Regulation of glycerol efflux in adipocytes. Structural and functional studies of the glycerol channel aquaporin 7

Abstract: Glycerol levels in adipocytes depend on the lipolysis, the hydrolysis of triglycerides into glycerol and free fatty acids, and the efflux of glycerol across the plasma membrane through glycerol channels. The aims of this work were to investigate how glycerol levels are regulated on a molecular level by PLIN1, a major lipid droplet-associated protein and a key regulator of the lipolysis by scaffolding for lipolytic proteins on the lipid droplet. Furthermore to elucidate the selectivity and conducting mechanisms of aquaporin 7 (AQP7), the major glycerol channel in adipocytes.PLIN1 was found to form micro domains in human primary adipocytes during basal conditions, and the micro domains dispersed after lipolytic stimulation. Interactions specifically with cholesteryl esters, DPPC and triglycerides on the lipid droplet formed the basis for the dynamic PLIN1 micro domains. The dispersion of PLIN1 along with bound lipolytic proteins results in a fully activated lipolysis. Further, PLIN1 was identified as an interacting partner to AQP7. Protein kinase A phosphorylation of the AQP7 N-terminus decreased the binding to PLIN1, resulting in translocation from the lipid droplet to the plasma membrane for glycerol efflux. The high-resolution crystal structure of AQP7 was determined with glycerol and water molecules lining the channel. A conducting mechanism was proposed in which the glycerol partly rotates as it travels along the pore, possibly facilitating the transition by altering the hydrogen bond network and releasing the glycerol from more tightly bound positions. Moreover, molecular dynamics simulations suggested that glycerol hinders water from diffusing through the pore at a high rate. Initial crystal growth of AQP7 for neutron diffraction studies was carried out, in order to be able to precisely locate hydrogen atoms and gain a deeper understanding of the selectivity and conducting mechanisms.Glycerol metabolism has implications in metabolic diseases and AQP7-knockout mice develop obesity and type 2 diabetes. This research has deepened our knowledge of how glycerol levels in adipocytes are regulated by PLIN1. Adipocyte lipolysis is upregulated by certain cancers in order to supply energy for rapid tumor growth. The structure of AQP7 unraveled the details of glycerol efflux through AQP7 and provides a structural basis for development of small- molecules drugs targeting AQP7.