C-reactive protein is directly related to plasminogen activator inhibitor type 1 (PAI-1) levels in diabetic subjects with the 4G allele at position -675 of the PAI-1 gene

Background and aimsC-reactive protein (CRP) has been identified as a possible factor able to promote atherosclerosis. “In vitro” studies have demonstrated that CRP induces plasminogen activator inhibitor type 1 (PAI-1) expression, suggesting a hypofibrinolytic role for CRP. As CRP and PAI-1 levels increase in type 2 diabetic subjects, we decided to study the relationship between CRP and PAI-1, and the role of the 4G/5G polymorphism of the PAI-1 gene on this relationship in a diabetic population without complications.Methods and resultsTwo hundred and ninety-five type 2 diabetic patients (age 60.9 ± 10.5 years) and 290 healthy controls (age 59.2 ± 11.5 years) were enrolled. A significant correlation between PAI-1 and CRP in diabetic subjects was found (r = 0.45, p < 0.001), whereas no relationship was evident in the control subjects between these inflammatory markers. Multiple regression analysis highlighted that CRP is the only one significant variable of PAI-1 antigen in diabetic subjects (partial r = 0.31, p < 0.01). Stratifying by genotype, a positive correlation between PAI-1 and CRP in 4G/4G (partial r = 0.64 p < 0.001) and 4G/5G (partial r = 0.47, p < 0.001) subjects was found, whereas no correlation in 5G/5G was present. Multiple regression analysis confirmed the presence of this correlation in 4G/4G (partial r = 0.45, p < 0.001) and in 4G/5G (partial r = 0.34, p = 0.007) diabetic patients.ConclusionsThese findings demonstrate that CRP plays an important role in the complex mechanism regulating PAI-1 antigen in 4G diabetic carriers.     

NO levels in diabetes mellitus: Effects of l-NAME and insulin on LCAT,Na+/K+ ATPase activity and lipid profile

ObjectivesDiabetes mellitus (DM) is a chronic disease and one of the most important health problems. Several factors may be responsible for the complications of diabetes mellitus including alterations in the activities of sodium–potassium adenosine triphosphatase (Na⁺/K⁺ ATPase) and lecithin:cholesterol acyltransferase (LCAT) and also levels of nitric oxide (NO). We have investigated the effects of alterations in serum NO levels on activities of erythrocyte membran Na/K ATPase and serum LCAT enzymes.Materials and methodsThe experiments were performed on male rats divided into four groups: group 1, control (standart diet); group 2, diabetic control (single dose of 65 mg/kg of streptozotocin (STZ), i.p); group 3, STZ + insulin (8 IU/kg/day s.c.); group 4 (STZ + l-NAME 5 mg/kg/day orally).ResultStreptozotocin-induced diabetic rats, showed a significant increase in blood glucose and serum cholesterol (C) and triglyceride (TG). Compared to the control group with diabetic group plasma LCAT concentrations and erythrocyte membrane Na⁺/K⁺ ATPase were found to be decreased. Activities of Na⁺/K⁺ ATPase and serum NO level were decreased with the administration of l-NAME. We observed that insulin was ameliorated in all parameters.ConclusionsSerum NO levels is related to erythrocyte membrane Na⁺/K⁺ ATPase activity. But serum NO levels did not affect the plasma LCAT activity and serum lipid profiles.     

Effects of hyper- and hypothyroidism on acetylcholinesterase, (Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>)- and Mg <Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-ATPase activities of adult rat hypothalamus and cerebellum

Thyroid hormones (THs) are recognized as key metabolic hormones, and the metabolic rate increases in hyperthyroidism, while it decreases in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na⁺, K⁺)- and Mg²⁺-ATPase in the hypothalamus and the cerebellum of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25μg/100 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. Neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE (−23%, p < 0.001) and the Na⁺, K⁺-ATPase activities (−26%, p < 0.001). Moreover, hypothyroidism had a similar effect on the examined enzyme activities: AChE (−17%, p < 0.001) and Na⁺, K⁺-ATPase (−27%, p < 0.001). Mg²⁺-ATPase activity was found unaltered in both the hyper- and the hypothyroid brain regions. In conclusion: neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE and the Na⁺, K⁺-ATPase activities. The decreased (by the THs) Na⁺, K⁺-ATPase activities may increase the synaptic acetylcholine release, and thus, could result in a decrease in the cerebellar AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems.     

