9-cyano-1-azapaullone (cazpaullone), a glycogen synthase kinase-3 (GSK-3) inhibitor activating pancreatic beta cell protection and replication.

Recently, the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) emerged as a regulator of pancreatic beta cell growth and survival. On the basis of the previous observation that GSK-3 inhibitors like 1-azakenpaullone promote beta cell protection and replication, paullone derivatives were synthesized including 1-aza-, 2-aza-, and 12-oxapaullone scaffolds. In enzymatic assays distinct 1-azapaullones were found to exhibit selective GSK-3 inhibitory activity. Within the series of 1-azapaullones, three derivatives stimulated INS-1E beta cell replication and protected INS-1E cells against glucolipotoxicity induced cell death. Cazpaullone (9-cyano-1-azapaullone), the most active compound in the protection assays, also stimulated the replication of primary beta cells in isolated rat islets. Furthermore, cazpaullone showed a pronounced transient stimulation of the mRNA expression of the beta cell transcription factor Pax4, an important regulator of beta cell development and growth. These features distinguish cazpaullone as a unique starting point for the development of beta cell regenerative agents which might be useful in the treatment of diabetes.

Inhibition of GSK3 promotes replication and survival of pancreatic beta cells.

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

Trypanosomes change their transferrin receptor expression to allow effective uptake of host transferrin.

In its mammalian host, Trypanosoma brucei covers its iron requirements by receptor-mediated uptake of host transferrin (Tf). The Tf-receptor (Tf-R) is a heterodimeric membrane protein encoded by expression site-associated gene (ESAG) 6 and 7 located promoter-proximal in a polycistronic expression site (ES). Each of the 20 ESs encodes a slightly different Tf-R; these differences strongly affect the binding affinity for Tfs of different hosts. The Tf-R encoded in the 221 ES has a low affinity for dog Tf. Transfer of trypanosomes with an active 221 ES to dilute dog serum leads to growth arrest, which they can overcome by switching to another ES encoding a Tf-R with higher affinity for dog Tf. Here we show that trypanosomes can also adapt to dilute dog serum without switching but by replacing the ESAG7 gene in the 221 ES by one from another ES, by deleting ESAG7 from the 221 ES with concomitant upregulation of transcription of ESAG7 in 'silent' ESs, by grossly overproducing the 221 Tf-R or by combinations of these alterations. Our results illustrate the striking genetic flexibility of trypanosomes.

Factors affecting the level and localization of the transferrin receptor in Trypanosoma brucei.

Transfer of bloodstream-form Trypanosoma brucei variant 221a from calf serum to dog serum-based medium induces acute iron starvation, as the transferrin receptor (Tf-R) of variant 221a binds dog Tf poorly. We show here that transfer to dog serum induces a 3-5-fold increase in Tf-R mRNA and protein within one doubling time (8 h). Because iron stores are still high 8 h after transfer, we infer that the signal for Tf-R overproduction is the decreased availability of cytosolic iron when cellular iron import drops. Up to 30% of the extra Tf-R spills out of the flagellar pocket onto the pellicular surface. Because the 5-fold increase in Tf-R is accompanied by a 5-fold increase in bovine Tf uptake, the up-regulation of Tf-R levels in response to Tf starvation helps the trypanosome to compete for limiting amounts of Tf. We noted that Tf-R levels also vary in calf serum medium. Cells in dense cultures contain up to 5-fold more Tf-R mRNA and protein than in dilute cultures. Only one-tenth of the extra Tf-R reaches the pellicular surface. The increase cannot be explained by a lack of Tf or to cell density sensing but is due to pericellular hypoxia. Our results show that bloodstream-form trypanosomes can regulate the expression of the two Tf-R subunit genes and the localization of their gene products in a flexible manner. This flexibility is made possible by the promoter-proximal position of the two genes in the variant surface glycoprotein expression site.

The expression level determines the surface distribution of the transferrin receptor in Trypanosoma brucei.

The transferrin receptor (TfR) of Trypanosoma brucei is a heterodimer attached to the surface membrane by a glycosylphosphatidylinositol (GPI) anchor. The TfR is restricted to the flagellar pocket, a deep invagination of the plasma membrane. The membrane of the flagellar pocket and the rest of the cell surface are continuous, and the mechanism that selectively retains the TfR in the pocket is unknown. Here, we report that the TfR is retained in the flagellar pocket by a specific and saturable mechanism. In bloodstream-form trypanosomes transfected with the TfR genes, TfR molecules escaped flagellar pocket retention and accumulated on the entire surface, even at modest (threefold) overproduction levels. Similar surface accumulation was observed when the TfR levels were physiologically upregulated threefold when trypanosomes were starved for transferrin. These results suggest that the TfR flagellar pocket retention mechanism is easily saturated and that control of the expression level is critical to maintain the restricted surface distribution of the receptor.

The physiological significance of transferrin receptor variations in Trypanosoma brucei.

Trypanosoma brucei escapes destruction by the host immune system by regularly replacing its Variant Surface Glycoprotein (VSG) coat. The VSG is expressed in a VSG expression site, together with expression site associated gene (ESAG) 6 and 7, encoding the heterodimeric transferrin receptor (Tf-R). There are around 20 VSG expression sites, and trypanosomes can change the site that is active. Since ESAG6 and 7 in different expression sites differ somewhat in sequence, expression site switching results in the production of a slightly different Tf-R. We have studied the physiological relevance of Tf-R variation for the survival of T. brucei in mammalian sera. Trypanosomes with an active 221 expression site, encoding a Tf-R with a very low affinity for canine Tf (Kd>1 microM), were cultured in canine serum based medium. This resulted in selection of trypanosomes that had switched to the VO2, the 223 or the bR-2 expression site, each encoding a Tf-R with higher affinity for canine Tf than the 221 site Tf-R. Adding bovine Tf to the medium could prevent the switch, indicating that the low uptake of Tf provided the selection against 221 trypanosomes. Horse serum based medium also induced switching to the VO2 expression site, but this was not prevented by bovine Tf. In the presence of physiological concentrations of anti-Tf-R antibody, only a high-affinity Tf allowed the growth of 221 Tf-R expressing trypanosomes. Our results suggest that a high-affinity Tf-R not only ensures efficient Tf uptake, but is also required to allow sufficient iron uptake by the trypanosome in the presence of anti-Tf-R antibodies.