Swim Training and the Genetic Expression of Adipokines in Monosodium Glutamate- Treated Obese Rats

Objective: The aim of this study was to evaluate the genetic expression of adipokines in the adipo-cytes of monosodium glutamate (MSG)-treated obese rats submitted to physical activity. Materials and methods: Obesity was induced by neonatal MSG administration. Exercised rats (MSG and control) were subjected to swim training for 30 min for 10 weeks, whereas their respective controls remained sedentary. Total RNA was obtained from sections of the mesenteric adipose tissue of the rats. mRNA levels of adiponectin (Adipoq), tumor necrosis factor alpha (Tnf), peroxisome proliferator-activated receptor alpha (Ppara), and peroxisome proliferator-activated receptor gamma (Pparg) adi-pokines were quantified by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Results: In the exercise-trained control group, the expression of Adipoq increased compared to the sedentary control, which was not observed in the MSG-obese rats. Increased levels of Tnf in MSG-obese rats were not reversed by the swim training. The expression of Ppara was higher in sedentary MSG-obese rats compared to the sedentary control. Swimming increased this adipokine expression in the exercise-trained control rats compared to the sedentary ones. mRNA levels of Pparg were higher in the sedentary MSG-rats compared to the sedentary control; however, the exercise did not influenced its expression in the groups analyzed. Conclusions: In conclusion, regular physical activity was not capable to correct the expression of proinflammatory adipokines in MSG-obese rat adipocytes.


INTRODUCTION
N eonatal treatment with monosodium glutamate (MSG) causes lesions in the arcuate nucleus, deregulating an important area involved in the body weight regulation and glycemic control (1,2).Rats treated with MSG in the first days after birth deve lop obesity in adult life and present insulin resistance, glucose intolerance, dyslipidemia and cardiovascular dysfunction, which are typical symptoms of metabolic syndrome carriers (3).Similar to obese subjects, MSG obese rats present lowgrade chronic inflammation, in particular on adipocyte tissue.Studies demonstrated that adipocytederived adipokine profile of MSGobese rats is altered, thereby contributing to insulin resistance in this obesity model (4).
Regular physical activity is an important tool in the obesity control and associated comorbidities, impro ving inflammatory condition typical of an obese indi vidual.There are no studies with this animal model (MSGobese rats) reporting the adipokines expression in mesenteric fat and physical activity.Thus, the aim of this study was to analyze the genetic expression profile of some adipokines of the mesenteric adipose tissue of MSGtreated obese rats subjected to swimming.Exercise and adipokines expression mals were weaned and divided in four groups (N = 8 rats per group): sedentary controls (CONSED), exercise trained controls (CONEXE), sedentary MSGtreated animals (MSGSED), and exercisetrained MSGtrea ted animals (MSGEXE).All of the exercisetrained animals were subjected to regular swimming 3 times per week and 30 min per day for 10 weeks, according to (5).Each rat had a load weight equivalent to 5% of body mass attached to the base of tail to ensure that animals were in constant swimming activity.Araujo and cols.(8) adapted the lactate minimum test to swimming in rats.Blood lactate concentrations of 5.5 mM are achieved at loads equivalent to 5% body weight, so this swimming program can be considered moderate exercise (8).The CONSED and MSGSED groups remained sedentary.

MATERIALS AND METHODS
At the age of 90 days, the nasoanal length (NAL) and body weight were measured to assess the obesity degree in the animals using the Lee index [ 3 √(body weight (g)/nasoanal length (cm)], which is an indica tor of obesity used for rodents (9).Additionally, viscer al fat (mesenteric) deposits were removed, washed and weighed.Data are reported considering body weight (g/100 g of body weight).All values are reported as means ± standard error (SEM).The Student Ttest was used with significance of P < 0.05.
For the analysis of genetic expression by qRTPCR, sections of mesenteric fat were used for total RNA ex traction and, subsequently cDNA synthesis.The am plified adipokines were: Adipoq, Tnf, Ppara and Pparg.The adipokines primer sequences were synthetized ac cording to (4).The 18S rDNA was used as an internal control.Relative change in genetic expression was pre sented as 2 ΔΔCt (10).