Suckling Rat Brain Regional Distribution of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity in the In Vitro Galactosaemia: The Effect of L-Cysteine and Glutathione

Inhibition of Na⁺,K⁺-ATPase activity causes edema and cell death in central nervous system. We determined the in vitro effects of galactose-1-phosphate (Gal-1-P), galactitol (Galtol) and galactose (Gal) (mix A = classical galactosaemia) or Galtol and Gal (mix B = galactokinase deficiency galactosaemia), on Na⁺,K⁺-ATPase activity in suckling rat brain frontal cortex, hippocampus or hypothalamus homogenates. Gal-1-P or Galtol alone at different concentrations, significantly inhibited Na⁺,K⁺-ATPase whereas Gal activated the enzyme in all investigated brain regions. Both mix A and mix B inactivated the enzyme by 20–30% (p < 0.001) in all studied areas. L-Cysteine (Cys) and glutathione (GSH) supplementation in mix B not only reversed the enzyme inhibition but also resulted in an activation of 50–60%, (p < 0.001) in all brain areas. Their presence in mix A also activated the inhibited Na⁺,K⁺-ATPase in hippocampus and hypothalamus to a lower degree, whereas Cys reversed the frontal cortex enzyme activity to control value only. These findings indicate that oxidation of the enzyme critical groups may be involved in galactosaemia, producing inhibitory effect. This phenomenon is reversed by antioxidants Cys and GSH, implying that free radicals may be implicated in the observed enzyme inactivation.     

The Protective Effect of L-Cysteine and Glutathione on the Adult and Aged Rat Brain (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-ATPase and Mg<Superscript>2+</Superscript>-ATPase Activities in Galactosemia In Vitro

The aim of this study was to evaluate whether the addition of the antioxidants L-cysteine (Cys) or the reduced glutathione (GSH) could reverse the alterations of brain total antioxidant status (TAS) and the modulated activities of the enzymes (Na⁺,K⁺)-ATPase, and Mg²⁺-ATPase in adult or aged rat brain homogenates induced by galactosemia in vitro. Mixture A [mix. A: galactose-1-phosphate (Gal-1-P, 2 mM) plus galactitol (Galtol, 2 mM) plus galactose (Gal, 4 mM) = classical galactosemia] or mixture B [mix. B: Galtol (2 mM) plus Gal (1 mM) = galactokinase deficiency galactosemia] were preincubated in the presence or absence of Cys (0.83 mM) or GSH (0.83 mM) with adult or aged brain homogenates at 37C for 1 h. TAS and the enzyme activities were determined spectrophotometrically. Mix. A or mix. B preincubation with the adult brain resulted in a significant (Na⁺,K⁺)-ATPase inhibition (–30%) and a Mg²⁺-ATPase stimulation (+300% and +33%, respectively), whereas lower modifications of the enzyme activities (p < 0.001) were found in the aged brain. Gal mixtures decreased TAS by 40% (p < 0.001) and by 20% (p < 0.01) in adult and aged samples, respectively. The antioxidants significantly increased TAS resulting in the reversion of (Na⁺,K⁺)-ATPase inhibition and Mg²⁺-ATPase stimulation by mix. B only. The inhibitory effect of Gal and its derivatives on brain (Na⁺,K⁺)-ATPase and their stimulatory effect on Mg²⁺-ATPase are being decreased with age, probably due to the producion of free radicals. Cys and GSH increased TAS resulting in a reversion of the inhibited (Na⁺,K⁺)-ATPase in both models of the in vitro galactosemia and the stimulated Mg²⁺-ATPase in galactokinase deficiency galactosemia only.     

