Inverse correlation of plasma leptin and soluble transferrin receptor levels in β-thalassemia patients

The aim of the study was to investigate the association of leptin with hematological parameters in beta-thalassemia patients in Greece. We measured plasma levels of soluble transferrin receptor (sTfR) and leptin by enzyme-linked immunosorbent assay (ELISA) in 40 beta-thalassemia patients (21 transfusion dependent and 19 not transfused or sporadically transfused), in 20 beta-thalassemia carriers, and in 30 healthy individuals (HI). The percentage of reticulocytes (RET) was measured by the NE 9500 Sysmex automated method. Body mass index (BMI) was calculated by dividing body weight (kg) by square height (m). Endocrine measurements including sex hormones were also determined. sTfR concentrations were significantly higher in both transfusion-dependent (females 10.5+/-2.9, males 9.1+/-3.1) and non-transfusion-dependent patients (females 15.8+/-5.4, males 19.8+/-13.7) as compared to carriers (females 3.1+/-2.5, males 3.8+/-1.8) and to HI (females 1.5+/-1.2, males 2.5+/-2.1). Leptin levels were lower both in female and in male transfusion-dependent patients (0.5+/-0.3 and 1.2+/-1, respectively) and in non-transfused males (1.9+/-2) compared to carriers (females 7.9+/-2.7, males 13.1+/-9.1) and HI (females 14.6+/-6, males 7.5+/-3). There was a negative correlation between leptin and sTfR levels in transfused patients (R=-0.61, p<0.05). A stronger negative correlation (R=-0.7, p=0.006) was found in hypogonadic men and women with beta-thalassemia. These findings enhance previous results indicating that leptin may play some role in hematopoiesis and could associate the pathophysiology of thalassemic patients with the triggering effect of leptin in reproductive ability.     

Prevention byα-difluoromethylornithine of skin carcinogenesis and immunosuppression induced by ultraviolet irradiation

Summary Administration of-difluoromethylornithine (DFMO) to mice was found to inhibit both the cutaneous carcinogenesis and the immunosuppression induced by ultraviolet B (UVB) irradiation. BALB/cAnNTacfBR mice were given 1% F₂MeOrn in their drinking water throughout the experiment. After 3 weeks, mice received UVB irradiation consisting of five 30-min exposures per week to banks of six FS40 Westinghouse sunlamps. In the photocarcinogenesis study, mice received a total dose of approximately 1273 kJ m^–2. Skin cancer incidence in UV-irradiated mice was 38% 28 weeks after the first UV exposure; DFMO reduced this incidence to 9% (P= 0.025, log-rank test). Although DFMO has been demonstrated to be chemopreventive of chemical carcinogenesis, this is the first report that it is effective against cancers induced by a physical carcinogen. The immunosuppression induced by UVB irradiation prevents the host from rejecting antigenic, syngeneic UV-induced tumors, which normal mice can reject. The level of immunosuppression in UV-irradiated mice treated with DFMO was measured by a passive-transfer assay. Splenocytes from UV-irradiated mice to naive mice prevented the recipients from rejecting 20/24 UV-induced tumor challenges, whereas splenocytes from UV-irradiated mice treated with DFMO did not prevent recipients from rejecting such challenges (2/24 grew). The difference between these values was significant (P<0.001, two-sample test for binomial proportions). Phenotypic analysis of splenocytes used in the passive transfer, using a biotin-avidin-immunoperoxidase technique, revealed that DFMO treatment prevented the reduction of Ia expression normally seen in UV-irradiated mice. Thus, administration of DFMO reduced skin carcinogenesis and immunosuppression induced by UVB irradiation.Abbreviations IL-1, -2 etc. interleukin-1, -2 et - DFMO -difluoromethylornithine     

Effects of bile salts on plasma concentration of β-endorphin-like immunoreactivity in men

