ARTICLE IN PRESS Prostaglandins, Leukotrienes and Essential Fatty Acids 79 (2008) 55–58
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Desaturase activities and metabolic control in type 2 diabetes G. Sartore a,, A. Lapolla a, R. Reitano a, S. Zambon a, G. Romanato a,b, R. Marin a, C. Cosma a, E. Manzato a,b, D. Fedele a a b
Department of Medical and Surgical Sciences, University of Padua, Via Dei Colli, 4, 35143 Padova, Italy CNR—Institute of Neuroscience, Aging Branch, Padova, Italy
a r t i c l e in fo
abstract
Article history: Received 28 March 2008 Received in revised form 27 June 2008 Accepted 11 July 2008
The aim of this study was to elucidate the effects of a poor glycemic control on fatty acid composition and desaturase activities in type 2 diabetic patients. Plasma phospholipid fatty acid composition and desaturase activities (estimated from fatty acid product to precursor ratios) were measured in 30 type 2 diabetic patients during poor metabolic control and after achieving a good metabolic control. Significant changes were recorded in the percentages of palmitic, stearic, dihomo-g-linolenic, docosatetraenoic and docosapentaenoic acid. The delta-5 desaturase activity was significantly higher with poor than with good metabolic control. The changes identified in plasma phospholipid fatty acid composition and the desaturase activity in type 2 diabetic patients go in the opposite direction to those described in similar conditions in type 1 diabetic patients and may be relevant to a better understanding of the role of metabolic control in the progression of chronic complications in type 2 diabetic patients. & 2008 Elsevier Ltd. All rights reserved.
1. Introduction Plasma fatty acid composition is an independent risk factor for coronary heart disease [1]. Fatty acid composition is abnormal in serum and several tissues in experimental diabetes [2] and in diabetic patients [3,4]. Plasma fatty acid composition is affected not only by dietary fat intake, but also by endogenous fatty acid metabolism, which is regulated by enzymes such as elongases and desaturases. Changes in desaturase activity are associated with insulin resistance [5] and may be involved in the pathophysiology of type 2 diabetes and metabolic syndrome [6,7], and they may also explain differences in coronary heart disease risk [8]. In streptozotocin-induced type 1 diabetes mellitus, a defect of delta-5 desaturase was found related to lower contents of arachidonic acid and higher contents of linoleic acid in almost all tissues except the brain [9–11]. In another report, Tilvis et al. [12] suggested that elongation and desaturation of essential fatty acids are decreased in women with type 1 diabetes. El Bustani et al. [13] showed that the activity of all the desaturases and of delta-5 desaturase, in particular, were depressed in type 1 diabetic patients and that insulin treatment restored their activity levels. Only two studies have examined the effects of a poor metabolic control on the fatty acid composition of plasma lipids and on desaturase activities in type 1 diabetes mellitus [14,15]. Both Corresponding author. Tel./fax: +39 4 9821 6848.
E-mail address:
[email protected] (G. Sartore). 0952-3278/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2008.07.001
studies showed that there is a change in plasma phospholipid fatty acid composition during short-lived diabetic ketosis, and that the desaturase activities are reduced in type 1 diabetic patients. Few studies have investigated desaturase activities in type 2 diabetes and the results contrast with those described in type 1 diabetes. In diabetes induced by a rich sucrose diet in rats, Brenner et al. [16] found a moderate increase in the delta-5 desaturase activity in the liver due to an insulin-activation or insulinresistance effect on the desaturase. Elongation and desaturation of essential fatty acids increase in type 2 diabetic patients [4], and exogenous insulin does not seem to significantly affect the desaturase activity in these patients [5]. No information is currently available on the effects of poor glycemic control on fatty acid composition and desaturase activities in type 2 diabetic subjects. The aim of this study was therefore to establish whether a metabolic derangement has any effect on the composition of plasma phospholipid fatty acids and desaturase activities in a group of type 2 diabetic patients.
