The effect of norepinephrine on insulin secretion and glucose effectiveness in non-insulin-dependent diabetes

It has previously been shown that in normal subjects, physiological elevation of norepinephrine (NE) impairs insulin sensitivity (Si) but does not influence insulin secretion. The aim of this study was to determine the effect of short-term physiological elevation of NE on insulin secretion, Si, and glucose-mediated glucose disposal, or the glucose effectiveness index (Sg), in non-insulin-dependent diabetes mellitus (NIDDM). Two intravenous glucose tolerance tests (IVGTTs) were performed in eight well-controlled NIDDM patients, using a supplemental exogenous insulin infusion to achieve an approximation of normal endogenous insulin secretion. The IVGTTs were performed in random order after 30 minutes of either the saline (SAL) or NE (25 ng/kg/min) infusions, which were continued throughout the 3-hour IVGTT. Sg and Si were estimated by minimal model analysis of the IVGTT data as previously described. Plasma C-peptide was used to estimate insulin secretion rate using the ISEC program. NE infusion produced approximately a threefold increase in plasma NE, associated with (1) a significant reduction in glucose disposal ([K_G] SAL v NE, 0.73 ± 0.06 v 0.61 ± 0.06 × 10⁻² · min⁻², P < .05), (2) no reduction in Si (2.33 ± 0.8 v 2.62 ± 0.9 × 10⁻⁴ · min⁻¹/mU/L, NS), (3) a reduced mean second-phase insulin secretion rate (1.21 ± 0.19 v 1.01 ± 0.16 × 10⁻³ pmol/kg/min per mmol/L glucose, P < .05), (4) a significant increase in Sg (0.89 ± 0.08 v 1.63 ± 0.2 × 10⁻² · min⁻¹ P < .05), and (5) a corresponding increase in glucose effectiveness at zero insulin ([GEZI] 0.55 ± 0.13 v 1.30 ± 0.33 × 10⁻² · min⁻¹, P < .05). These results show that in contrast to normal subjects, physiological elevation of NE in NIDDM does not result in a reduction in Si, but causes a reduction in glucose disposal related to inhibition of insulin secretion that is only partially compensated for by increased Sg.     

Association of β2-adrenoceptor Gln27Glu variant with body weight but not hypertension

β₂-Adrenoceptor (β₂-ADR)-mediated vasodilatation decreases vascular reactivity and blood pressure (BP) and chromosome 5 where its gene (ADRB2R) resides and shows linkage to hypertension (HT). A Gln27Glu ADRB2R variant confers resistance to agonist-induced desensitization and enhanced vasodilator response to isoprenaline. Therefore, we carried out a case-control study in a cohort of HT and normotensive (NT) Anglo-Celtic Australian white subjects whose parents had a similar BP status as the subjects. Glu27 frequency was 0.41 in 108 HT and 0.42 in 141 NT (χ² = 0.05, P = .82). Within the HT group, the Glu27 allele was more prevalent in 61 subjects who were overweight (body mass index [BMI] ≥ 25 kg/m²) compared with 41 who were lean (BMI <25 kg/m²); ie, 0.49 v 0.31, respectively (χ² = 6.4, P = .012). Furthermore, Glu27 tracked with elevation in BMI in these subjects: 24 ± 4 kg/m², 27 ± 5 kg/m², and 28 ± 5 kg/m² for Gln/Gln, Gln/Glu, and Glu/Glu, respectively (P = .0058 by one-way ANOVA). Thus, the Gln27Glu β₂-ADR variant is excluded in HT, but might influence body weight.     

Increased density of tolerogenic dendritic cells in the small bowel mucosa of celiac patients

AIM: To investigate the densities of dendritic cells(DCs) and FOXP3+ regulatory T cells(Tregs) and their interrelations in the small bowel mucosa in untreated celiac disease(CD) patients with and without type 1 diabetes(T1D).METHODS: Seventy-four patients(45 female, 29 male, mean age 11.1 ± 6.8 years) who underwent small bowel biopsy were studied. CD without T1 D was diagnosed in 18 patients, and CD with T1 D was diagnosed in 15 patients. Normal small bowel mucosa was found in two T1 D patients. Thirty-nine patients(mean age 12.8 ± 4.9 years) with other diagnoses(functional dyspepsia, duodenal ulcer, erosive gastritis, etc.) formed the control group. All CD patients had partial or subtotal villous atrophy according to the Marsh classification: Marsh grade Ⅲa in 9, grade Ⅲb in 21 and grade Ⅲc in 3 cases. Thirty-nine patients without CD and 2 with T1 D had normal small bowel mucosa(Marsh grade 0). The densities of CD11c+, IDO+, CD103+, Langerin(CD207+) DCs and FOXP3+ Tregs were investigated by immunohistochemistry(on paraffin-embedded specimens) and immunofluorescence(on cryostat sections) methods using a combination of mono- and double-staining. Sixtysixserum samples were tested for Ig A-tissue transglutaminase(t TG) using a fully automated Eli ATM Celikey Ig A assay(Pharmacia Diagnostics, Freiburg, Germany). RESULTS: The density of CD11c+ DCs was significantly increased in CD patients compared with patients with normal mucosa(21.67 ± 2.49 vs 13.58 ± 1.51, P = 0.007). The numbers of FOXP3+ cells were significantly higher in CD patients(10.66 ± 1.50 vs 1.92 ± 0.37, P = 0.0002) and in patients with CD and coexisting T1D(8.11 ± 1.64 vs 1.92 ± 0.37, P = 0.002) compared with patients with normal mucosa. The density of FOXP3+ cells significantly correlated with the histologicalgrade of atrophic changes in the small bowel mucosa according to the March classification(r = 0.62; P 0.0001) and with levels of Ig A antibody(r = 0.55; P 0.0001). The densities of IDO+ DCs were significantly higher in CD patients(21.6 ± 2.67 vs 6.26 ± 0.84, P = 0.00003) and in patients with CD and coexisting T1D(19.08 ± 3.61 vs 6.26 ± 0.84, P = 0.004) compared with patients with normal mucosa. A significant correlation was identified between the densities of IDO+ DCs and FOXP3+ T cells(r = 0.76; P = 0.0001). The mean values of CD103+ DCs were significantly higher in CD patients(10.66 ± 1.53 vs 6.34 ± 0.61, P = 0.01) and in patients with CD and associated T1D(11.13 ± 0.72 vs 6.34 ± 0.61, P = 0.00002) compared with subjects with normal small bowel mucosa. The mean value of Langerin+ DCs was higher in CD patients compared with persons with normal mucosa(7.4 ± 0.92 vs 5.64 ± 0.46, P = 0.04).CONCLUSION: The participation of diverse DC subsets in the pathological processes of CD and the possible involvement of tolerogenic DCs in Tregs development to maintain intestinal immunological tolerance in CD patients are revealed.     