The effects of bile salts on the release of -endorphin-like immunoreactivity ( -END-LI) were investigated in men using a specific radioimmunoassay developed by the authors. Plasma -END-LI was determined after extraction by the acid-acetone method (recovery: 73±5%). Oral administration of 400 mg of sodium taurocholate caused a rise in plasma -END-LI from 9.9±0.5 pmol/liter to 21.3±1.2 pmol/liter after 30 min and 18.1 ±0.5 pmol/liter after 60 min, with return to the initial value after 90 min. Oral administration of chenodeoxycholic acid (CDCA) also increased plasma -END-LI from a basal level of 8.4±0.7 pmol/liter to 18.7±0.8 pmol/liter after 30 min. Oral administration of ursodeoxycholic acid (UDCA) increased plasma -END-LI from 7.3±0.3 pmol/liter to 30.6±0.2 pmol/liter after 30 min. In gel chromatography, the -END-LI released after UDCA administration separated into two components, which eluted in the same positions as human -lipotropin and human -endorphin, respectively. These results suggested that bile salts may participate the release of -END-LI.     

Antioxidant activity and protection of pancreatic β-cells by embelin in streptozotocin-induced diabetes

Objectives: The aim of the present study was to evaluate the antioxidant potential of embelin in streptozotocin‐induced diabetes. Methods: Diabetes was induced in rats fasted overnight by the administration of a single dose of streptozotocin, and analyzed for blood, serum, and biological and histological pancreatic tissue parameters in intact control, diabetic, and embelin‐treated diabetic rats (n = 9) at the dose levels of 15, 25, and 30 mg/kg/day for 21 days. Results: Diabetes caused highly significant abnormalities in blood, serum, and pancreatic tissue biochemical parameters. Embelin and glibenclamide administration to diabetic rats caused a highly significant decline in the blood glycated hemoglobin and serum glucose levels and nitric oxide activity, with a concomitant increase in the serum insulin level (P < 0.001). Furthermore, embelin and glibenclamide treatment increased the pancreatic antioxidant enzyme status (superoxide dismutase, catalase, reduced glutathione, glutathione peroxidase, glutathione S‐transferase, and ascorbic acid), and also decreased the thiobarbituric acid reactive oxygen species contents (P < 0.001). The histoarchitecture of the diabetic rats typically showed a degenerated pancreas with reduced β‐cell counts, while embelin treatment was shown to significantly regenerate islet cells. Conclusion: The study proves the potent antioxidant activity of embelin, which has been found to be effective in managing severe hyperglycemia.     

Inhibition of succinate dehydrogenase and β-hydroxybutyrate dehydrogenase activities by methylmalonate in brain and liver of developing rats

Summary The effects of methylmalonate (MMA) on succinate dehydrogenase (SDH) and -hydroxybutyrate dehydrogenase (HBDH) activities in brain and liver of 15-day-old rats were studied. The apparentK _m of SDH for succinate was 0.45 mmol/L in brain and 0.34 mmol/L in liver. MMA inhibited the enzyme activity in both tissues withK _i values of 4.5 mmol/L and 2.3 mmol/L in brain and liver, respectively, and the inhibition was of the reversible competitive type. The calculatedK _m for HBDH with -hydroxybutyrate as substrate was 1.26 mmol/L in brain and 0.36 mmol/L in liver. MMA inhibited the enzyme with aK _i value of 0.015 mmol/L in brain and 0.275 mmol/L in liver. These results are probably relevant to our understanding of cerebral metabolism in methylmalonic acidaemic children, especially during ketoacidotic and hypoglycaemic crises, and may be related to the pathogenesis of cerebral dysfunction of methylmalonic acidaemia.     

