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Journal of Diabetes •• (2014) ••–••
O R I G I N A L A RT I C L E
In vitro effects of oil’s fatty acids on T cell function in gestational diabetic pregnant women and their newborns Farah DJELTI,1 Hafida MERZOUK,1 Sid Ahmed MERZOUK2 and Michel NARCE3 1
Laboratory of Physiology, Physiopathology and Biochemistry of Nutrition, Department of Biology, Faculty of Natural and Life Sciences, Department of Technical Sciences, Faculty of Engineering, University Abou-Bekr Belkaïd, Tlemcen, Algeria, and 3INSERM UMR 866, “Lipids Nutrition Cancer”, University of Burgundy, Faculty of Life, Earth and Environment Sciences, Dijon, France 2
Correspondence Hafida Merzouk, Laboratory of Physiology, Physiopathology and Biochemistry of Nutrition, Department of Biology, Faculty of Natural and Life Sciences, Earth and Universe, University Abou-Bekr Belkaïd, Tlemcen 13000, Algeria. Tel: +213 77830 3645 Fax: +213 4321 2145 Email: [email protected] Received 17 March 2014; revised 7 July 2014; accepted 11 August 2014. doi: 10.1111/1753-0407.12210
Abstract Background: The aim of this investigation was to determine the in vitro effects of linseed, olive and Nigel oils on T cell proliferation and function in gestational diabetes. Methods: Blood samples were collected from 40 control healthy and 32 gestational diabetic mothers and their newborns. Peripheral blood lymphocytes were isolated using a density gradient of Ficoll. T cell proliferation, interleukin-2 and -4 (IL-2, IL-4) secretion, fatty acid composition and intracellular oxidative status were investigated. Results: Mitogen (Concanavalin A) stimulated lymphocyte proliferation, IL-2 secretion, intracellular reduced glutathione levels, superoxide dismutase (SOD) and catalase activities were lower while intracellular malondialdehyde (MDA) and carbonyl proteins were higher in diabetic mothers and in their newborns as compared to their respective controls. Linseed oil induced a reduction in T-lymphocyte proliferation and IL-2 production, and alpha linolenic acid membrane enrichment in both diabetic and control groups. In the presence of Nigel oil, T-lymphocyte proliferation and IL-2 secretion, phospholipid linoleic and oleic acids were enhanced. Olive oil had no effect on lymphocyte proliferation in all groups. Linseed, olive and Nigel oils induced an increase in T cell levels of reduced glutathione levels and in activities of catalase and SOD with a concomitant decrease in MDA and carbonyl protein contents. Conclusion: Linseed, olive and Nigel oils had beneficial effects on T cell functions in gestational diabetes. Keywords: gestational diabetes, linseed oil, lymphocytes, Nigel oil, olive oil, oxidant/antioxidant status.
Significant findings of the study: Linseed, olive and Nigel oils improve lymphocyte intracellular oxidative status in gestational diabetes. What this study adds: The sensitivity of neonate lymphocytes to oils was similar and parallel to that in maternal lymphocytes.
Introduction Pregnant women with gestational diabetes have a reduction in insulin sensitivity, hyperglycemia, and hyperlipidemia.1–3 Gestational diabetes is associated with
oxidative stress, as a result of increased formation of reactive oxidative substances and reduced antioxidant defense mechanisms.4–6 In addition, the immunologic responses by the maternal immune system during pregnancy are not as well-
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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regulated in gestational diabetes patients as in healthy pregnant women.7–9 Abnormalities of T cell function are occurring during diabetes mellitus.10,11 Oxidative stress was found to influence the proliferation, activation, cytokine secretion and T-cell homeostasis.12 Enhanced intracellular free radical production or reduced antioxidant enzyme activity have been shown to induce T lymphocyte oxidative stress.13–15 Modulation of immune system by consumption of vegetable oils protected against some diseases. N-3 polyunsaturated fatty acids (PUFA) improved T cell intracellular oxidative status in diabetes.13 An important source of dietary n-3 PUFA is α-linolenic acid (ALA, C18:3 n-3), supplied by vegetable sources such as linseed oil. Previous research suggests that linseed oil modulates the immune response and may play a beneficial role in the clinical management of autoimmune diseases.16–18 Olive oil, the main source of fat in the Mediterranean diet, is rich in oleic acid (C18:1 n-9), and may have health benefits including immune system, insulin sensitivity and oxidative stress.19,20 Nigel oil, rich in linoleic acid (LA, C18:2 n-6) and oleic acid, possesses anti-inflammatory, immune and anti-oxidant effects through enhancing the oxidant scavenger system.21–23 Specific cellular immunity can be assayed in vitro by mitogen-induced lymphocyte proliferation. We have previously used this methodology to test several chemical substances and the results were quite promising.13–15 The addition of fatty acids in culture medium of immune cells affects various parameters of the immune response.13 Immunomodulators are biological response modifiers that suppress or stimulate lymphocyte proliferation, modulate multiple intercellular and intracellular molecular signals and change Th1- (cell mediated) and Th2(humoral) associated cytokine secretion.13–15 N-3 PUFA have been found to decrease T cell proliferation, interleukin-2 (IL-2) secretion, intracellular enzyme activity, gene transcription, T cell-mediated cytotoxicity, natural killer cell activity, macrophage mediated cytotoxicity, monocyte and neutrophil chemotaxis.24–26 Other studies show that oleic and linoleic acids also modulate Con A stimulated proliferation of lymphocytes isolated from rodent tissues and from human peripheral blood.24,27 To our knowledge, there are no reports in the literature on the effects of gestational diabetes on the in vitro maternal and fetal T cell function, and regulation by fatty acids. Therefore, in the present study we sought to determine the in vitro effects of oil fatty acids on maternal and fetal lymphocyte ability to proliferate and to produce cytokines in response to mitogenic stimuli during gestational diabetes. Also, we described oil fatty acid capacity to 2
modulate the intracellular oxidant/antioxidant status. The oils tested are linseed oil, olive oil, and Nigel oil, which are largely consumed in Algeria. We have chosen to use oils in spite of purified fatty acids to mimic human consumption.
Methods Subjects Forty control healthy pregnant women and 32 pregnant women with established diagnosis of gestational diabetes mellitus and their newborns were selected at the Maternity department of Tlemcen Hospital, Tlemcen, Algeria. The study was approved by the Tlemcen Hospital Committee for Research on Human Subjects. Free and informed consent was obtained from each participant of the study. Gestational diabetes was diagnosed if a 2-h 75-g oral glucose tolerance test value was ≥9.00 mmol/L in capillary whole blood, or if fasting blood glucose was >6.10 mmol/L, between 24 to 28 weeks of gestation. Pregnant mothers with pre-existing diabetes (Type 1 or Type 2) were excluded. Control pregnant women had no previous history of diabetes and no evidence of diabetes in the current pregnancy. All women were routinely seen by an obstetrician and a diabetologist. Care was taken to ensure that all the pregnant women were of similar age, gestational age and parity. Gestational age was estimated by the last menstrual period and was confirmed by a first-trimester ultrasound scan. Newborn weight was recorded immediately after delivery. The characteristics of study population are given in Table 1. Blood samples and analysis Fasting maternal blood samples were obtained from the arm veins of the mothers. Cord blood samples were obtained from the umbilical vein immediately following delivery and after the cutting of the umbilical cord.
Table 1
Maternal and neonate characteristics
Characteristics
Control
Diabetes
Number Age (years) Maternal prepregnancy BMI (kg/m2) Parity Gestational age (weeks) Birth weight (g) 5 min Apgar score M/F sex ratio
40 27.35 ± 4.93 22.80 ± 2.32 3±1 38.40 ± 0.50 3445 ± 390 9.2 ± 0.3 20 / 20
32 28.25 ± 3.01 21.14 ± 1.70 2±1 38.50 ± 0.40 3980 ± 350 8.7 ± 0.5 15 / 17
Values are means ± standard deviation (SD). BMI, body mass index (weight/height2); M/F, males/females.
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
F. DJELTI et al.
Blood samples were collected in two heparinized tubes from each patient. One blood sample was used for hematological parameters with an automated method using hematology auto analyzer Sysmex KX-21N, and plasma was collected for analysis of glucose, glycosylated hemoglobin (Hb A1C) and lipids. The other blood sample was used for lymphocyte separation. Plasma glucose, triglycerides and total cholesterol were determined by enzymatic methods (Kits Sigma Chemical Company, St Louis, MO, USA). Glycosylated hemoglobin was quantified using cation- exchange resin (Kit Fortress Diagnostics, UK). Lymphocyte proliferation assay Peripheral lymphocytes were separated from venous blood by Histopaque 1077 (Sigma Aldrich), as previously reported.13–15 The cells were washed and suspended in complete RPMI 1640 medium with or without mitogen, Concanavalin, ConA (Sigma Aldrich). Cultures were established in triplicate in microtiter plates and incubated at 37°C in a 5% CO2 humidified atmosphere for 48 h. To test the in vitro effects of oils, lymphocytes were incubated with linseed, olive and Nigel oils (final concentration 30 μM TG). The fatty acid composition of linseed oil was 10% saturated fatty acid (SFA), 18% C18:1 n-9, 12% C18:2 n-6 and 58% C18:3 n-3. The major fatty acids of Nigel oil were 16% SFA, 23% C18:1 n-9 and 58% C18:2 n-6. Those of olive oil were 14% SFA, 73% C18:1 n-9 and 9% C18:2 n-6. At the end of the incubation, the cells were removed by centrifugation at 1500 rpm for 10 min; the supernatants were collected for cytokine content. The viable cell number was determined by counting trypan blue unstained cells in a hemocytometer and was over 80% for all cultures. The proliferation of T cells was measured by colorimetric reading of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide reduction, as described previously.14,28 Stimulation index (SI) was expressed as percentage of the control. Interleukin-2 and −4 contents in the supernatants The concentration of IL-2, and IL-4 in the supernatants, was tested using ELISA kits specific for human cytokines (R & D Systems, Oxford, UK), as detailed in the manufacturer’s instructions. Lymphocyte phospholipid fatty acids Lymphocyte phospholipids fatty acid composition was analyzed by gas–liquid chromatography as previously reported.13,29
Oils and lymphocytes in gestational diabetes
Lymphocyte oxidative stress determination Determination of GSH content T cell reduced glutathione (GSH) content was assayed using a Bioxytech GSH-400 kit (OXIS International, Inc., Portland, OR, USA). Determinations of lymphocyte antioxidant enzyme activities Lymphocyte catalase (CAT, EC 1.11.1.6) activity was measured by spectrophotometric analysis of the rate of hydrogen peroxide decomposition at 240 nm.30 The activity of superoxide dismutase (SOD) was measured by the NADPH oxidation procedure.31 Determination of lymphocyte malondialdehyde Lymphocyte malondialdehyde (MDA) levels, a marker of lipid peroxidation, were determined by the reaction of MDA with thiobarbituric acid.32 Determination of lymphocyte carbonyl proteins Lymphocyte carbonyl proteins (markers of protein oxidation) were assayed by the 2,4-dinitrophenyl hydrazine reaction.33 Statistical analysis All values are given as means ± SD. Data were analyzed using the student’s t-test to compare between two groups. For multiple comparisons, a one way analysis of variance (ANOVA) followed by the LSD (least significant difference) test were used. The level of statistical significance was set at P < 0.05. All analyses were performed with STATISTICA (Statsoft, Paris, France).