Age-related decrease in plasma growth hormone: Response to growth hormone-releasing hormone, arginine, and l-dopa in obesity

Aging is associated with a reduction in plasma growth hormone (GH) secretion in non-obese subjects. To determine whether or not age-related changes in plasma GH secretion exist in obese subjects, we measured (a) plasma GH response to growth hormone-releasing hormone (GRH; 1 μg/kg body wt), arginine (0.5 g/kg body wt), l-dopa (500mg), and (b) plasma glucose, insulin, and free fatty acids (FFAs) in 26 fasted obese subjects of various ages ranging from 16 to 71 years. Only subjects with a body mass index (BMI; kg/m²) between 30.0 and 39.0 were studied. Six subjects were adolescents, 9 were in their 20s, and 11 were 30 years or older. The mean peak levels of plasma GH in response to GRH, arginine, and l-dopa in obese subjects were 11.3 ± 2.1, 21.9 ± 4.4, and 5.2 ± 0.3 ng/mL in adolescents, 8.2 ± 1.6, 9.1 ± 1.5, and 3.1 ± 0.6 ng/mL in those in their 20s, and 4.5 ± 0.4, 7.3 ± 1.4, and 2.8 ± 0.3 ng/mL in those 30 years or older, respectively, showing a significant decrease in peak GH level with advancing age (P < .05 to P < .01). There was a negative correlation between the logarithmic increase in age and the peak GH response to GRH (r = −.635, P < .01), arginine (r = −.564, P < .01), and l -dopa (r = −.630, P < .01), and between the logarithmic increase in age and the integrated GH response to GRH (r = −.564, P < .01), arginine (r = −.612, P < .01), and l-dopa (r = −.551, P < .01) in all subjects. Plasma glucose, insulin, and FFAs did not change with age. There was no correlation between the peak GH level or the integrated GH response to these three stimuli and the plasma levels of glucose, insulin, and FFAs, respectively. Our findings suggest that in obese subjects advancing age reduces the secretory responsiveness of pituitary somatotropes to these three stimuli.     

Effects of low-dose recombinant human insulin-like growth factor-I on insulin sensitivity, growth hormone and glucagon levels in young adults with insulin-dependent diabetes mellitus

Despite recent interest in the therapeutic potential of recombinant human insulin-like growth factor-I (rhIGF-I) in the treatment of diabetes mellitus, its mechanism of action is still not defined. We have studied the effects of low-dose bolus subcutaneous rhIGF-I (40 μg/kg and 20 μg/kg) on insulin sensitivity, growth hormone (GH) and glucagon levels in seven young adults with insulin-dependent diabetes mellitus (IDDM) using a randomized double-blind placebo-controlled crossover study design. Each was subjected to a euglycemic clamp (5 mmol/L) protocol consisting of a variable-rate insulin infusion clamp (6:00 to 8:00 ) followed by a two-dose hyperinsulinemic clamp (insulin infusion of 0.75 mU · kg⁻¹ · min⁻¹ from 8 to 10 and 1.5 mU · kg⁻¹ · min⁻¹ from 10 to 12 noon) incorporating [6,6 ²H₂]glucose tracer for determination of glucose production/utilization rates. Following rhIGF-I administration, the serum IGF-I level (mean ± SEM) increased (40 μg/kg, 655 ± 90 ng/mL, P < .001; 20 μg/kg, 472 ± 67 ng/mL, P < .001; placebo, 258 ± 51 ng/mL). Dose-related reductions in insulin were observed during the period of steady-state euglycemia (1 to 8 ) (40 μg/kg, 48 ± 5 pmol/L, P = .01; 20 μg/kg, 58 ± 8 pmol/L, P = .03; placebo, 72 ± 8 pmol/L). The mean overnight GH level (40 μg/kg, 9.1 ± 1.4 mU/L, P = .04; 20 μg/kg, 9.6 ± 2.0 mU/L, P = .12; placebo, 11.3 ± 1.7 mU/L) and GH pulse amplitude (40 μg/kg, 18.8 ± 2.9 mU/L, P = .04; 20 μg/kg, 17.0 ± 3.4 mU/L, P> .05; placebo, 23.0 ± 3.7 mU/L) were also reduced. No differences in glucagon, IGF binding protein-1 (IGFBP-1), acetoacetate, or β-hydroxybutyrate levels were found. During the hyperinsulinemic clamp conditions, no differences in glucose utilization were noted, whereas hepatic glucose production was reduced by rhIGF-I 40 μg/kg (P = .05). Our data demonstrate that in subjects with IDDM, low-dose subcutaneous rhIGF-I leads to a dose-dependent reduction in the insulin level for euglycemia overnight that parallels the decrease in overnight GH levels, but glucagon and IGFBP-1 levels remain unchanged. The decreases in hepatic glucose production during the hyperinsulinemic clamp study observed the following day are likely related to GH suppression, although a direct effect by rhIGF-I cannot be entirely discounted.     

