Substituting dietary linoleic acid with α-linolenic acid improves insulin sensitivity in sucrose fed rats

Biochimica et Biophysica Acta 1733 (2005) 67 – 75 http://www.elsevier.com/locate/bba

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Substituting dietary linoleic acid with a-linolenic acid improves insulin sensitivity in sucrose fed rats Ghafoorunissa*, Ahamed Ibrahim, Saravanan Natarajan Department of Biochemistry, National Institute of Nutrition, Indian Council of Medical Research, Jamai Osmania P.O., Hyderabad-500 007, A.P., India Received 31 August 2004; received in revised form 27 November 2004; accepted 1 December 2004 Available online 29 December 2004

Abstract This study describes the effect of substituting dietary linoleic acid (18:2 n-6) with a-linolenic acid (18:3 n-3) on sucrose-induced insulin resistance (IR). Wistar NIN male weanling rats were fed casein based diet containing 22 energy percent (en%) fat with ~6, 9 and 7 en% saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) respectively for 3 months. IR was induced by replacing starch (ST) with sucrose (SU). Blends of groundnut, palmolein, and linseed oil in different proportions furnished the following levels of 18:3 n-3 (g/100 g diet) and 18:2 n-6/18:3 n-3 ratios respectively: ST-220 (0.014, 220), SU-220 (0.014, 220), SU-50 (0.06, 50), SU-10 (0.27, 10) and SU-2 (1.1, 2). The results showed IR in the sucrose fed group (SU-220) as evidenced by increase in fasting plasma insulin and area under the curve (AUC) of insulin in response to oral glucose load. In SU-220, the increase in adipocyte plasma membrane cholesterol/phospholipid ratio was associated with a decrease in fluidity, insulin stimulated glucose transport, antilipolytic effect of insulin and increase in basal and norepinephrine stimulated lipolysis in adipocytes. In SU-50, sucrose induced alterations in adipocyte lipolysis and antilipolysis were normalized. However, in SU-2, partial corrections in plasma insulin, AUC of insulin and adipocyte insulin stimulated glucose transport were observed. Further, plasma triglycerides and cholesterol decreased in SU-2. In diaphragm phospholipids, the observed dose dependent increase in long chain (LC) n-3 PUFA was associated with a decrease in LC-n-6 PUFA but insulin stimulated glucose transport increased only in SU-2. Thus, this study shows that the substitution of one-third of dietary 18:2 n-6 with 18:3 n-3 (SU-2) results in lowered blood lipid levels and increases peripheral insulin sensitivity, possibly due to the resulting high LCn-3 PUFA levels in target tissues of insulin action. These findings suggest a role for 18:3 n-3 in the prevention of insulin resistant states. The current recommendation to increase 18:3 n-3 intake for reducing cardiovascular risk may also be beneficial for preventing IR in humans. D 2004 Elsevier B.V. All rights reserved. Keywords: Adipose tissue; Dietary sucrose; Diaphragm; Fatty acid composition; Glucose transport; Insulin resistance; Linoleic acid; a-Linolenic acid; n-6/n-3 PUFA ratio

1. Introduction IR is a common metabolic abnormality that is implicated in the development of obesity, type 2 diabetes and coronary heart disease [1,2]. The development of IR is linked to both genetic and environmental factors. The amount of dietary fat and its fatty acid composition are important environmental determinants of IR [3] and atherothrombogenic risk [4]. * Corresponding author. Tel.: +91 40 27001866; fax: +91 40 27019074. E-mail addresses: [email protected], [email protected] (Ghafoorunissa). 1388-1981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2004.12.003

While dietary SFA aggravate, PUFA of both n-6 and n-3 series have beneficial effects on key factors of atherothrombogenic risk. However, the LC n-3 PUFA have greater antiatherothrombogenic effects as compared to LC n-6 PUFA [4,5]. The PUFA composition of membranes depend on dietary fatty acids and delicate competitive interactions in the metabolism of 18:2 n-6 and 18:3 n-3 to LC n-6 PUFA and LC n-3 PUFA respectively. Therefore, the relative and absolute amounts of 18:2 n-6 and 18:3 n-3 in the diet affects the membrane lipid composition, which in turn modulates a wide range of eicosanoid and non-eicosanoid mediated effects on various metabolic processes. In view of the above, dietary recommendations should make a distinction