RESULTS
According to table 1, body weight and NAL were 27.7 and 13.4% lower in the MSGSED group, respectively, compared to the CONSED group (P < 0.05).As for the Lee index, the value was 4.7% higher in the MSG SED animals when compared to the CONSED ones (P < 0.05).MSGSED rats presented a significantly in crease of 109.9% of mesenteric fat in comparison with CONSED rats.Swimming had no effect in rat NAL.However, there was a reduction of approximately 8.7 and 6.7% of body weight and 18.5 and 20.6% of mes enteric fat accumulation in CONEXE and MSGEXE, respectively, when compared to their respective seden tary groups (P < 0.05).Physical activity significantly reduced the Lee index in 4.2% only in animals treated with MSG compared to the MSGSED.
There was no statistical difference in the expression of Adipoq between MSGSED and CONSED groups.The expression level of adiponectin was approximate ly 6fold higher in CONEXE rats compared to the CONSED rats (P < 0.05) (Figure 1A).Tnf expres sion significantly increased approximately 4 times in the MSGSED group in comparison with the CONSED group.Nevertheless, in both groups, exercise did not alter the expression of Tnf in the mesenteric adipose tissue (Figure 1B).The expression levels of Ppara were on average 3.7 times higher in the MSGSED group in comparison with the CONSED group (P < 0.05).The practice of physical activity resulted in a 4.1fold increase in the expression of Ppara in CONEXE rats when compared to the CONSED rats (P < 0.05) (Fi gure 1C).The levels of Pparg mRNA were around 4.5 times higher in the MSGSED group, compared to the CONSED (P < 0.05).However, exercise did not in fluenced the expression of Pparg in the groups assessed (Figure 1D).