Biomarkers involved in evaluation of platelets function in South-Eastern Romanian patients with hematological malignancies subtypes

At present, various researches presented how subtypes of hematological malignancies are related to stages of the immune response, because the activated immune system represents a promising form in cancer treatment. This study explores the relationship between the adaptive immune system (T cells), and the coagulation system (platelets, platelet membrane glycoproteins, platelets derivate microparticles) which seems to play an important role in host immune defense of patients with acute myeloblastic leukemia (AML) or B cell lymphoma (BCL), 2 of the most common hematological malignancies subtypes.Blood samples (n = 114) obtained from patients with AML or BCL were analyzed for platelet membrane glycoproteins (CD42b, CD61), glycoprotein found on the surface of the T helper cells (CD4⁺), protein complex-specific antigen for T cells (CD3⁺), platelet-derived microparticles (CD61 PMP) biomarkers by flow cytometry, and hematological parameters were quantified by usual methods.In patients with AML, the means of the percentage of the expressions of the molecules on platelet surfaces (CD61 and CD42b, P < .01; paired T test) were lower as compared to both control subgroups. The expression of cytoplasmic granules content (CD61 PMP) had a significantly higher value in patients with AML reported to controlling subgroups (P < .01; paired T test), which is suggesting an intravascular activation of platelets.The platelet activation status was presented in patients with low stage BCL because CD61 and CD42b expressions were significantly higher than control subgroups, but the expression of CD 61 PMP had a significantly decreased value reported to control subgroups (all P < .01; paired T test). T helper/inducer lineage CD4⁺ and T lymphoid lineage CD3⁺ expressions presented significant differences between patients with AML or low stage BCL reported to control subgroups (all P < .01; paired T test).Platelet–lymphocyte interactions are involved in malignant disorders, and CD61, CD42b present on platelet membranes, as functionally active surface receptors mediate the adhesion of active platelets to lymphocytes, endothelial cells, and cancer cells.     

Anti-hypertrophic effect of NHE-1 inhibition involves GSK-3β-dependent attenuation of mitochondrial dysfunction

Although Na⁺–H⁺ exchanger 1 (NHE-1) inhibition has been demonstrated to have anti-hypertrophic effect indirectly through mitochondria, the detailed cellular mechanisms mediating this effect remain elusive. In this study we sought to determine whether NHE-1 inhibition exerts an anti-hypertrophic effect by modulating the mitochondrial permeability transition pore (mPTP) opening through the AMP-activated protein kinase (AMPK)/glycogen synthase kinase 3β (GSK-3β) pathway during hypertrophy in cardiomyocytes. An in vivo model of hypertrophy was induced in male Sprague–Dawley rats by subjecting them to 3, 7 or 28 days of coronary artery ligation (CAL). To induce hypertrophy in vitro, cardiomyocytes isolated from hearts of neonatal (1–3 days) Sprague–Dawley rats were exposed to endothelin-1 (ET-1, 10 nM) in the presence or absence of various treatments. The results demonstrate that CAL affected both AMPKα and GSK-3β phosphorylation in a time-dependent manner. In cultured cardiomyocytes, ET-1 increased phosphorylation of AMPKα₁/α₂^{Ser485/Ser491} and GSK-3β^Ser9 by 80% (P < 0.05) and 225% (P < 0.05) respectively, both of which were significantly blunted by the NHE-1 inhibitor AVE-4890 (5 μM). ET-1-induced phosphorylation of GSK-3β^Ser9 was attenuated by inhibitors of phosphatidylinositol 3-kinase (LY294002), Akt (Akt inhibitor VIII), ERK1/2 (PD98059) and by the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). Prevention of GSK-3β^Ser9 phosphorylation was also accompanied by suppression of ET-1-induced increases in cell surface area, ANP and α-skeletal actin gene expression. Co-immunoprecipitation studies revealed that GSK-3β interacts with components of the mPTP, voltage-dependent anion channel (VDAC) and adenine nucleotide translocase. Furthermore, ET-1 reduced phosphorylation of VDAC, which was associated with both mPTP opening and mitochondrial membrane depolarization. These effects were mimicked by the GSK-3β inhibitor SB216763, thus showing that modulation of mPTP formation is GSK-3β-dependent. In conclusion, anti-hypertrophic effect of NHE-1 inhibition can be mediated through activation of GSK-3β which in turn induces inhibition of mPTP opening due to VDAC phosphorylation.     

Selective block of delayed rectifying potassium current in the rabbit sinoatrial node by a novel class III antiarrhythmic agent MS-551

Summary Electrophysiological actions of MS-551, a novel class III antiarrhythmic agent, were studied using small preparations (0.2 × 0.2 × 0.1mm) of the rabbit sinoatrial (SA) node. MS-551 (0.1–3 µM) exerted a negative chronotropic action by prolonging the action potential duration and diastolic interval. Automaticity was completely suppressed in 5 of 6 preparations at 1–3 µM. Voltage clamp experiments using double microelectrode techniques revealed that MS-551 (0.1–10µM) blocked the delayed rectifying K⁺ current (I_K) in a concentration-dependent manner, and the block was almost saturated at >1 µM, attaining 60% ± 10% at 10 µM (n = 5). The MS-551-sensitive I_K tail (K_d = 0.4µM, Hill r = 1.4,n = 5) had fast and slow components of deactivation. MS-551 (1 µM) reduced the amplitudes of control I_K fast and I_K slow from 20 ± 4 and 11 ± 4 nA to 8 ± 3 and 5 ± 3 nA, respectively (P < 0.01,n = 4). Although the fast deactivation time constant at –60mV remained unaltered (127 ± 12 vs 113 ± 13ms), the slow deactivation time constant was prolonged from 1,117 ± 130 to 1,555 ± 407ms by 1µM MS-551 (P < 0.05). This agent shifted the steady-state activation curve for I_K from –21 ± 2 to –26 ± 4mV and increased the slope factor from 8 ± 1 to 9 ± 1mV (P < 0.05,n = 4). The fully-activated I_K exhibited prominent inward rectification and was reduced by MS-551. These results suggest that (1) MS-551 prolongs the action potential duration and diastolic interval, and exerts a negative chronotropic action by blocking I_K, (2) MS-551 has a higher affinity for the activated than the resting state K⁺ channel, and (3) this agent may either preferentially block one type of I_K, or stabilize a single population of I_K in a subconductance state in the rabbit SA node.     

