ISSN No. 1606-7754                   Vol.17 No.3 December 2009
Melatonin effects on macrophage in diabetic rats and the maternal hyperglycemic implications for newborn rats
Adenilda Cristina Honório França1,4, Karina Aparecida Silva2, Nagilla Daliane Feliciano3, Iracema Mattos Paranhos Calderon4, Marilza Vieira Cunha Rudge4, Eduardo Luzía França1.
University of Araguaia, Federal University of Mato Grosso, Pontal do Araguaia, Brazil1 Institute of Health Sciences, University Center of Planalto de Araxá, Araxá, Brazil2, Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, Brazil3 Post Graduate Program in Gynecology, Obstetrics and Mastology, Botucatu Medical School, Unesp, São Paulo, Brazil4
Abstract

The modulatory effects of melatonin (MLT) on maternal and fetal macrophages in diabetic rats and the repercussion of maternal hyperglycemia on fetus-placenta parameters were studied. This was achieved by determining maternal and fetal blood glucose, weight and superoxide release by macrophages. Placental weight, protein, DNA and RNA concentration were also verified. Superoxide levels in macrophages isolated from pregnant healthy rats were higher than those obtained from diabetic animals. Melatonin increased significantly in the macrophages of control animals (18.7 ± 2.8 with MLT compared to 14.2 ± 1.6 without MLT) but decreased with melatonin stimulation in diabetic rats (8.8 ± 1.4 with MLT compared to 12.9 ± 2.1 without MLT). Melatonin significantly decreased superoxide levels in newborns of diabetic mothers (7.3 ± 3.4) compared to those of healthy (14.6 ± 3.5) mothers. Blood glucose levels were significantly higher (p<0.05) in newborn rats of diabetic mothers (108.3 ± 7.8) compared to blood glucose levels in newborn control rats (81.2 ± 10.7). Body weight was significantly higher (p <0.05) in the offspring of rats with alloxan-induced diabetes. No statistical difference (p> 0.05) was observed in the placenta weight, total protein concentration and DNA of rats. The RNA concentration was significantly lower (p <0.05) in the placentas of rats with alloxan-induced diabetes (156.1 ± 71.8), when compared to the concentration of RNA in the placentas of control rats (239.5 ± 77.3). In conclusion, maternal hyperglycemia modified the fetus-placental parameters and melatonin modulated the macrophages activation in maternal and fetal diabetic rats.

Keywords:

diabetes, hormone, macrophages, melatonin, pregnancy, superoxide.

Introduction

Diabetes mellitus consists of a group of chronic disorders characterized by hyperglycemia or diminished insulin secretion, or both, and has been associated to certain infections.1 Phagocytic disorders and changes in superoxide release are major factors for infectious diseases.2 Diabetic patients have reduced phagocytic activity, low microbicidal activity in lysosomal enzyme release and production of reactive oxygen species, owing to changes in antioxidant systems.2 The reduction in phagocytic and microbicidal activity of leukocytes is likely related to an increase in blood glucose levels.3 Phagocyte functional activity can be examined through indirect mechanisms to determine oxygen consumption4 or derivatives of cell oxidative metabolism, such as superoxide anion.5,6

Rats with diabetes induced by chemicals agents (alloxan or streptozotocin) have neutrophil quiomitaxia,7 low phagocytic activity,8 low hydrogen peroxide production9 and increased glucose levels. Diabetic pregnant rats have low antibody levels and decreased T-lymphocyte activity. These alterations are also found in their newborns.10

Diabetes may change pregnancy conditions, thus affecting the mother and even more so the fetus. Gestational diabetes is the most frequent metabolic disorder of pregnancy and occurs in 1% to 10% of gestations.11 The main pathophysiological consequences for the fetus are macrosomia and oxidative stress.12 Oxidative stress plays an important role in the pathogenesis and complications of diabetes. Some studies have shown that melatonin (N-acetyl-5-methoxy tryptamine) reduces oxidative stress induced in diabetic rats.13 Melatonin is an endogenous neurohormone produced by the pineal gland in mammals, and its actions are related to its capability to scavenge free radicals, increase the antioxidant activity of enzymes and beneficial effects for controlling diabetes complications,15 mainly due to its scavenging capacity against reactive oxygen species in diabetic rats.14 Furthermore, melatonin at physiological concentrations can stimulate the phagocytic activity of macrophages in animals.16

Phagocytes are considered the first line of the body's immune defense and the reduction in their functional activity contributes to increased susceptibility and severity in diabetes infections.17 Several immunological functions are affected by diabetes,1 but little is known about its influence of hormones on maternal and fetal macrophages, and the possible implications of maternal hyperglycemia for physiopathological disorders in newborns.

