotes angiogenesis, we investigated whether vasoconstriction and/or microvascular rarefaction could explain hypertension in these mice. METHODS: Immunohistochemistry with mouse monoclonal smooth muscle alpha-actin antibody was used to detect arterioles, and quantification of arteriolar density was performed in the left ventricle and in the gracilis muscle of 12-week-old male eNOS+/+ and eNOS-/- mice. Haemodynamic parameters - mean arterial pressure (MAP), cardiac index (CI), total peripheral résistance (TPR), myocardial blood flow, muscular blood flow and corresponding resistances - were measured or calculated using the fluorescent microsphere method in basal conditions and after infusion of sodium nitroprusside (SNP) (5 to 150 microg/kg per min) in eNOS-/- mice, compared with eNOS+/+ mice. RESULTS: We evidenced a significant decrease in arteriolar density in the heart (-16%, P < 0.02) and in the gracilis muscle (-22%, P < 0.05) in eNOS-/- mice. In basal conditions, eNOS-/- mice developed significant hypertension (MAP = 127 +/- 14 versus 77 +/- 14 mmHg, P < 0.001) associated with decreased CI (-29%, P < 0.001) and increased TPR (+ 125%, P < 0.001). Coronary and gracilis muscular resistances were increased (by 75 and 89% respectively, P < 0.001) compared with eNOS+/+ mice, whereas myocardial and skeletal muscle tissue blood flows were not affected. After SNP administration (10 microg/kg per min), a dose that did not significantly modify haemodynamic parameters in eNOS+/+ mice, MAP, TPR and regional resistances were normalized in eNOS-/- mice, showing that vasodilation may correct hypertension in eNOS-/- mice. However, under maximal vasodilating conditions, TPR and regional resistances remained significantly higher in eNOS-/- mice than those of eNOS+/+ mice. CONCLUSION: Anatomical and functional results show that both vasoconstriction and arteriolar rarefaction are involved in hypertension of eNOS-/- mice. Indeed, under maximal vasodilation, arterial pressure and TPR remained significantly higher in eNOS-/- mice than in eNOS+/+ mice, evidencing a major role of microvascular rarefaction in this model of hypertension.

Haemodynamic effects of dual blockade of the renin-angiotensin system in spontaneously hypertensive rats: influence of salt.

OBJECTIVE: To elucidate the mechanisms responsible for the adverse renal effects induced by dual blockade of the renin-angiotensin system (RAS) and the role of salt therein. METHODS: The effects of enalapril, losartan and their combination on blood pressure, renal haemodynamics, renal function and RAS were investigated over a wide range of doses in spontaneously hypertensive rats fed either a low-sodium or a high-sodium diet. RESULTS: In rats fed the low-sodium diet, the losartan-enalapril combination induced the same dose-dependent haemodynamic and hormonal changes as did three- to 10-fold greater doses of enalapril or losartan alone. When a strong decrease (> 50%) in blood pressure was achieved (with 10 mg/kg enalapril plus 10 mg/kg losartan, 100 mg/kg enalapril or 100 mg/kg losartan), a massive renal vasoplegia occurred and renal insufficiency developed. In addition, because of the huge release of renin, angiotensinogen concentrations were reduced, leading to a decrease in intrarenal angiotensins. In rats fed the high-sodium diet, those treated with the enalapril 30 mg/kg plus losartan 30 mg/kg combination, despite complete functional RAS blockade, exhibited smaller decreases in blood pressure and renal resistance, lesser release of renin and angiotensinogen consumption, and a normal renal function. These effects were similar to those produced by 100 mg/kg of enalapril or losartan in rats fed the high-salt diet, or by 10 mg/kg of enalapril or of losartan in rats fed the low-salt diet. CONCLUSIONS: Dual RAS blockade could be either beneficial, when sodium intake is unrestricted, or dangerous, when sodium intake is restricted.

Reperfusion duration paradox with late myocardial preconditioning in rabbits.

