Effect of insulin on lipolysis in adipose tissue of rats of different ages

Several authors have not been able to find any antilipolytic effect of insulin in adipose tissue "in vitro". We investigated the possible role of cell size and/or age of donors on this phenomenon. The lipolytic rates (glycerol release per cell) were lower in the small cells of the 4-6 weeks old rats than in the larger cells of the 25-30 weeks old animals; however, the difference disappeared when the data were expressed per unit of cell surface area. Insulin (0.5-50 ng/ml) failed to inhibit both maximally and submaximally noradrenaline stimulated lipolysis in the adipocytes of the young rats, but its antilipolytic action was fully restored by using glucose-free medium. Therefore, at our experimental conditions, a glucose dependent factor, possibly involving the preferential hydrolysis of newly synthetized triglycerides, seems to blunt or to mask the insulin induced inhibition of glycerol release. Relatively higher rates of glucose metabolism and a lower lipolysis in small fat cells might explain the difference in the action of insulin on glycerol release in the adipose tissue of young rats as compared to the older ones.     

Amphiphysin II (SH3P9; BIN1), a Member of the Amphiphysin/Rvs Family, Is Concentrated in the Cortical Cytomatrix of Axon Initial Segments and Nodes of Ranvier in Brain and around T Tubules in Skeletal Muscle

Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homologue of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. We report here the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasmamembrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrin_G), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function.     

A simple test for predisposition to LDL oxidation based on the fluorescence development during copper-catalyzed oxidative modification

When human low density lipoprotein (LDL) obtained from 10 volunteers was incubated in air at 37 degrees C in the presence of various concentrations of copper, an increase in fluorescence was observed with emission maximum at 430 nm when excitation was performed at 360 nm. The fluorescence increase was inhibited by ethylenediamine tetraacetic acid and by 4-methyl-2,6-di-tert-butylphenol. The fluorescence was found to be tightly bound to the protein moiety. Furthermore, Cu2+ modification of LDL was associated with a decrease in the reactive amino groups of apolipoprotein B and in the uptake of the lipoprotein by rabbit fibroblasts. Under our conditions, the fluorescence increase showed two consecutive periods; an inhibition period during which the fluorescence increased only weakly, and a propagation period with a rapid increase in fluorescence that was linear for at least 5 h. Both periods were influenced by copper concentration. The study also shows that the extent of fluorescence generated upon LDL oxidation varied greatly in the volunteers. Thus, while the results demonstrate that the fluorescence increase may likely monitor the extent of the apoB derivatization, the calculation of the fluorescence development rate of the propagation period together with the duration of the inhibition period may constitute a quantitative measurement of the susceptibility of apoB to be derivatized.     

Effect of Kainic Acid, Glutamate, and Aspartate on CO2 Production by Goldfish Tectal Slices

For a study of the excitatory effect of kainate, glutamate, and aspartate in the goldfish optic tectum, these substances were tested on the production of CO2 from radioactive glucose in tectal slices incubated in Krebs-Ringer medium for fish. Kainate increased the rate of CO2 production for up to 30 min in a dose-related manner, the effect being maximum at 0.1 mM concentration and decreasing at higher doses. The effect was blocked by ouabain (1 mM) as well as by the substitution of choline for Na+ in the incubation medium. Glutamate and aspartate exerted a less pronounced excitatory effect on CO2 production at higher concentration than kainate. This effect was also abolished by ouabain. Glutamate, added to the medium at a concentration at least 100-fold higher than kainate, partially reversed the increase in CO2 production induced by kainic acid. No similar effect was noticed for aspartate. The supposed glutamate antagonists glutamic acid diethylester (1 mM) and proline (5 mM) did not affect the excitatory action of kainic acid or exert an antagonistic effect towards glutamate. At higher concentration (10 mM) glutamic acid diethylester increased CO2 production, an effect that was, however, ouabain insensitive. Methyltetrahydrofolic acid (1 mM), a substance reported to compete for the kainate receptor, did not inhibit the effect of kainic acid or increase CO2 production.     

An Intracellular Threonine of Amyloid-β Precursor Protein Mediates Synaptic Plasticity Deficits and Memory Loss

Mutations in Amyloid-ß Precursor Protein (APP) and BRI2/ITM2b genes cause Familial Alzheimer and Danish Dementias (FAD/FDD), respectively. APP processing by BACE1, which is inhibited by BRI2, yields sAPPß and ß-CTF. ß-CTF is cleaved by gamma-secretase to produce Aß. A knock-in mouse model of FDD, called FDD_KI, shows deficits in memory and synaptic plasticity, which can be attributed to sAPPß/ß-CTF but not Aß. We have investigated further the pathogenic function of ß-CTF focusing on Thr⁶⁶⁸ of ß-CTF because phosphorylation of Thr⁶⁶⁸ is increased in AD cases. We created a knock-in mouse bearing a Thr⁶⁶⁸Ala mutation (APP^TA mice) that prevents phosphorylation at this site. This mutation prevents the development of memory and synaptic plasticity deficits in FDD_KI mice. These data are consistent with a role for the carboxyl-terminal APP domain in the pathogenesis of dementia and suggest that averting the noxious role of Thr⁶⁶⁸ is a viable therapeutic strategy for human dementias.     

Prognostic Role of Ventricular Ectopic Beats in Systemic Sclerosis: A Prospective Cohort Study Shows ECG Indexes Predicting the Worse Outcome

BackgroundArrhythmias are frequent in Systemic Sclerosis (SSc) and portend a bad prognosis, accounting alone for 6% of total deaths. Many of these patients die suddenly, thus prevention and intensified risk-stratification represent unmet medical needs. The major goal of this study was the definition of ECG indexes of poor prognosis.MethodsWe performed a prospective cohort study to define the role of 24h-ECG-Holter as an additional risk-stratification technique in the identification of SSc-patients at high risk of life-threatening arrhythmias and sudden cardiac death (SCD). One-hundred SSc-patients with symptoms and/or signs suggestive of cardiac involvement underwent 24h-ECG-Holter. The primary end-point was a composite of SCD or need for implantable cardioverter defibrillator (ICD).ResultsFifty-six patients (56%) had 24h-ECG-Holter abnormalities and 24(24%) presented frequent ventricular ectopic beats (VEBs). The number of VEBs correlated with high-sensitive cardiac troponin T (hs-cTnT) levels and inversely correlated with left-ventricular ejection fraction (LV-EF) on echocardiography. During a mean follow-up of 23.1±16.0 months, 5 patients died suddenly and two required ICD-implantation. The 7 patients who met the composite end-point had a higher number of VEBs, higher levels of hs-cTnT and NT-proBNP and lower LV-EF (p = 0.001 for all correlations). All these 7 patients had frequent VEBs, while LV-EF was not reduced in all and its range was wide. At ROC curve, VEBs>1190/24h showed 100% of sensitivity and 83% of specificity to predict the primary end-point (AUROC = 0.92,p<0.0001). Patients with VEBS>1190/24h had lower LV-EF and higher hs-cTnT levels and, at multivariate analysis, the presence of increased hs-cTnT and of right bundle branch block on ECG emerged as independent predictors of VEBs>1190/24h. None of demographic or disease-related characteristics emerged as predictors of poor outcome.ConclusionsVEBS>1190/24h identify patients at high risk of life-threatening arrhythmic complications. Thus, 24h-ECG-Holter should be considered a useful additional risk-stratification test to select SSc-patients at high-risk of SCD, in whom an ICD-implantation could represent a potential life-saving intervention.