Neonatal metformin short exposure inhibits male reproductive dysfunction caused by a high-fat diet in adult rats.

Metformin (Met) is widely used to control blood glucose levels and acts on various organs, including reproductive tissues, to improve reproductive and lifespan. This study evaluated whether neonatal Met exposure prevented male reproductive dysfunction caused by being overweight during adulthood. Randomized Wistar rat pups received an intraperitoneal injection from postnatal days (PNDs) 1 to 12of saline (Sal; 0.9% NaCl/day in 2mL/kg) or Met (100 mg/kg/day in 2 mL/kg). From PNDs 60 to 90, the animals received a regular (R; 4.5% fat; Sal R and Met R groups) or a high-fat (HF; 35% fat; Sal HF and Met HF groups) diet. At PND 90, all animals were euthanized to evaluate their biometric and reproductive parameters. The Sal and Met groups with R showed similar body weights, however, the HF diet increased the body weight in both groups. The Sal HF group showed testicular damage regarding in antioxidant status and inflammatory profile in the epididymal cauda. The HF diet reduced Leydig and Sertoli cells numbers, with lower sperm quality. The Met R animals showed positive reproductive programming, due to improved antioxidant defense, inflammatory biomarkers, and sperm morphology. Met HF prevented HF diet damage to reproductive organs and sperm morphology, but not to sperm motility. Early Met exposure positively affected the male reproductive system of adult rats, preventing reproductive HF disorders. STATEMENT OF NOVELTY AND SIGNIFICANCE: Metformin is used to control type 2 diabetes mellitus and can act to improve metabolism and lifespan. Metformin avoidance is recommended during pregnancy, but there is no information regarding its use when breastfeeding. For the first time, we showed in this current study that metformin positively acts in the male reproductive tissues and helps involved in later life. These data showed a better antioxidant defense and anti-inflammatory profile of Metformin animals than Saline animals and might directly improve reproductive organs morphophysiology and sperm morphology. Also, the neonatal Met application programs the male reproduction to counterbalance damages from an obesogenic environment in later life.

Early weaning induces short- and long-term effects on pancreatic islets in Wistar rats of both sexes.

KEY POINTS: The World Health Organization recommends exclusive breastfeeding until 6 months of age as an important strategy to reduce child morbidity and mortality. Studies have associated early weaning with the development of obesity and type 2 diabetes in adulthood. In our model, we demonstrated that early weaning leads to increased insulin secretion in adolescent males and reduced insulin secretion in adult offspring. Early weaned males exhibit insulin resistance in skeletal muscle. Early weaning did not change insulin signalling in the muscle of female offspring. Taking into account that insulin resistance is one of the primary factors for the development of type 2 diabetes mellitus, this work demonstrates the importance of breastfeeding in the fight against this disease. ABSTRACT: Early weaning (EW) leads to short- and long-term obesity and diabetes. This phenotype is also observed in experimental models, in which early-weaned males exhibit abnormal insulinaemia in adulthood. However, studies regarding the effect of EW on pancreatic islets are rare. We investigated the mechanisms by which glycaemic homeostasis is altered in EW models through evaluations of insulin secretion and its signalling pathway in offspring. Lactating Wistar rats and their pups were divided into the following groups: non-pharmacological EW (NPEW): mothers were wrapped with an adhesive bandage on the last 3 days of lactation; pharmacological EW (PEW): mothers received bromocriptine to inhibit prolactin (1 mg/kg body mass/day) on the last 3 days of lactation; and control (C): pups underwent standard weaning at PN21. Offspring of both sexes were euthanized at PN45 and PN180. At PN45, EW males showed higher insulin secretion (vs. C). At PN170, PEW males exhibited hyperglycaemia in an oral glucose tolerance test (vs. C and NPEW). At PN180, EW male offspring were heavier; however, both sexes showed higher visceral fat. Insulin secretion was lower in EW offspring of both sexes. Males from both EW groups had lower glucokinase in islets, but unexpectedly, PEW males showed higher GLUT2, than did C. EW males exhibited lower insulin signalling in muscle. EW females exhibited no changes in these parameters compared with C. We demonstrated distinct alterations in the insulin secretion of EW rats at different ages. Despite the sex dimorphism in insulin secretion in adolescence, both sexes showed impaired insulin secretion in adulthood due to EW.

