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Effects of perinatal protein restriction on the oxidative balance in the hypothalamus of 60-day-old rats

Efeitos da restrição proteica perinatal sobre o balanço oxidativo no hipotálamo de ratos de 60 dias de idade

ABSTRACT

Objective

Evaluate the effects of maternal low-protein diet on the oxidative stress in the hypothalamus of 60-day-old rats.

Methods

Male Wistar rats were divided into two experimental groups according to the mother’s diet during pregnancy and lactation; control group (NP:17% casein n=6) and a malnourished group (LP:8% casein n=6). At 60 days of life, the rats were sacrificed for the collection of the hypothalamus for further biochemical analysis.

Results

Our results showed an increase in oxidative stress in malnourished group, observed through an increase in carbonyl content (p=0.0357), a reduction in the activity of the glutathione-S-transferase enzyme (p=0.0257), and a reduction in the non-enzymatic antioxidant capacity evidenced by the decrease in the ratio reduced glutathione/oxidized glutathione (p=0.0406) and total thiol levels (p=0.0166).

Conclusion

A low-protein diet during pregnancy and lactation is closely associated with increased oxidative stress and reduced antioxidant capacity in the hypothalamus of sixty-day-old rats.

Keywords
Hypothalamus; Oxidative stress; Protein-energy malnutrition

RESUMO

Objetivo

Avaliar os efeitos da restrição proteica materna sobre o estresse oxidativo no hipotálamo de ratos de 60 dias de idade.

Métodos

Ratos Wistar machos foram divididos em dois grupos experimentais de acordo com a dieta da mãe durante a gestação e lactação: grupo controle (NP: 17% caseína n=6) e grupo desnutrido (LP: 8% caseína n=6). Aos 60 dias de vida, os ratos foram sacrificados para coleta do hipotálamo para posterior análise bioquímica.

Resultados

Os resultados demonstraram aumento do estresse oxidativo no grupo desnutrido, observado através do aumento do conteúdo de cabonilas (p=0,0357) e redução da atividade da enzima glutationa-S-transferase (p=0,0257) e da capacidade antioxidante não enzimática, evidenciada pela queda da razão glutationa reduzida/glutationa oxidada (p=0,0406) e dos níveis de tióis totais (p=0,0166).

Conclusão

Uma dieta com baixo teor de proteínas durante a gestação e lactação está intimamente associada ao aumento do estresse oxidativo e à redução da capacidade antioxidante no hipotálamo de ratos de 60 dias de vida.

Palavras-chave
Hipotálamo; Estresse oxidativo; Desnutrição proteico-calórica

INTRODUCTION

According to the Origins of Health and Disease Development theory, have been proposed that adverse conditions such as malnutrition, during critical periods of the development, can predispose the offspring to chronic diseases in adulthood [11 Gluckman PD, Hanson MA, Mitchell MD. Developmental origins of health and disease: reducing the burden of chronic disease in the next generation. Genome Med. 2010;2(14).

2 Lillycrop KA, Burdge GC. Epigenetic mechanisms linking early nutrition to long term health. Best Pract Res Clin Endocrinol Metab. 2012;26:667-76.

3 Hales CN, Barker DJP. The thrifty phenotype hypothesis: Type 2 diabetes. Br Med Bull. 2001;60:5-20.

4 Lee HS. Impact of Maternal Diet on the Epigenome during In Utero Life and the Developmental Programming of Diseases in Childhood and Adulthood. Nutrients. 2015;7:9492-507.

5 Wang G, Walker SO, Hong X, Bartell TR, Wang X. Epigenetics and early life origins of chronic noncommunicable diseases. J Adolesc Health. 2013;52:14-21.
-66 Fall CHD. Fetal programming and the risk of noncommunicable disease. Indian J Pediatrics. 2013;80:13-20.]. In this sense, experimental evidence demonstrates that maternal protein restriction during pregnancy is related to different outcomes in adulthood, resulting in mitochondrial dysfunction and oxidative stress in the heart, brainstem and kidney [77 Silva PAA, Bernardo EM, Pereira AR, Andrade SSC, Lima TA, Moura FC, et al. Moderate offspring exercise offsets the harmful effects of maternal protein deprivation on mitochondrial function and oxidative balance by modulating sirtuins. Nutr Metab Cardiovasc Dis. 2021;31(5):1622-34.

8 Ferreira DJS, Pedroza AA, Braz GRF, Fernandes MP, Lagranha CJ. Mitochondrial dysfunction: maternal protein restriction as a trigger of reactive species overproduction and brainstem energy failure in male offspring brainstem. Nutr Neurosci. 2019;22(11):778-88.
-99 Pedroza A, Ferreira DS, Santana DF, Silva PT, Aguiar JF, Sellitti D, et al. A maternal low-protein diet and neonatal overnutrition result in similar changes to glomerular morphology and renal cortical oxidative stress measures in male Wistar rats. Appl Physiol Nutr Metab. 2019;44(2):164-71.]. From a clinical point of view, the Dutch Famine of 1944 is perhaps the main parallel to the study of malnutrition in humans. In a cohort of 2,414 people aged 50 to 58 years, born in Amsterdam during the famine period, were observed in men greater glucose intolerance, microalbuminuria, atherogenic lipid profile, coronary artery disease, and, for women was observed, a greater risk of breast cancer [1010 Roseboom T, Rooij S, Painter R. The Dutch famine and its long-term consequences for adult health. Early Hum Dev. 2006;82(8):485-91.].