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between 18:2 n-6 and 18:3 n-3, which are obtained from plant foods and vegetable oils, and their respective LC PUFA, which have higher biological effects and are directly obtained from animal foods. Recent studies indicate that compared to SFA and fats rich in 18:2 n-6, fish oils (high LCn-3 PUFA) and perilla oil (high 18:3 n-3) reduce white fat pad mass [6]. Further, diets containing high SFA or n-6 PUFA induce IR [7], but LC n-3 PUFA present in fish oils prevent IR induced by diets containing high fat [8,9] or high sucrose [10–13]. The present study was designed to investigate the effects of substituting different levels of 18:2 n-6 with 18:3 n-3 without changing total PUFA on insulin sensitivity in adipose tissue and diaphragm in sucrose-induced IR rats.

2. Materials and methods 2.1. Materials [1,2-3H]-2-deoxy glucose, HEPES, insulin, phloretin, norepinephrine and fatty acid free bovine serum albumin were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. Collagenase was obtained from Gibco (Invitrogen, U.S.A). Fatty acid standards were obtained from NuChek, MN, U.S.A. 2.2. Animals and diets All the procedures involved in animal experiments were approved by institutional animal ethical committee (National Institute of Nutrition), Hyderabad, India. Fifty six male Wistar NIN weanling rats were obtained from the animal house facility of National Institute of Nutrition (Hyderabad, India). The animals were divided equally into four groups, housed individually in polypropylene cages at 21F1 8C with a 12 h light and dark cycle and fed different experimental diets for 3 months. The diet composition (g/100 g diet) was as follows: carbohydrate 54.5, casein 25, fat 10, cellulose 5, salt mixture 4.0, vitamin mixture 1.0, l-cystine 0.3, choline chloride 0.2. The salt and vitamin mixtures were prepared according to the AIN-93 [14]. Insulin resistance was induced by replacing starch (ST) with sucrose (SU). Vegetable oil blends were formulated by mixing groundnut, palmolein and linseed oils and used as a source of dietary fat. The fatty acid composition of the dietary fats were determined by gas chromatography (Table 1) as described earlier [15]. The total SFA, MUFA and PUFA (18:2 n-6+18:3 n-3) was similar in all the groups. The 18:3 n-3 (en%) and the ratios of 18:2 n-6/18:3 n-3 in various groups were: ST and SU-0.03, 220, SU-0.6, 10 and SU-2.3, 2 respectively. Animals had free access to food and water. The food intake of individual animals was recorded daily and the body weight was recorded once in a week. In a separate experiment, forty male weanling WNIN rats were divided equally into five groups. In this experiment,

Table 1 Fatty acid composition of dietary fats (g/100g diet) Fatty acids

ST-220

SU-220

SU-50

SU-10

SU-2

Sum SFA Sum MUFA Sum PUFA 18:2 n-6 18:3 n-3 PUFA/SFA 18:2 n-6/18:3 n-3

3.0 3.9 3.1 3.1 0.014 1.0 220

3.0 3.9 3.1 3.1 0.014 1.0 220

3.0 3.9 3.1 3.0 0.06 1.0 50

3.0 3.9 3.1 2.8 0.27 1.0 10

2.9 3.8 3.3 2.2 1.1 1.1 2

the adipocyte number, size, lipolytic activity of adipocytes and glucose transport in adipocytes and diaphragm were studied. The experimental design was same as the earlier experiment except that an additional group which provided 0.06 g (0.13 en%) 18:3 n-3 and 18:2 n-6/18:3 n-3 ratio 50 was included. Blood was collected from the retro-orbital sinus in EDTA tubes after overnight fasting and plasma was separated and stored at
70 8C for the analysis of glucose, insulin, triglycerides and cholesterol. The animals were sacrificed by CO2 asphyxia. Epididymal fat pads and diaphragms were removed. A portion of the epididymal fat pad was put immediately in Bouin’s fixative and cell size was measured by the procedure of Ashwell and Priest [16]. 2.3. Analysis of glucose, insulin, cholesterol and triglycerides in plasma Plasma glucose, triglycerides and cholesterol were estimated using enzymatic kit method (Biosystems, Spain) and insulin was determined using an RIA kit (BRIT, India). Oral glucose tolerance test (OGTT) was done 1 week before the termination of the experiment. Rats were administered d-glucose (3 g/kg body weight) after fasting for 18 h through gastric cavage and blood samples were collected from retro-orbital sinus in EDTA tubes at 0, 60 and 120 min. Plasma was separated and stored at
70 8C until analysis was performed. The area under the curve for glucose and insulin was calculated by trapezoidal rule. 2.4. Adipocyte isolation Adipocytes were isolated from epididymal fat pad by collagenase digestion by the Rodbell method [17]. Briefly, the fat pads were minced with scissors and placed in a plastic tube containing Krebs Ringer HEPES buffer (KRH, pH 7.4, 130 mM NaCl, 1.4 mM MgSO4, 5.2 mM KCl, 1.4 mM CaCl2, 1 mM KH2PO4, 10 mM HEPES) with 1% bovine serum albumin (fraction V, essentially fatty acid free), 2 mM pyruvate and 1 mg collagenase/g adipose tissue. Following collagenase digestion at 37 8C in shaking water bath for 1 h, the cells were washed three times in fresh buffer and allowed to separate from the infranatant by flotation.

