Fatty sh in the diet of patients with type 2 diabetes: comparison of the metabolic effects of foods rich in n23 and

n26 fatty acids1,2

Brita E Karlstro Anette E Ja m, rvi, Liisa Byberg, Lars G Berglund, and Bengt OH Vessby
ABSTRACT Background: Dietary advice, including modication of dietary fat quality, is the basis of treatment of diabetes, but there is some uncertainty about the optimal amount of polyunsaturated fatty acids of the n26 (omega-6) and n23 (omega-3) series. Objective: The objective was to compare the effects of diets rich in n23 or n26 fatty acids on glucose and lipoprotein metabolism in type 2 diabetes. Design: In a crossover study during 2 consecutive 3.5-wk periods, the participants were provided diets with identical nutrient compositions containing either a high proportion of n23 (n23 diet) or n26 (n26 diet) fatty acids through the inclusion of fatty sh or lean sh and fat containing linoleic acid, respectively. Results: Blood glucose concentrations at fasting and during the day were lower with the n26 than with the n23 diet (P = 0.009 and P = 0.029, respectively), and the area under the insulin curve during the day was signicantly higher (P = 0.03) with the n26 diet. Both diets showed similar effects on insulin sensitivity and plasminogen activator inhibitor 1 concentrations. The reductions in VLDLs and serum apolipoprotein B concentrations were more pronounced after the n23 diet. Conclusions: The risk related to the moderately higher blood glucose concentrations with the n23enriched diet may be counteracted by positive effects with regard to lipoprotein concentrations. An increase in long-chain n23 fatty acids from fatty sh, and of n26 fatty acids from linoleic acid, may be recommended for patients with type 2 diabetes. Am J Clin Nutr 2011;94:2633.

INTRODUCTION

Diet modication is the basis of the treatment of diabetes mellitus (1, 2). One important recommendation is to try to substitute unsaturated for saturated fat. A high proportion of monounsaturated fatty acids is compatible with good metabolic control in type 2 diabetes (35). It is difcult to make any rm recommendations regarding the optimal amounts and proportions of polyunsaturated fatty acids of the n26 and n23 series. Earlier studies have shown that a diabetic diet enriched in linoleic acid may reduce serum cholesterol and improve glucose sensitivity (6). Indications of the delayed development of retinopathy (7) and of improved glucose tolerance or insulin secretion, or both, have been observed. In contrast, it has been suggested that a high intake of linoleic acid may increase lipid peroxidation (8). The role of n23 fatty acids in the diabetic diet has also been questioned (9, 10). Many studies have shown an improvement in

serum triglycerides after supplementation with sh oil, but with a simultaneous moderate increase in LDL-cholesterol concentrations (11, 12). An impairment of blood glucose concentrations in patients with type 2 diabetes, especially after rather high doses of sh oil, was seen in some studies. Two meta-analyses investigating the effect of sh-oil supplementation on glycemic control in diabetes concluded that the effect on blood glucose is marginal (11) or nonsignicant (12) without any impairment of glycated hemoglobin (Hb A1c) concentrations. In a more recent meta-analysis of randomized trials restricted to type 2 diabetes, the pooled effect of n23 fatty acid supplementation was to moderately, but not signicantly, increase both fasting blood glucose and Hb A1c (13). An examination of the association between dietary long-chain n23 fatty acids and incidence of type 2 diabetes in 3 prospective cohorts of women and men showed that higher intakes may modestly increase the incidence of the disease (14). An increase in plasminogen activator inhibitor 1 (PAI-1) activity after sh oil has been reported in type 2 diabetes (15)an effect not observed in most studies in healthy subjects (16, 17). Nearly all studies of n23 fatty acids in diabetes have used sh oil or concentrates of long-chain n23 fatty acids from sh oil. The current study compared the effects on glucose homeostasis and the serum lipoprotein composition of 2 diabetic diets containing ordinary food with different proportions of n23 and n26 fatty acids. In addition, the aim was to compare the inuence on brinolysis, lipid oxidation, and serum tocopherol concentrations. One of the diets contained more n23 fatty acids (n23 diet) because of a higher content of fatty sh, whereas the other contained lean sh and more linoleic acid (n26 diet) because of a higher proportion of linoleic acid in edible fats. The proportions of macronutrients were closely similar.

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From Clinical Nutrition and Metabolism, Department of Public Health and Caring Sciences (AEJ and BOHV), Department of Food, Nutrition and Dietetics (BEK), and the Department of Surgical Sciences, Orthopaedics and Uppsala Clinical Research Centre (LB) and Uppsala Clinical Research Centre (LGB), Uppsala University, Uppsala, Sweden. 2 Address correspondence to BOH Vessby, Clinical Nutrition and Metabolism, Department of Public Health and Caring Sciences, Uppsala University, Uppsala Science Center, S-751 85 Uppsala, Sweden. E-mail: bengt. vessby@pubcare.uu.se. Received October 13, 2010. Accepted for publication April 19, 2011. First published online May 25, 2011; doi: 10.3945/ajcn.110.006221.