According to data already published in literature, increased oxidative stress is among the main molecular outcomes associated with low-protein diets, which ultimately may be associated with functional changes in different tissues [77 Silva PAA, Bernardo EM, Pereira AR, Andrade SSC, Lima TA, Moura FC, et al. Moderate offspring exercise offsets the harmful effects of maternal protein deprivation on mitochondrial function and oxidative balance by modulating sirtuins. Nutr Metab Cardiovasc Dis. 2021;31(5):1622-34.

8 Ferreira DJS, Pedroza AA, Braz GRF, Fernandes MP, Lagranha CJ. Mitochondrial dysfunction: maternal protein restriction as a trigger of reactive species overproduction and brainstem energy failure in male offspring brainstem. Nutr Neurosci. 2019;22(11):778-88.
-99 Pedroza A, Ferreira DS, Santana DF, Silva PT, Aguiar JF, Sellitti D, et al. A maternal low-protein diet and neonatal overnutrition result in similar changes to glomerular morphology and renal cortical oxidative stress measures in male Wistar rats. Appl Physiol Nutr Metab. 2019;44(2):164-71.]. In this sense, oxidative stress is understood as a chronic imbalance between the production of pro-oxidant agents and the ability to remove them by the antioxidant systems, with such agents coming mainly from the mitochondria [1111 Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol. 2003;552:335-44.]. The central nervous system, is easily affected by the deleterious effects of oxidative stress, due to its high lipid content, high energy demand and low antioxidant capacity [1212 Salim S. Oxidative Stress and the Central Nervous System. J Pharmacol Exp Ther. 2017;360(1):201-05.]. In a protein restriction model, Santana et al. (2019) demonstrated that the brainstem of Wistar rats exposed to two consecutive generations of low-protein diet during the gestation and lactation periods showed an overproduction of reactive species added to an impairment of mitochondrial bioenergetics [1313 Santana DF, Ferreira DS, Braz G, Sousa S, Silva T, Gomes DA, et al. Maternal Protein Restriction in Two Successive Generations Impairs Mitochondrial Electron Coupling in the Progeny’s Brainstem of Wistar Rats From Both Sexes. Front Neurosci. 2019;13(203).]. In addition, increased lipid and protein oxidation along with impaired mitochondrial function has also been demonstrated in the brainstem of Wistar rats submitted to a low-protein diet during pregnancy and lactation [1414 Ferreira DS, Liu Y, Fernandes MP, Lagranha CJ. Perinatal low-protein diet alters brainstem antioxidant metabolism in adult offspring. Nutr Neurosci. 2016;19(8):369-75.,1515 Ferreira DJS, Silva PAA, Braz GRF, Silva-Filho RC, Lima TA, Fernandes MP, et al. Mitochondrial bioenergetics and oxidative status disruption in brainstem of weaned rats: Immediate response to maternal protein restriction. Brain Res. 2016;1642:553-61.].

It’s well known that the hypothalamus regulates energy balance through neurons that respond to hormonal and nutritional variation, therefore, can be affected by protein restriction during critical periods of development [1616 Bouret SG. Nutritional programming of hypothalamic development: critical periods and windows of opportunity. Int J Obes Suppl. 2012;2:19-24.]. Specifically in the hypothalamus, few data are available in the literature to demonstrate the effects of a maternal low-protein diet on oxidative balance. Due to this lack of literature associating the impact of a low-protein diet during gestation and lactation period on oxidative balance, we hypothesize that maternal protein restriction during pregnancy and lactation is associated with an impairment in the hypothalamic oxidative balance of the offspring. To test this hypothesis, after undergoing protein restriction, the evaluation of biomarkers of oxidative stress and enzymatic and non-enzymatic antioxidant defenses in the hypothalamus of 60-day-old rats was performed.

