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European Journal of Clinical Nutrition (2006) 60, 1195–1200

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ORIGINAL ARTICLE

Dietary fatty acids and insulin secretion: a population-based study G Rojo-Martıńez, I Esteva, MS Ruiz de Adana, JM Garcıá-Almeida, F Tinahones, F Cardona, S Morcillo, E Garcıá-Escobar, E Garcıá-Fuentes and F Soriguer Endocrinology and Nutrition Service, Civil Hospital, Carlos Haya University Hospital, Malaga, Spain

Objective: Few epidemiological studies have examined the relationship of dietary fatty acids, especially MUFA, with the interrelation between insulin secretion and insulin resistance. We assessed the relation of dietary fatty acids with insulin secretion in a free-living population. Design and setting: This cross-sectional, population-based study was undertaken in Pizarra, a small town in Spain. Subjects and methods: Anthropometrical data were collected for 1226 persons selected randomly from the municipal census, 538 of whom (randomly chosen) were given a prospective, quantitative, 7-day nutritional questionnaire. The fatty acid composition of the serum phospholipids was used as a biological marker of the type of fat consumed. Beta-cell function (bCFI) and insulin-resistance index (IRI) were estimated by the Homeostasis Model Assessment. Results: To determine which factors influence the variability of the bCFI, we analyzed the variance of the bCFI according to sex, the presence of carbohydrate metabolism disorders and the different components of the diet, adjusting the models for age, body mass index (BMI) and IRI. The dietary MUFA and polyunsaturated fatty acids (PUFA) contributed to the variability of the bCFI, whereas only the proportion of serum phospholipid MUFA, but neither the saturated fatty acids nor the PUFA accounted for part of the variability of the bCFI in a multiple regression analysis. Conclusion: The results of this population-based study corroborate the results of other clinical and experimental studies suggesting a favorable relationship of MUFA with b-cell insulin secretion. Sponsorship: Fondo de Investigacioń Sanitaria, Junta de Andalucıá and the Asociacioń Maimońides.

European Journal of Clinical Nutrition (2006) 60, 1195–1200. doi:10.1038/sj.ejcn.1602437; published online 26 April 2006 Keywords: beta-cell function; homeostasis model assessment; monounsaturated fatty acids

Introduction Raised plasma glycemia levels may be the result of a defect in b-cell insulin secretion or increased resistance to the action of insulin. Type 2 diabetes mellitus appears when insulin secretion is insufficient to compensate for the resistance to Correspondence: Dr G Rojo-Martıńez, Endocrinology and Nutrition Service, Civil Hospital, Carlos Haya University Hospital, Plaza del Hospital Civil, 29009 Malaga, Spain. E-mail: [email protected] Guarantor: G Rojo-Martıńez. Contributors: IE, FS designed the study. GRM and FS wrote the original manuscript and performed the statistical analyses. GRM, IE and FS coordinated the trial. IS, MSRA, JMGA were the responsible for collection of clinical and anthropometrical data. JMGA, FT and EGF were the responsible for collection of nutritional data. FC, SM and EGE analyzed blood samples. All authors contributed to interpretation of data and critically revised the manuscript. Received 14 February 2005; revised 15 November 2005; accepted 16 January 2006; published online 26 April 2006

the peripheral action of insulin (DeFronzo et al., 1992). Diabetes mellitus is the last stage in a process which could start with normoglycemia at the expense of a compensating hyperinsulinemia, passing later through the stages of impaired fasting glucose (IFG) or impaired glucose tolerance (IGT), which are both recognized as risk groups in the current classification of diabetes mellitus (Alberti and Zimmet, 1998; Rojo-Martıńez et al., 2004), to finalize in clinical diabetes mellitus. The balance between insulin secretion and insulin resistance is determined both genetically (Lillioja et al., 1987) and by baseline environmental conditions. Diet is one of the most controversial of these environmental factors (Lundgren et al., 1989; Feskens et al., 1995). Most population-based studies agree that the intake of fats, especially saturated fats, has an adverse effect on the risk of diabetes. This adverse effect has also been shown in vitro (Hunnicutt et al., 1994). Furthermore, there is also general agreement regarding the increase in insulin resistance with

