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Experimental Studies |
Steno Diabetes Center and Hagedorn Research Institute (S.A.U., T.H., L.B.J., L.H., O.P.), Copenhagen, Denmark; Center of Preventive Medicine (J.C.), Glostrup University Hospital, Copenhagen, Denmark; and Division of Endocrinology and Metabolism (K.C.C.), University of California Los Angeles School of Medicine, Los Angeles, California
Address all correspondence and requests for reprints to: Søren A. Urhammer, M.D., Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. E-mail: sau{at}novo.dk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Association studies were performed in 246 Danish Caucasian NIDDM patients recruited from the outpatient clinic at Steno Diabetes Center, Copenhagen, and 242 age-matched and glucose-tolerant Danish Caucasian control subjects traced in the Danish Central Population Register and living in the same area of Copenhagen as the NIDDM patients. By a questionnaire, it was determined that the study participants were unrelated, i.e. no participants were full or half-sibs. NIDDM was diagnosed by World Health Organisation criteria, and all control subjects underwent a standard 75-g oral glucose tolerance test (OGTT).
For studies of insulin and C-peptide release, insulin sensitivity index (Si), and glucose effectiveness (Sg), 380 subjects were recruited randomly from a population of young individuals, 18–32 yr old (15). All were Danish Caucasians by self-identification. Physiological characteristics of the present population sample have been presented previously (15). Before participation in the study, informed consent was obtained from all subjects. The study was approved by the Ethical Committee of Copenhagen and was in accordance with the principles of the Declaration of Helsinki.
Clinical and biochemical variables
Body mass index (BMI), waist-to-hip ratio, fat mass, plasma concentration of glucose, serum levels of triglyceride, total cholesterol, and high-density lipoprotein cholesterol (Boehringer Mannheim GmbH, Diagnostica, Mannheim, Germany) were analyzed as described (15). Serum insulin and C-peptide were analyzed using Steno Diabetes Centers routine procedures (15).
Measurements of insulin secretion, Si, and Sg
After a 12-h overnight fast, each subject underwent an iv glucose tolerance test. Baseline values of plasma glucose, serum insulin, and C-peptide were obtained. Glucose was injected iv into the contralateral antecubital vein over a period of 1 min (0.3 g/kg BW of 50% glucose). At 20 min after the end of the glucose injection, a bolus of 3 mg tolbutamide/kg BW (Rastinon, Hoechst, Germany) was injected during 5 sec to elicit a secondary pancreatic ß-cell response. Venous blood was sampled at 2, 4, 8, 19, 22, 30, 40, 50, 70, 90, and 180 min (timed from the end of the glucose injection) for measurements of plasma glucose, serum insulin, and C-peptide. Glucose-induced acute serum insulin and C-peptide responses (0–8 min) were calculated by means of the trapezoidal rule, as the incremental values (area under the curve when expressed above basal values). Si and Sg were calculated using the Bergman MINMOD computer program developed specifically for the combined iv glucose and tolbutamide tolerance test (16).
Detection of the G-to-A -258 liver glucokinase promoter variant
The DNA segment containing the variant was PCR-amplified using sense primer 5'-CAGACCCCTGGATTGTATGAAATG-3' and antisense primer 5'-GGCTGCCTTGGCCACAGTA-3'. The PCR (model 9600, Perkin Elmer/Cetus, Norwalk, CT) started with denaturation at 94 C for 2 min, followed by 35 cycles of denaturation (94 C, 1 min), annealing (62 C, 1 min), and extension (72 C, 1 min) with a final extension at 72 C for 10 min. The gene variant occurs within a region that is not cut by any restriction enzyme. Thus, a de novo AccI site was created by replacing one of the nucleotides in the antisense primer. The amplified product was digested at 37 C for 3 h with 1 U of AccI and analyzed on a 3% agarose gel (1% GTG Nusieve, 2% Seakem Agarose).
Statistics
-square analysis was applied to test for significance of
differences in allelic and genotype frequencies. A Mann-Whitney test
was used for comparison between groups. Data are means ±
SD. A P-value less than 0.05 (two-tailed) was
considered significant. Multiple regression analysis was used to test
for interaction between the glucokinase promoter variant and obesity.