METHODS

Animals and diet

The experimental procedures followed what is recommended by the guidelines of the Institutional Ethics Committee for Animal Research (Approval Protocol nº 0060/2018), meeting the “Principles of Care for Laboratory Animals” described by the National Institutes of Health, Bethesda, MD, USA. Female Wistar rats (n=8), between 80 and 90 days old, were paired in a 2 to 1 male ratio and were evaluated daily for pregnancy detection. When pregnant, they were randomly divided into two groups, based on the amount of casein protein in the diet offered: control group (NP, 17% casein); and malnourished group (LP, 8% casein) (Table 1). The diet was carried out at the Laboratório de Técnica Dietética do Centro Acadêmico de Vitória-Universidade Federal de Pernambuco, and both the normal diet and the low-protein diet had the same energy value, being offered during pregnancy and lactation, as already described [88 Ferreira DJS, Pedroza AA, Braz GRF, Fernandes MP, Lagranha CJ. Mitochondrial dysfunction: maternal protein restriction as a trigger of reactive species overproduction and brainstem energy failure in male offspring brainstem. Nutr Neurosci. 2019;22(11):778-88.,1313 Santana DF, Ferreira DS, Braz G, Sousa S, Silva T, Gomes DA, et al. Maternal Protein Restriction in Two Successive Generations Impairs Mitochondrial Electron Coupling in the Progeny’s Brainstem of Wistar Rats From Both Sexes. Front Neurosci. 2019;13(203).,1717 Nascimento L, Freitas CM, Silva-Filho R, Leite AC, Silva AB, Silva AI, et al. The effect of maternal low-protein diet on the heart of adult offspring: role of mitochondria and oxidative stress. Appl Physiol Nutr Metabol. 2014;39:880-7.,1818 Reeves PG, Nielsen FH, Fahey JR, George C. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993;123(11):1939-51.]. One day after birth, litter sizes were standardized by eight pups per dams to avoid litter size interference in milk production. To avoid “litter effect”, only 2-3 males from each litter were chosen to continue in the study, which were fed with commercial chow (Labina; Purina Agriband, Brazil). At 60 days after birth, the rats were sacrificed to collect the hypothalamus for further biochemical analysis.

Table 1
Composition of the diets (g/100 g diet).

Tissue homogenization

The hypothalamus was homogenized in a buffer containing 50 mM-TRIS, 1 mM-EDTA [pH 7.4], 1 mM-sodium orthovanadate, 1.1 mM PMSF, and 0.1% NP-40. The samples were homogenized (Tecnal, Sao Paulo, Brazil), the homogenates were centrifuged at 1180 x g for ten minutes at four degrees Celsius, and stored at -80°C. Protein concentration was determined by the Bradford method [1919 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem. 1976;72:248-54.].

Evaluation of malondialdehyde production

A total of 150 µg of hypothalamic protein was used to evaluate malondialdehyde (MDA), a product generated after the reaction with thiobarbituric acid (TBA) according Draper et al. [2020 Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M. A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radic Biol Med. 1993;15:353-63.]. The reaction was initiated by adding 30% trichloroacetic acid (TCA) and Tris-HCl (3 mM) to the samples, after were subjected to centrifugation (10 min at 2500 x g). In the final step, the supernatant was transferred to another tube and mixed with 0.8% of TBA (v/v), and boiled for 30 min. After let the samples cool down at room temperature and read at 535 nm using a spectrophotometer. The results was expressed in mmol/mg of protein [2020 Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M. A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radic Biol Med. 1993;15:353-63.].

Evaluation of protein oxidation

Used as a marker for oxidative damage to the protein, the carbonyl content was measured according to Reznick and Packer [2121 Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol. 1994;233:357-63.]. 30% TCA was mixed with the sample and centrifuged at 1180 x g for 15 min. Then, the pellet was suspended in 10 mM 2,4-dinitrophenylhydrazine and incubated for one hour in the dark, shaking every fifteen minutes. Finally, the samples were centrifuged and washed three times with ethyl/acetate, with the resulting pellet suspended in 6M guanidine hydrochloride and incubated for five minutes at 37oC. The final solution was read at 370 nm and the results expressed in mol/mg of protein [2121 Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol. 1994;233:357-63.].

Measurement of superoxide dismutase (EC 1.15.1.1) activity

The total measurement of Superoxide Dismutase (SOD) enzyme activity was measured using the methodology previous described by Misra and Fridovich [2222 Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972;247:3170-5.]. 150 µg of protein from hypothalamic supernatants was incubated with 880 ml of sodium carbonate (0.05%, pH 10.2, 0.1 mM EDTA) under a temperature of 30oC and then the reaction was started with 30 mM epinephrine (0.05% acetic acid). The kinetics of epinephrine autooxidation inhibition was evaluated for 90 seconds at 480 nm and the measure of its activity expressed in U/mg of protein.

Measurement of catalase (EC 1.11.1.6) activity

To assess measurement of Catalase (CAT) activity, we follow Aebi [2323 Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121-6.] protocol. Briefly, the reaction mixture containing 50 mM of phosphate buffer at pH 7.0, 300 mM H2O2 and 150 µg of hypothalamic homogenate. The rate constant was obtained at 240 nm during 4 min at 30oC. The activity of this enzyme was demonstrated as U/mg of protein [2323 Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121-6.].

Measurement of glutathione-S-transferase (EC 2.5.1.18) activity

For the evaluation of GST activity, we applied the protocol from Habig [2424 Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249:7130-9.]. Initially, 150 µg of samples were placed in 0.1 M phosphate buffer, pH 6.5, with 1 mM EDTA (30oC), and the reaction was started with 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 1 mM-GSH. Finally, the 2,4-dinitrophenyl-S-glutathione product was monitored at 340 nm [2424 Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249:7130-9.].

