QTL-based association analyses reveal novel genes influencing pleiotropy of metabolic syndrome (MetS).

OBJECTIVE: Metabolic Syndrome (MetS) is a phenotype cluster predisposing to type 2 diabetes and cardiovascular disease. We conducted a study to elucidate the genetic basis underlying linkage signals for multiple representative traits of MetS that we had previously identified at two significant QTLs on chromosomes 3q27 and 17p12. DESIGN AND METHODS: We performed QTL-specific genomic and transcriptomic analyses in 1,137 individuals from 85 extended families that contributed to the original linkage. We tested in SOLAR association of MetS phenotypes with QTL-specific haplotype-tagging SNPs as well as transcriptional profiles of peripheral blood mononuclear cells (PBMCs). RESULTS: SNPs significantly associated with MetS phenotypes under the prior hypothesis of linkage mapped to seven genes at 3q27 and seven at 17p12. Prioritization based on biologic relevance, SNP association, and expression analyses identified two genes: insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) at 3q27 and tumor necrosis factor receptor 13B (TNFRSF13B) at 17p12. Prioritized genes could influence cell-cell adhesion and adipocyte differentiation, insulin/glucose responsiveness, cytokine effectiveness, plasma lipid levels, and lipoprotein densities. CONCLUSIONS: Using an approach combining genomic, transcriptomic, and bioinformatic data we identified novel candidate genes for MetS.

Serotonin (5-HT) receptor 5A sequence variants affect human plasma triglyceride levels.

Neurotransmitters such as serotonin (5-hydroxytryptamine, 5-HT) work closely with leptin and insulin to fine-tune the metabolic and neuroendocrine responses to dietary intake. Losing the sensitivity to excess food intake can lead to obesity, diabetes, and a multitude of behavioral disorders. It is largely unclear how different serotonin receptor subtypes respond to and integrate metabolic signals and which genetic variations in these receptor genes lead to individual differences in susceptibility to metabolic disorders. In an obese cohort of families of Northern European descent (n = 2,209), the serotonin type 5A receptor gene, HTR5A, was identified as a prominent factor affecting plasma levels of triglycerides (TG), supported by our data from both genome-wide linkage and targeted association analyses using 28 publicly available and 12 newly discovered single nucleotide polymorphisms (SNPs), of which 3 were strongly associated with plasma TG levels (P < 0.00125). Bayesian quantitative trait nucleotide (BQTN) analysis identified a putative causal promoter SNP (rs3734967) with substantial posterior probability (P = 0.59). Functional analysis of rs3734967 by electrophoretic mobility shift assay (EMSA) showed distinct binding patterns of the two alleles of this SNP with nuclear proteins from glioma cell lines. In conclusion, sequence variants in HTR5A are strongly associated with high plasma levels of TG in a Northern European population, suggesting a novel role of the serotonin receptor system in humans. This suggests a potential brain-specific regulation of plasma TG levels, possibly by alteration of the expression of HTR5A.

INSIG1 influences obesity-related hypertriglyceridemia in humans.

In our analysis of a quantitative trait locus (QTL) for plasma triglyceride (TG) levels [logarithm of odds (LOD) = 3.7] on human chromosome 7q36, we examined 29 single nucleotide polymorphisms (SNPs) across INSIG1, a biological candidate gene in the region. Insulin-induced genes (INSIGs) are feedback mediators of cholesterol and fatty acid synthesis in animals, but their role in human lipid regulation is unclear. In our cohort, the INSIG1 promoter SNP rs2721 was associated with TG levels (P = 2 x 10(-3) in 1,560 individuals of the original linkage cohort, P = 8 x 10(-4) in 920 unrelated individuals of the replication cohort, combined P = 9.9 x 10(-6)). Individuals homozygous for the T allele had 9% higher TG levels and 2-fold lower expression of INSIG1 in surgical liver biopsy samples when compared with individuals homozygous for the G allele. Also, the T allele showed additional binding of nuclear proteins from HepG2 liver cells in gel shift assays. Finally, the variant rs7566605 in INSIG2, the only homolog of INSIG1, enhances the effect of rs2721 (P = 0.00117). The variant rs2721 alone explains 5.4% of the observed linkage in our cohort, suggesting that additional, yet-undiscovered genes and sequence variants in the QTL interval also contribute to alterations in TG levels in humans.

