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Unit of Lipids and Arteriosclerosis, Hospital Universitario Reina Sofia, 14004 Córdoba, Spain

(Requests for offprints should be addressed to J López-Miranda; Email: jlopezmir{at}uco.es)

	Abstract

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Impaired insulin action has been associated with diabetes, dyslipidemia and atherosclerotic vascular disease. The expression of insulin resistance results from the interaction of environmental and genetic factors. Human hepatic lipase (HL) is a lipolytic enzyme that plays a role in the metabolism of several lipoproteins, while insulin up-regulates the activity of HL via insulin-responsive elements in the HL promoter. We have examined the influence of –514 C/T polymorphism in the hepatic lipase gene promoter on insulin sensitivity in 59 healthy young subjects (30 males and 29 females). The volunteers were subjected to three dietary periods, each lasting four weeks. During the first period all subjects consumed a saturated fat (SFA)-enriched diet with 38% as fat (20% SFA, 12% monounsaturated fatty acids (MUFA) and 6% polyunsaturated fatty acids (PUFA)). In the second and third dietary periods, a randomized crossover design was used, consisting of a low fat, high carbohydrate diet (CHO diet) (< 10% SFA, 12% MUFA and 6% PUFA) and a high-MUFA, or Mediterranean diet, with < 10% SFA, 22% MUFA and 6% PUFA. We determined the in vivo insulin resistance using the insulin suppression test with somatostatin. Steady-state plasma glucose (SSPG) concentrations (a measure of insulin sensitivity) were significantly higher in men carriers of the –514T allele after the consumption of the SFA diet than after the CHO diet and the Mediterranean diet. This effect was not observed in women. Moreover, there were no significant differences in insulin sensitivity after the three diets in men and women with the CC genotype. In summary, our results show an improvement in insulin sensitivity in men with the –514T allele of the HL promoter polymorphism, when MUFA and carbohydrates are consumed instead of SFA fat.

	Introduction

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Impaired insulin action has been associated with diabetes, obesity, dyslipidemia, hypertension and also with atherosclerotic vascular disease. Insulin resistance is determined by the interaction between genetic and environmental factors. In general, high intake of dietary fat has been associated with obesity and its comorbid conditions, including heart disease and diabetes. All these factors are related to the global process of ‘Westernization’ of lifestyles and dietary habits, especially the high intake in calories from saturated fat. Insulin resistance usually precedes the diagnosis of type 2 diabetes mellitus by decades and, in most cases, the clinical expression of the disease could be prevented by dietary and lifestyle modification (Mayer-Davis et al. 1997). On the other hand, the genetic base of type 2 diabetes mellitus is very heterogeneous and has been related to various genetic mutations that codify proteins connected with glucose and insulin metabolism, such as the insulin receptor (Kusari et al. 1991), insulin receptor substrate-1 (Almind et al. 1993), the Rad protein (Reynet & Kahn 1993), the glycogen synthase (Bjorbaek et al. 1994) and the ß-3-adrenergic receptor (Walston et al. 1995), to mention the most well-known. However, the genetic background of insulin resistance and type 2 diabetes mellitus is more complex and it can also involve other genes seemingly unrelated with carbohydrate metabolism.

Hepatic lipase (HL) is an enzyme anchored to the vascular endothelium in the liver as well as to the surface of hepatocytes, where it catalyzes the hydrolysis of various lipids in lipoprotein particles. Several studies have reported that the –514T allele of the HL gene promoter appears to be associated with decreased HL activity, increased high density lipoprotein cholesterol (HDL-C) (Guerra et al. 1997, Zambon et al. 1998), and increased low density lipoprotein (LDL) buoyancy (Zambon et al. 1998). However, at present, the effects of this common hepatic lipase variant –514C/T on lipid and lipoprotein levels are not well known. Moreover, as HL is involved in the clearance of triglyceride-rich lipoproteins, the primary effects of the variant genes influence the metabolism of triglyceride-rich lipoproteins which may, secondarily, affect glucose homeostasis. Also, insulin has been assumed to upregulate the activity of the HL promoter. This has been proposed to explain the associations between hyperinsulinemia and high HL activity (Romano et al. 1997). Recently, in a Finnish population, an association of the HL variant with insulin resistance was observed in patients with familial combined hyperlipidemia (Pihlajamaki et al. 2000).

Whether the HL promoter variant contributes to impaired insulin sensitivity in the normal population has not been investigated. In the present study we have explored the possible influence of the –514 C/T variant in the promoter of the HL gene on insulin sensitivity in a healthy young population. In addition, the interaction between this polymorphism and diet on insulin sensitivity was evaluated.