Measurement of REDOX state (GSH and GSSG)

The Reduced Glutathione (GSH) content was evaluated in a medium with 0.1 M phosphate buffer together with 5 mM EDTA (pH 8.0), and 100 µg supernatant protein. After that, at room temperature, 1 mg/ml of o-phthaldialdehyde was added to the mixture and incubated for fifteen minutes; then fluorescence was measured at 350 excitation and 420 nm emission wavelengths. To measure the amount of oxidized glutathione (GSSG), 200 µg of protein was incubated with N-ethylmaleimide (0.04 M) for thirty minutes at RT and then 0.1 M of NaOH buffer was added to make 0.2 ml. Similarly, for GSH, protein aliquots were added with o-phthaldialdehyde, and fluorescence was measured in the same way as oxidized glutathione. Results for the two glutathiones were compared with standard curves and the REDOX state defined as the ratio of GSH/GSSG [2525 Hissin PJ, Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 1976;74:214-26.,2626 Braz GRF, Emiliano AS, Sousa SM, Pedroza AAS, Santana DF, Fernandes MP, et al. Maternal low-protein diet in female rat heart: possible protective effect of estradiol. J Dev Orig Health Dis. 2017;8:322-30.].

Total thiol content

According to the methodology previously described [2727 Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82:70-7.], using 5,5-dithio-bis (2-nitrobenzoic acid (DTNB). Briefly, part of the hypothalamic supernatant (450 µg) was incubated in the dark with DTNB (30 ml, at 10 mM) and extraction buffer (pH 7.4) was used to reach a final volume of 1 ml. Final samples were measured at 412 nm of absorbance, and the results are expressed in mmol/mg protein.

Considering the normality of data distribution, the difference between the groups was evaluated by Student’s t test and all data were expressed as mean and standard error of the mean (SEM). Considering significant only with p<0.05, the analyzes were conducted using the GraphPad Prism 6.0® software (GraphPad Software, Inc).

RESULTS

Oxidative stress biomarkers

The evaluation of protein and lipid oxidation, were performed in the hypothalamus of both groups (Figure 1). Our results demonstrate that in low-protein group no difference in lipoperoxidation (NP: 26.76±1.119 N=4 vs LP: 28.13±1.217 N=6, p=0.4583), although in protein damage we observed twice more damage in LP group (NP: 1.705±0.4592 N=4 vs LP: 4.091±0.6992 N=6, p=0.0357) than the NP group, Figure 1A and 1B, respectively.

Figure 1
Malondialdehyde and Carbonyl concentration.

Enzymatic antioxidant system

Was performed the activity of the antioxidant enzymes SOD, CAT and GST in hypothalamus (Figure 2) [2828 Halliwell B, Gutteridge JM. Free radicals in biology and medicine. Oxford: United Kingdom; 2007.]. No difference was observed in SOD activity (NP: 67.16±6.149 N=4 vs LP: 95.78±12.10 N=7, p=0.1261) and CAT (NP: 413.6±173.6 N=3 vs HP: 512.5±34.33 N=4, p=0.5409). However, in GST activity we observed a significant decrease in LP group (NP: 0.2108±0.05301 N=4 vs LP: 0.05185±0.03226 N=6, p=0.0257) in this same group.

Figure 2
Activity of superoxide dismutase, catalase and glutathione-S-transferase enzymes.

Non-enzymatic antioxidant system

Additionally to the enzymatic system we evaluate the non-enzymatic antioxidant system. Thus, reduced glutathione and total thiol groups are the key non-enzymatic molecules that act to reduce oxidative stress. Our data showed (Figure 3), that protein malnutrition induce in the hypothalamus a reduction in the REDOX state (NP: 2.663±0.09223 N=5 vs LP: 1.727±0.3725 N=5, p=0.0406), and total thiol levels (NP: 0.0362±0.004329 N=5 vs LP: 0.0230±0.002017 N=6, p=0.0166), corroborating with the establishment of oxidative stress.

Figure 3
REDOX state and Total thiols groups.

DISCUSSION

Previous experimental data in literature have shown that maternal protein restriction is associated with altered food intake and disease in the adult offspring [2929 Breton C, Lukaszewski MA, Risold PY, Enache M, Guillemot J, Rivière G, et al. Maternal prenatal undernutrition alters the response of POMC neurons to energy status variation in adult male rat offspring. Am J Physiol Endocrinol Metab. 2009;296:E462-E472.

30 Palou M, Priego T, Sánchez J, Palou A, Picó C. Sexual dimorphism in the lasting effects of moderate caloric restriction during gestation on energy homeostasis in rats is related with fetal programming of insulin and leptin resistance. Nutr Metab. 2010;7:e69.
-3131 Manuel-Apolinar L, Rocha L, Damasio L, Tesoro-Cruz E, Zarate A. Role of prenatal undernutrition in the expression of serotonin, dopamine and leptin receptors in adult mice: Implications of food intake. Mol Med Rep. 2014;9:407-12.]. Here we demonstrate that maternal protein restriction disturbs the offspring’s hypothalamic oxidative balance at 60 days of life, which is observed through the increase in carbonyl content, reduction in GST activity, in the GSH/GSSG ratio and in the levels of total thiols.