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2.5. Preparation of adipocyte plasma membrane and measurement of fluidity

the cell layer was removed and the glycerol content was estimated.

Plasma membranes were prepared from adipocytes by density gradient centrifugation using percoll gradient [18]. Adipocyte membrane fluidity was estimated by steady state fluorescence polarization with the fluorescence probe 1,6 diphenyl 1,3,5-hexatriene (DPH). Results are expressed as fluorescence anisotropy [19].

2.8. Adipocyte and diaphragm glucose transport

2.6. Lipid analysis Adipocyte plasma membrane and skeletal muscle (diaphragm) lipids were extracted by the Folch method [20]. Neutral lipids were separated from phospholipids by thin layer chromatography on silica gel G with hexane/diethyl ether/acetic acid (80:20:1 v/v). The fatty acid profile of the membrane phospholipids was determined after methylation [15]. Fatty acid methyl esters were analyzed by gas chromatography using a SP-2330 capillary column (30 m0.32 mm id, Supelco, USA). Individual fatty acids were identified using authentic standards. Heptadecanoic acid was used as an internal standard. Membrane total cholesterol and phospholipids were determined by the Zlatkis [21] and Bartlett [22] methods respectively. Diaphragm triglycerides were quantitated using an enzymatic assay kit (Biosystems, Spain). Membrane protein was estimated by Lowry’s method [23]. 2.7. Adipocyte lipolysis and antilipolysis Lipolysis was assayed by measuring glycerol release into the incubation medium [24]. Adipocytes (~2105 cells/ml) were incubated at 37 8C for 2 h in KRH buffer (pH 7.4) in a final volume of 0.5 ml. Basal lipolysis was measured without the addition of hormone. Hormone stimulated lipolysis was assessed by adding various concentrations of norepinephrine. The antilipolytic effect of insulin on adipocytes was assessed by incubating adipocytes with 1 AM norepinephrine (maximum stimulating concentration) and various concentrations of insulin [25]. After 2 h of incubation, the cells were separated from the medium by brief centrifugation. The infranatant below

Basal and insulin stimulated glucose transport was measured as described [26]. Adipocytes (~2105 cells/ml) were pre-incubated with and without various concentrations of porcine insulin at 37 8C for 45 min. The cells were incubated with 2-[1,2-3H] deoxyglucose at a concentration of 0.1 mM in KRH buffer (pH 7.4). The assay was terminated at the end of 3 min by transferring the assay mixture to a microcentrifuge tube containing silicone oil. The tubes were centrifuged at 4000 rpm for 3 min. The top layer containing adipocytes were transferred to liquid scintillation vial and radioactivity associated with adipocytes was measured in liquid scintillation counter. All data were corrected for non-specific transport by measuring glucose transport in the presence of 0.3 mM phloretin. Glucose transport in the diaphragm was determined by the incubation of hemi-diaphragms with 400 nM of insulin [27]. Basal glucose transport was measured in the absence of insulin. After 15 min of pre-incubation, hemidiaphragms were incubated for 45 min in the presence or absence of insulin. Subsequently, the hemidiaphragms were incubated at 37 8C in KRH buffer containing 0.1 mM 2-[1,2-3H]deoxyglucose for 30 min. After the incubation, the hemidiaphragms were washed with ice-cold Tris buffer (0.1 M Tris HCl; 0.9% NaCl, pH 7.4) and released from the rib cage, blotted on a filter paper and weighed. The muscle tissue was homogenized in 20% TCA and centrifuged at 2000 rpm for 5 min. The radioactivity in the supernatant was determined by liquid scintillation counter (LKB). 2.9. Statistical analysis Statistical analysis was done using SPSS statistical programme. All values are presented as meansFS.E. Oneway ANOVA was used to test significant differences between the dietary groups. Post hoc comparisons were performed using LSD test. A value of Pb0.05 was considered statistically significant.

