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Deficiency Increases the Risk of Maternal Abortion and Neonatal Mortality in Murine Pregnancy with or without Diabetes Mellitus: Modulation of T Cell Differentiation
Department of Physiology (A.Y., A.H., N.A.K.), University of Burgundy, Unité Propre de Recherche de l’Enseignement Supérieur Lipids and Nutrition, Faculty of Life Sciences, 21000 Dijon, France; Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l’Alimentation (P.B.), University of Burgundy, Dijon, France; and Laboratory of Cell Biology and Physiology (A.Y., K.M.), Faculty of Sciences and Techniques, University of Abomey-Calavi, 01BP 526 Cotonou, Bénin
Address all correspondence and requests for reprints to: Prof. Naim Akhtar Khan, Head, Department of Physiology, Unité Propre de Recherche de l’Enseignement Supérieur Lipides and Nutrition, Université de Bourgogne, Faculté des Sciences de la vie, 6 Boulevard Gabriel, 21000 Dijon, France. E-mail: Naim.Khan{at}u-bourgogne.fr.
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deficiency in pregnancy outcome and neonatal survival and in the modulation of T cell differentiation in murine diabetic pregnancy and their offspring. Pregnant wild-type (WT) and PPAR-null mice of C57BL/6J genetic background were rendered diabetic by five low doses of streptozotocin. We observed that, in the absence of diabetes, PPAR deficiency resulted in an increase in abortion rate, i.e. 0% in WT mice vs. 20% in PPAR-null mice [odds ratio (OR) = 14.33; P = 0.013]. Under diabetic conditions, the abortion rate was enhanced, i.e. 8.3% in WT mice vs. 50% in PPAR-null mice (OR = 4.28; P = 0.011). In the pups born to diabetic dams, the offspring mortality, due to the absence of PPAR, was enhanced, i.e. 27.7% in WT mice vs. 78.9% in PPAR-null animals (OR = 11.48; P < 0.001). Moreover, we observed that T helper (Th) 1/Th2 balance was shifted to a pregnancy protecting Th2 phenotype in WT diabetic dams and to a noxious Th1 phenotype in PPAR-null mice with diabetic pregnancy. Furthermore, offspring born to diabetic WT dams were hyperinsulinemic and hyperglycemic, and they exhibited up-regulated profile of Th2 cytokines, whereas those born to diabetic PPAR-null dams were hypoinsulinemic and hyperglycemic, and they showed down-regulated profile of Th2 cytokines. However, IFN-
, a Th1 cytokine, was up-regulated in the offspring of both diabetic WT and PPAR-null dams. Altogether, our results suggest that PPAR deficiency in mice may be implicated in the increase in maternal abortion, neonatal mortality, and T cell differentiation. |
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in the incidence of abortion and neonatal mortality during DP.
PPARs are ligand-activated transcriptional factors that regulate a large number of genes by transcriptional activation and repression (6). The three isotypes have been identified in lower vertebrates and mammals (7). PPAR, PPARß (
), and PPAR exhibit different tissue distribution as well as different ligand specificities and functions (8). PPAR is highly expressed in the liver and brown adipose tissue and regulates lipid homeostasis. PPAR is activated by natural ligands, such as fatty acids, as well as the lipid-lowering fibrates, which are used clinically for the treatment of hypertriglyceridemia (9, 10). These agents have been shown to exert beneficial effects in autoimmune diseases and atherosclerosis (11, 12, 13, 14, 15). PPAR controls positively the fatty acid transport and oxidation in the liver (7). Thus, PPAR plays an important role in the regulation of chronic diseases such as diabetes, obesity, and atherosclerosis.