Early metformin treatment improves pancreatic function and prevents metabolic dysfunction in early overfeeding male rats at adulthood.

NEW FINDINGS: What is the central question of this study? Studies reported the efficacy of metformin as a promising drug for preventing or treating of metabolic diseases. Nutrient stresses during neonatal life increase long-term risk for cardiometabolic diseases. Can early metformin treatment prevent the malprogramming effects of early overfeeding? What is the main finding and its importance? Neonatal metformin treatment prevented early overfeeding-induced metabolic dysfunction in adult rats. Inhibition of early hyperinsulinaemia and adult hyperphagia might be associated with decreased metabolic disease risk in these animals. Therefore, interventions during infant development offer a key area for future research to identify potential strategies to prevent the long-term metabolic diseases. We suggest that metformin is a potential tool for intervention. ABSTRACT: Given the need for studies investigating the possible long-term effects of metformin use at crucial stages of development, and taking into account the concept of metabolic programming, the present work aimed to evaluate whether early metformin treatment might program rats to resist the development of adult metabolic dysfunctions caused by overnutrition during the neonatal suckling phase. Wistar rats raised in small litters (SLs, three pups per dam) and normal litters (NLs, nine pups per dam) were used as models of early overfeeding and normal feeding, respectively. During the first 12 days of suckling, animals from SL and NL groups received metformin, whereas the controls received saline injections. Food intake and body weight were monitored from weaning until 90 days of age, when biometric and biochemical parameters were assessed. The metformin treatment decreased insulin concentrations in pups from SL groups, and as adults, these animals showed improvements in glucose tolerance, insulin sensitivity, body weight gain, white fat pad stores and food intake. Low-glucose insulinotrophic effects were observed in pancreatic islets from both NL and SL groups. These results indicate that early postnatal treatment with metformin inhibits early overfeeding-induced metabolic dysfunctions in adult rats.

Malnutrition during late pregnancy exacerbates high-fat-diet-induced metabolic dysfunction associated with lower sympathetic nerve tonus in adult rat offspring.

Objectives: We aimed to assess the effects of a maternal protein-caloric restriction diet during late pregnancy on the metabolism of rat offspring fed a high-fat diet (HFD) during adulthood.Methods: During late pregnancy, rat dams received either a low-protein (4%; LP group) or normoprotein (23%; NP group) diet. After weaning, the offspring were fed a standard diet (Control; C). Male offspring (60 days old) from both groups were then fed either the C diet or HFD until they were 90 days old. The adult offspring and maternal metabolic parameters and autonomic nervous system (ANS) were then evaluated.Results: Dams exhibited low body weight gain and food intake during the LP diet consumption. At lactation, these dams showed high body weight gain, hypoinsulinemia and hyperglycemia. The maternal LP diet resulted in low body weights for the pups. There were also no differences in the metabolic parameters between the adult LP offspring that were fed the C diet and the NP group. Adults of both groups that were fed the HFD developed obesity associated with altered insulin/ glucose homeostasis and altered ANS activity; however, the magnitudes of these parameters were higher in the LP group than in the NP group.Conclusions: Maternal protein malnutrition during the last third of pregnancy malprograms the metabolism of rat offspring, resulting in increased vulnerability to HFD-induced obesity, and the correlated metabolic impairment might be associated with lower sympathetic nerve activity in adulthood.

Assessment of bendamustine-induced genotoxicity in eukaryotic cells.

