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Nutrition and Genomics Laboratory (D.C., L.Q., O.C., J.M.O.) and the Obesity and Metabolism Laboratory (A.S.G.), Jean Mayer–United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts; the Genetic and Molecular Epidemiology Unit (D.C., J.V.S., O.P.), Department of Preventive Medicine, University of Valencia, Valencia, Spain; Department of Internal Medicine (J.V.S., D.G.), University General Hospital, Valencia, Spain; and Department of Computer Languages and Systems (O.C.), University Jaume I, Castellón, Spain
Address all correspondence and requests for reprints to: J. M. Ordovas, Nutrition and Genomics Laboratory, Jean Mayer–United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, Massachusetts 02111. E-mail: jose.ordovas{at}tufts.edu.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Objective: The objective of the study was to examine the association of several polymorphisms at the perilipin (PLIN) locus with obesity and weight reduction in response to a low-energy diet in obese patients.
Design: This study was a 1-yr randomized (depending on the PLIN genotype) trial with three follow-up evaluations.
Setting: The study was conducted at a university research center.
Subjects: One hundred fifty obese patients (body mass index, 42 ± 8 kg/m2) at baseline and 48 patients who completed the dietary follow-up treatment for weight loss participated in the study.
Interventions: Subjects completed a 1-yr low-energy diet.
Main Outcomes Measurements: Body weight (BW) at baseline and 3, 6, and 12 months was measured.
Results: The minor A-allele at the PLIN 11482G>A polymorphism was associated with lower baseline BW. Moreover, we found a gene-diet interaction (P = 0.015) between this polymorphism and weight loss in patients that completed the 1-yr dietary treatment. Diet resulted in significant decreases in BW (from 114.3 ± 3.9 kg at baseline to 105.5 ± 3.5 kg at 1 yr; P lineal trend, 0.020) in GG patients (n = 33). Conversely, carriers of the minor A allele (n = 15) did not show significant changes in BW (from 105.0 ± 4.6 kg at baseline to 104.3 ± 4.4 kg at 1 yr; P lineal trend, 0.985). This gene-diet interaction remained statistically significant, even after adjustment for differences in BW at baseline and for other potential confounders.
Conclusions: PLIN11482A carriers were resistant to weight loss, suggesting that this polymorphism may predict outcome of BW reduction strategies based on low-energy diets.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In this regard, adipose tissue plays a central role in regulating the storage and mobilization of energy, and it has been the focus of efforts to identify candidate genes for obesity and weight management. Perilipins are phosphorylated proteins in adipocytes that are localized at the surface of the lipid droplet (4, 5). Experimental studies have shown that these proteins are essential in the regulation of triglyceride deposition and mobilization (6, 7, 8). After activation of protein kinase A, perilipin is phosphorylated, resulting in translocation of the protein away from the lipid droplet and allows hormone-sensitive lipase to hydrolyze the adipocyte triglycerides to release nonesterified fatty acids (9, 10). Perilipin functions to increase cellular triglycerides storage by decreasing the rate of triglyceride hydrolysis and serves an additional role in controlling the release of triglycerides at times of need. Two independent laboratories produced perilipin knockout mice (11, 12) and showed that these animals were lean, had increased basal lipolysis, and were resistant to diet-induced obesity. A recent study examining perilipin expression in humans found that perilipin was elevated in obese subjects (13). Moreover, in the first large population-based study, we demonstrated that variations at the perilipin (PLIN) locus are associated with obesity risk (14). These findings prompted us to initiate the present study aimed to analyze the influence of PLIN polymorphisms on anthropometrical measures in massively obese subjects as well as examine the potential gene-diet interaction between PLIN polymorphisms and an energy-restricted diet on the ability to lose weight during a 1-yr follow-up intervention.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The present study included 150 obese patients (29 men and 121 women aged 18–68 yr) referred to the Endocrinology Unit of the University General Hospital in Valencia, Spain, for diagnostic and weight reduction treatments related to obesity. These patients were randomly selected among those obese subjects referred consecutively from May 2001 to September 2002 and who had normal thyroid function and no concomitant renal, hepatic, cardiac, or Cushing disease. Pregnant or nursing women were also excluded. Sixty-two women (51%) were postmenopausal. All patients were Caucasian and the mean age was 48 ± 14 yr. Body mass index (BMI) ranged from 30 to 79 kg/m2, with 88% of patients having a BMI 35 kg/m2 or greater. All participants provided informed consent, and the study protocol was approved by the Ethics Committees of the Valencia University and the University General Hospital.
