Improvement of diabetic control by continuous subcutaneous insulin infusion therapy changes fatty acid composition of serum lipids and erythrocytes in Type 1 (insulin-dependent) diabetes

Diabetologia, Oct 1986

The influence of improved diabetic control on the fatty composition of serum lipids, erythrocytes and platelets was investigated in 24 patients with Type 1 (insulin-dependent) diabetes treated for 6 months with either continuous subcutaneous insulin infusion (n = 14) or conventional insulin therapy (n = 10). The groups were matched for age, sex, body mass index, serum lipids, duration of diabetes, glycosylated haemoglobin and insulin dose. Glycaemic control improved, and the contents of dihomogammalinolenic acid and arachidonic acid but not linoleic acid rose significantly (p < 0.05), in serum lipids of patients treated with continuous infusion. No changes were observed in the group treated with insulin injections. Both in serum and erythrocytes the n-6 polyunsaturated fatty acid ratios rose consistently in the patients, with improvement of control regardless of the mode of treatment. Furthermore, the change of HbA1 was negatively correlated with that of arachidonic acid in erythrocytes. No changes were found in the platelet fatty acid compositions. The findings suggest that improved diabetic control enhances the conversion of linoleic acid to arachidonic acid, probably by activating enzymes needed for chain elongation and desaturation.

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Improvement of diabetic control by continuous subcutaneous insulin infusion therapy changes fatty acid composition of serum lipids and erythrocytes in Type 1 (insulin-dependent) diabetes

Diabetologia Improvement of diabetic control by continuous subcutaneous insulin infusion therapy changes fatty acid composition of serum lipids and erythrocytes in Type 1 (insulin-dependent) diabetes R. S. Tilvis 0 E. Helve 0 T. A. Miettinen 0 0 Second and Third Departments of Medicine, University of Helsinki , Helsinki , Finland Summary.The influence of improved diabetic control on the fatty composition of serum lipids, erythrocytes and platelets was investigated in 24patients with Type 1 (insulin-dependent) diabetes treated for 6 months with either continuous subcutaneous insulin infusion (n = 14) or conventional insulin therapy (n = 10). The groups were matched for age, sex, b o d y mass index, serum lipids, duration of diabetes, glycosylated haemoglobin and insulin dose. Glycaemic control improved, and the contents of dihomogammalinolenic acid and arachidonic acid but not linoleic acid rose significantly (p < 0.05), in serum lipids of patients treated with continuous infusion. No changes were observed in the group treated with insulin injections. Both in serum and erythrocytes the n-6 polyunsaturated fatty acid ratios rose consistently in the patients, with improvement of control regardless of the mode of treatment. Furthermore, the change of HbA1 was negatively correlated with that of arachidonic acid in erythrocytes. No changes were found in the platelet fatty acid compositions. The findings suggest that improved diabetic control enhances the conversion of linoleic acid to arachidonic acid, probably by activating enzymes needed for chain elongation and desaturation. Diabetes mellitus; fatty acids; serum lipids; erythrocytes; platelets - H u m a n diabetes mellitus is often associated with disturbances of lipid metabolism. As c o m p a r e d to serum triglyceride and cholesterol levels and lipoprotein metabolism, relatively little attention has been paid to the fatty acid composition of serum lipids and blood cell membranes in h u m a n Type 1 (insulin-dependent) diabetes. However, both h u m a n studies [ 1-3 ] and animal experiments [ 4-10 ] have indicated that the insulin deficit may affect the metabolism of essential fatty acids. The conversion of essential fatty acids like linoleic acid (18:2,n-6) to longer-chain polyunsaturated fatty acids (PUFA), e.g. d i h o m o g a m m a l i n o l e n i c acid (DHGLA, 20:3,n-6) and arachidonic acid (ARA 20:4,n-6), is of particular interest because of their role as prostanoid precursors [1l]. In fact, several abnormalities of the platelet function and prostaglandin metabolism have been described in h u m a n diabetes [ 12-15 ]. Cross-sectional studies have shown that ARA-content of diabetic platelets may be increased [ 17, 18 ], normal [ 19 ] or decreased [ 20 ], the latter