Citrus aurantium L. and synephrine improve brown adipose tissue function in adolescent mice programmed by early postnatal overfeeding.

Introduction and aims: Obesity is a multifactorial condition with high health risk, associated with important chronic disorders such as diabetes, dyslipidemia, and cardiovascular dysfunction. Citrus aurantium L. (C. aurantium) is a medicinal plant, and its active component, synephrine, a ß-3 adrenergic agonist, can be used for weight loss. We investigated the effects of C. aurantium and synephrine in obese adolescent mice programmed by early postnatal overfeeding. Methods: Three days after birth, male Swiss mice were divided into a small litter (SL) group (3 pups) and a normal litter (NL) group (9 pups). At 30 days old, SL and NL mice were treated with C. aurantium standardized to 6% synephrine, C. aurantium with 30% synephrine, isolated synephrine, or vehicle for 19 days. Results: The SL group had a higher body weight than the NL group. Heart rate and blood pressure were not elevated. The SL group had hyperleptinemia and central obesity that were normalized by C. aurantium and synephrine. In brown adipose tissue, the SL group showed a higher lipid droplet sectional area, less nuclei, a reduction in thermogenesis markers related to thermogenesis (UCP-1, PRDM16, PGC-1α and PPARg), and mitochondrial disfunction. C. aurantium and synephrine treatment normalized these parameters. Conclusion: Our data indicates that the treatment with C. aurantium and synephrine could be a promising alternative for the control of some obesity dysfunction, such as improvement of brown adipose tissue dysfunction and leptinemia.

Low protein diet during lactation programs hepatic metabolism in adult male and female rats.

The liver is an essential regulator of energy metabolism, and its function can be disrupted by nutritional alterations. Since liver development continues during breastfeeding nutritional challenges during this period predispose patients to diseases throughout life. A maternal protein-restricted (PR) diet during lactation promotes reductions in the body weight, adiposity, and plasma glucose and insulin, leptin resistance and an increase in corticosterone and catecholamines in adult male rat offspring. Here, we investigated hepatic metabolism in the offspring (both sexes) of PR (8% protein diet during lactation) and control (23% protein diet) dams. Both male and female offspring were evaluated at 6 months of age. PR males had no liver steatosis and manifested a reduction in lipids in hepatocytes adjacent to the vasculature. These animals had lower levels of esterified cholesterol in hepatocytes, suggesting higher biliary excretion, unchanged glycolysis and gluconeogenesis, and lower contents of the markers of mitochondrial redox balance and endoplasmic reticulum (ER) stress response and estrogen receptor alpha. PR females showed normal hepatic morphology associated with higher uptake of cholesterol esters, normal glycolysis and gluconeogenesis, and lower ER stress parameters without changes in the key markers of the redox balance. Additionally, these animals had lower content of estrogen receptor alpha and higher content of androgen receptor. The maternal PR diet during lactation did not program hepatic lipid accumulation in the adult progeny. However, several repair homeostasis pathways were altered in males and females, possibly compromising maintenance of normal liver function.

Pregnancy and lactation after Roux-en-Y gastric bypass worsen nonalcoholic fatty liver disease in obese rats and lead to differential programming of hepatic de novo lipogenesis in offspring.