In addition to adipocytes and liver, it has been shown that cells of monocyte/macrophage lineage express both PPAR and PPAR, indicating a possible role of these receptors in immune function (16, 17, 18, 19). Several investigators have reported that PPAR is expressed in B and T cells, and its expression wanes soon after lymphocyte activation (20, 21). Indeed, PPAR ligands have been shown to regulate inflammatory responses because they can inhibit production of IL-2, a T helper (Th) 1 cytokine, and T cell proliferation (11). PPAR ligands have also been shown to increase IL-4 expression, a Th2 cytokine (21). Most of these results argue for an immunosuppressive effect of PPAR that may promote Th2 immunity, necessary for a successful pregnancy (22). Moreover, the T cell-derived cytokines are involved in the autoimmune destruction of pancreatic islet cells leading to type 1 diabetes (5), and evidence from human and experimental models suggests that a shift between Th1 and Th2 cells may modulate the severity of this disease (5, 23). It is also known that maternal type 1 diabetes during pregnancy is an important risk factor for fetal overnutrition and macrosomia and may increase susceptibility to obesity and diabetes in the offspring (24, 25). Experimental studies have demonstrated that the incidence of macrosomia in offspring born to diabetic mothers could even reach 64% (26, 27, 28, 29). Besides, evidence from studies on murine and human pregnancy shows a strong association between maternal Th2 immunity and successful pregnancy and between Th1 immune reactivity and pregnancy loss (22).
Our initial interest, in this study, was to examine the implication of PPAR in the induction of macrosomia in DP in mice because we have previously demonstrated the incidence of macrosomia in DP in rats (26, 27, 28). Surprisingly, we have observed no sign of macrosomia in both the groups of animals, i.e. wild-type (WT) and PPAR-null mice. Besides, we have observed high levels of abortion and neonatal mortality in PPAR-null mice. Because type 1 diabetes is an autoimmune disease strongly associated with Th1/Th2 balance and PPAR being immunosuppressive plays a key role in T cell differentiation (11, 12, 13), we further investigated the Th1/Th2 immune status in the spleen and blood of these animals at parturition and in their offspring during adulthood.
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-null (PPAR-knockout) mice of C57BL/6J genetic background (30) (The Jackson Laboratory, Bar Harbor, ME). After mating, the 1st day of gestation was determined by the presence of spermatozoids in vaginal smears. Pregnant mice were housed individually in wood chip-bedded plastic cages at constant temperature (25 C) and humidity (60 ± 5%) with a 12-h light-dark cycle.
Pregnant WT (n = 12) and PPAR-null (n = 30) mice were rendered diabetic by five consecutive daily ip injections of STZ (40 mg/kg body weight) in 0.1 M citrate buffer (pH 4.5) starting on d 5 of gestation (31). Pregnant WT (n = 12) and PPAR-null (n = 30) mice were also injected with the vehicle alone and considered as control groups. To follow the hyperglycemia during gestation, maternal blood was collected on d 8 and 16 of gestation, by cutting off the tip of tail and squeezing it gently. Glycemia was measured by One Touch ULTRA Glucometer (LifeScan, Johnson & Johnson, Piscataway, NJ). All diabetic dams included in the study had the fasting blood glucose levels above 5.55 mM on all two occasions.
Table 1
shows the total number of mice and pups, delivered in each group of mice and the percentage of abortion, and neonatal mortality. At 3 wk of age, pups of each group of mice were weaned and weighed throughout the study until 3 months of age. Only the male offspring were included in the study. All experiments were performed at parturition in mothers and at 3 months of age in offspring.
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Blood, liver, spleen, and adipose tissue samples
After overnight fasting, eight dams at parturition or 15 male offspring at age of 3 months, in each group, were anesthetized with pentobarbital (60 mg/kg body weight). The abdominal cavity was opened, and whole blood was drawn from the abdominal aorta. Serum was obtained by low-speed centrifugation (1000 x g x 20 min) and immediately stored at –80 C for insulin, lipid, and cytokine determinations. The spleen and the liver were removed and immediately frozen in liquid nitrogen and used, respectively, for total RNA extraction and lipid determinations. The uterine white adipose tissue (WAT) in mothers and epididymal WAT in male offspring were removed, washed with cold saline, weighted, and frozen in liquid nitrogen.