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.     

Reduced DIDS-sensitive chloride conductance in Ae1-/- mouse erythrocytes

The resting membrane potential of the human erythrocyte is largely determined by a constitutive Cl⁻ conductance  100-fold greater than the resting cation conductance. The 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive electroneutral Cl⁻ transport mediated by the human erythroid Cl⁻/HCO₃⁻ exchanger, AE1 (SLC4A1, band 3) is > 10,000-fold greater than can be accounted for by the Cl⁻ conductance of the red cell. The molecular identities of conductive anion pathways across the red cell membrane remain poorly defined. We have examined red cell Cl⁻ conductance in the Ae1^−/− mouse as a genetic test of the hypothesis that Ae1 mediates DIDS-sensitive Cl⁻ conductance in mouse red cells. We report here that wildtype mouse red cell membrane potential resembles that of human red cells in the predominance of its Cl⁻ conductance. We show with four technical approaches that the DIDS-sensitive component of erythroid Cl⁻ conductance is reduced or absent from Ae1^−/− red cells. These results are consistent with the hypothesis that the Ae1 anion exchanger polypeptide can operate infrequently in a conductive mode. However, the fragile red cell membrane of the Ae1^−/− mouse red cell exhibits reduced abundance or loss of multiple polypeptides. Thus, loss of one or more distinct, DIDS-sensitive anion channel polypeptide(s) from the Ae1^−/− red cell membrane cannot be ruled out as an explanation for the reduced DIDS-sensitive anion conductance.     

Beneficial effect of a moderately energy-restricted diet on fibrinolytic factors in non-obese men

Impaired fibrinolytic activity has been reported in the elderly and is thought to play a role in the etiology of cardiovascular disease, one of the leading causes of death in most Western countries. Since restriction of energy intake has been demonstrated to act beneficially on the aging process in a variety of species, we studied the effect of a 10-week moderately energy-restricted (ER) regimen (80% of habitual) on plasminogen activator inhibitor (PAl) activity, PAl-1 antigen, tissue plasminogen activator (tPA) activity, and tPA antigen in non-obese, middle-aged men. Moreover, the relationship between these fibrinolytic markers and glucose tolerance was investigated. Weight loss in the ER group (n = 16) was considerable (−7.4 ± 1.7 kg, P < .001). Subjects in the control group (n = 8) also lost some weight (−2.1 ± 1.5 kg, P < .01). Fasting glucose levels decreased in the ER group (−0.31 ± 0.48 mmol/L, P < .05), which was correlated with the extent of weight loss (P < .01). Baseline insulin levels at 2 hours after an oral glucose load correlated with baseline PAl activity (P < .001) and PAl-1 antigen levels (P < .001). PAl activity decreased in the ER group (−2.94 ± 2.90 IU/mL, P < .001), particularly in subjects with a high baseline PAl activity (>9 IU/mL). Furthermore, energy restriction led to decreased PAl-1 antigen concentration (P < .05), a nonsignificant increase in tPA activity, and a decrease in tPA antigen concentration (P < .001). All these changes were more clear in subjects with a high baseline PAl activity: These results suggest that 10 weeks of moderate energy restriction has a profibrinolytic effect in non-obese, middle-aged men, at least in subjects with higher baseline PAl activity (>9 IU/mL). Moreover, in line with the suggestion that high PAl activity goes together with insulin resistance, a relationship between insulin concentration after a glucose load and PAl activity was found.     

Effects of short-term acid and aluminum exposure on the parr-smolt transformation in Atlantic salmon (Salmo salar): disruption of seawater tolerance and endocrine status

Episodic acidification resulting in increased acidity and inorganic aluminum (Al_i) is known to interfere with the parr-smolt transformation of Atlantic salmon (Salmo salar), and has been implicated as a possible cause of population decline. To determine the extent and mechanism(s) by which short-term acid/Al exposure compromises smolt development, Atlantic salmon smolts were exposed to either control (pH 6.7–6.9) or acid/Al (pH 5.4–6.3, 28–64 μg l⁻¹ Al_i) conditions for 2 and 5 days, and impacts on freshwater (FW) ion regulation, seawater (SW) tolerance, plasma hormone levels and stress response were examined. Gill Al concentrations were elevated in all smolts exposed to acid/Al relative to controls confirming exposure to increased Al_i. There was no effect of acid/Al on plasma ion concentrations in FW however, smolts exposed to acid/Al followed by a 24 h SW challenge exhibited greater plasma Cl⁻ levels than controls, indicating reduced SW tolerance. Loss of SW tolerance was accompanied by reductions in gill Na⁺,K⁺-ATPase (NKA) activity and Na⁺,K⁺,2Cl⁻ (NKCC) cotransporter protein abundance. Acid/Al exposure resulted in decreased plasma insulin-like growth factor (IGF-I) and 3,3′,5′-triiodo-l-thyronine (T₃) levels, whereas no effect of treatment was seen on plasma cortisol, growth hormone (GH), or thyroxine (T₄) levels. Acid/Al exposure resulted in increased hematocrit and plasma glucose levels in FW, but both returned to control levels after 24 h in SW. The results indicate that smolt development and SW tolerance are compromised by short-term exposure to acid/Al in the absence of detectable impacts on FW ion regulation. Loss of SW tolerance during short-term acid/Al exposure likely results from reductions in gill NKA and NKCC, possibly mediated by decreases in plasma IGF-I and T₃.     