In this study, pharmacological late preconditioning was induced in 53 rabbits with an adenosine A(1) receptor agonist (2-chloro-N(6)-cyclopentyladenosine, CCPA, 100 microg/kg), or a NO-donor (S-nitroso-N-acetyl-penicillamine, SNAP, 2.5 microg/kg/min; 75 min) vs. saline as control. Later, after 24 h, rabbits underwent a 30-min coronary occlusion and subsequent reperfusion. After 3 h of coronary artery reperfusion, infarct size was reduced with CCPA (43+/-4%) and SNAP (27+/-4%) vs. saline (56+/-4%). However, after 72 h of coronary artery reperfusion, infarct sizes were similar in all groups, demonstrating an only transient effect of late preconditioning against myocardial infarction. Combined administration of CCPA and SNAP failed to induce sustained cardioprotection.

Is it important to correct apparent drug tissue concentrations for blood contamination in the dog?

The goal of this study was to quantify in the dog the error that is made in assessing drug tissue concentrations when no correction for blood contamination is performed and hence to determine in which organs such a correction should be made. The organs investigated were the heart, the brain, the liver and the skeletal muscle, and the test drug used was the H1-antihistamine, cetirizine (0.1 or 0.6 mg/kg/day for 3 days, orally, n = 6 dogs). Radiolabelled serum albumin was used to quantitate blood trapped in the tissues. Blood and tissue samplings were performed 2 h after the last drug administration. Mean (+/-SEM) volumes of blood trapped in the liver, heart, muscle and brain were 263 +/- 12, 91 +/- 3, 27 +/- 1 and 20 +/- 2 microL/g, respectively. Apparent tissue/blood concentration ratios of cetirizine were 2.39 +/- 0.33, 1.11 +/- 0.09, 0.77 +/- 0.07 and 0.37 +/- 0.05 in the four organs. When correction for residual blood is not performed, cetirizine concentrations are underestimated (-13.6 +/- 3.2%) in the liver, slightly overestimated (+4.7 +/- 1.5 to +6.3 +/- 2.8%) in the brain, and neither over nor underestimated in the heart and muscle. Simulation data over a wide range of theoretical drug tissue/blood concentration ratios indicate that in the dog: (a) for the liver, correction of apparent tissue concentration for residual blood should be performed when the drug tissue/blood concentration ratio achieved is <0.8 or >4, (b) for the heart, correction should be made when this ratio is < or =0.4 and (c) for the brain and muscle, no correction is necessary unless the ratio is < or =0.1.

Effects of age, malnutrition and refeeding on the expression and secretion of ghrelin.

BACKGROUND & AIMS: Anorexia is frequent in the malnourished elderly. We studied the effects of age, nutritional status and refeeding on the expression and secretion of the orexigenic peptide ghrelin. METHODS: Four groups were prospectively enrolled: 11 undernourished elderly (80+/-6 y, BMI: 17.4+/-1.9 [Mean+/-SD]), nine well-nourished elderly (76+/-9 y, 23.5+/-2.0), 10 undernourished young (26+/-6 y, 15.1+/-1.9) and 10 well-nourished young (34+/-8 y, 22.2+/-2.7). Fasting and postprandial plasma ghrelin and other hormones (every 30 min) were measured at baseline and after a 21-day enteral nutrition in malnourished patients. Gastric ghrelin mRNA levels were measured by RT-PCR at baseline in all subjects. RESULTS: Ghrelin was significantly higher in undernourished (2151+/-871 ng/L) than in well-nourished (943+/-389 ng/L) adults, whereas there were no differences between undernourished (1544+/-758 ng/L) and well-nourished (1154+/-541 ng/L) elderly. Refeeding did not influence ghrelin levels. Gastric ghrelin mRNA levels were similar in all groups. CONCLUSIONS: There is an absence of malnutrition-induced increase of plasma ghrelin levels in elderly subjects. This feature, post-transcriptional, may be important in the lack of adaptation of elderly subjects to malnutrition.

