Stabilization of insulin mRNA in response to glucose is a significant component of insulin production, but the mechanisms governing this process are unknown. We presently observe that insulin mRNA is a highly abundant messenger and that the content of this mRNA is mainly controlled by changes in messenger stability. We also demonstrate specific binding of the polypyrimidine tract-binding protein to a pyrimidine-rich sequence located in the 3-untranslated region (3-UTR) of insulin mRNA. This binding was increased in vitro by dithiothreitol and in vivo by glucose. Inhibition of polypyrimidine tract-binding protein binding to the pyrimidinerich sequence by mutation of the core binding site resulted in a destabilization of a reporter gene mRNA. Thus, glucose-induced binding of polypyrimidine tractbinding protein to the 3-UTR of insulin mRNA could be a necessary event in the control of insulin mRNA levels.

Background: Recent reports identify the 3Ј-UTR of insulin mRNA as crucial for control of insulin messenger stability. This region contains a pyrimidine-rich sequence, which is similar to the hypoxia-responsive mRNA-stabilizing element of tyrosine hydroxylase. This study aimed to determine whether hypoxia affects insulin mRNA levels. Materials and Methods: Rat islets were incubated at normoxic or hypoxic conditions and with or without hydrogen peroxide and a nitric oxide donor. Insulin mRNA was determined by Northern hybridization. Islet homogenates were used for electrophoretic mobility shift assay with an RNA-oligonucleotide, corresponding to the pyrimidinerich sequence of the 3Ј-UTR of rat insulin I mRNA. The expression of reporter gene mRNA, in islets transfected with reporter gene constructs containing the wild-type or mutated insulin mRNA pyrimidine-rich sequences, was measured by semiquantitive RT-PCR. Results:Insulin mRNA was increased in response to hypoxia. This was paralleled by increased binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3Ј-UTR of insulin mRNA, which was counteracted by hydrogen peroxide. The reporter gene mRNA level containing the wild-type binding site was not increased in response to hypoxia, but mutation of the site resulted in a destabilization of the mRNA. Conclusions:The complete understanding of different diabetic conditions requires the elucidation of mechanisms that control insulin gene expression. Our data show that hypoxia may increase insulin mRNA levels by promoting the binding of PTB to the insulin mRNA 3Ј-UTR. Hydrogen peroxide abolishes the hypoxic effect indicating involvement of reactive oxygen species and/or the redox potential in the oxygen-signaling pathway.

Despite considerable knowledge on the regulation of insulin gene transcription, little is known about the post-transcriptional control mechanisms of this gene. We have recently reported glucose- and hypoxia-regulated binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-untranslated insulin mRNA (ins-PRS), an event which may control insulin mRNA stability. The present aim was to probe for the signaling pathways that control this binding activity. Rat islets were exposed to pharmacological inhibitors against several molecules, previously shown to be involved in glucose signaling. The inhibitors used were; LY 294002 (PI3 kinase), Rp-cAMP triatylamine (the cAMP-dependent protein kinase PKA), bisindolylmaleimide I hydrochloride (PKC), PD 098059 (ERK1/ERK2), SB 203580 (p38/SAPK2a), rapamycin (mTOR) and okadaic acid (PP1/2A). PTB-binding activity to the ins-PRS was then analyzed by elecrophoretic mobility shift assay (EMSA). The glucose-induced PTB-binding was only inhibited by the mTOR inhibitor rapamycin. Rapamycin also reduced glucose-induced insulin mRNA expression. Thus, our results suggest an involvement of mTOR in glucose-induced PTB/ins-PRS binding and insulin mRNA stability.

Although extensively studied, there are still many unanswered questions regarding the regulation of insulin gene expression. This is important to further investigate since it will help us understand the pathophysiology of some types of diabetes. The insulin mRNA has a long half-life and changes in insulin mRNA stability, induced by glucose, are likely to be regulated through specific mechanisms. Recent findings indicate that the polypyrimidine tract binding protein (PTB), also named hnRNP I, by binding to the 3'-UTR (untranslated region) of the insulin mRNA molecule, stabilizes the messenger thereby participating in the glucose-induced increase in insulin mRNA. This review will focus on recent findings pertinent to PTB subcellular localization and function. It appears that PTB shuttles between the nucleus and the cytosol, and that protein kinase A (PKA)-mediated PTB phosphorylation promotes PTB translocation to the cytosol, an event that might enhance insulin mRNA stability. We will also review beta-cell signaling events that may control the mRNA stabilizing effect of PTB.

Increased levels of prolactin often coincide with an increased risk for thromboembolic events, but it is unclear whether a direct causal relation exists. Our aim was to examine the effect of prolactin on platelet function. In addition to using recombinant prolactin for experiments in vitro, we analyzed platelet function by flow cytometry in a group of 13 females with hyperprolactinaemia and 18 healthy female controls. Platelet activation was measured by P-selectin expression and by the amount of platelet-bound fibrinogen after stimulation with adenosine di phosphate (ADP), collagen-related peptide and the protease activated receptor (thrombin receptor) (PAR)-activating peptides PAR4-AP and PAR1-AP. Free oscillation rheometry was used to measure clotting time in whole blood. No significant effect on platelet activation or clotting time could be seen in in vitro experiments by adding recombinant prolactin. However, significantly lower P-selectin expression was found in the hyperprolactinemic group when platelets were activated by ADP (5 and 10 μM) or PAR4-AP. The expression of fibrinogen did not differ between the two groups for any of the activators used. For all samples, inverse significant correlations between P-selectin expression and prolactin concentration were found for both 5 μM ADP (r = - 0.61, p < 0.01), 10 μM ADP (r = - 0.62, p < 0.001) and PAR4-AP (r = - 0.69, p < 0.001). Thrombin cleavage of recombinant prolactin resulting in a 16 kDa C-terminal fragment did not alter the P-selectin expression upon activation. We found an indirect inhibitory effect of prolactin on platelets in hyperprolactinemic patients, suggesting that prolactin might have a protective role in thromboembolic disease.