	Materials and methods

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Subjects and diets

Fifty-nine healthy normolipemic subjects, 39 homozygous for the most common allele (C/C) and 20 carriers of the T allele (CT/TT), attending the University of Cordoba volunteered to participate in the study. They ranged in age from 22.6 ± 1.4 years. None of them had diabetes, or liver, renal or thyroid disease. All underwent a comprehensive medical history, physical examination, and clinical chemistry analysis before enrollment. None of the subjects was taking medication or vitamins known to affect plasma lipids. Dietary information, including alcohol consumption, was collected over seven consecutive days. Individual energy requirements were calculated by taking into consideration each subject’s weight and physical activity. Subjects were encouraged to maintain their regular physical activity and life-style and were asked to record in a diary any event that could affect the outcome of the study, such as stress, change in smoking habits, and alcohol consumption or foods not included in the experimental design as described in our previous studies (Fuentes et al. 2001, Pérez-Jiménez et al. 2001, Pérez-Martínez et al. 2001).

The study design included an initial 28-day period during which all the subjects consumed a saturated fat (SFA)-enriched diet with 15% of energy as protein, 47% as carbohydrate, and 38% as fat (20% SFA, 12% monounsaturated fatty acids (MUFA) and 6% poly-unsaturated fatty acids (PUFA)). All participants were then randomized in a crossover design and exposed to two new dietary periods: a low fat, high carbohydrate diet (CHO diet), and a high-MUFA diet, with a typical ‘Mediterranean diet’ enriched with olive oil. The two groups of subjects were assigned one of the two dietary regimes for 28 days each. Group 1 (30 subjects) was placed on a Mediterranean diet followed by a CHO diet. For group 2 (29 subjects) the order was reversed. Assignment of volunteers to the sequence of diets was carried out at random. The CHO diet (National Cholesterol Education Program 1994) contained 15% of energy as protein, 57% as CHO, and 28% as fat (< 10% SFA, 12% MUFA, and 6% PUFA). The Mediterranean diet contained 15% of energy as protein, 47% as CHO, and 38% as fat (< 10% SFA, 22% MUFA, and 6% PUFA). Olive oil provided 75% of total MUFA consumed during this last dietary period. Dietary cholesterol was maintained constant in our experimental design and the mean cholesterol intake was 115 mg/1000 kcal during the three periods. The Human Investigation Review Committee approved this study at the Reína Sofia University Hospital. Informed consent was obtained from all participants.

The composition of the experimental diets was calculated using the United States Department of Agriculture (USDA) (Human Nutrition Information Service of Department of Agriculture 1987) food tables, or the Spanish food composition tables for local foodstuffs. Fourteen menus, prepared with regular solid foods, were rotated during the experimental period. We used virgin olive oil for cooking and salad dressing during the Mediterranean diet, and palm oil and butter for the high-SFA diet. During the CHO diet period, biscuits, bread and jam replaced some olive oil or palm oil. Lunch and dinner were consumed in the hospital dining room, whereas breakfast and an afternoon coffee break were eaten in the medical school cafeteria. A dietician supervised all meals. Duplicate samples from each menu were collected, homogenized, and stored at –80 °C. Protein, fat and CHO content of the diet were analyzed using standard methods (Association of Official Analytical Chemists, Arlington 1990). Evaluation of dietary compliance was also performed by examining the food diaries and by analyzing the fatty acid content of the cholesterol ester fraction in LDL (Ruiz-Gutierrez et al. 1993).

Blood sampling and biochemical determinations
Venous blood for insulin, glucose, lipid and lipoprotein analysis was collected in EDTA-containing tubes from the subjects after a 12-h overnight fast at the end of each dietary period. Each analysis was performed three times. Total cholesterol (TC) and triglycerides (TG) were assayed by enzymatic procedures (Allain et al. 1974, Bucolo & David et al. 1973). HDL-C was measured by analyzing the supernatant obtained after precipitation of a plasma aliquot with dextran sulfate-Mg²⁺ (Warnick et al. 1982). The LDL-cholesterol (LDL-C) level was calculated from total cholesterol, triglyceride, and HDL-C values using the Friedewald formula (Friedewald et al. 1972). Unesterified free fatty acid (FFA) levels were determined by an enzymatic colorimetric assay (Boehringer Mannheim) as described by Shimizu et al.(1979). To reduce inter-assay variation, plasma for biochemical determinations was stored at –80 °C and analyzed in duplicate at the end of the study.