Comparison of linkage disequilibrium patterns between the HapMap CEPH samples and a family-based cohort of Northern European descent.

The International HapMap Consortium has determined the linkage disequilibrium (LD) patterns of four major human populations. The aim of our investigation was to compare the LD patterns of the HapMap CEPH (Centre d'Etude du Polymorphisme Humain) samples with a family-based cohort of similar ancestry to determine its usefulness as a reference population for disease association studies. We examined four genomic regions on chromosomes 7q, 12p, and 14q totaling 14.3 Mb, initially identified in our linkage study of obesity and the metabolic syndrome. Near identical patterns of LD were detected in both populations. Furthermore, tagSNPs selected based on the HapMap CEPH cohort data capture over 98% of the variants at an r2 > 0.8 in the disease cohort. This confirms the usefulness of the CEPH cohort of the HapMap as a reference sample for further investigations into the genomic variation of populations of Northern European descent.

The mitochondrial rhomboid protease PSARL is a new candidate gene for type 2 diabetes.

AIMS/HYPOTHESIS: This study aimed to identify genes that are expressed in skeletal muscle, encode proteins with functional significance in mitochondria, and are associated with type 2 diabetes. METHODS: We screened for differentially expressed genes in skeletal muscle of Psammomys obesus (Israeli sand rats), and prioritised these on the basis of genomic localisation and bioinformatics analysis for proteins with likely mitochondrial functions. RESULTS: We identified a mitochondrial intramembrane protease, known as presenilins-associated rhomboid-like protein (PSARL) that is associated with insulin resistance and type 2 diabetes. Expression of PSARL was reduced in skeletal muscle of diabetic Psammomys obesus, and restored after exercise training to successfully treat the diabetes. PSARL gene expression in human skeletal muscle was correlated with insulin sensitivity as assessed by glucose disposal during a hyperinsulinaemic-euglycaemic clamp. In 1,031 human subjects, an amino acid substitution (Leu262Val) in PSARL was associated with increased plasma insulin concentration, a key risk factor for diabetes. Furthermore, this variant interacted strongly with age to affect insulin levels, accounting for 5% of the variation in plasma insulin in elderly subjects. CONCLUSIONS/INTERPRETATION: Variation in PSARL sequence and/or expression may be an important new risk factor for type 2 diabetes and other components of the metabolic syndrome.

Genotype-by-smoking interaction for leptin levels in the Metabolic Risk Complications of Obesity Genes project.

RATIONALE: Recently, we identified a genotype-by-smoking status interaction with serum leptin levels in a sample of Mexican Americans. However, it is unknown whether this phenomenon occurs in other populations as well. OBJECTIVE: The goal of this study was to examine the genetic architecture of the response to smoking in leptin levels using data from Midwestern Caucasian subjects participating in the Metabolic Risk Complications of Obesity Genes project. METHODS: We employed a variance decomposition analysis using maximum likelihood methods to model genotype-by-smoking interactions for leptin levels and examined the impact of the exclusion of smokers in a subsequent linkage analysis. RESULTS: We found significant evidence (p-value=0.027) for a genotype-by-smoking status interaction for serum leptin levels. In the subsequent linkage analysis with smokers excluded, we obtained a maximum LOD score of 3.4 (P=0.00004) near D8S1128. CONCLUSIONS: These results suggest that a QTL on chromosome 8 may have a differential effect on the expression of leptin in smokers vs nonsmokers, as first identified in Mexican Americans.

Plasma leptin concentrations during extended fasting and graded glucose infusions: relationships with changes in glucose, insulin, and FFA.

Despite numerous studies, the in vivo regulation of plasma leptin levels in response to nutritional factors continues to remain unclear. We investigated temporal and dose-response relationships of plasma leptin in response to physiological changes in insulin/glucose. After an overnight fast of 10 h, lean, healthy subjects were investigated for an additional 16 h of either extended fasting or one of three levels of glycemia/insulinemia induced by stepwise increasing iv glucose infusions. During extended fasting, plasma leptin values declined steadily and significantly. Plasma leptin levels remained constant at glucose concentrations between 5.8-6.5 mmol/liter, which maintained normoinsulinemia at 41.5-45.4 pmol/liter and FFA at 106-123 mg/liter, but leptin concentrations were increased at higher rates of glucose infusion (with plasma glucose rising to 8.7 mmol/liter). Concentrations of serum leptin were inversely related to FFA levels during extended fasting and at all levels of glycemia. Our data indicate that in lean healthy subjects, physiological changes in glycemia and insulinemia significantly alter plasma FFA and leptin concentrations. The increases in leptin concentrations demonstrate dose-dependent relationships that appear to relate to changes in FFA levels as well as to changes in glycemia/insulinemia.