At baseline, anthropometric, biochemical, and clinical characteristics were determined in all patients. In addition, genomic DNA was isolated from blood and stored for further genetic analysis. Weight reduction treatments including diet, drugs, or surgery were recommended to each obese patient according to standard clinical guidelines (15). Bariatric surgery (16) was recommended for 13 patients. Forty-two patients received weight-loss medications (orlistat, sibutramine, antidepressants, or fiber) combined with diet, and 92 patients were prescribed to receive an energy-restricted diet. Patients assigned to the energy-restricted diet with no medication for weight loss (61% of patient at baseline) were invited to participate in the 1-yr follow-up study to investigate whether PLIN polymorphisms modulate the weight loss in response to diet. Because PLIN genotypes in these patients were determined at the end of the follow-up, the design of this study can be classified as a double-blinded, paralleled, randomized trial because no one had previous information about the group assignment. The randomization of individuals was provided by Mendelian randomization, the term applied to the random assortment of alleles at the time of gamete formation (17).
Dietary intervention
Forty-eight motivated patients (nine men and 39 women) completed the 1-yr dietary follow-up treatment and had complete data set at each time point. All patients started with a 2-wk very low-energy diet (Modifast; Novartis Nutrition, Bern, Switzerland) providing 603 kcal/d (lipids, 13.5 g; proteins, 52.5 g; and carbohydrates, 67.5 g) under highly controlled hospital conditions. Thereafter, conventional food was introduced and patients were advised by a registered dietitian to consume a standard hypocaloric diet with an energy content being approximately 1200 kcal/d (lipids, 52 g; proteins, 62 g; and carbohydrates, 121 g) for 1 yr. The patients were given dietary instructions based on an education system consisting of isoenergetic interchangeable units. Three follow-up evaluations were performed at 3, 6, and 12 months. All evaluations were conducted at the Endocrinology Unit of the University General Hospital. Adherence to diet was confirmed in these evaluations. None of the obese patients was involved in an exercise program.
Measurements
Anthropometrical measurements were taken using standard techniques (18): weight with light clothing by digital scales; height without shoes by fixed stadiometer. In the follow-up study, the subjects were weighed when they visited the endocrinology unit at baseline and 3, 6, and 12 months of the study. All measurements were done on the same equipment by the same personal each time.
Venous blood was collected into EDTA-containing glass tubes. Plasma total cholesterol, fasting triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein-cholesterol, and fasting glucose were measured at baseline as previously described (18). A baseline questionnaire was used to obtain demographic information, education, health status, menopausal status, medication, tobacco smoking, alcohol consumption, physical activity, and weight history for the prior years. Education was classified into three categories: primary, secondary (high school), and university. Current smokers were defined as those smoking at least one cigarette per day. Subjects with any amount of alcohol consumed were classified as drinkers. Physical activity was estimated from questions about regular leisure-time physical sports, and subjects were categorized as sedentary (no physical exercise) or active (18). Subjects were classified as having type 2 diabetes if they were on hypoglycemic drug therapy for diagnosed type 2 diabetes of if they had fasting plasma glucose levels greater than 126 mg/dl (19).
PLIN genotyping
Four polymorphisms at the PLIN locus were genotyped: PLIN 6209T>C (intron 2), PLIN 11482G>A (intron 6), PLIN 13041A>G (exon 8, synonymous), and PLIN 14995A>T (exon 9, untranslated region). Genotyping was carried out using single nucleotide extension as previously reported (14) using the ABI Prism SnaPshot multiplex system on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Standard good laboratory practices were undertaken to assure the accuracy of genotype data. Final success rate for PLIN genotyping in the study participants was 100%.
