Neonatal nicotine exposure affects adult rat hepatic pathways involved in endoplasmic reticulum stress and macroautophagy in a sex-dependent manner.

Nonalcoholic fatty liver disease (NAFLD) involves changes in hepatic pathways, as lipogenesis, oxidative stress, endoplasmic reticulum (ER) stress, and macroautophagy. Maternal nicotine exposure exclusively during lactation leads to fatty liver (steatosis) only in the adult male offspring, not in females. Therefore, our hypothesis is that neonatal exposure to nicotine sex-dependently affects the signaling pathways involved in hepatic homeostasis of the offspring, explaining the hepatic lipid accumulation phenotype only in males. For this, between postnatal days 2 and 16, Wistar rat dams were implanted with osmotic minipumps, which released nicotine (NIC; 6 mg/Kg/day) or vehicle. The livers of offspring were evaluated at postnatal day 180. Only the male offspring that had been exposed to nicotine neonatally showed increased protein expression of markers of unfolded protein response (UPR), highlighting the presence of ER stress, as well as disruption of the activation of the macroautophagy repair pathway. These animals also had increased expression of diacylglycerol O-acyltransferase 1 and 4-hydroxynonenal, suggesting increased triglyceride esterification and oxidative stress. These parameters were not altered in the female offspring that had been neonatally exposed to nicotine, however they exhibited increased phospho adenosine monophosphate-activated protein kinase pAMPK expression, possibly as a protective mechanism. Thus, the disturbance in the hepatic homeostasis by UPR, macroautophagy, and oxidative stress modifications seem to be the molecular mechanisms underlying the liver steatosis in the adult male offspring of the nicotine-programming model. This highlights the importance of maternal smoking cessation during breastfeeding to decrease the risk of NAFLD development, especially in males.

Nicotine exposure during breastfeeding alters the expression of endocannabinoid system biomarkers in female but not in male offspring at adulthood.

Early nicotine exposure compromises offspring's phenotype at long-term in both sexes. We hypothesize that offspring exposed to nicotine during breastfeeding show deregulated central and peripheral endocannabinoid system (ECS), compromising several aspects of their metabolism. Lactating rats received nicotine (NIC, 6 mg/Kg/day) or saline from postnatal day (PND) 2 to 16 through implanted osmotic minipumps. Offspring were analyzed at PND180. We evaluated protein expression of N-acylphosphatidylethanolamide-phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), diacylglycerol lipase (DAGL), monoacylglycerol lipase (MAGL) and cannabinoid receptors (CB1 and/or CB2) in lateral hypothalamus, paraventricular nucleus of the hypothalamus, liver, visceral adipose tissue (VAT), adrenal and thyroid. NIC offspring from both sexes did not show differences in hypothalamic ECS markers. Peripheral ECS markers showed no alterations in NIC males. In contrast, NIC females had lower liver DAGL and CB1, higher VAT DAGL, higher adrenal NAPE-PLD and higher thyroid FAAH. Endocannabinoids biosynthesis was affected by nicotine exposure during breastfeeding only in females; alterations in peripheral tissues suggest lower action in liver and higher action in VAT, adrenal and thyroid. Effects of nicotine exposure during lactation on ECS markers are sex- and tissue-dependent. This characterization helps understanding the phenotype of the adult offspring in this model and may contribute to the development of new pharmacological targets for the treatment of several metabolic diseases that originate during development.

Neonatal nicotine exposure changes insulin status in fat depots: sex-related differences.

