1 Human Genomics Laboratory, Pennington Biomedical Research Center, Baton Rouge, Louisiana
2 Department of Pediatrics, "F. Fede" Second University of Naples, Napoli, Italy
3 Lipid Research Center, CHUL Medical Research Center, Québec, Canada
4 Division of Biostatistics, Washington University School of Medicine, St. Louis, Missouri
5 Laboratory of Physiological Hygiene and Exercise Science, School of Kinesiology, University of Minnesota, Minneapolis, Minnesota
6 Departments of Genetics and Psychiatry, Washington University School of Medicine, St. Louis, Missouri
7 Department of Kinesiology, Indiana University, Bloomington, Indiana
8 Keck School of Medicine, University of Southern California, Los Angeles, California
9 Quebec Heart Institute, Laval Hospital Research Center, Sainte-Foy, Québec, Canada
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ABSTRACT |
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Hepatic lipase hydrolyzes triglyceride and phospholipids from high-, intermediate-, and low-density lipoproteins, transforming them into smaller and denser particles, and promotes the cellular uptake of HDL cholesterol (1). The hepatic lipase gene (LIPC), located on chromosome 15q21-q23, is expressed in the liver. Hepatic lipase has been the subject of recent reviews for its molecular (1) and pathophysiologic significance in obesity, dyslipidemia, and atherogenesis (2). There is familial resemblance for plasma postheparin lipoprotein lipase (LPL) and postheparin hepatic lipase activities (3), and exercise traininginduced improvement in insulin sensitivity (Si) correlates with decreased postheparin hepatic lipase activity.
Genetic polymorphisms in the LIPC gene are associated with postheparin hepatic lipase activity (46). Four frequently reported genetic variants in the LIPC promoter are in almost complete linkage disequilibrium, forming two haplotypes (2). The less common T allele in the 514C>T variant has been associated with a 3040% decrease in postheparin hepatic lipase activity and higher HDL cholesterol levels (4). However, the relevance of this polymorphism for the transcriptional regulation of the LIPC gene remains controversial (710).
The benefits of regular physical activity in decreasing the risk of developing insulin resistance and coronary heart disease (CHD) are well documented, although individual responses vary considerably (11,12). Regular exercise affects lipid-lipoprotein profiles and peripheral glucose utilization (12,13). We investigated associations between the LIPC 514C>T variant and plasma lipoprotein levels, Si and postheparin hepatic lipase, and postheparin LPL activities in a biracial cohort under sedentary-state conditions and their responses to endurance exercise training.
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RESEARCH DESIGN AND METHODS |
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Exercise training program.
The exercise training program has been described in detail previously (14,15). Briefly, the exercise intensity of the 20-week program was customized for each participant based on the heart rateoxygen uptake relationship measured at baseline. During the first 2 weeks, the subjects trained at a heart rate corresponding to 55% of VO2max for 30 min per session. Duration was gradually increased to 50 min per session and intensity to the heart rate associated with 75% of the baseline VO2max. These conditions were then sustained for the last 6 weeks. Training frequency was three times per week, and all training sessions were performed under supervision on cycle ergometers at the participating clinical centers. The subjects were instructed not to change their diet during the intervention.
Phenotype measurements.
All phenotypes were measured at baseline and after the 20-week exercise training program.
Measurement of Si.
A frequently sampled intravenous glucose tolerance test was performed as described (16) in the morning after a 12-h overnight fast and no less than 24 h after the last exercise session. From the frequently sampled intravenous glucose tolerance test data, Si was derived using the MINMOD Millennium computer program (17). Si measures the ability of plasma insulin to enhance the net disappearance of glucose from plasma. These analyses were described previously (16).
Postheparin lipase activities, lipids, and lipoproteins.
Postheparin LPL and postheparin hepatic lipase activities were measured by the modified Nilsson-Ehle and Ekman method, as previously reported (13). Activity is expressed as nanomoles of free fatty acid released divided by milliliters of plasma divided by minutes. Lipid, lipoprotein, and apolipoprotein (apo) assays have been reported previously in detail (3).
Genetic analyses.
Genomic DNA was extracted from lymphoblastoid cell lines using standard procedures. Genotyping for the LIPC 514C>T polymorphism (dbSNP rs#1800588) was performed using the TaqMan allelic discrimination method. Details for PCR conditions and primer/probe sequences are available upon request.
Statistical analyses.
All statistical analyses were performed with the SAS Statistical Software Package (release 8.2; SAS Institute, Cary, NC). A 2 test was used to compare allele frequencies and to test whether the genotype distributions were in Hardy-Weinberg equilibrium. Associations between the LIPC 514C>T genotypes and phenotypes were analyzed using a MIXED procedure. Nonindependence among family members was adjusted using a "sandwich estimator" as described in previous HERITAGE publications. Because of the skewed distributions, baseline plasma triglyceride, VLDL cholesterol, and HDL cholesterol were normalized with log transformation. The Si data derived from the MINMOD Millennium model required a square root transformation to approximate a normal distribution. All analyses were done separately in blacks and whites. All baseline data were adjusted for age, sex, and BMI, whereas the training responses were also adjusted for the respective baseline values.