Dietary fatty acids and insulin secretion G Rojo-Martıńez et al

1196 saturated fats (Hunnicutt et al., 1994) and the reduction in insulin resistance with n-3 polyunsaturated fatty acids (PUFA) (Rizkalla et al., 1996). However, the effect of n-6 PUFA on insulin resistance is more controversial (Berry, 2001). Some studies have shown a reduction in insulin resistance with MUFA-rich diets or a Mediterranean-style diet (Ryan et al., 2000, Esposito et al., 2004), whereas others have not found this effect (Lovejoy et al., 2002). Although the type of fat consumed can modify the fatty acid composition of plasma and tissues, the way dietary modification of the whole pool of fatty acids affects insulin secretion is not well understood (Lands, 1995). In vitro, the release of insulin after stimulation with glucose is more pronounced the longer the fat chain and the higher the degree of fatty acid saturation in the culture medium (Stein et al., 1997; Tinahones et al., 1999). Stearic acid and palmitic acid have a marked stimulatory effect on glucose-mediated insulin secretion, both in the perfused pancreas (Stein et al., 1997) and in islets cultured in vitro (Tinahones et al., 1999). Beysen et al. (2002) recently reported the greater effect of MUFA on insulin secretion in humans. However, epidemiological studies of the influence of dietary fatty acids on the relationship between insulin resistance and insulin secretion are few, especially studies examining the role of MUFA. We evaluated the influence of the dietary fatty acids on insulin secretion, as assessed by the Homeostasis Model Assessment (HOMA) (Mattews et al., 1985), in a general Mediterranean population from the south of Spain with a high intake of MUFA in their usual diet.

JMGA). Weight and height were measured, and the body mass index (BMI ¼ weight/height2) calculated (WHO Expert Committee, 1995). To determine the presence of carbohydrate metabolism disorders each person received an oral glucose tolerance test (OGTT) with 75 g of glucose. Venous blood samples were taken fasting and 120 min after the OGTT, and the serum stored at 701C for later study of insulin and fatty acid composition in serum phospholipids. Subjects with known diabetes mellitus were excluded from the analysis.

Dietary survey A prospective, 7-day, quantitative questionnaire was administered to a random subset of 538 persons generated by the ‘Random sample of cases’ facility of SPSS (55% of cases). With this sample size, the estimations of the means of the nutritional variables presented a sample error below 3% in all cases. The questionnaire was administered by experienced dietitians, specially trained for this project. After arranging an appointment by telephone, the dietitians visited the families in their homes, explained the nature of the study and handed over the questionnaire. After 7 days these questionnaires were collected after resolving any doubts concerning the recording of data. The conversion to nutrients was carried out with a computer program expressly designed for this study and incorporating details about the composition of all local food, based on data commonly used in this region of Spain (Moreiras et al., 1992).

Subjects and methods Subjects The study was undertaken in Pizarra, a small town in the province of Malaga, Andalusia, southern Spain. Details of the study design and sample have been reported previously (Soriguer et al., 2003b; Rojo-Martıńez et al., 2004; Soriguer et al., 2004). A total of 1226 persons aged 18–65 years were selected randomly from the municipal census. All institutionalized persons, for whatever reason, were excluded from the study, as were pregnant women, and those persons with a severe clinical problem or psychological disorder; basically, persons were excluded if they were unable to come without assistance to the clinic where the study was undertaken or if they did not have the legal ability to sign the informed consent. The subjects were requested by mail to attend their local health center for a medical examination. Those who failed to attend their first appointment were sent a second letter giving them another appointment, and all those still not attending were visited at home in order to ascertain the reason. The final sample distribution by age and sex was not significantly different from the population distribution. Procedures All participants were interviewed and given a standardized clinical examination by the same physicians (IE, MSRA, European Journal of Clinical Nutrition

Laboratory measurements Baseline insulinemia was measured by radioimmunoassay (Coat A Count Insulin, DPC, Los Angeles, CA, USA). Insulin resistance was estimated by the HOMA method (Mattews et al., 1985), according to the formula:

Insulin resistance index ðIRIÞ ¼ ðFasting insulin ðmU=mlÞ Fasting glucose ðmmol=lÞÞ=22:5 b-cell function index ðbCFIÞ ¼ ð20Fasting insulin ðmU=mlÞÞ =ðFasting glucose ðmmol=lÞ 3:5Þ

Those persons with fasting glucose values p3.5 mmol/l (0.4% of all the subjects) were excluded from the final analysis to avoid including data from bCFI-negative persons. The fatty acid composition of serum phospholipids was determined by extraction of the serum fat with chloroform: methanol 2:1 and BHT at 0.025% (Hamilton et al., 1992) and phospholipid separation by TLC. Fatty acid methyl esters were formed by heating the extracted fat for 30 min with H2SO4 0.61 M in anhydrous methanol (Hamilton et al.,


