Nutritional research and fetal programming: parental nutrition influences the structure and function of the organs / Investigación nutricional y programación fetal: la nutrición parental influye en la estructura y función de los órganos

SUMMARY: Rodents are animals extensively used in biomedical and nutrition research, a necessary step before the research in humans. The composition and type of administration of the experimental diets are relevant and should be thought, considering each type of animal used in the research. It is particularly important to consider, among others, the metabolic differences between species and food needs in macro- and micronutrients to avoid possible bias. The American Institute of Nutrition (AIN) made recommendations for rodents, adapted to the period of growth (AIN-93G), which are pivotal in fetal programming studies. The experiments can be compared among different studies and better translated into humans, considering these limitations in the nutrition of parents and offspring. The review addresses different compositions of experimental food for rodents during development with the ability to induce fetal programming in the offspring and chronic diseases in adulthood due to the nutrition of the mother and father. The 'developmental origins of health and disease' (DOHaD) concept due to maternal nutrition is commented considering the protein restriction, vitamin D restriction, obesity, and intake of fructose or fish-oil. The 'paternal origins of health and disease transmission' (POHaD), because of the nutritional state of the father, were also analyzed in the review, primarily considering the obesity of the father. The review proposes some diet compositions to experimental research considering varied nutritional situations, hoping to assist young researchers or researches not familiar with experimental diet manipulations in the elaboration of the projects.

RESUMEN: Los roedores son animales utilizados frecuentemente en la investigación biomédica y nutricional, un paso necesario antes de la investigación en humanos. La composición y el tipo de administración de las dietas experimentales son relevantes y se debe considerar cada tipo de animal utilizado en los estudios. Es particularmente importante considerar las diferencias metabólicas entre las especies y las necesidades alimentarias de macro y micronutrientes para evitar posibles sesgos. El Instituto Americano de Nutrición (AIN) estableció recomendaciones para los roedores, adaptadas al período de crecimiento (AIN-93G), que son fundamentales en los estudios de programación fetal. Los experimentos se pueden comparar entre diferentes estudios y aplicar en humanos, considerando estas limitaciones en la nutrición de padres e hijos. La revisión aborda diferentes composiciones de alimentos para estudios experimentales en roedores durante su desarrollo, con la capacidad de inducir programación fetal en la descendencia y enfermedades crónicas en la adultez, considerando la nutrición de los padres. El concepto de 'orígenes del desarrollo de la salud y la enfermedad' (DOHaD) debido a la nutrición materna se comenta considerando la restricción de proteínas, la restricción de vitamina D, la obesidad y la ingesta de fructosa o aceite de pescado. Los 'orígenes paternos de la salud y transmisión de enfermedades' (POHaD), debido al estado nutricional del padre, también fueron analizados considerando principalmente la obesidad del padre. La revisión propone algunas composiciones dietéticas a la investigación experimental considerando situaciones nutricionales variadas, con la esperanza de ayudar a jóvenes investigadores o investigadores no familiarizados con las manipulaciones experimentales de la dieta en la elaboración de los proyectos.

Morphological and morphometric study of the muscle fibers and neuromuscular junctions of the extensor digitorum longus in aged rats submitted to maternal protein restriction / Estudio morfológico y morfométrico de las fibras musculares y de las intersecciones neuromusculares del músculo extensor largo de los dedos de ratas de edad avanzada sometidas a la restricción proteica materna

Protein restriction implies the functional involvement of several systems and organs, including the skeletal muscle, because it is a protein reservoir in the body. This study sought to analyze the morphological and morphometric features of the muscle fibers and neuromuscular junctions (NMJs) of the extensor digitorum longus (EDL) muscle in rats at 365 days of age, submitted to maternal protein restriction during the gestation and lactation periods. Wistar rats were divided into two groups: a Control Group - mothers fed a normal-protein diet (17 % protein) during pregnancy and lactation; and a Restricted Group - mothers fed a low-protein diet (6 % protein) during pregnancy and lactation. The pups were kept with the mother throughout the lactation period (21 days), after which the offspring received a normal protein diet until 365 days of age. Histological (HE) and histoenzymological (NADH-TR) studies were conducted on the muscle fibers. The muscle was subjected to Nonspecific Esterase reaction to stain the Neuromuscular Junctions. Regarding the animals from the restricted group: the histologic analysis of the muscle fibers showed the presence of centralized nuclei and a diminished area; the histoenzymological study showed the different types of muscle fibers were randomly distributed in the EDL muscle and the area of the Type IIa muscle fiber was smaller; the ultrastructural study revealed disorganization of the Z line, and the presence of lipid droplets and vacuoles containing myelin figures in subsarcolemmal and intramiofibrilar regions; while the analysis of the NMJs exhibited no significant differences between the groups. Protein restriction in the pregnancy and lactation period may have affected the development of skeletal muscle, producing a permanent muscle-fiber deficit in the EDL muscle of the offspring.