Maternal obesity increases the risk of nonalcoholic fatty liver disease (NAFLD) in offspring. The Roux-en-Y gastric bypass (RYBG) is effective for achieving weight loss and ameliorates NAFLD. To determine whether these benefits are maintained after pregnancy and/or lactation, and whether they modulate hepatic morphofunction in the next generation, we evaluated hepatic lipid metabolism in Western diet (WD)-obese female rats that underwent RYGB and in their F1 offspring at adulthood. Female Wistar rats consumed a WD from 21 to 130 days of age, before being submitted to RYGB (WD-RYGB-F0) or SHAM (WD-SHAM-F0) operations. After 5 weeks, these females were mated with control male breeders, and the male and female F1 offspring were identified as WD-RYGB-F1 and WD-SHAM-F1. WD-RYGB-F0 dams exhibited lower serum lipids levels, but severe hepatic steatosis and pathological features of advanced liver injury. The hepatic proteins involved in lipogenesis were reduced in WD-RYGB-F0, as were the genes related to ß-oxidation and bile acids (BAs). Although the female and male WD-RYGB-F1 groups did not exhibit hepatic steatosis, the livers of female WD-RYGB-F1 demonstrated higher amounts of lipogenic genes and proteins, while male WD-RYGB-F1 presented a similar downregulation of lipogenic factors to that seen in WD-RYGB-F0 dams. In contrast, maternal and offspring groups of both sexes displayed reductions in the expressions of genes involved in BAs physiology and gluconeogenesis. As such, RYGB aggravates NAFLD after pregnancy and lactation and induces a gender-dependent differential expression of the hepatic lipogenesis pathway in offspring, indicating that female WD-RYGB-F1 may be an increased risk of developing NAFLD.

Late effects of early weaning on food preference and the dopaminergic and endocannabinoid systems in male and female rats.

Early weaning (EW) is associated with obesity later in life. Here, using an EW model in rats, we investigated changes in feeding behavior and the dopaminergic and endocannabinoid systems (ECS) in the adult offspring. Lactating Wistar rats were divided into two groups: EW, dams were wrapped with a bandage to interrupt suckling during the last 3 days of breastfeeding; CONT; dams fed the pups throughout the period without hindrances. EW animals were compared with CONT animals of the same sex. At PN175, male and female offspring of both groups could freely self-select between high-fat and high-sugar diets (food challenge test). EW males preferred the high-fat diet at 30 min and more of the high-sugar diet after 12 h compared to CONT males. EW females did not show differences in their preference for the palatable diets compared to CONT females. Total intake of standard diet from PN30-PN180 was higher in both male and female EW animals, indicating hyperphagia. At PN180, EW males showed lower type 2 dopamine receptor (D2r) in the nucleus accumbens (NAc) and dorsal striatum, while EW females had lower tyrosine hydroxylase in the ventral tegmental area and NAc, D1r in the NAc, and D2r in the prefrontal cortex. In the lateral hypothalamus, EW males had lower fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase, whereas EW females showed lower N-arachidonoyl-phosphatidylethanolamine phospholipase-D and increased FAAH. Early weaning altered both the dopaminergic and ECS parameters at adulthood, contributing to the eating behavior changes of the progeny in a sex-dependent manner.

Maternal Roux-en-Y gastric bypass surgery reduces lipid deposition and increases UCP1 expression in the brown adipose tissue of male offspring.

Maternal obesity induced by cafeteria diet (CAF) predisposes offspring to obesity and metabolic diseases, events that could be avoided by maternal bariatric surgery (BS). Herein we evaluated whether maternal BS is able to modulate brown adipose tissue (BAT) morphology and function in adult male rats born from obese female rats submitted to Roux-en-Y gastric bypass (RYGB). For this, adult male rat offspring were obtained from female rats that consumed standard diet (CTL), or CAF diet, and were submitted to simulated operation or RYGB. Analysis of offspring showed that, at 120 days of life, the maternal CAF diet induced adiposity and decreased the expression of mitochondrial Complex I (CI) and Complex III (CIII) in the BAT, resulting in higher accumulation of lipids than in BAT from offspring of CTL dams. Moreover, maternal RYGB increased UCP1 expression and prevented excessive deposition of lipids in the BAT of adult male offspring rats. However, maternal RYGB failed to reverse the effects of maternal diet on CI and CIII expression. Thus, maternal CAF promotes higher lipid deposition in the BAT of offspring, contributing to elevated adiposity. Maternal RYGB prevented obesity in offspring, probably by increasing the expression of UCP1.

Pancreatic steatosis in adult rats induced by nicotine exposure during breastfeeding.

