Key Points• Inositol hexakisphosphate kinase 1 (IP6K1) knockout mice display lower inorganic polyphosphate levels in platelets.• Low platelet polyphosphate leads to lengthened clotting time, altered clot architecture, and protection against pulmonary thromboembolism.Polyphosphate (polyP), a polymer of orthophosphate moieties released from the dense granules of activated platelets, is a procoagulant agent. Inositol pyrophosphates, another group of phosphate-rich molecules, consist of mono-and diphosphates substituted on an inositol ring. Diphosphoinositol pentakisphosphate (IP 7 ), the most abundant inositol pyrophosphate, is synthesized on phosphorylation of inositol hexakisphosphate (IP 6 ) by IP 6 kinases, of which there are 3 mammalian isoforms (IP6K1/2/3) and a single yeast isoform. Yeast lacking IP 6 kinase are devoid of polyP, suggesting a role for IP 6 kinase in maintaining polyP levels. We theorized that the molecular link between IP 6 kinase and polyP is conserved in mammals and investigated whether polyP-dependent platelet function is altered in IP6K1 knockout (Ip6k1) mice. We observe a significant reduction in platelet polyP levels in Ip6k1 2/2 mice, along with slower platelet aggregation and lengthened plasma clotting time. Incorporation of polyP into fibrin clots was reduced in Ip6k1 2/2 mice, thereby altering clot ultrastructure, which was rescued on the addition of exogenous polyP. In vivo assays revealed longer tail bleeding time and resistance to thromboembolism in Ip6k1 2/2 mice. Taken together, our data suggest a novel role for IP6K1 in regulation of mammalian hemostasis via its control of platelet polyP levels.

Autophagy is a quality-control mechanism that helps to maintain cellular homeostasis by removing damaged proteins and organelles through lysosomal degradation. During autophagy, signaling events lead to the formation of a cup-shaped structure called the phagophore that matures into the autophagosome. Recruitment of the autophagy-associated Atg12-5-16L1 complex to Wipi2-positive phagophores is crucial for producing microtubule-associated protein 1 light chain 3-II (LC3-II), which is required for autophagosome formation. Here, we explored the role of the autophagy receptor optineurin (Optn) in autophagosome formation. Fibroblasts from Optn knock-out mouse showed reduced LC3-II formation and a lower number of autophagosomes and autolysosomes during both basal and starvation-induced autophagy. However, the number of Wipi2-positive phagophores was not decreased in Optn-deficient cells. We also found that the number of Atg12/16L1-positive puncta and recruitment of the Atg12-5-16L1 complex to Wipi2-positive puncta are reduced in Optn-deficient cells. Of note, Optn was recruited to Atg12-5-16L1-positive puncta, and interacted with Atg5 and also with Atg12-5 conjugate. A disease-associated Optn mutant, E478G, defective in ubiquitin binding, was also defective in autophagosome formation and recruitment to the Atg12-5-16L1-positive puncta. Moreover, we noted that Optn phosphorylation at Ser-177 was required for autophagosome formation but not for Optn recruitment to the phagophore. These results suggest that Optn potentiates LC3-II production and maturation of the phagophore into the autophagosome, by facilitating the recruitment of the Atg12-5-16L1 complex to Wipi2-positive phagophores.

WD-repeat proteins are very diverse, yet these are structurally related proteins that participate in a wide range of cellular functions. WDR13, a member of this family, is conserved from fishes to humans and localizes into the nucleus. To understand the in vivo function(s) of Wdr13 gene, we have created and characterized a mutant mouse strain lacking this gene. The mutant mice had higher serum insulin levels and increased pancreatic islet mass as a result of enhanced beta cell proliferation. While a known cell cycle inhibitor, p21, was downregulated in the mutant islets, over expression of WDR13 in the pancreatic beta cell line (MIN6) resulted in upregulation of p21, accompanied by retardation of cell proliferation. We suggest that WDR13 is a novel negative regulator of the pancreatic beta cell proliferation. Given the higher insulin levels and better glucose clearance in Wdr13 gene deficient mice, we propose that this protein may be a potential candidate drug target for ameliorating impaired glucose metabolism in diabetes.

Aim/hypothesis Type 2 diabetes is a complex disease characterised by hyperglycaemia, hyperinsulinaemia, dyslipidaemia and insulin resistance accompanied by inflammation. Previously, we showed that mice lacking the Wdr13 gene had increased islet mass due to enhanced beta cell proliferation. We hypothesised that introgression of a Wdr13-null mutation, a beta cell-proliferative phenotype, into Lepr db/db mice, a beta cell-destructive phenotype, might rescue the diabetic phenotype of the latter. Methods Wdr13-deficient mice were crossed with Lepr db/db mice to generate mice with the double mutation. We measured various serum metabolic variables of Wdr13Lepr db/db and Wdr13Lepr db/db mice. Further, we analysed the histopathology and gene expression of peroxisome proliferator-activated receptor (PPAR)γ and, activator protein (AP)1 targets in various metabolic tissues. Results Lepr db/db mice with the Wdr13 deletion had a massively increased islet mass, hyperinsulinaemia and adipocyte hypertrophy. The increase in beta cell mass in Wdr13 −/0 Lepr db/db mice was due to an increase in beta cell proliferation. Hypertrophy of adipocytes may be the result of increase in transcription of Pparg and its target genes, leading in turn to increased expression of several lipogenic genes. We also observed a significant decrease in the expression of AP1 and nuclear factor κ light chain enhancer of activated B cells (NFκB) target genes involved in inflammation.Conclusions/interpretation This study provides evidence that loss of WD repeat domain 13 (WDR13) protein in the Lepr db/db mouse model of diabetes is beneficial. Based on these findings, we suggest that WDR13 may be a potential drug target for ameliorating hyperglycaemia and inflammation in diabetic conditions.