This study investigated immunomodulatory effects of melatonin (MLT) on macrophage activity in maternal and fetal rats with alloxan-induced diabetes and the repercussion of maternal hyperglycemia on fetus-placenta parameters.

Materials and methods
Animals and operation procedures

Adult female Wistar rats, weighing between 200g and 250g (10-12 weeks of age) and still in their reproductive phase, were housed under constant conditions of light (photoperiod L:D 12:12) and temperature (25oC) with ad libitum access to water and food. The animals were separated into two groups: control pregnancy (n=10) and alloxan-induced diabetic pregnancy (n=10). For diabetic induction, 12-h fasted animals received a single intravenous injection of alloxan (42 mg/kg body weight) in the medial portion of the tail. Control animals were given an equivalent volume of 0.9% saline.

For diabetes confirmation, blood glucose levels were determined by an Accu-Check Advantage (Roche, Sao Paulo, Brasil) glucometer 15 d after alloxan injection (mean glucose blood control group = 74 ± 6.5 and alloxan-induced diabetic =146 ± 30.3). Animals of both groups were paired for mating. The fertilization day was assessed by the vaginal presence of sperm.

In the morning at days 1, 7, 14 and 21 of pregnancy, maternal blood glucose levels were determined by puncture of the tail vein of rat. The mean maternal blood glucose level was calculated using all glycaemic values obtained during pregnancy.

On the morning of day 21 of pregnancy the animals were anesthetized with sodium phentobarbital (50 mg/kg body weight, intraperitonealy) and submitted to a surgical procedure for newborn removal. The spleen was removed and placed in phosphate buffered saline (PBS) solution. The newborn rats were weighed and decapitated and blood samples were collected and pooled all puppies for determination of glucose.

The spleens were also removed, pooled and stored in PBS for further superoxide determination. The placentas were removed, weighed and conserved at -20oC for further analysis of total protein, RNA and DNA. The research project was evaluated institutionally and approved in July, 2005.

Isolation, Purification and Identification of Macrophages

Maternal and newborn spleens were macerated in PBS. The cells were fractioned by centrifugation (160 x g, 30 min.) through gradient density (density 1.077 g/l) using Ficoll-Paque (Pharmacia, Uppsala, Sweden). Purified macrophages were resuspended independently in serum-free medium 199 (Gibco, MD, USA) and washed twice. Cell count (using Newbauer hemocytometer) and macrophage suspension were adjusted to a final concentration of 1x106cells/ml.6 Before undergoing superoxide release measurements, the macrophages were identified under light microscope and their viability was measured by the trypan blue exclusion test.  

Release of Superoxide Anion

To measure superoxide release, cytochrome C (Sigma) reduction was determined as previously described.5,6 The macrophages were centrifuged twice at 160 x g for 10 min at 4oC, and 500 mL of the phagocytes was resuspended in PBS containing 2.6 mM CaCl2 and 2.0mM MgCl2 and cytochrome C (Sigma-2 mg/ml) for verified the spontaneous superoxide release. Phorbol myristate acetate (PMA- Sigma) stimulation was performed as positive control at final concentration of 10-7M. To evaluated the melatonin effect the macrophages were stimulated with melatonin hormone (Sigma, final concentration of 10-7M).16 The suspensions (100 μL) were incubated for 60 min at 37oC in culture plates. The reaction rates were measured by absorbance at 450 nm and the results were expressed as nmol/O2-. All the experiments were performed in duplicate or triplicate.

Total protein, ribonucleic acid (RNA) and desoxiribonucleic acid (DNA) in the placenta

The placentas were preserved in liquid nitrogen, macerated in PBS and filtered to determine total protein concentration, DNA and RNA.18,19,20,21 The results were expressed in mg%.

Statistical analysis

Analysis of variance (ANOVA) with F statistics was used to evaluate the quantitative data on maternal and newborn blood glucose, weight, superoxide release, placental weight, protein concentration, DNA and RNA; significant differences were evaluated by Tukey’s test.22 All the results were expressed as mean ± SD. Statistically significant differences were considered for alpha error below 0.05 (p <0.05).