Statistical analysis
2 Tests were used to test differences between observed and expected frequencies, assuming Hardy-Weinberg equilibrium, to test linkage disequilibrium and differences in percentages. Pairwise linkage disequilibrium coefficients were estimated by the LINKAGE program. D and D' coefficients were calculated. Normal distribution for all continuous variables was tested and triglycerides were logarithmically transformed. Three genotype groups were first considered to check dominant or codominant allelic effects. Then carriers of the less common allele were grouped and compared with wild-type homozygotes. At baseline, Student’s t test for independent groups was applied to compare crude means between genotypes. In addition, to estimate and compare adjusted means, analysis of covariance was used to test the null hypotheses of no association between genetic variants and obesity-related phenotypes. The main covariates were gender, age, tobacco smoking, alcohol consumption, physical activity, type 2 diabetes, education, baseline BMI, and menopausal status in women. Homogeneity of allelic effects according to gender or other factors was tested by introducing the corresponding terms of interaction in the more parsimonious multivariate model. Standard regression diagnostic procedures were used to ensure the appropriateness of these models. In the dietary intervention follow-up study, analysis of covariance for repeated measures (at baseline, 3, 6, and 12 months) was used to test the gene-diet interaction in determining weight loss as well as control for the potential confounders. The Statistical Package for Social Sciences (version 11.5; SPSS, Inc., Chicago, IL) was used for statistical analyses.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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shows anthropometric, biochemical, clinical, and lifestyle characteristics of the study subjects according to the PLIN 11482G>A polymorphism. At baseline, we observed that carriers of the A allele had significantly less body weight and BMI than GG homozygotes (P < 0.05). This association remained statistically significant, even after additional control for potential confounders (smoking, drinking, physical activity, education, type 2 diabetes, and menopausal status in women). Due to the high linkage disequilibrium between the PLIN 11482G>A and PLIN 6209T>C polymorphisms, lower mean body weight was also observed in carriers of the 6209 C allele; however, the difference did not reach the statistical significance (P = 0.071).
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). Forty-two patients received weight-loss medications combined with diet, and 92 patients were prescribed to receive only an energy-restricted diet. Patients assigned to the energy-restricted diet and who were not receiving medication for weight loss were invited to participate in the 1-yr follow-up study. Forty-eight patients (nine men and 39 women) were followed up for the entire 1-yr diet period and had complete data set at each time point (3, 6, and 12 months). After the dietary intervention, we found a statistically significant (P = 0.015) gene-diet interaction between the PLIN 11482G>A polymorphism and the energy restriction in body weight decrease in response to diet. No statistically significant interaction terms were found for the other PLIN polymorphisms.
Table 1 also shows baseline characteristics of the 48 patients that completed the 1-yr energy-restricted follow-up study according to the PLIN 11482G>A polymorphism. No statistically significant differences were observed in baseline variables between the two genotype groups. Smoking, drinking, education, physical activity, diabetes, and menopausal status in women did not differ either. Interestingly, although both genotype groups received the same energy-restricted diet, weight loss differed significantly between the two groups. Figure 1 shows body weight means (gender and age adjusted) in the 48 obese subjects who completed the study for weight reduction under the energy-restricted diet according to the PLIN 11482G>A polymorphism.
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Moreover, because prevalence of diabetes was higher (although not statistically significant) in carriers of the A allele, the potential modulation of diabetes status in the gene-diet interaction was also tested. We did not observe a statistically significant interaction term between the PLIN 11482G>A polymorphism, the low-energy diet and diabetes status in determining body weight (P = 0.902). When the stratified analysis of the gene-diet interaction was carried out in nondiabetic and diabetic patients, we observed this homogeneity of the effect. In both nondiabetic and diabetic subjects, body weight decreased over the course of the intervention in subjects homozygous for the PLIN 11482G allele (from 111.9 ± 4.5 kg at baseline to 103.4 ± 4.3 kg at 1 yr in nondiabetic patients and from 125.3 ± 7.5 kg at baseline to 115.7 ± 6.4 kg at 1 yr in diabetic patients). However, in carriers of the variant allele (A), no decreases in body weight were observed. This gene-diet interaction effect was statistically significant (P = 0.041) in nondiabetic subjects. In diabetic subjects, although the magnitude of the effect was similar, the interaction term did not reach the statistical significance due to limitations in sample size (P = 0.203)