Bendamustine, an anticancer drug with alkylating properties, is widely used to treat hematological malignancies. Since the nitrogen mustard family alkylators induce DNA damages and have been associated with an elevated risk of second malignancy, current study evaluates the cytotoxic, mutagenic, and recombinogenic effects of bendamustine by using, respectively the mitotic index assay, the in vitro mammalian cell micronucleus test (Mnvit) and the chromosome aberration (CA) test in human peripheral lymphocytes, and the in vivo homozygotization assay in Aspergillus nidulans, which detects the loss of heterozygosity (LOH) due to somatic recombination. Bendamustine (6.0 µg/ml, 9.0 µg/ml, and 12.0 µg/ml) induced a statistically significant concentration-related increase in the frequencies of micronuclei and a significant reduction in the cytokinesis block proliferation index (CBPI) rates when compared to negative control. In the CA test, bendamustine significantly increased the frequencies of structural aberrations at the three tested concentrations when compared to the negative control. Aspergillus nidulans diploids, obtained after bendamustine treatment (6.0 µg/ml, 12.0 µg/ml, and 24.0 µg/ml), produced, after haploidization, homozygotization index (HI) rates higher than 2.0 and significantly different from the negative control. Since bendamustine showed genotoxic effects in all tested concentrations, two of them corresponding to the peak plasma concentrations observed in cancer patients treated with bendamustine, data provided in the current research work may be useful to identify the most appropriate dosage regimen to achieve the efficacy and safety of this anticancer medication.

Methylglyoxal treatment in lactating mothers leads to type 2 diabetes phenotype in male rat offspring at adulthood.

PURPOSE: Environmental and nutritional disorders during perinatal period cause metabolic dysfunction in the progeny and impair human health. Advanced glycation end products (AGEs) are primarily produced during metabolism of excess blood glucose, which is observed in diabetes. Methylglyoxal (MG) is a precursor for the generation of endogenous AGEs, which disturbs the metabolism. This work aimed to investigate whether the maternal MG treatment during lactation programs the progeny to metabolic dysfunction later in life. METHODS: Female Wistar rats were divided into two groups: control group (C) treated with saline and MG group treated with MG (60 mg/kg/day) by gavage throughout the lactation period. Both mothers and offspring were fed a standard chow. At weaning, breast milk composition was analyzed and mothers euthanized for blood and tissue sample collections. At 90 days of age, offspring were submitted to glucose tolerance test (ivGTT) and euthanized for blood and tissue samples collection. RESULTS: MG mothers showed increase in glucose and fructosamine levels; however, they showed low insulin levels and failure in ß-cell function (p < 0.05). MG mothers also showed dyslipidemia (p < 0.05). Moreover, breast milk had elevated levels of glucose, triglycerides, cholesterol and fructosamine and low insulin (p < 0.05). Interestingly, MG offspring had increased body weight and adipose tissue at adulthood, and they also showed glucose intolerance and failure in ß-cell function (p < 0.05). Besides, MG offspring showed dyslipidemia (p < 0.05) increasing cardiovascular diseases risk. CONCLUSIONS: Maternal MG treatment negatively affects the male rat offspring, leading to type 2 diabetes and dyslipidemia in later life, possibly by changes in breast milk composition.

Treatment with soy isoflavones during early adulthood improves metabolism in early postnatally overfed rats.

PURPOSE: The incidences of obesity and related diseases have reached epidemic proportions, and new therapeutic approaches are needed. Soy isoflavones have been identified as an important dietary factor for preventing and treating metabolic dysfunction. This study examined the effects of high doses of isoflavone on glucose and fat metabolism in a model of programmed obesity and evaluated its effects on the autonomic nervous system. METHODS: Litters of Wistar rats were standardized at nine pups per dam in normal litters (NL) or reduced to three pups per dam at the third day of life (P3) in small litters (SL) to induce postnatal overfeeding. Gavage with a soy bean isoflavone mixture (1 g/day) diluted in water was started at P60 and continued for 30 days. The control animals received vehicle gavage. At P90, biometric and metabolic parameters as well as direct autonomic nerve activity were measured. RESULTS: Increases in glycaemia and insulinaemia observed in SL rats were reduced by isoflavone treatment, which also caused lower glucose-induced insulin secretion by pancreatic islets. Sympathetic activity in the major splanchnic nerve was increased, while vagus nerve activity was reduced by isoflavone treatment. The dyslipidaemia induced by overfeeding in SL rats was restored by isoflavone treatment. CONCLUSION: The present study shows that treatment with isoflavone reduces adiposity and improves glucose and lipid metabolism. Collectively, these effects may depend on autonomic changes.