Table 2 Effect of dietary a-linolenic acid on food intake, body weight gain and adipose depot weights in rats (90 days) Parameters Food intake (g/day) Body weight gain (g) Epididymal fat weight (g/100 g body weight) Retroperitoneal fat weight (g/100 body weight) Adipocyte number (106/g tissue)* Adipocyte size (Am)*

ST-220

SU-220 a

12.2F0.5 249F4.7a 1.2F0.5a 2.3F0.19a 5.0F0.4a 88F5.2a

SU-50 a

12.4F0.6 273F6.8b 1.4F0.08b 2.9F0.14a 3.6F0.3a 86F2.1a

– – – 2.8F0.18a,b 5.0F0.4a 93F3.9a

SU-10

SU-2 a

13.1F0.4 283F9.1b 1.5F0.1b 2.7F0.19a,b 4.1F0.3a 91F5.3a

12.8F0.6a 289F8.2b 1.3F0.04a,b 2.8F0.18a,b 4.1F0.5a 90F3.3a

Values represent meansFS.E. of 14 rats. a,b Values in a row not sharing a common superscript differ significantly at Pb0.05. * These parameters were analyzed in a separate set of experimental animals (n=8) with a similar dietary strategy except the inclusion of SU-50 diet.

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3. Results

Glucose#

190 170

3.1. Characteristics of animals mg/dl

150

Food intake, body weight gain and adipose tissue weights of rats fed different diets are shown in Table 2. Feeding sucrose increased body weight gain and adipose tissue weight. Substituting 18:2 n-6 with 18:3 n-3 did not alter body weight gain and adipose tissue weights. Neither sucrose feeding nor dietary 18:3 n-3 altered adipocyte number and size (Table 2). 3.2. Plasma glucose, insulin and lipids

AUC Glucose*

3.3. Adipocyte plasma membrane lipid composition and fluidity In SU-220, plasma membrane cholesterol/phospholipid (C/P) molar ratio was higher than in ST-220 (Table 4). SU10 and SU-2 had higher levels of phospholipid long chain (n-3) PUFA. Membrane fluidity was low (higher fluorescence anisotropy) in SU-220 as compared to ST-220.

Table 3 Effect of dietary a-linolenic acid on plasma glucose, insulin and lipids in rats Parameters

Sampling ST-220 day

Glucose (mg/dl) Insulin (AU/ml) Cholesterol (mg/dl) Triglycerides (mg/dl)

45 90 45 90 45 90 45 90

SU-220

SU-10

90F2.2a 96F3.2a 92F3.6a 86F3.0a 89F3.1a 86F2.4a a b 71F6.4 93F9.8 90F8.0b 63F4.0a 79F6.4b 76F5.4a,b a a 97F3.0 94F3.3 95F2.5a 90F1.9a,b 92F3.4a 93F3.8a a b 112F9.3 138F11.3 134F13.5b 75F7.0a,c 93F9.0a,b 98F10b

SU-2 91F4.1a 87F2.2a 82F5.2a 63F4.2a 76F3.5b 82F3.2b 95F8.5a 68F6c

Values represent meansFS.E. of 14 rats. a,b,c Values in a row not sharing a common superscript differ significantly at Pb0.05.

110 90 70 50 0hr

µU/ml

The data on plasma parameters are shown in Table 3. At the end of both 45 and 90 days of feeding, the sucrose fed group (SU-220) showed significantly higher plasma insulin levels as compared to ST-220. Substituting 2.3 en% 18:2 n-6 with 18:3 n-3 (SU-2) decreased fasting plasma insulin to the levels comparable to starch fed groups (ST-220). The data on OGTT showed higher AUC of insulin in SU-220 group with no change in AUC of glucose (Fig. 1). Compared to the SU-220 group, the AUC of insulin was low in both the SU-10 and SU-2 groups but statistical significance was missed in the SU-2 group. Sucrose fed groups (SU220 and SU10) had higher triglycerides as compared to the starch fed group (ST220). The SU-2 group had lower levels of triglycerides and cholesterol as compared to other sucrose fed groups (SU-220 and SU-10).