In general, the brain of offspring from mothers submitted to protein malnutrition is subject to several negative modulations, including reticulum stress, oxidative damage, and downregulation of the growth factors [3232 Fernandes MS, Pedroza AA, Andrade Silva SC, Lemos MDT, Bernardo EM, Pereira AR, Santos TM, Lagranha C. Undernutrition during development modulates endoplasmic reticulum stress genes in the hippocampus of juvenile rats: Involvement of oxidative stress. Brain Res. 2022;23:1797.,3333 Fragoso J, Carvalho JSG, Silva HT, Loizon E, Oliveira NSV, Vidal H, et al. Effects of maternal low-protein diet and spontaneous physical activity on the transcription of neurotrophic factors in the placenta and the brains of mothers and offspring rats. J Dev Orig Health Dis. 2021;12(3):505-12.]. Knowing the important relationship between clock genes and the regulation of energy metabolism [3434 Kalsbeek A, Scheer FA, Perreau-Lenz S, La Fleur SE, Yi CX, Fliers E, et al. Circadian disruption and SCN control of energy metabolism. FEBS Lett. 2011;585(10):1412-26.], Crossland et al. [3535 Crossland RF, Balasa A, Ramakrishnan R, Mahadevan SK, Fiorotto ML, Van den Veyver IB. Chronic Maternal Low-Protein Diet in Mice Affects Anxiety, Night-Time Energy Expenditure and Sleep Patterns, but Not Circadian Rhythm in Male Offspring. Plos One. 2017;12(1).] demonstrated a time-effect difference in the expression of Per 1, Clock, and Per 2 in the hypothalamus of rats submitted to a low-protein maternal diet. The results together show that maternal protein restriction during the developmental period can negatively influence several aspects of the hypothalamic milieu.

Our data show increased protein oxidation, with no difference in lipid oxidation. Similar to what we found here for biomarkers of oxidative stress, Ferreira et al. [1515 Ferreira DJS, Silva PAA, Braz GRF, Silva-Filho RC, Lima TA, Fernandes MP, et al. Mitochondrial bioenergetics and oxidative status disruption in brainstem of weaned rats: Immediate response to maternal protein restriction. Brain Res. 2016;1642:553-61.] using a similar experimental design with low-protein diet in Wistar rats during pregnancy and lactation, also found increased carbonyl content and no change in MDA levels in brainstem from LP animals when compared to their normoproteic counterparts. The preferential oxidative damage to proteins, instead to lipids, is not fully understood, but previous data in literature showed damage to the amino acids tyrosine and tryptophan in CNS regions from rats with 60-day-old submitted to a low-protein diet [3636 Feoli AM, Siqueira IR, Almeida L, Tramontina AC, Vanzella C, Sbaraini S, et al. Effects of protein malnutrition on oxidative status in rat brain. Nutrition. 2006;22:160-5.,3737 Torres N, Bautista CJ, Tovar AR, Ordaz G, Rodriguez-Cruz M, Ortiz V, et al. Protein restriction during pregnancy affects maternal liver lipid metabolism and fetal brain lipid composition in the rat. Am J Physiol Endocrinol Metab. 2010;298:E270-7.]; these studies can support the hypothesis that proteins can be more sensitive to oxidative damage depending on the amino acids that compose them [3838 Stadtman ER, Van RH, Richardson A, Wehr NB, Levine RL. Methionine oxidation and aging. Biochim Biophys Acta. 2005;1703:135-40.].

In addition, we observed that in the LP group a strong reduction in GST activity, without any significant changes in SOD and CAT activity. Once again, data from Ferreira et al. (2016) showed an similar result, where animals submitted to a low-protein diet exhibited lower GST activity, without any change in SOD, CAT, Glutathione peroxide (GPx) and Glucose-6-Phosphate Dehydrogenase (G6PDH) [1515 Ferreira DJS, Silva PAA, Braz GRF, Silva-Filho RC, Lima TA, Fernandes MP, et al. Mitochondrial bioenergetics and oxidative status disruption in brainstem of weaned rats: Immediate response to maternal protein restriction. Brain Res. 2016;1642:553-61.]. Considering, therefore, the ability of GST to detoxify lipid peroxidation products and electrophilic compounds [3939 Singhal SS, Singh SP, Singhal P, Horne D, Singhal J, Awasthi S. Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling. Toxicol Appl Pharmacol. 2015;289:361-70.