DISCUSSION
Rodents treated with MSG develop several obesity char acteristics (13), probably caused by the reduction in the energy metabolism (11) and a dysfunction in the auto nomic nervous system, which is characterized by a high parasympathetic and a low sympathetic activities, as con sequences of hypothalamic lesion caused by MSG (12).The increase in the Lee index associated with a major accumulation of mesenteric fat in MSGtreated obese rats confirmed the efficacy of MSG neonatal administra tion in obesity induction in the animals of our study.However, MSGobese rats presented reduction in body weight and in NAL due to the delay in bone and muscle development, which is a consequence of a reduction in the growth hormone (GH) levels resulting from the ar cuate nucleus lesion (13).
Corroborating with previous reports (5,6), our stu dy showed that swimming 30 min/3 times per week with a load weight equivalent to 5% of body mass was effective in promoting reduction in mesenteric fat deposits in MSGtreated animals, without causing collateral effects.Regular swimming corrects hyperin sulinemia and resistance to insulin and favors lipolysis triggered by the activation of the sympathetic nervous system (5).Similarly, Shima and cols.( 14) using run in rats showed that frequency of 2 to 3 times/week is sufficient to prevent development of type 2 diabetes in OLETF rats (spontaneous model of type 2 diabetes).
The training protocol used in this study began imme diately after weaning, when rodents may be treated in terms of development like children.Atlantis and cols.(15) revised the beneficial effects of body fat reduction in obesity/overweight children submitted to exercise and showed that good results in training can be ob tained with exercises in lower intensity and frequency, compared with those recommended in the literature.
Regular physical activities, even if there is no loss of weight, are related to a substantially reduction of total and visceral fat, besides leading to a significantly improvement in obesity (16,17).MSGSED rats have hypertrophic adipocytes (5), which presented an altered inflammatory profile directly related to insulin resistan ce.Carvalho Leite and cols.( 5) demonstrated that swim training program was effective in attenuating morpho logical alterations in the adipose tissue and pancreatic islets in MSGtreated obese rats.Histological analyzes in adipose tissue deposits, demonstrated that physical training reduced the adipocyte diameter and increased the number of adipocytes in MSGEXE and CONEXE groups, compared with their sedentary counterparts (5).
It is known that the expression of adiponectin mRNA is inversely related to obesity, type 2 diabetes mellitus (T2DM) and cardiovascular diseases (18).Our study showed that there was no difference in adipo nectin expression between MSGobese rats and control animals.Other studies have already shown that MSG obesity model did not reflect hyperadiponectinemia or adiponectin resistance, a phenomenon observed in other obesity models (4,19).Our research showed that swimming stimulated the genetic expression of adipo nectin in adipocytes of CONEXE rats.High plasma Arch Endocrinol Metab.2015;59/3 Exercise and adipokines expression levels of adiponectin were also found in humans sub jected to aerobic exercises (20).On the other hand, exercised MSGobese rats did not present alteration in adiponectin expression, which may reinforce that adi ponectin levels in the MSG model is normal.
MSG rats presented a higher expression of Tnf, when compared to control animals, corroborating with RomanRamos and cols.(4).Indeed, expression levels of Tnf increased in obesity, T2DM and cardiovascular disease conditions (18).TNF promotes reduction in adiponectin expression and secretion (18), contribu ting to metabolic syndrome development.Together, these data indicate that high levels of Tnf expression found in adipocytes of MSGobese rats are probably due to chronic inflammation of the adipose tissue with macrophage infiltration (21).Probably this adipokine contributes to insulin resistance, which is typical for this model.According to our data, exercise does not correct the inflammatory profile in this obesity model.
Corroborating RomanRamos and cols.( 4), Ppara and Pparg had their mRNA levels increased in the MSGtreated animals, which may indicate an inflamma tory condition due to obesity.Pparg is mainly expressed in the adipose tissue and its activation in MSGtreated rat adipose tissue may lead to the increase of adipocyte size, probably causing hypertrophy (5).RomanRamos and cols.(4) suggested that the activation of PPARs is responsible for the deregulated inflammatory profi le of MSG animals; however, some authors reported that Ppara and Pparg activation may improve insulin resistance induced by obesity (22).Our results indicate that Pparg may participate directly in the increase of the visceral fat content and adipocyte size that occurs in MSGobese animals (5).On the other hand, exercise was not able to correct its levels in obese animals.Pro bably, Pparg function is not only metabolic, but it may also be involved in the inflammatory control, partici pating in the proinflammatory cytokine pathway and in the modulation of acute phase of the inflammatory response by independent mechanisms of lipoprotein changes (23).
Genes regulated by Ppara participate in the keypro tein regulation involved in lipid metabolism, fatty acid oxidation, hemostasis and inflammation (24).High levels of Ppara mRNA may be related to the increase of body weight in MSGtreated animals.Physical activ ity did not have influence over this gene expression in these animals.The higher expression of Ppara mRNA in CONEXE animals can be explained by a high mi tochondrial activity and peroxisomal betaoxidation.PPARA is capable of upregulating the expression of genes to enzymes such as carnitinepalmitoyltransfera seI (CPTI), which transports fatty acids into the mi tochondria for betaoxidation.Expression of PPARA also increases expression of acetyilCoA synthase, a mi tochondrial enzyme necessary for betaoxidation (25).However, more studies are needed to determine which exactly the role of Ppara is and its relation to obesity.
Regular physical activity was not capable to correct proinflammatory adipokine expression in adipocytes of MSGtreated obese rats.Other factors or adipoki nes cannot be discarded.More studies are necessary to determine which adipokines have altered expression by physical activity and the resulting effect in energy homeostasis.Furthermore, studies to analyze the re sulting proteins of the analyzed genes are necessary, although cytokines may have autocrine and paracrine actions, which may affect the metabolism of adipose tis sue without necessarily affecting plasma levels.

3 Exercise and adipokines expressionFigure 1 .
Figure 1. mRNA expression levels of the mesenteric fat tissue samples of MSG-obese and control rats submitted at swimming training (N = 8 rats per group).(A) Adipoq, (B) Tnf, (C) Ppara and, (D) Pparg.Letters above the bars represent statistical difference in Student T-test (P < 0.05).a CON-SED; b CON-EXE; c MSG-SED; d MSG-EXE.
Experiments were approved by the Ethical Commit tee for Animal Experiments of Universidade Estad ual de Ponta Grossa (protocol number 02860).The obese group received subcutaneous injections of MSG (4 mg/g of body weight) during the first 5 days of life, whereas the control group received equimolar saline, a protocol adapted by (57).At the age of 21 days, ani Arch Endocrinol Metab.2015;59/3

Table 1 .
Biometric parameters in MSG-obese and control rats submitted at swimming training