2. Patients and methods 2.1. Subjects Thirty type 2 diabetic subjects (15 men, 15 women; mean age 5676 years, with a mean disease duration of 2.370.6 years) were studied during a period of poor metabolic control and after
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achieving a good metabolic control thanks to 3 months of intensive therapy. They were enrolled from among those consecutively seen as outpatients at the Diabetes Clinic of the Department of Medical and Surgical Sciences at the University of Padova. At the baseline, all subjects were being treated with a controlled diet and oral hypoglycemic agents (sulphonylureas, metformin). None of the cases included in the study were on any medication influencing lipid and fatty acid metabolism or insulin therapy, and none had any evidence of hepatic, renal or myocardial dysfunction, nor any other endocrine diseases or chronic complications of diabetes. To rapidly improve glycemic control, all subjects were initially treated with intensified insulin therapy (consisting of three or more daily injections of insulin with dose adjustments based on at least four self-monitored glucose measurements per day) [17]. The sulphonylureas were suspended at the beginning of insulin therapy, while metformin was continued. The fatty acid composition of the patients’ diet was similar before their metabolic control was lost and during the time it took to restore a good metabolic control. The proportions of carbohydrates, fats and proteins were 50%, 30% and 20%, respectively. The dietary regimens were carefully matched and remained the same during the study period. Diabetes self-management education for individuals with type 2 diabetes on glycemic control interventions (including advices on dietary risk, physical activity, and basic diabetic knowledge) were provided. Informed consent was obtained from each subject. 2.2. Biochemical measurements On the day of the study, blood samples were taken to measure fasting plasma glucose, HbA1c, total cholesterol, HDL-cholesterol, triglycerides and plasma phospholipid fatty acids. Plasma glucose was determined by a glucose oxidase method [18]. HbA1C was measured by an LC method (Bio-Rad, Milan, Italy) [19]. Total cholesterol and HDL-cholesterol were measured by enzymatic analytical chemistry (CHOD-PAP method, Roche, Milan, Italy) [20,21], as plasma triglycerides (GPO-PAP colorimetric enzyme test, Roche, Milan, Italy) [22]. Plasma phospholipid fatty acid composition was determined after lipid extraction using the Folch et al. [23] method. Phospholipids were isolated by applying the lipid extract to a column of silica coupled with aminopropyl groups (Bond Elute NH2, Analytichem International, Harbor City, CA, USA) [24]. After transmethylation using the Morrison and Smith [25] method, fatty acids were assayed by gas chromatography using a PerkinElmer 8320 gas chromatograph equipped with a 30 m capillary column (internal diameter 0.32 mm) (Omegawax 320, Supelco, Bellefonte, PA, USA). The column conditions were 200 1C, injection port 250 1C, and flame ionization detector 260 1C. Helium was used as the carrier gas (linear velocity 0.25 m/s); the split ratio was 100:1. Fatty acid peaks were identified by comparison with standard mixtures of fatty acids supplied by Supelco, Bellefonte, PA, USA. The amounts of individual fatty acids were calculated as the relative absorption percentage in the chromatographic result, with the fatty acids evaluated as 100%. Only fatty acid percentages higher than 0.2% are shown in the results. We used fatty acid product-to-precursor ratios to estimate desaturase activities [5,26]. 2.3. Statistical analysis Data are expressed as means7SD. Statistical comparisons were drawn using Student’s t-test. Any relationship between paired variables was evaluated by linear regression analysis. Pearson’s
correlation coefficient r was used to quantify the strength of the relationship. Statistical significance was set at po0.05.