Determination of serum insulin and cytokine levels
The determination of insulin and cytokines was performed in samples that were stored at –80 C. Serum cytokine (IFN- and IL-2 for Th1 and IL-4 and IL-10 for Th2) levels were determined by ELISA using the eBioscience (San Diego, CA) mouse Th1/Th2 ELISA kit, Ready-Set-Go, according to the manufacturer’s instructions. Serum insulin was determined using an ELISA kit (LINCO Research Inc., St. Charles, MO), according to the manufacturer’s instructions.
Determination of serum and liver lipids
After total lipid extraction, according to the method of Bligh and Dyer (32), serum or liver triglyceride (TG) and free fatty acids (FFAs) were separated on silica gel by thin-layer chromatography using the following solvent: hexane/diethyl ether/acetic acid/methanol at 90:20:2:3 (vol/vol). The purified fractions of FFA and TG were quantified by gas liquid chromatography (33) using an internal standard, C17:0 for FFA and TriC15:0 for TG, with a Becker gas chromatograph (Becker Instruments, Downers Grove, IL) equipped with a 50-m capillary glass column packed with carbowax 20 m (Spiral-RD, Couternon, France).
Real-time RT-PCR quantification assay
Total RNA was prepared from spleen using Trizol reagent (Invitrogen Life Technologies, Groningen, The Netherlands) according to the manufacturer’s instructions. The integrity of RNA was electrophoretically checked by ethidium bromide staining and by the OD absorption ratio OD260nm/OD280nm more than 1.9. One microgram of total RNA was reverse transcribed with Superscript II RNAse H-reverse transcriptase using oligo (dT) according to the manufacturer’s instructions (Invitrogen Life Technologies).
Real-time PCR was performed on an iCycler iQ real-time detection system (Bio-Rad, Hercules, CA), and amplification was done by using SYBR Green I detection (SYBR Green JumpStart, Taq ReadyMix for Quantitative PCR, Sigma-Aldrich, St. Louis, MO). Oligonucleotide primers, used for mRNA analysis, were based on the sequences of mice gene in the GenBank database. The sequences of the PCR primers used are as follows: ß-actin, forward, 5'-AGAGGGAAATCGTGCGTGAC-3' and reverse, 5'-CAATAGTGATGACCTGGCCGT-3'; IL-2, forward, 5'-CCTGAGCAGGATGGAGAATTACA-3' and reverse, 5'-TCCAGAACATGCCGCAGAG-3'; IL-4, forward, 5'-ACAGGAGAAGGGACGCCAT-3' and reverse, 5'-GAAGCCCTACAGACGAGCTCA-3'; IL-10, forward, 5'-GGTTGCCAAGCCTTATCGGA-3' and reverse, 5'-ACCTGCTCCACTGCCTTGCT-3'; and IFN-, forward, 5'-TCAAGTGGCATAGATGTGGAAGAA-3' and reverse, 5'-TGGCTCTGCAGGATTTTCATG-3'. The amplification was carried out in a total volume of 25 µl containing 12.5 µl SYBR Green Taq ReadyMix, 0.3 µM each primer and diluted cDNA. Cycling conditions consisted of an initial denaturation step of 95 C for 5 min as a hot start followed by 40 cycles of 95 C for 30 sec/60 C for 30 sec with a single fluorescence detection point at the end of the relevant annealing or extension segment. At the end of the PCR, the temperature was increased from 60–95 C at a rate of 2 C/min, and the fluorescence was measured every 15 sec to draw the melting curve. The standard curves were generated for each protein or ß-actin using serial dilutions of positive control template to establish PCR efficiencies. All determinations were performed, at least, in duplicates using two dilutions of each assay to achieve reproducibility.