40 Awasthi YC, Ansari GA, Awasthi S. Regulation of 4-hydroxynonenal mediated signaling by glutathione S-transferases. Methods Enzymol. 2005;401:379-407.
-4141 Gajewska B, Kazmierczak B, Kuzma-Kozakiewicz M, Jamrozik Z, Baranczyk-Kuzma A. GSTP1 polymorphisms and their association with glutathione transferase and peroxidase activities in patients with motor neuron disease. CNS Neurol Disord Drug Targets. 2015;14(10):1328-33], it can be suggested that the LP diet results in an impairment of hypothalamic function, due to the modulation in the ROS production and an impair in antioxidant system.

Related to non-enzymatic defense, the ratio between reduced and oxidized glutathione (GSH/GSSG) is one of the main indicators of the REDOX state of the cell, since this molecule is the main thiols related to intracellular antioxidant capacity [4040 Awasthi YC, Ansari GA, Awasthi S. Regulation of 4-hydroxynonenal mediated signaling by glutathione S-transferases. Methods Enzymol. 2005;401:379-407.,4242 Circu ML, Aw TY. Glutathione and modulation of cell apoptosis. Biochim Biophys Acta. 2012;1823(10):1767-77.]. Considering the similarity of different brain regions, such as high O2 consumption and polyunsaturated fatty acid content, this organ is particularly vulnerable to oxidative stress and allows a comparison between the hypothalamus and the brainstem [4343 Halliwell B. Oxidative stress and neurodegeneration: where are we now? J Neurochem. 2006;97(6):1634-58.]. In this sense, data published previously in brainstem [1414 Ferreira DS, Liu Y, Fernandes MP, Lagranha CJ. Perinatal low-protein diet alters brainstem antioxidant metabolism in adult offspring. Nutr Neurosci. 2016;19(8):369-75.,1515 Ferreira DJS, Silva PAA, Braz GRF, Silva-Filho RC, Lima TA, Fernandes MP, et al. Mitochondrial bioenergetics and oxidative status disruption in brainstem of weaned rats: Immediate response to maternal protein restriction. Brain Res. 2016;1642:553-61.] demonstrate that perinatal protein malnutrition impairs, at different stages of life, mitochondrial bioenergetics, increases in oxidative stress markers and reduces the antioxidant capacity, which was justified by the reduction of the GSH/GSSG ratio – corroborating with our result, where we observed a reduction in malnourished animals when compared to their normonourished controls.

Finally, in line with the reduction in the REDOX state, we observed that the total thiol content was significantly reduced in animals from malnourished mothers. In the same sense, in terms of brain tissues (brainstem), was previously demonstrated that maternal protein restriction reduce both the level of Reduced Glutathione (GSH) in male Wistar rats and the content of total thiols in females at 22 days old [1515 Ferreira DJS, Silva PAA, Braz GRF, Silva-Filho RC, Lima TA, Fernandes MP, et al. Mitochondrial bioenergetics and oxidative status disruption in brainstem of weaned rats: Immediate response to maternal protein restriction. Brain Res. 2016;1642:553-61.,4444 Sousa SM, Braz GRF, Freitas CM, Santana DF, Sellitti DF, Fernandes MP, et al. Oxidative injuries induced by maternal low-protein diet in female brainstem. Nutr Neurosci. 2018;21(8):580-8.]. This effect in female rats, varying according to age, seems to be established due to the protective role of female estrogens, found in higher levels at the reproductive ages of rats [4444 Sousa SM, Braz GRF, Freitas CM, Santana DF, Sellitti DF, Fernandes MP, et al. Oxidative injuries induced by maternal low-protein diet in female brainstem. Nutr Neurosci. 2018;21(8):580-8.,4545 Braz GRF, Emiliano AS, Sousa SM, Pedroza AAS, Santana DF, Fernandes MP, et al. Maternal low-protein diet in female rat heart: possible protective effect of estradiol. J Dev Orig Health Dis. 2017;8(3):322-30.]. Thus, the increased oxidative stress biomarkers and reduced antioxidant systems may be the initial mechanisms of neurodegenerative disease observed in adulthood, although initiated during the development due to the perinatal protein restriction.

Regardless of our manuscript demonstrating that perinatal protein deficient induces an increase in the key markers of oxidative stress in the hypothalamus, and this oxidative stress could be related to negative consequences in the tissue functionality, our study has the limitation of not having performed behavioral studies and performing a direct correlation between increased levels of oxidative stress with possible negative modulations in behaviors linked to hypothalamic function, mainly related to satiety eating behavior, among others effect in hypothalamic function.

CONCLUSION

In conclusion, we observed that a low-protein diet during development is closely associated with boosted oxidative stress and impair in antioxidant system in the hypothalamus of sixty-day-old rats. Thus, considering the central role of this tissue in energy homeostasis and, therefore, in metabolic regulation, greater focus should be given to the control of malnutrition, making more studies necessary to investigate this condition in other stages of life in order to better preventive and therapeutic interventions can be implemented. In addition, the need for additional work is justified due to the scarcity of data in the scientific community regarding the study of the hypothalamus under protein restriction conditions.