R56865, a Na- and Ca-Overload Inhibitor, Reduces Myocardial Ischemia-Reperfusion Injury in Blood-Perfused Rabbit Hearts

The cardioprotective effects of R56865 were studied in isolated rabbit hearts, blood-perfused with a support rabbit system. The effect on ischemic injury was evaluated by comparing myocardial contracture and contents of ATP catabolites and of lactate during 60 min of normothermic ischemia in untreated hearts (group I) and in hearts treated with 0.63 mg/kg of R56865 starting 20 min before ischemia (group II; n = 5 in each group). R56865 delayed the onset, and decreased the extent of ischemic contracture, but had no effect on the myocardial content of ATP, of its catabolites or of lactate. The effect on reperfusion injury was studied by monitoring left ventricular function during 80-min reperfusion after the 60-min ischemia in three groups (n = 6 in each): an untreated group (group I) and two groups treated with R56865 given either before (group II) or after ischemia (group III). Ultrastructural changes and cellular calcium distribution after reperfusion were also studied. R56865 improved the recovery of function and prevented contracture during reperfusion. Left ventricular end-diastolic pressure was 13.2 ± 2.8 mmHg in group II and 31.3 ± 8.1 mmHg in group III vs 45.0 ± 2.6 mmHg in group I (P < 0.0001 for II vs I; P > 0.05 for III vs I). Left ventricular developed pressure, maximum dP/dt and minimum dP/dt recovered to 71.0 ± 5.4%, 98.9 ± 6.1%, 85.3 ± 4.8% of baseline values, respectively, in group II, to 64.5 ± 3.0% (P > 0.05), 76.8 ± 3.0%, 70.2 ± 4.0% in group III, vs 52.0 ± 6.5%, 58.9 ± 6.9% and 53.6 ± 5.8% in untreated hearts (P < 0.05 for II or III vs I). Coronary flow was 24.5 ± 2.2 ml/min and 19.8 ± 1.8 ml/min in groups II and III vs 14.8 ± 0.7 ml/min (P < 0.05) in the untreated group. On histology the myocardium in hearts treated either before after ischemia was well protected and calcium distribution was almost normal after reperfusion, while in untreated hearts, most of the myocardium displayed irreversible damage accompanied by massive intracellular calcium accumulation. We conclude that R56865 could attenuate Ca²⁺-overload, thereby reducing myocardial ischemia-reperfusion injury after an extended period of ischemia.     

Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state

The sodium pump (Na⁺, K⁺-ATPase, NKA) is vital for animal cells, as it actively maintains Na⁺ and K⁺ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF₃⁻ complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K⁺-antagonism and suggest a route to isoform specific CTSs.

