Cintia R. Pinheiro

Neonatal nicotine exposure causes insulin and leptin resistance and inhibits hypothalamic leptin signaling in adult rat offspring

Maternal nicotine (NIC) exposure during lactation leads to overweight, hyperleptinemia, and hypothyroidism in adult rat offspring. In this model, we analyzed adipocyte morphology, glucose homeostasis (serum insulin and adiponectin; liver and muscle glycogen), serum lipid, and the leptin signaling pathway. After birth, osmotic minipumps were implanted in lactating rats, which were divided into the groups NIC (6 mg/kg per day s.c. for 14 days) and control (C, saline). NIC and C offspring were killed at the age of 180 days. Adult NIC rats showed higher total body fat (+10%, P<0.05), visceral fat mass (+12%, P<0.05), and cross-sectional area of adipocytes (epididymal: +12% and inguinal: +43%, P<0.05). Serum lipid profile showed no alteration except for apolipoprotein AI, which was lower. We detected a lower adiponectin:fat mass ratio (-24%, P<0.05) and higher insulinemia (+56%, P<0.05), insulin resistance index (+43%, P<0.05), leptinemia (+113%, P<0.05), and leptin:adiponectin ratio (+98%, P<0.05) in the adult NIC group. These rats presented lower hypothalamic contents of the proteins of the leptin signaling pathway (leptin receptor (OB-R): -61%, janus tyrosine kinase 2: -41%, and p-signal transducer and activator of transcription 3: -56%, P<0.05), but higher suppressor of cytokine signaling 3 (+81%, P<0.05). Therefore, NIC exposure only during lactation programs rats for adipocyte hypertrophy in adult life, as well as for leptin and insulin resistance. Through the effects of NIC, perinatal maternal cigarette smoking may be responsible for the future development of some components of the metabolic syndrome in the offspring.

Effects of tobacco smoke exposure during lactation on nutritional and hormonal profiles in mothers and offspring

Exposure to tobacco smoke is related to changes in energy balance regulation and several endocrine dysfunctions. Previously, we showed that maternal nicotine (the main addictive compound of tobacco) exposure exclusively during lactation affects biochemical profiles in mothers, milk, and pups. As the possible consequences for mothers and offspring of maternal smoking during lactation are still unknown, we evaluated the effects of tobacco smoke exposure on nutritional, biochemical, and hormonal parameters in dams and pups at weaning. After 72 h from birth, lactating rats were divided into two groups: smoke-exposed (S) in a cigarette-smoking machine, 4 × 1 h per day throughout the lactation period without pups; control (C), rats were treated the same as the experimental group but exposed to filtered air. Dams and pups were killed at weaning (21 days of lactation). Body weight and food intake were evaluated. Milk, blood, visceral fat, adrenal, and carcass were collected. S dams showed hyperprolactinemia (+50%), hypoinsulinemia (-40%), hypoleptinemia (-46%), as well as lower triglycerides (-53%) and very low-density lipoprotein cholesterol (-50%). Milk of S dams had higher lactose (+52%) and triglycerides (+78%). S pups presented higher body protein (+17%), lower total (-24%) and subcutaneous fat contents (-25%), hypoglycemia (-11%), hyperinsulinemia (+28%), hypocorticosteronemia (-40%), lower adrenal catecholamine content (-40%), hypertriglyceridemia (+34%), higher high-density lipoprotein cholesterol (+16%), and lower low-density lipoprotein cholesterol (-45%). In conclusion, tobacco smoke exposure leads to changes in nutritional, biochemical, and hormonal parameters in dams and, passively through the milk, may promote several important metabolic disorders in the progeny.

Endocrine effects of tobacco smoke exposure during lactation in weaned and adult male offspring