Nicotine is the main psychoactive substance present in cigarette smoke that is transferred to the baby by breast milk. In rats, maternal nicotine exposure during breastfeeding induces obesogenesis and hormone dysfunctions in adult male offspring. As glucocorticoid (GC), insulin, and vitamin D change both adipogenesis and lipogenesis processes, we assessed parameters related to metabolism and action of these hormones in visceral and subcutaneous adipose tissues (VAT and SAT) of adult male and female rats in a model of neonatal nicotine exposure. At postnatal (PN) day 2, dams were kept with six pups (three per sex) and divided into nicotine and control groups for implantation of osmotic minipumps that released 6 mg/kg nicotine or saline, respectively. At PN180, fat mass, hormone levels, and protein contents of biomarkers of the GC activation and receptor (11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor alpha), insulin signaling pathway [insulin receptor beta (IRß), phosphorylated insulin receptor substrate 1, insulin receptor substrate 1 (IRS1), phosphorylated serine/threonine kinase (pAKT), serine/threonine kinase, glucose transporter type 4 (GLUT4)], and vitamin D activation and receptor (1α-hydroxylase and vitamin D receptor) were evaluated. While nicotine-exposed males showed increased fat mass, hypercorticosteronemia, hyperinsulinemia, and higher 25-hydroxyvitamin D, these alterations were not observed in nicotine-exposed females. Nicotine-exposed males only showed lower IRS1 in VAT, while the females had hyperglycemia, higher pAKT in VAT, while lower IRß, IRS1, and GLUT4 in SAT. Parameters related to metabolism and action of GC and vitamin D were unaltered in both sexes. We evidence that exposure exclusively to nicotine during breastfeeding affects the hormone status and fat depots of the adult progeny in a sex-dependent manner.

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.

Early life nicotine exposure alters mRNA and microRNA expressions related to thyroid function and lipid metabolism in liver and BAT of adult wistar rats.

In rats, maternal nicotine exposure during lactation induces obesity, thyroid dysfunction, brown adipose tissue (BAT) hypofunction and liver alterations in adult offspring. Both thyroid function and lipid metabolism are influenced by gene silencing mediated by microRNAs (miRNAs). Here we investigated long-term effects of early nicotine exposure on molecular and epigenetic mechanisms closely related to thyroid and lipid metabolism, through the expression of mRNAs and miRNAs in BAT and liver of adult male and female offspring. At postnatal day 2 (PND2), lactating control (CON) or nicotine (NIC) dams were subcutaneously implanted with osmotic minipumps containing, respectively, saline or 6 mg/kg nicotine. Litters were adjusted to 3 males and 3 females. Offspring's euthanasia occurred at PND180. In the BAT, NIC females showed higher Dio2 mRNA expression, while miR-382* expression was not altered in both sexes. In the liver, NIC offspring of both sexes showed lower Dio1 mRNA expression and higher miR-224 expression, while only NIC females had higher miR-383 and miR-21 expressions. NIC offspring of both sexes showed higher mRNA expression of SCD1 in the liver; NIC males had decreased CPT1 expression, whereas NIC females had increased FASN, miR-370 and miR-122 expressions. Regardless of sex, alterations in liver Dio1, miR-224 and SCD1 expressions are involved in the disturbances caused by maternal nicotine exposure during breastfeeding. Interestingly, females had more altered miRs in the liver. Early nicotine exposure induces a sex dimorphism, particularly regarding hepatic lipid metabolism, through miRs expression.

Thyroid redox imbalance in adult Wistar rats that were exposed to nicotine during breastfeeding.

Maternal nicotine exposure causes several consequences in offspring phenotype, such as obesity and thyroid dysfunctions. Nicotine exposure can increase oxidative stress levels, which could lead to thyroid dysfunction. However, the mechanism by which nicotine exposure during breastfeeding leads to thyroid gland dysfunction remains elusive. We aimed to investigate the long-term effects of maternal nicotine exposure on redox homeostasis in thyroid gland, besides other essential steps for thyroid hormone synthesis in rats from both sexes. Lactating Wistar rats were implanted with osmotic minipumps releasing nicotine (NIC, 6 mg/kg/day) or saline (control) from postnatal day 2 to 16. Offspring were analyzed at 180-day-old. NIC males showed lower plasma TSH, T3 and T4 while NIC females had higher T3 and T4. In thyroid, NIC males had higher sodium-iodide symporter protein expression, whereas NIC females had higher thyroid-stimulating hormone receptor (TSHr) and thyroperoxidase (TPO) protein expression. TPO activity was lower in NIC males. Hydrogen peroxide generation was decreased in NIC males. Activities of superoxide dismutase, catalase and glutathione peroxidase were compromised in NIC animals from both sexes. 4-Hydroxynonenal was higher only in NIC females, while thiol was not affected in NIC animals from both sexes. NIC offspring also had altered expression of sex steroid receptors in thyroid gland. Both sexes showed similar thyroid morphology, with lower follicle and colloid size. Thyroid from female offspring exposed to nicotine during breastfeeding developed oxidative stress, while the male gland seemed to be protected from redox damage. Thyroid dysfunctions seem to be associated with redox imbalance in a sex-dependent manner.