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Am J Clin Nutr 2011;94:2633. Printed in USA. 2011 American Society for Nutrition

FATTY FISH IN THE DIET IN TYPE 2 DIABETES SUBJECTS AND METHODS

27

Study design Sixteen patients with type 2 diabetes were included in the study, which was conducted over 2 consecutive 3.5-wk periods in a crossover model. The participants were admitted to a metabolic ward at the beginning and at the end of the treatment periods. On admission and in the morning immediately after the last day of each treatment period, a clinical examination and laboratory tests were performed in the fasting state. In addition, blood samples were drawn at xed time points throughout the last day of each dietary period from 0730 to 1800 (prole day) for assay of plasma glucose and insulin at fasting and at 60, 120, 180, 240, 300, 360, 480, and 540 min after breakfast. The subjects followed the study diets on the prole day, with breakfast at 0800, lunch at 1215, dinner at 1615, and snacks at 1000 and 1400. Throughoutthestudytheparticipantswerefree-living,continuing their customarylife and activities. Three times aweek, they collected their food at the metabolic ward. A 1-wk menu, including food items that were already weighed and packed, was planned for each participant. All prepared dishes were packed in special cool-boxes and wereready tobeheatedina microwave oven. Instructions weregiven on how to store the food, and in the written 1-wk menu the participants were told what food items shouldbe eaten for breakfast, lunch, and dinner and in between meals. No other food items except water, mineral water, coffee, and tea were allowed during the study period. The participants started in a randomized order on either of the 2 diets and switched over to the other diet after 24 d. Subjects The patients were referred to the metabolic outpatient clinic because of type 2 diabetes. All subjects had earlier received dietary advice. A total of 16 patients, 8 men and 8 women with a mean age of 58.9 y (range: 3775 y) entered the study. The mean body mass index (BMI; kg/m2) was 26.1 (range: 20.228.7). To be included, blood glucose control should have been fairly stable during the last months and there should have been no major changes in body weight. None of the patients had suffered from an episode of coronary heart disease or other major diseases during the last 6 mo. All had liver and renal functions within the reference ranges. None of the participants were receiving insulin treatment, but 13 subjects were treated with oral antidiabetic drugs (sulfonylureas, metformin, or a combination of both). Four subjects were receiving antihypertensive treatment. Two of these subjects were also taking digitalis and loop diuretics. One woman received thyroxine substitution and was euthyroid on ongoing treatment. All drug treatment remained unchanged throughout the study period. All patients gave their informed written consent to participate in the study, and the protocol was approved by the Ethical Committee of the Medical Faculty, Uppsala University, Uppsala, Sweden. Diets The diets were based on ordinary food items included in the traditional Swedish diet. The diets were designed to have the same nutrient composition (Table 1) meeting requirements of a diabetic diet with 29% of the energy derived from fat and with a similar proportion of saturated, monounsaturated, and poly-

TABLE 1 Calculated nutrient composition of 2 diabetic diets enriched with n26 or n23 fatty acids n26 Diet Protein (% of energy) Fat (% of energy) Saturated fatty acids Monounsaturated fatty acids Polyunsaturated fatty acids Carbohydrates (% of energy) Dietary ber (g/1600 kcal) Cholesterol (mg/1600 kcal) 16 29 9.6 9.7 9.7 55 26 114 n23 Diet 16 29 8.9 10.2 9.9 55 26 142