3. Results 3.1. Metabolic parameters The clinical and metabolic characteristics of the diabetic subjects during poor metabolic control and after achieving good metabolic control are shown in Table 1. Fasting plasma glucose and HbA1c were 303781 mg/dl and 12.771.7%, respectively, during poor metabolic control and 142738 and 7.271.1 at the end of the study. Total cholesterol and triglyceride concentrations were significantly higher during poor metabolic control than with good control. No significant differences in BMI and HDL-cholesterol concentrations were observed. 3.2. Plasma phospholipid fatty acids Table 2 shows the percentage content of each fatty acid in the plasma phospholipids and the percentage content of the most important families of fatty acids during poor metabolic control and after achieving good control. The percentage of palmitic acid was higher and the percentage of stearic acid was lower during poor metabolic control than during good control (30.571.7 vs. 29.471.5, p ¼ 0.002, and 12.471.1 vs. 13.570.9, p ¼ 0.005, respectively). Poor metabolic control coincided with significantly lower percentages of dihomo-g-linolenic and docosatetraenoic acid by comparison with the situation during good metabolic control (2.970.9 vs. 3.770.8, p ¼ 0.001, and 0.370.1 vs. 0.470.1, p ¼ 0.001, respectively). The percentage of arachidonic acid remained unchanged in the two observation periods, while docosapentaenoic acid levels were lower during metabolic derangement (0.670.1 vs. 0.770.1, p ¼ 0.001). 3.3. Desaturase activities The estimated elongase and desaturase activities during poor metabolic control and at the end of the study are given in Table 2. The elongase activity and delta-9 desaturase activity indexes showed no significant differences in the two periods considered, while delta-5 desaturase activity was significantly higher during severe hyperglycemia (3.571.3 vs. 2.871.0; po0.004) than in situations of good metabolic control. Delta-5 desaturase activity was the only variable significantly correlating with HbA1c: this positive correlation was only seen Table 1 Clinical and metabolic characteristics during poor metabolic control and after achieving a good metabolic control in type 2 diabetic patients (mean7SD) Variables
Poor metabolic control
Good metabolic control
p
Men/women (n) Age (years) Duration of diabetes (years) BMI (kg/m2) Fasting plasma glucose (mg/dl) HbA1c (%) Total cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl)
15/15 5676 2.370.6 30.276.9 303781 12.771.7 240748 43710 2247138
– – – 30.177.1 142738 7.271.1 216742 46713 161764
– – – ns o0.000 o0.000 0.003 ns 0.041
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Table 2 Plasma phospholipid fatty acids (% of total fatty acids) and enzyme activities during poor metabolic control and after achieving good metabolic control (mean7SD) Good metabolic control
p
Saturated 14:0 (myristic) 16:0 (palmitic) 18:0 (stearic)
0.670.3 30.571.7 12.471.1
0.670.1 29.471.5 13.570.9
ns 0.002 0.005
Monounsaturated 16:1 (n-7) (palmitoleic) 18:1 (n-9) (oleic)
1.270.7 15.173.1
1.070.3 14.272.2
ns ns
Polyunsaturated n-6 18:2 (n-6) (linoleic) 20:3 (n-6) (dihomo-g-linolenic) 20:4 (n-6) (arachidonic) 22:4 (n-6) (docosatetraenoic)
22.173.7 2.970.9 9.572.1 0.370.1
21.872.5 3.770.8 9.872.1 0.470.1
ns 0.001 ns 0.001
Polyunsaturated n-3 18:3 (n-3) (a-linolenic) 20:5 (n-3) (eicosapentaenoic) 22:5 (n-3) (docosapentaenoic) 22:6 (n-3) (docosahexaenoic)
0.470.2 0.770.5 0.670.1 3.371.1
1.474.4 0.970.6 0.770.1 3.471.1
ns ns 0.001 ns
Saturated Monounsaturated Polyunsaturated n-6 Polyunsaturated n-3
43.171.8 15.772.3 35.373.2 5.071.5
42.871.6 15.272.3 35.672.7 6.374.5
ns ns ns ns
Elongase (22:5/20:5) Desaturase D9-16 (16:1 [n-7]/ 16:0) Desaturase D9-18 (18:1 [n-9]/ 18:0) Desaturase D5 (20:4 [n-6]/20:3 [n-6])
0.9670.42 0.0370.01
1.0470.45 0.0470.02
ns ns
1.0770.21
0.1870.23
ns
3.571.3
2.871.0
Delta 5 desaturase
Poor metabolic control
8 7 6 5 4 3 2 1 0
0.004
Good metabolic control Poor metabolic control
0
5
10 HbA1c
15
20
Fig. 1. Correlation between delta-5 desaturase and HbA1c in type 2 diabetic patients in periods of good (r ¼ 0.057, p ¼ 0.764) and poor (r ¼ 0.505, p ¼ 0.004) metabolic control.