Results were evaluated by iCycler iQ software including standard curves, amplification efficiency (E) and threshold cycle (Ct). Relative quantitation of cytokine mRNA in different groups was determined using the 
Ct method (34, 35), in which Ct = Ct of cytokine of interest – Ct of ß actin. Ct = Ct of interest group - Ct of control group. Relative quantity (RQ) was calculated as follows: RQ = (1 + E)(–Ct).
Statistical analysis
The JMP 5.01 and R software packages were used to analyze the data of Table 1
. We have calculated the odds ratio (OR) of aborted pregnancies in mothers and the OR of pups dying in diabetic condition in WT and PPAR-deficient mice. Differences were considered significant at P < 0.05. Pregnancy success in female mice was encoded as a binary response variable (aborted pregnancy, 1; successful pregnancy, 0). The effects of PPAR deficiency and diabetes on the likelihood to abort were then investigated using a general linear model with a logit link and a binomial error distribution. Mice groups (PPAR-null vs. WT), diabetes effect (diabetic vs. control), and the two-way interaction were included as fixed factors. Model was built following a step-wise backward procedure, where the least significant terms were sequentially removed (36). The final model included only significant explanatory terms. Explanatory terms that significantly improved the fit of the model are given with their corresponding estimates, ORs, Wald statistics, and P values.
Neonatal mortality ratio for a litter (number of dead pups per litter) in relation to mice groups and diabetes effect was investigated using the general linear model. Model was fitted with the number of dead pups at parturition in a litter as the response variable and litter number as a binomial denominator, using a logit link and a binomial error distribution. Quasilikelihood estimations were used to determine the scale parameter, and the significance of explanatory terms was tested using F tests (37). Explanatory terms that significantly improved the fit of the model are given with their corresponding estimates, ORs, F statistics, and P values.
Results in Tables 2–5 and Figs. 1 and 2 are shown as means ± SEM. The significance of the differences between mean values was determined by two-way ANOVA (STATISTICA, Version 4.1, Statsoft, Paris, France), followed by the least significant difference (LSD) test. Differences were considered significant at P < 0.05.
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-null mice aborted, whereas the percentage of abortion in WT ones was not observed (OR = 14.33; P = 0.013). When the mice were rendered diabetic, the abortion rate in PPAR-deficient animals was 50%, whereas it was only 8.3% in WT mice (OR = 4.28; P = 0.011) (Table 1). Statistical analysis has shown that the interaction relative to diabetes effect between the two groups of mice was not statistically different, suggesting that diabetes effect on the abortion rate in the group of PPAR-deficient mice, as well as in the WT group animals, was not significantly different.
Similarly, 13.3% of the neonates of nondiabetic PPAR-null mice died, whereas the mortality rate in offspring of control WT animals was 5.08% (OR = 3.34; P = 0.005). In diabetic condition, the mortality rate increased from 27.7% in the offspring of diabetic WT mice to 78.9% (OR = 11.48; P < 0.001) in the offspring of diabetic PPAR-deficient mice (Table 1). The statistical analysis has revealed that the interaction relative to diabetes effect between the two groups of animals was not significantly different. The increases in the mortality rate in the offspring due to diabetes effect were not statistically different between the two groups of mice.
Th1 and Th2 cytokine expression in spleen of mice with DP
Figure 1 shows that expression of mRNA of Th1 cytokines, IL-2 and IFN-, was down-regulated in spleen of WT animals with DP. The expression of Th2 cytokine, IL-4 mRNA, was also down-regulated in both WT and PPAR-null mice with DP in comparison with their corresponding controls. However, the expression of mRNA of IL-10, another Th2 cytokine, was either up-regulated in WT mice or remained unchanged in PPAR-null animals with DP compared with control animals. The expression of IL-2 mRNA did not differ in PPAR-null mice with DP compared with their respective controls (Fig. 1).