Mutation I810N in the α3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS

In a mouse mutagenesis screen, we isolated a mutant, Myshkin (Myk), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk/+ mice carry a mutation (I810N) which renders the normally expressed Na⁺,K⁺-ATPase α3 isoform inactive. Total Na⁺,K⁺-ATPase activity was reduced by 42% in Myk/+ brain. The epilepsy in Myk/+ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na⁺,K⁺-ATPase α3 by transgenesis, which also rescued Na⁺,K⁺-ATPase activity. Our findings reveal the functional significance of the Na⁺,K⁺-ATPase α3 isoform in the control of epileptiform activity and seizure behavior.     

Cardiovascular autonomic function and vibration perception threshold in type 2 diabetes mellitus

ObjectiveThe aim of this study was to compare cardiovascular autonomic function tests (AFT) and vibration perception threshold (VPT) of patients with type 2 diabetes mellitus (T2DM) with controls.Research Design/MethodsThe study was conducted on 60 diabetic patients comparing with 30 controls. The cardiovascular AFT and VPT were assessed in both groups.ResultsAmong cardiovascular AFT, E:I ratio [1.24 (1.2–1.32) vs 1.3 (1.24–1.4), p = 0.001], and Valsalva ratio [1.28 (1.22–1.4) vs 1.6 (1.5–1.73), p = 0.001], the indicators of parasympathetic reactivity were reduced in T2DM. Rise in DBP during handgrip, an indicator of sympathetic reactivity was lower in T2DM [12 (10–14) vs 16 (14–18) mmHg, p = 0.001] whereas, fall in SBP during head up tilt [4 (4–8) vs 5 (4–8) mmHg] was comparable. VPT (somatic sensation) was comparable between T2DM and control.ConclusionAutonomic involvement is more marked than somatic, and parasympathetic involvement is more marked than the sympathetic, possibly reflecting severity and chronological pattern of their involvement.     

Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes

Assays for complete quantification of Na⁺, K⁺-and Ca²⁺-ATPase in crude homogenates of rat ventricular myocardium by determination of K⁺-and Ca²⁺-dependentp-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K⁺-and Ca²⁺-dependentpNPPase activities in ventricular myocardium of 11–12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 mol×min^–1×g^–1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na⁺, K⁺-and Ca²⁺-ATPase concentrations were estimated to 2 and 10 nmol×g^–1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca²⁺-dependentpNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K⁺-dependentpNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K⁺-and Ca²⁺-dependentpNPPase activity gradually increased to a plateau. The present assay for Na⁺, K⁺-ATPase quantification has the advantage over [³H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K⁺-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications thepNPPase assay allows quantification of Ca²⁺-ATPase on crude myocardial homogenates. Age-dependent changes in K⁺-and Ca²⁺-dependentpNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na⁺, K⁺-and Ca²⁺-ATPase in excitable tissues.Abbreviations Na⁺, K⁺-ATPase sodium, potassium-dependent ATPase - Ca²⁺-ATPase caldium-dependent ATPase - pNP p-nitrophenyl - pNPP p-nitrophenyl phosphate - 3-0-MFP 3-0 methylfluorescein phosphate - DOC sodium deoxycholate     

Insulin does not regulate vascular smooth muscle Na+, K+-ATPase activity in rabbit aorta