Glucose suppression test
At the end of each dietary period all subjects underwent a modified insulin suppression test (Harano et al. 1977, Laws et al. 1994). The technique used in the present study to quantify insulin sensitivity was the insulin suppression test, a simple and cost-effective test for the measurement of insulin resistance, which has been used increasingly often in recent years. The study began at 0800 h, after a 12-h fast. A continuous infusion of somatostatin (214 nmol/h), insulin (180 pmol/m²/min), and glucose (13.2 mmol/m²/min) was administered in the same vein. Somatostatin was used to inhibit endogenous insulin secretion. Blood was sampled every 30 min for the first 2.5 hours, by which time steady-state plasma glucose (SSPG) and steady-state plasma insulin (SSPI) levels were achieved. Blood was then sampled at 10-min intervals for the last 30 min (at 150, 160, 170 and 180 min) for measurement of plasma glucose and insulin concentrations. These four values determined the SSPG and SSPI concentrations. Since SSPI levels were similar in all subjects, SSPG levels provided a measure of the ability of insulin to promote disposal of infused glucose. Subjects with high SSPG are relatively more insulin-resistant than those with lower SSPG.

Genotyping of hepatic lipase gene polymorphism
DNA was extracted from 10 ml EDTA-containing blood. Amplification of a 299-bp region of the hepatic lipase gene was carried out by polymerase chain reaction (PCR) with 250 ng genomic DNA and 0.2 µmol of each oligonucleotide primer (P1; 5'-AAGAAGTGTGTTTA CTCTAAGGATCA-3', and P2, 5'-GGTGGCTTCCA CGTGGCTGCCTAAG-3') in 50 µl. DNA was denaturated at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 58 °C for 1.5 min, and extension at 72 °C for 2 min. The PCR product (10 µl) was digested with 5 units of restriction enzyme NlaIII (BRL, Baltimore, MA, USA) in a total volume of 35 µl. Digested DNA was separated by electrophoresis in an 8% non-denaturing polyacrylamide gel at 150 V for 2 h. Bands were visualized after silver staining. Samples containing the T allele were amplified a second time to verify the genotype.

Statistical analysis
Statistical analyses were carried out using the SPSS statistical package (SPSS, Chicago, IL, USA). ANOVA for repeated measures was used to analyze the differences in plasma lipid, glucose, SSPG levels and basal glucose and insulin-stimulated glucose uptake between dietary phases. When statistically significant effects were demonstrated, Tukey’s post-hoc test was used to identify between-group differences. The general linear models for repeated measures procedure was used to test gene and diet interactions. A value of P<0.05 was considered significant.

	Results

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Dietary composition was analyzed in duplicate meals portions, and the results are shown in Table 1. The results were in good agreement with values obtained from the food composition tables. Analysis of the cholesterol ester fraction of plasma LDL showed good adherence to the different diets. During consumption of the SFA diet, there was a significant increase in palmitic acid (16:0) compared with that recorded during the high CHO and Mediterranean dietary periods (27.2 ± 1.4 vs 18.9 ± 3.9, P<0.004 and 27.2 ± 1.4 vs 15.1 ± 0.4, P<0.004 respectively). We also observed a significant increase in oleic acid (18:1) when subjects switched from the CHO diet to the Mediterranean diet (38.5 ± 9.0 vs 49.7 ± 4.7, P<0.05).

View larger version (15K): [in this window] [in a new window]   Figure 1 Steady state plasma glucose concentrations in relation to the –514C/T HL promoter polymorphism after different diets in (A) men and (B) women. The saturated fat enriched diet (SFA) is represented by solid bars; the low-fat, high-carbohydrate diet (CHO diet) is represented by shaded bars and the monounsaturated fat-enriched Mediterranean diet is represented by open bars. Multivariate ANOVA for repeated measures are shown: P1, diet effect; P2, genotype effect; P3, genotype by diet interaction. Bars with different letters are significantly different (P < 0.05).

	Discussion

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The saturated fatty acids reduce insulin sensitivity in type 2 diabetes patients (Moller et al. 1996, Purnell & Brunzell 1997) and in healthy subjects as shown in a recent study carried out by our group (Pérez-Jiménez et al. 2001). Furthermore, it has also been suggested that hypertriglyceridemia and an SFA diet might favor insulin resistance (Steiner et al. 1991, Storlien et al. 1993), increasing FFAs, which may inhibit glucose utilization by peripheral cells, thus reducing insulin sensitivity (Bjontorp 1994). In addition, there is a stronger interindividual variability in the response to dietary fat, as we show in our present study. This suggests that differences in gene products involved in candidate metabolic pathways produce phenotypic differences in response to dietary changes. Although it is well known that nutrients are involved in modulating the metabolism of lipoproteins, these aspects have been poorly investigated as regards HL. Previous studies in rats have shown that HL activity is inhibited by diets rich in saturated fats (Summerfield et al. 1984).

