ENPP1 affects insulin action and secretion: evidences from in vitro studies

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.     

Impaired cytoprotective function of muscle in human gallbladders with cholesterol stones

Acute cholecystitis develops in gallbladders (GB) with excessive bile cholesterol (Ch). Increased membrane Ch content affects membrane function and may affect PGE(2) receptors involved in the cytoprotection against acute inflammation. This study was aimed at determining whether the cytoprotective response to PGE(2) is affected by lithogenic bile with Ch. Muscle cells from human GB with cholesterol stones (ChS) or pigment stones (PS) were obtained by enzymatic digestion. PGE(2) levels were measured by radioimmunoassay, and activities of superoxide dismutase (SOD) and catalase were assayed by spectrophotometry. The contraction in response to H(2)O(2) in muscle cells from PS was 14 +/- 0.3%, not different from normal controls, and decreased after the cells were incubated with Ch-rich liposomes (P < 0.05), which increase the Ch content in the plasma membranes. In muscle cells from GB with ChS, H(2)O(2)-induced contraction was only 9.2 +/- 1.3% and increased to 14 +/- 0.2% after Ch-free liposome treatment to remove Ch from the plasma membranes (P < 0.01). H(2)O(2) caused a similar increase in the levels of lipid peroxidation and PGE(2) content in muscle cells from GBs with ChS and PS. However, the activities of SOD and catalase were significantly lower in muscle cells from GBs with ChS compared with those with PS. The binding capacity of PGE(2) receptors was also significantly lower in muscle cells from GBs with ChS compared with those with PS. In conclusion, the cytoprotective response to reactive oxygen species is reduced in muscle cells from GBs with ChS despite a normal increase in the cellular levels of PGE(2). This impaired cytoprotective response may be due to a dysfunction of PGE(2) receptors with decreased binding capacity resulting from excessive Ch levels in the plasma membrane.     

Extremely low-frequency electromagnetic fields do not affect DNA damage and gene expression profiles of yeast and human lymphocytes

We studied the effects of extremely low-frequency (50 Hz) electromagnetic fields (EMFs) on peripheral human blood lymphocytes and DBY747 Saccharomyces cerevisiae. Graded exposure to 50 Hz magnetic flux density was obtained with a Helmholtz coil system set at 1, 10 or 100 microT for 18 h. The effects of EMFs on DNA damage were studied with the single-cell gel electrophoresis assay (comet assay) in lymphocytes. Gene expression profiles of EMF-exposed human and yeast cells were evaluated with DNA microarrays containing 13,971 and 6,212 oligonucleotides, respectively. After exposure to the EMF, we did not observe an increase in the amount of strand breaks or oxidated DNA bases relative to controls or a variation in gene expression profiles. The results suggest that extremely low-frequency EMFs do not induce DNA damage or affect gene expression in these two different eukaryotic cell systems.     

Misfolding pathways of the prion protein probed by molecular dynamics simulations

Although the cellular monomeric form of the benign prion protein is now well characterized, a model for the monomer of the misfolded conformation (PrP(Sc)) remains elusive. PrP(Sc) quickly aggregates into highly insoluble fibrils making experimental structural characterization very difficult. The tendency to aggregation of PrP(Sc) in aqueous solution implies that the monomer fold must be hydrophobic. Here, by using molecular dynamics simulations, we have studied the cellular mouse prion protein and its D178N pathogenic mutant immersed in a hydrophobic environment (solution of CCl4), to reveal conformational changes and/or local structural weaknesses of the prion protein fold in unfavorable structural and thermodynamic conditions. Simulations in water have been also performed. Although observing in general a rather limited conformation activity in the nanosecond timescale, we have detected a significant weakening of the antiparallel beta-sheet of the D178N mutant in CCl4 and to a less extent in water. No weakening is observed for the native prion protein. The increase of beta-structure in the monomer, recently claimed as evidence for misfolding to PrP(Sc), has been also observed in this study irrespective of the thermodynamic or structural conditions, showing that this behavior is very likely an intrinsic characteristic of the prion protein fold.     

Crystal structure of the mononuclear complex [Mn(mesalim)2Cl] and electrochemical studies of manganese complexes of the ligand Hmesalim

The complex [Mn(mesalim)₂Cl] (1), (Hmesalim = methyl salicylimidate) has been synthesized and fully characterized. The manganese(III) complex is formed by the reaction of the ligand Hmesalim with manganese(II) chloride. Complex 1 is mononuclear and crystallizes in the space group . Electrochemical studies were performed for complex 1, as well as for the related complexes [Mn(mesalim)₂(OAc)(MeOH] · MeOH (2) and [Mn₂(etsalim)₄(Hetsalim)₂](ClO₄)₂ (3), (Hetsalim = ethyl salicylimidate). The complexes display intricate oxidation-reduction behaviour, and coulometric analyses in combination with electrochemical analyses have been used to understand the electron transfer mechanisms occurring at the electrodes.