Under physiological conditions, Na⁺,K⁺-ATPase (NKA) actively extrudes three cytoplasmic Na⁺ ions in exchange for two extracellular K⁺ ions per ATP hydrolyzed (see Fig. 1 for a simplified reaction diagram). The established gradients for Na⁺ and K⁺ are pivotal for generating a membrane potential, regulation of cell volume, and providing chemical energy for various secondary active transporters. They are expressed in all animal cells and are finely tuned. In humans, the catalytic α-subunit exists in four isoforms. α1 is ubiquitous and best studied. α2 is most abundant in skeletal and heart muscle, whereas α3 is found in brain cells and α4 in cells of the testis. The α-subunit complexes with a β-subunit (isoforms β1–4 in humans) and a tissue specific regulatory protein FXYD (1–7 in humans) (for a recent general review, see, e.g., refs. 1, 2).Open in a separate windowFig. 1.A reaction diagram of Na⁺,K⁺-ATPase with special emphasis on the backward phosphorylation with P_i and inhibition by CTSs. CTSs can bind to at least three E2P species with different affinities. The states demonstrated to allow high-affinity binding of CTSs appear in purple letters, and those supposed to allow high-affinity binding but not demonstrated are in red letters; the state that allows low-affinity binding is in orange letters. E2P^ATP is the physiological E2P ground state (path A); E2P^Pi can be formed in the backward reaction starting from E2 with P_i (path B). This reaction places Mg²⁺ at the phosphorylation site of NKA but likely to incorporate another Mg²⁺ at site II (E2P^Pi·Mg²⁺). If the backward phosphorylation reaction starts from E2·2K⁺, E2P·2K⁺ will be (transiently) formed in which two K⁺ bind to NKA in E2P with high affinity (E2P·2K⁺_high). CTSs can stabilize a different state, in which two K⁺ are bound with (presumably) low affinity (E2P^Pi·2K⁺_low; path C). Crystal structures available are boxed and phosphate analogs used are shown below the boxes. The crystal structures obtained in this study are highlighted (yellow boxes). PDB ID codes for published crystal structures are: E1∼P·ADP·3Na⁺, 3WGU; E2P^Pi·Mg²⁺(OBN), 4HYT; E2P^Pi·Mg²⁺(DGX), 4RET; E2P^Pi·2K⁺ (BUF), 4RES; E2·P_i·2K⁺, 2ZXE; E2·P_i·2K⁺(OBN), 3A3Y.Cardiotonic glycosides, such as digoxin (DGX) and digitoxin (DTX), are specific inhibitors of NKA and have been prescribed for patients with heart failure for centuries. Canonical cardiotonic glycosides, including ouabain (OBN), the best studied member, have a tripartite structure: a central steroid core, a five-membered or six-membered lactone ring, and a carbohydrate moiety of one to four residues. Each part appears to have a different role in binding. As summarized by Glynn (3), critical features of high affinity cardiac glycosides are: 1) The unsaturated lactone ring attached in the correct configuration at C17; 2) the cis configuration of the AB and CD ring junctions in the steroid nucleus; 3) the presence of a hydroxyl group at C14; and 4) the presence of an appropriate sugar at C3. A wide range of cardiotonic steroids (CTSs), including aglycones, as the sugar at C3 does not necessarily improve the affinity, showing vastly different inhibitory properties, have been developed in order to improve their usability in the clinical setting.Indeed, several new members, such as rostafuroxin (ROS) (4) and istaroxime (IST) (5), now under clinical trials, have distinct chemical structures. ROS is proposed as a potent antihypertensive compound in ouabain-dependent models of hypertension (4). It is reported to be capable of displacing OBN from NKA at a concentration 10 times lower than that expected from its K_I, which is 1,000 times greater than that of OBN (6). IST has only a carbonyl group instead of the unsaturated lactone, and an aminoalkyloxime group instead of the sugar, but shows an inhibitory potency similar to that of digoxin (5). It is reported to have a significant inotropic effect but with a lower risk of causing cardiac arrhythmia compared to digoxin (5). Why these compounds can replace OBN at a much lower concentration than that expected from their binding affinities is paradoxical and addressed in this study.Reflecting their very long history, the accumulated literature on CTSs is huge. Numerous studies report on their inhibitory activities, but they appear rather inconsistent, partly due to differences in experimental conditions (7). It is well established that CTSs preferentially bind to NKA in the E2P ground state from the extracellular face (8). However, the E2P states formed in different routes show distinct properties. In the physiological route, in the presence of Mg²⁺ and Na⁺, the E2P state is reached through phosphorylation by ATP (path A in Fig. 1) and denoted here as E2P^ATP. The E2P state can be reached by backward phosphorylation by P_i in the presence of Mg²⁺ (path B in Fig. 1) and denoted as E2P^Pi [denoted previously as E′2P (9)]. These states show different kinetic properties. In particular, dephosphorylation of E2P^ATP is fast if K⁺ is present, whereas that of E2P^Pi is slow and hardly accelerated by K⁺ (9, 10). As this insensitivity is due to the binding of a second Mg²⁺ to the ATPase in E2P^Pi (10), it would be more appropriate to denote this state as E2P^Pi·Mg²⁺ (Fig. 1). As the affinity of Mg²⁺ in E2P^Pi is ∼0.5 mM (10), the majority of the ATPase molecules phosphorylated by P_i will be in this state. E2P^ATP has a low affinity for Mg²⁺ (not saturated at 6 mM) (10). Therefore, the transmembrane cation binding sites and, accordingly, the CTS-binding cavity will be different in the two E2P states. Indeed, the signal from RH421, a voltage-sensitive styryl dye, is clearly different (9). Then, the inhibitory properties of CTSs will also be different in these two E2P states (type I and II complexes in refs. 11, 12). Furthermore, if phosphorylation by P_i + Mg²⁺ is performed in the presence of K⁺, another type of E2P form with loosely occluded K⁺, termed E2P^Pi·2K⁺, is generated (path C in Fig. 1). This form has a high rate of dephosphorylation (9, 10). OBN is well known to have a much-reduced affinity in the presence of K⁺ (K⁺ antagonism) (e.g., ref. 13), but other CTSs have not been well characterized in this regard. Indeed, Laursen et al., reported that bufalin (BUF) requires K⁺ for high-affinity binding (14). In a recent report (15), the difference in K⁺ antagonism is attributed to the lactone ring. Therefore, systematic measurements on the inhibitory potency in the three E2P states are clearly required, in addition to the one under turnover conditions.Confusion in the literature is apparent even in structural studies. There are several crystal structures published for NKA with bound CTSs: those in E2·P_i·2K⁺ with ouabain at low affinity (2.8-Å resolution) (16), BUF in E2P^Pi·2K⁺ (3.4-Å resolution) (14), and those in E2P^Pi·Mg²⁺ with ouabain at high affinity (3.4 Å) (17) or digoxin (3.9 Å) (14). All of the crystals of the high-affinity complexes are generated in the presence of a high concentration (>100 mM) of Mg²⁺, and indeed, Mg²⁺ is observed to occupy site II for K⁺. Therefore, the E2P state stabilized by CTSs should be denoted as E2P^Pi·Mg²⁺ (Fig. 1). These crystal structures have established that the high affinity of CTSs primarily arises from complementarity between the M5 helix and the α-face of the steroid core, consistent with mutagenesis studies (18–22). However, other than this, there seems to be serious discrepancies between biochemical and structural data. For instance, ouabagenin (OBG), which lacks rhamnose attached to C3, has a 300-fold reduced affinity in binding to NKA in E2P^Pi·Mg²⁺, but Laursen et al. (17) describes that the sugar moiety in ouabain does not interact with the ATPase. Mutagenesis studies have identified residues responsible for isoform dependence (23, 24), but the crystal structure failed to explain why (24). We really do not know if any structural changes are caused by CTS binding to NKA, because no structure is available for the E2P ground state without CTS.We answer this question in this report, as we now have crystal structures of the BeF₃⁻ complex of NKA, an E2P ground state analog, free of CTS. Systematic measurements of the inhibitory properties of various CTSs, including ROS and IST, under four different conditions provide a basis for addressing their structure-activity relationships. One striking finding is that ROS shows a much higher affinity under turnover conditions than in E2P^Pi·Mg²⁺, in marked contrast to OBN. Such differences, as well as the K⁺ antagonism, are nicely explained by the crystal structures of NKA with various CTSs. The crystal structures also explain the isoform dependence unambiguously and suggest ways to confer α2 specificity on CTSs.     

