Normalization of prandial blood glucose and improvement of glucose tolerance by liver-specific inhibition of SH2 domain containing inositol phosphatase 2 (SHIP2) in diabetic KKAy mice: SHIP2 inhibition causes insulin-mimetic effects on glycogen metabolism, gluconeogenesis, and glycolysis

Type 2 diabetes is characterized by a progressive resistance of peripheral tissues to insulin. Recent data have established the lipid phosphatase SH2 domain-containing inositol phosphatase 2 (SHIP2) as a critical negative regulator of insulin signal transduction. Mutations in the SHIP2 gene are associated with type 2 diabetes. Here, we used hyperglycemic and hyperinsulinemic KKA(y) mice to gain insight into the signaling events and metabolic changes triggered by SHIP2 inhibition in vivo. Liver-specific expression of a dominant-negative SHIP2 mutant in KKA(y) mice increased basal and insulin-stimulated Akt phosphorylation. Protein levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase were significantly reduced, and consequently the liver produced less glucose through gluconeogenesis. Furthermore, SHIP2 inhibition improved hepatic glycogen metabolism by modulating the phosphorylation states of glycogen phosphorylase and glycogen synthase, which ultimately increased hepatic glycogen content. Enhanced glucokinase and reduced pyruvate dehydrogenase kinase 4 expression, together with increased plasma triglycerides, indicate improved glycolysis. As a consequence of the insulin-mimetic effects on glycogen metabolism, gluconeogenesis, and glycolysis, the liver-specific inhibition of SHIP2 improved glucose tolerance and markedly reduced prandial blood glucose levels in KKA(y) mice. These results support the attractiveness of a specific inhibition of SHIP2 for the prevention and/or treatment of type 2 diabetes.     

G1-Cdk activity is required for both proliferation and viability of cytokine-dependent myeloid and erythroid cells.

Hematopoietic cytokines are critically required for survival and cell proliferation of myeloid and erythroid progenitors. It is poorly understood how the apoptotic machinery of progenitor cells senses the absence of specific cytokines. Here we show that G1-Cdk activity is essential for cytokine-mediated viability of myeloid and erythroid progenitors. Cytokine deprivation is associated with rapid downregulation of G1-Cdk activity, cell cycle arrest, and apoptosis. Specific inhibition of G1-Cdk activity results in apoptotic cell death in the presence of saturating cytokine levels. In contrast, specific cell cycle arrest in G2/M does not affect viability. When cell proliferation is arrested by cytokine withdrawal, primary erythroid progenitors expressing v-ErbA maintain G1-Cdk activity and undergo delayed apoptosis. Cdk-inhibitors strongly enhance apoptosis in starved v-ErbA cells, indicating that sustained Cdk activity is required for protection from apoptosis by v-ErbA.     

Endogenous ligands of a putative LSD-serotonin receptor in the cerebrospinal fluid: Higher level of LSD-displacing factors (LDF) in unmedicated psychotic patients

In human cerebrospinal fluid substances were detected that are capable of reversibly displacing the hallucinogen d-lysergic acid diethylamide (LSD) from its high-affinity binding sites in neuronal membranes. The binding sites may represent a specific type of LSD-serotonin receptor protein. The LSD-displacing factors (LDF) occur physiologically in concentrations high enough to interact with the putative LSD-serotonin receptors. The LDF can be separated from proteins and inorganic salts. LDF was found to be clearly different from endogenous indoleamine ligands such as serotonin, being anionic at pH 7.4.The LDF concentration was assayed in the cerebrospinal fluid of 49 nonpsychotic and of 19 acute psychotic patients before 30 days therapy with the antipsychotic drugs haloperidol or clozapine. LDF concentration was found to be significantly higher (P<0.001) in the group of unmedicated acute psychotic patients (5.55 U/ml) in comparison to the control group (3.56 U/ml). Within this group of acute psychotic patients, a high positive correlation was found between concentration of LDF and clinical improvement (r=0.650). Thus, a nosological subgroup was traced out, characterized by both a higher concentration of LDF and a higher responsiveness to antipsychotic drugs (P<0.01). Ratings of clinical improvement and determinations of the concentration of LDF before drug therapy were performed in a doubleblind study. Since antipsychotic drugs act on dopamine receptors and LDF acts on putative serotonin receptors, dopamine and serotonin receptors may both be affected in the psychotic state. A working hypothesis is offered that links the dopamine and the serotonin hypotheses.     