The genetic basis of plasma variation in adiponectin, a global endophenotype for obesity and the metabolic syndrome.

Here we present the first genetic analysis of adiponectin levels, a newly identified adipocyte-derived protein. Recent work has suggested that adiponectin may play a role in mediating the effects of body weight as a risk factor for coronary artery disease. For this analysis we assayed serum levels of adiponectin in 1100 adults of predominantly northern European ancestry distributed across 170 families. Quantitative genetic analysis of adiponectin levels detected an additive genetic heritability of 46%. The maximum LOD score detected in a genome wide scan for adiponectin levels was 4.06 (P = 7.7 x 10(-6)), 35 cM from pter on chromosome 5. The second largest LOD score (LOD = 3.2; P = 6.2 x 10(-5)) was detected on chromosome 14, 29 cM from pter. The detection of a significant linkage with a quantitative trait locus on chromosome 5 provides strong evidence for a replication of a previously reported quantitative trait locus for obesity-related phenotypes. In addition, several secondary signals offer potential evidence of replications for additional previously reported obesity-related quantitative trait loci on chromosomes 2 and 10. Not only do these results identify quantitative trait loci with significant effects on a newly described, and potentially very important, adipocyte-derived protein, they also reveal the emergence of a consistent pattern of linkage results for obesity-related traits across a number of human populations.

Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome.

Recent research has emphasized the importance of the metabolic cluster, which includes glucose intolerance, dyslipidemia, and high blood pressure, as a strong predictor of the obesity-related morbidities and premature mortality. Fundamental to this association, commonly referred to as the metabolic syndrome, is the close interaction between abdominal fat patterning, total body adiposity, and insulin resistance. As the initial step in identifying major genetic loci influencing these phenotypes, we performed a genomewide scan by using a 10-centiMorgan map in 2,209 individuals distributed over 507 nuclear Caucasian families. Pedigree-based analysis using a variance components linkage model demonstrated a quantitative trait locus (QTL) on chromosome 3 (3q27) strongly linked to six traits representing these fundamental phenotypes [logarithm of odds (lod) scores ranged from 2.4 to 3.5]. This QTL exhibited possible epistatic interaction with a second QTL on chromosome 17 (17p12) strongly linked to plasma leptin levels (lod = 5.0). Situated at these epistatic QTLs are candidate genes likely to influence two biologic precursor pathways of the metabolic syndrome.

Plasma leptin and insulin levels in weight-reduced obese women with normal body mass index: relationships with body composition and insulin.

Obesity is a complex disease with multiple features that has confounded efforts to unravel its pathophysiology. As a means of distinguishing primary from secondary characteristics, we compared levels of fasting plasma leptin and insulin in a cohort of weight-reduced obese women who have attained and maintained a normal BMI for more than 1 year with the levels in cohorts of never-obese and currently obese women. Weight-reduced obese women showed decreased plasma concentrations of leptin and insulin compared with obese women, but these levels remained significantly higher than those of never-obese women. Plasma leptin levels were highly correlated with plasma insulin levels (r = 0.60, P < 0.001). To further explore relationships with body composition, total body fat was determined by dual-energy X-ray absorptiometry and body fat distribution by computed tomography in subsets of these groups. Weight-reduced obese women had a significantly greater percent body fat and subcutaneous abdominal fat mass than did the never-obese women, and these were highly correlated with plasma leptin (r = 0.90, P < 0.001, and r = 0.52, P < 0.001, respectively). In these weight-reduced obese women, visceral fat mass was similar to that of the never-obese. The insulin sensitivity index and first-phase insulin response were also comparable. These results demonstrate that higher leptin levels in weight-reduced obese women are related to the higher total fat and particularly the subcutaneous fat masses. Normalization of visceral fat mass in the weight-reduced obese was accompanied by normalization of insulin sensitivity index and first-phase insulin response. This study suggests that increases in plasma leptin and insulin in obesity are secondary features of the obese state.

Identification and functional analysis of novel human melanocortin-4 receptor variants.

