Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression

Abstract

Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.

Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

“…3 and 5) (Table 2) in contrast to Mahajan et al (40), who observed decreased protein expression of the Leu219 variant relative to Val219. The explanation for this difference is unclear, but we observe the same result using G6PC2 expressed using the pcDNA3.1D vector in COS cells (22) and the pJPA5 vector in 832/13 (Figs. 3 and 5) (Table 2) and βTC-3 cells (Fig.…”

“…Interestingly, a comparison of these results with our published data examining the effect of some of these same SNPs on mouse G6PC1 protein expression (22) suggests that several of these SNPs differentially affect the stability of mouse and human G6PC1. For example, Ser34Phe, Trp63Arg, Thr111Arg, and Tyr209Ser had limited effects on mouse G6PC1 protein expression (22) but markedly reduced human G6PC1 protein expression (Tables 1 and 2). A direct, simultaneous comparison of the effects of these SNPs on mouse and human G6PC1 protein expression broadly replicated these observations (Fig.…”

“…This transient transfection assay uses a combination of a glucoseresponsive G6pc1-luciferase fusion gene and the rat 832/ 13 cell line. We previously showed that luciferase activity is induced by elevated glucose in this assay but repressed by cotransfection with a plasmid encoding G6PC1 (22). We hypothesized that G6PC1 reduces intracellular G6P, the key metabolite in glucose-regulated gene transcription (22,29).…”

“…We previously showed that luciferase activity is induced by elevated glucose in this assay but repressed by cotransfection with a plasmid encoding G6PC1 (22). We hypothesized that G6PC1 reduces intracellular G6P, the key metabolite in glucose-regulated gene transcription (22,29). Again, taking advantage of the high G6PC2 protein expression conferred by the pJPA5 vector, this assay was used to study G6PC2 activity in intact cells.…”

“…Measurement of glucose-6-phosphatase (G6Pase) activity in situ G6Pase activity was measured in situ as previously described (22). Briefly, semiconfluent 832/13 cells in 3.5 cm diameter dishes were cotransfected with 2 μg of a G6pc1-firefly luciferase fusion gene construct, 0.5 μg of SV40-Renilla luciferase (Promega) and 1 μg of an expression vector encoding WT or variant G6PC2, using the lipofectamine reagent (InVitrogen).…”