Low concentrations of UD-CG 212 enhance myocyte contractility by an increase in calcium responsiveness in the presence of inorganic phosphate

Recent data show that UD-CG 212 in nanomolar concentrations increases myofibrillar Ca⁺⁺ responsiveness of chemically skinned cardiac preparations in the presence of elevated inorganic phosphate. We studied the effects of UD-CG 212 on cell shortening of intact myocytes and in addition measured the intracellular calcium transients with the aid of INDO-1 fluorescence in the presence of 5 mM inorganic phosphate.The validity of our experimental system was first tested with the calcium channel opener Bay k 8644. Bay k 8644 at 10^–8 M did not significantly influence myocyte shortening ( + 13.9 ± 4.6%, n = 9) but at 10^–7 M and 10^–6 M significantly increased contraction by 40.1 +- 13.6%and52.5 ± 17.0% respectively. Bay k 8644 at 10^–8 M increased the INDO-1 fluorescence ratio by 17.3 ± 4.7% (P < 0.01; n = 9), and at 10^–7 M by 21.5 + 4.3% (P < 0.01; n = 9), whereas 10^–6 M Bay k 8644 had no significant effect on peak INDO-1 ratio. However, 10^–7and 10^–6 M Bay k 8644 accelerated and broadened the calcium transients.Cell shortening of guinea pig ventricular myocytes electrically stimulated at 1 Hz was significantly increased by UD-CG 212 (10^–9-10^–7 M) and isoprena line(3 × 10^–8 M). An increase of 37.0 ± 14.0% (P < 0.05; n = 9) was observed at 10^–9 M UD-CG 212, 90.5±18.2% (P<0.05; n=9) at 10^–8 M UD-CG 212, 164.0 ± 34.9% (P < 0.05; n = 9) at 10^–7 M UD-CG 212, and 258.2 ± 67.4% (P < 0.05; n = 9) at 3 × 10-8 M isoprenaline. Peak INDO-1 fluorescence ratios were not significantly (P > 0.05) influenced after addition of 10^–9 M and 10^–8 M UD-CG 212, but significantly increased by 19.4 ± 4.9%(P < 0.05; n = 9) at 10^–7 MUD-CG 212 and by 81.1 ± 11.1% (P < 0.05; n = 9) at 3 x 10^–8 M isoprenaline.In conclusion, UD-CG 212 (10^–9 - 10^–7 M) Concentration-dependently increased myocyte shortening in the presence of 5 mM inorganic phosphate. Low concentrations of 10^–9 and 10^–8 M UD-CG 212 increased myocyte contractility without altering the peak INDO-1 fluorescence ratio whereas 10^–7 M UD-CG 212 and 3 × 10^–8 M isoprenaline increased cell shortening as well as peak INDO-1 fluorescence ratio. These data suggest that low concentrations of UD-CG 212 increase myocyte contractility by enhancing myofibrillar calcium responsiveness whereas higher concentrations elevate intracellular calcium probably via increased intracellular CAMP brought about by phosphodiesterase inhibition.     

Scopolamine and lorazepam exert different patterns of effects in a test battery assessing stages of information processing

The effects of a single dose of scopolamine (0.5 mg) SC and of lorazepam (2.5 mg) PO were tested in two independent studies for their effects on performance in a psychometric battery which measured functions related to different stages of information processing. Attention and vigilance were measured by a continuous attention task and a vigilance task, respectively. Working memory and reasoning were evaluated by the rapid information processing and logical reasoning task; memory acquisition and storage were measured by pre- and post-drug immediate and delayed recall using visual material. The following pattern of effects was revealed: both scopolamine and lorazepam impaired performance in attentional and vigilance tasks as well as in the rapid information processing task significantly (P<0.05) when compared with their own placebo; in the logical reasoning task lorazepam significantly prolonged the time required to solve a problem; scopolamine did not have any effect on this task. Scopolamine impaired performance in the immediate recall but left delayed recall unaffected; lorazepam impaired only delayed recall, immediate recall remaining unaffected. These data suggest that scopolamine at this dose impaired mostly attention and early stages of information processes; lorazepam at the dose tested impaired also the later acquisition and encoding aspects of memory.This article is part of the Doctoral Thesis of B. Redemann     

Single amino acid exchanges in the finger domain impair the function of the Drosophila gene Krüppel (Kr).

Mutations in the Drosophila gene Krüppel (Kr) cause deletions of segments in the embryo and also affect Malpighian tubule development. In the hypomorphic Kr alleles that were analyzed, the defects in the segment pattern and the Malpighian tubules are parallel in strength. We have sequenced the DNA of four Kr alleles that show normal spatial and temporal patterns of Kr-encoded protein expression. Three of the four alleles have single base pair mutations that result in a single amino acid change. The exchanges occur in the putative DNA-binding domain of the Kr protein, which is characterized by four repeats of the zinc finger motif. Sequence comparison of the finger motifs helped to define the structural requirements for the folding of the finger domain to some extent. Our data on the Kr mutants support the view that has emerged from the evolutionary analysis.