unsaturated fatty acids in the 2 diets. The menu was calculated at an energy level of 6.7 MJ. To achieve the different energy levels required, all of the ingredients of the basic diet were multiplied by 1.25, 1.50, and 1.75 to provide 8.4 MJ (2000 kcal), 10.1 MJ (2400 kcal), and 11.8 MJ (2800 kcal), respectively. The estimated energy intake of the participants was calculated on the basis of their initial body weight in an attempt to maintain the body weight unchanged during the study. The database from the Swedish Food Administration (18) was used for calculation of the diets. An example of a 1-d menu is given in Table 2. Three duplicate portions of each daily menu were collected and homogenized to be analyzed for the total fat content and for the fatty acid composition. The analyzed average fatty acid compositions of the diets are shown in Table 3. The 2 diets differed mainly with regard to the content of the long-chain n23 fatty acids and linoleic acid. The sum of the cholesterol-elevating saturated fatty acids (12:0, 14:0, and 16:0) was identical, as was the estimated amount of trans fatty acids. The ratio between n26 and n23 fatty acids in the 2 diets was 6.1 in the n26 and 2.5 in the n23 diet (Table 4). The ratio between n26 and long-chain n23 fatty acids differed by 10 in the 2 diets. A higher content of n23 fatty acids in the n23 diet (3.3 g long-chain n23 fatty acids/ 1600 kcal) than in the n26 diet (0.4 g/1600 kcal) was achieved by incorporating more fatty sh in the diet. The content of n26 fatty acids acid was increased in the n26 diet (14.7 g/1600 kcal) compared with the n23 diet (12.9 g/1600 kcal) by using lean sh and a higher proportion of linoleic acid in the edible fat. Dietary instructions were given by a dietitian before entering the study. The participants received information regarding the food items that were included in the menu to ensure that all food was acceptable. They were also instructed to eat everything that was served. If, for some reason, the participants were not able to eat a particular food item, he or she was asked to write down what was left and also to take the food back to the metabolic ward to be weighed and subtracted from the calculated amount of the diet. Laboratory analyses Lipoprotein concentrations in serum were measured after an overnight fast. VLDLs, LDLs, and HDLs were isolated by a combination of preparative ultracentrifugation (19) and precipitation with sodium phosphotungstate/magnesium chloride solution (20). Triglyceride and cholesterol concentrations were measured in serum and in the isolated lipoprotein fractions by enzymatic methods by using the IL Test Cholesterol Method 18161810 and IL Test Triglyceride Enzymatic Colorimetric Method 18161060 in a Monarch apparatus (Instrumentation Laboratory, Lexington, MA).

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28

KARLSTROM ET AL
TABLE 3 Analyzed fatty acid composition of 2 diabetic diets enriched with n26 or n23 fatty acids (n = 14)1 Fatty acid 8:0 10:0 12:0 14:0 16:0 16:1n27 18:0 18:1 18:2n26 18:3n23 20:4n26 20:5n23 22:5n23 22:6n23 P:S ratio n26:n23 ratio n26:long-chain n23
1 2

TABLE 2 Example of a 1-d menu for diets enriched with n26 or n23 fatty acids at the 1600-kcal (6.7-MJ) level Meal/foodstuff Breakfast Cultured milk, 0.5 g fat Breakfast cereals Raspberries, frozen Bread, graham and oat our Crisp bread, rye Margarine spread, 80% fat Orange, unpeeled Cucumber Cheese, 17% fat Herring, pickled Lunch Cod, fried and pickled Baltic herring, fried and pickled Rye our Margarine for frying, 80% fat Potatoes Crisp bread, rye Carrots Dressing, corn oil Fresh lemon juice Low-alcohol beer Margarine spread, 80% fat Dinner Chili con carne White beans, dried Minced meat, beef Onion Tomato, canned Wheat our Margarine, 80% fat Rice, parboiled uncooked Mixed salad Tomato Sweet peppers, green Lettuce Dessert Bilberries, frozen Sugar Low-fat milk, 0.5% fat Snacks Sweet wheat bread Banana, unpeeled Crisp bread, rye Sweet peppers, green Ham, boiled Sardines in tomato sauce Margarine spread, 80% fat Low-fat milk, 0.5% fat n26 Diet g 150 20 25 30 10 6 97 40 10 95 3 11 75 15 50 4 4 150 3 n23 Diet g 150 20 25 30 4 97 40 25 100 3 11 75 15 50 150 3

n26 Diet 0.5 1.0 6.9 3.6 13.6 0.6 7.4 34.8 27.4 3.7 0.2 0.3 % 6 0.22 6 0.2 6 0.9 6 0.3 6 0.7 6 0.1 6 0.4 6 1.5 6 2.0 6 0.4 6 0.1 6 0.0 0.5 6 0.2 0.97 6.1 36.8

n23 Diet % 0.5 6 0.2 0.8 6 0.2 5.4 6 0.8 4.5 6 0.7 14.4 6 0.6 2.1 6 0.5 6.4 6 0.4 32.1 6 1.8 24.1 6 2.2 3.6 6 0.4 0.2 6 0.1 2.3 6 0.7 0.5 6 0.2 3.4 6 0.9 1.07 2.5 3.9

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P:S, ratio of polyunsaturated to saturated fatty acids. Mean 6 SD (all such values).