during poor metabolic control (r ¼ 0.505; p ¼ 0.004), not after achieving good control (r ¼ 0.057; p ¼ 0.764) (Fig. 1). No other correlations were observed.
4. Discussion This study addressed the influence of metabolic control on fatty acid composition in the plasma and on desaturase activities in type 2 diabetic patients. We used fatty acid ratios as indicators of desaturase activity as other Authors reported [5,26], even if it should be considered that these ratios do not directly inform about desaturase activity, since many other factors are involved in determining fatty acid composition of plasma phospholipids,
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including for example substrate availability. Another weakness of this work is that we have not studied the fatty acid composition of cell membranes (e.g. red blood cells), which contain the functional pool of arachidonic acid for the eicosanoid synthesis. This objective needs further investigation. Our findings show that the fatty acid composition of plasma phospholipids is different during metabolic decompensation from the situation during good metabolic control, and that delta-5 desaturase activity is affected by the degree of metabolic control. Type 2 diabetic patients have lower linoleic acid levels and higher levels of highly unsaturated fatty acids [4]. Faas et al. [27] found no change in plasma phospholipid fatty acid composition before and after treatment with glyburide in patients with type 2 diabetes, but they suggested that a more severe metabolic impairment was needed in humans to produce the fatty acid abnormalities described in type 2 diabetic rats. In our patients, the percentage of palmitic acid was higher and that of stearic acid was lower during periods of poor metabolic control as compared with periods of better metabolic control. Palmitic acid inhibits beta-cell growth, impairs insulin secretion and induces apoptosis [28,29]. Moreover, palmitate oxidation in cell cultures is involved in apoptotic process in cardiomyocytes [30]. These data suggest that the higher percentage of palmitic acid that we observed might contribute to an impaired beta-cell function and promote cardiac complications in such patients. Lower levels of all the desaturases were found in experimental type 1 diabetes [11], including delta-5 desaturase, suggesting a defect in this enzyme. Moreover, insulin treatment failed to correct delta-5 desaturase activity in the streptozotocin-induced diabetic rat [9]. A study on type 1 diabetic patients has highlighted a change in the fatty acid composition in the plasma phospholipids in shortlived diabetic ketosis, with a significant decrease in the percentage of arachidonic acid content and a parallel significant drop in n-6 total polyunsaturated fatty acids. The arachidonic acid content returned to baseline values after recovery from the ketosis [14]. In the same study, a significant inverse correlation was observed between the change in the C20:4/C20:3 ratio (the product/ precursor ratio for the reaction catalyzed by delta-5 desaturase) and the HbA1c level from baseline conditions to ketosis. In another study, a significant increase in arachidonic acid levels during recovery from diabetic ketoacidosis was seen in type 1 diabetic children [15]: this study found that both arachidonic acid levels and delta-5 desaturase activity were significantly lower during than after an episode of diabetic ketoacidosis. The authors also suggested that the disruptions in essential fatty acid metabolism in diabetic children are related to hypoinsulinemia. Data on type 1 diabetic patients seem to indicate that only continuous insulin treatment over several days results in a normal delta-5 desaturase activity, suggesting the insulin dependence of this enzyme’s activity in humans [13]. These results in type 1 diabetes go in the opposite direction from those described in type 2 diabetes. In fact, desaturase activity increased in sucrose-induced diabetic rats [16] and type 2 diabetic patients [4]. In