Children from pregnant smokers show more susceptibility to develop obesity in adult life. Previously, we failed to demonstrate a program for obesity in rat offspring only when the mothers were exposed to tobacco smoke during lactation. Here, we studied the short- and long-term effects of smoke exposure (SE) to both dams and their pups during lactation on endocrine and metabolic parameters. For this, we designed an experimental model where nursing rats and their pups were divided into two groups: SE group, exposed to smoke in a cigarette smoking machine (four times/day, from the third to the 21st day of lactation), and group, exposed to filtered air. Pups were killed at 21 and 180 days. At weaning, SE pups showed lower body weight (7%), length (5%), retroperitoneal fat mass (59%), visceral adipocyte area (60%), and higher subcutaneous adipocyte area (95%) with hypoinsulinemia (-29%), hyperthyroxinemia (59%), hypercorticosteronemia (60%), and higher adrenal catecholamine content (+58%). In adulthood, SE offspring showed higher food intake (+10%), body total fat mass (+50%), visceral fat mass (retroperitoneal: 55%; mesenteric: 67%; and epididymal: 55%), and lower subcutaneous adipocyte area (24%) with higher serum glucose (11%), leptin (85%), adiponectin (1.4-fold increase), total triiodothyronine (71%), free thyroxine (57%), TSH (36%), triglycerides (65%), VLDL cholesterol (+66%), and HDL cholesterol (91%) levels and lower corticosteronemia (41%) and adrenal catecholamine content (57%). Our present findings suggest that tobacco SE to both dams and their pups during lactation causes malnutrition in early life that programs for obesity and hormonal and metabolic disturbances in adulthood, only if the pups are submitted to the same smoke environment as the mother.

Neonatal nicotine exposure alters leptin signaling in the hypothalamus-pituitary-thyroid axis in the late postnatal period and adulthood in rats.

AIMS Postnatal nicotine exposure causes precocious primary hypothyroidism and programs for overweight, hyperleptinemia and secondary hypothyroidism in adulthood. As leptin and thyroid hormones share the ability to increase energy expenditure, we studied the effects of maternal nicotine exposure during lactation on the leptin signaling in the hypothalamus-pituitary-thyroid axis of suckling and adult offspring. MAIN METHODS Two days after delivery, osmotic minipumps were implanted in lactating rats, and nicotine (NIC, 6 mg/kg/day s.c.) or saline (C) was administered for 14days. Offspring were killed at 15 and 180 days-old. Proteins belonging to leptin signaling were analyzed by Western blot. Significant differences had p<0.05. KEY FINDINGS In the hypothalamus, NIC offspring showed higher OB-R and pSTAT-3 content (+58%,+1.34x) at 15 days, and lower OB-R, JAK-2 and pSTAT-3 (-61%, -42%, -56%) at 180 days. In the pituitary gland, NIC offspring showed lower JAK-2 content (-52%) at 15 days, but no differences in adulthood. In the thyroid gland, the NIC group presented lower OB-R, JAK-2 and STAT-3 (-44%, -50%, -47%) and higher pSTAT-3 expression (+80%) at 15 days. At 180 days-old, NIC offspring presented higher thyroid OB-R (+1.54x) and lower pSTAT-3 content (-34%). SIGNIFICANCE Neonatal primary hypothyroidism induced by maternal nicotine exposure during lactation may be partially explained by decreased leptin signaling in the thyroid, though the early stimulation of the central leptin pathway did not prevent the thyroid dysfunction. Long-term effects of postnatal nicotine exposure on leptin signaling in the hypothalamus and thyroid appear to involve central and peripheral leptin resistance in adulthood.

Programming of rat adrenal medulla by neonatal hyperleptinemia: adrenal morphology, catecholamine secretion, and leptin signaling pathway

Leptin serum concentration in early life is an important factor for adequate future development of the offspring. Previously, we demonstrated that hyperleptinemia on lactation programmed for hyperleptinemia, central leptin resistance with lower expression of the long form of leptin receptor at hypothalamus, and higher medullary catecholamine levels with cardiovascular consequences at adulthood. The central objective of this study was to determine the direct effect of leptin on adrenal medullary function of adult rats that were leptin treated during lactation. Adrenal morphology was also accessed. Recombinant murine leptin was injected in the pups during the first 10 days of life (group L, leptin-programmed) or at adulthood during 6 days (group LC). The controls of both experiments received saline (groups C and CC). Both treatments resulted in hyperleptinemia at 150 days old (+78% and 2-fold increase, respectively; P < 0.05). Programmed animals showed hypertrophy of adrenal and higher adrenal catecholamine content at 150 days old (3-fold increase, P < 0.05), and no changes were observed in the LC group. However, LC rats had lower adrenal content of tyrosine hydroxylase (-17%, P < 0.05). Leptin-programmed rats had a lower response to leptin in vitro stimulation (-22%, P < 0.05) and lower expression of key proteins of the leptin signaling pathway, leptin receptor and janus tyrosine kinase 2 in the medullas (-61% and -29%, respectively, P < 0.05). However, they presented higher expression of phosphorylated signal transducer and activator of transcription 3 (+2-fold, P < 0.05). Leptin treatment at adulthood did not affect these parameters. The higher catecholamine synthesis and secretion in the leptin-programmed rats observed in our previous study does not seem to be a consequence of the direct effect of leptin on the medullas. We suggest that the hyperleptinemia of the programmed animals increases adrenal medullary function through sympathetic nervous system activation. In conclusion, high leptin levels on lactation program the activity of the sympathoadrenal system at adulthood that may contribute to the development of adult chronic diseases such as hypertension.