1197 1992). After extraction with hexane, the fatty acid methyl esters were analyzed in a Hewlett-Packard chromatograph, equipped with a flame ionization detector and using a BPX75 fused-silica capillary column (SGE, Villebon, France). The American Diabetes Association 1998 criteria were used for classification of persons with diabetes and carbohydrate metabolism disorders (Alberti and Zimmet, 1998). A normal OGTT was considered to be a baseline capillary glycemia lower than 5.6 mmol/l and post OGTT glycemia lower than 7.8 mmol/l. Obesity was set at a BMI of 30 or above.

Statistical study Statistical differences between means of continuous variables were studied with the Student t or ANOVA test and w2 test was used for qualitative variables. Non-normally distributed variables were transformed by natural log to achieve normality. Pearson coefficients of correlation were calculated for assessment relations between variables. ANOVA and multiple regression were used to study which variables were associated with the variability of the bCFI. In all cases, the rejection level for a null hypothesis was a ¼ 0.05 for two tails. Analyses were made using SPSS v10 (SPSS Inc., Chicago, IL, USA).

Ethical considerations All subjects were informed of the nature of the study and gave their written consent to participate. The study was approved by the Ethics and Clinical Investigation Committee of Carlos Haya Hospital.

Results Table 1 summarizes the variables studied. No differences were seen between sex and age or BMI, but the men had a higher mean IRI and a lower bCFI than the women. The distribution of carbohydrate metabolism disorders also differed according to sex: IFG and diabetes mellitus were more common among the men and IGT was more common among the women. The only significant differences in the fatty acid composition of the plasma phospholipids were seen in the proportion of saturated fatty acids and n-6 PUFA. Regarding the food pattern of the study population, the men consumed a greater amount of energy than the women, although the proportions of energy in the form of proteins, carbohydrates and fats were similar for both sexes. The proportions of saturated fatty acids and n-3 PUFA were similar for both men and women, but whereas the men consumed more n-6 PUFA, the women consumed more MUFA. The intake of MUFA represented just over 50% of the total amount of dietary fat and about 18% of the energy consumed. In our sample, 53.3% of the subjects usually consumed olive oil, 25.3% sunflower oil, and 21.4% both. The fatty acid concentration in the serum phospholipids was significantly associated with the type of oil usually consumed (Table 2). To determine which factors influenced the variability of the bCFI, which can vary widely with the same IRI, we analyzed the variance of the bCFI according to sex, the presence of carbohydrate metabolism disorders and the different components of the diet, adjusting the models for age, BMI and IRI (Table 3). Among the dietary factors

Table 1 Characteristics of the subjects, prevalence of carbohydrate metabolism disorders, concentration of serum phospholipid fatty acids and macronutrient intake according to sex

Age (years) BMI (kg/m2) Insulin resistance index Beta-cell function index Impaired fasting glycemia – prevalence (%) Impaired glucose tolerance – prevalence (%) Unknown diabetes mellitus – prevalence (%) Serum phospholipid saturated fatty acids (%) Serum phospholipid monounsaturated fatty acids (%) Serum phospholipid polyunsaturated n-6 fatty acids (%) Serum phospholipid polyunsaturated n-3 fatty acids (%) Total energy in the diet (kJ) Dietary proteins (% total energy) Dietary carbohydrates (% total energy) Dietary fats (% total energy) Dietary saturated fatty acids (% lipids) Dietary monounsaturated fatty acids (% lipids) Dietary polyunsaturated n-6 fatty acids (% lipids) Dietary polyunsaturated n-3 fatty acids (% lipids)

Men (N ¼ 215)

Women (N ¼ 323)

42.1713.8 26.773.8 2.671.8 90.9756.6 22.9 10.8 13.9 46.776.2 12.072.7 35.975.0 5.371.8 1154872958 15.172.7 45.776.5 39.275.7 30.275.2 52.375.8 16.275.2 1.370.6

40.6713.1 28.175.6 2.371.6* 105.1770.5* 12.0 17.5 9.8 45.976.7* 12.072.5 36.675.6* 5.472.6 840972418** 15.372.7 45.176.7 39.675.7 29.674.7 54.375.0* 14.874.7* 1.370.4

Data are means7s.d. or prevalence (%). *Po0.01 between men and women, **Po0.001 between men and women. Comparisons by Student’s t-test (normal or normalized continuous variables) or w2 test (qualitative variables).

