The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 8 2993-2996
Copyright © 1998 by The Endocrine Society
Variations in the Vitamin D-Binding Protein (Gc Locus) Are Associated with Oral Glucose Tolerance in Nondiabetic Pima Indians
Leslie J. Baier,
Angela M. Dobberfuhl,
Richard E. Pratley,
Robert L. Hanson and
Clifton Bogardus
Phoenix Epidemiology and Clinical Research Branch, National
Institute of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Phoenix, Arizona 85016
Address all correspondence and requests for reprints to: Dr. Leslie Baier, Clinical Diabetes and Nutrition Section, National Institutes of Health, 4212 North 16th Street, Phoenix, Arizona 85016. E-mail:
lbaier{at}phx.niddk.nih.gov
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Abstract
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Electrophoretic variants of the vitamin D-binding protein (DBP) have
been associated with type 2 diabetes as well as with metabolic
characteristics that predispose to type 2 diabetes in several
populations. The Gc gene that encodes DBP maps to
chromosome 4q12, a region that has recently been found to be
potentially linked to plasma glucose and insulin concentrations in Pima
Indians. Therefore, the gene that encodes DBP was analyzed as a
candidate gene for our linkage findings in the Pima Indians. Sequence
analysis of the coding exons identified two previously described
missense polymorphisms at codons 416 and 420, which are the genetic
basis for the three common electrophoretic variants of DBP (Gc1f, Gc1s,
and Gc2). These variants in DBP were associated with differences in
oral glucose tolerance in nondiabetic Pima Indians.
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Introduction
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WE HAVE recently completed a genomic
scan for loci linked to prediabetic phenotypes in Pima Indians (1).
Four genetic markers suggestively linked to fasting insulin
concentrations [log of the odds ratio (LOD) = 1.3–2.8] and
two markers suggestively linked to fasting glucose concentrations
(LOD = 1.4–1.9) were all clustered on chromosome 4p15-q12 (1).
The Gc gene, which encodes the vitamin D-binding protein
(DBP), maps to chromosome 4q12. Polymorphisms in this gene give rise to
three major electrophoretic variants of the DBP serum glycoprotein,
which differ by amino acid substitutions as well as attached
polysaccharide structures (2). These variants of DBP are termed Gc1
fast (Gc1f), Gc1 slow (Gc1s), and Gc2, because before the
identification of its function as a transporter of vitamin D (3), this
plasma protein was known as group-specific component (Gc). Several
groups have reported associations between alleles encoding the variants
of DBP and either type 2 diabetes or prediabetic metabolic
characteristics. In seven Polynesian Island populations, DBP is
associated with type 2 diabetes (4), and alleles encoding DBP variants
are associated with elevated plasma glucose in the ethnically mixed,
Hispanic-American/Anglos population of the San Luis Valley in Colorado
(5) and with both fasting insulin and glucose concentrations in Dogrib
Indians of the Northwest Territories (6, 7). Therefore, we investigated
DBP as a candidate for the linkage to plasma glucose and insulin
concentrations at chromosome 4q12 observed in Pima Indians.
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Experimental Subjects
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DNA samples from a group of 912 out-patient Pima Indians from
the Gila River Indian Community were genotyped for the polymorphisms in
the gene for DBP. This group included both diabetic (n = 578) and
nondiabetic subjects (n = 334), as determined by a 3-h, 75-g oral
glucose tolerance test (8). In addition, a second group of 261
nondiabetic Pima in-patient volunteers were genotyped for these
polymorphisms. The nondiabetic status of these subjects was confirmed
by a 3-h, 75-g oral glucose tolerance test (OGTT), which followed a
minimum of 3 days of a weight-maintaining diet. All OGTT results were
assessed using the criteria of the WHO (8). The 261 nondiabetic
subjects underwent multiple metabolic tests at the Clinical Research
Center, including underwater weighing to determine body composition and
a two-step hyperinsulinemic clamp at physiologic (MLow;
mean plasma insulin concentration, 865 ± 10 pmol/L) and maximally
stimulating (MHigh; mean plasma insulin concentration,
14,790 ± 335 pmol/L) insulin concentrations (9). Tritiated
glucose was infused to calculate glucose appearance rates before and
during the clamp. Glucose disposal rates were normalized to kilograms
of estimated metabolic body size (fat-free mass + 17.7) (9). These
studies were approved by the institutional review board of the NIDDK
and the Tribal Council of the Gila River Indian Community, and written
informed consent was obtained before all tests.