  • How to cite this article: Silva DGM, Santana JH, Rodrigues TO, Bernardo EM, Pedroza AAS, Lagranha CJ. Effects of perinatal protein restriction on the oxidative balance in the hypothalamus of 60-day-old rats. Rev Nutr. 2023;36:e220181. https://doi.org/10.1590/1678-9865202336e220181
  • Support

    Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Process: APQ-0765-4.05/10; 1026-4.09/12; Universal-408403/2016). Universidade Federal de Pernambuco (UFPE), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) that provided scholarships.

REFERENCES

  • 1
    Gluckman PD, Hanson MA, Mitchell MD. Developmental origins of health and disease: reducing the burden of chronic disease in the next generation. Genome Med. 2010;2(14).
  • 2
    Lillycrop KA, Burdge GC. Epigenetic mechanisms linking early nutrition to long term health. Best Pract Res Clin Endocrinol Metab. 2012;26:667-76.
  • 3
    Hales CN, Barker DJP. The thrifty phenotype hypothesis: Type 2 diabetes. Br Med Bull. 2001;60:5-20.
  • 4
    Lee HS. Impact of Maternal Diet on the Epigenome during In Utero Life and the Developmental Programming of Diseases in Childhood and Adulthood. Nutrients. 2015;7:9492-507.
  • 5
    Wang G, Walker SO, Hong X, Bartell TR, Wang X. Epigenetics and early life origins of chronic noncommunicable diseases. J Adolesc Health. 2013;52:14-21.
  • 6
    Fall CHD. Fetal programming and the risk of noncommunicable disease. Indian J Pediatrics. 2013;80:13-20.
  • 7
    Silva PAA, Bernardo EM, Pereira AR, Andrade SSC, Lima TA, Moura FC, et al. Moderate offspring exercise offsets the harmful effects of maternal protein deprivation on mitochondrial function and oxidative balance by modulating sirtuins. Nutr Metab Cardiovasc Dis. 2021;31(5):1622-34.
  • 8
    Ferreira DJS, Pedroza AA, Braz GRF, Fernandes MP, Lagranha CJ. Mitochondrial dysfunction: maternal protein restriction as a trigger of reactive species overproduction and brainstem energy failure in male offspring brainstem. Nutr Neurosci. 2019;22(11):778-88.
  • 9
    Pedroza A, Ferreira DS, Santana DF, Silva PT, Aguiar JF, Sellitti D, et al. A maternal low-protein diet and neonatal overnutrition result in similar changes to glomerular morphology and renal cortical oxidative stress measures in male Wistar rats. Appl Physiol Nutr Metab. 2019;44(2):164-71.
  • 10
    Roseboom T, Rooij S, Painter R. The Dutch famine and its long-term consequences for adult health. Early Hum Dev. 2006;82(8):485-91.
  • 11
    Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol. 2003;552:335-44.
  • 12
    Salim S. Oxidative Stress and the Central Nervous System. J Pharmacol Exp Ther. 2017;360(1):201-05.
  • 13
    Santana DF, Ferreira DS, Braz G, Sousa S, Silva T, Gomes DA, et al. Maternal Protein Restriction in Two Successive Generations Impairs Mitochondrial Electron Coupling in the Progeny’s Brainstem of Wistar Rats From Both Sexes. Front Neurosci. 2019;13(203).
  • 14
    Ferreira DS, Liu Y, Fernandes MP, Lagranha CJ. Perinatal low-protein diet alters brainstem antioxidant metabolism in adult offspring. Nutr Neurosci. 2016;19(8):369-75.
  • 15
    Ferreira DJS, Silva PAA, Braz GRF, Silva-Filho RC, Lima TA, Fernandes MP, et al. Mitochondrial bioenergetics and oxidative status disruption in brainstem of weaned rats: Immediate response to maternal protein restriction. Brain Res. 2016;1642:553-61.
  • 16
    Bouret SG. Nutritional programming of hypothalamic development: critical periods and windows of opportunity. Int J Obes Suppl. 2012;2:19-24.
  • 17
    Nascimento L, Freitas CM, Silva-Filho R, Leite AC, Silva AB, Silva AI, et al. The effect of maternal low-protein diet on the heart of adult offspring: role of mitochondria and oxidative stress. Appl Physiol Nutr Metabol. 2014;39:880-7.
  • 18
    Reeves PG, Nielsen FH, Fahey JR, George C. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993;123(11):1939-51.
  • 19
    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem. 1976;72:248-54.
  • 20
    Draper HH, Squires EJ, Mahmoodi H, Wu J, Agarwal S, Hadley M. A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radic Biol Med. 1993;15:353-63.
  • 21
    Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol. 1994;233:357-63.
  • 22
    Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972;247:3170-5.
  • 23
    Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121-6.
  • 24
    Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249:7130-9.