Genotoxic investigation of a thiazolidinedione PPARγ agonist using the in vitro micronucleus test and the in vivo homozygotization assay.

Pioglitazone (PTZ) is an oral antidiabetic agent whose anti-cancer properties have been described recently. Since PTZ increases the production of reactive oxygen species in mammalian cells, the aim of current study was to evaluate the cytotoxic, mutagenic and recombinogenic effects of PTZ using respectively the in vitro mitotic index assay and the in vitro mammalian cell micronucleus test in human peripheral lymphocytes, and the in vivo homozygotization assay in Aspergillus nidulans, which detects the loss of heterozygosity due to somatic recombination. Although the lowest PTZ concentrations (4-36 µM) did not show any significant rise in the micronucleus production, the higher PTZ concentration (108 µM) produced a statistically higher number of micronuclei than the negative control and significantly altered the cell-proliferation kinetics, demonstrating the mutagenic and antiproliferative effects of PTZ, respectively. The recombinogenic activity of PTZ, demonstrated here for the first time, was observed at the highest tested concentration (400 µM) through the homozygotization index rates significantly different from the negative control. Taken together, our results show that PTZ is genotoxic at a concentration higher than the therapeutic plasma concentration. This PTZ genotoxicity may be a potential benefit to its previously described antitumour activity.

Protective effect of metformin against walker 256 tumor growth is not dependent on metabolism improvement.

BACKGROUND/AIMS: The objective of the current work was to test the effect of metformin on the tumor growth in rats with metabolic syndrome. METHODS: We obtained pre-diabetic hyperinsulinemic rats by neonatal treatment with monosodium L-glutamate (MSG), which were chronically treated every day, from weaning to 100 day old, with dose of metformin (250 mg/kg body weight). After the end of metformin treatment, the control and MSG rats, treated or untreated with metformin, were grafted with Walker 256 carcinoma cells. Tumor weight was evaluated 14 days after cancer cell inoculation. The blood insulin, glucose levels and glucose-induced insulin secretion were evaluated. RESULTS: Chronic metformin treatment improved the glycemic homeostasis in pre-diabetic MSG-rats, glucose intolerance, tissue insulin resistance, hyperinsulinemia and decreased the fat tissue accretion. Meanwhile, the metformin treatment did not interfere with the glucose insulinotropic effect on isolated pancreatic islets. Chronic treatment with metformin was able to decrease the Walker 256 tumor weight by 37% in control and MSG rats. The data demonstrated that the anticancer effect of metformin is not related to its role in correcting metabolism imbalances, such as hyperinsulinemia. However, in morphological assay to apoptosis, metformin treatment increased programmed cell death. CONCLUSION: Metformin may have a direct effect on cancer growth, and it may programs the rat organism to attenuate the growth of Walker 256 carcinoma.

Glycolytic and mitochondrial metabolism in pancreatic islets from MSG-treated obese rats subjected to swimming training.

BACKGROUNDS/AIMS: Obese rats obtained by neonatal monosodium glutamate (MSG) administration present insulin hypersecretion. The metabolic mechanism by which glucose catabolism is coupled to insulin secretion in the pancreatic ß-cells from MSG-treated rats is understood. The purpose of this study was to evaluate glucose metabolism in pancreatic islets from MSG-treated rats subjected to swimming training. METHODS: MSG-treated and control (CON) rats swam for 30 minutes (3 times/week) over a period of 10 weeks. Pancreatic islets were isolated and incubated with glucose in the presence of glycolytic or mitochondrial inhibitors. RESULTS: Swimming training attenuated fat pad accumulation, avoiding changes in the plasma levels of lipids, glucose and insulin in MSG-treated rats. Adipocyte and islet hypertrophy observed in MSG-treated rats were attenuated by exercise. Pancreatic islets from MSG-treated obese rats also showed insulin hypersecretion, greater glucose transporter 2 (GLUT2) expression, increased glycolytic flux and reduced mitochondrial complex III activity. CONCLUSION: Swimming training attenuated islet hypertrophy and normalised GLUT2 expression, contributing to a reduction in the glucose responsiveness of pancreatic islets from MSG-treated rats without altering glycolytic flux. However, physical training increased the activity of mitochondrial complex III in pancreatic islets from MSG-treated rats without a subsequent increase in glucose-induced insulin secretion.