Effects of angiotensin II type 1 receptor blockade in ApoE-deficient mice with post-ischemic heart failure.

We investigated in mice whether atherosclerosis exacerbates the development of post-ischemic heart failure and alters the beneficial effects of long-term angiotensin II type 1 receptor blockade in this model. ApoE-deficient (ApoE(-/-)) and C57BL/6J (C57) mice with myocardial infarction (coronary ligation) received vehicle (C57 and ApoE(-/-)) or irbesartan (Ir, 50mg/kg/d orally, C57-Ir and ApoE(-/-)-Ir). Ten months post myocardial infarction, survival rates were similar in C57 (58%) and ApoE(62%). Atherosclerosis induced no significant alteration in blood pressure, cardiac output (fluospheres), total peripheral resistance, or shortening fraction (echocardiography) but increased renal resistance (+50%, P<0.05). Chronic Ir treatment significantly improved survival to a similar extent in both C57-Ir (85%) and ApoE(-/-)-Ir (86%). It also decreased blood pressure to a similar extent in both strains (-16% and -18%, both P<0.05). In C57-Ir mice, Ir did not modify cardiac output or total peripheral resistance, but it decreased renal resistance (-28%, P<0.001) and left-ventricular weight (-28%, P<0.05). In ApoE(-/-)-Ir mice, Ir limited atherosclerotic lesions (-13%, P<0.05), increased cardiac output (+28%, P<0.05) and shortening fraction (+24%, P<0.05), and decreased total peripheral resistance (-33%, P<0.01), renal resistance (-61%, P<0.001), and left-ventricular weight (-27%, P<0.001). In conclusion, atherosclerosis does not worsen heart failure development in mice and, although the beneficial cardiovascular effects of AT1 receptor blockade are greater in ApoE(-/-) than in C57, reduction in mortality is similar in both strains.

Heart rate reduction during exercise-induced myocardial ischaemia and stunning.

AIMS: The respective contributions of reduction in heart rate and inotropism in the beneficial effects of beta-blockade in ischaemic heart disease remains debated. The effects of selective heart rate reduction by ivabradine (If inhibitor) were compared to those of atenolol on exercise-induced ischaemia and stunning. METHODS AND RESULTS: In seven instrumented dogs, coronary stenosis was calibrated to suppress increase in coronary blood flow during a 10-min treadmill exercise. When administered before exercise, atenolol and ivabradine similarly reduced heart rate versus saline at rest and during exercise (154+/-2 and 155+/-9 vs 217+/-13 beats/min, respectively). During exercise, left ventricular wall thickening (LVWth) was reduced to 2+/-1% from 23+/-4% under saline but ivabradine limited this effect (10+/-3%) and reduced the subsequent myocardial stunning vs saline. Atenolol also limited LVWth decrease during exercise (17+/-4%) but had no effect during recovery. When administered after exercise, ivabradine attenuated stunning and this effect disappeared when heart rate reduction was corrected by atrial pacing. Atenolol administered after exercise severely depressed LVWth vs saline. CONCLUSION: Selective heart rate reduction not only provides an anti-ischaemic effect but also per se improves contractility of the stunned myocardium. Additional negative inotropism is protective against ischaemia but deleterious during stunning.

Adenosine A(1)-receptor induced late preconditioning and myocardial infarction: reperfusion duration is critical.