Summary To determine whether insulin regulates vascular smooth muscle Na⁺, K⁺-ATPase activity and if impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity could be a cause of increased vascular reactivity to norepinephrine and angiotensin II in diabetic states, the effects of insulin on Na⁺, K⁺-ATPase activity were examined in normal rabbit aortic intima-media incubated with normal plasma glucose and myo-inositol levels for 30 min. Insulin at 100 U/ml (600 pmol/l) had no effect on Na⁺, K⁺-ATPase activity. At 250 U/ml it caused a 4.2±0.8% increase, and at 500 U/ml insulin caused a 17.7±1.4% increase in Na⁺, K⁺-ATPase activity that was completely inhibited by amiloride (1 mmol/l). Human insulin-like growth factor I (600 pmol/l) caused an 18.0±1.0% increase in Na⁺, K⁺-ATPase activity that was inhibited by amiloride. Insulin does not regulate (stimulate) aortic vascular smooth muscle Na⁺, K⁺-ATPase activity. Supraphysiological insulin concentrations, probably acting through an insulin-like growth factor I receptor, stimulate Na⁺/H⁺ exchange in aortic vascular smooth muscle and cause small secondary increases in Na⁺, K⁺-ATPase activity. In aortic intima-media incubated with normal plasma glucose and myo-inositol levels, endogenously released adenosine stimulates and maintains a component of resting Na⁺, K⁺-ATPase activity and stimulates acute increases in activity when norepinephrine (1 mol/l) or angiotensin II (100 nmol/l) is added. These adenosine-stimulated components of Na⁺, K⁺-ATPase activity are selectively inhibited when the medium glucose is raised to 30 mmol/l during a 30-min equilibration and 30-min incubation. Insulin (100 U/ml) added during the incubation had no effect on the alterations in Na⁺, K⁺-ATPase activity induced by glucose at an elevated plasma level. Impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity is not a possible cause for alterations in vascular reactivity in diabetes.     

Medication non-adherence and poor glycaemic control in patients with type 2 diabetes mellitus

AimsHyperglycemia causes generation of free radicals which leads to oxidative stress and apoptosis in various cells. The present study was undertaken to investigate the correlation between oxidative stress and apoptotic markers in lymphocytes of diabetic patients with chronic non healing wounds.MethodsThirty healthy, thirty uncontrolled type 2 diabetes mellitus (T2DM) and thirty uncontrolled T2DM with chronic, non healing, neuropathic diabetic foot patients were included in this study. Indices of oxidative stress inside the lymphocyte lysate were estimated by measuring content of superoxide dismutase (SOD), Catalase, Glutathione and malonaldialdehyde (MDA). Protein expression studies of pro and anti apoptotic markers were carried out to elucidate their possible involvement in diabetic context.ResultsSOD and MDA activity was significantly higher in the lymphocytes of diabetic patients having chronic, non healing diabetic wound as compared with healthy (p < 0.001); whereas catalase and GSH activity was significantly reduced (p < 0.001) in the same group. Expressions of pro apoptotic markers (Caspase-3, Fas and Bax) were significantly higher whereas reduced expression of anti-apoptotic marker (Bcl-2) were obtained in lymphocytes of diabetic and non diabetic individuals.ConclusionsHyperglycemia confers pro apoptotic manifestations which are mostly through altered indices of oxidative stress within lymphocytic milieu.     

Platelet Na+,K+-ATPase activity as a possible peripheral marker for the neurotoxic effects of phenylalanine in phenylketonuria

Thein vitro effects of phenylalanine or alanine alone or combined on Na⁺,K⁺-ATPase activity in membranes from human platelets were investigated. The enzyme activity was assayed in membranes prepared from platelet-rich plasma of healthy donors. Phenylalanine or alanine were added to the assay to final concentrations of 0.3 to 1.2 mM, similar to those found in plasma of phenylketonuric patients. Phenylalanine inhibited Na⁺,K⁺-ATPase activity by 20–50% [F(4,25)=11.47; p<0.001]. Alanine had no effect on Na⁺,K⁺-ATPase activity but when combined with phenylalanine prevented the enzyme inhibition. These results, allied to others previously reported on brain Na⁺,K⁺-ATPase activity, may reflect a general inhibitory effect of phenylalanine on this important enzyme activity. Therefore, it is possible that measurement of Na⁺,K⁺-ATPase activity in platelets from PKU patients may be a useful peripheral marker for the neurotoxic effects of phenylalanine.     

