Cellular characterisation of the GCKR P446L variant associated with type 2 diabetes risk

Abstract

Aims/Hypothesis Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intra-cellular localization. Methods Fluorescently-tagged rat and human wild-type or P446L-GKRP and GCK were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low (5.5mmol/l) or high (25mmol/l) glucose conditions. Interaction between GCK and GKRP were measured by sensitized emission-based FRET efficiency (FRETN). Results P446L-GKRP had a decreased degree of nuclear localization, ability to sequester GCK, and direct interaction with GCK as measured by FRET compared to WT-GKRP. Decreased interaction was observed between WT-GKRP and GCK at high (25mmol/l) compared to low glucose (5.5mmol/l) but not between P446L-GKRP and GCK. Rat WT-GKRP and P446L-GKRP behaved quite differently: both variants responded to high glucose by diminished sequestration of GCK but showed no effect of the P446L variant on nuclear localization or GCK sequestration. Conclusions Our study suggests the common human GKRP-P446L variant results in elevated hepatic glucose uptake and disposal by increasing active, cytosolic GCK. This would increase hepatic lipid biosynthesis but decrease fasting plasma glucose concentrations and provides a potential mechanism for the protective effect of this allele on type 2 diabetes risk.

Correlation of rare coding variants in the gene encoding human glucokinase regulatory protein with phenotypic, cellular, and kinetic outcomes

“…In accordance with previous observations, GCK exhibited similar subcellular localization patterns for multiple glucose concentrations tested (data not shown) (43). Therefore, images from 1 glucose concentration (25 mM) are presented and representative of other concentrations.…”

“…Second, consistent with our previous findings, common variant p.Pro446Leu localized to both the cytoplasm and nucleus ( Figure 2B and ref. 43). Rare variants p.Ile396Asn and p.Ile500Ser also displayed this behavior.…”

“…Further genome-wide analyses have documented associations of this region with risk of type 2 diabetes (T2D), total cholesterol, fasting insulin, C peptide, homeostasis model assessment of insulin resistance (HOMA-IR), and concentrations of a number of other metabolites, including C-reactive protein (CRP), mannose, the liver enzyme γ-glutamyl transferase, and urate (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Functionally, p.Pro446Leu-GKRP has been shown to decrease F6P-mediated inhibition of GCK and reduce nuclear sequestration of GCK (13,43). Decreased inhibition and sequestration of GCK may lead to increased GCK activity in the liver, resulting in increased de novo triglyceride and cholesterol synthesis and export, as suggested by recently observed associations of GCKR with VLDL particle concentrations (44).…”

Association of the GCKR rs780094 polymorphism with metabolic traits including carotid intima-media thickness in Japanese community-dwelling men, but not in women

“…However, the molecular mechanism has been partially clarified by which GCKR rs1260326 (P446L) variant, an SNP with a strong linkage disequilibrium to GCKR rs780094, is related to glucose and lipid metabolism [26]. Wild type GCKR is supposed to bind GCK and inhibit GCK enzyme activity in the nucleus of the liver at a physiological concentration of fructose-6-phosphate (F6P), while the GKRP P446L is likely to dissociate with GCK even at the same concentration of F6P [27,28]. This means that the GKRP P446L may provide for increased activity of GCK compared to wild-type GKRP.…”

Discrete associations of the GCKR variant with metabolic risk in a Chinese population: longitudinal change analysis

“…Second, and more importantly, the GCKR rs780092 might have a direct effect on glucose metabolism. GKRP also regulates concentrations of fructose 6-phosphate (F6P), which can enhance GCK-CKRP complex formation [14,16]. The GCKR variant (rs120326 [c.1337C> T; p.P446L]) produces GKRP that has reduced nuclear sequestration and F6P-mediated inhibition of GCK, which leads to increased GCK activity, resulting in increased hepatic glucose disposal and the subsequent decreased plasma glucose concentrations [17].…”