La restricción proteica implica compromiso funcional de diversos sistemas y órganos, entre ellos, el músculo estriado esquelético, por ser una reserva de proteína del organismo. De esa forma, el presente trabajo procuró analizar las características morfológicas y morfométricas de las fibras musculares y de las intersecciones neuromusculares (JNMs) del músculo extensor largo de los dedos (EDL) en ratas de 365 días de edad, sometidas a restricción proteica materna durante los periodos de gestación y lactancia. Las ratas Wistar fueron separadas en dos grupos: El grupo Control - madres alimentadas durante la gestación y lactancia con ración normoproteica (17 % de proteína) y Grupo con restricción ­ madres alimentadas durante la gestación y lactancia con ración hipoproteica (6 % de proteína). Las crías permanecieron con la madre durante todo el periodo de lactancia (21 días) y después de este periodo la prole recibió ración normoproteica hasta los 365 días de edad. Se realizó un estudio histológico (HE) e histoenzimológico (NADH-TR) de las fibras musculares. Para la marcación de las JNMs, el músculo fue sometido a la reacción de Esterasa Inespecífica. El análisis histológico de las fibras musculares de los animales del Grupo con restricción mostró la presencia de núcleos centralizados y una disminución del área en el grupo con restricción. En el estudio histoenzimológico, el músculo EDL presentó una distribución aleatoria de los diferentes tipos de fibras musculares y el área de las fibras musculares del tipo IIa fue menor en el grupo con restricción. En relación al estudio ultraestructural, en los animales del grupo con restricción se observó desorganización de la línea Z, presencia de pequeñas gotas de lípidos y vacuolas que abrigaban figuras de mielina en las regiones subsarcolemal e intramiofibrilar. En el análisis de las JNMs no hubo diferencias significativas. La restricción proteica impuesta en el periodo de gestación y lactancia puede haber afectado el desarrollo del músculo esquelético, produciendo un déficit permanente en las fibras musculares del músculo EDL de la prole.

Maternal protein restriction during lactation modulated the expression and activity of rat offspring hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2, and CYP2E1 during development

Early nutrition plays a long-term role in the predisposition to chronic diseases and influences the metabolism of several drugs. This may happen through cytochromes P450 (CYPs) regulation, which are the main enzymes responsible for the metabolism of xenobiotics. Here, we analyzed the effects of maternal protein restriction (MPR) on the expression and activity of hepatic offspring’s CYPs during 90 days after birth, using Wistar rats as a mammal model. Hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2 and CYP2E1 mRNA and protein expression, and associated catalytic activities (ECOD, EROD, MROD, BROD, PROD and PNPH) were evaluated in 15-, 30-, 60-, and 90-day-old offspring from dams fed with either a 0% protein (MPR groups) or a standard diet (C groups) during the 10 first days of lactation. Results showed that most CYP genes were induced in 60- and 90-day-old MPR offspring. The inductions detected in MPR60 and MPR90 were of 5.0- and 2.0-fold (CYP1A2), 3.7- and 2.0-fold (CYP2B2) and 9.8- and 5.8– fold (CYP2E1), respectively, and a 3.8-fold increase of CYP2B1 in MPR90. No major alterations were detected in CYP protein expression. The most relevant CYP catalytic activities’ alterations were observed in EROD, BROD and PNPH. Nevertheless, they did not follow the same pattern observed for mRNA expression, except for an induction of EROD in MPR90 (3.5-fold) and of PNPH in MPR60 (2.2-fold). Together, these results suggest that MPR during lactation was capable of altering the expression and activity of the hepatic CYP enzymes evaluated in the offspring along development.

Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02 vs 0.15 ± 0.02), medial (0.30 ± 0.06 vs 0.14 ± 0.01) and distal (0.43 ± 0.07 vs 0.07 ± 0.02 U·g-1·min-1) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11 vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g-1·min-1) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring.

Análise biométrica e morfologia do rim de ratos submetidos à restrição proteica gestacional / Biometric analysis and kidney morphologic of rats submitted to gestational protein restriction

Restrição proteica gestacional em ratos está associada ao desenvolvimento de doenças crônicas como diabetes e hipertensão na idade adulta. O objetivo deste estudo foi desenvolver modelo em ratos para o desenvolvimento de hipertensão através da restrição proteica durante a gestação e avaliar a relação da mesma com o baixo peso ao nascer e a morfologia renal. Foram estudados filhotes de ratos, machos e fêmeas, divididos em grupos controle e restrito nas seguintes idades: dia do nascimento, 10 dias, 3 e 6 meses pós-natais. Os animais restritos durante a gestação nasceram com massa corporal e volume renal menor do que os animais que tiveram dieta normoproteica durante a gestação, além de apresentar lesão renal na idade adulta o que provocou desenvolvimento da hipertensão arterial.