130

130 120 110 100 90 80 70 60 50

1hr

2hr

ST-220

SU-220

SU-10

298 ± 31a

291 ± 17a

266 ± 8a 282 ± 8.9a

Insulin #

b b

SU-2

b

ab

b b

a a

0hr ST-220 AUC Insulin* 143 ± 9.6a

1hr SU-220 203 ± 15b

2hr SU-10 170 ± 13.2a,c

SU-2 184 ± 7.0b,c

Fig. 1. Plasma glucose and insulin levels alter oral glucose load in rats fed diets with different n-6/n-3 ratios. Values represent meansFS.E. of 14 rats. a,b,c Values in a row not sharing a common superscript differ significantly at Pb0.05. #At a particular time point values with different letters differ significantly at Pb0.05. -w- ST-220; -5- SU-220; -4- SU-10; -o- SU-2.

Substituting 18:2 n-6 with 18:3 n-3 did not prevent the sucrose-induced alteration in membrane fluidity. 3.4. Adipocyte lipolysis, insulin mediated antilipolysis and glucose transport In SU-220 group, both basal and norepinephrine mediated lipolysis increased (Fig. 2a) and insulin stimulated antilipolysis (Fig. 2b) decreased as compared to ST-220. Compared to SU-220, both basal and norepinephrine mediated lipolysis decreased, and basal and insulin mediated antilipolysis increased to the same extent in SU-50, SU10 and SU-2 groups. The data on adipocyte glucose transport showed that basal transport was similar in all the groups (Fig. 3). The dose response curve showed a decrease in glucose transport in the sucrose fed groups (SU-220, SU-50 and SU-10) as compared to ST-220. The insulin required for half maximal stimulation of glucose transport (EC-50) was not altered (Table 5). Sucrose feeding (SU-220, SU-50 and SU-10) decreased maximal glucose uptake. In the SU-2 group, the sucrose-induced inhibition of glucose transport and decrease in maximal glucose uptake were partially corrected.

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ST-220

Cholesterol 134F6a (nmol/mg protein) Phospholipid 224F8a (nmol/mg protein) Cholesterol/ 0.59F0.03a Phospholipid

SU-220

SU-10

SU-2

169F10a,b

180F14b

147F16a,b

244F13a

238F15a

243F29a

0.69F0.02b,c 0.75F0.02b

Phospholipid fatty acid composition (nmol%) Sum SFA 38.3F1.0a 38.0F0.8a a Sum MUFA 25.5F1.1 26.5F1.5a Sum PUFA 36.2F0.7a 35.0F1.3a a,b 18:2 n-6 18.6F0.2 17.5F0.7a 20:4 n-6 17.5F0.8a 16.7F0.1a 22:5 n-3 ND ND 22:6 n-3 ND 1.3F0.12a a Fluorescence 0.13F0.006 0.16F0.004b anisotropy

37.0F0.7a 25.7F1.1a 37.3F1.4a,b 18.9F0.7a,b 16.5F1.2a 0.62F0.1a 1.3F0.01a 0.16F0.004b

0.61F0.05a,c

37.0F0.6a 23.3F0.7a 40.2F0.8b 20.0F1.2b 17.3F0.8a 0.83F0.15a 2.1F0.3a 0.15F0.01a,b

Values represent meansFS.E. of 7 rats. a,b,c Values in a row not sharing a common superscript differ significantly at Pb0.05.

3.5. Diaphragm muscle lipid composition and glucose transport The data presented in Table 6 showed that the total SFA and total MUFA were similar in all the groups. Rats fed different levels of 18:3 n-3 showed a dose dependent increase in LCn-3 PUFA with concomitant decrease in 20:4 n-6. Neither sucrose nor 18:3 (n-3) affected the diaphragm triglycerides content. Insulin stimulated glucose transport was higher in SU-2 as compared to other groups (Fig. 4).