Results Maternal and neonate hematological and biochemical parameters Hematological and biochemical parameters are shown in Table 2. No significant differences in white and red blood cell, platelet, haemoglobin, granulocyte and monocyte values were detected between diabetic pregnant women and healthy subjects. These values were also similar in both newborn groups. Lymphocytes were significantly low and Hb A1C levels were significantly high among the diabetic group compared to controls. As expected, significant increase in plasma glucose levels were found in diabetic group compared to controls. No significant differences were found concerning plasma cholesterol and triglyceride concentrations between maternal and neonate groups.
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Oils and lymphocytes in gestational diabetes Table 2
F. DJELTI et al.
Maternal and neonate hematological and biochemical parameters Control group
WBC (103/mL) RBC (106/mL) Platelet (103/mL) Hemoglobin (g/dL) Hb A1c (%) Monocytes (%) Lymphocytes (%) Granulocytes (%) Glucose (g/L) Triglycerides (g/L) Cholesterol (g/L)
Diabetic group
Mothers
Newborns
Mothers
Newborns
7.97 ± 1.56 4.44 ± 0.43 282.70 ± 52.11 12.01 ± 0.88 4.43 ± 0.25 5.12 ± 1.67 25.47 ± 4.98 66.99 ± 4.39 0.77 ± 0.05 1.93 ± 0.13 2.43 ± 0.18
14.36 ± 1.90 4.25 ± 0.38 290.41 ± 43.81 13.40 ± 0.96 2.71 ± 0.48 4.37 ± 1.25 33.62 ± 3.66 61.70 ± 3.60 0.66 ± 0.03 0.62 ± 0.04 0.53 ± 0.07
8.53 ± 1.01 4.13 ± 0.49 219 ± 53.34 11.64 ± 1.22 7.11 ± 0.42** 7.07 ± 2.47 17.90 ± 2.38** 73.75 ± 4.01 1.42 ± 0.19** 1.95 ± 0.11 2.47 ± 0.17
16.59 ± 2.28 4.36 ± 0.29 238 ± 49.73 12.55 ± 0.85 4.35 ± 0.37** 6.76 ± 1.96 24.77 ± 1.09* 64.27 ± 3.32 0.75 ± 0.03* 0.64 ± 0.05 0.58 ± 0.05
Values are means ± standard deviation (SD). HBA1c, glycosylated hemoglobin; RBC, red blood cells; WBC, white blood cells. Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test. *P < 0.01; **P < 0.001.
Figure 1 T cell proliferation in the presence of oil fatty acids in mothers and their newborns. The values are means ± standard deviation (SD) of triplicate assays. Multiple comparisons were performed using ANOVA followed by the least significant difference (LSD) test. a, b, c . . . indicate significant differences obtained with different incubations in the same group (P < 0.05). Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test: *P < 0.01; **P < 0.001.
In vitro T cell proliferation in the presence of different oil fatty acids Con A stimulated cell proliferation, reflected by stimulation index (SI), was found to be significantly decreased in gestational diabetic mothers and in their newborns, compared to their respective controls (Fig. 1). The presence of insulin in the culture potentiated T cell proliferation in all groups; which remained significantly diminished in diabetic group in comparison with control group. Lymphocyte proliferation stimulated with con A showed a significant increase when treated with Nigel oil for both diabetic and control groups. Addition of Olive oil had no effects on stimulation index. Nonetheless, T 4
cell proliferation was always reduced in diabetic mothers and in their newborns compared to their respective controls. In the presence of linseed oil, lymphocyte proliferative response to Con A stimulation was reduced in both diabetic and control groups. With linseed oil, diabetic mothers and their newborns showed similar T cell proliferative response to Con A stimulation compared with their respective controls. Interleukin release in the presence of oil fatty acids After incubations, the supernatant IL-2 content was measured as Th1 cytokine, while IL-4 was measured as Th2 cytokine (Fig. 2). Lymphocytes isolated from
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
F. DJELTI et al.
Oils and lymphocytes in gestational diabetes
Figure 2 T cell interleukin production in mothers and their newborns. The values are means ± standard deviation (SD) of triplicate assays. Multiple comparisons were performed using ANOVA followed by the least significant difference (LSD) test. a, b, c . . . indicate significant differences obtained with different incubations in the same group (P < 0.05). Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test: *P < 0.01; **P < 0.001.
diabetic mothers and their newborns secreted less IL-2 after mitogen stimulation compared to their respective controls. No significant differences were found between IL-4 secretions by Con A stimulated lymphocytes from diabetic and control groups. The addition of Nigel oil induced a significant increase in IL-2 production by Con A stimulated lymphocytes but no effect on IL-4 release in both diabetic and control groups. Nonetheless, IL-2 secretion was still significantly reduced in diabetic groups in comparison with controls. The capacity of T cells to produce IL-2 after stimulation with Con A was markedly reduced after exposure to linseed oil for all groups. IL-4 release did not change in response to linseed oil. We observed that lymphocytes from diabetic mothers and from their newborns, when cultured with linseed oil, secreted similar IL-2 and IL-4 amounts than control mothers and their newborns. The addition of olive oil to the medium had no effect on IL-2 and IL-4 secretions in all groups. Lymphocyte fatty acid profile in the presence of oil fatty acids The fatty acid composition of lymphocyte phospholipids showed several alterations in diabetic mothers compared to control mothers (Table 3). In fact, a statistical rise in C18:2n-6 proportions and a significant fall in C20:4n-6 proportions were observed in gestational diabetic
mother lymphocytes compared to control values. Fatty acid profile was restored by insulin in diabetic mothers. Fatty acid composition of lymphocytes did not differ significantly between the two groups of newborns. As expected, linseed oil induced a significant increase in C18:3 n-3 and C20:5 n-3 associated to a significant decrease in C18:1 n-9 and C20:4 n-6 proportions in all groups. Nigel oil induced a significant increase in the proportions of C18:1 n-9, C18:2 n-6 and C20:4 n-6 with a reduction in SFA levels. In the presence of olive oil, the proportion of C18:1 n-9 was increased while SFA proportion was decreased in all groups. Lymphocyte oxidative stress variables As shown in Fig. 3, MDA and carbonyl protein levels were significantly increased in T lymphocytes from diabetic mothers and their newborns compared to control values. In the presence of insulin, MDA and carbonyl protein levels were higher than values in Con A stimulated lymphocytes from all groups. Addition of Nigel oil, linseed oil or olive oil in the culture medium produced a significant decrease in MDA and carbonyl protein levels in all groups. In the presence of these oils, MDA and carbonyl protein levels became similar in diabetic and control groups.
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Oils and lymphocytes in gestational diabetes Table 3