Gestational protein restriction in rats is associated with development of chronic diseases like diabetes and hypertension in adulthood. The aim of this study was to develop model in mice to develop hypertension by protein restriction during pregnancy and assess similar relationship with low birth weight and renal morphology. We studied rats, male and female, divided into control group and low protein to the following day of birth, 10 days, 3 and 6 months postnatal. The restricted animals during pregnancy were born with body mass and renal volume smaller than the animals that had normal protein diet during pregnancy, besides having kidney damage in adults what causes the development of hypertension.

Restrição protéica na prenhez: efeitos relacionados ao metabolismo materno / Protein restriction in pregnancy: effects related to dam metabolism

Foram avaliadas as alterações no metabolismo materno durante a prenhez em ratas Wistar, prenhes e não-prenhes, submetidas à restrição protéica, que receberam dietas isocalóricas (15,74 kJ/g), controle ou hipoprotéica (17 por cento versus 6 por cento), distribuídas em quatro grupos (n = 7), quais sejam: controle não-prenhe (CNP) e prenhe (CP) e hipoprotéico não-prenhe (HNP) e prenhe (HP), do 1º ao 18º dia de prenhez. Parâmetros bioquímicos, hormonais e relacionados à síntese de lipídios foram considerados. Utilizou-se ANOVA a duas vias seguido de teste Tukey-HSD e teste t de Student, significância de p < 0,05. A restrição protéica elevou a síntese de lipídios e a atividade da enzima málica (EM) no fígado (FIG) e reduziu a massa ( por cento) e a razão lipí+dio/glicogênio nesse tecido, bem como reduziu a ingestão protéica (total e por cento), o conteúdo ( por cento) de lipídios na glândula mamária (GMA), as proteínas e a albumina séricas, com consequente redução nas massas da placenta e fetos. A prenhez reduziu a proteinemia, a albuminemia, a síntese de lipídios, a atividade da EM, os lipídios e o glicogênio no FIG. Mas elevou a massa corporal final, a massa ( por cento) do tecido adiposo gonadal (GON), do FIG e da GMA, e reduziu a massa ( por cento) da carcaça (CARC), a síntese e o conteúdo de lipídios no GON e, na GMA, o conteúdo de lipídios. A insulinemia elevou-se na prenhez, com glicemia reduzida, caracterizando resistência hormonal. A leptina e a prolactina também se elevaram na prenhez, sendo o aumento maior no HP. A restrição protéica na prenhez modificou o metabolismo materno, alterando a síntese de lipídios no FIG e o perfil hormonal, além de reduzir a massa da placenta e dos fetos.

Metabolism alterations were evaluated in female Wistar rats (dams) during pregnancy. Pregnant and non-pregnant dams submitted to protein restriction, were fed isocaloric (15.74 kJ/g), control or hypoproteic (17 percent vs. 6 percent) diets, and distributed in four Groups (n=7) as follows: non-pregnant control (NPC), pregnant control (PC), non-pregnant hypoproteic (NPH), and pregnant hypoproteic (PH); from Day 1 to Day 18 of pregnancy. Biochemical, hormonal and metabolic parameters related to lipid synthesis were assessed. The two-way ANOVA, followed by Tukey-HSD and Student-t tests were used, with a significance of p< 0.05. Protein restriction elevated lipid synthesis and malic enzyme (ME) activity in the liver, and reduced mass and the lipid/glycogen ratio in this tissue; it also lowered protein ingestion (total and percent), lipid content ( percent) in the mammary gland (MAG), serum proteins and albumin, with consequent reduction of placenta and fetal masses. Pregnancy reduced serum protein and albumin concentrations, lipid synthesis, ME activity, hepatic lipid and glycogen content. However, it increased final body mass; increased relative masses of gonad (GON), liver and MAG; but reduced lipid synthesis and content of GON, lipid content of MAG and the relative mass of carcass. Pregnancy Insulinemia increased during pregnancy with reduced glycemia, characterizing hormonal resistance. Leptin and prolactin were also increased during pregnancy, being the highest increase in observed in HP rats. Protein restriction in pregnancy modified maternal metabolism, altering lipid synthesis in the liver and hormonal profile and decreasing the placenta and fetus masses.

