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Division of Endocrinology, Diabetes, and Metabolism (M.O.G.) and Medical Genetics Institute (M.O.G., K.D.T., X.G., J.C., Y.-D.I.C., J.I.R.), Cedars-Sinai Medical Center, Los Angeles, California 90048; Department of Medicine (M.O.G., Y.-D.I.C., J.I.R.), David Geffen School of Medicine at University of California, Los Angeles, California 90095; Department of Preventive Medicine and Biometrics (J.E.H.), University of Colorado Health Sciences Center, Denver, Colorado 80262; Department of Medicine (S.M.H.), University of Texas Health Sciences Center at San Antonio, San Antonio, Texas 78229; Department of Public Health Sciences (L.E.W.), Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157; and Department of Physiology and Biophysics (R.N.B.), Keck School of Medicine, University of Southern California, Los Angeles, California 90033
Address all correspondence and requests for reprints to: Mark O. Goodarzi, M.D., Ph.D., Cedars-Sinai Medical Center Division of Endocrinology, Diabetes, and Metabolism, 8700 Beverly Boulevard, Becker B-131, Los Angeles, California 90048. E-mail: mark.goodarzi{at}cshs.org.
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Abstract |
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Objective: In this study of Hispanics in the Insulin Resistance Atherosclerosis Study Family Study, we sought to replicate LPL haplotype association with insulin sensitivity/resistance.
Design: LPL haplotypes based on 12 single nucleotide polymorphisms were analyzed for association with indexes of insulin sensitivity and other metabolic and adiposity measures.
Setting: This study was conducted in the general community of San Antonio, Texas, and San Luis Valley, Colorado.
Participants: Participants in this study were 978 members of 86 Hispanic families.
Main Outcome Measures: LPL haplogenotype, metabolic phenotypes, and adiposity were measured in this study.
Results: The haplotype structure was identical with that observed in prior studies. Among 978 phenotyped subjects, haplotype 1 was associated with decreased fasting insulin (P = 0.01), and haplotype 4 was associated with increased fasting insulin (P = 0.02) and increased visceral fat mass (P = 0.002). Insulin sensitivity, derived from iv glucose tolerance testing, tended (P > 0.1) to be higher with haplotype 1 (SI = 1.72) and lower with haplotype 4 (SI=1.38). Haplotype 2 was associated with increases in fasting insulin, triglycerides (TGs), TG to high-density lipoprotein-cholesterol ratio, and apolipoprotein B (P = 0.01–0.04).
Conclusions: This study independently replicates our prior results of LPL haplotypes 1 and 4 as associated with measures of insulin sensitivity and resistance, respectively. Haplotype 4 may confer insulin resistance by increasing visceral fat. Haplotype 2 was identified as a new risk haplotype, suggesting the complex nature of LPL’s effect on features of the insulin resistance syndrome.
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Introduction |
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We have focused on the 3' end of LPL, distal to a recombination hotspot in intron 6 (2). We found that haplotypes based on six haplotype-tagging single nucleotide polymorphisms (SNPs), spanning introns 7–9, were associated with prevalent coronary artery disease (CAD) in Mexican Americans with a family history of CAD in the Mexican-American Coronary Artery Disease (MACAD) study (3). In MACAD, these haplotypes were associated with insulin sensitivity/resistance (4); a consistent pattern emerged, with the most common haplotype associated with protection against CAD and insulin resistance and the fourth haplotype predisposing to these conditions. We hypothesized that these effects were due to linkage disequilibrium with functional variants in the 3'-untranslated region (UTR) of LPL, encoded by exon 10; in rodents, LPL 3' UTR sequences influence translation of LPL (5). To test this hypothesis, we sequenced exon 10; additional polymorphisms discovered in exon 10 were combined with the original six SNPs and genotyped in the entire cohort, resulting in haplotypes based on 19 SNPs (6). The extended fourth most common haplotype (designated 19-4) showed association with postheparin plasma LPL activity, suggesting the presence of functional variants (6).