Results
Confirmation of diabetes

Mean glucose level, determined four times over 21 days of pregnancy, was significantly higher (p < 0.05) in diabetic rats treated with alloxan than those of untreated rats (Table 1). There was no difference (p > 0.05) in body weight between diabetic and control animals.

The effect of diabetes on maternal macrophages

Diabetes not influenced significantly (p > 0.05) the several parameters of isolated macrophages. The number of maternal macrophages retrieved as well as their viability. The functional activity also were not affected by diabetes (p > 0.05), according to analysis of spontaneous superoxide release (control group =14.2 ± 1.6 – diabetic group 12.9 ± 2.1; Table 1).

Influence of melatonin on the superoxide release from macrophages of pregnancy rats

In control pregnant rats, melatonin at 10-7 M significantly (p < 0.05) stimulated superoxide release from macrophages compared to non-stimulated macrophages (18.7 ± 2.8 versus 14.2 ± 1.6) (Fig. 1). There was no significant difference (p > 0.05) between macrophage stimulation by melatonin and PMA (21.1 ± 1.8).

In diabetic pregnant rats, melatonin stimulation failed to induce superoxide release (8.8 ± 1.4 compared to 12.9 ± 2.1 of control group). Melatonin decreased macrophage superoxide release in diabetic pregnant rats (8.8 ± 1.4; (p < 0.005) compared to control pregnant rats (18.7 ± 2.8; Fig. 1).

Influence of maternal hyperglycemia on fetus-placenta parameters

Blood glucose levels were significantly higher (p < 0.05) in the newborn rats of diabetic mothers (108.3 ± 7.8) compared to blood glucose levels in newborn control rats (81.2 ± 10.7). Body weight was significantly higher (p < 0.05) in the offspring of rats with alloxan-induced diabetes compared to control (Table 2). No statistical difference (p > 0.05) was observed in placental weight, total protein concentration and DNA of rats with alloxan-induced diabetes, when compared with the placentas of control rats (Table 2). The RNA concentration was significantly lower (p < 0.05) in the placentas of rats with alloxan-induced diabetes (156.1 ± 71.8), compared to the concentration of RNA in the placentas of control rats (239.5 ± 77.3) (Table 2).

The effect of diabetes on the number and function of newborn macrophages

Diabetes influenced (p > 0.05) neither the number nor the viability of spleen macrophages in newborn rats. In contrast, the functional activity of the offspring of rats with alloxan-induced diabetes decreased significantly (p < 0.05), as shown by the lower superoxide release by non-stimulated macrophages in the group with alloxan-induced diabetes (Table 2).

Influence of melatonin on the superoxide release of macrophages in newborn rats.

Melatonin at a concentration of 10-7 M, did not stimulate (p > 0.05) superoxide release from macrophages of offspring of control rat (14.6 ± 3.5 in MLT-stimulated versus 13.3 ± 2.9 innon-stimulated macrophages) (Fig. 2). Spleen macrophages from newborns of both diabetic and control mothers had higher superoxide release when stimulated by PMA (16.6 ± 1.8), more than that of non-stimulated macrophages (13.3 ± 2.9) and melatonin-stimulated macrophages (14.6 ± 3.5). Melatonin failed to stimulate spleen macrophages of newborns of diabetic mothers (7.3 ± 3.4 compared to 8.3 ± 1.9 without melatonin). Melatonin significantly decreased (p < 0.005) superoxide release by newborn macrophages of diabetic rats (7.3 ± 3.4) compared to newborn macrophages of control rats (14.6 ± 3.5) (Fig.2).

Discussion

The present study evaluated, in vitro, the effect of melatonin on the release of superoxide anion by macrophages isolated from the spleen of alloxan-induced diabetic pregnant rats and their pups. This work also studies the repercussion of maternal hyperglycemia fetus-placental parameters.

The pregnant rats with alloxan-induced diabetes had moderate hyperglycemia accompanied by low body weight (although not significant) (Table 1). On the other hand, there was no change in the count or viability of spleen macrophages in both diabetic and control groups. The activation ability of these cells, indicated by superoxide release, was also similar in the two groups, suggesting that diabetes does not affect these cells during pregnancy in rats. Similar results regarding the functional activity of leukocytes23 and phagocytic activity of macrophages16 were observed in patients with diabetes and in experimental studies on diabetic animals.

