From the Division of Kinesiology, Department of Social and Preventive Medicine, Faculty of Medicine, Laval University, Ste-Foy, Québec, Canada.
Address correspondence and reprint requests to Denis R. Joanisse, PhD, Division of Kinesiology, Department of Social and Preventive Medicine, Faculty of Medicine, Room 0223 PEPS, Laval University, Ste-Foy, Québec, Canada, G1K 7P4. Email: denis.joanisse{at}kin.msp.ulaval.ca .
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ABSTRACT |
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INTRODUCTION |
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As recently described for other populations (Swedish [0%] and Danish [0.4%]
[13]), the genotype frequency
of the Met416Val mutation was relatively low (only 1.3%) in our cohort of
French-Canadian subjects (116 men and 41 women) when compared with Finnish
(17%) (5) or Japanese
(10%) (8) groups. Only two
subjects in our cohort were heterozygous for this mutation (i.e., a carrier
genotype frequency of 1.3%). Because of this low number of carrier subjects,
no further investigation was pursued with this mutation. Interestingly, none
of a cohort of 130 French individuals carried the Met416Val mutation (J.S.-O.,
J.-A.S., C. Bouchard, unpublished data). Given its low frequency in many
different populations, it is unlikely that the Met416Val mutation contributes
significantly to insulin resistance in the general population. In addition, a
recent report showed no effect of Met416Val on GS activity
(13), further casting doubt on
its potential role in insulin resistance. However, the genotype frequency of
the XbaI variant in French-Canadians (17.2%) was similar to that
previously reported in other ethnic groups
(4,7,9,11,12).
Of the 157 subjects, 27 were identified as carrying the A1A2 alleles, whereas
all others were homozygous for the A1 allele.
To determine if the XbaI polymorphism influences GS protein content in skeletal muscle (measured by dot blot, Fig. 1), vastus lateralis muscle biopsies (14) were taken from 21 carriers and 21 noncarriers pair-matched for sex, age, BMI, and level of physical activity. As shown in Fig. 2, no difference was found under basal conditions in GS protein content measured in muscle from carriers of the XbaI variant allele (46.9 ± 1.2 U/g) and noncarriers (44.9 ± 10.2 U/g), which is in agreement with the work of Groop et al. (4).
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The originality of our study lies in the fact that we verified if carriers and noncarriers of the XbaI polymorphism responded differently to a standardized environmental stimulus recognized to alter GS protein content in skeletal muscle. The knee extensor muscles of seven matched pairs of carriers and noncarriers were subjected to neuromuscular electrical stimulation lasting 6 weeks combined with vastus lateralis muscle biopsies taken before and after the protocol. Neuromuscular electrical stimulation (NMES) leads to significant alterations in skeletal muscle characteristics in humans (15). In addition to functional and contractile property changes, the content or activity of markers of different metabolic pathways, including GS activity, has been substantially modulated after as little as 4 weeks of chronic low-frequency NMES (16). Not only was the NMES-induced increase in skeletal muscle GS protein content recently confirmed in humans, the increase was shown to occur concomitantly with improved insulin-stimulated whole-body glucose uptake (17). However, the magnitude of the changes varied substantially among individuals.
As illustrated in Fig. 3, the key finding of the present study is that the significant stimulation-induced increase (23%) in skeletal muscle GS protein content seen in individuals having the XbaI A1A1 wild-type genotype was impaired in the carriers of the A2 variant allele (genotype effect and interaction P < 0.05). This result does not arise from global differences in the response of skeletal muscle from the two groups of subjects to NMES. This is evidenced by the similar behaviors of creatine kinase (CK) activity (unchanged) and cytochrome c oxidase (COX) activity (increased by 22%; NMES effect P < 0.05); both of these results are consistent with previous findings (15). Therefore, the difference measured in GS content after NMES appears to be genotype related.
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Our results are particularly interesting in light of the recent report of Orho-Melander et al. (6), which showed increased susceptibility of XbaI polymorphism carriers to hypertension, insulin resistance, and earlier onset of type 2 diabetes. Our data provide clues to a mechanism that may explain their results. It is unlikely that the XbaI polymorphism, located in untranslated intron 14, causes any alteration in the activity of GS itself. Other than direct effects on mRNA stability, this genetic variant may be in linkage disequilibrium with other yet unidentified mutations within the coding or flanking regions of the GYS1 gene. Our results suggest that whatever the regulatory mechanisms involved, the differences in GS content become manifest only under certain specific conditions. Thus, the present study supports the thrifty genotype hypothesis proposed by Neel (18), stipulating that some individuals have a survival advantage during periods of restrictive energy intake but, when exposed to periods of decreased energy expenditure and food abundance, favor fat deposition. Insulin resistance would be a major factor involved in the channeling of ingested fat towards tissues such as adipose tissue and skeletal muscle for storage rather than skeletal muscle for oxidation (19). Indeed, the results of our study show that individuals differ in their capacity to alter the content of an important metabolic marker of skeletal muscle insulin-stimulated glucose storage in response to a challenge (NMES) based on their different genetic background. If, in the nonresponder individuals, a similar lack of adjustment is taking place in response to environmental conditions, such as a positive energy balance or a diabetogenic diet, it follows that this could increase their likelihood to develop skeletal muscle insulin resistance and type 2 diabetes over time.