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.

Supplementation of suckling rats with cow's milk induces hyperphagia and higher visceral adiposity in females at adulthood, but not in males.

In humans, complementary feeding should be started after 6 months-old; the introduction of any food or water before this time is considered early weaning, which is associated with health problems in adulthood. Cow's milk is a common food introduced to children less than 6 months that has inadequate nutritional composition mainly due to a worse casein: whey protein ratio compared to human milk. We hypothesized that suckling rats fed with cow's milk, rich in bioactive peptides, develop further metabolic dysfunctions. From postnatal day (PN) 14 to 20, Wistar rat pups were divided into 3 groups: rat milk (RM) - pups received rat milk orally in a syringe; cow's milk (CM), pups received cow's milk; CM with high protein (CM-H), CM with twice protein amount of rat milk. Pups were killed on PN21 and PN180. At PN21, CM males had lower visceral fat mass compared with other groups. Serum corticosterone was higher in CM-H males, despite no change in glucocorticoid metabolism in liver and visceral fat. At PN180, CM and CM-H females had greater fat depots and hyperphagia, although no alteration in leptinemia and leptin signaling in hypothalamus. CM-H females had a trend of hypoinsulinemia and significant decrease in HOMA-ß, suggesting lower insulin secretion. Males from CM-H group had only lower total body protein mass. CM males had hypercorticosteronemia associated with lower expression of 11ßHDS1 in visceral fat. In conclusion, early introduction of cow's milk in neonate rats leads to gender-dependent differences in metabolic and endocrine parameters in the short- and long-term.

Effects of cigarette smoke exposure during suckling on food intake, fat mass, hormones, and biochemical profile of young and adult female rats.

PURPOSE: Children from smoking mothers have a higher risk of developing obesity and associated comorbidities later in life. Different experimental models have been used to assess the mechanisms involved with this increased risk. Using a rat model of neonatal nicotine exposure via implantation of osmotic minipumps in lactating dams, we have previously shown marked sexual dimorphisms regarding metabolic and endocrine outcomes in the adult progeny. Considering that more than four thousand substances are found in tobacco smoke besides nicotine, we then studied a rat model of neonatal tobacco smoke exposure: adult male offspring had hyperphagia, obesity, hyperglycemia, hypertriglyceridemia, secondary hyperthyroidism and lower adrenal hormones. Since litters were culled to include only males and since sexual dimorphisms had already been identified in the nicotine exposure model, here we also evaluated the effects of tobacco smoke exposure during lactation on females. METHODS: Wistar rat dams and their pups were separated into two groups of 8 litters each: SMOKE (4 cigarettes per day, from postnatal day 3 to 21) and CONTROL (filtered air). Offspring of both sexes were euthanized at PN21 and PN180. RESULTS: Changes in male offspring corroborated previous data. At weaning, females showed lower body mass gain and serum triglycerides, but no alterations in visceral fat and hormones. At adulthood, females had higher body mass, hyperphagia, central obesity, hyperleptinemia, hypercholesterolemia, hypercorticosteronemia, but no change in serum TSH and T3, and adrenal catecholamine CONCLUSIONS: Sexual dimorphisms were observed in several parameters, thus indicating that metabolic and hormonal changes due to smoke exposure during development are sex-dependent.