Moderate exercise restores pancreatic beta-cell function and autonomic nervous system activity in obese rats induced by high-fat diet.

BACKGROUND/AIMS: Metabolic syndrome has been identified as one of the most significant threats to human health in the 21(st) century. Exercise training has been shown to counteract obesity and metabolic syndrome. The present study aimed to investigate the effects of moderate exercise training on pancreatic beta-cell function and autonomic nervous system (ANS) activity in rats fed a high-fat diet (HFD). METHODS: Weaning rats were divided into four groups: rats fed a standard chow or HFD (sedentary, Control-SED and HFD-SED; or exercised, Control-EXE and HFD-EXE, respectively). Exercised rats ran (from 21- to 91-days-old) for 60 minutes (3 times/week) over a 10-week period. Glucose and insulin tolerance tests were performed. Pancreatic islets were isolated to study glucose-induced insulin secretion (GIIS). Parasympathetic and sympathetic nerve electrical signals were measured, and liver samples were processed and histologically analyzed. RESULTS: Exercise prevented obesity, insulin resistance, and liver steatosis as well as improved total cholesterol, ALT, and AST levels. Islets from HFD rats showed insulin hypersecretion which was ameliorated by exercise. Exercise decreased vagal nerve activity in the HFD-EXE group and increased the activity of the sympathetic nervous system in both exercised groups. CONCLUSION: Exercise prevents obesity and liver steatosis and restores pancreatic beta-cell function and ANS activity in HFD-obese rats.

Protein restriction during peripubertal period impairs endothelial aortic function in adult male Wistar rats.

Protein restriction during early phases of body development, such as intrauterine life can favor the development of vascular disorders. However, it is not known if peripubertal protein restriction can favor vascular dysfunction in adulthood. The present study aimed to evaluated whether a protein restriction diet during peripubertal period favors endothelial dysfunction in adulthood. Male Wistar rats from postnatal day (PND) 30 until 60 received a diet with either 23% protein (CTR group) or with 4% protein (LP group). At PND 120, the thoracic aorta reactivity to phenylephrine, acetylcholine, and sodium nitroprusside was evaluated in the presence or absence of: endothelium, indomethacin, apocynin and tempol. The maximum response (Rmax) and pD2 (-log of the concentration of the drug that causes 50% of the Rmax) were calculated. The lipid peroxidation and catalase activity were also evaluated in the aorta. The data were analyzed by ANOVA (one or two-ways and Tukey's) or independent t-test; the results were expressed as mean ± S.E.M., p < 0.05. The Rmax to phenylephrine in aortic rings with endothelium were increased in LP rats when compared with the Rmax in CTR rats. Apocynin and tempol reduced Rmax to phenylephrine in LP aortic rings but not in CTR. The aortic response to the vasodilators was similar between the groups. Aortic catalase activity was lower and lipid peroxidation was greater in LP compared to CTR rats. Therefore, protein restriction during the peripubertal period causes endothelial dysfunction in adulthood through a mechanism related to oxidative stress.

Protein-caloric restriction induced HPA axis activation and altered the milk composition imprint metabolism of weaned rat offspring.