In conclusion, the allelic frequency of the GYS1 XbaI variant observed in the present study was similar to previously reported results and significantly more frequent than the Met416Val mutation, which was relatively rare in our cohort of French-Canadian subjects. Confirming previous results, muscle biopsies taken under basal conditions from the carriers and noncarriers of the XbaI polymorphism contained similar GS protein content. However, the novel and important finding of the present study is that the stimulation-induced increase in skeletal muscle GS protein content normally seen in individuals having the wild-type genotype is completely impaired in those carrying the XbaI mutation. These data demonstrate that some individuals, because of their genetic background, are unable to stimulate the processes of GS protein accumulation in skeletal muscle. These results could explain why some individuals appear to be genetically pre-disposed to develop skeletal muscle insulin resistance when exposed to unfavorable metabolic environments.
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RESEARCH DESIGN AND METHODS |
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Determination of the XbaI and Met416Val polymorphisms. The XbaI polymorphism in intron 14 of the GYS1 gene was genotyped using the previously described polymerase chain reaction (PCR) method (7). The A1 and A2 alleles were determined by the absence or recognition of the XbaI restriction site formed by the substitution of a cytosine for a thymine in the GYS1 nucleotide sequence of intron 14. The Met416Val polymorphism was genotyped using the previously described PCR method (20). The wild-type and mutated alleles were determined by the presence or absence of the NlaIII restriction site that occurs from a nucleotide substitution changing a methionine to a valine in the amino acid sequence of the GS protein.
NMES protocol. The procedures used were previously described (15). Briefly, knee extensor muscles of both thighs were subjected to 3 h of NMES per day, 6 days per week for 6 weeks. NMES was delivered at a low frequency (8 Hz) using a portable battery-powered stimulator (Respond II; EMPI) and 3-in diameter round adhesive electrodes (Pals Plus 9000; EMPI).
GS protein content. Muscle samples of the subjects, frozen in liquid
nitrogen and kept at -70°C until used, were homogenized (1:40 wt:vol) in
buffer (Na-K-PO4 100 mmol/l, EDTA 2 mmol/l, pH 7.2). The total
protein content of the muscle homogenates was determined using the Bio-Rad
Protein Assay method. Quantification of the GS protein content was done with
the use of an antibody provided by J. C. Lawrence at the University of
Virginia School of Medicine, Charlottesville, VA. Mini-PROTEAN II and
Trans-Blot Cell equipment (Bio-Rad) were used for sodium dodecyl
sulfatepolyacrylamide gel electrophoresis and Western blotting
following the procedures recommended by the manufacturer. Of the total
protein, 8 µg was electrophoretically separated (0.75-mm thick 10% SDS
gels) and electrically transferred (60 min at 100 V) to polyvinylidene
fluoride membranes (Immobilon; Millipore). After blocking in 5% powdered milk,
membranes were incubated for 1 h with 0.05 µg/ml affinity-purified GS
antibody, washed three times in Tris-buffered saline with 0.1% Tris-buffered
saline with Tween-20 (TBST) solution, and incubated with rabbit anti-chicken
antibodies conjugated to alkaline phosphatase (5,000-fold dilution). Membranes
were washed again three times in TBST before antibody binding was detected
using the alkaline phosphatase reaction. A single major band was found at
84 kDa corresponding to the molecular weight of GS
(Fig. 1A). Dot blots
(Fig. 2B) (5 µg of
total protein per well) were subsequently used to quantify protein levels, and
these were made in triplicate for each sample. The reaction product of each
blot was scanned and analyzed twice with the use of National Institutes of
Health Image analysis software. A gradient concentration of human latissimus
dorsi muscle proteins (from 2 to 18 µg) was deposited on each membrane and
served as internal standard. The results were expressed as arbitrary units of
protein per gram of wet weight muscle (U/g).
CK and COX enzyme activities. The activities of CK and COX from pre- and poststimulated muscles were spectrophotometrically determined by standard methods (21) and expressed in U (µmol substrate consumed/min)/g wet wt muscle.
Statistical analysis. Unpaired Student's t tests were used to test for significant differences among genotypes. Two-factor analyses of variance were used to test for differences in the stimulation-induced changes in muscle GS protein content and CK and COX enzyme activities between carriers and noncarriers of the XbaI gene polymorphism. Data are presented as means ± SD.
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ACKNOWLEDGMENTS |
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The cooperation of our research volunteers is gratefully acknowledged. The collaboration of Yves Gélinas, MSc; Rémy Thériault, PhD; and Germain Thériault, MD is also appreciated. We would like to give special thanks to Dr. David E. Kelley for his critical comments.
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FOOTNOTES |
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CK, creatine kinase; COX, cytochrome c oxidase; GS, glycogen synthase; NMES, neuromuscular electrical stimulation; PCR, polymerase chain reaction; TBST, Tris-buffered saline with Tween.
Received for publication April 13, 2000 and accepted in revised form September 8, 2000
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REFERENCES |
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