Inactivation of the melanocortin-4 receptor (MC4-R) by gene-targeting results in mice that develop maturity-onset obesity, hyperinsulinemia, and hyperglycemia. These phenotypes resemble common forms of human obesity, which are late-onset and frequently accompanied by NIDDM. It is not clear whether sequence variation of the MC4-R gene contributes to obesity in humans. Therefore, we examined the human MC4-R gene polymorphism in 190 individuals ascertained on obesity status. Three allelic variants were identified, including two novel ones, Thr112Met and Ile137Thr. To analyze possible functional alterations, the variants were cloned and expressed in vitro and compared with the wild-type receptor. One of the novel variants, Ile137Thr, identified in an extremely obese proband (BMI 57), was found to be severely impaired in ligand binding and signaling, raising the possibility that it may contribute to development of obesity. Furthermore, our results also suggest that sequence polymorphism in the MC4-R coding region is unlikely to be a common cause of obesity in the population studied, given the low frequency of functionally significant mutations.

Glucose utilization in muscle fiber types: use of the partial pancreatectomized rat model to distinguish effects of glucose and insulin on insulin resistance.

We have used the partially pancreatectomized infusion model in order to examine individual and combined effects of glucose and insulin on insulin resistance in rat skeletal muscles. Infusing glucose or insulin can produce animals which are hyperglycemic, hyperinsulinemic, or both. Individual and combined effects of chronic hyperglycemia and hyperinsulinemia on basal and insulin-mediated glucose utilization indices in glycolytic and oxidative muscle fibers were examined by 2-deoxyglucose uptake. Hyperglycemia reduced the basal glucose utilization index by 49% and hyperinsulinemia by 55%, while combined hyperglycemia + hyperinsulinemia diminished 2-deoxyglucose uptake by 69%. Maximally insulin-stimulated utilization was diminished only 28% under hyperglycemia but by 81% in the hyperinsulinemic state. In order to assess utilization in individual muscle fibers, uptake was examined in three tissues of differing fiber composition. The slow-twitch oxidative soleus muscle demonstrated greater basal uptake than the fast-twitch gastrocnemius (glycolytic) and quadriceps (oxidative) muscles. In addition basal (though not maximally insulin-stimulated) glucose utilization in the fast-twitch fibers was affected by chronic glucose and insulin to a greater extent than the slow-twitch soleus muscle, indicating that chronic hyperglycemia is more likely to precipitate insulin resistance in fast-twitch muscles. Significant differences in glucose metabolism among muscle fiber types suggests that results from insulin resistance studies in mixed muscles may be skewed according to their fiber composition.

Simultaneous gradient-echo/spin-echo EPI of graded ischemia in human skeletal muscle.

The goal of this study was to evaluate the usefulness of blood oxygenation level-dependent (BOLD) methodologies to provide temporal and spatial information about skeletal muscle perfusion. A simultaneous gradient echo (GE) and spin-echo (SE) imaging sequence (GE/SE) with alternating TE was used to acquire images of leg skeletal muscle throughout a stepped reactive hyperemia paradigm. The change in both the GE and SE relaxation rates (deltaR2*, deltaR2) measured during ischemia and reactive hyperemia scaled with the duration of cuff inflation (the ischemic period) plateaued for cuff inflations lasting longer than 120 seconds and were greater in soleus muscle than in gastrocnemius. The ratio deltaR2*/deltaR2 was found to be less during the reactive hyperemia period relative to ischemia. Considering that a greater proportion of capillary vessels are perfused during reactive hyperemia than during ischemia, this finding suggests that magnetic susceptibility methodologies, with their dependence on compartment size, may provide a measure of the relative distribution of small and large vessels in skeletal muscle.

Mechanisms of insulin-resistant glucose utilization in rat skeletal muscle.