European Journal of Clinical Nutrition


1198 Table 2 Serum phospholipid fatty acids according to the type of oil consumed

Table 3

ANOVA of beta-cell function index (log transformed)

Source of variation Oil consumed

Serum phospholipid fatty acids

Saturated (%) Monounsaturated (%) Polyunsaturated n-6 (%) Polyunsaturated n-3 (%)

Olive (N ¼ 287)

Mix (N ¼ 115)

Sunflower (N ¼ 136)

45.876.8a 12.772.8a

44.775.5a 11.772.0b

46.576.3a 11.072.1b

NS o0.0001

35.975.7b

37.974.5a

37.175.5a

0.009

5.4 71.8a

5.371.8a

5.271.8a

NS

In each row, data with the same letter do not differ significantly (P40.05).

studied, the proportion of MUFA (model 1), n-6 PUFA (model 2) and n-3 PUFA (model 3) determined the bCFI. A statistically significant interaction was found between the OGTT results and the dietary MUFA and the dietarty n-3 PUFA, but not between the OGTT results and the dietary n-6 PUFA (Table 3). Multiple regression analysis showed that 70% of the variance of the bCFI was explained by the IRI (P ¼ 0.0001). Inclusion of the BMI, age, sex and the serum phospholipid MUFA increased the R2 to 77%, but whereas the age and BMI were related negatively with the bCFI, the serum phospholipid MUFA were related positively (Table 4). The serum phospholipid saturated fatty acids and the PUFA were not related with bCFI.

Discussion The main finding of this study is that b-cell insulin secretion is associated with the dietary intake of fatty acids. Type 2 diabetes mellitus generally appears when b-cell insulin secretion is unable to counterbalance the preceding normoglycemic insulin resistance (DeFronzo et al., 1992). The rupture of this balance between insulin resistance and insulin secretion is the result of genetic and environmental factors (Lillioja et al., 1987). Age, obesity and a sedentary lifestyle are some of the better studied environmental variables, but the influence of diet is more controversial. The composition of fatty acids in serum reflects the composition of fatty acids in the diet, over both the short and the long term (Lands, 1995). Previous studies in the same population (Soriguer et al., 2003b; Soriguer et al., 2004) have shown that the amount of oleic acid in the phospholipids depends on the composition of the frying oil used. In this study, persons who consumed olive oil had greater concentrations of MUFA than those who consumed sunflower oil, but these latter had higher concentrations of n-6 PUFA. Numerous experimental studies in animals and controlled clinical trials in humans have demonstrated an association European Journal of Clinical Nutrition

Mean square

P

0.6 56.9 0.06

o0.0001 o0.0001 0.09

0.32 2.1 0.07

o0.0001 o0.0001 0.02

0.05

0.01

1.0 92.5 0.07

o0.0001 o0.0001 0.05

0.34 3.8 0.07

o0.0001 o0.0001 0.03

0.02

40.05

P Model 1 Covariates Age Log (IRI) BMI Main effects Sex OGTT resultsa MUFA proportion in diet (quartiles) Interaction OGTT resultsa MUFA Model 2 Covariates Age Log (IRI) BMI Main effects Sex OGTT resultsa PUFA n-6 proportion in diet (quartiles) Interaction OGTT resultsa PUFA n-6 Model 3 Covariates Age Log (IRI) BMI Main effects Sex OGTT resultsa PUFA n-3 proportion in diet (quartiles) Interaction OGTT resultsa PUFA

0.3 54.8 0.07

o0.0001 o0.0001 0.07

0.21 1.9 0.08

0.001 o0.0001 0.02

0.05

0.03

a OGTT results are incorporated in the ANOVA as OGTT normal, IFG, IGT and DM.

Table 4

Multiple regression analysis

Variables

b

SEb

P

Model 1 (R2 ¼ 0.70) Log (IRI)

þ 0.77

0.02

Model 2 (R2 ¼ 0.77) Log (IRI) Sex (Male ¼ 1, Female ¼ 2) Age BMI MUFA proportion in serum phospholipids

þ 0.84 þ 0.11 0.005 0.005 þ 0.01

0.02 o0.0001 0.02 o0.0001 0.0005 o0.0001 0.001 0.008 0.003 0.0001

o0.0001

Dependent variable is b-cell function index log-transformed.

