The role of hnRNPK in pancreatic beta cells

Project Summary Type 2 diabetes (T2D) pathology is complex and multifactorial. Typically, individuals develop insulin resistance and beta cell compensation that ultimately succumbs to chronic hyperglycemia leading to beta cell failure and death. By increasing our understanding of the mechanisms and predispositions leading to beta cell demise, we can begin to develop better strategies and medicines to treat people living with this debilitating disease. It is well known that beta cells harness transcriptional and post transcriptional controls to increase both the gene and protein expression of critical proteins that support insulin secretion in response to nutrient stimulation. However, due to the technical limitations in assessing translational control as opposed to high throughput methods used to study transcriptional control such as RNA-seq, we have a limited understanding of the mechanisms that coordinate the rapid translational response of proteins after beta cell stimulation. Our lab has shown that glucolipotoxicity to mimic the metabolic environment of individuals with T2D stimulates a robust increase in the translation but not transcription of the transcription factor JUND. This is dependent upon the activity of the RNA binding protein, hnRNPK, that binds the RNA helicase DDX3X stimulating 18S and preinitiation complex recruitment and translation initiation. Therefore, the goals of this proposal are to determine the in vivo function of hnRNPK in beta cells and to decipher the mechanism by which beta cell hnRNPK stimulates translation in response to metabolic stress. In the first aim, we will utilize our beta cell hnRNPK deletion mouse line to elucidate the metabolic outcomes of hnRNPK deficiency using glucose and insulin tolerance tests to determine glucose homeostasis. We will also perform morphometric analyses to determine any effects on beta cell mass or survival following hnRNPK deletion. In the second aim, we will use co-immunoprecipitation, immunofluorescence, and gene deletion or reduction to understand how hnRNPK and its interactors regulate the translatome in response to metabolic stress. Our preliminary data show that male mice devoid of hnRNPK are glucose intolerant and appear to have reduced beta cell mass suggesting an important role for hnRNPK in beta cells. Furthermore, hnRNPK and DDX3X share 10 common interactors determined by mass spectrometry upregulated in glucolipotoxicity conditions that are closely linked to mRNA metabolism and translation in the cell. Using polysome profiling, we will determine how hnRNPK controls translation under metabolic stress and identify translationally upregulated transcripts by dissociating mRNA from polysome fractions followed by RNA sequencing. Using CRISPR-Cas9 and shRNAs in MIN6 cells and isolated islets we can determine the functional impact hnRNPK or interactor removal has on beta cell translation. Together, these proposed aims will uncover novel mechanisms underlying protein translation in beta cells mediated by the RNA binding protein hnRNPK. This will have important implications in our understanding of beta cell function that could be leveraged to aid the development of better treatments focused on improving beta cell insulin output and survival.