Defects in glucose uptake are among the primary defects associated with peripheral insulin resistance, but fundamental mechanisms leading to this state are poorly understood. In order to elucidate mechanisms leading toward defects in glucose transport, we have used a partially pancreatectomized infusion (PxI) animal model with infusions of saline, glucose, or insulin to examine individual and combined effects of hyperglycemia and hyperinsulinemia on skeletal muscle glucose utilization. Moderate hyperglycemia induced by pancreatectomy reduced basal hindlimb muscle glucose utilization by 57% without affecting maximal insulin-stimulated glucose utilization; insulin administered in an amount sufficient to correct this hyperglycemia did not alter basal glucose utilization, but maximal insulin-stimulated glucose utilization was sharply diminished (75%); hyperglycemia with hyperinsulinemia similarly reduced basal and maximal insulin-stimulated glucose utilization. In order to establish the role of the glucose transporter protein in these insulin-resistant states, we quantified GLUT 4 content by immunoblotting and GLUT 4 mRNA by solution hybridization/RNAse protection assays. Hyperglycemia (2 weeks) reduced total muscle GLUT 4 protein content (53%) and mRNA (46%), while subsequent hyperinsulinemia (72 h) with either normo- or hyperglycemia partially restored both total GLUT 4 protein and mRNA levels. As insulin-stimulated GLUT 4 content in plasma membranes was not diminished by combined hyperglycemia/hyperinsulinemia, these results indicate functional GLUT 4 translocation in this model and suggest suppression of GLUT 4 transporter activity.

Relationships of plasma leptin levels to changes in plasma free fatty acids in women who are lean and women who are abdominally obese.

Regulation of leptin production by the hormonal and metabolic milieu is poorly understood. Because abdominal obesity is commonly associated with elevated plasma free fatty acid (FFA) flux, we examined the effects of augmenting FFA on plasma leptin levels in women who were lean and of suppressing FFA in women with abdominal obesity. In study 1, nine subjects who were lean, after a 12-hour overnight fast, received either intravenous saline or Intralipid plus heparin to increase the plasma FFA concentration to approximately 1000 mumol/ L. After 3 hours of additional fasting, subjects underwent 3-hour hyperglycemic clamps. In study 2, seven subjects with abdominal obesity were evaluated by a similar protocol, but lipolysis and plasma FFA flux were instead maximally suppressed by acipimox. In the individuals who were lean, leptin levels were unchanged during clamping. Increasing plasma FFA reduced plasma leptin from 7.66 +/- 0.66 to 7.05 +/- 0.66 (p = 0.03), but 3 hours of hyperglycemia plus hyperinsulinemia had no additional effect on leptin levels (7.15 +/- 0.71). Basal leptin levels, 4-fold higher in the subjects with obesity, were reduced from 34.6 +/- 2.4 micrograms/L to 32.3 +/- 1.1 micrograms/L (p = 0.004) during the clamp period. When plasma FFA flux was suppressed, however, plasma leptin levels after clamped hyperglycemia/hyperinsulinemia were increased to 38.9 +/- 1.2 micrograms/L (p = 0.014 vs. time 0 and 0.001 vs. saline protocol). Changes in leptin concentrations are not correlated with changes in FFA. These results suggest that plasma FFA concentration does not regulate plasma leptin levels in basal, extended fasting, or hyperglycemic/hyperinsulinemic states.

Effects of free fatty acids and glucose on splanchnic insulin dynamics.

The mechanism of hyperinsulinemia that accompanies insulin resistance in some abdominally obese and diabetic individuals is poorly understood. Both increased secretion of insulin and decreased clearance have been demonstrated. The present study was undertaken to examine the role of free fatty acids (FFAs) and glucose in regulating splanchnic insulin dynamics in vivo. Plasma FFA levels were raised approximately twofold via an intralipid/heparin infusion in eight lean women. Insulin dynamics were assessed using the individual's C-peptide kinetic coefficients. Studies were performed in the basal state and during two levels of glycemia, 7 and 11 mmol/l. Studies were repeated using saline, and thus each subject served as her own control. Under basal conditions, raising FFA flux resulted in a modest increase in plasma insulin concentration (PIC) secondary to an increase in insulin secretion rate (ISR); however, endogenous insulin clearance (EIC) was not influenced. During the 7 mmol/l hyperglycemic clamp, maintaining a high FFA flux resulted in a 30% increase in PIC above the effect produced by glucose alone. This represents the cumulative effects of stimulation of ISR and inhibition of EIC. Clamping plasma glucose at 11 mmol/l while maintaining a high FFA flux increased PIC twofold above that produced by glucose alone. This increase in PIC was mainly due to a significant reduction in EIC without an accompanying increase in ISR (392 +/- 159 and 787 +/- 187 ml/min with and without intralipid infusion, respectively). Analysis of variance indicated that the suppressive effect of FFA on EIC was independent of the effect of glucose. The effect of the two substrates seems to be additive.