Transgenic overexpression of translationally controlled tumor protein induces systemic hypertension via repression of Na+,K+-ATPase

Inhibition of Na⁺,K⁺-ATPase has been implicated in the pathogenesis of hypertension via its effect on smooth muscle reactivity and myocardial contractility. We recently demonstrated that translationally controlled tumor protein (TCTP) interacts with the 3rd cytoplasmic domain of Na⁺,K⁺-ATPase α₁-subunit and acts as its cytoplasmic repressor. Therefore, we hypothesized that repression of Na⁺,K⁺-ATPase by overexpressed TCTP might underlie the development of hypertension. In the present study, we confirmed that transgenic mice overexpressing TCTP developed systemic arterial hypertension at about 6 weeks after birth. Vascular smooth muscle of TCTP-overexpressing transgenic mice also displayed augmented contractile response to vasoconstrictors and attenuated relaxation response to vasodilators. These responses seem to be caused by reduced Na⁺,K⁺-ATPase activity and increased intracellular calcium, suggesting that inhibition of Na⁺,K⁺-ATPase by overexpression of TCTP is involved in the pathogenesis of hypertension. This study provides a new link between alteration of sodium pump activity and hypertension in vivo, and suggests that TCTP might be a therapeutic target for the treatment of hypertension.     

Protein and glutamine kinetics during counter-regulatory failure in type 1 diabetes

Background and aimsHealthy individuals counteract insulin-induced hypoglycaemia by increasing glutamine utilization but not proteolysis. Glucagon is important to this response because it increases glutamine uptake. In type 1 diabetes (T1DM) glucagon and epinephrine responses to hypoglycaemia are defective. We investigated whether glutamine and amino acid utilization during hypoglycaemia is altered in T1DM with defective counter-regulatory responses.Methods and resultsEight T1DM patients (duration of diabetes 14 ± 4 years and therefore with presumed defective counter-regulatory response) and eight controls (CON) received a 3 h hypoglycaemic hyperinsulinaemic (0.65 mU/kg per min) clamp coupled to [6,6-²H₂]glucose, [1-¹³C]leucine and [2-¹⁵N]glutamine to trace the relative kinetics.Post-absorptive plasma glucose and glucose uptake were increased in T1DM (9.09 ± 0.99 vs 5.01 ± 0.22 mmol/l and 19.5 ± 0.9 vs 12.6 ± 0.8 μmol/kg per min, p < 0.01). During the clamp T1DM but not CON required exogenous glucose (4.4 ± 1.7 μmol/kg per min) to maintain the hypoglycaemic plateau because the endogenous glucose production was significantly suppressed (p < 0.01). In T1DM the leucine and phenylalanine concentrations were less suppressed from basal (p < 0.05) despite a similar insulin suppression of proteolysis (−16 ± 2 vs −20 ± 4%, p = ns) indicating a defective stimulation of leucine metabolic clearance from basal (+18 ± 3% vs +55 ± 9%, p < 0.01). Glutamine concentration remained unchanged from basal (−7 ± 3% vs −35 ± 3%, p < 0.01) and the clearance of glutamine was markedly defective in T1DM (+6 ± 2%) in comparison with controls (+22 ± 4%; p = 0.02).ConclusionsIn T1DM, the counter-regulatory failure to hypoglycaemia seems to be associated with a defective glutamine utilization. The failure to clear circulating amino acids, specifically glutamine, during hypoglycaemia may adversely affect gluconeogenesis.     