Desensitization pattern of cardiac β-adrenoceptor subtypes by prolonged in vivo infusion of pindolol and celiprolol in rats

Summary The regulatory effects of pindolol and celiprolol on cardiac -adrenoceptor density were studied in vivo in order to assess the subtype selectivity of their partial agonistic activity (PAA). The substances were continuously administered to rats for 1 week by means of implanted osmotic minipumps. The density of -adrenoceptor subtypes were estimated from ICYP saturation binding experiments performed on cardiac ventricular plasma membranes in the presence of a highly selective antagonist (CGP 20172 A or ICI 118,551). Both antagonists were employed at concentrations as high as to block one subtype only without affecting the complementary subtype. For control purposes, rats were also treated with isoprenaline (0.4 mg/kg/h) and propranolol (0.15 mg/kg/h), or vehicle. Pindolol (0.036 mg/kg/h) and celiprolol (0.36 mg/kg/h) reduced the density of ventricular ₂-adrenoceptors by 46% and 23%, respectively, which — in the case of pindolol — was significant when compared to the non-treated controls. Both compounds, however, produced a small, but distinct increase in the number of ₁-adrenoceptors by approximately 26%. This finding is in contrast to the propranolol-induced upregulation of both ₁- and ₂-adrenoceptors by approximately 80%. Since supramaximal doses of each drug were administered, a significant smaller increase of ₁-adrenoceptors by pindolol and celiprolol —as compared to the increase produced by propranolol — can be interpreted as evidence for a PAA of pindolol and celiprolol on ₁-adrenoceptors as well. Isoprenaline as a full agonist caused a marked loss of of both -adrenoceptor subtypes. Although it exhibits equal affinity at both subtypes the decrease amounted to 80% of the ₂- but only to 54% of the ₁-adrenoceptors density. This indicates that the down-regulation of cardiac -adrenoceptors in general seems to be more pronounced at the ₂-than at the ₁-adrenoceptors population. We conclude that the subtype desensitization pattern of agents with intrinsic activity precludes the determination of subtype-selectivity, since ₁- and ₂-adrenoceptors appear to differ in their sensitivity presumably as a result of subtype specific baseline desensitization produced by endogenous catecholamines.     

Induction of glycogenolysis in cultured Ewing's sarcoma cells by dopamine and β-adrenergic agonists

Summary This study describes hormonal regulation of glycogen metabolism in Ewing's sarcoma cells. ³H-Glycogen synthesized in cultured Ewing's sarcoma WE-68 cells from ³H-glucose was hydrolyzed in a concentration-dependent manner by various catecholamines. The order of potency for the glycogenolytic effects of catecholamines was isoproterenol dopamine > norepinephrine > epinephrine. The concentrations giving half-maximal effectiveness (EC₅₀) were about 2×10^-8 M, 3×10^-8 M, 8×10^-8 M, and 5×10^-7 M for isoproterenol, dopamine, norepinephrine, and epinephrine, respectively. Higher concentrations of each of the catecholamines were necessary to elicit EC₅₀ stimulation of cyclic AMP production in Ewing's sarcoma cells. Glycogenolysis induced by dopamine was blocked by chlorpromazine, a dopamine D₁-receptor antagonist, but not by haloperidol, a dopamine D₂-receptor antagonist. The glycogenolytic action of norepinephrine was markedly reduced by propranolol, a -adrenoreceptor antagonist, and was not affected by yohimbine, an -adrenoreceptor antagonist. In addition, chlorpromazine also antagonized the glycogenolytic response to norepinephrine. Dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, and the diterpene forskolin were also found to induce ³H-glycogen hydrolysis. Our data indicate that cate-cholamines exert their glycogenolytic effects in Ewing's sarcoma cells by stimulation of cyclic AMP formation via -adrenergic recepetors and dopamine D₁-receptors.     

Association of β-cell function and insulin sensitivity with fasting and 2-h plasma glucose in a large Chinese population