Th1 and Th2 cytokine expression in spleen of offspring born to mice with DP
The expression of Th1 cytokine, IFN- mRNA in spleen, was up-regulated in offspring of both groups of mice with DP compared with control offspring (Fig. 2). The expression of Th2 cytokine (IL-4 and IL-10) mRNA was up-regulated in offspring of WT mice with DP and down-regulated in offspring of PPAR-null mice with DP compared with their respective controls. IL-2 expression remained unchanged in offspring of both groups of mice with DP compared with control offspring.
Serum level of Th1/Th2 cytokines in mice with DP and their offspring
Serum concentrations of IFN-, IL-2, and IL-4 were significantly decreased, whereas the IL-10 concentration was increased in WT mice with DP compared with their respective controls (Table 2). In PPAR-null mice with DP, circulating IL-4 and IL-10 were significantly decreased, whereas IFN- concentration was increased, in comparison with their corresponding control animals. Table 2 reveals the shift of the Th1/Th2 ratio to a Th2 phenotype in WT mice with DP and to a Th1 phenotype in PPAR-null animals with DP.
Table 3 shows that the concentrations of IFN- were significantly increased in offspring of both the groups of mice with DP compared with their respective controls. The concentrations of IL-4 and IL-10 were significantly increased in offspring of WT mice with DP, whereas they were significantly decreased in offspring of PPAR-null mice with DP compared with their respective controls. The shift of the Th1/Th2 ratio, to a Th2 phenotype in the offspring of WT mice with DP and to a Th1 phenotype in the offspring of PPAR-null mice with DP, is once more visible in Table 3.
Serum glycemia, insulin, and lipids in mice with DP and their offspring
After overnight fasting, PPAR-null control mice were hypoglycemic at each experimental point during gestation, compared with WT control animals. Diabetic mice of both WT and PPAR-null groups showed the high level of hyperglycemia on d 16 of gestation, i.e. 1 wk after the last injection of STZ. The glycemia decreased in both the groups of animals at parturition (Table 4).
The WT and PPAR-null mice are of the C57BL/6J genetic background. At the same age, the weight gain was greater in PPAR-null mice than in their counterpart WT animals. PPAR-null mice (control and diabetic) exhibited a high adiposity and high lipidemia. DP induced hyperlipidemia in both the groups of mice. The lipidemia was higher in PPAR-null diabetic mice than WT diabetic animals (Table 4). The offspring of PPAR-null mice (control and diabetic) were larger than their homologous age-matched WT animals, both at birth and during their growth. Besides, DP failed to induce macrosomia in the progenies of both groups of mice (Table 5). However, the 3-month-old offspring of WT diabetic dams developed hyperglycemia, hyperinsulinemia, and hyperlipidemia with no increase in body weight compared with their corresponding controls. Although the 3-month-old offspring of PPAR-null diabetic mice were hyperglycemic and hyperlipidemic, they were, in contrast, hypoinsulinemic with a mean body weight lower than their corresponding controls (Table 5).
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-deficient mice. This is specific to this mouse strain; female aged animals develop a more pronounced obesity than males (48).
The WT and PPAR-null mice are of the C57BL/6J genetic background. The specificity related to this mouse strain is that, at the same age, the weight gain is greater in PPAR-null mice than in their counterpart WT animals. Indeed, the offspring of PPAR-null mice (control and diabetic) were larger than age-matched WT mice. Moreover, the PPAR-null mice (control and diabetic) exhibited a high adiposity and high lipidemia. Nondiabetic PPAR-null mice developed hypoglycemia after overnight fasting. In fact, PPAR controls positively the mitochondrial ß-oxidation of fatty acids. PPAR-deficient mice (30) exhibit a reduced capacity to metabolize long-chain fatty acids (49), which likely contributes to dyslipidemia (50, 52) and larger adipose stores observed in these mice with aging (48). Under fasting conditions, the hypoglycemia in nondiabetic PPAR-null mice has been also observed by other investigators (49).