Other studies indicate that neurohormones pocess immunomodulatotory effects.24 Melatonin is an endogenous neurohormone produced by the pineal gland in mammals, and its beneficial action has been linked to its ability to scavenge different free radicals and increase the antioxidant activity of enzymes.14,15,25 Many studies have postulated that melatonin has a stimulatory action on the immune cells.16,26 In the present study, melatonin stimulated the cellular oxidative metabolism of control pregnant rats. This was evidenced by the increased superoxide release by macrophages, which reached values similar to those obtained in PMA-stimulated macrophages (positive control). Moreover, the cells of diabetic pregnant rats were not stimulated by melatonin. Similar results were found by Pawlak et al,16 who observed that melatonin stimulates phagocytosis in normal rats, but failed to stimulate this activity in cells isolated from diabetic rats.

During oxidative stress, cells generate high contents of superoxide radicals.28 Free radical generation has been reported as an important mechanism for body protection in infectious processes, mainly intestinal infections.6,29 In the present study, these radicals were not released in diabetic pregnant animals. In contrast to control rats, superoxide release by macrophages from diabetic rats was not modified by melatonin and was only weakly stimulated by PMA, which indicates that diabetes may partially affect the function of these cells. Diabetes mellitus is known to affect the immune system and some studies have demonstrated that blood neutrophils and B and T lymphocytes from diabetic patients show impaired immune function17,23 Similar results were reported in the leukocytes of diabetic patients23 and macrophages from diabetic rats.16

The association between diabetes and pregnancy may cause severe immunological changes because pregnancy alone makes diabetes control difficult, accentuating maternal hyperglycemia levels. Some studies demonstrate that immune alterations due to maternal hyperglycemia are reflected in alterations of immunological and biochemical parameters in the fetus, increased frequency of embryonic death as well as malformations and changes in placental parameters.10,11,30,31

Diabetic pregnancy represents an important risk factor for overnutrition and the development of obesity in the offspring..32 Some literature reports have associated fetal growth disturbances with maternal blood glucose and weight with the placenta. In the present study, the offspring of diabetic rats had increased blood glucose and body weight. A physiological state of insulin resistance is required to better direct maternal nutrients toward the fetus-placenta unit, allowing adequate fetal growth. Women with gestational diabetes mellitus have more a severe insulin resistance, which disrupts the intrauterine milieu, resulting in accelerated fetal development with increased risk of macrosomia. In the present paper, maternal blood glucose was characterized by mild hyperglycemia, which probably interfered in the weight of the newborn rats.  As a natural interface between mother and fetus, the placenta is the obligatory target of such environmental changes.33 The maternal metabolic alterations associated with profound diabetes in the uterine environment leads to an abnormal pattern of fetal growth.34

Studies that investigated the impact of placental alterations on fetal development revealed that rats with alloxan-induced diabetes had lower placental RNA and cell count. Abnormal placental structure and function in diabetic pregnancies are also related to increased oxidative stress and reduced antioxidant capacity in diabetic rats.35 Oxidative stress occurs in diabetic pregnancy, likely compromising antioxidant defense mechanisms and increasing free radical production.36

In contrast, no alterations were observed in the count and viability of the spleen macrophages of offspring from both control and diabetic groups. Offspring macrophages, however, had compromised functional activity. The decrease in spontaneous superoxide release by newborn macrophages shows the effects of maternal hyperglycemia on the activity of these cells. Melatonin also failed to stimulate offspring macrophages to release superoxide, indicating that maternal hyperglycemia may affect macrophage function in newborn rats. The role that melatonin plays as an immunomodulator is explained in the literature. Many studies have reported melatonin effects in the scavenging of free radicals14 and diabetes control,15 whereas others found that melatonin does not scavenge superoxide radicals.37 Phagocytic activity and superoxide alterations have been considered major factors of infectious diseases, mainly the bacterial infections,2,6 and are likely associated to higher infection rates  in children of diabetic mothers.

The maternal hyperglycemia modified the fetus-placental parameters and the melatonin modulated the macrophages activation in control and diabetics maternal rats and the newborn of the diabetic mothers. Melatonin can stimulate macrophage superoxide release in healthy pregnant rats, but not in the macrophages of newborn rats. When diabetes is induced in these animals, spontaneous superoxide release remains unaffected, but melatonin treatment decreases superoxide release in both mother and offspring.