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Materials and Methods
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Genomic DNA from 40 Pimas was initially screened for
substitutions in the sequences that encode DBP. Each coding exon was
individually amplified by PCR using primers that recognize flanking
intron regions (2), and the PCR products were sequenced by double
stranded DNA cycle sequencing (Life Technologies, Gaithersburg, MD).
Two single base missense substitutions in exon 11, at codons 416 and
420, were identified. These 2 polymorphisms were further genotyped in
DNA samples from the group of 912 out-patient Pimas and the group of
261 nondiabetic inpatient Pimas, where a region of exon 11 was
specifically amplified and sequenced with the primers: forward,
5'-TGTAGTAAGACCTTACATTTAAATGG-3'; and reverse,
5'-TACGTTCTTAAAAGATTCTGCCATG-3'.
Values for plasma insulin concentrations and insulin action in
vivo were log10 transformed before analysis. The
general linear modeling program (SAS Institute, Cary, NC) was used to
perform repeated measures of ANOVA and to compare groups after
adjusting for covariates. This analysis takes into account multiple
measurements of a single individual and adjusts the P value
for these repeated measures. As many of the subjects in this study were
siblings, these analyses also included nuclear family membership as a
class variable to adjust for familial effects not attributable to
variations in DBP.
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Results
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Two missense polymorphisms in exon 11 of the gene that encodes DBP
were identified among the 40 Pimas screened for variants. A
GAT to GAG polymorphism (allele frequency, 0.59
and 0.41, respectively) that predicts an aspartic acid to a glutamic
acid substitution was identified at codon 416. In addition, an
ACG to AAG polymorphism (allele frequency
= 0.88 and 0.12, respectively) that predicts a threonine to a lysine
substitution was identified at codon 420. These polymorphisms
differentiate 3 of the most common DBP variants. The frequencies of
these polymorphisms in Pimas did not differ from Hardy-Weinberg
equilibrium (Table 1
). In the 912 DNA
samples from full-blooded Pima subjects (578 diabetic; 334
nondiabetic), these 2 polymorphisms were in linkage disequilibrium
(
2 = 198.6; df = 1; P < 0.001). No
association was found between DBP variants and the prevalance of type 2
diabetes (2 = 5.4; df = 5; P =
0.37).
Polymorphisms in the gene encoding DBP were also analyzed in 261
nondiabetic Pima Indians who had undergone metabolic testing (Table 2). No association was found between DBP
and basal rate of endogenous glucose production, MLow or
MHigh, or in mean fasting plasma insulin concentrations.
The mean fasting plasma glucose concentration was initially different
based on DBP type, as previously reported (5, 7), but after adjusting
for age, sex, percent body fat, and nuclear family membership, this
difference was no longer significant. In contrast, the DBP phenotypic
groups significantly differed in their plasma glucose concentration
during the OGTT (P < 0.028, by repeated measures
ANOVA). This difference was not due to variation in fasting plasma
glucose concentrations, because the differences persisted after
comparing the incremental glucose responses (above fasting
concentrations) between groups (P < 0.015, by repeated
measures ANOVA). These differences in incremental glucose persisted
after adjusting for age, sex, percent body fat, and nuclear family
membership (P < 0.05, by repeated measures of ANOVA;
Fig. 1a). Individuals homozygous for the
Gc1f allele had the highest mean incremental glucose
concentrations at 30 and 60 min as well as the lowest mean incremental
glucose concentration at 180 min. Individuals homozygous for the
Gc2 allele had significantly lower mean incremental glucose
concentrations at 60 and 120 min. Despite these differences in glucose
responses to oral glucose, the mean fold increases in insulin
concentrations (insulin response to OGTT/basal insulin) did not differ
significantly (Fig. 1b).