Growth hormone-binding protein related immunoreactivity is regulated by the degree of insulinopenia in diabetes mellitus

OBJECTIVE Derangements in the GH/IGF axis are common in patients with diabetes mellitus. In insulin-dependent diabetes mellitus (IDDM), these disturbances seem to be due to a partial defect in GH action on its own receptor or via a post-receptor defect. In non-insulin-dependent diabetes mellitus (NIDDM), data are limited, and the regulation of the GH receptor (GHR) remains unclear. However, animal studies with diabetic rats demonstrated that the GHR density may be influenced by insulin disposal at the hepatocyte. With respect to this hypothesis we studied the relation between peripheral insulin status and the serum GH-binding protein (GHBP), which reflects indirectly the GHR density in the tissues. Patients with IDDM were compared to a NIDDM group as well as to a group of healthy subjects. DESIGN AND PATIENTS Basal blood samples for the determination of serum GHBP, GH, and IGF-I were obtained from patients with IDDM (n = 27), subjects with NIDDM (n = 112) and healthy controls (n = 42). Insulin, proinsulin, C-peptide and IGF-binding protein 1 (IGFBP-1) serum levels were used to estimate the insulin status in diabetic patients. RESULTS GHBP serum levels were significantly lower in patients with IDDM than in either NIDDM or controls (P < 0.001). Conversely, the IGF-I levels were reduced in both groups of diabetics. A subgroup of hypoinsulinaemic NIDDM patients showed significantly decreased GHBP concentrations (P < 0.05) compared to the NIDDM subgroup with hyperinsulinaemia. Furthermore, GHBP levels were significantly decreased in insulin-treated patients with NIDDM compared to either non-insulin-requiring subjects or normal controls (P < 0.05). A significant direct relation was found between levels of GHBP and total insulin dose (P < 0.01) in patients with IDDM. In the NIDDM group, GHBP was correlated with proinsulin (P < 0.001), C-peptide (P < 0.01), insulin (P < 0.05) and inversely with IGFBP-1 (P < 0.001). Multiple linear regression analysis indicated a significant contribution of proinsulin and IGFBP-1 to the variation of GHBP. CONCLUSIONS Decreased GHBP levels in IDDM as well as in NIDDM correlate with insulinopenia. Since the degree of insulinopenia depends on the capability of the β-cells to secrete proinsulin, C-peptide and insulin, we hypothesize that these hormones at least partially influence the serum level of GHBP. Low GHBP levels may reflect a reduced GH receptor density and a concomitant GH insensitivity, which leads to an impaired IGF generation in insulin-deficient patients.     

The influence of sex, body composition, and nonesterified fatty acids on serum adipokine concentrations

Serum adiponectin concentrations are higher in women than men. The sexual dimorphism for adiponectin has been attributed to the direct effects of testosterone on adipose tissue adiponectin secretion. However, serum testosterone and adiponectin concentrations are generally lower in obese men than lean men, suggesting that sex steroids may not be the only factor that contributes to sex differences in serum adiponectin. The primary objective of this study was to examine the influence of sex, body composition, and nonesterified fatty acids (NEFAs) on serum adiponectin concentrations. Women and men between the ages of 18 and 35 years were consecutively accrued into the study. Sixty-one participants were partitioned into normal-weight (15 female and 16 male) or obese (14 female and 16 male) groups. Blood samples were obtained after a 12-hour fast. Differences between groups were determined by analysis of variance with Tukey-Kramer post hoc testing. Serum adiponectin was 26% higher in women compared with men. Body mass index was associated with total serum adiponectin in men (r = −0.63, P < .05) but not women. Adiponectin was correlated with the homeostasis model assessment index in women (r = −0.56, P < .05) and men (r = −0.58, P < .05) and with NEFAs (r = −0.68, P < .05) in men only. After partitioning men and women into normal-weight and obese groups, serum adiponectin was lower and NEFAs were higher in obese men only. Homeostasis model assessment was similar between obese women and men despite higher NEFAs in the obese men. Leptin and plasminogen activator inhibitor–1 were higher in obese participants but were not associated with serum NEFAs. These results suggest that serum NEFAs may reduce adiponectin concentrations independent of their effects on insulin sensitivity in obese young men.     

Pharmacokinetics,pharmacodynamics and glucose counterregulation following subcutaneous injection of the monomeric insulin analogue [Lys(B28),Pro(B29)] in IDDM

Summary The aim of these studies was to compare the pharmacokinetics, pharmacodynamics, counterregulatory hormone and symptom responses, as well as cognitive function during hypoglycaemia induced by s. c. injection of 0.15 IU/kg of regular human insulin (HI) and the monomeric insulin analogue [Lys(B28),Pro (B29)] (MI) in insulin-dependent-diabetic (IDDM) subjects. In these studies glucose was infused whenever needed to prevent decreases in plasma glucose below 3 mmol/l. After MI, plasma insulin increased earlier to a peak (60 vs 90 min) which was greater than after HI (294±24 vs 255±24 pmol/l), and plasma glucose decreased earlier to a 3 mmol/l plateau (60 vs 120 min) (p<0.05). The amount of glucose infused to prevent plasma glucose falling below 3 mmol/l was three times greater after MI than HI (293±26 vs 90±25 mol · kg^–1 · 60–375 min^–1, p<0.05). After MI, hepatic glucose production was more suppressed (0.7±1 vs 5.9±0.54 mol · kg^–1 · min^–1) and glucose utilization was less suppressed than after HI (11.6±0.65 vs 9.1±0.11mol · kg^–1 · min^–1) (p<0.05). Similarly, plasma NEFA, glycerol, and -OH-butyrate were more suppressed after MI than HI (p<0.05), whereas plasma lactate increased only after MI, but not after HI. Responses of counterregulatory hormones, symptoms and deterioration in cognitive function during plasma glucose plateau of 3 mmol/l were superimposable after MI and HI (p=NS). Post-hypoglycaemia hyperglycaemia was greater after MI than HI (at 480 min 12.1±1 vs 11±1 mmol/l) because of greater hepatic glucose production during insulin waning which occurred at least 135 min earlier with MI as compared to HI (p<0.05). It is concluded that counterregulatory hormones, symptoms and deterioration in cognitive function during hypoglycaemia respond similarly after MI and HI. The biological effect of MI appears greater than that of HI for at least 4 h after the s.c. injection and appears as a good candidate for achieving optimal post-prandial glucose control in IDDM.Abbreviations HI Human insulin - MI monomeric insulin - NEFA non-esterified fatty acid - HGO hepatic glucose production rate - -OH-butyrate -hydroxy-butyrate - IDDM insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus     