The progression of type 1 diabetes has been demonstrated to be closely associated with high expression of mRNA of Th1 cytokines, particularly of IFN- (51, 52). The role of IL-2 is somewhat controversial (53). In normal pregnancy, Th1 cytokines are down-regulated, whereas Th2 cytokines are up-regulated (22, 54). Astonishingly, we observed, in both WT and PPAR-null diabetic mice, a diminished expression of IL-4 mRNA and low concentrations of circulating IL-4. This may be responsible for the induction of diabetes in female mice. Our idea can be supported with the observations of Müller et al. (23) who have shown that diabetes susceptibility was more associated with reduction of IL-4 than with induction of IFN- in mice administered the mild doses of STZ. Similarly, Wood et al. (38) have reported diminished expression of IL-4 mRNA in thymocytes of mice, administered with multiple doses of STZ. Hence, administration of exogenous IL-4 before STZ treatment prevented the STZ-induced pancreatic islet destruction and hyperglycemia in these animals (38). However, we observed a decreased expression of IL-2 and IFN- mRNA in spleen of WT mice with DP, whereas PPAR-null animals with DP exhibited an increased expression of IFN- mRNA without any change in IL-2 mRNA expression, compared with that in control animals. Similarly, WT mice with DP showed diminished concentrations of circulating IFN- and IL-2, whereas PPAR-null exhibited significant increased concentrations of circulating IFN-. Serum IL-2 concentrations did not change in PPAR-null mice with DP, compared with control mice. Hence, low expression of Th1 cytokines in WT mice with DP may be due to the fact that, during pregnancy, Th1/Th2 ratios revealed a shift to a pronounced Th2 phenotype (22). Kruse et al. (55) have reported a significantly reduced IL-2 and IFN- expression during normal human pregnancy. In fact, the shift of Th1 phenotype to Th2 during pregnancy has been shown to encourage vigorous production of antibodies that not only combat infections during pregnancy but also offer passive immunity to the fetus (56). Hence, in WT animals with DP, the diminished Th1 cytokines and the increased mRNA expression and serum concentration of IL-10 (Th2) may be implicated in maintaining the pregnancy in these mice. Regarding the Th1/Th2 profile, the present results in WT mice corroborate our recent findings on Wistar rats and their offspring. Hence, the Th1/Th2 balance was shifted to a Th2 phenotype in pregnant diabetic rats (57). The fact that PPAR-null mice with DP had increased IFN- (expression and circulation) and failed to have increased IL-10 (expression and circulation), which may be protective, may explain the high degree of abortions (50%) observed in pregnant diabetic PPAR-null animals. Our results suggest that the presence of PPAR seems to play an important role in the shift of Th1/Th2 ratio to a Th2 phenotype by increasing IL-10 expression and production for protecting pregnancy in WT mice with DP. Our suggestion is consistent with the studies by Maruyama et al. (58) who have shown, by using fenofibrate, a PPAR agonist, an increased in vivo expression of IL-10 in rats with autoimmune myocarditis. Indeed, several recent studies have reported the Th1-suppressing effect and Th2-promoting effect of PPAR. Using PPAR ligands, Marx et al. (11) have demonstrated a diminished expression of IFN- and IL-2 in human CD4+ T cell cultures. Cunard et al. (12) have noticed impaired production of IFN- in splenocytes of mice fed with WY14,643, a PPAR agonist. They have, moreover, observed that PPAR ligands elicited augmented IL-4 expression in cultures of mitogen-activated splenocytes (21). Lovett-Racke et al. (13) have also reported that gemfibrozil and fenofibrate, other PPAR ligands, can shift the cytokine secretion of human T cell lines by inhibiting IFN- and promoting IL-4 secretion. The results of our study clearly demonstrate that the ratio Th1/Th2, which must be shifted to a protective Th2 phenotype, had been shifted to a Th1 profile in PPAR-null mice with DP. This profile is in favor of abortions because studies on murine and human beings have demonstrated a strong association between maternal Th1 immune reactivity and pregnancy loss (22). In nondiabetic dams, the effect of PPAR deficiency on the increased abortion rate was consistent with the shift of Th1/Th2 ratio (relative to IL-2) to a noxious Th1 phenotype for the pregnancy. However, in diabetic dams, the effect of the lack of PPAR on the increased abortion rate was confirmed by the shift of the Th1/Th2 ratio (relative to IL-2 and IFN-) to a deleterious Th1 immunity, suggesting that the deleterious effect of PPAR deficiency on the pregnancy might be potentiated by diabetes mellitus, although the statistical calculations of the data did not confirm this effect.