Aim: Our aim was to provide a quantitative analysis of the changes in the principal determinants of insulin sensitivity and secretion in relation to fasting plasma glucose (FPG) or 2‐h plasma glucose (2h PG) in a Chinese population with a wide range of glucose tolerance. Methods: A total of 5728 adults spanning the entire range of glucose tolerance were included. Insulin sensitivity was measured by Matsuda insulin sensitivity index (ISI_M) and homeostasis model assessment of 1/homeostasis model assessment of insulin resistance (HOMA‐IR). β‐Cell function adjusted by insulin sensitivity was assessed from disposition index (DI) at early‐phase DI₃₀ and total DI₁₂₀. The exponential curve was established as the best fit for the relationship between insulin sensitivity or β‐cell function and FPG or 2h PG. Results: Relative to the trend classified as increasing 2h PG, hepatic insulin sensitivity and insulin secretion showed a decreasing trend to a substantial degree as FPG increased. A 1 mmol/l increase in FPG and 2h PG concentration was associated with a ?22 and ?21% decline in ISI_M, ?16 and ?4% in 1/HOMA‐IR, ?38 and ?35% in DI₃₀ and ?36 and ?26% in DI₁₂₀. The decay constant of ISI_M and DI₃₀ in IFG or ISI_M, 1/HOMA‐IR, DI₃₀ and DI₁₂₀ in IGT was lower than that in normal glucose tolerance. Significant interactions between sex and glucose levels determining DI were found. Conclusions: We conclude that impairment of insulin sensitivity and insulin secretion contributes to both FPG or 2h PG hyperglycaemia in a Chinese population, but that the decline in insulin secretion is more pronounced with increasing fasting than 2h PG.     

Acid β-mannosidase of human plasma: Influence of age and sex on enzyme activity

Summary Optimum conditions for assay of human plasma -mannosidase activity were determined. Maximum activity was observed at pH 3.0–3.4, the apparentK _m was 0.9 mmol L^–1 and enzymatic hydrolysis was linear for at least 60 min. Assays were performed on plasma from two siblings with the recently described condition -mannosidosis and their parents and controls. Both -mannosidosis patients showed a marked deficiency of -mannosidase activity. Control samples showed no variation between sexes (p>0.05) but enzyme activity varied significantly with age (p<0.05). Highest activity was observed in the 0–1 year age group, activity then decreasing to adulthood, Both mother and father of the affected individuals showed reduced activity (34% and 53%, respectively) compared to age-matched controls. This suggests that heterozygote detection in this condition is possible using plasma, provided the variation of activity with age is taken into consideration.     

Repair of diverse diabetic defects of β-cells in man and mouse by pharmacological glucokinase activation

Glucokinase activators (GKAs) are being developed and clinically tested for potential antidiabetic therapy. The potential benefits and limitations of this approach continue to be intensively debated. To contribute to the understanding of experimental pharmacology and therapeutics of GKAs, we have tested the efficacy of one of these agents (Piragliatin) in isolated islets from humans with type 2 diabetes mellitus (T2DM), from mice with glucokinase (GK) mutations induced by ethyl-nitroso-urea (ENU) as models of Maturity Onset Diabetes of the Young linked to GK and Permanent Neonatal Diabetes Mellitus linked to GK (PNDM-GK) and finally of islets rendered glucose insensitive by treatment with the sulphonyl urea compound glyburide in organ culture. We found that the GKA repaired the defect in all three instances as manifest in increased glucose-induced insulin release and elevated intracellular calcium responses. The results show the remarkable fact that acute pharmacological activation of GK reverses secretion defects of β-cells caused by molecular mechanism that differ vastly in nature, including the little understood multifactorial lesion of β-cells in T2DM of man, the complex GK mutations in mice resembling GK disease and acute sulphonylurea failure of mouse β-cells in tissue culture. The implications of these results are to be discussed on the theoretical basis underpinning the strategy of developing these drugs and in light of recent results of clinical trials with GKAs that failed for little understood reasons.     

Inhibition of experimental pulmonary metastasis in mice by β-cyclodextrin-benzaldehyde

Summary The effect of -cyclodextrin-benzaldehyde (CDBA) on experimental pulmonary metastasis in C3H/He mice was examined. In an in vitro assay, the growth of RCT(+) cells was inhibited by 1200 g/ml CDBA using unrenewed media, and by 600 g/ml CDBA in that using daily renewed media. When mice were treated daily with CDBA, 3 weeks later the number of lung nodules developing after i.v. injection of 1×10⁶ RCT(+) cells was significantly decreased in a dose-dependent manner, i.e., 73.8%, 85.6%, and 95.7% inhibition was observed following 0.5, 5, and 25 mg CDBA/mouse per day p.o. administration, respectively. The same mice showed almost as much natural killer (NK) activity as normal mice. Therefore, experiments were designed to evaluate the effect of CDBA on the NK activity of tumor-free mice whose immunity had been suppressed by 5-fluorouracil (5FU). Injections of 5FU only suppressed this activity to about 50% of normal mice, but the combined treatment with CDBA negated the suppressive effect of 5FU on NK activity. The results suggested that the inhibition of experimental pulmonary metastasis might be induced by the possible combined effects of CDBA; that is, the direct inhibition of tumors and the augmentation of NK cell activity.     