PURPOSE: Maternal nicotine exposure negatively impacts offspring's health and metabolism, leading to obesity and insulin resistance. Here we investigated the pancreatic islet function, glycemic homeostasis, and insulin signaling in adult rat offspring that were nicotine-exposed during breastfeeding. METHODS: For this, lactating Wistar rat dams were divided into two groups: Nicotine (implanted with osmotic minipumps containing 6 mg/Kg, NIC) and Control (saline, CON). Solutions were released from postnatal (PN) day 2-16. At PN110 and PN170, 10 offspring per litter/sex/group were submitted to the oral glucose tolerance test (OGTT). PN180 offspring were killed and glycemia, insulinemia, adiponectinemia, pancreas morphology as well as pancreatic islet protein expression (related to insulin secretion) and skeletal muscle (related to insulin action) were evaluated. Males and females were compared to their respective controls. RESULTS: Adult NIC offspring of both sexes showed glucose intolerance in the OGTT. Despite normoglycemia, NIC males showed hyperinsulinemia while females, although normoinsulinemic, had hyperglycemia. Both sexes showed increased IRI, reduced adiponectin/visceral fat mass ratio and higher ectopic deposition of lipids in the pancreatic tissue adipocytes. In pancreatic islets, NIC males showed lower PDX-1 expression while females had higher PDX-1 and GLUT2 expressions plus lower α2 adrenergic receptor. In the muscle, NIC offspring of both sexes showed reduction of GLUT4 expression; NIC males also had lower insulin receptor and pAKT expressions. CONCLUSIONS: Thus, glycemic homeostasis and peripheral insulin signaling in adult offspring of both sexes are affected by nicotine exposure through the milk, increasing the risk for type 2 diabetes development.

Hepatic lipid metabolism in adult rats using early weaning models: sex-related differences.

Non-pharmacological early weaning (NPEW) induces liver damage in male progeny at adulthood; however, pharmacological early weaning (PEW) does not cause this dysfunction. To elucidate this difference in liver dysfunction between these two models and determine the phenotype of female offspring, de novo lipogenesis, ß-oxidation, very low-density lipoprotein (VLDL) export, and gluconeogenesis in both sexes were investigated in the adult Wistar rats that were weaned after a normal period of lactation (control group) or early weaned either by restriction of access to the dams' teats (NPEW group) or by reduction of dams' milk production with bromocriptine (PEW group). The offspring received standard diet from weaning to euthanasia (PN180). NPEW males had higher plasma triglycerides and TyG index, liver triglycerides, and cholesterol by de novo lipogenesis, which leads to intracellular lipids accumulation. As expected, hepatic morphology was preserved in PEW males, but they showed increased liver triglycerides. The only molecular difference between PEW and NPEW males was in acetyl-CoA carboxylase-1 (ACC-1) and stearoyl-CoA desaturase-1 (SCD-1), which were lower in PEW animals. Both early weaning (EW) females had no changes in liver cholesterol and triglyceride contents, and the hepatic cytoarchitecture was preserved. The expression of microsomal triglyceride transfer protein was increased in both the female EW groups, which could constitute a protective factor. The changes in hepatic lipid metabolism in EW offspring were less marked in females. EW impacted in the hepatic cytoarchitecture only in NPEW males, which showed higher ACC-1 and SCD-1 when compared to the PEW group. As these enzymes are lipogenic, it could explain a worsened liver function in NPEW males.

Body Adiposity and Endocrine Profile of Female Wistar Rats of Distinct Ages that were Early Weaned.