Our study clearly shows that gender interacts with genotype and diet to determine dietary modifications in insulin resistance. Likewise, in the present study, while the female carriers of the T allele displayed a tendency towards lower insulin resistance following the three dietary periods, no significant differences were observed when compared with the females homozygous for the C allele. The lack of effect of the T allele on insulin sensitivity in women is similar to the phenomenon observed in plasma HDL-C levels in response to changes in diet (Gómez et al. 2004). The association of the –514C/T polymorphism with HDL-C levels in a specific gender may be due to differences in exogenous administration or endogenous levels of sex hormones, which may differentially modulate lipoprotein metabolism between males and females. Moreover, because HL activity is regulated by sex-steroid hormones and because HL activity is higher in men than in women, divergent findings could be caused by gender difference, as we observed in our study. The gender difference in HL activity has led some to hypothesize that HL activity is a major determinant of the more atherogenic lipoprotein profile in men compared with women.

Insulin has been assumed to upregulate the activity of HL via insulin-responsive elements in the HL promoter. This has been proposed to explain the associations between hyperinsulinemia and high HL activity (Jansen et al. 1997, Romano et al. 1997). Jansen et al.(1997) observed a positive correlation between plasma levels of insulin and HL activity in non-carriers of the –514T allele, whereas no such relation was noted in carriers of the mutation. The same effect was recorded by Pihlajamaki et al.(2000) who observed an association between insulin resistance and the –250 G/A polymorphism in the HL promoter region. Therefore, variants in HL promoter activity may abolish the ability of insulin to stimulate HL activity. However, whether the promoter polymorphisms of the HL gene could regulate other actions of insulin is not known. Theoretically, changes in serum FFA levels could regulate the expression of peroxisome proliferator-activator receptors and, therefore, insulin sensitivity. Alternatively, the effect of HL on insulin sensitivity could be partly mediated via changes in the amount or distribution of body lipid storage. A third possibility is that changes in HL activity primarily alter serum lipids and secondarily lead to changes in intramyocellular lipid storage; therefore, the HL promoter variant could affect insulin sensitivity in skeletal muscle. Finally, HL may have other currently unknown functions that could affect the ability of insulin to stimulate glucose uptake. In addition, it is noteworthy that the –514 site is at the center of a CAC*GGG sequence, almost analogous to the CACGTG motif characteristic of an E-box onto which the upstream stimulatory factors (USF) 1/2 can bind. The latter are transcription factors involved in the regulation of glucose and lipid metabolism in the liver. For instance, USFs are part of the insulin responsive complexes that interact with the fatty acid synthase gene (Wang & Sul 1997). It is tempting to speculate that the –514C/T substitution would disrupt the E-box analogous sequence and impair the stimulatory regulation exerted by insulin. Interestingly, it has recently been reported that USF proteins can bind to the –514 region, and that the affinity is reduced fourfold by the –514C/T substitution (Botma et al. 2001).

In summary, after ingestion of a saturated fat-enriched diet, male carriers of the –514T allele in the promoter region of the HL gene show decreased insulin sensitivity which improves on consuming a carbohydrate-rich diet and a Mediterranean diet. Our results suggest that male carriers of the –514T allele are at greater risk of developing the insulin resistance syndrome and type 2 diabetes mellitus when they ingest a diet rich in saturated fat. Detection of this group of subjects may be one way to inhibit the development of type 2 diabetes.

	Acknowledgements

 
This work was supported by research grants from the CICYT (SAF96/0060, OLI 96/2146 to F P-J, SAF 01/2466-C05 04 to F P-J, SAF 01/03666 to J L-M), the Spanish Ministry of Health (FIS, 98/1531, 01/0449 to J L-M, FIS 99/0949 to F P-J), Fundación Cultural ‘Hospital Reina Sofía-Cajasur’ (P G), Consejería de Salud, Servicio Andaluz de Salud (PAI 97/58, 98/126, 99/116, 00/212 and 01/243 to J L-M, PAI 97/57, 98/132, 99/165, 00/39 to F P-J, and PAI 01/239 to F F-J), Diputación Provincial de Córdoba (to C M) and Patrimonio Comunal Olivarero (to F P-J). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received 8 October 2004
Accepted 21 December 2004

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Gómez, P. Articles by López-Miranda, J. Search for Related Content PubMed PubMed Citation Articles by Gómez, P. Articles by López-Miranda, J.