Construction of pETNF-P16 plasmid and its expression properties in EC9706 cell line induced by X-ray irradiation

AIM:Recombined plasmid pETNF-P16 was constructed toinvestigate its expression properties in esophagealsquamous carcinoma cell line EC9706 induced by X-rayirradiation and the feasibility of gene-radiotherapy foresophageal carcinoma.METHODS:Recombined plasmid pETNF-P16 was constructedand transfected into EC9706 cells with lipofectamine.ELISA,Western blot,and immunocytochemistry were performedto determine the expression properties of pETNF-P16 inEC9706 after transfection induced by X-ray irradiation.RESULTS:Eukaryotic expression vector pETNF-P16 wassuccessfully constructed and transfected into EC9706 cells.TNFα expressions were significantly increased in thetransfected cells after different doses of X-ray irradiation thanin those after 0Gy irradiation(1 192.330-2 026.518 pg/mL,P<0.05-0.01),and the TNFα expressions and P16 weresignificantly higher 6-48 h after 2 Gy X-ray irradiation(358.963-585.571 pg/mL,P<0.05-0.001).No P16 expressionwas detected in normal EC9706 cells.However,there wasstrong expression in the transfected and irradiation groups.CONCLUSION:X-ray irradiation induction could significantlyenhance TNFα and P16 expression in EC9706 cellstransfected with pETNF-P16 plasmid.These results mayprovide important experimental data and therapeuticpotential for gene-radiotherapy of esophageal carcinoma.     

Determination of rivaroxaban,apixaban and edoxaban in rat plasma by UPLC–MS/MS method

To establish a rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for the determination of rivaroxaban, apixaban and edoxaban in rat plasma. The analytes and the internal standard (diazepam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1 mm × 50 mm, 1.7 μm) using gradient elution with a mobile phase of acetonitrile and 0.1 % formic acid in water at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 436.1 → 145.1 for rivaroxaban, m/z 460.0 → 443.1 for apixaban, m/z 548.2 → 366.1 for edoxaban and m/z 285.2 → 193.1 for diazepam (IS) using a positive electrospray ionization interface. The method was validated over a concentration range of 1.0–200 ng/mL for rivaroxaban, 1.0–100 ng/mL for apixaban and 1.0–500 ng/mL for edoxaban. Total time for each chromatograph was 3.5 min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations <10.5 % and the accuracy values ranged from ?9.9 to 11.3 %. The method was successfully applied to a pharmacokinetic study of rivaroxaban, apixaban and edoxaban in rats.     

Antiaging gene Klotho enhances glucose-induced insulin secretion by up-regulating plasma membrane levels of TRPV2 in MIN6 β-cells

Klotho is a recently discovered antiaging gene. Klotho is expressed in mouse pancreatic islets and in insulinoma β-cells (MIN6 β-cells). The purpose of this study was to investigate whether Klotho plays a role in the regulation of insulin secretion in MIN6 β-cells by overexpression and silencing of Klotho. It is interesting that overexpression of Klotho increased glucose-induced insulin secretion in MIN6 β-cells. Overexpression of mouse Klotho protein also significantly increased plasma membrane levels of transient receptor potential V2 (TRPV2), calcium entry, and the glucose-induced increase in intracellular calcium. On the other hand, knockdown of Klotho by siRNA significantly decreased plasma membrane levels of TRPV2 and attenuated glucose-induced calcium entry and insulin secretion. Tranilast, a selective inhibitor of TRPV2, abolished the promoting effects of overexpression of Klotho on glucose-induced calcium entry and insulin secretion in MIN6 cells. Thus, TRPV2 lies in the downstream of Klotho in the regulation of glucose-induced insulin secretion. This study demonstrated, for the first time, that Klotho may enhance glucose-induced insulin secretion by up-regulating plasma membrane levels of TRPV2 and thus glucose-induced calcium responses. These findings reveal a previously unidentified role of Klotho in the regulation of glucose-induced insulin secretion in MIN6 β-cells.     

