Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(3), 191-194
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Relationship between plasma insulin and erythrocyte fatty acid composition P. M. Clifton, 1 P. J. Nestel 2 1Division of Human Nutrition, Adelaide, South Australia 2Baker Medical Research Institute, Melbourne, Victoria, Australia
Summary Insulin resistance is an important condition which underlies much of the coronary artery disease in affluent societies. We have related insulin resistance, as assessed by fasting plasma insulin, to erythrocyte membrane composition in 54 healthy men and women on a low fat diet. We found a inverse relationship (r = -0.41, P = 0.002) between fasting plasma insulin and the percentage of arachidonic acid in erythrocyte fatty acids. An inverse relationship of similar strength was found with total n-6 fatty acids and a positive relationship was found with the percentage of saturated fatty acids (r = 0.39, P < 0.01). No relationship was found with n-3 fatty acids. We would suggest that n-6 fatty acids, and in particular arachidonic acid, modify the membrane environment of the insulin receptor (or the glucose transporters) so that lower levels of insulin are required for glucose homeostasis.
INTRODUCTION
Insulin resistance underlies a considerable proportion of coronary artery disease 1; and is significantly related to two of the major risk factors: low HDL cholesterol and high fasting triglyceride.3 Insulin resistance is exacerbated by obesity, patXicularly central obesity,4 but the cellular defect which interferes with the action of insulin is not clearly understood. In animal studies, increasing the content of polyunsaturated fatty acids in the cell membrane enhances insulin binding and insulin action. 5 The fatty acid composition of skeletal muscle phospholipids is strongly related both to fasting insulin in normoglycemic subjects and to estimates of insulin sensitivity as derived from euglycemic clamp studies? Decreased insulin sensitivity is associated with a decreased content of long chain polyunsaturate, in particular arachidonic acid, and the total percentage of C20-C22 polyunsaturates. Insulin secretion and insulin action has also been related ~0 the ratio of n-6 polyunsaturated fatty acids to saturated fatty acids in serum phospholipids. 7 We speculated that differences in muscle phospholipid Received 22 June 1998 Accepted 17 July 1998 Correspondence to: Dr Peter Clifton, CSIRO, Division of Human Nutrition, PO Box 10041, Gouger Street, Adelaide, South Australia 5000. Tel: +61 883038826; Fax: +61 883038899; E-mail:
[email protected]
composition would be reflected in alterations in the phospholipid composition in all cell types, not just in the major targets of insulin action, such as muscle. We chose to examine an easily accessible ceil, the circulating erythrocyte, in normoglycemic subjects on a controlled, low fat diet, and measure the total fatty acid composition of the erythrocyte and a plasma fasting insulin on the same day. PATIENTS AND METHODS
Subjects were normoglycemic men and women who were undergoing a dietary intervention trial, the details of which have been previously published. 8 The study was approved by the CSIRO Human Ethics Committee and the volunteers gave their informed consent. The last 54 subjects (31 men and 23 women) recruited into this trial had blood collected and analysed for insulin and erythrocyte fatty acid composition at the end of a 2 week low fat (25% of calories as fat) baseline, isocaloric period. No weight loss occurred during this baseline period. In this trial, insulin levels measured at the end of this period were not significantly different from those measured at the end of a high fat/high cholesterol supplement period or a high carbohydrate supplement period. As fatty acid composition of the diet was not calculated, intakes of individual fatty acids, in particular linoleic and 191
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Clifton et al
Table I Baseline characteristics of subjects Sex
Age
BMI
TC
TG
HDL
Insulin
Women (n = 23) Men (n = 31 )
51.4± 9.8 54.4± 7.6
25.8± 2.5 26.0± 2.7
5.43± 0.91 5.42± 0.86
1.24± 0.52 1.28+ 0.57
1.17± 0.30 0.86+ 0.20
53± 39 53± 20
Data are mean + SD, mmol/L except insulin pmol/L.