F. DJELTI et al.
Fatty acid composition of maternal and neonatal ConA-stimulated T lymphocytes Control group
Fatty acids SFA Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil C18:1 n-9 Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil C18:2 n-6 Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil C18:3 n-3 Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil C20:4 n-6 Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil C20:5 n-3 Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil
Diabetic group
Mothers
Newborns
Mothers
Newborns
51.20 ± 1.15a 49.50 ± 2.61a 50.11 ± 2.11a 40.42 ± 1.76c 46.33 ± 1.12b
48.67 ± 1.33a 49.02 ± 2.19a 49.73 ± 2.52a 42.33 ± 1.74b 43.76 ± 1.41b
52.30 ± 2.33a 51.18 ± 1.85a 51.43 ± 1.66a 39.03 ± 1.58c 45.05 ± 1.75b
48.06 ± 1.21a 49.04 ± 2.38a 49.75 ± 1.42a 42.65 ± 1.77b 44.06 ± 1.63b
16.14 ± 1.24c 17.48 ± 1.33c 11.74 ± 1.02d 20.06 ± 1.26b 23.55 ± 1.04a
17.03 ± 1.22c 17.74 ± 1.53c 13.03 ± 1.32d 20.11 ± 1.15b 24.95 ± 1.28a
16.63 ± 1.56c 17.22 ± 1.72c 10.68 ± 1.01d 21.02 ± 1.35b 23.06 ± 1.20a
17.32 ± 1.52c 16.98 ± 1.42c 13.98 ± 1.37d 20.73 ± 1.51b 24.84 ± 1.38a
10.58 ± 1.07b 10.74 ± 1.65b 9.93 ± 0.86b 16.77 ± 1.27a 9.07 ± 1.02b
8.11 ± 0.56b 8.56 ± 0.57b 8.71 ± 0.44b 12.33 ± 0.83a 8.82 ± 0.66b
15.20 ± 1.03*b 12.20 ± 2.15c 13.35 ± 1.11*c 18.83 ± 1.04*a 15.55 ± 1.21*b
9.46 ± 0.44b 9.11 ± 0.63b 8.88 ± 0.51b 12.83 ± 0.67a 9.02 ± 0.34b
1.38 ± 0.39b 1.36 ± 0.40b 9.43 ± 1.33a 1.55 ± 0.41b 1.33 ± 0.37b
1.54 ± 0.11b 1.63 ± 0.23b 7.62 ± 0.38a 1.52 ± 0.23b 1.68 ± 0.37b
1.27 ± 0.21b 1.30 ± 0.28 9.66 ± 0.85a 1.52 ± 0.33b 1.35 ± 0.35b
1.44 ± 0.25b 1.56 ± 0.35b 7.18 ± 0.28a 1.47 ± 0.36b 1.50 ± 0.31b
15.83 ± 1.45b 16.62 ± 1.82b 11.48 ± 1.03c 18.05 ± 1.16a 14.33 ± 1.89b
17.04 ± 1.53b 17.81 ± 1.32b 13.43 ± 1.27c 19.33 ± 1.21a 16.88 ± 1.33b
10.16 ± 0.66*c 14.37 ± 1.61b 8.44 ± 0.65*d 15.83 ± 1.05*a 11.22 ± 0.75*c
16.08 ± 1.11b 17.65 ± 1.34b 13.17 ± 1.26c 19.52 ± 1.21a 16.73 ± 1.36b
2.85 ± 0.48b 2.80 ± 0.55b 4.56 ± 0.73a 2.52 ± 0.33b 2.46 ± 0.31b
1.66 ± 0.31b 1.54 ± 0.26b 5.11 ± 0.42a 1.72 ± 0.28b 1.75 ± 0.34b
2.07 ± 037b 2.65 ± 0.34b 4.93 ± 0.83a 2.31 ± 0.55b 2.22 ± 0.40b
1.57 ± 0.29b 1.58 ± 0.33b 5.25 ± 0.38a 1.60 ± 0.31b 1.64 ± 0.28b
The values are means ± standard deviation (SD). Con A, concanavalin A, mitogen; SFA, saturated fatty acids. Multiple comparisons were performed using ANOVA followed by the least significant difference (LSD) test. a, b, c . . . indicate significant differences obtained with different incubations in the same group (P < 0.05). Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test: *P < 0.01; **P < 0.001.
Reduced glutathione (GSH) levels in Con A stimulated lymphocytes from diabetic mothers and their newborns were lower than control values (Fig. 3). The addition of Nigel oil or linseed oil or olive oil in the culture produced a significant increase in GSH levels in lymphocytes from diabetic mothers and from their newborns, but had no effects on lymphocyte GSH from controls. In the presence of these oils, no significant differences in GSH contents were noted between diabetic and control groups. 6
Con A stimulated lymphocyte catalase and SOD activities were significantly lower in diabetic mothers and their newborns when compared with their respective controls (Table 4). In the presence of insulin, catalase and SOD activities were enhanced in lymphocytes from diabetic and control groups, but were still decreased in diabetics when compared to controls. The treatment with linseed or Nigel or olive oil induced a significant increase in enzyme activities in all groups. Catalase and SOD activities in lymphocytes cultured
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
F. DJELTI et al.
Oils and lymphocytes in gestational diabetes
Figure 3 Lymphocyte malondialdehyde (MDA), glutathione (GSH) and Carbonyl protein levels in mothers and their newborns. The values are means ± standard deviation (SD) of triplicate assays. CARP, protein carbonyls. Multiple comparisons were performed using ANOVA followed by the least significant difference (LSD) test. a, b, c . . . indicate significant differences obtained with different incubations in the same group (P < 0.05). Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test: *P < 0.01; **P < 0.001.
Table 4
Superoxide dismutase (SOD) and catalase activities in maternal and neonatal T lymphocytes Control group
Enzyme Catalase (U/mg) Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil SOD (U/mg) Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil
Mothers
Diabetic group Newborns
Mothers
Newborns
20.13 ± 1.88c 38.06 ± 1.53a 25.82 ± 2.26b 25.61 ± 1.12b 24.13 ± 2.13b
18.53 ± 1.02b 32.96 ± 2.76a 18.15 ± 2.92b 19.26 ± 1.06b 19.54 ± 2.44b
16.34 ± 1.49*b 26.40 ± 1.46*a 27.41 ± 1.92a 26.97 ± 1.23a 24.54 ± 2.77a
14.67 ± 1.19*c 25.75 ± 1.61*a 20.75 ± 1.19b 20.59 ± 1.31b 21.41 ± 2.92b
80.32 ± 2.54b 123.35 ± 6.11a 128 ± 7.42a 125.57 ± 7.17a 128.33 ± 9.31a
44.34 ± 1.13b 72.38 ± 2.42a 70.77 ± 3.17a 73.63 ± 2.88a 71.39 ± 4.15a
56.27 ± 2.31**c 81.03 ± 5.27*b 132.25 ± 8.33a 133.61 ± 9.22a 125.54 ± 6.66a
35.63 ± 1.18*c 56.99 ± 2.11*b 68.06 ± 4.19a 71.72 ± 3.36a 70.62 ± 1.14a
The values are means ± standard deviation (SD). Multiple comparisons were performed using ANOVA followed by the least significant difference (LSD) test. a, b, c . . . indicate significant differences obtained with different incubations in the same group (P < 0.05). Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test. *P < 0.01; **P < 0.001.
with these three oils did not differ between diabetic and control groups. Discussion The results obtained in this in vitro study demonstrate that olive, linseed and Nigel oils modulated maternal and
fetal lymphocyte function after mitogen stimulation with an improvement in some defects observed during gestational diabetes. In the current study, diabetic mothers and their newborns showed similar plasma triglyceride and cholesterol levels compared to their respective controls. However, high fasting glucose concentrations were
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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observed in diabetic mothers and in their newborns compared to controls. Indeed, hematological parameters were normal except for lymphocyte number, which was reduced and HbA1C levels, which were increased in diabetic mothers and also in their newborns compared to their respective controls. Hyperglycemia and elevated concentration of glycosylated hemoglobin are well documented in gestational diabetes.1,34 Maternal hyperglycemia resulted in increased levels of glucose transported across the placenta to the fetus, thus maternal hyperglycemia is fetal hyperglycemia.35 The lower levels of cord blood hemoglobin glycosylation compared with that of maternal hemoglobin is the result of several factors including the shorter fetal red blood cell life span than maternal erythrocyte, the fetal hemoglobin acetylation at the N-terminus of the gamma chain, the lower fetal glucose levels than those in the mother maintaining a maternal-fetal gradient. Serum cholesterol and triglyceride levels in diabetic pregnant women were not statistically significantly different compared to those seen in normal pregnant women.36,37 Previous studies reported a decrease in naïve T cells in diabetic pregnancy.38 Newborns of diabetic mothers have a reduction in NK lymphocytes than control values.8 Our results showed that linseed and Nigel oils have in vitro important modulatory effects on T cell function, with similar kinetic profiles in maternal and neonate lymphocytes. The immunomodulatory effect of olive oil was not evident on maternal and fetal lymphocyte proliferation and cytokine secretion but significant in reducing intracellular oxidative stress. We observed that mitogen-activated lymphocyte proliferation was lower in diabetic mothers and their newborns than that in controls. While IL-4 production by T cells after mitogen stimulation was normal, IL-2 production was reduced in diabetic mothers and in their newborns. These findings were in agreement with previous reports showing that gestational diabetes induced depressed lymphocyte proliferation and IL-2 secretion compared to normal pregnancy.7,8,38 Modulation of T cell function in the presence of insulin was in agreement with our previous findings.13 In the current study, fatty acids from linseed oil significantly reduced in vitro lymphocyte proliferation in both diabetic and control mothers and their newborns, while fatty acids from Nigel oil significantly enhanced it. However, olive oil’s fatty acids had no effects on lymphocyte proliferation in both diabetic and control groups. The effects of oil’s fatty acids on in vitro cytokine production supported those on lymphocyte proliferation. IL-2 is a well characterized Th1 cytokine with central roles in inflammation and immune signaling. IL-4 8
is a Th2 cytokine associated with humoral immunity. The suppressed lymphocyte proliferative response of linseed oil is consistent with previous data showing that n-3 PUFA significantly reduced mitogen lymphocyte proliferation.14,15,24,25,39 Possible explanation for inhibitory effects of linseed oil could be due to the reduction of IL-2 secretion by stimulated lymphocytes, knowing that IL-2 is required for the proliferation of T cells. The alterations in lymphocyte proliferation after oil exposure in the current study could be attributed to the modification of lymphocyte phenotype proportions. We noted that while linseed oil induced a decrease in IL-2 secretion within lymphocytes, it did not affect IL-4 secretion. Th2-like response was nevertheless dominant reflecting probably the anti-inflammatory effect of linseed oil. We have previously shown that n-3 PUFA shift the Th1/Th2 balance toward the Th2 pole by suppression of Th1 development rather than enhancement of Th2 development in type 1 diabetic patients.13 Nigel oil induced a significant increase in lymphocyte proliferation with a concomitant increase in IL-2 secretion in both diabetic and control groups. Since IL-4 levels were not affected and IL-2 levels were increased, Nigel oil appeared to have generated a Th1-like phenotype. Our findings are in agreement with previous studies showing immunostimulation with Nigel oil in healthy and in diabetic patients.22,23 Olive oil had no effects on cytokine production in both control and diabetic groups. Previous studies did not find significant differences in the proliferation of lymphocytes from humans or animals fed with a diet containing olive oil.40,41 Similar immune effects have been observed with in vitro olive oil showing no effect on lymphocyte proliferation and the production of IL-2.42 The levels of C18:1 n-9 were increased while SFAs were decreased in olive oil treated lymphocytes compared with non-treated lymphocytes in all groups. Our results showed also that a high level of C18:3 n-3 in linseed oil induced an increase of C18:3 n-3 and C20:5 n-3 acids with a concomitant decrease of C18:1 n-9 and C20:4 n-6 acids in the lymphocyte phospholipid membranes of mothers and their newborns. High levels of linoleic and oleic acids in Nigel oil induced an increase in linoleic, oleic and arachidonic acids with a concomitant decrease in SFAs in the phospholipid membranes. Previous studies found a significant correlation between lymphocyte function and the oleic acid present in the phospholipids of lymphocyte membranes.43 Our results suggest that the immunosuppressive effect of linseed oil might be due to a decrease of oleic acid in the lymphocyte membrane. Suppressed proliferation response in the presence of linseed oil may be also due to arachidonic acid (AA) depletion in lymphocyte membrane phospho-