4. Discussion Sucrose diet induced IR in comparison to starch diet (ST) as evidenced by the increase in the fasting levels of plasma insulin, triglycerides and increase in AUC of insulin after oral glucose load without changes in either fasting plasma glucose or the AUC of glucose (Table 3 and Fig. 1). The magnitude of increase in fasting levels of plasma insulin and triglycerides were similar at the end of 45 and 90 days of feeding sucrose diet (Table 3). Del Parto et al. [28] showed that hyperinsulinemia induced either by exogenous insulin infusion or by endogenous insulin secretion induced insulin resistance. Studies in both humans and rats showed inverse correlation between insulin sensitivity and fasting plasma triglycerides [29,30]. Further, the increase in plasma triglycerides decrease insulin receptors [31] and impair insulin signal transduction [32].

a. nmoles glycerol/106 cells/1hr

Parameters

LCn-3 PUFA has been shown to prevent IR in high sucrose [11–13] and high fat [9] fed rats. In adult rats fed high fat diets, the substitution of 18:2 n-6 with 18:3 n-3 (n-6/n-3 ratio ~7) over a period of 4 weeks did not prevent IR [9]. In the present study, in weanling rats over a period of 12 weeks, although dietary n-6/n-3 ratios of 50 or 10 had no effect, a ratio of 2 (SU-2) partially corrected sucroseinduced IR. Studies in animals and humans have documented dose dependent effects of LC n-3 PUFA on VLDL production [33–36]. Studies in rats showed that 18:3 n-3 has hypotriglyceridemic and hypocholesterolemic effects [37– 40]; maximum effects were observed with a 18:2 n-6/18:3 n-3 ratio of 0.33 [38]. The hypolipidemic effects of 18:3 n-3 may be due to an increase in LC n-3 PUFA in membrane

b

500 450 400 350 300 250 200 150 100 50 0

b

b a a,c a,c

b

a a,c

a a,c a,c a,c

a,c a,c

a,c

a a a,a

0.1

0.3

1.0

Norepinephrine (nM)

b. 600 nmoles glycerol/106 cells/hr

Table 4 Effect of dietary a-linolenic acid on adipocyte plasma membrane lipid composition and fluidity in rats

71

500 400 a,c

300 200

b

a a,c a,c

b

b

a,c

b b

a,c a,c ca a a,c

a,c a

a,c a,c

a,c a

a,c a,c

a,c a a,c a,c

100 0

0.5

1.0 Insulin (nM)

10.0

100

Fig. 2. (a) Dose response curves of norepinephrine stimulated lipolysis in isolated epididymal adipocytes from rats fed different diets. Adipocytes were incubated at 37 8C for 2 h in the absence and presence of indicated concentrations of norepinephrine. Glycerol released by adipocytes into the incubation medium was taken as index of lipolysis. At a particular concentration of norepinephrine the values with different letter differ statistically significantly at Pb0.05. Values represent meansFS.E. of 8 rats. -w- ST-220; -5- SU-220; -4- SU-50; -X- SU-10; -o- SU-2. (b) Antilipolytic effect of insulin on norepinephrine induced lipolysis in epididymal adipocytes isolated from rats fed different diets. Adipocytes were incubated at 37 8C for 2 h with norepinephrine (1 AM) in the presence of indicated concentrations of insulin. Glycerol released by adipocytes into the incubation medium was taken as an index of lipolysis. At a particular concentration of insulin the values with different letter differ significantly at Pb0.05. Values represent meansFS.E. of 8 rats. -w- ST-220; -5- SU-220; -4- SU-50; -X- SU-10; -o- SU-2.

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pmoles/106cells/3min

72

200 180 160 140 120 100 80 60 40 20 0

Table 6 Effect of dietary a-linolenic acid on diaphragm lipid composition in rats

a

a

Parameters a b b b b

a

a

a

b b

b b

Triglycerides (mg/mg protein)

b b

b

0.10 Insulin (nM)

SU-220 a

SU-10 a

0.13F0.01 0.15F0.02

SU-2 a

0.15F0.02

0.16F0.02a

40.5F0.6c,b 8.2F0.3a 23.1F0.3cb 1.5F0.3a 15.2F0.4b 1.4F0.1a 0.6F0.02b 17.2F0.6b 0.2F0.05a 0.4F0.06a 2.2F0.54b 8.6F0.30b 10.2F0.4b