Early weaning (EW) is a risk factor for metabolic syndrome. Male rats that were precociously weaned present neonatal malnutrition and, in adulthood, developed overweight, accumulation of body fat, dyslipidemia, changes in glycemic homeostasis, hyperleptinemia, and increase of vitamin D. As metabolic profile of early-weaned females is not known, we investigated the endocrine-metabolic parameters in adolescence and adult female rats of 2 different EW models. Wistar lactating rats and pups from both sexes were separated into 3 groups: non-pharmacological EW (NPEW), dams were involved with a bandage interrupting suckling in the last 3 days of lactation; pharmacological EW (PEW), dams were bromocriptine-treated (0.5 mg/twice a day via intraperitoneal injection) for 3 days before weaning; and control, dams whose pups ate milk throughout lactation. At 21 days-old, NPEW and PEW females had lower body weight. At 180 days-old, NPEW and PEW females showed higher feed efficiency, weight gain, body fat percentage, and greater accumulation of gonadal and retroperitoneal fat depots associated with adipocyte hypertrophy. NPEW females also showed hyperphagia. Only NPEW females presented hyperleptinemia. Plasma thyroid hormones and vitamin D were unchanged among EW females. Regarding sex hormones, at 45 days-old, no change was found in EW females, while at 180 days-old, PEW females had hypoestrogenemia. EW increases the risk for obesity in female rats in adulthood, as already demonstrated for males, although through distinct mechanisms involving some hormones.

Early weaning leads to specific glucocorticoid signalling in fat depots of adult rats.

PURPOSE: Early weaning (EW) is a stressful condition that programmes a child to be overweight in adult life. Fat mass depends on glucocorticoids (GC) to regulate adipogenesis and lipogenesis. We hypothesised that the increased adiposity in models of EW was due to a disturbed HPA axis and/or disrupted GC function. METHODS: We used two experimental models, pharmacological early weaning (PEW, dams were bromocriptine-treated) and non-pharmacological early weaning (NPEW, dams' teats were wrapped with a bandage), which were initiated during the last 3 days of lactation. Offspring from both genders was analysed on postnatal day 180. RESULTS: Offspring in both models were overweight with increased visceral fat mass, but plasma corticosterone was increased in both genders in the PEW group but not the NPEW group. NPEW males had increased GRα expression in visceral adipose tissue (VAT), and GRα expression decreased in PEW males in subcutaneous adipose tissue (SAT). Females in both EW groups had increased 11ßHSD1 expression in SAT. PEW males had increased C/EBPß expression in SAT. PEW females had lower PPARy and FAS expression in VAT than the NPEW females. We detected a sex dimorphism in VAT and SAT in the EW groups regarding 11ßHSD1, GRα and C/EBPß expression. CONCLUSIONS: The accumulated adiposity induced by EW exhibited distinct mechanisms depending on gender, specific fat deposition and GC metabolism and action. The higher proportion of VAT/SAT in both sets of EW males may be related to the action of GC in these tissues, and the higher conversion of GC in SAT in females may explain the differences in the fat distribution.

Maternal Roux-en-Y gastric bypass impairs insulin action and endocrine pancreatic function in male F1 offspring.

PURPOSE: Obesity is predominant in women of reproductive age. Roux-en-Y gastric bypass (RYGB) is the most common bariatric procedure that is performed in obese women for weight loss and metabolic improvement. However, some studies suggest that this procedure negatively affects offspring. Herein, using Western diet (WD)-obese female rats, we investigated the effects of maternal RYGB on postnatal body development, glucose tolerance, insulin secretion and action in their adult male F1 offspring. METHODS: Female Wistar rats consumed a Western diet (WD) for 18 weeks, before being submitted to RYGB (WD-RYGB) or SHAM (WD-SHAM) operations. After 5 weeks, WD-RYGB and WD-SHAM females were mated with control male breeders, and the F1 offspring were identified as: WD-RYGB-F1 and WD-SHAM-F1. RESULTS: The male F1 offspring of WD-RYGB dams exhibited decreased BW, but enhanced total nasoanal length gain. At 120 days of age, WD-RYGB-F1 rats displayed normal fasting glycemia and glucose tolerance but demonstrated reduced insulinemia and higher glucose disappearance after insulin stimulus. In addition, these rodents presented insulin resistance in the gastrocnemius muscle and retroperitoneal fat, as judged by lower Akt phosphorylation after insulin administration, but an increase in this protein in the liver. Finally, the islets from WD-RYGB-F1 rats secreted less insulin in response to glucose and displayed increased ß-cell area and mass. CONCLUSIONS: RYGB in WD dams negatively affected their F1 offspring, leading to catch-up growth, insulin resistance in skeletal muscle and white fat, and ß-cell dysfunction. Therefore, our data are the first to demonstrate that the RYGB in female rats may aggravate the metabolic imprinting induced by maternal WD consumption, in their male F1 descendants. However, since we only used male F1 rats, further studies are necessary to demonstrate if such effect may also occur in female F1 offspring from dams that underwent RYGB operation.