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
F. DJELTI et al.
lipids as observed in our study. T cell activation implicates AA liberation and its metabolism.44 In the same way, the activation of lymphocyte proliferation by Nigel oil could be linked to oleic acid and AA membrane enrichment. However, in the case of olive oil, despite the increase in oleic acid, there was no effect on lymphocyte proliferation. Then, the concomitant enrichment in n-6 fatty acids and oleic acid of lymphocyte phospholipids was important to exert a stimulatory effect. In this study, lymphocyte redox markers were consistent with the presence of an oxidative stress in gestational diabetes. Lymphocyte GSH levels, catalase and SOD activities were significantly reduced in diabetic mothers and their newborns compared to controls. These findings reflected a diminished antioxidant defense in gestational diabetes, in agreement with previous studies.4,5,45 MDA and carbonyl protein levels were increased in lymphocytes from diabetic mothers and their newborns resulting probably from overproduction of free radicals and disturbance of the scavenging system. Our results were similar to findings reported in earlier studies showing an imbalance in the pro and anti-oxidant status in gestational diabetes.4,5,45 In the current study, we found that MDA and protein carbonyl contents were decreased while catalase and SOD activities were increased in linseed oil treated lymphocytes from control and diabetic groups. No changes in the levels of GSH were observed in control T cells cultured in the presence of linseed oil. However, in diabetic mothers and their newborns, addition of linseed oil to culture induced an increase in T cell levels of GSH. We observed that in the presence of linseed oil, the T cell redox status from diabetic groups was normalized to control values. We suggested that linseed oil regulated T cell redox status during gestational diabetes. Similar findings were reported in previous studies showing that linseed oil enhanced antioxidant capacity in diabetic rat tissue cells.18 Linseed oil reduced MDA and GSSG level and maintained GSH level in diabetic erythrocytes.46 In the presence of Nigel and olive oils, oxidant/ antioxidant status was also improved in lymphocytes from diabetic mothers and their newborns. In fact, Nigel and olive oils induced a significant reduction in lymphocyte MDA and carbonyl protein levels with a concomitant increase in lymphocyte GSH and catalase and SOD activities in gestational diabetes. Our results are in agreement with earlier reports showing beneficial effects of Nigel and olive oils on oxidative stress.19–22 In diabetes, Nigel oil prevents lipid peroxidation and increases antioxidant defense system activity.47 Olive oil decreased the diabetes-induced increases in tissue pro-oxidants and restored the antioxidant status.48
Oils and lymphocytes in gestational diabetes
Interestingly, the sensitivity of neonate lymphocytes to the oils used was similar and parallel to that in maternal lymphocytes. Linseed, olive and Nigel oils had then beneficial effects on lymphocytes from mothers and their newborns. It has been demonstrated that oxidative modification of cellular macromolecules can induce cell apoptosis and cell necrosis.46 Increased oxidative stress may suppress insulin receptor activation or decrease the translocation of glucose transporter-4 (GLUT-4) on the cell membrane.18 Linseed, olive and Nigel oils may have then protective effect on cell damage and beneficial effect on insulin sensibility in gestational diabetes through the scavenging of free radicals and increase in antioxidant capacity. In conclusion, the results obtained in this research showed that gestational diabetes affected T lymphocyte proliferation, IL-2 production, fatty acid composition and oxidant/antioxidant status in mothers and their newborns. Linseed oil possessed immunosuppressive properties while Nigel oil had immunostimulatory effect on T cells. Olive oil had no effects on lymphocyte proliferation or cytokine secretion. The three oils used had positive effects on oxidative stress, especially by reducing lymphocyte MDA and carbonyl protein levels and enhancing GSH and antioxidant enzyme activities. Linseed, olive and Nigel oils improved T cell intracellular oxidative status in gestational diabetes. The beneficial effects of these oils were apparent in mothers and in their newborns. Acknowledgments This work was supported by the Algerian Research Project (PNR, 2011) from the Algerian Health investigation office (ATRSS). Disclosure None declared. References 1. Lindsay RS. Gestational diabetes: Causes and consequences. Br J Diabet Vasc Dis. 2009; 9: 27–31. 2. Buchanan TA, Xiang AH. Gestational diabetes mellitus. J Clin Invest. 2005; 115: 485–91. 3. Butte NF. Carbohydrate and lipid metabolism in pregnancy: Normal compared with gestational diabetes mellitus. Am J Clin Nutr. 2000; 71: 1256–61. 4. Djordjevic A, Spasic S, Jovanovic-Galovic A, Djordjevic R, Grubor-Lajsic G. Oxidative stress in diabetic pregnancy: SOD, CAT and GSHPx activity and lipid peroxidation products. J Matern Fetal Neonat Med. 2004; 16: 367–72.
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5. Lappas M, Hiden U, Desoye G et al. The role of oxidative stress in the pathophysiology of gestational diabetes mellitus. Antioxid Redox Signal. 2011; 15: 3061– 100. 6. Coughlan MT, Vervaart PP, Permezel M, Georgiou HM, Rice GE. Altered placental oxidative stress status in gestational diabetes mellitus. Placenta. 2004; 25: 78–84. 7. Mahmoud F, Abul H, Omu A, Haines D. Lymphocyte sub-populations in gestational diabetes. Am J Reprod Immunol. 2005; 53: 21–9. 8. Lapolla A, Dalfrà MG, Sanzari M et al. Lymphocyte subsets and cytokines in women with gestational diabetes mellitus and their newborn. Cytokine. 2005; 31: 280–7. 9. Shah BR, Hux JE. Quantifying the risk of infectious diseases for people with diabetes. Diabetes Care. 2003; 26: 510–13. 10. Otton R, Mendonca JR, Curi R. Diabetes causes marked changes in lymphocyte metabolism. J Endocrinol. 2002; 174: 55–61. 11. Lindley S, Dayan CM, Bishop A, Roep BO, Peakman M, Tree TIM. Defective suppressor function in CD4_ CD25_T-Cells from patients with type 1 diabetes. Diabetes. 2005; 54: 92–9. 12. Cope AP. Studies of T-cell activation in chronic inflammation. Arthritis Res. 2002; 4: 197–211. 13. Merzouk SA, Saker M, Briksi Reguig K et al. N-3 Polyunsaturated fatty acids modulate in-vitro T cell function in type I diabetic patients. Lipids. 2008; 43: 485–97. 14. Medjdoub A, Merzouk SA, Merzouk H, Chiali FZ, Narce M. 2011). Effects of Mancozeb and Metribuzin on in vitro proliferative responses and oxidative stress of human and rat spleen lymphocytes stimulated by mitogens. Pestic Biochem Physiol. 2011; 101: 27–33. 15. Baba Hamed Y, Medjdoub A, Mostefa Kara B, Merzouk H, Villemin D, Narce M. 5,6-Dihydro-2H-pyranones and 5,6-dihydro-2H-pyridones and their derivatives modulate in vitro human T lymphocyte function. Mol Cell Biochem. 2012; 360: 23–33. 16. Park HJ, Park JS, Hayek MG, Reinhart GA, Chew BP. Dietary fish oil and flaxseed oil suppress inflammation and immunity in cats. Vet Immunol Immunopathol. 2011; 141: 301–6. 17. Conners WE. Importance of n-3 fatty acids in health and disease. Am J Clin Nutr. 2000; 71: 171–5. 18. Hussein J, El-Khayat Z, Taha M, Morsy S, Drees E, Khateeb S. Insulin resistance and oxidative stress in diabetic rats treated with flaxseed oil. J Med Plants Res. 2012; 42: 5499–506. 19. Covas MI, Ruiz-Gutiérrez V, De la Torre R et al. Minor components of olive Oil: Evidence to date of health benefits in humans. Nutr Rev. 2006; 64: 20–30. 20. Bogani P, Galli C, Villa M, Visioli F. Postprandial antiinflammatory and antioxidant effects of extra virgin olive oil. Atherosclerosis. 2007; 190: 181–6. 21. Burits M, Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytother Res. 2000; 14: 323–8. 22. Salem ML. Immunomodulatory and therapeutic properties of the Nigella sativa L. seed. Int Immunopharmacol. 2005; 5: 1749–70. 10
23. Ali BH, Blunden G. Pharmacological and toxicological properties of Nigella sativa. Phytother Res. 2003; 17: 299– 305. 24. Calder PC. Polyunsaturated fatty acids, inflammation, and immunity. Lipids. 2001; 36: 1007–24. 25. Stulnig TM. Immunomodulation by polyunsaturated fatty acids: Mechanisms and effects. Int Arch Allergy Immunol. 2003; 132: 310–21. 26. Raza Shaikh S, Edidin M. Immunosuppressive effects of polyunsaturated fatty acids on antigen presentation by human leukocyte antigen class I molecules. J Lipid Res. 2007; 48: 127–38. 27. Calder PC. Effects of fatty acids and dietary lipids on cells of the immune system. Proc Nutr Soc. 1996; 55: 127– 50. 28. Mossman T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65: 55–63. 29. Merzouk S, Hichami A, Sari A et al. Impaired oxidant/ antioxidant status and LDL-fatty acid composition are associated with increased susceptibility to peroxidation of LDL in diabetic patients. Gen Physiol Biophys. 2004; 23: 387–99. 30. Aebi H. Catalase. In: Bergmeyer HU (ed.). Methods of Enzymatic Analysis, 2nd edn. Verlag Chemie GmbH, Weinheim, 1974, 673–84. 31. Elstner EF, Youngman RJ, Obwald W. Superoxide dismutase. In: Bergmeyer HU (ed.). Methods of Enzymatic Analysis, 3rd edn. Verlag Chemie GmbH, Weinheim, 1983, 293–302. 32. Draper H, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 1990; 186: 421–31. 33. Levine RL, Garland D, Oliver CN et al. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990; 186: 464–78. 34. Khan R, Ali K, Khan Z, Ahmad T. Lipid profile and glycosylated hemoglobin status of gestational diabetic patients and healthy pregnant women. Indian J Med Sci. 2012; 66: 149–54. 35. Persaud ODD. Maternal Diabetes and the consequences for her Offspring. J Dev Disabil. 2007; 13: 101–33. 36. Vitoratos N, Kassanos D, Salamalekis E, Sirisratidis CH, Baimacou E, Creatsas G. Maternal homocysteine levels and plasma lipids in gestational diabetes: Is there any relationship? J Obstet Gynaecol. 2002; 22: 366–9. 37. Bartha JL, Comino-Delgado R, Martinez-Del-Fresno P, Fernandez-Barrios M, Bethencourt I, Moreno-Corral L. Insulin-sensitivity index and carbohydrate and lipid metabolism in gestational diabetes. J Reprod Med. 2000; 45: 185–9. 38. Mahmoud F, Abul H, Dashti A, Al-Jassar W, Omu A. Trace elements and cell-mediated immunity in gestational and pre-gestational diabetes mellitus at third trimester of pregnancy. Acta Med Acad. 2012; 41: 175–85. 39. Thies F, Nebe-von-Caron G, Powell JR, Yaqoob P, Newsholme EA, Calder PC. Dietary supplementation with g-linolenic acid or fish oil decreases T lymphocyte proliferation in healthy older humans. J Nutr. 2001; 131: 1918–27.