addition, exogenous insulin does not seem to significantly affect phospholipid fatty acid composition or desaturase activity in patients with type 2 diabetes [5,31]. We found that delta-5 desaturase activity was significantly higher during severe hyperglycemia than at times of good metabolic control, and that this enzyme’s activity significantly correlated with HbA1c in times of poor metabolic control. In our patients with type 2 diabetes, the percentages of arachidonic acid were unaffected by the degree of metabolic control: this may mean that arachidonic acid metabolism is not affected in conditions of poor metabolic control in type 2 diabetes, as in type 1 diabetes, i.e. that the concentration of arachidonic acid is
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preserved in type 2, but not in type 1 diabetes in poor metabolic control. Arachidonic acid may have a crucial role in maintaining the appropriate mass and function of islet beta cells by influencing cell proliferation rates and insulin secretion. This regulatory effect may be disrupted by high circulating levels of glucose and/or palmitic acid, which has a lipotoxic effect on the pancreatic beta cells [28,29]. Our results seem to indicate that delta-5 desaturase activity in type 2 diabetes is not only regulated by long-chain polyunsaturated fatty acid, insulin and physical activity [5], but it also depends on the degree of metabolic control, being particularly affected by conditions of poor metabolic control. Conversely, the activity of elongase and delta-9 desaturase does not seem to be influenced by the level of metabolic control. In conclusion, changes in plasma phospholipid fatty acid composition and desaturase activity in type 2 diabetic patients go in the opposite direction to those seen in similar conditions in type 1 diabetic patients. These differences may be important for a better understanding of the role of metabolic control in the betacell physiology and the progression of chronic complications in patients with type 2 diabetes. References [1] T.A. Miettinen, V. Naukkarinen, J.K. Huttunen, S. Mattila, T. Kumlin, Fatty-acid composition of serum lipids predicts myocardial infarction, Br. Med. J. 285 (1982) 993–996. [2] R.R. Brenner, A.M. Bernasconi, H.A. Garda, Effect of experimental diabetes on the fatty acid composition, molecular species of phosphatidyl-choline and physical properties of hepatic microsomal membranes, Prostaglandins Leukot. Essent. Fatty Acids 63 (2000) 167–176. [3] A. Kalofoutis, J. Lekakis, Changes of platelet phospholipids in diabetes mellitus, Diabetologia 21 (1981) 540–543. [4] T. Pelikanova, M. Kohout, J. Valek, J. Base, Z. Stefka, Fatty acid composition of serum lipids and erythrocyte membranes in type 2 (non-insulin-dependent) diabetic men, Metabolism 40 (1991) 175–180. [5] B. Vessby, I.-B. Gustaffsson, S. Tengblad, M. Boberg, A. Andersson, Desaturation and elongation of fatty acids and insulin action, Ann. N. Y. Acad. Sci. 967 (2002) 183–195. [6] M. Borkman, L.H. Storlien, D.A. Pan, A.B. Jenkins, D.J. Chisholm, L.V. Campbell, The relation between insulin sensitivity and the fatty-acid composition of skeletal-muscle phospholipids, N. Engl. J. Med. 328 (1993) 238–244. [7] E. Warensjo¨, U. Rise´rus, B. Vessby, Fatty acid composition of serum lipids predicts the development of the metabolic syndrome in men, Diabetologia 48 (2005) 1999–2005. [8] L. Wang, A.R. Folsom, J.H. Eckfeldt, For the ARIC Study Investigators, plasma fatty acid composition and incidence of coronary heart disease in middleaged adults: the Atherosclerosis Risk in Communities (ARIC) Study, Nutr. Metab. Cardiovasc. Dis. 13 (2003) 256–266. [9] A.Q. Dang, K. Kemp, F.H. Faas, W.J. Carter, Effects of dietary fats on fatty acid composition and D-5 desaturase in normal and diabetic rats, Lipids 24 (1989) 882–889. [10] S. Nishida, T. Kanno, S. Nakagawa, Diabetes-induced and age-related changes in fatty acid proportions of plasma lipids in rats, Lipids 33 (1998) 251–259.
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