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RESULTS |
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DISCUSSION |
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The LIPC 514TT homozygotes had 30% lower postheparin hepatic lipase activity compared with the CC homozygotes, and the genotype accounted for 78% of the variation in postheparin hepatic lipase activity. This association remained after exercise training. Blacks had lower postheparin hepatic lipase activity, an ethnic difference found in both sexes and concordant with previous observations (19,20). Several in vitro studies have reported a 3040% reduction in transcriptional activity in cells transfected with the T allele compared with the C allele (rev. in 2). Other studies found no functional effects of this allelic variant, perhaps because of differences in cell lines, transfection efficiencies, and reporter vectors used (7,8,10). Overall, our data strengthen reports that associate postheparin hepatic lipase activity with the LIPC 514C>T polymorphism (4,7,9). Recently, a prospective cohort study found that the TT genotype had increased susceptibility to CHD in sedentary or moderately physically active subjects but not among the vigorously active (21).
Individuals carrying the LIPC 514T allele had higher postheparin LPL activity at baseline and after exercise training. We have reported earlier higher postheparin LPL activity in blacks in the sedentary state (22) and that increased LPL activity was associated with an improved lipoprotein-lipid profile in response to an exercise training program (13). Here we report that the TT homozygotes had 25% higher LPL activity after exercise training than the CC homozygotes. The increase in LPL activity after endurance exercise training in combination with the higher HDL cholesterol observed in the TT homozygotes could impact CHD risk.
Hepatic lipase deficiency modifies the lipoprotein profile. It has been related to increased plasma cholesterol and trigylceride levels and mass redistribution within HDL particles (2). The most consistent finding in hepatic lipase deficiency is increased HDL2. The LIPC 250G>A variant, which is in complete linkage disequilibrium with the LIPC 514C>T, also leads to decreased hepatic lipase activity in the A allele carriers and to buoyant LDL particles and high HDL cholesterol (4). We found associations between the LIPC 514C>T polymorphism and the lipoprotein profile in the sedentary state, with higher HDL cholesterol and apoA-1 levels in the TT homozygotes. Although there were no significant associations with other lipoproteins, the association for HDL cholesterol levels remained significant in the trained state. Increased risk of atherosclerosis has been associated with the LIPC 514T allele, and CC homozygotes benefit the most from intensive lipid-lowering therapy (4,23).
This study is the first to examine the associations between the LIPC 514C>T genotypes and Si and the responses of postheparin lipase to an exercise training intervention. The LIPC 514C>T genotypes showed associations with Si and postheparin hepatic lipase activity after exercise training. The enhanced Si after exercise training, primarily in the CC homozygotes, was associated with higher postheparin hepatic lipases activity. Remarkably, these Si differences appeared only in response to exercise training. The second European Atherosclerosis Research Study reported that T allele carriers have the lowest glucose tolerance (24). Recently, the LIPC-205G>A polymorphism, which is in complete linkage disequilibrium with the LIPC 514C>T, has been reported to predict the conversion from impaired glucose tolerance to type 2 diabetes in a 3-year follow-up lifestyle intervention (25).
The mechanisms behind the association of the LIPC 514C>T polymorphism and Si are unclear. Changes in blood lipids in response to decreased postheparin hepatic lipase activity and increased postheparin LPL activity may accompany changes in intramyocellular lipid storage, affecting Si (26). Several regulatory elements have been identified in the rat LIPC promoter, and consensus analysis with the human sequence suggests that the LIPC 514C>T variant may be in a DNA motif containing the sequences necessary for insulin/glucose responses. Insulin regulation of the LIPC gene could be mediated by either upstream stimulatory factor or sterol regulatory elementbinding protein-1c transcription factors, which bind to the E-box motif located in the LIPC promoter region at 514 bp, although this has not been demonstrated (9). One could speculate that human variation in Si response to exercise training associated with the LIPC genotype is the consequence of transcriptional effects on the LIPC gene by insulin and the binding (or lack of) of transcription factors to the E-box motif.
Finally, the present study suggests that regular exercise may not influence the lipoprotein profile or improve Si at the same rate for all individuals and that the efficacy of regular exercise could be related in part to the LIPC 514C>T genotype. C allele carriers may not only have more clinical benefit from intensive lipid-lowering therapy, as reported (4), but are more likely to benefit from an exercise training program. The improvement in Si with regular exercise in individuals with the LIPC 514C allele has significant clinical applications due to its high frequency in various populations (4,5,7). Arguably, screening for T allele carriers may allow the identification of subjects at elevated risk requiring individualized therapeutic strategies to decrease cardiovascular and metabolic disease risks.
In summary, results from the HERITAGE Family Study indicate that the C allele at 514 in the promoter region of the LIPC gene is associated with higher postheparin hepatic lipase activity and better Si response to regular exercise. The benefits on Si from an exercise program could be of considerable importance in the general population given the high frequency of the LIPC 514C allele.
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ACKNOWLEDGMENTS |
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Gratitude is expressed to Dr. Jack H. Wilmore for the critical reading of the manuscript and his comments. We thank Anik Boudreau for her expert contribution to the single nucleotide polymorphism genotyping. Thanks are expressed to Nina Laidlaw for her editorial support.
Address correspondence and reprint requests to Claude Bouchard, Human Genomics Laboratory, Pennington Biomedical Research Center, 6400 Perkins Rd., Baton Rouge, LA 70808. E-mail: bouchac{at}pbrc.edu
Received for publication July 26, 2004 and accepted in revised form April 1, 2005
apo, apolipoprotein; CHD, coronary heart disease; HERITAGE, Health, Risk Factors, Exercise Training, and Genetics; LPL, lipoprotein lipase
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REFERENCES |
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