Programming of hepatic lipid metabolism in a rat model of postnatal nicotine exposure - Sex-related differences.

Maternal nicotine exposure during lactation induces liver damage in adult male rats. However, the mechanism in males is unknown and females have not been tested. Here, we determined the liver lipid composition and lipogenic enzymes in male and female offspring at two ages in a model of postnatal nicotine exposure. Osmotic minipumps were implanted in lactating Wistar rat dams at postnatal day (PND) 2 to release 6 mg/kg/day of nicotine (NIC group) or saline (CON group) for 14 days. Offspring received a standard diet from weaning until euthanasia at PND120 (1 pup/litter/sex) or PND180 (2 pups/litter/sex). At PND120, NIC males showed lower plasma triglycerides (TG), steatosis degree 1, higher hepatic cholesterol (CHOL) ester, free fatty acids, monoacylglycerol content as well as acetyl-coa carboxylase-1 (ACC-1) and fatty acid synthase (FAS) protein expression in the liver compared to CON males. At this age, NIC females had preserved hepatocytes architecture, higher plasma CHOL, higher CHOL ester and lower total CHOL content in the liver compared to CON females. At PND180, NIC males showed steatosis degrees 1 and 2, higher TG, lower free fatty acids and total CHOL content in the liver and an increase in ACC-1 hepatic protein expression. NIC females had higher plasma TG and CHOL levels, no change in hepatic morphology, lower CHOL ester and free fatty acids in the liver, which also showed higher total ACC-1 and FAS protein expression. Maternal nicotine exposure induces long-term liver dysfunction, with an alteration in hepatic cytoarchitecture that was aggravated with age in males. Concerning females, despite unchanged hepatic cytoarchitecture, lipid metabolism was compromised, which deserves further attention.

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.

Short and long-term effects of bisphenol S (BPS) exposure during pregnancy and lactation on plasma lipids, hormones, and behavior in rats.

Bisphenol S (BPS) has replaced bisphenol A (BPA), a known non-persistent endocrine disrupting chemical, in several products. Considering that little is known regarding BPS effects, especially during critical windows of ontogenetic development, and that BPA, which is quite similar to BPS, is know to be transferred to the offspring via the placenta and milk, in the present study we investigated the behavioral, biochemical and endocrine profiles of Wistar rats born from dams that were BPS-exposed [groups: BPS10 (10 µg/kg/day), BPS50 (50 µg/kg/day)] during pregnancy and lactation. Due to the non-monotonic dose-response effect of bisphenol, the data of both BPS groups were directly compared with those of the controls, not to each other. Males and females were analyzed separately. At weaning, male BPS50 offspring had hypotriglyceridemia and hyperthyroxinemia, whereas BPS50 females showed higher 25(OH)D levels. At adulthood, BPS offspring of both sexes had lower food intake. BPS males showed lower visceral adiposity. BPS50 females had smaller fat droplets in brown adipocytes. BPS males showed higher anxiety and higher locomotor activity, while BPS10 females showed lower exploration. During a food challenge test at adulthood, BPS males consumed more high-fat diet at 30 min. BPS10 females initially (at 30 min) consumed more high-fat diet but, after 12 h, less of this diet was consumed. BPS50 males had hypertriglyceridemia and lower plasma T3, while BPS females showed lower plasma T4. BPS10 females had lower progesterone, whereas BPS50 females had higher plasma 25(OH)D. Maternal BPS exposure has adverse effects on the triacylglycerol, hormones levels and behavior of the progeny. Furthermore, the increased preference for the fat-enriched diet suggests an increased risk for obesity and its health consequences in the long term.