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40. Yaqoob P, Knapper JA, Webb DH, Williams CM, Newsholme EA, Calder PC. Effect of olive oil on immune function in middle-aged men. Am J Clin Nutr. 1998; 67: 129 –35. 41. De Pablo MA, Puertollano MA, Álvarez de Cienfuegos G. Olive oil and immune system functions: Potential involvement in immunonutrition. Grasas Aceites. 2004; 55: 42–51. 42. Granato D, Blum S, Rossle C, Le Boucher J, Malnoe A, Dutot G. Effects of parenteral lipid emulsions with different fatty acid composition on immune cell functions in vitro. JPEN J Parenter Enteral Nutr. 2000; 24: 113–18. 43. Moussa M, Tkaczuk J, Ragab J et al. Relationship between the fatty acid composition of rat lymphocytes and immune functions. Br J Nutr. 2000; 83: 327–33. 44. Raza Shaikh S, Edidin M. Polyunsaturated fatty acids, membrane organization, T cells, and antigen presentation. Am J Clin Nutr. 2006; 84: 1277–89.
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45. Biri A, Onan A, Devrim E, Babacan F, Kavutcu M, Durak I. Oxidant status in maternal and cord plasma and placental tissue in gestational diabetes. Placenta. 2006; 27: 327–32. 46. Yang W, Fu J, Yu M et al. Effects of flaxseed oil on anti-oxidative system and membrane deformation of human peripheral blood erythrocytes in high glucose level. Lipids Health Dis. 2012; 11: 88–98. 47. Mathura ML, Gaura J, Sharmaa R, Haldiyaa KR. Antidiabetic properties of a spice plant Nigella sativa. J Endocrinol Metab. 2011; 1: 1–8. 48. Abo Ghanema II, Sadek KM. Olive Leaves Extract Restored the antioxidant perturbations in red blood cells hemolysate in streptozotocin induced diabetic rats. Eng Technol. 2012; 6: 134–40.
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Journal of Diabetes •• (2014) ••–••
O R I G I N A L A RT I C L E
In vitro effects of oil’s fatty acids on T cell function in gestational diabetic pregnant women and their newborns Farah DJELTI,1 Hafida MERZOUK,1 Sid Ahmed MERZOUK2 and Michel NARCE3 1
Laboratory of Physiology, Physiopathology and Biochemistry of Nutrition, Department of Biology, Faculty of Natural and Life Sciences, Department of Technical Sciences, Faculty of Engineering, University Abou-Bekr Belkaïd, Tlemcen, Algeria, and 3INSERM UMR 866, “Lipids Nutrition Cancer”, University of Burgundy, Faculty of Life, Earth and Environment Sciences, Dijon, France 2
Correspondence Hafida Merzouk, Laboratory of Physiology, Physiopathology and Biochemistry of Nutrition, Department of Biology, Faculty of Natural and Life Sciences, Earth and Universe, University Abou-Bekr Belkaïd, Tlemcen 13000, Algeria. Tel: +213 77830 3645 Fax: +213 4321 2145 Email: [email protected] Received 17 March 2014; revised 7 July 2014; accepted 11 August 2014. doi: 10.1111/1753-0407.12210
Abstract Background: The aim of this investigation was to determine the in vitro effects of linseed, olive and Nigel oils on T cell proliferation and function in gestational diabetes. Methods: Blood samples were collected from 40 control healthy and 32 gestational diabetic mothers and their newborns. Peripheral blood lymphocytes were isolated using a density gradient of Ficoll. T cell proliferation, interleukin-2 and -4 (IL-2, IL-4) secretion, fatty acid composition and intracellular oxidative status were investigated. Results: Mitogen (Concanavalin A) stimulated lymphocyte proliferation, IL-2 secretion, intracellular reduced glutathione levels, superoxide dismutase (SOD) and catalase activities were lower while intracellular malondialdehyde (MDA) and carbonyl proteins were higher in diabetic mothers and in their newborns as compared to their respective controls. Linseed oil induced a reduction in T-lymphocyte proliferation and IL-2 production, and alpha linolenic acid membrane enrichment in both diabetic and control groups. In the presence of Nigel oil, T-lymphocyte proliferation and IL-2 secretion, phospholipid linoleic and oleic acids were enhanced. Olive oil had no effect on lymphocyte proliferation in all groups. Linseed, olive and Nigel oils induced an increase in T cell levels of reduced glutathione levels and in activities of catalase and SOD with a concomitant decrease in MDA and carbonyl protein contents. Conclusion: Linseed, olive and Nigel oils had beneficial effects on T cell functions in gestational diabetes. Keywords: gestational diabetes, linseed oil, lymphocytes, Nigel oil, olive oil, oxidant/antioxidant status.
Significant findings of the study: Linseed, olive and Nigel oils improve lymphocyte intracellular oxidative status in gestational diabetes. What this study adds: The sensitivity of neonate lymphocytes to oils was similar and parallel to that in maternal lymphocytes.
Introduction Pregnant women with gestational diabetes have a reduction in insulin sensitivity, hyperglycemia, and hyperlipidemia.1–3 Gestational diabetes is associated with
oxidative stress, as a result of increased formation of reactive oxidative substances and reduced antioxidant defense mechanisms.4–6 In addition, the immunologic responses by the maternal immune system during pregnancy are not as well-
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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regulated in gestational diabetes patients as in healthy pregnant women.7–9 Abnormalities of T cell function are occurring during diabetes mellitus.10,11 Oxidative stress was found to influence the proliferation, activation, cytokine secretion and T-cell homeostasis.12 Enhanced intracellular free radical production or reduced antioxidant enzyme activity have been shown to induce T lymphocyte oxidative stress.13–15 Modulation of immune system by consumption of vegetable oils protected against some diseases. N-3 polyunsaturated fatty acids (PUFA) improved T cell intracellular oxidative status in diabetes.13 An important source of dietary n-3 PUFA is α-linolenic acid (ALA, C18:3 n-3), supplied by vegetable sources such as linseed oil. Previous research suggests that linseed oil modulates the immune response and may play a beneficial role in the clinical management of autoimmune diseases.16–18 Olive oil, the main source of fat in the Mediterranean diet, is rich in oleic acid (C18:1 n-9), and may have health benefits including immune system, insulin sensitivity and oxidative stress.19,20 Nigel oil, rich in linoleic acid (LA, C18:2 n-6) and oleic acid, possesses anti-inflammatory, immune and anti-oxidant effects through enhancing the oxidant scavenger system.21–23 Specific cellular immunity can be assayed in vitro by mitogen-induced lymphocyte proliferation. We have previously used this methodology to test several chemical substances and the results were quite promising.13–15 The addition of fatty acids in culture medium of immune cells affects various parameters of the immune response.13 Immunomodulators are biological response modifiers that suppress or stimulate lymphocyte proliferation, modulate multiple intercellular and intracellular molecular signals and change Th1- (cell mediated) and Th2(humoral) associated cytokine secretion.13–15 N-3 PUFA have been found to decrease T cell proliferation, interleukin-2 (IL-2) secretion, intracellular enzyme activity, gene transcription, T cell-mediated cytotoxicity, natural killer cell activity, macrophage mediated cytotoxicity, monocyte and neutrophil chemotaxis.24–26 Other studies show that oleic and linoleic acids also modulate Con A stimulated proliferation of lymphocytes isolated from rodent tissues and from human peripheral blood.24,27 To our knowledge, there are no reports in the literature on the effects of gestational diabetes on the in vitro maternal and fetal T cell function, and regulation by fatty acids. Therefore, in the present study we sought to determine the in vitro effects of oil fatty acids on maternal and fetal lymphocyte ability to proliferate and to produce cytokines in response to mitogenic stimuli during gestational diabetes. Also, we described oil fatty acid capacity to 2
modulate the intracellular oxidant/antioxidant status. The oils tested are linseed oil, olive oil, and Nigel oil, which are largely consumed in Algeria. We have chosen to use oils in spite of purified fatty acids to mimic human consumption.