Evaluación del ffecto modulador del factor de cecimiento similar a la insulinaa tipo i (igf-i) en linfocitos t de bazo de ratas sometidas a estrés nutricional / Evaluation of the modulating effect of type-i insulin-like growthfactor (igf-i) in rat spleen-t lymphocytes under nutritional stress / Avaliacáo doefeiito modulador do factor de cescimento similar a insulina tiipo i (igf-i) em linfócitos t de baço de ratos submetidos a stress nutricional

En este trabajo se estudió el papel del factor de crecimiento similar a la insulina tipo I (IGF-I) como factormodulador del estrés inducido por una restricción proteica en la dieta, en linfocitos T de bazo de rata. Se encontró que la restricción proteica disminuye el peso corporal en un 15%, el peso del bazo en un 32% y la población total de linfocitos T en un 42%. Igualmente, se observó en la población restringida un incremento en los porcentajes de las subpoblaciones T-CD4, T-CD8 y linfocitos B, y una relación T-CD4/T-CD8 disminuida, lo que sugiere una función inmune afectada por la malnutrición. También se halló que los cultivos de linfocitos provenientes de bazo de ratas en restricción proteica presentanmenor proliferación que los provenientes de ratas bien alimentadas; dicha proliferación se incrementa al adicionar IGF-I de manera dependiente de la dosis. Esta respuesta depende, también, del contenido de proteína en la dieta, observándose una mayor y más pronta respuesta a IGF-I en el grupo bien nutrido. La concanavalina A incrementó, igualmente, la proliferación en ambos grupos de animales, y al combinarla con IGF-I se presentó un sinergismo en la respuesta. En este trabajo se encontró, también, que la restricción proteica incrementa la apoptosis de los linfocitos T, y que la adición de IGF-I logra proteger dichas células de la apoptosis soportando el papel inmunomodulador de esta hormona en estrés nutricional.

In this work we studied the role of Insulin- like Growth Factor-I (IGF-I) as stress modulator in spleen T-lymphocytes from protein restricted rats. In protein restriction we found 15% lowered body weight, 32% lowered spleen weight and a reduction of 42% in total T-cell population. Also, increased percentages of T-CD4, T-CD8 and B cell populations and lowered T-CD4/T-CD8 ratio was observed, suggesting an impaired immune function due to malnutrition. Cultures of spleen T-lymphocytes from protein restricted rats showed less proliferation than the ones from well fed rats, this proliferation was increased after the addition of IGF-I in a dose depending manner. This answer depends, also, from the protein content of the diet, since the well fed animals showed a higher and faster answer. Concanavaline A, also, increased proliferation in both groups of rats and when combined with IGF-I a synergistic answer was found. In this work we also found that protein restriction increased apoptosis of T-lymphocytes, and the addition of IGF-I helped in the recovery of those cells supporting the immunomodulator role of IGF-I in nutritional stress.

Neste trabalho foi estudado o papel de Factor de Crescimento Similar à Insulina Tipo I (IGF-I) como factor modulador do stress induzido por uma restrição proteica em linfócitos T de baço de rato. Encontrou- se que a restrição proteica diminui o peso corporal em 15%, o peso do baço dos animais em 32% e a população total de linfócitos T em 42%. Igualmente, se observou uma alteração nas percentagens das subpopulações T-CD4, T-CD8 e linfócitos B, e uma relação T-CD4/T-CD8 diminuída que sugere uma função imune afectada pela má nutrição. Foi encontrado que os cultivos de linfócitos provenientes do baço de rato em restrição proteica apresentam menor proliferação que os provenientes de ratos bem alimentados; dita proliferação incrementa ao adicionar IGF-I de maneira dependente da dose. Esta resposta depende, também, do conteúdo de proteína na dieta, observando- se uma maior e mais rápida resposta a IGF-I no grupo bem nutrido. A concavalina A incrementou, igualmente, a proliferação em ambos grupos de animais, e ao combiná-la com IGF-I apresentou-se um sinergismo na resposta. Nesta investigação também foi encontrado que a restrição proteica incrementa a apoptose dos linfócitos T e que a adição de IGF-I permite proteger estas células da apoptose dando suporte ao papel imunomodulador desta hormona em stress nutricional.

Effect of different levels of the dietary protein on long term prognosis of experimental renal damage

We examined the effect of various levels of dietary protein on long term prognosis of Adriamycinnephropathy of S-D rat, fed with high protein (30%), intermediately low (10%), and strictly low (5%) protein diet for 15 weeks. 1) In rats fed with strictly low protein diets (5%), proteinuria and serum creatinine decreased and creatinine clearance and histological changes were relatively well preserved. But hypoproteinemia and weight loss were more marked and 2 rats died due to severe ascites and pleural effusion in cachexic state. 2) In rats fed with high protein diets (30%), general health condition and weight gain were relatively well preserved. But there were massive proteinuria, progressive increase in serum creatinine and progressive decrease in creatinine clearance. Focal glomerular sclerosis and severe tubulointerstitial change on histologic examination were marked. 3) With intermediately low protein diet (10%), renal function and pasma protein levels were relatively well preserved compared with high protein diet group. But weight gain did not increase normally. 4) We tentatively conclude that appropriately restricted dietary protein can prevent functional and histological renal damage. But too strict protein restriction aggravate nutitional state and general condition.