In the current study, we turned to Hispanics in the Insulin Resistance Atherosclerosis Study (IRAS) Family Study (7). Our goal was to replicate our principal result that LPL 3' end haplotypes are associated with insulin sensitivity/resistance. We also evaluated haplotype association with other phenotypes. We found that the same two LPL haplotypes (haplotypes 1 and 4) associated with insulin sensitivity/resistance in MACAD were also associated with indexes of insulin sensitivity/resistance in the IRAS Hispanics. Furthermore, the insulin resistance haplotype 4 was associated with visceral adiposity. We also discovered in IRAS that haplotype 2 was associated with increased fasting insulin and adverse effects on lipid parameters, representing a new risk haplotype.
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Subjects and Methods |
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Participants were members of Hispanic families recruited for the IRAS Family Study from two clinical sites (San Antonio, Texas, and the San Luis Valley, Colorado). For the study design and recruitment strategies, see Henkin et al. (7). All subjects gave informed consent.
Genotyping and haplotype determination
Twelve SNPs were genotyped in LPL, including the original six 3' end SNPs, rs312, rs319, rs320, rs327, rs328, rs330 [previously designated 7315, 8292, 8393, 8852, 9040, 9712 (3, 4, 8)]. We also genotyped the following exon 10 variants identified from prior sequencing (6): rs4922115, rs3289, rs3200218, rs1059611, rs15285, and rs3866471. These six variants were predicted to tag the common haplotypes in exon 10.
The 12 SNPs were genotyped in 1424 subjects from 90 families using the 5'-exonuclease assay (TaqMan MGB) and primers and probes described previously (3, 6, 9).
The program Haploview was used to determine haplotype frequencies as well as delineate haplotype blocks (10). Haploview constructs haplotypes using an accelerated expectation maximization algorithm. Haploview was used to calculate linkage disequilibrium (LD, D', and r2) between each pair-wise combination of SNPs.
Haplotypes were constructed as the most likely set (determined by maximum likelihood) of fully determined parental haplotypes of the marker loci for each individual, using the simulated annealing algorithm implemented in Simwalk2 (11). This allowed us to assign a haplogenotype to 1262 of the 1424 genotyped subjects.
Phenotyping
Indexes of glucose homeostasis were assessed by the frequently sampled iv glucose tolerance test (IVGTT), with minimal model analyses (12). The IVGTT protocol was modified as previously described (13). Insulin sensitivity index (SI) and glucose effectiveness were calculated using the minimal model. The acute insulin response to glucose was the mean insulin increment in the plasma insulin concentration above the basal in the first 8 min after the administration of glucose. Disposition index was calculated as DI = AIR x SI. Plasma glucose was obtained using standard methods, and insulin was measured by a single-antibody RIA (14). Glucose and insulin values were used to derive the homeostasis model assessment (HOMA) index of insulin resistance (15). Of the genotyped subjects, 978 subjects from 86 families were haplotyped and had measures of insulin sensitivity.
Lipid parameters, blood pressure, and anthropometry were measured as previously described (13, 16). Computed tomographic evaluation of visceral and sc fat at the L4-L5 level was performed (17).
Data analysis
Association was evaluated by quantitative transmission disequilibrium testing using the QTDT program (18). Age, gender, and body mass index (BMI) were specified as covariates. The within-family component of association was evaluated to eliminate any effects of population stratification. Log-transformed or square-root transformed trait values were used as appropriate to reduce skewness. For illustrative purposes, trait values by haplotype are presented as the median values in carriers of a particular haplotype vs. noncarriers.
The primary phenotypes for association were indexes of insulin sensitivity (fasting insulin, HOMA, and SI), given our main goal of replicating association of LPL haplotypes with insulin sensitivity/resistance. Overall P values and haplotype-specific P values were calculated for the primary traits. Secondary phenotypes included the acute insulin response to glucose, DI, glucose effectiveness, lipid traits, measures of adiposity [BMI, waist-to-hip ratio, sc and visceral adipose tissue (VAT)], and blood pressure. Only haplotypes showing association with primary traits were analyzed for association with secondary traits.
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Results |
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. The haplotypes observed in this Hispanic population were also observed in our prior studies of Hispanics in the MACAD study, with modest differences in frequency (Table 1).
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). Haplotypes 2 (P = 0.0096) and 4 (P = 0.022) were associated with insulin resistance, i.e. higher fasting insulin. Association results for HOMA values tracked exactly as the fasting insulin results. SI was not statistically significantly associated with these haplotypes; however, median values agreed, with haplotype 1 associated with insulin sensitivity (higher SI) and haplotypes 2 and 4 with insulin resistance (lower SI) (Table 2).