Methods Subjects Forty control healthy pregnant women and 32 pregnant women with established diagnosis of gestational diabetes mellitus and their newborns were selected at the Maternity department of Tlemcen Hospital, Tlemcen, Algeria. The study was approved by the Tlemcen Hospital Committee for Research on Human Subjects. Free and informed consent was obtained from each participant of the study. Gestational diabetes was diagnosed if a 2-h 75-g oral glucose tolerance test value was ≥9.00 mmol/L in capillary whole blood, or if fasting blood glucose was >6.10 mmol/L, between 24 to 28 weeks of gestation. Pregnant mothers with pre-existing diabetes (Type 1 or Type 2) were excluded. Control pregnant women had no previous history of diabetes and no evidence of diabetes in the current pregnancy. All women were routinely seen by an obstetrician and a diabetologist. Care was taken to ensure that all the pregnant women were of similar age, gestational age and parity. Gestational age was estimated by the last menstrual period and was confirmed by a first-trimester ultrasound scan. Newborn weight was recorded immediately after delivery. The characteristics of study population are given in Table 1. Blood samples and analysis Fasting maternal blood samples were obtained from the arm veins of the mothers. Cord blood samples were obtained from the umbilical vein immediately following delivery and after the cutting of the umbilical cord.
Table 1
Maternal and neonate characteristics
Characteristics
Control
Diabetes
Number Age (years) Maternal prepregnancy BMI (kg/m2) Parity Gestational age (weeks) Birth weight (g) 5 min Apgar score M/F sex ratio
40 27.35 ± 4.93 22.80 ± 2.32 3±1 38.40 ± 0.50 3445 ± 390 9.2 ± 0.3 20 / 20
32 28.25 ± 3.01 21.14 ± 1.70 2±1 38.50 ± 0.40 3980 ± 350 8.7 ± 0.5 15 / 17
Values are means ± standard deviation (SD). BMI, body mass index (weight/height2); M/F, males/females.
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Blood samples were collected in two heparinized tubes from each patient. One blood sample was used for hematological parameters with an automated method using hematology auto analyzer Sysmex KX-21N, and plasma was collected for analysis of glucose, glycosylated hemoglobin (Hb A1C) and lipids. The other blood sample was used for lymphocyte separation. Plasma glucose, triglycerides and total cholesterol were determined by enzymatic methods (Kits Sigma Chemical Company, St Louis, MO, USA). Glycosylated hemoglobin was quantified using cation- exchange resin (Kit Fortress Diagnostics, UK). Lymphocyte proliferation assay Peripheral lymphocytes were separated from venous blood by Histopaque 1077 (Sigma Aldrich), as previously reported.13–15 The cells were washed and suspended in complete RPMI 1640 medium with or without mitogen, Concanavalin, ConA (Sigma Aldrich). Cultures were established in triplicate in microtiter plates and incubated at 37°C in a 5% CO2 humidified atmosphere for 48 h. To test the in vitro effects of oils, lymphocytes were incubated with linseed, olive and Nigel oils (final concentration 30 μM TG). The fatty acid composition of linseed oil was 10% saturated fatty acid (SFA), 18% C18:1 n-9, 12% C18:2 n-6 and 58% C18:3 n-3. The major fatty acids of Nigel oil were 16% SFA, 23% C18:1 n-9 and 58% C18:2 n-6. Those of olive oil were 14% SFA, 73% C18:1 n-9 and 9% C18:2 n-6. At the end of the incubation, the cells were removed by centrifugation at 1500 rpm for 10 min; the supernatants were collected for cytokine content. The viable cell number was determined by counting trypan blue unstained cells in a hemocytometer and was over 80% for all cultures. The proliferation of T cells was measured by colorimetric reading of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide reduction, as described previously.14,28 Stimulation index (SI) was expressed as percentage of the control. Interleukin-2 and −4 contents in the supernatants The concentration of IL-2, and IL-4 in the supernatants, was tested using ELISA kits specific for human cytokines (R & D Systems, Oxford, UK), as detailed in the manufacturer’s instructions. Lymphocyte phospholipid fatty acids Lymphocyte phospholipids fatty acid composition was analyzed by gas–liquid chromatography as previously reported.13,29
Oils and lymphocytes in gestational diabetes
Lymphocyte oxidative stress determination Determination of GSH content T cell reduced glutathione (GSH) content was assayed using a Bioxytech GSH-400 kit (OXIS International, Inc., Portland, OR, USA). Determinations of lymphocyte antioxidant enzyme activities Lymphocyte catalase (CAT, EC 1.11.1.6) activity was measured by spectrophotometric analysis of the rate of hydrogen peroxide decomposition at 240 nm.30 The activity of superoxide dismutase (SOD) was measured by the NADPH oxidation procedure.31 Determination of lymphocyte malondialdehyde Lymphocyte malondialdehyde (MDA) levels, a marker of lipid peroxidation, were determined by the reaction of MDA with thiobarbituric acid.32 Determination of lymphocyte carbonyl proteins Lymphocyte carbonyl proteins (markers of protein oxidation) were assayed by the 2,4-dinitrophenyl hydrazine reaction.33 Statistical analysis All values are given as means ± SD. Data were analyzed using the student’s t-test to compare between two groups. For multiple comparisons, a one way analysis of variance (ANOVA) followed by the LSD (least significant difference) test were used. The level of statistical significance was set at P < 0.05. All analyses were performed with STATISTICA (Statsoft, Paris, France).
Results Maternal and neonate hematological and biochemical parameters Hematological and biochemical parameters are shown in Table 2. No significant differences in white and red blood cell, platelet, haemoglobin, granulocyte and monocyte values were detected between diabetic pregnant women and healthy subjects. These values were also similar in both newborn groups. Lymphocytes were significantly low and Hb A1C levels were significantly high among the diabetic group compared to controls. As expected, significant increase in plasma glucose levels were found in diabetic group compared to controls. No significant differences were found concerning plasma cholesterol and triglyceride concentrations between maternal and neonate groups.
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Oils and lymphocytes in gestational diabetes Table 2
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Maternal and neonate hematological and biochemical parameters Control group
WBC (103/mL) RBC (106/mL) Platelet (103/mL) Hemoglobin (g/dL) Hb A1c (%) Monocytes (%) Lymphocytes (%) Granulocytes (%) Glucose (g/L) Triglycerides (g/L) Cholesterol (g/L)
Diabetic group
Mothers
Newborns
Mothers
Newborns
7.97 ± 1.56 4.44 ± 0.43 282.70 ± 52.11 12.01 ± 0.88 4.43 ± 0.25 5.12 ± 1.67 25.47 ± 4.98 66.99 ± 4.39 0.77 ± 0.05 1.93 ± 0.13 2.43 ± 0.18
14.36 ± 1.90 4.25 ± 0.38 290.41 ± 43.81 13.40 ± 0.96 2.71 ± 0.48 4.37 ± 1.25 33.62 ± 3.66 61.70 ± 3.60 0.66 ± 0.03 0.62 ± 0.04 0.53 ± 0.07
8.53 ± 1.01 4.13 ± 0.49 219 ± 53.34 11.64 ± 1.22 7.11 ± 0.42** 7.07 ± 2.47 17.90 ± 2.38** 73.75 ± 4.01 1.42 ± 0.19** 1.95 ± 0.11 2.47 ± 0.17
16.59 ± 2.28 4.36 ± 0.29 238 ± 49.73 12.55 ± 0.85 4.35 ± 0.37** 6.76 ± 1.96 24.77 ± 1.09* 64.27 ± 3.32 0.75 ± 0.03* 0.64 ± 0.05 0.58 ± 0.05
Values are means ± standard deviation (SD). HBA1c, glycosylated hemoglobin; RBC, red blood cells; WBC, white blood cells. Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test. *P < 0.01; **P < 0.001.
Figure 1 T cell proliferation in the presence of oil fatty acids in mothers and their newborns. The values are means ± standard deviation (SD) of triplicate assays. Multiple comparisons were performed using ANOVA followed by the least significant difference (LSD) test. a, b, c . . . indicate significant differences obtained with different incubations in the same group (P < 0.05). Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test: *P < 0.01; **P < 0.001.