Leptin: a significant indicator of total body fat but not of visceral fat and insulin insensitivity in African-American women.

The recently cloned adipose tissue hormone leptin has been proposed to be involved in the neuroendocrine regulation of adiposity and its metabolic sequelae. Visceral fat is known to predict reduced insulin sensitivity and associated adverse metabolic profiles. In this study, we report the first evaluation of the relationships between leptin levels and total body fat, visceral fat, and insulin sensitivity in a cohort of premenopausal African-American women. Thirty-four subjects were analyzed for total fat mass and visceral fat by dual-energy X-ray absorptiometry and computerized axial tomography, respectively. Insulin sensitivity (SI) was assessed using Bergman's minimal model. Results showed that fasting leptin levels strongly correlated with total body fat mass (r = 0.797, P < 0.001). Correlations of leptin with visceral fat (r = 0.54, P < 0.001) and SI (r = -0.419, P = 0.02) were dependent on total body fat. In conclusion, leptin levels reflect total body fat mass, and although visceral fat is known to predict reduced insulin sensitivity independently, leptin did not. Our data thus suggest that diverse mechanisms are responsible for the regulation of total body versus visceral fat distribution, with its metabolic and health risks.

Influence of androgenicity on adipocytes and precursor cells in female rats.

We examined the effects of overexposure of testosterone (T) on fat cell morphology and adipocyte precursor pools in inguinal and retroperitoneal fat depots of ovariectomized rats. In both tissues peripubertal T decreased weights without affecting adipocyte mean cell size or the size distribution profiles, but adipocyte number was decreased by 65% in the inguinal and by 38% in the retroperitoneal depots. Immunofluorescent flow cytometry utilizing a specific antibody to rat adipose tissue lipoprotein lipase was used to quantify regional precursor cell populations. T sharply reduced the percentages of differentiated and undifferentiated preadipocytes in the inguinal depot, from 43.2 +/- 5.3 to 23.5 +/- 2.1% and from 57.7 +/- 4.0 to 43.6 +/- 5.3%, respectively, with a concomitant increase in fibroblasts from 1.6 to 32.9%. On the other hand, T had no effect on retroperitoneal preadipocyte pools. Perinatal androgenization exacerbated the decline in the inguinal weight (1.4 +/- 0.1 vs. 2.2 +/- 0.1g) but otherwise did not influence the actions of peripubertal T. Androgens may thus act in a tissue-specific manner to regulate fat cell growth potential in the femoral region in the female.

Dietary fish oil decreases low-density-lipoprotein clearance in nonhuman primates.

To assess whether fish oil-induced alterations in low-density-lipoprotein (LDL) composition have distinct and important effects on LDL metabolism, we evaluated LDL kinetic behavior in cynomolgus macaques fed an atherogenic diet supplemented with either fish oil (1.6 g n-3 fatty acids; n = 10) or olive oil (n = 9) for > or = 6 mo. LDL from monkeys supplemented with fish oil or olive oil was isolated, labeled with either 125I or 131I, and simultaneously reinjected so that each monkey received its own (autologous injection) and donor (homologous injection) LDL. For LDL injected autologously (monkeys that received their own LDL), the LDL fractional clearance rate (FCR) was reduced in fish oil-supplemented monkeys compared with the olive oil-supplemented controls (0.42 +/- 0.03 compared with 0.56 +/- 0.05 pools/d, P = 0.04). The cholesteryl ester content of fish oil LDL increased compared with olive oil LDL (43 +/- 2% and 36 +/- 3%, respectively, P = 0.03), and the LDL cholesteryl ester content was strongly correlated with autologous LDL clearance (r = -0.76, P = 0.0001). Compared with olive oil LDL, fish oil LDL had a reduced dissociation constant (KD) for binding to the LDL receptor in vitro (KD for fish oil LDL compared with olive oil LDL: 13.9 +/- 1.8 and 7.4 +/- 1.0 mg LDL protein/L, P = 0.03). When both fish oil LDL and olive oil LDL were simultaneously injected into fish oil-supplemented monkeys, the FCR of fish oil LDL was decreased compared with olive oil LDL (0.42 +/- 0.03 and 0.52 +/- 0.04 pools/d, P = 0.006). These data suggest that dietary supplementation with fish oil decreases LDL clearance, and that this effect is mediated, at least in part, by altering LDL structure and reducing the affinity of LDL for its receptor.