especially in cases with severe retinopathy [201.We have recently observed that in w o m e n with Type 1 diabetes the content of linoleic acid of ser u m lipids was higher and that of A R A and other n-6 P U F A lower than in controls [ 3 ]. Furthermore, glycosylated haemoglobin (HbAD was positively correlated with the linoleic acid content but, if anything, negatively with the proposed n-6 P U F A metabolites of linoleic acid, suggesting that altered linoleic acid metabolism was related to diabetic control. The present study was designed to explore whether improved diabetic control with continuous subcutaneous insulin infusion therapy (CSII) affects the fatty acid composition of serum and cell membrane lipids in Type 1 diabetes. Subjects and methods Subjects Twenty-four patients with Type 1 diabetes were selected from the outpatient diabetes clinic of Helsinki University Central Hospital (Table 1). The patients were participants of the WHO Multicenter Study on the feasibility of continuous subcutaneous insulin infusion therapy (CSI1). Ten patients were randomized to continue conventional insulin injection therapy (CIT) for 6 months, whereas 14patients started CSII for 6 months. The two groups were matched for sex, age, serum lipids, duration and severity of diabetes, glycosylated haemoglobin (HbAD and daily insulin dose. CSII was performed using the Nordisk Insulin Infuser-pump (Nordisk Insulin Laboratories, Gentofte, Denmark). Approximately 50% of the daily dose of insulin was given as a continuous infusion and the rest as boluses before meals. CIT consisted of two or three R. S. Tilvis et al.: Insulin infusion a n d fatty acids daily injections. Velosulin 100 U (Nordisk Insulin Laboratories, G e n tofte, D e n m a r k ) was u s e d in CSII, a n d a mixture o f intermediate-acting a n d short-acting insulins (Lente a n d Actrapid; N o v o A / S , C o p e n hagen, D e n m a r k ) in CIT. T h e diet was controlled before a n d during the t r e a t m e n t period a n d s h o w e d no detectable change. T h e energy intake (30 kcal. kg -1. day 1) contained 45% carbohydrates, 20% protein a n d 35% fat, the n a t u r e o f which was kept constant. Methods Following an overnight fast, blood samples were d r a w n into two 10-ml tubes a n d erythrocytes were centrifuged at 125 g a n d w a s h e d twice with 0.15 m m o l / 1 saline. Serum cholesterol a n d triglycerides were m e a s u r e d by s t a n d a r d hospital laboratory m e t h o d s [ 21, 22 ]. A portion o f s e r u m was centrifuged at 250 g for 15 rain to prepare platelet-rich plasma. Platelet-rich p l a s m a was t h e n recentrifuged (1950 g), a n d the platelet pellet was w a s h e d twice with 0.15 m m o l / 1 saline. Erythrocytes a n d platelets were frozen immediatedly. Glycosylated h a e m o g l o b i n Aa was m e a s u r e d by the m i c r o c o l u m n m e t h o d [ 23 ] after overnight dialysis of h a e m o l y s a t e in saline [ 24 ]. The n o r m a l values o f HbA1 range f r o m 6.0% to 9.0%. Serum lipids were extracted with c h l o r o f o r m - m e t h a n o l (2:1, v/v). Triglycerides, cholesterol esters a n d p h o s p h o l i p i d s were separated on plastic silica gel chromatoplates using h e p t a n e : ethyl ether: acetic acid (80: 20: 2 v / v / v ) as a solvent. After addition o f trimargarin as an internal s t a n d a r d isolated s e r u m lipids, erythrocytes a n d platelets were saponified with 1 N N a O H in 90% ethanol for I h at 80 °C. After removal o f nonsaponitiable lipids a n d acidification o f the saponification mixture, free fatty acids were extracted with n - h e p t a n e a n d methylated with 5% methanol-HC1 for 2 h at 85 °C. T h e fatty acids were analyzed with a Vat±an 2100 gas chromatog r a p h (Varian Associates, Palo Alto, Calif., USA) e q u i p p e d with a 3 5 - m m glass capillary BDS (butane-I,4-diol succinate) column. The temperature p r o g r a m m e was 2 ° C / m i n , f r o m 130°C to 200°C. The peaks were identified on the basis o f retention times recorded for different standards a n d m e a s u r e d with an electronic integrator. Statistical analysis T h e differences o f the fatty acid c o m p o s i t i o n between the groups were tested with an analysis o f variance (Anova). The changes o f individual fatty acids were tested with a paired two-tailed Student's t-test. Results CSII significantly reduced HbA1 (p < 0.05) and insulin requirement, whereas no changes were f o u n d in the C I T group (Table 1). Prior to the treatment period, the content and composition o f fatty acids o f serum lipids, erythrocytes and platelets were virtually similar in the two groups (Table 2). Values are the m e a n + SD. CSII = c o n t i n u o u s s u b c u t a n e o u s insulin infusion, C I T = conventional injection therapy. a p < 0 . 