In this context, obesity was defined as BMI 
25
kg/m2. Interaction terms BMI and the variant in its
homozygous and heterozygous form were constructed and included in the
analysis. Statistical Package of Social Science for Windows, version
7.0, was used for statistical analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The observed genotype frequencies were in
Hardy-Weinberg equilibrium. The clinical and biochemical data of the
242 middle-aged subjects comprising the control group in the
association study are listed in Table 2 according to the
genotype. During the OGTT, the values of plasma glucose and serum
insulin did not differ among the three genotype groups of normal
subjects. Neither did fasting serum values of lipids nor the clinical
characteristics of the subjects differ between the groups. There was no
interaction between obesity and the liver glucokinase variant,
considering serum insulin or serum C-peptide responses during the OGTT
(data not shown).
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). In particular, no
associations between the -258 gene variant and whole-body Si or the
pancreatic ß-cell function were observed (Table 3). Neither was there
any interaction between obesity and the glucokinase variant,
considering Si or ß-cell function.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Within the control group of middle-aged subjects the, variant was not related to any alterations in circulating glucose or insulin levels after an oral glucose challenge. Neither were we able to demonstrate any relationships between the -258 variant and the Si, Sg, pancreatic ß-cell function, or fasting serum lipids in the cohort of 380 young healthy Danish Caucasians.
Previous studies have shown associations between dinucleotide markers at the glucokinase locus and NIDDM in American blacks and in Mauritian Creoles, suggesting that the glucokinase gene might play a role in the common form of NIDDM in these ethnic groups (17, 18). However, mutational analysis of the coding region of the glucokinase gene, including the tissue-specific exon 1a of the pancreas and exon 1b of the liver, have not revealed common NIDDM-associated mutations in this gene (19). In the liver-specific glucokinase promoter, the -258 variant was identified in American black women with gestational diabetes (13). The -258 G-to-A substitution of the liver glucokinase promoter occurs within a conserved segment, which is highly homologous to the insulin regulatory sequence of the phosphoenolpyruvate carboxykinase (PEPCK) gene (20). PEPCK governs the rate limiting-step in gluconeogenesis (20). Thus, both PEPCK and glucokinase are key enzymes in the glucose metabolism in the liver. Therefore, it has been suggested that the -258 variant may affect the transcription rate of glucokinase and thereby reduce the insulin sensitivity of the liver, leading to hyperinsulinemia (14). The hypothesis that the -258 promoter variant may cause insulin resistance was further supported by transfection experiments, which showed that the variant causes a 58% reduction in the glucokinase promoter activity (14). However, there is no information available in the literature showing to what degree the promoter activity should be reduced in order to provoke insulin resistance. Furthermore, it is not possible to extrapolate directly from in vitro transfection experiments, which are simple models, to the very complex metabolic network in the human liver cell.
Previously, a 50% reduction in liver glucokinase activity was demonstrated in NIDDM patients, compared with nondiabetic subjects, indicating a pathogenic role of the liver glucokinase in common NIDDM (12). However, it is not known whether this finding may be explained by a secondary metabolic dysfunction. Similarly, observations in transgenic mice with disruption of one allel of the liver and the pancreatic glucokinase genes showed both decreased glucose tolerance and abnormal liver glucose metabolism, but it was not possible to determine whether an impaired liver glucokinase activity was implicated as a primary pathogenic factor in the disease development (21).
In the present study, which is the first study of the -258 glucokinase variant in a Caucasian population, we were not able to confirm the results from black American subjects. The study in blacks showed that only homozygous carriers of the variant had higher serum values of insulin at 60 and 90 min during an OGTT, compared with wild-type and heterozygous carriers (14). Because these results (14) are based on only three homozygous carriers, whereas, respectively, 8 and 10 homozygous carriers were detected in the present study populations, false positive results are possible.
In our protocol, we measured insulin sensitivity after iv glucose and tolbutamide injections, with data interpretation in accordance to Bergmann’s minimal model. This index of insulin sensitivity comprises both the effect of insulin on hepatic glucose output and glucose uptake in peripheral tissue. To obtain a more precise estimate of the potential role of the -258 liver glucokinase variant on insulin-controlled hepatic glucose release, however, consideration might be given to studying homozygous carriers of the variant, applying a euglycemic clamp at several physiological steady-state levels of hyperinsulinemia.
We conclude that the common nucleotide substitution, at position -258 of the liver glucokinase promoter gene is not associated with NIDDM among Caucasian subjects. Nor does it seem that the variant is related to altered Si or insulin secretion in healthy subjects.
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Acknowledgments |
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Footnotes |
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Received December 9, 1996.
Accepted February 19, 1997.
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