In vitro T cell proliferation in the presence of different oil fatty acids Con A stimulated cell proliferation, reflected by stimulation index (SI), was found to be significantly decreased in gestational diabetic mothers and in their newborns, compared to their respective controls (Fig. 1). The presence of insulin in the culture potentiated T cell proliferation in all groups; which remained significantly diminished in diabetic group in comparison with control group. Lymphocyte proliferation stimulated with con A showed a significant increase when treated with Nigel oil for both diabetic and control groups. Addition of Olive oil had no effects on stimulation index. Nonetheless, T 4
cell proliferation was always reduced in diabetic mothers and in their newborns compared to their respective controls. In the presence of linseed oil, lymphocyte proliferative response to Con A stimulation was reduced in both diabetic and control groups. With linseed oil, diabetic mothers and their newborns showed similar T cell proliferative response to Con A stimulation compared with their respective controls. Interleukin release in the presence of oil fatty acids After incubations, the supernatant IL-2 content was measured as Th1 cytokine, while IL-4 was measured as Th2 cytokine (Fig. 2). Lymphocytes isolated from
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
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Oils and lymphocytes in gestational diabetes
Figure 2 T cell interleukin production in mothers and their newborns. The values are means ± standard deviation (SD) of triplicate assays. Multiple comparisons were performed using ANOVA followed by the least significant difference (LSD) test. a, b, c . . . indicate significant differences obtained with different incubations in the same group (P < 0.05). Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test: *P < 0.01; **P < 0.001.
diabetic mothers and their newborns secreted less IL-2 after mitogen stimulation compared to their respective controls. No significant differences were found between IL-4 secretions by Con A stimulated lymphocytes from diabetic and control groups. The addition of Nigel oil induced a significant increase in IL-2 production by Con A stimulated lymphocytes but no effect on IL-4 release in both diabetic and control groups. Nonetheless, IL-2 secretion was still significantly reduced in diabetic groups in comparison with controls. The capacity of T cells to produce IL-2 after stimulation with Con A was markedly reduced after exposure to linseed oil for all groups. IL-4 release did not change in response to linseed oil. We observed that lymphocytes from diabetic mothers and from their newborns, when cultured with linseed oil, secreted similar IL-2 and IL-4 amounts than control mothers and their newborns. The addition of olive oil to the medium had no effect on IL-2 and IL-4 secretions in all groups. Lymphocyte fatty acid profile in the presence of oil fatty acids The fatty acid composition of lymphocyte phospholipids showed several alterations in diabetic mothers compared to control mothers (Table 3). In fact, a statistical rise in C18:2n-6 proportions and a significant fall in C20:4n-6 proportions were observed in gestational diabetic
mother lymphocytes compared to control values. Fatty acid profile was restored by insulin in diabetic mothers. Fatty acid composition of lymphocytes did not differ significantly between the two groups of newborns. As expected, linseed oil induced a significant increase in C18:3 n-3 and C20:5 n-3 associated to a significant decrease in C18:1 n-9 and C20:4 n-6 proportions in all groups. Nigel oil induced a significant increase in the proportions of C18:1 n-9, C18:2 n-6 and C20:4 n-6 with a reduction in SFA levels. In the presence of olive oil, the proportion of C18:1 n-9 was increased while SFA proportion was decreased in all groups. Lymphocyte oxidative stress variables As shown in Fig. 3, MDA and carbonyl protein levels were significantly increased in T lymphocytes from diabetic mothers and their newborns compared to control values. In the presence of insulin, MDA and carbonyl protein levels were higher than values in Con A stimulated lymphocytes from all groups. Addition of Nigel oil, linseed oil or olive oil in the culture medium produced a significant decrease in MDA and carbonyl protein levels in all groups. In the presence of these oils, MDA and carbonyl protein levels became similar in diabetic and control groups.
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Oils and lymphocytes in gestational diabetes Table 3
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Fatty acid composition of maternal and neonatal ConA-stimulated T lymphocytes Control group
Fatty acids SFA Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil C18:1 n-9 Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil C18:2 n-6 Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil C18:3 n-3 Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil C20:4 n-6 Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil C20:5 n-3 Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil
Diabetic group
Mothers
Newborns
Mothers
Newborns
51.20 ± 1.15a 49.50 ± 2.61a 50.11 ± 2.11a 40.42 ± 1.76c 46.33 ± 1.12b
48.67 ± 1.33a 49.02 ± 2.19a 49.73 ± 2.52a 42.33 ± 1.74b 43.76 ± 1.41b
52.30 ± 2.33a 51.18 ± 1.85a 51.43 ± 1.66a 39.03 ± 1.58c 45.05 ± 1.75b
48.06 ± 1.21a 49.04 ± 2.38a 49.75 ± 1.42a 42.65 ± 1.77b 44.06 ± 1.63b
16.14 ± 1.24c 17.48 ± 1.33c 11.74 ± 1.02d 20.06 ± 1.26b 23.55 ± 1.04a
17.03 ± 1.22c 17.74 ± 1.53c 13.03 ± 1.32d 20.11 ± 1.15b 24.95 ± 1.28a
16.63 ± 1.56c 17.22 ± 1.72c 10.68 ± 1.01d 21.02 ± 1.35b 23.06 ± 1.20a
17.32 ± 1.52c 16.98 ± 1.42c 13.98 ± 1.37d 20.73 ± 1.51b 24.84 ± 1.38a
10.58 ± 1.07b 10.74 ± 1.65b 9.93 ± 0.86b 16.77 ± 1.27a 9.07 ± 1.02b
8.11 ± 0.56b 8.56 ± 0.57b 8.71 ± 0.44b 12.33 ± 0.83a 8.82 ± 0.66b
15.20 ± 1.03*b 12.20 ± 2.15c 13.35 ± 1.11*c 18.83 ± 1.04*a 15.55 ± 1.21*b
9.46 ± 0.44b 9.11 ± 0.63b 8.88 ± 0.51b 12.83 ± 0.67a 9.02 ± 0.34b
1.38 ± 0.39b 1.36 ± 0.40b 9.43 ± 1.33a 1.55 ± 0.41b 1.33 ± 0.37b
1.54 ± 0.11b 1.63 ± 0.23b 7.62 ± 0.38a 1.52 ± 0.23b 1.68 ± 0.37b
1.27 ± 0.21b 1.30 ± 0.28 9.66 ± 0.85a 1.52 ± 0.33b 1.35 ± 0.35b
1.44 ± 0.25b 1.56 ± 0.35b 7.18 ± 0.28a 1.47 ± 0.36b 1.50 ± 0.31b
15.83 ± 1.45b 16.62 ± 1.82b 11.48 ± 1.03c 18.05 ± 1.16a 14.33 ± 1.89b
17.04 ± 1.53b 17.81 ± 1.32b 13.43 ± 1.27c 19.33 ± 1.21a 16.88 ± 1.33b
10.16 ± 0.66*c 14.37 ± 1.61b 8.44 ± 0.65*d 15.83 ± 1.05*a 11.22 ± 0.75*c
16.08 ± 1.11b 17.65 ± 1.34b 13.17 ± 1.26c 19.52 ± 1.21a 16.73 ± 1.36b
2.85 ± 0.48b 2.80 ± 0.55b 4.56 ± 0.73a 2.52 ± 0.33b 2.46 ± 0.31b
1.66 ± 0.31b 1.54 ± 0.26b 5.11 ± 0.42a 1.72 ± 0.28b 1.75 ± 0.34b
2.07 ± 037b 2.65 ± 0.34b 4.93 ± 0.83a 2.31 ± 0.55b 2.22 ± 0.40b
1.57 ± 0.29b 1.58 ± 0.33b 5.25 ± 0.38a 1.60 ± 0.31b 1.64 ± 0.28b
The values are means ± standard deviation (SD). Con A, concanavalin A, mitogen; SFA, saturated fatty acids. Multiple comparisons were performed using ANOVA followed by the least significant difference (LSD) test. a, b, c . . . indicate significant differences obtained with different incubations in the same group (P < 0.05). Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test: *P < 0.01; **P < 0.001.
Reduced glutathione (GSH) levels in Con A stimulated lymphocytes from diabetic mothers and their newborns were lower than control values (Fig. 3). The addition of Nigel oil or linseed oil or olive oil in the culture produced a significant increase in GSH levels in lymphocytes from diabetic mothers and from their newborns, but had no effects on lymphocyte GSH from controls. In the presence of these oils, no significant differences in GSH contents were noted between diabetic and control groups. 6
Con A stimulated lymphocyte catalase and SOD activities were significantly lower in diabetic mothers and their newborns when compared with their respective controls (Table 4). In the presence of insulin, catalase and SOD activities were enhanced in lymphocytes from diabetic and control groups, but were still decreased in diabetics when compared to controls. The treatment with linseed or Nigel or olive oil induced a significant increase in enzyme activities in all groups. Catalase and SOD activities in lymphocytes cultured
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
F. DJELTI et al.
Oils and lymphocytes in gestational diabetes
Figure 3 Lymphocyte malondialdehyde (MDA), glutathione (GSH) and Carbonyl protein levels in mothers and their newborns. The values are means ± standard deviation (SD) of triplicate assays. CARP, protein carbonyls. Multiple comparisons were performed using ANOVA followed by the least significant difference (LSD) test. a, b, c . . . indicate significant differences obtained with different incubations in the same group (P < 0.05). Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test: *P < 0.01; **P < 0.001.
Table 4
Superoxide dismutase (SOD) and catalase activities in maternal and neonatal T lymphocytes Control group
Enzyme Catalase (U/mg) Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil SOD (U/mg) Con A Con A + Insulin Con A + Linseed oil Con A + Nigel oil Con A + Olive oil
Mothers
Diabetic group Newborns
Mothers
Newborns
20.13 ± 1.88c 38.06 ± 1.53a 25.82 ± 2.26b 25.61 ± 1.12b 24.13 ± 2.13b
18.53 ± 1.02b 32.96 ± 2.76a 18.15 ± 2.92b 19.26 ± 1.06b 19.54 ± 2.44b
16.34 ± 1.49*b 26.40 ± 1.46*a 27.41 ± 1.92a 26.97 ± 1.23a 24.54 ± 2.77a
14.67 ± 1.19*c 25.75 ± 1.61*a 20.75 ± 1.19b 20.59 ± 1.31b 21.41 ± 2.92b
80.32 ± 2.54b 123.35 ± 6.11a 128 ± 7.42a 125.57 ± 7.17a 128.33 ± 9.31a
44.34 ± 1.13b 72.38 ± 2.42a 70.77 ± 3.17a 73.63 ± 2.88a 71.39 ± 4.15a
56.27 ± 2.31**c 81.03 ± 5.27*b 132.25 ± 8.33a 133.61 ± 9.22a 125.54 ± 6.66a
35.63 ± 1.18*c 56.99 ± 2.11*b 68.06 ± 4.19a 71.72 ± 3.36a 70.62 ± 1.14a
The values are means ± standard deviation (SD). Multiple comparisons were performed using ANOVA followed by the least significant difference (LSD) test. a, b, c . . . indicate significant differences obtained with different incubations in the same group (P < 0.05). Statistical comparisons between diabetic and control mothers and between their newborns were performed by Student’s t-test. *P < 0.01; **P < 0.001.