Importance of glycemic control on the course of glomerular filtration rate in type 2 diabetes with hypertension and microalbuminuria under tight blood pressure control

Background and aimsTo evaluate the role of glycemic control on the evolution of glomerular filtration rate (GFR) in type 2 diabetes (T2DM) with mild-moderate hypertension under tight blood pressure control, and to address the current controversy whether diabetic nephropathy worsens, independently of blood pressure, proportionally to HbA1c at any physiological level or only when HbA1c is above a 7.5–8% threshold.Methods and resultsT2DM (N = 127) during early stage diabetic nephropathy characterized by microalbuminuria were followed during a 2 year multicenter study. Individual GFR profiles were accurately obtained by ⁵¹Cr – EDTA bolus injections and analyzed with linear statistical mixed-effects models. GFR at baseline was significantly negatively correlated with age and plasma creatinine concentration (P ≤ 0.0001), and GFR declined, on average, by 4.0 ml/min 1.73 m²/year (P = 0.001). A significant correlation was found between individual GFR decline rate and average systolic (SBP) and diastolic (DBP) blood pressures (−0.254 (0.736) ml/min 1.73 m²/year per mmHg increase in SBP (DBP), P = 0.041 (0.014)) and % of glycated hemoglobin (HbA1c) (−1.78 ml/min 1.73 m²/year per % increase in HbA1c, P = 0.048). This implies a 44% increase/reduction in GFR decline rate for 1% HbA1c increase/reduction around 7.0% (i.e. 5.79 and 2.24 ml/min 1.73 m²/year at 8% and 6% HbA1c, respectively).ConclusionsThis study demonstrates that, despite tight blood pressure control, an accurate glycemic control till very low patterns of HbA1c (from 10–11% to 5–6%) is needed to delay the progression of GFR decay in Mediterranean T2DM in south Europe with microalbuminuria.     

Changes in Antioxidant Status,Protein Concentration,Acetylcholinesterase, (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-, and Mg<Superscript>2+</Superscript>-ATPase Activities in the Brain of Hyper- and Hypothyroid Adult Rats

It is a common knowledge that metabolic reactions increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how the metabolic reactions could affect the total antioxidant status (TAS), protein concentration (PC) and the activities of acetylcholinesterase (AChE), (Na⁺,K⁺)-ATPase and Mg²⁺-ATPase in the brain of hyper- and hypothyroid adult male rats. Hyperthyroidism was induced in rats by subcutaneous administration of thyroxine (25 g/100 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. TAS, PC, and enzyme activities were evaluated spectrophotometrically in the homogenated brain of each animal. TAS, PC, and Mg²⁺-ATPase activity were found unaffected in hyperthyroidism, while AChE and Na⁺,K⁺-ATPase activities were reduced by 25% (p < 0.01). In contrast, TAS, (Na⁺,K⁺)-ATPase and Mg²⁺-ATPase activities were found to be increased (approx. 23–30%, p < 0.001) in the hypothyroid brain, while AChE activity and PC were shown to be inhibited (approx. 23–30%, p < 0.001). These changes on brain enzyme activities may reflect the different metabolic effects of hyper- and hypothyroidism. Such changes of the enzyme activities may differentially modulate the brain intracellular Mg²⁺, neural excitability, as well as the uptake and release of biogenic amines.     

Isoform-specific alterations in cardiac and erythrocyte Na ,K-ATPase activity induced by norepinephrine

Background: Myocardial Na⁺,K⁺-ATPase activities are decreased in congestive heart failure because of an increase in plasma norepinephrine levels, but it is difficult to monitor the activities in the clinical setting.Methods and Results: This study investigated whether erythrocyte Na⁺,K⁺-ATPase activity can reflect myocardial enzyme activity and whether isoform-specific alterations occur in the presence of catecholamine. Na⁺,K⁺-ATPase activity was measured by the colorimetric method by using the left ventricular myocardium and erythrocytes prepared from eight rabbits given norepinephrine for 7 days and from eight control rabbits that received saline. The protein levels of total catalytic subunit and α₁ - or α₃-isoform of Na⁺,K⁺-ATPase were determined by Western blot analysis. Na⁺,K⁺-ATPase activity was lower in both myocardium and erythrocytes from norepinephrine-treated rabbits than control rabbits (P < .01 and P < .01, respectively). There was a close correlation in Na⁺,K⁺-ATPase activity between myocardium and erythrocytes (r = .963). Total catalytic subunit protein level was lower in myocardium from norepinephrine-treated rabbits than control rabbits, but the α₁-isoform level was similar between the two groups. The α₃-isoform level was lower in norepinephrinetreated rabbits than control rabbits. In erythrocytes, α₁-isoform was lower in norepinephrinetreated rabbits than control rabbits.Conclusions: Na⁺,K⁺-ATPase activity in myocardium could be reflected in erythrocyte membrane, although there was a difference in isoform-specific regulation between the two.     