40.3F0.5b 8.3F0.2a 23.1F0.3c,b 1.5F0.2a 12.7F0.4c 1.2F0.3a 0.5F0.02c 14.7F0.9c 0.6F0.23a 0.6F0.12a 3.2F0.56c 10.2F0.43c 12.8F0.6c

b

b

a aa

0.05

ST-220

c

c

1.0

10

Fig. 3. Dose response curves of insulin stimulated glucose uptake in isolated epididymal adipocytes from rats fed different diets. Adipocytes were pre incubated at 37 8C for 45 min in the absence and presence of indicated concentrations of insulin. Glucose uptake was measured as described in Materials and methods. At a particular concentration of insulin the values with different letter significantly different ( Pb0.05). Values represent meansFS.E. of 8 rats. -w- ST-220; -5- SU-220; -4- SU-50; -XSU-10; -o- SU-2.

lipids [41–44] and possibly to PPAR a activation and downregulation of SREBP-1 [45,46]. Short term studies on high sucrose diets induced IR without increase in visceral adiposity [47,48]. However, in long term feeding studies, IR was associated with an increase in visceral adiposity [49]. In the present study, IR induced in sucrose fed rats was associated with increase in both body weight and visceral adiposity but adipocyte number and size were not altered (Table 2). The substitution of one-third of 18:2 n-6 with 18:3 n-3 did not reduce either body weight or visceral fat weight (Table 2). However, LC n-3 PUFA supplementation has been shown to reduce both body weight gain and visceral adiposity [50]. The observation that sucrose feeding increased both basal and norepinephrine stimulated lipolysis and decreased antilipolytic action of insulin in adipocytes is in agreement with other studies [51,52]. The aforementioned sucrose induced alterations in the lipolysis and antilipolytic action of insulin were reversed in all the three groups receiving higher dietary 18:3 n-3 (SU-50, SU-10 and SU-2). These finding suggest that ~0.06 g 18:3 n-3 (0.13 en%, SU-50) may be adequate to prevent the sucrose-induced changes in lipid mobilization and deposition in adipose tissue. Membrane fluidity influences several cellular functions including hormone-

Phospholipid fatty acid composition (nmol%) Sum SFA 42.4F0.8a 42.1F0.6a,c Sum MUFA 8.2F0.4a 8.9F0.3a a 18:2 n-6 24.7F0.7 23.9F0.4a,c 20:3 n-6 1.5F0.3a 1.2F0.2a a 20:4 n-6 18.6F0.3 19.1F0.3a 22:5 n-6 1.1F0.1a 0.9F0.1a a 20:4 n-6/18:2 n-6 0.7F0.02 0.8F0.01a Sum LC-n-6 PUFA 20.6F0.5a 20.8F0.4a 18:3 n-3 ND ND 20:5 n-3 ND ND 22:5 n-3 0.6F0.04a 0.4F0.03a 22:6 n-3 2.0F0.06a 1.7F0.05a Sum LC n-3 PUFA 2.5F0.07a 2.0F0.07a

Values represent meansFS.E. of 14 rats. a,b,c Values in a row not sharing a common superscript differ significantly at Pb0.05. ND—non-detectable. Sum LCn-6 PUFA=20:3 n-6, 20:4 n-6, 22:5 n-6. Sum LCn-3 PUFA=20:5 n-3, 22:5 n-3, 22:6 n-3.

mediated responsiveness. The fatty acid composition of the phospholipid bilayer, cholesterol/phospholipid molar ratio, size and shape of phospholipid head groups determine membrane fluidity [53–55]. The increase in adipocyte plasma membrane cholesterol/phospholipid molar ratio and decrease in fluidity could have contributed to the observed decrease in glucose transport in SU-220. A recent study demonstrated that sucroseinduced impairment of adipocyte glucose transport can be prevented by LC n-3 PUFA which in turn was associated with elevated GLUT-4 protein and mRNA levels [11]. The data presented in Table 4 shows that high levels of LCn-3 PUFA in adipocyte plasma membrane phospholipids as observed in the SU-2 group may be needed for improvement in insulin stimulated glucose transport (Fig. 4). It therefore appears that diets providing 18:3 n-3 improve insulin action in adipocytes by enhancing glucose transport and antilipolysis and inhibiting lipolysis. High proportions of LCPUFA in skeletal muscle membrane [9,56,57] and intramyocellular triglyceride content

Table 5 Effect of dietary a-linolenic acid on adipocyte insulin response to glucose transport ST220 EC50 (nM) Maximal uptake (pmol/106 cells/3 min)

SU220 a

0.066F0.009 174F3.0a

SU50 a

0.058F0.008 106F4.4b

SU10 a

0.065F0.013 105F4.4b

SU2 a

0.076F0.023 115F6.1b

0.075F0.009a 139F5.1c

EC50—Insulin required for half maximal stimulation of glucose transport. Values calculated from individual values from dose responsive curve. Values represent meansFS.E. of 8 rats. a,b,c Values in a row not sharing a common superscript differ significantly at Pb0.05.