We investigated the influence of coronary artery reperfusion (CAR) duration on the infarct-limiting properties of adenosine A(1)-receptor stimulation-induced delayed preconditioning (A(1)-DPC) compared with ischemia-induced delayed preconditioning (I-DPC). Sixty-one chronically instrumented conscious rabbits successfully underwent the following protocol. On day 1, rabbits were randomly divided into four groups: control (saline, iv), I-DPC (six 4-min coronary artery occlusion/4-min reperfusion cycles), A(1)-DPC(100) (N(6)-cyclopentyladenosine, 100 microg/kg iv), and A(1)-DPC(400) (N(6)-cyclopentyladenosine, 400 microg/kg iv). On day 2 (i.e., 24 h later), rabbits underwent a 30-min coronary artery occlusion after which CAR was started and maintained for either 3 or 72 h. Infarct size (percentage of the area at risk) was determined by triphenyltetrazolium chloride staining. After 3 h of CAR, I-DPC, A(1)-DPC(100), and A(1)-DPC(400) significantly decreased infarct size (36 +/- 5, 41 +/- 4, 38 +/- 5%, respectively) compared with control (55 +/- 3%). After 72 h of CAR, infarct sizes were not significantly different among the four groups. This result was confirmed by histologic analysis. Thus A(1)-DPC at the two investigated doses, as well as I-DPC, decreased infarct size after 3 h but not 72 h of CAR.

Contributions of heart rate and contractility to myocardial oxygen balance during exercise.

The respective contributions of heart rate (HR) reduction and left ventricular (LV) negative inotropy to the effects of antianginal drugs are debated. Accordingly, eight instrumented dogs were investigated during exercise at spontaneous and paced HR (250 beats/min) after administration of either saline, atenolol, or ivabradine (selective pacemaker current channel blocker). During exercise, atenolol and ivabradine (both 1 mg/kg iv) similarly reduced HR (-30% from 222 +/- 5 beats/min), and LV mean ejection wall stress was not altered. LV dP/dt(max) was reduced by atenolol but not ivabradine. Diastolic time (DT) was increased by atenolol versus saline (195 +/- 6 vs. 123 +/- 4 ms, respectively) and to a greater extent by ivabradine (233 +/- 11 ms). Myocardial oxygen consumption (MVo(2)) was lower under ivabradine and atenolol versus saline (6.7 +/- 0.6 and 4.7 +/- 0.4 vs. 8.1 +/- 0.6 ml/min, respectively, P < 0.05). Under pacing, DT and MVo(2) were similar between ivabradine and saline but significantly reduced with atenolol. Thus HR reduction and negative inotropy equally contribute to the reduction in MVo(2) during exercise in the normal heart. The negative inotropy limits the increase in DT afforded by HR reduction.

Differential effects of heart rate reduction and beta-blockade on left ventricular relaxation during exercise.

Left ventricular (LV) relaxation is crucial for LV function, especially during exercise. We compared the effects of increasing doses of ivabradine, a selective inward hyperpolarization-activated current inhibitor, and atenolol on the rate and extent of LV relaxation (best fit method: time constant tau(BF), pressure asymptote P(BF)) at rest and during exercise. Eight dogs were chronically instrumented to measure LV pressure and LV wall stresses. During exercise under saline, heart rate increased from 108 +/- 5 to 220 +/- 6 beats/min and tau(BF) was significantly reduced from 22 +/- 1 to 14 +/- 2 ms. At rest, atenolol but not ivabradine increased tau(BF). For similar heart rate reductions during exercise, atenolol impeded the shortening of tau(BF) (23 +/- 2 ms) whereas ivabradine had no effect (15 +/- 2 ms). The extent of the relaxation process (P(BF)) at peak exercise was increased by ivabradine, and to a greater extent by atenolol, compared with saline. Thus, for a similar reduction in heart rate at rest and during exercise, ivabradine, in contrast with atenolol, does not exert any negative lusitropic effect.

Comparative pharmacokinetics and pharmacodynamics of intravenous and oral nefopam in healthy volunteers.