OBJECTIVES: Maternal protein-caloric restriction during lactation can malprogram offspring into having a lean phenotype associated with metabolic dysfunction in early life and adulthood. The aim of this study was to investigate the relationships between nutritional stress, maternal behavior and metabolism, milk composition, and offspring parameters. Additionally, we focused on the role of hypothalamus-pituitary-adrenal axis hyperactivation during lactation. METHODS: Dams were fed a low-protein diet (4% protein) during the first 2 wk of lactation or a normal-protein diet (20% protein) during all lactation. Analyses of dams, milk, and offspring were conducted on postnatal days (PD) 7, 14, and 21. RESULTS: Body weight and food intake decreased in dams, which was associated with reduced fat pad stores and increased corticosterone levels at PD 14. The stressed low-protein diet dams demonstrated alterations in behavior and offspring care. Despite nutritional deprivation, dams adapted their metabolism to provide adequate energy supply through milk; however, we demonstrated elevated corticosterone and total fat levels in milk at PD 14. Male offspring also showed increased corticosterone at PD 7, associated with a lean phenotype and alterations in white and brown adipose tissue morphology at PD 21. CONCLUSION: Exposure to protein-caloric restriction diet of dams during lactation increased the glucocorticoid levels in dams, milk, and offspring, which is associated with alterations in maternal behavior and milk composition. Thus, glucocorticoids and milk composition may play an important role in metabolic programming induced by maternal undernutrition.

Puberty as a DOHaD programming window: high-fat diet induces long-term hepatic dysfunction in male rats.

The aim of this study was to evaluate whether high-fat (HF) diet intake during puberty can program obesity as well as generate glucose imbalance and hepatic metabolic dysfunctions in adult life. Male Wistar rats were randomly assigned into two groups: rats fed standard chow (NF) and rats fed a HF from postnatal 30-day-old (PND30) until PND60. Then, both groups were fed a standard chow from PND60 until PND120. Euthanasia and samples collections occurred at PND120. HF animals were overweight (+11%) and had increased adiposity, hyperphagia (+12%), hyperglycaemia (+13%), hyperinsulinemia (+69%), and hypertriglyceridemia (+34%). Plasma glucose levels during intravenous glucose tolerance test (ivGTT) and intraperitoneal insulin tolerance test (ipITT) were also higher in the HF group, whereas Kitt was significantly lower (-34%), suggesting reduced insulin sensitivity. In the same sense, HF animals present pancreatic islets hypertrophy and high ß-cell mass. HF animals also had a significant increase in blood glucose levels during pyruvate tolerance test, indicating increased gluconeogenesis. Hepatic morphology analyses showed an increase in lipid inclusion in the HF group. Moreover, PEPCK and FAS protein expression were higher in the livers of the HF animals (+79% and + 37%, respectively). In conclusion, HF during puberty causes obese phenotype leading to glucose dyshomeostasis and nonalcoholic fatty liver disease, which can be related to the overexpression of proteins PEPCK and FAS.

High-fructose diet during puberty alters the sperm parameters, testosterone concentration, and histopathology of testes and epididymis in adult Wistar rats.

The consumption of fructose has increased in children and adolescents and is partially responsible for the high incidence of metabolic diseases. The lifestyle during postnatal development can result in altered metabolic programming, thereby impairing the reproductive system and fertility during adulthood. Therefore, the aim of this study was to evaluate the effect of a high-fructose diet in the male reproductive system of pubertal and adult rats. Male Wistar rats (30 d old) were assigned to four different groups: Fr30, which received fructose (20%) in water for 30 d and were euthanized at postnatal day (PND) 60; Re-Fr30, which received fructose (20%) for 30 d and were euthanized at PND 120; and two control groups C30 and Re-C30, which received water ad libitum and were euthanized at PND 60 and 120, respectively. Fructose induced an increase in abnormal seminiferous tubules with epithelial vacuoles, degeneration, and immature cells in the lumen. Moreover, Fr30 rats showed altered spermatogenesis and daily sperm production (DSP), as well as increased serum testosterone concentrations. After discontinuing high-fructose consumption, DSP and sperm number decreased significantly. We observed tissue remodeling in the epididymis, with a reduction in stromal and epithelial compartments that might have influenced sperm motility. Therefore, we concluded that fructose intake in peripubertal rats led to changes in the reproductive system observed both during puberty and adulthood.