arachidonic acids, is not known. Characteristics of these subjects are shown in Table 1. Men and women had very similar baseline characteristics and most had normal cholesterol and triglyceride levels. Insulin levels ranged from 29 to 215 pmol/L. Fasting plasma insulin was measured by radioimmunoassay using Pharmacia kits (Phadedas Pharmacia, Uppsala, Sweden). Erythrocytes were washed twice with normal saline then haemolysed with an equal volume of distilled water. Lipids were extracted from the cells using a chloroform/methanol solution added to the erythrocyte solution in a ratio of 4:2:1. The lipids were methylated and fatty acids were determined with a vitreous silica (50 M x 0.53 mm i.d.) cross-linked free fatty acid phase gas chromatography column using a Hewlett-Packard 5711A gas chromatograph (Hewlett-Packard, Avondale, PA, USA). Because of several small unidentified peaks on the chromatogram the fatty acids in Table 2 do not add up to 100%. RESULTS
Thirty-one men and 23 women had erythrocyte composition measured. Their characteristics are shown in Table 1. Only five subjects had abnormal triglyceride ('92.0 mmol/L) and total cholesterol (>6.5 mmol/L), but these proportions are quite typical of the healthy population who volunteer for nutritional studies. Two subjects only had a plasma insulin >100 pmol/L (the 95th percentile in our normal population, n = 170) and thus had a minor degree of insulin resistance. In the other 52 subjects insulin ranged from 29-99 pmol/L All subjects had a normal fasting glucose. Weight loss did not occur during the baseline period so the insulin levels were measured in a stable state. Fatty acid composition of the erythrocyte is shown in Table 2. Men and women differed in only three minor fatty acids; 14:1, 24:0, 24:1 and one major fatty acid 18:1. They have been pooled for further analysis as none of these fatty acids were related to insulin or cholesterol levels. Total cholesterol was significantly related to the percentage of total saturates in the membrane (r=0.37, P < 0.01). The total saturates and monounsaturates were strongly related to each other (r--0.69, P<0.001). The
proportion of arachidonic acid varied from 9.9 to 16.8% and the total n-6 varied from 23 to 36% while the total saturates were more tightly controlled varying only between 31.5 and 39%. Fasting plasma insulin was related only to one individual fatty acid-arachidonic acid (20:4 n-6), r---0.41, P--0.002 (Table 2). The relationship between insulin and the total n-6 polyunsaturated fatty acid amount (r=-0.39, P<0.01) and insulin and total saturated fat (r= 0.39, P < 0.01) and insulin and the n-6/saturated fat ratio (r=-0.44, P = 0.001) was very similar. Adjustment for BMI or family history of diabetes did not alter these relationships. There was no relationship between total or individual n-3 fatty acids or the n-6/n-3 ratio and plasma insulin. The ratio of 20:4 (n-6) to 20:3 (n-6) and the sum of all 20-22 carbon polyunsaturates were only weakly inversely related to plasma insulin (r =-0.28, P = 0.04). Thus even in the group with normal plasma insulin levels and no apparent insulin resistance the n-6 polyunsaturates appear to be related positively to insulin sensitivity while membrane saturated fatty acids appear to have the opposite effect. DISCUSSION
Our results confirm the findings of Borkman et al,6 Vessby et al9 and Pan et all0 that membrane composition is related to fasting serum insulin which is a good surrogate measure of insulin resistance. We showed for the first time that arachidonic acid and the sum of all n-6 fatty acids in erythrocyte membranes was inversely related to fasting plasma insulin. Thus invasive muscle biopsy is not required to demonstrate these relationships so that membrane composition can be examined repeatedly after dietary or other interventions, akhough the correlations with erythrocyte membranes are not as strong as those seen with muscle membrane phospholipids in some of the studies. In addition, it shows that in normal healthy subjects more direct assessments of insulin resistance, such as clamps or glucose tolerance tests, are not required to demonstrate the relationship. We did not separate phospholipid classes as virtually all the relevant data in the literature is based on the total phospolipid pool. Our results differ from those of Pelikanova et al7 who examined fasting serum phospholipids and insulin secretion with an oral glucose tolerance test and insulin action in a euglycemic clamp in normal men. In their studies, fasting insulin was only related to 18:3 (inversely) but integrated insulin levels after the oral glucose tolerance test were inversely related to total n-6 polyunsaturated fatty acids, while glucose infusion rates in the low dose euglycemic clamp were positively related to total n-6 polyunsaturated fatty acids, indicating greater insulin sensitivity with an increased n-6 polyunsaturated content of serum phospho-
Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(3), 191-194
© Harcourt Brace & Co. Ltd 1998
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lipids. Arachidonic acid levels were also positively related to the clamp glucose infusion rates, again indicating greater insulin sensitivity with more arachidonic acid. Saturated fatty acid levels, in particular palmitic (C 16:0), were positively related to fasting insulin (as in our study), integrated post-glucose insulin and negatively to insulin action, as assessed by glucose infusion rates. Agostoni et al '1 found in 12 obese adolescents that the products of deka 6 desaturase (using the 18:3/18:2 ratio as an index), but not arachidonic acid, levels were inversely related to peak insulin levels (and area) during an oral glucose tolerance test. Saturated fatty acids were positively related to insulin peak and area. In patients with type 2 diabetes the data on erythrocyte fatty acid composition is conflicting:2-15 with some evidence of enhanced delta 6 desaturase activity in two studiesJ 2,~s In type 1 diabetes the evidence of impaired elongation of linoleic acid to arachidonic acid is much more consistent '6,17with improvement in delta 6 desaturase with better glucose control. Thus it is reasonable to conclude that membrane composition in cells generally, including cells not targets of insulin action, is related to insulin action. Animal studies have shown that n-6 polyunsaturates increase insulin receptor number and binding while saturated fat decreases binding and transport. 5 Limited clinical studies are suggestive of a similar effect. TM The reverse scenario where increased insulin levels change membrane composition seems less likely as the erythrocyte, although it possesses insulin receptors, does not respond metabolically to insulin. However, as insulin treatment stimulates the activity of A6 and A5 desaturase in diabetic rats (where the activity is low compared with normal rats), 19 enhanced insulin sensitivity may be associated with greater elongation and desaturation of linoleic acid to arachidonic acid within the liver with secondary peripheral membrane changes, which have been demonstrated to lag behind liver membrane changes. 2° However, mild insulin resistance or a high normal insulin level is far removed metabolically from true type 1 diabetes, so it is unlikely the membrane change is secondary to differences in desaturase activity. There have been many clear demonstrations that erythrocyte membrane composition can be altered by diet z~,22 and this has been used as a marker of dietary intake of linoleic acid. 2s,24However, despite large increases in linoleic acid in the diet and in the erythrocyte or platelet membrane, the content of arachidonic acid does not usually change, nor is the level of oleic acid in the membrane sensitive to the level of dietary oleic acid. 25 The factors which determine the level of oleic and arachidonic acid in the erythrocyte membrane are unknown. Elevated saturated fatty acids in hypercholesterolemic subjects has been noted previously 26 and may relate to a higher intake of dietary saturates in this group. Prostaglandins, Leukotrienes and Essential FattyAcids (1998) 59(3), 191-194
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In c o n c l u s i o n , e r y t h r o c y t e m e m b r a n e fatty a c i d c o m p o sition h a s t h e s a m e r e l a t i o n s h i p w i t h i n s u l i n r e s i s t a n c e as d o e s m u s c l e p h o s p h o l i p i d f a t t y acids a n d c o u l d b e u s e d in e p i d e m i o l o g i c a l a n d i n t e r v e n t i o n s t u d i e s i n s t e a d o f m u s c l e b i o p s y in n o n - d i a b e t i c subjects. T h e r e l a t i o n s h i p b e t w e e n fasting i n s u l i n a n d f a t t y a c i d c o m p o s i t i o n is a p p a r e n t in n o r m a l s u b j e c t s w i t h n o o v e r t i n s u l i n resistance.
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Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 59(3), 191-194
© Harcourt Brace & Co. Ltd 1998