To determine the pharmacokinetic, subjective effects of a single 20 mg dose of nefopam administered either intravenously or orally in healthy volunteers, twenty-four healthy Caucasian men received 20 mg nefopam orally+placebo intravenous infusion and placebo orally+intravenous infusion of 20 mg nefopam with one week interval, in a double-blind, double-dummy cross-over study. Nefopam and desmethyl-nefopam plasma concentrations were measured by HPLC with UV detection up to 48 hr after drug administration. Self-rating questionnaires (Mood and vigilance Visual Analogue Scales, Addiction Research Centre Inventory) and drug safety were investigated. The F value (bioavailability) of the parent drug was 0.36+/-0.13. The AUCoral/AUCiv ratio of nefopam+desmethyl-nefopam was 0.62+/-0.23. The half-life of nefopam was similar whether administered orally (5.1+/-1.3 hr) or intravenously (5.1+/-0.6 hr). The half-life of desmethyl-nefopam was two to three times longer than that of the parent molecule (orally: 10.6+/-3.0 versus 5.1+/-1.3 hr, P<10(-4) and intravenously: 15.0+/-2.4 versus 5.1+/-0.6 hr, P<10(-4)). As assessed by the Addiction Research Centre Inventory, no evidence of abuse liability in healthy, drug-naive volunteers was observed. On visual analogue scales, volunteers rated themselves as more drowsy, less alert, less energetic and less anxious after oral compared to intravenous administration. The AUC0-->24 hr of anxiety and energy parameters were not different after oral and intravenous administration: 90+/-142 versus 35+/-84 (P=0.27) and 66+/-74 versus 46+/-54 mm x hr (P=0.36), respectively. The AUC0-->24 hr of drowsiness and alertness parameters were significantly greater after oral than after intravenous administration: 68+/-65 versus 27+/-30 (P=0.005) and 54+/-63 versus 28+/-48 mm x hr (P=0.03), respectively. A clockwise hysteresis loop was observed for drowsiness in 16 out of 24 volunteers after oral administration. The results suggest that in healthy volunteers desmethyl-nefopam may contribute to the pharmacodynamic effects of single dose nefopam solution administered orally. This study shows a rather low bioavailability of nefopam given in intravenous solution when administered orally. Nevertheless, when the main metabolite desmethyl-nefopam is taken into account, the ratio of the areas under the curves is almost doubled.

Human glycated albumin affects glucose metabolism in L6 skeletal muscle cells by impairing insulin-induced insulin receptor substrate (IRS) signaling through a protein kinase C alpha-mediated mechanism.

Nonenzymatic glycation is increased in diabetes and leads to increased levels of glycated proteins. Most studies have focused on the role of glycation products in vascular complications. Here, we have investigated the action of human glycated albumin (HGA) on insulin signaling in L6 skeletal muscle cells. Exposure of these cells to HGA inhibited insulin-stimulated glucose uptake and glycogen synthase activity by 95 and 80%, respectively. These effects were time- and dose-dependent, reaching a maximum after 12 h incubation with 0.1 mg/ml HGA. In contrast, exposure of the cells to HGA had no effect on thymidine incorporation. Further, HGA reduced insulin-stimulated serine phosphorylation of PKB and GSK3, but did not alter ERK1/2 activation. HGA did not affect either insulin receptor kinase activity or insulin-induced Shc phosphorylation on tyrosine. In contrast, insulin-dependent IRS-1 and IRS-2 tyrosine phosphorylation was severely reduced in cells preincubated with HGA for 24 h. Insulin-stimulated association of PI3K with IRS-1 and IRS-2, and PI3K activity were reduced by HGA in parallel with the changes in IRS tyrosine phosphorylation, while Grb2-IRS association was unchanged. In L6 myotubes, exposure to HGA increased PKC activity by 2-fold resulting in a similar increase in Ser/Thr phosphorylation of IRS-1 and IRS-2. These phosphorylations were blocked by the PKC inhibitor bisindolylmaleimide (BDM). BDM also blocked the action of HGA on insulin-stimulated PKB and GSK3 alpha. Simultaneously, BDM rescued insulin-stimulation of glucose uptake and glycogen synthase activity in cells exposed to HGA. The use of antibodies specific to PKC isoforms shows that this effect appears to be mediated by activated PKC alpha, independent of reactive oxygen species production. In summary, in L6 skeletal muscle cells, exposure to HGA leads to insulin resistance selectively in glucose metabolism with no effect on growth-related pathways regulated by the hormone.