0 5 Linoleic M e a n + S E M aThe sum of identified fatty acids was indicated by 1000. Significance between the groups: bp<0.05 or less. Effect of treatment: Cp <0.05 or less Serum fatty acids In the serum lipids the intensified insulin treatment by the pump increased only the contents o f n-6 P U F A and had no consistent effect on other fatty acids including linoleic acid (Table 2). Thus, the contents o f D H G L A and arachidonic acid rose by up to 25% in serum triglycerides, cholesterol esters and phospholipids of the CSII group, whereas no significant changes were ob served in the CIT group (Fig. 1). The changes of n-6 P U F A were analyzed further according to improvement of diabetic control by the treatments using the fluctuation o f glycosylated HbA1 as the indicator o f a change in the control. In the patients in w h o m HBA1 fell the contents o f arachidonic acid significantly rose (p < 0.05) in serum cholesterol esters, phospholipids and triglycerides, whereas no significant changes were found in patients with an unaltered or increased HbA1 level (Fig. 2). In serum phospholipids and triglycerides similar significant results were recorded for the D H G L A (p < 0.05), but no significant changes were found in linoleic acid of serum lipids. Poor diabetic control was associated with a significant increase in serum triglycerides (p <0.01) and non-significant in cholesterol esters and phospholipids. Owing to the fluctuations o f serum lipids neither significant differences nor changes were found in the absolute serum concentrations o f individual fatty acids among the groups. Erythrocytes In contrast to the serum lipids, n-6 P U F A rose signifi cantly (p < 0.05) in the erythrocytes o f both treatment groups (Table 3). However, in the patients in w h o m HbA1 was decreased the n-6 P U F A / L A ratio was in creased, whereas no consistent changes were found in other patients (Fig.3). Furthermore, the changes in HbA1 were negatively correlated with those o f n-6 PU FA, e.g. arachidonic acid (Fig. 4), but not with that o f linoleic acid. Thrombocytes CSII Before 2 + 0 146+ 4 12+ 1 189+ 5 192_+ 3 110+13 1 + 0 6 + 0 19_+ 1 233_+10 21_+ 4 19_+ 1 27_+ 1 27_+ 2 After 2 + 0 146+ 2 11-+ 1 182-+ 3 196+ 3 102_+10 1 + 0 6_+ 0 16_+ 1 242_+ 8 20_+ 2 2 0 + 1 28_+ 1 27_+ 1 CIT Before 2 + 0 150-+ 6 13+ 3 194+13 186_+ 4 109_+21 1_+ 0 6_+ 0 16_+ 2 229_+27 19_+ 2 20_+ 2 27_+ 1 27_+ 3 After 2 + 0 142+5 9 + 1 1 8 5 + 4 192+3 97_+5 1-+0 7-+0 17_+1 256+3 18_+1 21_+2 26_+1 28_+1 R. S. Tilvis et al.: Insulin infusion and fatty acids ~.. 1.5 +10 HbAi ~ HbAl f / : i A £ -3 r -2 i -1 HbAt (A%) I 0 oo o °o ' ~ . I • +1 I ÷2 The platelet fatty acid composition was not affected by CSII or CIT (Table 3). Our earlier findings [ 3 ] suggested that the low n-6 PUFA content o f serum lipids and cellular membranes in women with Type I diabetes, especially in those with poor diabetic control, was due to impaired conversion o f linoleic acid to the longer chain n-6 PUFA, i.e. DHGLA, arachidonic acid and docosatetraenoic acid. If this were true, improved diabetic control should be associated with an increase in n-6 PUFA. The present findings actually demonstrate that the intensified insulin treatment which improved diabetic control was associated with an increased contribution o f n-6 P U F A of serum lipids and erythrocytes. This increase was proportionate to the improvement of diabetic control as indicated by the negative correlations between the changes in the fatty acid contents and that in the HbA1 level of erythrocytes. Serum levels of n-6 P U F A are influenced by dietary intake of essential fatty acids, mainly linoleic acid and gamma-linolenic acid (GLNA; 18: 3,n-6), chain-elongation and desaturation of linoleic acid to other n-6 PUFA, and incorporation of n-6 P U F A into tissue membrane lipids. The similarity of the initial values of serum lipids and their fatty acids suggests that the diets of the two treatment groups had been similar. The linoleic acid content in serum lipids and in the diet was not changed detectably during the treatment period. Dietary intake of arachidonic acid