with these three oils did not differ between diabetic and control groups. Discussion The results obtained in this in vitro study demonstrate that olive, linseed and Nigel oils modulated maternal and
fetal lymphocyte function after mitogen stimulation with an improvement in some defects observed during gestational diabetes. In the current study, diabetic mothers and their newborns showed similar plasma triglyceride and cholesterol levels compared to their respective controls. However, high fasting glucose concentrations were
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observed in diabetic mothers and in their newborns compared to controls. Indeed, hematological parameters were normal except for lymphocyte number, which was reduced and HbA1C levels, which were increased in diabetic mothers and also in their newborns compared to their respective controls. Hyperglycemia and elevated concentration of glycosylated hemoglobin are well documented in gestational diabetes.1,34 Maternal hyperglycemia resulted in increased levels of glucose transported across the placenta to the fetus, thus maternal hyperglycemia is fetal hyperglycemia.35 The lower levels of cord blood hemoglobin glycosylation compared with that of maternal hemoglobin is the result of several factors including the shorter fetal red blood cell life span than maternal erythrocyte, the fetal hemoglobin acetylation at the N-terminus of the gamma chain, the lower fetal glucose levels than those in the mother maintaining a maternal-fetal gradient. Serum cholesterol and triglyceride levels in diabetic pregnant women were not statistically significantly different compared to those seen in normal pregnant women.36,37 Previous studies reported a decrease in naïve T cells in diabetic pregnancy.38 Newborns of diabetic mothers have a reduction in NK lymphocytes than control values.8 Our results showed that linseed and Nigel oils have in vitro important modulatory effects on T cell function, with similar kinetic profiles in maternal and neonate lymphocytes. The immunomodulatory effect of olive oil was not evident on maternal and fetal lymphocyte proliferation and cytokine secretion but significant in reducing intracellular oxidative stress. We observed that mitogen-activated lymphocyte proliferation was lower in diabetic mothers and their newborns than that in controls. While IL-4 production by T cells after mitogen stimulation was normal, IL-2 production was reduced in diabetic mothers and in their newborns. These findings were in agreement with previous reports showing that gestational diabetes induced depressed lymphocyte proliferation and IL-2 secretion compared to normal pregnancy.7,8,38 Modulation of T cell function in the presence of insulin was in agreement with our previous findings.13 In the current study, fatty acids from linseed oil significantly reduced in vitro lymphocyte proliferation in both diabetic and control mothers and their newborns, while fatty acids from Nigel oil significantly enhanced it. However, olive oil’s fatty acids had no effects on lymphocyte proliferation in both diabetic and control groups. The effects of oil’s fatty acids on in vitro cytokine production supported those on lymphocyte proliferation. IL-2 is a well characterized Th1 cytokine with central roles in inflammation and immune signaling. IL-4 8
is a Th2 cytokine associated with humoral immunity. The suppressed lymphocyte proliferative response of linseed oil is consistent with previous data showing that n-3 PUFA significantly reduced mitogen lymphocyte proliferation.14,15,24,25,39 Possible explanation for inhibitory effects of linseed oil could be due to the reduction of IL-2 secretion by stimulated lymphocytes, knowing that IL-2 is required for the proliferation of T cells. The alterations in lymphocyte proliferation after oil exposure in the current study could be attributed to the modification of lymphocyte phenotype proportions. We noted that while linseed oil induced a decrease in IL-2 secretion within lymphocytes, it did not affect IL-4 secretion. Th2-like response was nevertheless dominant reflecting probably the anti-inflammatory effect of linseed oil. We have previously shown that n-3 PUFA shift the Th1/Th2 balance toward the Th2 pole by suppression of Th1 development rather than enhancement of Th2 development in type 1 diabetic patients.13 Nigel oil induced a significant increase in lymphocyte proliferation with a concomitant increase in IL-2 secretion in both diabetic and control groups. Since IL-4 levels were not affected and IL-2 levels were increased, Nigel oil appeared to have generated a Th1-like phenotype. Our findings are in agreement with previous studies showing immunostimulation with Nigel oil in healthy and in diabetic patients.22,23 Olive oil had no effects on cytokine production in both control and diabetic groups. Previous studies did not find significant differences in the proliferation of lymphocytes from humans or animals fed with a diet containing olive oil.40,41 Similar immune effects have been observed with in vitro olive oil showing no effect on lymphocyte proliferation and the production of IL-2.42 The levels of C18:1 n-9 were increased while SFAs were decreased in olive oil treated lymphocytes compared with non-treated lymphocytes in all groups. Our results showed also that a high level of C18:3 n-3 in linseed oil induced an increase of C18:3 n-3 and C20:5 n-3 acids with a concomitant decrease of C18:1 n-9 and C20:4 n-6 acids in the lymphocyte phospholipid membranes of mothers and their newborns. High levels of linoleic and oleic acids in Nigel oil induced an increase in linoleic, oleic and arachidonic acids with a concomitant decrease in SFAs in the phospholipid membranes. Previous studies found a significant correlation between lymphocyte function and the oleic acid present in the phospholipids of lymphocyte membranes.43 Our results suggest that the immunosuppressive effect of linseed oil might be due to a decrease of oleic acid in the lymphocyte membrane. Suppressed proliferation response in the presence of linseed oil may be also due to arachidonic acid (AA) depletion in lymphocyte membrane phospho-
© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd
F. DJELTI et al.
lipids as observed in our study. T cell activation implicates AA liberation and its metabolism.44 In the same way, the activation of lymphocyte proliferation by Nigel oil could be linked to oleic acid and AA membrane enrichment. However, in the case of olive oil, despite the increase in oleic acid, there was no effect on lymphocyte proliferation. Then, the concomitant enrichment in n-6 fatty acids and oleic acid of lymphocyte phospholipids was important to exert a stimulatory effect. In this study, lymphocyte redox markers were consistent with the presence of an oxidative stress in gestational diabetes. Lymphocyte GSH levels, catalase and SOD activities were significantly reduced in diabetic mothers and their newborns compared to controls. These findings reflected a diminished antioxidant defense in gestational diabetes, in agreement with previous studies.4,5,45 MDA and carbonyl protein levels were increased in lymphocytes from diabetic mothers and their newborns resulting probably from overproduction of free radicals and disturbance of the scavenging system. Our results were similar to findings reported in earlier studies showing an imbalance in the pro and anti-oxidant status in gestational diabetes.4,5,45 In the current study, we found that MDA and protein carbonyl contents were decreased while catalase and SOD activities were increased in linseed oil treated lymphocytes from control and diabetic groups. No changes in the levels of GSH were observed in control T cells cultured in the presence of linseed oil. However, in diabetic mothers and their newborns, addition of linseed oil to culture induced an increase in T cell levels of GSH. We observed that in the presence of linseed oil, the T cell redox status from diabetic groups was normalized to control values. We suggested that linseed oil regulated T cell redox status during gestational diabetes. Similar findings were reported in previous studies showing that linseed oil enhanced antioxidant capacity in diabetic rat tissue cells.18 Linseed oil reduced MDA and GSSG level and maintained GSH level in diabetic erythrocytes.46 In the presence of Nigel and olive oils, oxidant/ antioxidant status was also improved in lymphocytes from diabetic mothers and their newborns. In fact, Nigel and olive oils induced a significant reduction in lymphocyte MDA and carbonyl protein levels with a concomitant increase in lymphocyte GSH and catalase and SOD activities in gestational diabetes. Our results are in agreement with earlier reports showing beneficial effects of Nigel and olive oils on oxidative stress.19–22 In diabetes, Nigel oil prevents lipid peroxidation and increases antioxidant defense system activity.47 Olive oil decreased the diabetes-induced increases in tissue pro-oxidants and restored the antioxidant status.48
Oils and lymphocytes in gestational diabetes
Interestingly, the sensitivity of neonate lymphocytes to the oils used was similar and parallel to that in maternal lymphocytes. Linseed, olive and Nigel oils had then beneficial effects on lymphocytes from mothers and their newborns. It has been demonstrated that oxidative modification of cellular macromolecules can induce cell apoptosis and cell necrosis.46 Increased oxidative stress may suppress insulin receptor activation or decrease the translocation of glucose transporter-4 (GLUT-4) on the cell membrane.18 Linseed, olive and Nigel oils may have then protective effect on cell damage and beneficial effect on insulin sensibility in gestational diabetes through the scavenging of free radicals and increase in antioxidant capacity. In conclusion, the results obtained in this research showed that gestational diabetes affected T lymphocyte proliferation, IL-2 production, fatty acid composition and oxidant/antioxidant status in mothers and their newborns. Linseed oil possessed immunosuppressive properties while Nigel oil had immunostimulatory effect on T cells. Olive oil had no effects on lymphocyte proliferation or cytokine secretion. The three oils used had positive effects on oxidative stress, especially by reducing lymphocyte MDA and carbonyl protein levels and enhancing GSH and antioxidant enzyme activities. Linseed, olive and Nigel oils improved T cell intracellular oxidative status in gestational diabetes. The beneficial effects of these oils were apparent in mothers and in their newborns. Acknowledgments This work was supported by the Algerian Research Project (PNR, 2011) from the Algerian Health investigation office (ATRSS). Disclosure None declared. References 1. Lindsay RS. Gestational diabetes: Causes and consequences. Br J Diabet Vasc Dis. 2009; 9: 27–31. 2. Buchanan TA, Xiang AH. Gestational diabetes mellitus. J Clin Invest. 2005; 115: 485–91. 3. Butte NF. Carbohydrate and lipid metabolism in pregnancy: Normal compared with gestational diabetes mellitus. Am J Clin Nutr. 2000; 71: 1256–61. 4. Djordjevic A, Spasic S, Jovanovic-Galovic A, Djordjevic R, Grubor-Lajsic G. Oxidative stress in diabetic pregnancy: SOD, CAT and GSHPx activity and lipid peroxidation products. J Matern Fetal Neonat Med. 2004; 16: 367–72.
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