Abstract.In the present work, we demonstrate that treatment with 1,25-dihydroxyvitamin D3 for 24 hours increased in a dose-dependent manner the levels of the two major insulin receptor (IR) mRNAs (11 and 8.5 Kb) present in U-937 human promonocytic cells. These levels reached maximum values (1.8-fold 11 Kb; 1.4-fold 8.5 Kb) with the addition of 108 M 1,25-dihydroxyvitamin D3. In these optimal conditions the stimulatory effect of 1,25-dihydroxyvitamin D3 was accompanied by increases in both IR capacity, and insulin responsiveness for glucose transport in these cells. Moreover, such increases appear to be mediated by an enhanced expression of the receptor for 1,25-dihydroxyvitamin D3, measured at the level of both RNA and protein. These results provide evidence of 1,25-dihydroxyvitamin D3 acting as genomic stimulator of the insulin response in the control of glucose transport.

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Treatment with 10(-8) M 1,25-dihydroxyvitamin D(3) for 24 h causes transcriptional activation of the human insulin receptor gene in U-937 human promonocytic cells. The activation seems to potentiate the response to insulin in terms of glucose oxidation. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, causes a greater inhibition of insulin-stimulated glucose oxidation in 1,25-dihydroxyvitamin D(3)-treated cells than in untreated cells. This suggests a stimulation of phosphatidylinositol 3-kinase activity by 1,25-dihydroxyvitamin D(3), which could mediate, at least in part, the potentiation of the insulin response.

SummaryThe PhaF is a nucleoid-associated like protein of Pseudomonas putida KT2442 involved in the polyhydroxyalkanoate (

Phasins are intracellular polyhydroxyalkanoat4e (PHA)-associated proteins involved in the stabilization of these bacterial carbon storage granules. Despite its importance in PHA metabolism and regulation, only few reports have focused so far on the structure of these proteins. In this work we have investigated the structure and stability of the PhaF phasin from Pseudomonas putida KT2440, a protein that is involved in PHA granule stabilization and distribution to daughter cells upon cell division. A structural, three-dimensional model of the protein was built from homology modeling procedures and consensus secondary structure predictions. The model predicts that PhaF is an elongated protein, with a long, amphipathic N-terminal helix with PHA binding capacity, followed by a short leucine zipper involved in protein oligomerization and a superhelical C-terminal domain wrapped around the chromosomal DNA. Hydrodynamic, spectroscopical and thermodynamic experiments validated the model and confirmed both that free PhaF is a tetramer in solution and that most part of the protein is intrinsically disordered in the absence of its ligands. The results lay a molecular basis for the explanation of the biological role of PhaF and, along with an exhaustive analysis of phasin sequence databases, suggest that intrinsic disorder and oligomerization through coiled-coils may be a widespread mechanism among these proteins.