and other long chain n-6 PUFA is low and probably does not vary remarkably [ 25, 26 ]. Thus, increased serum n-6 P U F A are most likely formed endogenously from linoleic acid, even though the decreased utilization o f n-6 P U F A to eicosanoids or conversion to peroxides during improved diabetic control can not be excluded. Some evidence has been presented that CSII might normalize the elevated thromboxane production in Type 1 diabetes [27]. The increase of n-6 PUFA in the erythrocytes of the present study and the unaltered platelet fatty acid composition suggest that the rise of serum n-6 PUFA, e. g. arachidonic acid, was not caused by a diminished incorporation o f serum PUFA to membrane lipids during the improvement o f the diabetic state. Thus, the increase of metabolic products of LA, n-6 PUFA, and the n-6 PUF A / L A ratios in serum lipids and erythrocytes concomitantly with the proportionate decrease of HbAt suggest that the chain-elongation and desaturation of linoleic acid had been activated in patients with improved diabetic control. The initial and rate-controlling step in the conversion of linoleic acid to arachidonic acid is the formation of 7/-linolenic acid (GLNA 18: 3,n-6) by the A6 desaturase enzyme [ 4-10 ]. G L N A is further converted to D H G L A by chain elongation and further by the A 5 desaturase to arachidonic acid. The activities o f these enzymes are decreased in experimental insulin deficiency [ 4-10 ]. In the present study the contents o f D H G L A and arachidonic acid were slightly increased in serum lipids and especially in erythrocytes of the diabetic patients proportionately to the decrease in glycosylated haemoglobin. No significant changes were observed in the contents of serum or erythrocyte eicosapentaenoic acid (20:5, n-3) and docosahexaenoic acid (22:6, n-3), even though the sum of n-3 P U F A tended to increase more in CSII than CIT. The n-3 P U F A are proposed to be the A 5 and A4 desaturation and chain-elongation products of a-linolenic acid (LNA 18:3, n-3). Our findings suggest that either the A 5 and A 4 desaturases and the chain elongation were not significantly activated by improved diabetic control or that only a negligible amount of tissue n-3 p U F A is derived from the dietary linoleic acid. The low n-3 P U F A in Type 1 diabetes [ 3 ] m a y be due to low dietary intake of these fatty acids or their enhanced consumption. On the other hand, if the A4 and A5 desaturases are not activated by improved diabetic balance, the rises of n-6 P U F A in serum lipids and erythrocytes could be secondary to the activation A6 desaturation and subsequently increased formation of G L N A and D H G L N A . Furthermore, no changes were observed in the concentrations of saturated and monoenoic fatty acids or their ratios, even though the activity o f A9 desaturase, responsible for formation of monoenoic fatty adds, is decreased in the experimental insulin deficiency [8]. It is quite possible that changes greater than found in the diabetic control of our study could result in alterations of saturated/monoenoic fatty acid ratios and of n-3 PUFA. The present experiments confirm the earlier results, suggesting the dependence of fatty acid composition of serum and erythrocyte membranes on the diabetic state in m a n [ 1-3 ], and imply that the insulin therapy enhances the conversion of linoleic acid to prostanoid precursor fatty adds. Further studies are needed to elucidate the degree to which alterations in prostanoid metabolism of diabetic patients [ 12-20 ] are based on disturbances in the synthesis of the precursor PUFA. Acknowledgements.Ms A. Salolainen, R Hoffstr6m and M. Aarnio are acknowledged for technical and secretarial help. This study was supported by Nordisk Insulin Laboratories, Gentofte, Denmark and by a grant from the Finnish State Council for Medical Research. Professor Tatu A. Miettinen, MD Second Department of Medicine University of Helsinki SF-00290 Helsinki Finland 1. Schrade W , Boehle E , Biegler R , Harmuth E ( 1963 ) Fatty acid composition of lipid fractions in diabetic serum . Lancet 1 : 285 - 290 2. 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R. S. Tilvis, E. Helve, Professor T. A. Miettinen MD. Improvement of diabetic control by continuous subcutaneous insulin infusion therapy changes fatty acid composition of serum lipids and erythrocytes in Type 1 (insulin-dependent) diabetes, Diabetologia, 1986, 690-694, DOI: 10.1007/BF00870277