25 35 40 30 3 15 25 35 25 35 75 5 50 75 103 15 25 15 6 100

25 35 40 30 3 15 25 35 25 35 75 5 50 75 103 15 25 20 4 100

Blood and urinary glucose concentrations were measured by using the glucose oxidase assay. Plasma insulin assays were performed by using the enzyme-immunologic test for quantitative determination of human insulin (enzyme-linked immunosorbent assay/1-step sandwich assay; Boehringher Mannheim Immunodiagnostics for the ES 300). Plasma C-peptide was measured by immunoassay (human enzyme-linked immunosorbent assay; Mercodia, Uppsala, Sweden). Insulin sensitivity was evaluated by using a euglycemic hyperinsulinemic clamp technique according to DeFronzo et al (22). Insulin sensitivity is expressed as the insulin sensitivity index (M/I60120), which is a measure of the tissue sensitivity to insulin expressed per unit of insulin obtained by dividing the mean glucose uptake (M) by the mean insulin concentration (I) during the last 60 min of the clamp study. The activity of tissue PAI-1 was measured with a 2-step indirect enzymatic assay with the use of Spectolyse/pL kits (Biopool AB, Umea, Sweden) (23). a- and c-Tocopherol concentrations in serum were analyzed as previously described (24). The tocopherol concentrations were corrected for the sum of serum cholesterol and serum triglycerides as suggested by Thurnham et al (25). An HPLC system was used to measure the plasma concentration of malondialdehyde (MDA) (26). Statistical analysis

The serum concentrations of apolipoprotein (apo) A-I and apo B were measured by immunochemical assay (Orion Diagnostica, Espoo, Finland) in the Monarch apparatus. Lipoprotein(a) [Lp(a)] was measured by the Pharmacia Apo(a) RIA method. The concentration is expressed in units (U/L), where 1 U of apo(a) is approximately equal to 0.7 mg Lp(a), according to the manufacturer. The fatty acid composition of the diets and of the plasma lipid esters was determined by gas-liquid chromatography as described previously (21).

The number of subjects included was estimated to allow 80% power for detecting a 0.33-mmol/L difference between the 2 test groups for changes in LDL cholesterol, a 0.46-unit difference in M/I, and 660 glucose area under the curve units at a signicance level of P , 0.05. The analysis was performed with SAS (version 9.1; SAS Institute, Cary, NC) by using the general linear model procedure. Values are presented as means 6 SDs. For variables with a skewed distribution, the data were log transformed before statistical analysis. The analyses were performed, with the change from baseline as the

FATTY FISH IN THE DIET IN TYPE 2 DIABETES

TABLE 4 Effects of 2 diabetic diets enriched with n26 or n23 fatty acids on serum lipoprotein lipid and apolipoprotein concentrations (n = 16) Admission Serum cholesterol (mmol/L) Change (%) Serum triglycerides (mmol/L) Change (%) VLDL cholesterol (mmol/L) Change (%) VLDL triglycerides (mmol/L) Change (%) LDL cholesterol (mmol/L) Change (%) LDL triglycerides (mmol/L) Change (%) HDL cholesterol (mmol/L) Change (%) HDL triglycerides (mmol/L) Change (%) Apolipoprotein B (g/L) Change (%) Apolipoprotein A-I (g/L) Change (%) Apolipoprotein(a) (g/L) Change (%)
1

29

n26 Diet 4.68 6 1.10 215 1.61 6 0.89 230 0.46 6 0.33 237 1.16 6 0.81 234 3.16 6 0.98 29 0.36 6 0.14 218 1.07 6 0.25 213 0.10 6 0.04 234 0.90 6 0.25 29 1.19 6 0.17 210 0.25 6 0.27 +38

n23 Diet 4.39 6 0.82 220 1.23 6 0.62 247 0.29 6 0.23 261 0.79 6 0.63 255 3.06 6 0.74 212 0.36 6 0.11 219 1.03 6 0.23 216 0.08 6 0.03 247 0.83 6 0.19 216 1.14 6 0.16 213 0.25 6 0.29 +41

P for difference between diets1 0.074 0.002 0.037 0.003 0.458 0.696 0.533 0.058 0.039 0.009 0.678

5.47 6 0.992 2.31 6 2.64 0.74 6 1.08 1.75 6 2.48 3.48 6 1.11 0.44 6 0.18 1.22 6 0.29 0.16 6 0.12 0.98 6 0.25 1.32 6 0.19 0.18 6 0.24

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Denotes differences between changes during the 2 dietary regimens. The analyses were performed by using ANCOVA appropriate for crossover design. 2 Mean 6 SD (all such values).

outcome using ANCOVA. Estimation of carryover effects and treatment differences for the change from baseline for the dependent variables was performed with a model, where the factors were sequence, patient nested within sequence, period, and treatment (= sequence period interaction) (27). Change in BMI from baseline was included as a covariate to take weight changes into account. Carryover effects were evaluated by comparing the treatment sequences with patient nested within sequence as the error term. These tests were 2-sided and were performed at the 10% signicance level. Because no signicant carryover effects were observed, the results from the same treatment periods could be combined, irrespective of the order of treatment. Comparisons were made of the changes during the 2 dietary periods. The tests for the treatment effects were 2-sided and were performed at the 5% signicance level. If the usual assumptions for t tests did not hold, or if the data were on an ordinal scale, the t test was replaced by a Mann-Whitneys U test. The results are presented as least-squares means because they form the basis of the statistical tests and estimates in the analysis and take imbalance into account. Imbalance inuenced only the levels of the marginal means, because only subjects with values from both diets were included in the estimation of treatment difference.

