Effects of a promoter variation in the hepatic glucokinase gene on promoter activity and glucose tolerance in Southern Chinese subjects

To the Editor: Glucokinase plays an important role in glucose homeostasis. In the pancreatic b-cell, it functions as the glucose sensor, whereas in the liver it facilitates glucose uptake during hyperglycaemia. A 258 G-to-A substitution in the liver promoter of the glucokinase gene was associated with diabetes and insulin resistance in African Americans and Asian Indians (1–3), but not in Danish Caucasians (4). In vitro transfection experiments showed that the G-to-A variant decreased the promoter activity to 42% of the wild-type promoter activity (2). We therefore investigated whether this promoter variation was associated with impaired glucose tolerance (IGT) in a Southern Chinese population. We studied 325 subjects with IGT and 351 subjects of similar age with normal glucose tolerance (NGT), as determined by using World Health Organization (WHO) diagnostic criteria (5), identified in the population-based Hong Kong Cardiovascular Risk Factor Prevalence Study (6). All subjects underwent a 75-g oral glucose tolerance test with measurement of fasting and 2-h plasma glucose and insulin levels. Homeostasis model assessment (HOMA) index [(fasting glucose fasting insulin) 22.5] was calculated to estimate insulin resistance. The 258 G-to-A polymorphism was determined by AccI restriction fragment length polymorphism analysis and its effect on glucokinase promoter activity by transfection experiments. Different lengths (0.5 kb, 1.2 kb and 1.7 kb) of wild-type (G/G) and mutated (A/A) glucokinase promoter subcloned into luciferase reporter plasmid vector pGL2enhance (Promega Corp., Madison, WI) were transfected into HepG2 cells. After 72 h of incubation, the cells were harvested for protein, luciferase and b-galactosidase assay. Each experiment was repeated at least three times. All data were presented as mean SD. The differences in genotype frequency were tested using the -test and comparisons between different genotypes by analysis of covariance (ANCOVA) with confounding variables (including age, gender and body mass index) as covariates. The allele frequency of the A variant was 25.5% in IGT subjects and 23.5% in NGT subjects, and there were no differences in the distribution of genotypes between the two groups (p1⁄4 0.5). There were no significant differences in the fasting or 2-h glucose and insulin levels between the different genotypes, regardless of the glucose tolerance status, and even after adjusting for potential confounding variables (Table 1). In transient transfection experiments, the mutant promoter (A/A) activity was only 73% of the wild-type promoter (p1⁄4 0.008) when the length of the cloned glucokinase promoter was 0.5 kb from the transcription start site (Fig. 1). However, when the cloned glucokinase promoter length was increased to 1.2 kb or to 1.7 kb, the wildtype and variant glucokinase promoters exhibited similar activities. In agreement with findings reported in Caucasian subjects (4), we did not find any differences in the allelic frequency of the variant between Chinese subjects with IGT andNGT, nor in the insulin levels and HOMA index amongst subjects with different genotypes. Hence, this polymorphism is unlikely to contribute significantly to glucose intolerance in Chinese people. We have also shown that the size of the glucokinase gene promoter used in transfection experiments affects the promoter activity. The mutant promoter (A/A) activity is reduced when the Clin Genet 2003: 63: 232–234 Copyright # Blackwell Munksgaard 2003 Printed in Denmark. All rights reserved CLINICAL GENETICS