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Discussion |
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Our prior work demonstrated association of haplotype 1 with insulin sensitivity and haplotype 4 with insulin resistance in the MACAD study (4). In this study of Hispanic families, we demonstrated association of haplotype 1 with decreased fasting insulin and haplotype 4 with increased fasting insulin. This represents confirmation of our initial findings in a separate population of similar ethnicity. In the genetic epidemiology of common disorders, independent replication of linkage or association results, achieved in only a fraction of all studies (19), provides convincing support for the effect of a gene on a phenotype. Variation in the 3' end of LPL appears to influence insulin sensitivity/resistance in Hispanics; whether it does so in other populations remains to be shown.
In the prior MACAD study, insulin resistance was quantified by fasting measures and the hyperinsulinemic-euglycemic clamp. In that study, the clamp-derived measures achieved statistical significance for associations with LPL haplotypes 1 and 4; the trait values for fasting insulin and HOMA were consistent (4). In the present study, statistical significance for association with LPL haplotypes was found with fasting insulin/HOMA but not SI from the IVGTT, although median SI values were consistent in terms of insulin sensitivity for haplotype 1 (higher SI) vs. insulin resistance for haplotype 4 (lower SI). Because there is no physiological reason to propose the clamp is superior to the IVGTT, we believe this discrepancy is a statistical phenomenon.
Overactivity of LPL may lead to excessive adipose accumulation. Excess adiposity, particularly in the visceral depot, may contribute to insulin resistance via altered secretion of adipokines such as adiponectin and leptin. Our prior work identified haplotype 4 as predisposing to insulin resistance and increased LPL activity. The present study suggests a possible mechanism unifying these findings, that the increased LPL activity with haplotype 4 is mainly expressed in VAT, leading to increased visceral fat mass and consequently insulin resistance.
Herein, haplotype 2 was associated with adverse effects on fasting insulin and lipid parameters, multiple facets of the metabolic syndrome. That Hispanic Americans have the highest age-specific prevalence of the metabolic syndrome (20) may in part be explained by their high frequency (
18%) of haplotype 2. Haplotype 2 was not associated with insulin resistance in MACAD, perhaps because of the smaller sample size of MACAD. This is the first detection of phenotypic effects of haplotype 2. Given the non-disease-specific ascertainment of IRAS families, haplotype 2 is likely to be important to Hispanics in general and warrants further investigation. Because haplotype 2 has very few sequence differences from haplotype 1 and no minor alleles in common with haplotype 4, the functional variants on haplotype 2 may lie in coding or regulatory regions outside the 3' UTR (exon 10) that we previously sequenced (6). This may explain the different phenotypic associations displayed by haplotypes 2 and 4. That haplotype 2 influenced lipid traits is not surprising, given LPL’s role in the metabolism of TGs in chylomicrons and very low-density lipoprotein particles. After interacting with LPL, these particles form remnants that then contribute to formation of HDL particles and Apo B-containing particles.
This work provides independent confirmation in two independent Hispanic populations of LPL as influencing insulin sensitivity/resistance, with the exact same haplotypes (haplotypes 1 and 4) displaying the previously identified opposing effects. We also have preliminary evidence for a mechanism connecting haplotype 4 to insulin resistance, that of increased visceral adiposity. In addition, a new risk haplotype (haplotype 2) in LPL was identified, which will lead to additional investigation and likely further elucidation of the complex role played by LPL in the insulin resistance syndrome and its multiple phenotypes.
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Footnotes |
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The authors have nothing to disclose.
First Published Online October 10, 2006
Abbreviations: Apo B, Apolipoprotein B; BMI, body mass index; CAD, coronary artery disease; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; IRAS, Insulin Resistance Atherosclerosis Study; IVGTT, iv glucose tolerance test; LPL, lipoprotein lipase; MACAD, Mexican-American Coronary Artery Disease; SI, insulin sensitivity index; SNP, single nucleotide polymorphism; TG, triglyceride; UTR, untranslated region; VAT, visceral adipose tissue.
Received June 2, 2006.
Accepted October 2, 2006.
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References |
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