74.9 6 10.0, and 74.7 6 10.0 kg, respectively. The magnitude of changes was identical during both treatment periods. Because body weight changes as such may have some inuence on lipid composition and glucose homeostasis, independent of the effects of the diet composition, the data presented have been adjusted for individual changes in body weight. Overall, this did not inuence the results in any important way, but there were some minor adjustments of the magnitude of changes recorded for example for blood lipids and blood glucose control. Serum lipoprotein and apolipoprotein concentrations A highly signicant improvement in the lipoprotein lipid prole was found during both treatment periods (Table 4), and a more pronounced reduction in serum triglyceride concentrations was observed, which corresponded to a larger decrease in VLDL lipid concentrations during the diet rich in n23 fatty acids, whereas LDL and HDL cholesterol decreased to a similar extent. Also, the reductions in apo B and apo A-I were signicantly larger with the n23 rich diet than with the diet with a higher content of n26 fatty acids. There was a tendency to increasing concentrations of Lp(a) on both diets with closely similar changes during both periods. PAI-1 activity The activity of PAI-1 decreased to the same extent during the experimental diets from 31.3 6 20.5 (mean 6 SD) U/mL before the study to 21.3 6 13.3 (232%) after the n26rich diet and to 22.2 6 13.5 (229%) U/mL after the diet containing fatty sh.

RESULTS

Body weight and BMI A slight but signicant reduction in mean body weight and BMI was observed during both dietary periods; mean body weights on admission and at the end of the n26 and n23 diets were 76.1 6 9.5,

30
Serum tocopherol and plasma malondialdehyde concentrations

KARLSTROM ET AL

Fatty acid composition of serum cholesterol esters Adherence to the diet was conrmed by the changes proportions of the essential fatty acids in serum lipids (Table 6). Thus, the proportion of linoleic acid was considerably higher at the end of the diet rich in linoleic acid, whereas the content of eicosapentaenoic acid (20:5n23) and docosahexaenoic acid (22:6n23) was much higher after the diet containing fatty sh. Estimations of changes in fatty acid desaturase activities during the treatment periods indicated more pronounced effects of the n23enriched diet than of the n26 diet, with signicantly larger reductions in the D9 and D6 desaturases. The D5 desaturase activity, which remained unchanged after the n26 diet, increased by 58% during the diet containing the fatty sh.

No changes in lipid-adjusted serum a-tocopherol concentrations were found. The concentration was 1.41 6 0.35 (mean 6 SD) before the study, 1.54 6 0.23 after the n26 diet (+9%), and 1.44 6 0.29 (+3%) after the n23 diet. The concentration of c-tocopherol, on the other hand, increased from 0.19 6 0.08 to 0.28 6 0.06 mg/mmol (+48%) during the n26 diet and to 0.23 6 0.04 mg/mmol (+22%) during the n23 diet, signicantly more (P , 0.001) during the diet rich in n26 fatty acids. No signicant changes were found in MDA concentrations, which were 0.84 6 0.22 lmol/L before the diet and 0.70 6 0.17 (227%) after the n26 diet period and 0.79 6 0.24 (26%) after the n23 diet period.

Glucose homeostasis The fasting glucose concentrations and the area under the curve for daily blood glucose concentration decreased signicantly during the n26 compared with concentrations after the n23 diet (Table 5). No improvements in blood glucose concentrations were found after the n23 diet. The effects on fasting serum insulin concentrations were similar during the 2 diets. The changes in plasma C-peptide concentrations were not signicantly different, but had a tendency (P = 0.052) to a reduction during the n26 diet relative to the n23 diet. When the effects of the diets on the insulin area under the curve during the day were compared, a signicantly increased area under the curve was found after the n26 diet compared with that after the n23 diet (Table 5). Insulin sensitivity measured with the euglycemic hyperinsulinemic clamp technique showed that peripheral insulin sensitivity increased to the same extent during both treatment periods (Table 5).

DISCUSSION

This study compared the metabolic effects of inclusion of different proportions of n26 and n23 fatty acids in diabetic diets The foods and the content of macronutrients in the 2 diets were closely similar. A high proportion of n23 fatty acids in the n23 diet was achieved by including fatty sh in the diet. The increased proportion of linoleic acid in the n26 diet was due to inclusion of lean sh and more linoleic acid in edible fats (Table 1). To our knowledge this was the rst controlled study in which the metabolic effects of ordinary foods rich in n23 and n26 fatty acids were compared in subjects with type 2 diabetes. In accordance with earlier studies of sh-oil supplements (11, 12), an increased content of n23 fatty acids supplied as fatty sh in an ordinary diet caused a signicant reduction in serum triglyceride and VLDL lipid concentrations. This was more pronounced than after the n26 diet (Table 4). The reduction in triglyceride concentrations was compatible with reduced VLDL