Maternal nicotine exposure during lactation alters food preference, anxiety-like behavior and the brain dopaminergic reward system in the adult rat offspring

The mesolimbic reward pathway is activated by drugs of abuse and palatable food, causing a sense of pleasure, which promotes further consumption of these substances. Children whose parents smoke are more vulnerable to present addictive-like behavior to drugs and food.We evaluated the association between maternal nicotine exposure during lactation with changes in feeding, behavior and in the dopaminergic reward system. On postnatal day (PN) 2,Wistar rat dams were implanted with minipumps releasing nicotine (N; 6 mg/kg/day, s.c.) or saline (C) for 14 days. On PN150 and PN160, offspring were divided into 4 groups for a food challenge: N and C that received standard chow(SC); and N and C that could freely self-select (SSD) between high-fat and high-sugar diets (HFD and HSD, respectively). Offspring were tested in the elevated plus maze (EPM) and open field (OF) arena on PN152–153. On PN170, offspring were euthanized for central dopaminergic analysis. SSD animals showed an increased food intake compared to SC ones and a preference for HFD. However, N-SSD animals consumed relatively more HSD than C-SSD ones. Regarding behavior, N animals showed an increase in the time spent in the EPM center and a reduction in relative activity in the OF center. N offspring presented lower dopamine receptor (D2R) and transporter (DAT) contents in the nucleus accumbens, and lower D2R in the arcuate nucleus. Postnatal exposure to nicotine increases preference for sugar and anxiety levels in the adult progeny possibly due to a decrease in dopaminergic action in the nucleus accumbens and arcuate nucleus.

Concurrent maternal and pup postnatal tobacco smoke exposure in Wistar rats changes food preference and dopaminergic reward system parameters in the adult male offspring.

Children from pregnant smokers are more susceptible to become obese adults and to become drug or food addicts. Drugs and food activate the mesolimbic reward pathway, causing a sense of pleasure that induces further consumption. Here, we studied the relationship between tobacco smoke exposure during lactation with feeding, behavior and brain dopaminergic reward system parameters at adulthood. Nursing Wistar rats and their pups were divided into two groups: tobacco smoke-exposed (S: 4times/day, from the 3rd to the 21th day of lactation), and ambient air-exposed (C). On PN175, both offspring groups were subdivided for a food challenge: S and C that received standard chow (SC) or that chose between high-fat (HFD) and high-sucrose diets (HSDs). Food intake was recorded after 30min and 12h. Offspring were tested in the elevated plus maze and open field on PN178-179; they were euthanized for dopaminergic analysis on PN180. SSD (self-selected diet) animals presented a higher food intake compared to SC ones. S-SSD animals ate more than C-SSD ones at 30min and 12h. Both groups preferred the HFD. However, S-SSD animals consumed relatively more HFD than C-SSD at 30min. No behavioral differences were observed between groups. S animals presented lower tyrosine hydroxylase (TH) content in the ventral tegmental area, lower TH, dopaminergic receptor 2, higher dopaminergic receptor 1 contents in the nucleus accumbens and lower OBRb in hypothalamic arcuate nucleus. Tobacco-smoke exposure during lactation increases preference for fat in the adult progeny possibly due to alterations in the dopaminergic system.

Developmental plasticity of endocrine disorders in obesity model primed by early weaning in dams.