between the type of fat in the diet and the sensitivity of the peripheral action of insulin (Hu et al., 2001). Diets rich in saturated fats generally favor insulin resistance, both in vitro and in vivo (Chattaway et al., 1990; Hunnicutt et al., 1994; Ryan et al., 2000) and enriching the diet with n-3 fatty acids increases this sensitivity (Rizkalla et al., 1996). However, the


1199 effect of n-6 fatty acids remains controversial (Berry, 2001). Recent studies of the role of MUFA in insulin resistance have resulted in different findings. Some studies have found that a MUFA-rich diet increases the peripheral sensitivity to the action of insulin, both in persons with diabetes (Salas et al., 1999; Ryan et al., 2000) and in healthy subjects (Vessby et al., 2001; Soriguer et al., 2004). Other studies, however, have found that the MUFA in the diet influence fat oxidation (Lovejoy et al., 2002) or other clinical parameters such as blood pressure (Lahoz et al., 1999) but not sensitivity to the action of insulin. In vivo studies in humans of the role of a particular fatty acid on insulin secretion are scarce. Studies by our group (Pareja et al., 1997; Tinahones et al., 2002; Soriguer et al., 2004) and others (Stein et al., 1997) have shown a direct effect of fatty acids on b-cells and that, at least during short periods of incubation of pancreas islets with different fatty acids, insulin secretion increases with the length of the fat chain and with the degree of saturation. However, few studies have examined the effect of a particular fatty acid on insulin secretion in vivo, partly because of methodological difficulties, and some studies which have examined this question found no effect of the MUFA on insulin secretion (Thomsen et al., 1999; Stefan et al., 2001). A study by Beysen et al. (2002), using a model of increasing plasma NEFA with heparin in healthy subjects, clearly showed that the dietary MUFA raise the in vivo secretion of insulin stimulated by glucose, independently of the effect on insulin sensitivity. The results of our study, undertaken in a large population-based sample, confirm that insulin secretion is directly related with the intake of MUFA-rich fats, independently of the level of insulin resistance. The results are specially relevant if we consider that variability of the serum MUFA was particularly small (in our study it was 22.6% as compared with a variability of 69.2% for the IRI). Several explanations may account for the rise in insulin secretion induced by a MUFA-rich diet. The MUFA may have a different effect on gastric emptying. Our group has recently found that rats fed with MUFA have a greater amount of fat in their feces and a lower calorie balance than rats fed with saturated fats or with n-6 or n-3 PUFA (Soriguer et al., 2003a), an effect which could be mediated by glucagon-like-peptide 1 (GLP-1) inhibition of gastric emptying (Wilms et al., 1996). GLP-1 is secreted by intestinal L cells in response to a meal and boosts the secretion of insulin stimulated by glucose in a dose-dependent fashion (Garcia-Flores et al., 2001). Several studies, both in healthy subjects and in persons with type 2 diabetes mellitus, have found that levels of GLP-1 are increased more by dietary MUFA than by dietary saturated fatty acids (Thomsen et al., 2003), and that the greater postprandial clearance of an oral overload of MUFA-rich fats is associated with a greater increase in postprandial incretins such as GLP-1 or gastric inhibitory polypeptide. Tinahones et al. (2003) recently showed that postprandial hypertriglyceridemia is independently associated with b-cell function in the metabolic syndrome, with numerous studies

demonstrating that the type of dietary fat influences the plasma clearance of postprandial lipemia (Thomsen et al., 2003). The main limitation of our study involves its crosssectional nature, which does not allow more robust associations of causality to be established. Thus, a prospective study, currently under way, will be necessary to confirm the results. In summary, the results of this study suggest that fatty acids somehow modulate secretion by the pancreas b cells, within the context of a population with a high intake of MUFA. Although the mechanisms by which a diet rich in MUFA favor insulin secretion require further investigation, the results of this population-based study corroborate the results of clinical and experimental studies suggesting a favorable relationship of a MUFA-rich diet with b-cell secretion of insulin.

Acknowledgements We are grateful to Marieta Catala´, M Jose´ Merelo, Araceli ã Cano, Gonza´lez, Isabel Garcıá, Salvador Alarcoń, Begon Patricia Soares, Manuela Beltrań, and Pablo Rodrı´guez-Bada for their inestimable technical assistance, and to Ian Johnstone for the English language version of the manuscript. This study was undertaken with finance from the Fondo de Investigacioń Sanitaria of the Instituto de Salud Carlos III (PI021311 and Metabolism and Nutrition Network RCMYN C03-08), Junta de Andalucıá (191/2001 and 3/03), and the Asociacioń Maimońides.

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