Finally, the allelic effect of the PLIN 11482G>A in determining body weight response to the 1-yr low-energy diet was tested in the whole sample. Table 2 shows adjusted means of body weight at baseline and 3, 6, and 12 months and percent of change depending on the PLIN genotypes (GG, GA, AA). This gene-diet interaction was statistically significant (P = 0.041) and showed that the A allele has a dominant effect because no significant differences between GA and AA carriers in the difficulty of losing weight were observed.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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9%) than in the general population (3.5%), suggesting a more prominent role of variations at the PLIN locus in morbidly obese subjects. The maximization of genetic effects of important candidate gene for obesity in severely obese subjects has been pointed out by Bell et al. (20) in their recent review about the genetics of human obesity. Another interesting contrast between morbidly obese patients and the general population is that this association was observed only in women from the general population (14), and we have shown similar gender-specific associations in white American (21) and Asian populations (22). Conversely, in obese patients, the PLIN 11482A variant allele was associated with lower body weight in both men and women. The reason for this discrepancy is unknown; however, it is likely that the higher adiposity observed in severely obese men as compared with men from the general population largely contribute to this association. The evidence from animal models suggests similar effects in both male and female mice (12). Therefore, more human studies analyzing PLIN variation in men are needed to confirm whether the effect of PLIN variation on anthropometric variables in men depends on the degree of obesity. In agreement with results from animal models (11, 12), the protective effects of the PLIN 11482G>A minor allele observed in obese patients at baseline as well as women from the general population are compatible with a reduced expression of the 11482A-variant allele as compared with GG homozygotes. Data from animal models have consistently shown that targeted disruption of the perilipin gene results in healthy mice that are much leaner and more muscular than wild-type controls and had increased levels of basal lipolysis (11, 12). In support of this hypothesis, a study by Kern et al. (13) carried out in 44 healthy subjects (five men and 39 women) demonstrated a significant positive relationship between perilipin expression and obesity. Although the PLIN 11482G>A polymorphism is located in an intron and it does not appears to be functional according to traditional standards, Mottagui-Tabar et al. (8), in a study carried out in human fat cells of obese women, demonstrated that the perilipin protein content was markedly decreased and lipolysis increased in carriers of the 11482A-variant allele, supporting the observed results.
Despite the apparent protective role of the 11482A-variant allele associated with lower body weight at baseline, our dietary intervention follow-up study has revealed that carriers of this allele at the PLIN locus are more resistant to weight loss in response to an energy-restricted diet than GG homozygotes. Therefore, patients with the 11482A variant-allele did not show significant changes in mean body weight. Conversely, subject homozygotes for the 11482G allele had a greater and statistically significant mean weight loss during the same 1-yr low-energy diet. In addition, it seems that this effect is dominant because we have found the same difficulty in losing weight in both GA and AA patients. Although the PLIN 6209T>C paralleled the effect of the PLIN 11482G>A single-nucleotide polymorphism due to the high degree of linkage disequilibrium, a haplotype analysis (results not shown) indicated that the observed effect was mainly due to the PLIN 11482G>A polymorphism. This is the first study on weight loss and PLIN variation in humans in response to a long-term energy-restricted diet, and the molecular mechanism to explain the observed results remains to be explained. However, considering the results obtained in perilipin knockout mouse that are resistant to diet-induced obesity (17), we can speculate that carriers of the 11482A-variant allele (associated with less perilipin expression) might experiment a buffer effect by which body weight regulation in these subjects is more independent of the energy intake than in GG homozygotes. Another aspect that remains to be investigated is macronutrient influence in the PLIN-diet interaction. In our study, the dietary target for fat content in the low-energy diet was relatively high, according to the typical Mediterranean diet, in which olive oil is the main fat consumed (23), and little is known about the ways in which macronutrients or energy restriction affect the regulation of adipose tissue gene expression (24).
In conclusion, our results show that the PLIN 11482G>A polymorphism may predict outcome of body weight reduction strategies based on low-energy diets. Carriers of the A allele might have higher stability in the mechanisms that control body weight and a higher difficulty in losing weight in response to a low-energy diet. This difficulty was consistently observed at 3, 6, and 12 months, reducing the likelihood that this finding was observed by chance However, because this is the first longitudinal investigation, there is a need to confirm the present findings in other interventional studies.
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Footnotes |
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First Published Online June 28, 2005
Abbreviations: BMI, Body mass index; PLIN, perilipin.
Received March 16, 2005.
Accepted June 22, 2005.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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to increase lipolysis in 3T3–L1 adipocytes. J Biol Chem 273:24665–24669This article has been cited by other articles:
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