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TABLE 5 Effects of 2 diabetic diets enriched with n26 or n23 fatty acids on fasting blood glucose and serum insulin concentrations, insulin sensitivity, and areas under the curve (AUCs) for blood glucose and serum insulin concentrations during the day (n = 16) Admission Fasting blood glucose (mmol/L) Change (%) Fasting serum insulin (mU/L) Change (%) Fasting C-peptide (nmol/L) Change (%) Glycated hemoglobin (%) Change (%) Insulin sensitivity (M/I)3 Change (%) Glucose AUC during the day Change (%) Insulin AUC during the day Change (%) 10.6 6 2.92 11.1 6 5.6 0.82 6 0.39 7.21 6 1.54 3.82 6 2.43 5957 6 1747 7084 6 3754 n26 Diet 9.3 6 2.0 212 13.4 6 6.0 +21 0.70 6 0.26 215 7.00 6 1.39 23 4.87 6 2.52 +27 5173 6 1444 213 8160 6 4179 +15 n23 Diet 10.2 6 2.6 24 12.0 6 5.0 +8 0.86 6 0.35 +5 6.96 6 1.23 24 4.72 6 2.58 +23 5601 6 1345 26 6743 6 3016 26 P for difference between diets1 0.009 0.303 0.052 0.710 0.451 0.029 0.030

1 Denotes differences between changes during the 2 dietary regimens. The analyses were performed by using ANCOVA appropriate for crossover design. 2 Mean 6 SD (all such values). 3 Insulin sensitivity is expressed as the insulin sensitivity index (M/I60120), which is a measure of the tissue sensitivity to insulin expressed per unit of insulin obtained by dividing the mean glucose uptake (M) by the mean insulin concentration (I) during the last 60 min of the clamp study.

FATTY FISH IN THE DIET IN TYPE 2 DIABETES

TABLE 6 Effects of 2 diabetic diets enriched with n26 or n23 fatty acids on the fatty acid composition of serum cholesterol esters (n = 16) Admission 18:2n26 (%) Change (%) 18:3n23 (%) Change (%) 20:5n23 (%) Change (%) 22:6n23 (%) Change (%) 16:1/16:0 (%)3 Change (%) 18:3n26/18:2n263 Change (%) 20:4n26/20:3n263 Change (%)
1

31

n26 Diet 55.1 6 2.3 +11 0.72 6 0.09 25 1.50 6 0.44 239 0.97 6 0.22 23 0.275 6 0.062 214 0.0127 6 0.0045 233 8.87 6 2.83 +4

n23 Diet 48.1 6 3.1 24 0.66 6 0.09 211 7.21 6 1.68 +191 1.72 6 0.29 +72 0.245 6 0.042 223 0.0085 6 0.0023 255 13.52 6 3.06 +58

P for difference between diets1 ,0.001 0.042 ,0.001 ,0.001 0.041 0.0153 ,0.001

49.8 6 3.42 0.75 6 0.14 2.48 6 1.27 1.00 6 0.34 0.320 6 0.080 0.0189 6 0.0088 8.54 6 2.19

Denotes differences between changes during the 2 dietary regimens. The analyses were performed by using ANCOVA appropriate for crossover design. 2 Mean 6 SD (all such values). 3 16:1/16:0, 18:3n26/18:2n26, and 20:4n26/20:3n26 denote the apparent activities of D9, D6, and D5 desaturase, respectively.

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triglyceride synthesis (28). The more pronounced reduction in the apo B concentration with the n23-rich diet was probably mainly due to reduced VLDL concentrations, because the effects of the diets on LDL lipid concentrations did not differ signicantly. This is in contrast with certain earlier studies indicating an increase in LDL cholesterol after sh-oil treatment in diabetes (11, 12), not seen by others (13). Fisher et al (29) studied the mechanisms behind the elevation in LDL cholesterol and LDL apo B on sh-oil supplementation and showed a decreased catabolism of intermediate-density lipoproteins in type 2 diabetes, which resulted in a larger mass of LDL apo B. On average, HDL cholesterol is not affected by n23 supplementation in type 2 diabetes (1113), and apo A-I usually remains unchanged (13, 26, 30). Whereas HDL concentrations were moderately reduced with both diets, apo A-I decreased more with the n23 diet (Table 4). The reduction in HDL may have related to a reduction in the total fat content of the diets, compared with before treatment. The increase in Lp(a) may have been due to the increased content of unsaturated fat (31). However, a moderate dose of sh oil, relative to the same amount of sunower oil, reduced Lp(a) in a group of men with type 2 diabetes (30). The reduction in fasting blood glucose and in the glucose area under the curve during the day was signicantly greater with the n26 than with the n23 diet (Table 5). An earlier study showed impairment of glycemic control after a diet rich in n23 fatty acids in type 2 diabetes (32). The data on effects of sh-oil supplementation on glycemia in diabetes are controversial, but indicate that the increase in fasting blood glucose and of Hb A1c, if any, is moderate (1113). In this study, no differences in Hb A1c were found between the diets, which may have been due to the rather short duration of the treatment periods. Both diets showed similar improvements in peripheral insulin sensitivity. Most earlier studies showed no specic effect of the addition of moderate amounts of n23 fatty acids to the diet on