Time-restricted feeding during embryonic development leads to metabolic dysfunction in adult rat offspring.

OBJECTIVES: Maternal circadian eating time and frequency are associated with altered glucose metabolism during pregnancy in humans. Research on long maternal fasting intervals is inconclusive, and little is known about the effect of maternal time feeding on offspring health. Therefore, the aim of the present study is to determine whether maternal time feeding influences the metabolic status of both male and female offspring. METHODS: Pregnant rats were provided ad libitum access to chow diet or fed during either the light phase (LP) or dark phase (DP) during embryonic development. At the age of 150 days, glucose tolerance, lipid concentrations, and insulin secretion were determined in adult male and female offspring. RESULTS: Both male and female offspring of LP and DP dams exhibited alterations in the lipid profile, and female offspring were glucose intolerant. Glucose-stimulated insulin secretion decreased in male and female offspring of LP and DP dams. Acetylcholine increased insulin secretion in male and female offspring. Islets from male and female offspring of DP dams exhibited less pronounced inhibition of insulin secretion by epinephrine, suggesting alterations in the cholinergic and adrenergic pathways in these animals. CONCLUSIONS: Our data suggest that a time-restricted feeding regimen during embryonic development could program rat offspring for metabolic dysfunction during adulthood.

Fecal Microbiota Transplantation During Lactation Programs the Metabolism of Adult Wistar Rats in a Sex-specific Way.

BACKGROUND: The intestinal microbiota is involved in many physiological processes. However, the effects of microbiota in metabolic programming still unknow. We evaluated whether the transplantation of fecal microbiota during early life can program health or disease during adulthood in a model of lean and obese male and female Wistar rats. METHODS: Parental obesity were induced using a small litter (SL, 3 pups/dam) model. At 90 d old, normal litter (NL, 9 pups/dam) and SL males and females (parents) from different litters were mated: NL male vs. NL female; SL male vs. SL female. After birth, male and female offspring rats were also standardized in normal litters or small litters . From the 10th until 25th d of life, the NL and SL male and female offspring received via gavage of a solution containing the diluted feces of the opposite dam (fecal microbiota, M) or saline solution (S). At 90 d of age, biometric and biochemical parameters were assessed. RESULTS: NLM male rats transplanted with obese microbiota showed increased body weight, and fat pad deposition, hyperinsulinemia, glucose intolerance and dyslipidemia. SLM male rats transplanted with lean microbiota had decreased retroperitoneal and mesenteric fat, triglycerides and VLDL levels and improvement of glucose tolerance. Despite SLM female rats showed higher visceral fat, microbiota transplantation in female rats caused no changes in these parameters compared with control groups. CONCLUSION: Fecal microbiota transplantation during lactation induces long-term effects on the metabolism of male Wistar rats. However, female rats were resistant to metabolic alterations caused by the treatment.

Protein restriction during lactation causes transgenerational metabolic dysfunction in adult rat offspring.

Introduction: Protein restriction during lactation can induce metabolic dysfunctions and has a huge impact on the offspring's phenotype later in its life. We tested whether the effects of a maternal low-protein diet (LP) in rats can be transmitted to the F2 generation and increase their vulnerability to dietary insults in adulthood. Methods: Female Wistar rats (F0) were fed either a low-protein diet (LP; 4% protein) during the first 2 weeks of lactation or a normal-protein diet (NP; 23% protein). The female offspring (F1 generation) were maintained on a standard diet throughout the experiment. Once adulthood was reached, female F1 offspring from both groups (i.e., NP-F1 and LP-F1) were bred to proven males, outside the experiment, to produce the F2 generation. Male F2 offspring from both groups (NP-F2 and LP-F2 groups) received a standard diet until 60 days old, at which point they received either a normal fat (NF; 4.5% fat) or a high fat diet (HF; 35% fat) for 30 days. Results: At 90 days old, LPNF-F2 offspring had increased lipogenesis and fasting insulinemia compared to NPNF-F2, without alteration in insulin sensitivity. HF diet caused increased gluconeogenesis and displayed glucose intolerance in LPHF-F2 offspring compared to LPNF-F2 offspring. Additionally, the HF diet led to damage to lipid metabolism (such as steatosis grade 3), higher body weight, fat pad stores, and hepatic lipid content. Discussion: We concluded that an F0 maternal protein restricted diet during lactation can induce a transgenerational effect on glucose and liver metabolism in the F2 generation, making the offspring's liver more vulnerable to nutritional injury later in life.