  • 25
    Hissin PJ, Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 1976;74:214-26.
  • 26
    Braz GRF, Emiliano AS, Sousa SM, Pedroza AAS, Santana DF, Fernandes MP, et al. Maternal low-protein diet in female rat heart: possible protective effect of estradiol. J Dev Orig Health Dis. 2017;8:322-30.
  • 27
    Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959;82:70-7.
  • 28
    Halliwell B, Gutteridge JM. Free radicals in biology and medicine. Oxford: United Kingdom; 2007.
  • 29
    Breton C, Lukaszewski MA, Risold PY, Enache M, Guillemot J, Rivière G, et al. Maternal prenatal undernutrition alters the response of POMC neurons to energy status variation in adult male rat offspring. Am J Physiol Endocrinol Metab. 2009;296:E462-E472.
  • 30
    Palou M, Priego T, Sánchez J, Palou A, Picó C. Sexual dimorphism in the lasting effects of moderate caloric restriction during gestation on energy homeostasis in rats is related with fetal programming of insulin and leptin resistance. Nutr Metab. 2010;7:e69.
  • 31
    Manuel-Apolinar L, Rocha L, Damasio L, Tesoro-Cruz E, Zarate A. Role of prenatal undernutrition in the expression of serotonin, dopamine and leptin receptors in adult mice: Implications of food intake. Mol Med Rep. 2014;9:407-12.
  • 32
    Fernandes MS, Pedroza AA, Andrade Silva SC, Lemos MDT, Bernardo EM, Pereira AR, Santos TM, Lagranha C. Undernutrition during development modulates endoplasmic reticulum stress genes in the hippocampus of juvenile rats: Involvement of oxidative stress. Brain Res. 2022;23:1797.
  • 33
    Fragoso J, Carvalho JSG, Silva HT, Loizon E, Oliveira NSV, Vidal H, et al. Effects of maternal low-protein diet and spontaneous physical activity on the transcription of neurotrophic factors in the placenta and the brains of mothers and offspring rats. J Dev Orig Health Dis. 2021;12(3):505-12.
  • 34
    Kalsbeek A, Scheer FA, Perreau-Lenz S, La Fleur SE, Yi CX, Fliers E, et al. Circadian disruption and SCN control of energy metabolism. FEBS Lett. 2011;585(10):1412-26.
  • 35
    Crossland RF, Balasa A, Ramakrishnan R, Mahadevan SK, Fiorotto ML, Van den Veyver IB. Chronic Maternal Low-Protein Diet in Mice Affects Anxiety, Night-Time Energy Expenditure and Sleep Patterns, but Not Circadian Rhythm in Male Offspring. Plos One. 2017;12(1).
  • 36
    Feoli AM, Siqueira IR, Almeida L, Tramontina AC, Vanzella C, Sbaraini S, et al. Effects of protein malnutrition on oxidative status in rat brain. Nutrition. 2006;22:160-5.
  • 37
    Torres N, Bautista CJ, Tovar AR, Ordaz G, Rodriguez-Cruz M, Ortiz V, et al. Protein restriction during pregnancy affects maternal liver lipid metabolism and fetal brain lipid composition in the rat. Am J Physiol Endocrinol Metab. 2010;298:E270-7.
  • 38
    Stadtman ER, Van RH, Richardson A, Wehr NB, Levine RL. Methionine oxidation and aging. Biochim Biophys Acta. 2005;1703:135-40.
  • 39
    Singhal SS, Singh SP, Singhal P, Horne D, Singhal J, Awasthi S. Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling. Toxicol Appl Pharmacol. 2015;289:361-70.
  • 40
    Awasthi YC, Ansari GA, Awasthi S. Regulation of 4-hydroxynonenal mediated signaling by glutathione S-transferases. Methods Enzymol. 2005;401:379-407.
  • 41
    Gajewska B, Kazmierczak B, Kuzma-Kozakiewicz M, Jamrozik Z, Baranczyk-Kuzma A. GSTP1 polymorphisms and their association with glutathione transferase and peroxidase activities in patients with motor neuron disease. CNS Neurol Disord Drug Targets. 2015;14(10):1328-33
  • 42
    Circu ML, Aw TY. Glutathione and modulation of cell apoptosis. Biochim Biophys Acta. 2012;1823(10):1767-77.
  • 43
    Halliwell B. Oxidative stress and neurodegeneration: where are we now? J Neurochem. 2006;97(6):1634-58.
  • 44
    Sousa SM, Braz GRF, Freitas CM, Santana DF, Sellitti DF, Fernandes MP, et al. Oxidative injuries induced by maternal low-protein diet in female brainstem. Nutr Neurosci. 2018;21(8):580-8.
  • 45
    Braz GRF, Emiliano AS, Sousa SM, Pedroza AAS, Santana DF, Fernandes MP, et al. Maternal low-protein diet in female rat heart: possible protective effect of estradiol. J Dev Orig Health Dis. 2017;8(3):322-30.

Edited by

Editor

Alceu Afonso Jordão

Publication Dates

  • Publication in this collection
    29 May 2023
  • Date of issue
    2023

History

  • Received
    31 Aug 2022
  • Reviewed
    18 Nov 2022
  • Accepted
    01 Feb 2023
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