Early weaning is associated with changes in the developmental plasticity. Here, we studied the adipocytes morphology, adipokines expression or content in adipose tissue as well as adrenal and thyroid function of neonate and adult offspring primed by early weaning. After birth, lactating rats were divided into 2 groups: EW (early weaning)--dams were wrapped with a bandage to block access to milk during the last 3 days of lactation, and Control--dams whose pups had free access to milk throughout lactation (21 days). At postnatal day (PN) 21, EW pups had lower visceral and subcutaneous adipocyte area (-67.7% and -62%, respectively), body fat mass (-26%), and leptin expression in visceral adipocyte (-64%) but higher leptin expression in subcutaneous adipocyte (2.9-fold increase). Adrenal evaluations were normal, but neonate EW pups presented lower serum T3 (-55%) and TSH (-44%). At PN 180, EW offspring showed higher food intake, higher body fat mass (+21.6%), visceral and subcutaneous adipocyte area (both 3-fold increase), higher leptin (+95%) and ADRβ3 (2-fold increase) content in visceral adipose tissue, and higher adiponectin expression in subcutaneous adipose tissue (+47%) but lower in visceral adipose tissue (-40%). Adult EW offspring presented higher adrenal catecholamine content (+31%), but no changes in serum corticosterone or thyroid status. Thus, early weaning primed for hypothyroidism at weaning, which can be associated with the adipocyte hypertrophy at adulthood. The marked changes in catecholamine adrenal content and visceral adipocyte ADRB3 are generally found in obesity, contributing to the development of other cardiovascular and metabolic disturbances.

Neonatal hypothyroidism caused by maternal nicotine exposure is reversed by higher T3 transfer by milk after nicotine withdraw

Maternal nicotine exposure leads to neonatal hypothyroidism that can be returned to euthyroidism after nicotine withdrawal. Here, we examined the transfer of iodine through milk, deiodinase activities (D1 and D2), and serum T3, T4 and TSH in rat offspring after maternal exposure to nicotine. One day after birth, a minipump was implanted to dams releasing nicotine (NIC), 6 mg/kg/day for 13 days or vehicle saline. Animals were killed at the day 15 and 21 of lactation. At day 15, NIC-treated dams showed decreased T4 and mammary 2h-radioiodine uptake (RAIU) and increase of TSH, thyroid 2h-RAIU, liver D1 and mammary D2. At the cessation of NIC-exposure, pups displayed decreased T3, T4 and thyroid 2h-RAIU and increased TSH. At weaning (21-postnatal day), NIC-treated dams recovered their T4 and TSH, but increased deiodinase level in the liver and mammary gland. Milk T3 content in NIC-treated dams was higher at both day 15 and 21, and thyroid function was recovered at the day 21. Thus, thyroid function was affected by nicotine in both mothers and pups, suggesting a primary hypothyroidism. After nicotine withdrawal, pups recovered thyroid function probably due to the increased lactational transfer of T3 in relation with increased mammary gland deiodinase activities.

Maternal nicotine exposure during lactation alters hypothalamic neuropeptides expression in the adult rat progeny

Maternal exposure to nicotine during lactation causes hyperleptinemia in the pups and, at adulthood, these animals are overweight and hyperleptinemic, while, in their hypothalamus, the leptin signaling pathway is reduced, evidencing a central leptin resistance. Then, we evaluated the expression of pro-opiomelanocortin (POMC), alpha-melanocyte stimulating hormone (α-MSH), cocaine and amphetamine-regulated transcript (CART), neuropeptide Y (NPY), agouti-related peptide (AgRP) and others in different hypothalamic nuclei in order to better understand the mechanisms underlying the obese phenotype observed in these animals at adulthood. On the 2nd postnatal day (P2), dams were subcutaneously implanted with osmotic minipumps releasing nicotine (NIC-6 mg/kg/day) or saline for 14 days. Offspring were killed in P180 and immunohistochemistry and Western blot analysis were carried out. Significance data had p<0.05. Adult NIC offspring showed more intense NPY staining in the paraventricular nucleus (PVN) (+21%) and increased number of POMC-positive cells in the: arcuate nucleus (+33%), as an increase in fiber density of α-MSH in PVN (+85%). However, the number of CART-positive cells was reduced in the PVN (-25%). CRH staining was more intense in NIC offspring (+136%). Orexins and AgRP were not altered. Thus, maternal nicotine exposure changes hypothalamic neuropeptides in the adult progeny that is partially compatible with leptin resistance.