Regulation of angiotensinogen gene expression and protein in neonatal rat cardiac fibroblasts by glucocorticoid and β-adrenergic stimulation

We previously demonstrated the presence of components for a renin-angiotensin system in fibroblasts cultured from neonatal rat ventricles, the regulation of expression of which has not been studied. Since glucocorticoids and β-adrenergic stimuli have been implicated in cardiac hypertrophy, and function as regulators of the circulating renin-angiotensin system, we examined the effects of dexamethasone and isoproterenol on angiotensinogen mRNA levels and protein secretion in cultured neonatal rat cardiac fibroblasts. Treatment of cardiac fibroblasts for 8 h with 10 μmol/l isoproterenol or 100 nmol/l dexamethasone increased angiotensinogen mRNA levels by 246 ± 7% and 1406 ± 207%, respectively. Over 24 h, dexamethasone and isoproterenol increased angiotensinogen secretion by 148 ± 32% and 123 ± 26%, respectively. Angiotensin II, which has been reported to be a positive regulator of angiotensinogen synthesis and secretion in liver, markedly attenuated the effects of dexamethasone and isoproterenol on angiotensinogen mRNA expression and secretion. In the presence of 1 μmol/l angiotensin II, the stimulation in angiotensinogen secretion observed with dexamethasone and isoproterenol was decreased by 62% and 76%, respectively. The negative feedback of angiotensin II on angiotensinogen expression was primarily mediated through the type one angiotensin II (AT₁) receptor (IC₅₀ = 0.30 ± 0.02 nmol/l). In summary, results from this study demonstrate that angiotensinogen mRNA levels and protein secretion in cardiac fibroblasts are positively regulated by glucocorticoid and β-adrenergic stimulation. In addition, angiotensinogen production by cardiac fibroblasts is under negative feedback control of angiotensin II. Received: 28 October 1999, Returned for revision: 24 November 1999, Revision received: 14 January 2000, Accepted: 26 January 2000     

Cardiovascular effects of splenomegaly and splenectomy in β-thalassemia

Splenomegaly is common in -thalassemia, bearing some particular hemodynamic features, while splenectomy is an established therapeutic intervention in these patients. Their effects, however, on systemic hemodynamics and thalassemia heart disease have not yet been assessed. The study included 32 consecutive patients, 13 with thalassemia major (TM) and 19 with thalassemia intermedia (TI), aged 23.4±4.2 years, requiring splenectomy. Assessment was performed before and 6 months after splenectomy and included hematological profile and resting echocardiography; total blood volume was also measured in 10 of 32 cases. Preoperative echocardiographic data were compared with those of 34 controls. Preoperative left ventricular diameters and mass, cardiac index, and systolic pulmonary artery pressure were all significantly higher in patients compared to controls (p<0.001), but did not differ significantly between TM and TI patients. Postoperatively, the mean hemoglobin level increased from 8.1±0.6 to 9.1±0.4 g/dl (p<0.001), total blood volume index declined from 2847±332 to 2310±276 ml/m² (p<0.001), blood transfusions were discontinued in 80% of TI patients and mean 6-monthly transfusion requirements in TM patients were reduced from 28±5 to 22±4 units (p<0.001). However, cardiac parameters were not significantly modified. It seems that left ventricular remodeling, high output state, and increased pulmonary artery pressure characterize both TM and TI patients who require splenectomy. Although these abnormalities remain unchanged after splenectomy, the removal of the spleen may contribute to the prevention of further cardiac damage by ameliorating the patients hematological status and reducing their transfusion needs.