insulin sensitivity in type 2 diabetes (33). Studies by Mostad et al (34), on the other hand, showed that a high intake of sh oil in type 2 diabetes increases blood glucose and decreases insulin sensitivity in a time-dependent matter. Although the area under the glucose curve was higher during the day with the n23 diet, the area under the insulin curve was lower. This apparent paradox, ie, a lower insulin response to blood glucose after supplementation of diabetic diets with n23 fatty acids, as discussed earlier (10), may indicate an effect on the glucose-mediated insulin release from the pancreas or an enhanced uptake of insulin by the liver. Whether a high intake of oily sh during long periods of time may compromise insulin secretion and increase the risk of developing type 2 diabetes (14), or, in contrast, the higher insulin secretion with a diet rich in linoleic acid might eventually increase the risk of b cell failure, remains a matter of speculation. The dietary n26:n23 ratio varied from 6.1 in the linoleic acid rich diet to 2.5 in the diet including more fatty sh. In healthy elderly subjects, no difference in fasting plasma glucose, insulin, or the HOMA index was found when diets with ratios of 11.4 to 2.4 were found between n26 and n23 polyunsaturated fatty acids (35). The improved brinolytic activity with both diets agrees with earlier ndings after the switch from a habitual, relatively fat-rich diet to a prudent diet (36) with a higher proportion of carbohydrates and dietary ber. The addition of n23 fatty acids to a diabetic diet, while the proportions of other nutrients were unchanged, increased the PAI-1 activity (15). Such an increase has not been seen in healthy subjects (16, 17). In the current study, the changes in PAI-1 did not differ between the diets, which indicated that polyunsaturated fatty acids of the n26 series may have effects on PAI-1 similar to those of the long-chain n23 fatty acids. Similar conclusions were drawn from in vitro studies (37) and in a cross-sectional study of elderly men (38). Adherence to the prescribed diet was conrmed by analysis of the fatty acid composition of the plasma lipid esters (Table 6).