Although in pregnant WT diabetic mice, the down-regulation of IL-2 (expression and circulating) may be related to the successful pregnancy, it was surprising to observe no change in this cytokine in PPAR-deficient mice (both diabetics and controls). Moreover, IL-2 levels were lower in PPAR-null mice than those in WT animals. These observations are surprising because PPAR agonists are known to have an immunosuppressive effect. Several investigations, using PPAR agonists, have demonstrated a down-regulation of IL-2 production in human T cell cultures (11, 21). This point on in vivo and in vitro production of IL-2 in PPAR-null mice with DP requires detailed investigations in future. Because the diabetic PPAR-null pregnant mice exhibited a high level of IFN- and a low level of IL-4 (due to diabetes induction), they failed to have increased IL-10 levels for maintaining the pregnancy. In this condition, the additional increases in IL-2 levels could have been more deleterious for the pregnancy. Thus, the low level of IL-2, in PPAR-deficient animals compared with WT mice, may contribute to the pregnancy viability.
The reasons of the death of pups in PPAR-null diabetic mice are not well understood. We did not perform postmortem studies. However, several reasons have been evoked in the literature and could furnish us with sound arguments to explain the mortality in our study. Hashimoto et al. (59) have recently observed that gemfibrozil and clofibrate, two PPAR agonists, down-regulate human chorionic gonadotrophin and up-regulate progesterone secretions in human trophoblast cells, suggesting that the lack of PPAR may be deleterious for the secretion of these hormones, essential for maintaining pregnancy. Michalik et al. (60) have reported that PPAR is expressed in the placenta in late gestation. On the other hand, Wang et al. (61) have observed that PPARs (, ß, and ) are expressed in junctional (endocrine functional) and labyrinth (barrier transport) zones of the rat placenta, from d 13–21 of gestation (i.e. 3rd trimester). PPAR has also been observed in human term placenta, suggesting its potential and critical role in placental fatty acid transfer to meet fetal requirements, necessary for the biosynthesis of biological membrane, myelin, and various signaling molecules (61). In our study, PPAR-deficient mice (control and diabetic) aborted between d 12 and 16, i.e. between 2nd and 3rd trimesters, and their offspring died at birth. These observations show that, even if PPAR-null mothers were able to accumulate lipids in the placenta labyrinth, due to the presence of PPAR, the lack of PPAR may result in severely impaired fetal-maternal nutrient exchanges, therefore, leading to the lethal effects in the offspring. This placental dysfunction may be more pronounced in diabetic conditions because diabetes increases the demand of energy. PPAR-null diabetic mice were hypoinsulinemic, unable to use either glucose or lipids, respectively, due to diabetes and PPAR deficiency. Therefore, in addition to the hormonal dysfunction of placenta and the high proinflammatory Th1 cytokine levels observed in PPAR diabetic and their offspring, the high rate of abortion and pups mortality could also be related to the defect in energy homeostasis and utilization.