Physical activity and sports participation in children and adolescents with type 1 diabetes mellitus

Background and aimRegular physical activity is of great importance in the management of type 1 diabetes mellitus (T1DM). We investigate here the levels of moderate/vigorous physical activity (MVPA) and participation in sporting activity in a sample of children and adolescents with T1DM and analyse whether they differed from healthy subjects. The family variables associated with MVPA or sports participation and the influence of exercise on metabolic parameters are also explored.Methods and resultsIn this cross-sectional case control study, 138 children and adolescents with T1DM (of which 67 were boys, age 13.6 ± 4.1 years; duration of diabetes 6.1 ± 3.8 years) and 269 (of which 120 were boys) healthy controls were studied. Weekly levels of MVPA and sports participation were investigated using a questionnaire. Body mass index standard deviation score (BMI-SDS) values, plasma total cholesterol, serum triglycerides and the mean glycated haemoglobin (A1c) levels over the past year were assessed in T1DM subjects. MVPA scores in T1DM patients were lower than in controls (p = 0.0004). MVPA was higher in boys than in girls, both in diabetic and control subjects; T1DM girls were less frequently engaged in MVPA than control girls. MVPA scores were significantly and independently correlated with sex, age and diabetic status. Lower triglyceride levels and fewer subjects with poor metabolic control were found more among physically active patients (MVPA > 5 days/week) than in inactive patients (weekly MVPA = 0). Sports participation was lower in T1DM patients than in controls (p = 0.002) and was significantly and independently correlated with sex, father's education level and diabetic status. Triglyceride levels and the percentage of subjects with poor metabolic control were significantly lower in sports participants than in non-participants.ConclusionsChildren and adolescents with T1DM appeared to spend less time in physical activity than their non-diabetic peers. Regular physical activity was associated with better metabolic control and lipid profile. Adolescents, particularly the girls, tended to be less active. Further efforts should be made to motivate patients with type 1 diabetes.     

Alterations of Na/K-ATPase function in caveolin-1 knockout cardiac fibroblasts

Recent studies have demonstrated that the Na⁺/K⁺-ATPase is not only an ion pump, but also a membrane receptor that confers the ligand-like effects of cardiotonic steroids (CTS) such as ouabain on protein kinases and cell growth. Because CTS have been implicated in cardiac fibrosis, this study examined the role of caveolae in the regulation of Na⁺/K⁺-ATPase function and CTS signaling in cardiac fibroblasts. In cardiac fibroblasts prepared from wild-type and caveolin-1 knockout [Cav-1(−/−)] mice, we found that the absence of caveolin-1 did not affect total cellular amount or surface expression of Na⁺/K⁺-ATPase α1 subunit. However, it did increase ouabain-sensitive ⁸⁶Rb⁺ uptake. While knockout of caveolin-1 increased basal activities of Src and ERK1/2, it abolished the activation of these kinases induced by ouabain but not angiotensin II. Finally, ouabain stimulated collagen synthesis and cell proliferation in wild type but not Cav-1(−/−) cardiac fibroblasts. Thus, we conclude that caveolae are important for regulating both pumping and signal transducing functions of Na⁺/K⁺-ATPase. While depletion of caveolae increases the pumping function of Na⁺/K⁺-ATPase, it suppresses CTS-induced signal transduction, growth, and collagen production in cardiac fibroblasts.     