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Figure 1. Glucose and insulin responses (mean ±
SE) to a 75-g OGTT. a, DBP groups significantly differed in
their incremental glucose responses to an OGTT (P
< 0.05, by repeated measures of ANOVA) after adjusting for age, sex,
percent body fat, and nuclear family membership. b, DBP groups did not
significantly differ in their fold increase in insulin concentration
during an OGTT.
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Discussion
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Two previously described missense polymorphisms in Gc,
the gene that encodes DBP, were detected, and the resulting DBP
variants were associated with differences in oral glucose tolerance in
Pima Indians. There was no association of DBP variants with the insulin
response to oral glucose. Variation in insulin secretory responses
would have been expected to parallel the glycemic variations across the
DBP groups. As no difference in insulin response was observed, and all
groups were similar in the degree of insulin sensitivity and the rate
of endogenous glucose production, our result is consistent with the
association of DBP variants with the glucose response resulting from
differences in the glucose sensitivity of the insulin secretory
response. To confirm this, more detailed measurements of insulin
secretory function in subjects with DBP variants are needed.
Norman et al. demonstrated in rats that vitamin D acts on B
cells and is essential for normal insulin secretion (10, 11). Vitamin D
deficiency inhibits the insulin secretory response, and dietary vitamin
D repletion can improve the insulin response in isolated rat perfused
pancreas (10). In vitamin D-deficient elderly Dutch men, serum
concentrations of 25-hydroxyvitamin D are inversely associated with the
1 h glucose level, the area under the glucose curve, and the total
insulin concentration during a standard 1-h OGTT (12). Similarly, in
East London Asians, a significant correlation between vitamin D levels
and plasma glucose, insulin secretion, and C peptide concentrations has
been reported (13).
If the difference we observed in glucose concentrations after an OGTT
in Pima Indians is indeed due to the effects of vitamin D on the
insulin secretory response to glucose, then DBP variants would be
expected to differentially affect the level of vitamin D metabolites
within the pancreas. The 1
,25-dihydroxyvitamin D3
[1,25-(OH)2D3] metabolite of
25-hydroxyvitamin D3 has been shown to have the greatest
effect on insulin secretion (14). It is possible that the DBP variants
differentially bind 1,25-(OH)2D3, thereby
affecting the amount of 1,25-(OH)2D3 that is
present in the ß-cell. Alternatively, the association between DBP
variants and insulin secretion may not be mediated by vitamin D
metabolites. DBP binds other ligands, such as fatty acids, which may be
important because increased concentrations of islet fatty acids induce
ß-cell abnormalities (15). It is also possible that the association
of DBP with glucose tolerance is not due to functional variation in
DBP, but results from variation in a closely linked gene on chromosome
4q12.
DBP variants have previously been associated with type 2 diabetes in
seven Polynesian populations and glucose and/or insulin regulation in
an Hispanic-American/Anglo population of Southern Colorado and Dogrib
Indians of Canada (4, 5, 6, 7). In addition, subsequent to our initial report
describing linkage of this locus to prediabetic phenotypes in Pima
Indians (16), the Gc locus has been reported to be
associated with noninsulin-dependent diabetes mellitus in the Japanese
(17), and a genetic marker near this locus has been found to be linked
to fasting glucose concentrations (P = 0.001) and
quintile of glucose response (P = 0.0002) in North
American Caucasians (18). Another study, using two-dimensional
electrophoresis to compare blood proteins from normal control subjects
and first degree relatives of individuals with type 2 diabetes,
identified a polypeptide spot subsequently identified as a fragment of
DBP that was present in 80% of the relatives and 32% of the controls
(19). We conclude that although variation in DBP is not a major
determinant of diabetes or glucose tolerance, our data in combination
with those of others (4, 5, 6, 7, 17, 18, 19) suggest that it may have a
contributory role in at least five ethnic groups.
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Acknowledgments
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We gratefully acknowledge the technical assistance of Pamela
Thuillez and Christopher Wiedrich, and the support of the research
volunteers from the Gila River Indian Community.
Received February 13, 1998.
Revised May 1, 1998.
Accepted May 8, 1998.
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Abstract
Introduction