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and apolipoprotein concentrations and insulin sensitivity in noninsulin-dependent diabetic patients. Am J Clin Nutr 1989;49:44856. Houtsmuller AJ, van Hal-Ferwerda J, Zahn KJ, Henkes KE. Favourable inuences of linoleic acid on the progression of diabetic micro- and macroangiopathy. Nutr Metab 1980;24(suppl 1):10518. Dimitriadis E, Grifn M, Owens D, Johnson A, Collins P, Tomkin GH. Oxidation of low-density lipoprotein in NIDDM: its relationship to fatty acid composition. Diabetologia 1995;38:13006. Malasanos TH, Stacpoole PW. Biological effects of w-3 fatty acids in diabetes mellitus. Diabetes Care 1991;14:116079. Vessby B. Dietary supplementation with n23 polyunsaturated fatty acids in type 2 diabeteseffects on glucose homeostasis. Ann N Y Acad Sci 1993;683:2449. Friedberg CE, Janssen MJFM, Heine RJ, Grobbee DE. Fish oil and glycemic control in diabetes. A meta-analysis. Diabetes Care 1998;21: 494500. Montori VM, Farmer A, Wollan PC, Dinneen SF. Fish oil supplementation in type 2 diabetes. A quantitative systematic review. Diabetes Care 2000;23:140715. MacLean CH, Mojica WA, Morton SC et al. Effects of omega-3 fatty acids on lipids and glycemic control in type II diabetes and the metabolic syndrome and on inammatory bowel disease, rheumatoid arthritis, renal disease, systemic lupus erythematosus, and osteoporosis. Evid Rep Technol Assess (Summ) 2004;89:14. Kaushik M, Mozaffarian D, Spiegelman D, Manson JE, Willett WC, Hu FB. Long-chain omega-3 fatty acids, sh intake, and the risk of type 2 diabetes mellitus. Am J Clin Nutr 2009;90:61320. Boberg M, Pollare T, Siegbahn A, Vessby B. Supplementation with n23 fatty acids reduces triglycerides but increases PAI-1 in noninsulin-dependent diabetes mellitus. Eur J Clin Invest 1992;22:64550. Hansen J, Grimsgaard S, Nordoy A, Bonaa KH. Dietary supplementation with highly puried eicosapentaenoic acid and docosahexaenoic acid does not inuence PAI-1 activity. Thromb Res 2000;98:12332. Sanders TA, Lewis F, Slaughter S, et al. Effect of varying the ratio of n26 to n23 fatty acids by increasing the dietary intake of alphalinolenic acid, eicosapentaenoic and docosahexaenoic acid, or both, on brinogen and clotting factors VII and XII in persons aged 45-70 Y: the OPTILIP study. Am J Clin Nutr 2006;84:51322. National Food Administration. PC-Kost database. Uppsala, Sweden: National Food Administration, 1996. Havel RJ, Eder HA, Bragdon JH. The determination and chemical composition ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:134553. Seigler L, Wu WT. Separation of serum high-density lipoprotein for cholesterol determination: ultracentrifugation vs precipitation with sodium phosphotungstate and magnesium chloride. Clin Chem 1981;27: 83441. Boberg M, Croon LB, Gustafsson IB, Vessby B. Platelet fatty acid composition in relation to fatty acid composition in plasma and to serum lipoprotein lipids in healthy subjects with special reference to the linoleic acid pathway. Clin Sci 1985;68:5817. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979;237: E21423. Eriksson E, Ranby M, Gyzander E, Risberg B. Determination of plasminogen activator inhibitor in plasma using t-PA and a chromogenic single-point poly-D-lysine stimulated assay. Thromb Res 1988;50:91101. Ohrvall M, Tengblad S, Vessby B. Lower tocopherol serum levels in subjects with abdominal obesity. J Intern Med 1993;234:5360. Thurnham DI, Davies JA, Crump BJ, Situnayake RD, Davis M. The use of different lipids to express serum tocopherol:lipid ratios for the measurement of vitamin E status. Ann Clin Biochem 1986;23:51420. Ohrvall M, Tengblad S, Ekstrand B, Siegbahn A, Vessby B. Malondialdehyde concentration in plasma is inversely correlated to the proportion of linoleic acid in serum lipoprotein lipids. Atherosclerosis 1994;108:10310. Jones B, Kenward MG. Design and analysis of cross-over trials. 2nd ed. London, United Kingdom: Chapman and Hall, 2003. Nestel PJ, Connor WE, Reardon MF, Connor S, Wong S, Boston R. Suppression by diets rich in sh oil of very low density lipoprotein production in man. J Clin Invest 1984;74:829. Fisher WR, Zech LA, Stacpoole PW. Apolipoprotein B metabolism in hypertriglyceridemic diabetic patients administered either a sh oil- or vegetable oil-enriched diet. J Lipid Res 1998;39:388401.

Whereas the proportion of linoleic acid increased during the n26 diet, the long-chain polyunsaturated n23 fatty acids increased considerably when fatty sh was included in the diet. A decrease in plasma arachidonic acid after the n23 (216%), compared with after the n26 (+3%), diet was seen in plasma phospholipids (P = 0.0001), whereas no difference in the cholesterol esters was observed. Interestingly, a signicant (P = 0.012) decrease in palmitoleic acid in the serum phospholipids was found after the n23 diet (26%), compared with after the n26 rich diet (+7%), despite a slightly higher intake of palmitoleic acid during the n23 diet period (Table 3). This nding indicates a reduction in D9 desaturase (stearoyl desaturase) activity (39). A comparison of the effects on desaturase activities during the 2 test periods (Table 6) showed that the n23 diet had more pronounced effects, whether studied in cholesterol esters or phospholipids (data not shown). A pronounced increase in D5 desaturase activity was recorded only during the n23 rich diet. High D9 and low D5 desaturase activity have been associated with the development of the metabolic syndrome (40) and with total and cardiovascular mortality (41). The data regarding a- and c-tocopherol and MDA are reassuring and do not support the suggestions that an increased content of polyunsaturated fat could increase the tendency to lipid peroxidation (8). A recent study showed that supplementation with n23 fatty acids in type 2 diabetes reduced MDA concentrations, whereas antioxidant enzymes increased or were unchanged (42). In type 2 diabetes patients, no indication of an increased concentration of lipid oxidation products was found after a diet rich in linoleic acid (43). In summary, the current study showed that metabolic control improved considerably with both diets. The risk related to higher blood glucose concentrations with the n23enriched diet were probably counteracted by the positive effects of lipoprotein concentrations. An increase in long-chain n23 fatty acids from fatty sh, and in n26 fatty acids from linoleic acid, may be recommended as a part of the dietary advice given to persons with type 2 diabetes.
The authors responsibilities were as followsBOHV and BEK: designed the research and had primary responsibility for the nal content; AEJ, BEK, and BOHV: conducted the research; LB, BOHV, and BEK: analyzed the data; and LGB: performed the statistical analyses. All authors contributed to writing the manuscript and read and approved the manuscript. None of the authors declared any conicts of interest related to this manuscript

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