Basis of Rise in Intracellular Sodium in Airway Hyperresponsiveness and Asthma

The aim of this study was to investigate the basis of disturbances in sodium transport in asthma and in airway hyperresponsiveness without symptoms of asthma (asymptomatic AHR). We measured the intracellular sodium (Na_i); activity of Na⁺/K⁺-ATPase in unstimulated cells (resting activity) and in cell homogenate under optimal conditions (maximal activity); and sodium influx, in mixed leukocytes of 15 normal subjects, 12 subjects with asymptomatic AHR, and 26 asthmatics with or without active symptoms. Resting Na⁺/K⁺-ATPase activity was the same as sodium influx, consistent with homeostasis. Compared with normal subjects, those with asymptomatic AHR or asthma with controlled symptoms had a twofold increase in sodium influx and Na_i. Symptomatic asthmatics also had a twofold increase in sodium influx but a fourfold elevation of Na_i. Maximal Na⁺/K⁺-ATPase activity was reduced by half in symptomatic asthmatics compared with normal subjects. The reduction of maximal Na⁺/K⁺-ATPase activity was associated with a significant decrease in ATP turnover per Na⁺/K⁺-ATPase molecule but not number of Na⁺/K⁺-ATPase molecules per cell. In summary, airway hyperresponsiveness with or without asthma is associated with increased sodium influx and Na in leukocytes. Resting activity of Na⁺/K⁺-ATPase is also increased as a compensatory response to the increased sodium influx, but it is achieved at the expense of higher Na_i. Symptomatic asthma is additionally associated with reduction in maximal activity of Na⁺/K⁺-ATPase, resulting in reduced capacity to handle the increase in sodium influx and consequent severe elevations in Na_i.     

Usefulness of lipoprotein ratios in assessing carotid atherosclerosis in Japanese type 2 diabetic patients

ObjectiveIt is indicated that total/HDL cholesterol and LDL/HDL cholesterol ratios have more predictive power for cardiovascular disease compared to classic lipid parameters. However, there have been few reports about the usefulness of these indices for the assessment of early stage atherosclerosis in Japanese type 2 diabetic subjects.MethodsWe examined the relation between various lipid parameters and carotid atherosclerosis in 934 type 2 diabetic subjects without apparent atherosclerotic diseases (males, 71.7%; age, 59.6 ± 10.5 years (mean ± SD)). Serum concentrations of total cholesterol (TC), HDL cholesterol (HDL-C), and triglyceride were measured. LDL cholesterol (LDL-C) level was calculated using the Friedewald formula. The presence of carotid plaque and intima-media thickness (IMT) were evaluated by ultrasonography.ResultsA stepwise multivariate regression analysis demonstrated that HDL-C (β = ?0.110, p < 0.001), TC/HDL-C (β = 0.132, p < 0.001) and LDL-C/HDL-C ratios (β = 0.132, p < 0.001) were independent determinants of IMT even after adjustment of other conventional risk factors. However, there was no significant correlation between IMT and TC, triglyceride, LDL-C, and non-HDL-C levels. TC/HDL-C and LDL-C/HDL-C ratios and non-HDL-C levels were significantly higher, but HDL-C levels were significantly lower in patients with carotid plaque than those without it (p < 0.05). There was no significant difference between the groups regarding TC, LDL-C, and triglyceride levels. Furthermore, TC/HDL-C (OR; 1.34, p < 0.001) and LDL-C/HDL-C (OR; 1.54, p < 0.001) ratios showed a positive and linear relationship with the prevalence of carotid plaque, whether covariates were adjusted or not.ConclusionsTC/HDL-C and LDL-C/HDL-C ratios are useful as a tool to assess the risk of early stage atherosclerosis in Japanese type 2 diabetic patients.