title: RNAi screening in primary human hepatocytes of genes implicated in genome-wide association studies for roles in type 2 diabetes identifies roles for CAMK1D and CDKAL1, among others, in hepatic glucose regulation.
abstract: Genome-wide association (GWA) studies have described a large number of new candidate genes that contribute to of Type 2 Diabetes (T2D). In some cases, small clusters of genes are implicated, rather than a single gene, and in all cases, the genetic contribution is not defined through the effects on a specific organ, such as the pancreas or liver. There is a significant need to develop and use human cell-based models to examine the effects these genes may have on glucose regulation. We describe the development of a primary human hepatocyte model that adjusts glucose disposition according to hormonal signals. This model was used to determine whether candidate genes identified in GWA studies regulate hepatic glucose disposition through siRNAs corresponding to the list of identified genes. We find that several genes affect the storage of glucose as glycogen (glycolytic response) and/or affect the utilization of pyruvate, the critical step in gluconeogenesis. Of the genes that affect both of these processes, CAMK1D, TSPAN8 and KIF11 affect the localization of a mediator of both gluconeogenesis and glycolysis regulation, CRTC2, to the nucleus in response to glucagon. In addition, the gene CDKAL1 was observed to affect glycogen storage, and molecular experiments using mutant forms of CDK5, a putative target of CDKAL1, in HepG2 cells show that this is mediated by coordinate regulation of CDK5 and PKA on MEK, which ultimately regulates the phosphorylation of ribosomal protein S6, a critical step in the insulin signaling pathway.
authors.affiliation: Target Generation Unit, Pfizer Research Technology Center, Cambridge, Massachusetts, USA. haney_steven_a@lilly.com
authors.forename: Steven
authors.initials: S
authors.lastname: Haney
authors.forename: Juan
authors.initials: J
authors.lastname: Zhao
authors.forename: Shiwani
authors.initials: S
authors.lastname: Tiwari
authors.forename: Kurt
authors.initials: K
authors.lastname: Eng
authors.forename: Lin T
authors.initials: LT
authors.lastname: Guey
authors.forename: Eric
authors.initials: E
authors.lastname: Tien
mesh_terms: D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1 / Q000235:genetics*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics* / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D055785:Gene Knockdown Techniques; D015894:Genome, Human; D055106:Genome-Wide Association Study; D005934:Glucagon / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D006003:Glycogen / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D020641:Polymorphism, Single Nucleotide; D061251:Primary Cell Culture; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D019289:Pyruvic Acid / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D012359:tRNA Methyltransferases
chemical_list: C493245:CRTC2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; D019289:Pyruvic Acid; D006003:Glycogen; D005934:Glucagon; D012359:tRNA Methyltransferases; D051360:Cyclin-Dependent Kinase 5; D019893:Ribosomal Protein S6 Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C517601:CAMK1D protein, human; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; C495905:CDK5 protein, human; D048049:Extracellular Signal-Regulated MAP Kinases; C525110:CDKAL1 protein, human; C521898:PCK1 protein, human; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D005947:Glucose