Our results showed that the offspring of WT mice with DP were hyperglycemic and hyperinsulinemic when compared with those of WT control animals. Moreover, it is interesting to distinguish that in WT mice with DP, the mRNA expression and serum concentration of IFN- were down-regulated, whereas in their offspring, IFN- levels were up-regulated. Both WT mothers with DP and their offspring are associated with hyperglycemia. The difference in Th1 phenotype may be due to their different physiological status in this pathology. The low Th1 profile in mothers with DP, associated with successful pregnancy, may be contributed by elevated levels of reproductive hormones like human chorionic gonadotrophin, whose administration is known to diminish the production of Th1 cytokines (62). The up-regulated Th1 profile in their offspring may be due to their diabetogenic status, associated with hyperglycemia and hyperinsulinemia (26). However, these diabetic offspring showed increased mRNA expression and serum concentration of Th2 cytokines (IL-4 and IL-10). This fact may be responsible for their survival. These observations are contradictory to those obtained in offspring of PPAR-null diabetic mice, which exhibited low mRNA expression and circulating concentration of IL-4 and IL-10 and increased mRNA expression and serum concentration of IFN-, although they were hyperglycemic and hypoinsulinemic. These observations suggest two aspects: firstly, PPAR, known to up-regulate Th2 profile (11, 12, 13), is absent in these animals, and, therefore, its absence may be responsible for the down-regulation of Th2 cytokines; and secondly, PPAR seems to be mandatory for insulin secretion. Indeed, Bihan et al. (63) have suggested that PPAR is needed to ensure appropriate insulin secretion in situation of short-term hyperglycemia, likely by maintaining islet lipid homeostasis. In nondiabetic animals, the effect of the absence of PPAR on the pups mortality (13.3%) was not consistent with the shift of the Th1/Th2 ratio. However, in diabetic mice, the effect of the lack of PPAR on the pups mortality (78.9%) was confirmed by the shift of Th1/Th2 balance to a noxious Th1 immunity, suggesting that the deleterious effect of PPAR deficiency on the neonate mortality might be potentiated by diabetes mellitus, although the statistical observations on the data of Table 1 did not confirm this effect.
The molecular mechanism of how PPAR induces the shift of Th1/Th2 to Th2 phenotype and exerts beneficial effects remains unclear. Some studies have suggested that there is a cross-talk between PPAR signaling pathways with the STAT and GATA transcription factors (64) because it is known that differentiation of T cells to Th2 phenotype involves activation of STAT-6, which is translocated to the nucleus, resulting in expression of GATA-3 (65). GATA-3 is thought to be the key regulator of Th2 differentiation (66). Other investigators have suggested that PPAR also play a physiological role in regulating T-bet, an inducible transcription factor important for the initiation of cytokine gene transcription (67). They have demonstrated that PPAR was able to down-regulate T-bet transcription, which influenced the amount of IFN- produced by T cell (67). This regulation occurred independent of DNA binding, suggesting that there may be several mechanisms of how PPAR can regulate T cell activation and cytokine gene transcription (13).
To our knowledge, ours is the first study that demonstrates that, during pregnancy, PPAR may play an important role in the prevention of maternal abortion, neonatal survival, and T cell differentiation. These observations may allow us to investigate, in the future, whether PPAR is implicated in fetoplacental transport and the production and transmission of antibodies from mothers to fetus.
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Acknowledgments |
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All of the authors have nothing to declare as far as the conflict of interest is concerned.
First Published Online June 8, 2006
Abbreviations: Ct, Threshold cycle; DP, diabetic pregnancy; FFA, free fatty acid; LSD, least significant difference; OR, odds ratio; PPAR, peroxisome proliferator-activated receptor; STZ, streptozotocin; TG, triglyceride; Th, T helper; WAT, white adipose tissue; WT, wild type.
Received January 18, 2006.
Accepted for publication May 25, 2006.
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 J.-M. Ategbo, O. Grissa, A. Yessoufou, A. Hichami, K. L. Dramane, K. Moutairou, A. Miled, A. Grissa, M. Jerbi, Z. Tabka, et al. Modulation of Adipokines and Cytokines in Gestational Diabetes and Macrosomia J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 4137 - 4143. [Abstract] [Full Text] [PDF]
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, P < 0.05.