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.
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.
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennesse, 37232, United States of America.
Kayla A
KA
Boortz
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennesse, 37232, United States of America.
Kristen E
KE
Syring
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennesse, 37232, United States of America.
Lynley D
LD
Pound
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennesse, 37232, United States of America.
Yingda
Y
Wang
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennesse, 37232, United States of America.
James K
JK
Oeser
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennesse, 37232, United States of America.
Richard M
RM
O'Brien
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001786:Blood Glucose; D002460:Cell Line; D003062:Codon; D017124:Conserved Sequence; D004789:Enzyme Activation; D005215:Fasting / Q000097:blood; D015870:Gene Expression; D005952:Glucose-6-Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D051381:Rats
D001786:Blood Glucose; D003062:Codon; D005952:Glucose-6-Phosphatase