Ghafoorunissa et al. / Biochimica et Biophysica Acta 1733 (2005) 67–75 b

% Stimulation over basal

300 250 200

a

a

ST220

SU220

a,b

a,b

150 100 50 0 SU50

SU10

SU2

Fig. 4. Insulin stimulated glucose uptake in rat diaphragm muscle from rats fed different diets. Hemidiaphragms were pre-incubated for 15 min at 37 8C followed by incubation for 45 min in the presence and absence of insulin (400 nM). Glucose uptake was measured as described in Materials and methods. Values are meanFS.E. (n=8). Values with different letters are statistically different ( Pb0.05). ST-220; SU-220; SU-50; SU-10; n SU-2.

influences insulin sensitivity [58]. Storlien et al. [59] reported a positive correlation between SFA in skeletal muscle plasma membrane phospholipids and triglyceride content. Studies in our laboratory showed that an increase in trans fatty acids in muscle phospholipids increases intramyocellular triglycerides and induce IR in rats (unpublished observations, Saravanan et al.). In the present study, neither high dietary sucrose (SU-220) nor 18:3 n-3 increased triglyceride content in the diaphragm. However, in diaphragm phospholipids, the substitution of varying levels of 18:2 n-6 with 18:3 n-3 increased LC n-3 PUFA (eicosapentaenoic acid, 20:5 n-3, and docosahexaenoic acid, 22:6 n-3) with a concomitant decrease in arachidonic acid (20:4 n-6). These observations are consistent with the known competitive interactions and inhibition of the desaturation and elongation of 18:2 n-6 to LC n-3 PUFA and preferential conversion of 18:3 n-3 to LC n-3 PUFA. Animal studies showed that dietary 18:3 n-3 increases LC n-3 PUFA in various tissues [39,40,60]. In humans, while some studies did not show an effect, in other studies bioequivalence has been reported by providing 18:3 n-3 at levels 7–10 times that of LC n-3 PUFA [44,61–63]. However, a critical issue is whether dietary 18:3 n-3 can increase LC n-3 PUFA levels needed for optimal physiological function in various tissues. The observation that insulin stimulated glucose transport in the diaphragm increased only in SU-2 suggests that ~12 nmol% LC n-3 PUFA (LC n-6 PUFA/LC n-3 PUFA ratio ~1:1) in diaphragm phospholipids (possibly in the entire skeletal muscle) may be conducive for effective glucose disposal. Giron et al. [64] showed that high levels of LC n-3 PUFA in the diaphragm increases GLUT4mRNA and protein whereas high 20:4 n-6 decreases GLUT4 mRNA and protein. This study has demonstrated the effectiveness of 18:3 n-3 for lowering blood lipids and increasing peripheral insulin sensitivity in muscle and adipose tissue. These effects can be ascribed to the resulting high levels of 20:5 n-3 and 22:6 n-3 in the structural lipids of adipose tissue and muscle.

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Epidemiological studies documented an inverse association between dietary 18:3 n-3 and mortality from CHD [63,65]. Studies in apparently normal Indian adults showed that daily intake of ~2 g 18:3 n-3 (~1 en% 18:3 n-3 and 18:2 n-6/18:3 n-3 ratio 6–9) for a period of 4 months increased LC n-3 PUFA in plasma and platelet phospholipids and decreased ADP-induced platelet aggregation [44]. However, it remains to be determined whether a long term increase in dietary 18:3 n-3 would also prevent IR and dyslipidemia in Indian subjects. Increasing dietary 18:3 n-3 is a component of dietary advice to reduce cardiovascular risk. Since the present study suggests a role for 18:3 n-3 in the prevention of IR, the dietary advice to increase 18:3 n-3 should be reinforced to also achieve reduction in IR.

Acknowledgements This research was supported by a grant from the Department of Science and Technology (DST), Government of India. The authors wish to thank Swarupa Rani, Laxmi Rajkumar and Vani Acharya for their technical assistance.

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