Protein Restriction in the Peri-Pubertal Period Induces Autonomic Dysfunction and Cardiac and Vascular Structural Changes in Adult Rats.

Perturbations to nutrition during critical periods are associated with changes in embryonic, fetal or postnatal developmental patterns that may render the offspring more likely to develop cardiovascular disease in later life. The aim of this study was to evaluate whether autonomic nervous system imbalance underpins in the long-term hypertension induced by dietary protein restriction during peri-pubertal period. Male Wistar rats were assigned to groups fed with a low protein (4% protein, LP) or control diet (20.5% protein; NP) during peri-puberty, from post-natal day (PN) 30 until PN60, and then all were returned to a normal protein diet until evaluation of cardiovascular and autonomic function at PN120. LP rats showed long-term increased mean arterial pressure (p = 0.002) and sympathetic arousal; increased power of the low frequency (LF) band of the arterial pressure spectral (p = 0.080) compared with NP animals. The depressor response to the ganglion blocker hexamethonium was increased in LP compared with control animals (p = 0.006). Pulse interval variability showed an increase in the LF band and LF/HF ratio (p = 0.062 and p = 0.048) in LP animals. The cardiac response to atenolol and/or methylatropine and the baroreflex sensitivity were similar between groups. LP animals showed ventricular hypertrophy (p = 0.044) and increased interstitial fibrosis (p = 0.028) compared with controls. Reduced protein carbonyls (PC) (p = 0.030) and catalase activity (p = 0.001) were observed in hearts from LP animals compared with control. In the brainstem, the levels of PC (p = 0.002) and the activity of superoxide dismutase and catalase (p = 0.044 and p = 0.012) were reduced in LP animals, while the levels of GSH and total glutathione were higher (p = 0.039 and p = 0.038) compared with NP animals. Protein restriction during peri-pubertal period leads to hypertension later in life accompanied by sustained sympathetic arousal, which may be associated with a disorganization of brain and cardiac redox state and structural cardiac alteration.

Maternal postnatal early overfeeding induces sex-related cardiac dysfunction and alters sexually hormones levels in young offspring.

Postnatal early overfeeding (PO) is a risk factor for cardiometabolic disorders. However, remains unknown the cardiac effects in the second generation from postnatal overfed dams. Our aim was to investigate the effects of maternal PO on cardiac parameters in second generation (F2) offspring. For this, pregnant Wistar rats (F0) were divided into two groups: normal litter (NL, 9 pups) and small litter (SL, 3 pups). At P70, female offspring (F1) of both groups were mated with non-PO male rats. At P21 male and female F2 offspring (NLO and SLO) were weaned, and at P45 they were euthanized to evaluate the cardiac function and sample collection. Male and female SLO showed increased body weight, food intake and adiposity. Blood estradiol levels were increased in the male SLO and decreased in the female SLO. Blood testosterone levels increased in SLO females, but not change in SLO male rats. Although SLO offspring presented cardiac hypertrophy, only males had ex vivo functional impairments, such as reduction of the intraventricular systolic pressure and dP/dt. Male and female SLO had increased interstitial fibrosis; however, only the male SLO had increased perivascular fibrosis. In addition, only male rats from SLO group had decreased AKT and Type 2 Ang-2 receptor, increased catalase and type alpha estrogenic receptor protein levels. Maternal PO leads to obese phenotype and alters sex-steroid levels in both male and female offspring. Although both sexes showed cardiac hypertrophy, only male offspring showed cardiac dysfunction, which may be related with Ang2 and AKT signaling impairments.