Different concentrations of various radiopharmaceuticals in the two main liver lobes: a preliminary study in clinical patients

Background At clinical scintigraphic examinations of the abdomen using single photon emission computed tomography (SPECT), we have observed a different distribution between the left and right main liver lobes of various radiopharmaceuticals. This was studied retrospectively in clinical patients.Methods Examinations with [¹²³I]-metaiodobenzylguanidine MIBG; (n = 19), a ^99mTc-labelled monoclonal antibody against granulocytes (n = 18), and ¹¹¹In-pentetreotide (n = 26) were assessed. There was no known history of, or risk factor for liver disease, and all lobes showed a uniform activity distribution. Twenty healthy volunteers underwent consecutive examinations with ^99mTc-dimethyliminodiacetic acid (HIDA). The activity ratios between the left and right main liver lobes were calculated from the transverse tomographic (SPECT) sections.Results The left : right lobar activity ratio for [¹²³I]-MIBG was (mean ± SD) 1.25 ± 0.21 (null hypothesis = 1.00; P < 0.001); for the antibody, acquisition after 3–5 h was 0.98 ± 0.06 (NS) and after 20–24 h, 0.99 ± 0.11 (NS); for ¹¹¹In-pentetreotide, 0.90 ± 0.09 (P < 0.001); for ^99mTc-HIDA, immediate acquisition, 0.68 ± 0.12 (P < 0.001) and acquisition at 7 min, 0.66 ± 0.12 (P < 0.001).Conclusions The differences in tracer uptake between the liver lobes cannot be caused only by differences in blood flow. One explanation of the higher uptake of [¹²³I]-MIBG by the left lobe may be a greater presence of catecholamines and a higher sympathetic nerve density in this liver portion. Consequently, there may be a functional difference between the two main liver lobes.     

Nutritional risk factors for the development of hypertension in diabetic patients

The aim of this cross-sectional study was to determine the significant limitations, sensitivity, specificity, partial correlations, and odds ratios of nutrient intake in patients with and without hypertension with Type 2 diabetes mellitus.Diabetic patients (n=220) with clinical diagnosis of hypertension and diabetic patients (n=230) without hypertension were included in this study. The questionnaire form included a list of 65 food items formed from five main food groups (grain, meat and alternatives, dairy products, vegetables–fruits and fat) and 25 dietary habits.When both groups were compared and analyzed by logistic regression, black tea consumption (OR=0.823, P<.001), vegetables–fruits scores (OR=0.853, P<.001), triglycerides (OR=0.726, P<.05), waist-to-hip ratio (WHR) (OR=0.777, P<.01) and high-density lipoprotein cholesterol (HDL-C) (OR=0.526, P<.001) made significant differences. In ROC curves, the area under the curve of black tea (0.921), vegetables–fruits (0.906), triglycerides (0.889), WHR (0.881) and HDL-C (0.820) provided high accuracy to distinguish between patients with and without hypertension (P<.001).In diabetic patients without hypertension, significant partial correlations were observed between blood pressure and dairy products (systolic: r=)0.14; diastolic: r=)0.14, P<.05), vegetables–fruits groups (systolic: r=)0.18; diastolic: r=)0.17, P<.01) and black tea intake (systolic: r=)0.23; diastolic: r=)0.22, P<.001).It has been found that higher intake of black tea and vegetables–fruits consumption in diabetic patients protect against developing hypertension.     

Blood pressure changes in diabetes in urban Tanzania

Little is known of the natural history of blood pressure (BP) levels in diabetic patients from sub-Saharan Africa. BP levels were therefore recorded in such patients in Dar es Salaam, Tanzania, over 2, 5, and 7 years. Hypertension was found in 5% of insulin-treated diabetes mellitus (IDDM) and 29.2% of non-insulin-dependent diabetes mellitus (NIDDM) patients at presentation with diabetes. Hypertension developed in a further 2 IDDM (3.7%) and 27 NIDDM (15.6%) patients at 2 years, and in 3 IDDM (13.0%) and 9 NIDDM (9.8%) patients at 5 years. Seven NIDDM (18.4%) patients had developed hypertension by 7 years. In NIDDM patients with normal BP initially, the mean systolic BP rose from 131 to 141 mmHg (P<0.001) 2 years later (n=146); from 131 to 138 mmHg (P<0.001) for those followed for 5 years (n=82); and from 131 to 138 mmHg (P<0.05) for those followed for 7 years (n=31). The mean diastolic BP was 83 mmHg initially and 84 mmHg (NS) for those followed for 2 years (n=146). There was no observed rise in mean diastolic BP at 5 or 7 years of follow-up. In IDDM patients without hypertension, only the systolic BP rose significantly by 5 years, from 124 to 132 mmHg (P<0.001;n=20). These changes were independent of age, sex, body mass index, and proteinuria. We conclude that: (1) in black Tanzanians, as in other ethnic groups, it is likely that hypertension is significantly associated with diabetes; (2) rates of hypertension and BP levels continue to increase with time, particularly in NIDDM subjects; and (3) BP measurements should be a regular feature of diabetes care in the African diabetic population as in other populations.