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Epigenomic and metabolic responses of hypothalamic POMC neurons to gestational nicotine exposure in adult offspring

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epidemiological and animal studies have reported that prenatal nicotine exposure (PNE) leads to obesity and type-2 diabetes in offspring. Central leptin-melanocortin signaling via hypothalamic arcuate proopiomelanocortin (POMC) neurons is crucial for the regulation of energy and glucose balance. Furthermore, hypothalamic POMC neurons were recently found to mediate the anorectic effects of nicotine through activation of acetylcholine receptors. Here, we hypothesized that PNE impairs leptin-melanocortinergic regulation of energy balance in first-generation offspring by altering expression of long non-coding RNAs (lncRNAs) putatively regulating development and/or function of hypothalamic POMC neurons.

Methods: C57BL/6J females were exposed ad libitum to nicotine through drinking water and crossed with C57BL/6J males. Nicotine exposure was sustained during pregnancy and discontinued at parturition. Offspring development was monitored from birth into adulthood. From the age of 8 weeks, central leptin-melanocortin signaling, diabetes, and obesity susceptibility were assessed in male offspring fed a low-fat or high-fat diet for 16 weeks. Nicotine-exposed and non-exposed C57BL/6J females were also crossed with C57BL/6J males expressing the enhanced green fluorescent protein specifically in POMC neurons. Transgenic male offspring were subjected to laser microdissections and RNA sequencing (RNA-seq) analysis of POMC neurons for determination of nicotine-induced gene expression changes and regulatory lncRNA/protein-coding gene interactions.

Results: Contrary to expectation based on previous studies, PNE did not impair but rather enhanced leptin-melanocortinergic regulation of energy and glucose balance via POMC neurons in offspring. RNA-seq of laser microdissected POMC neurons revealed only one consistent change, upregulation of Gm15851, a lncRNA of yet unidentified function, in nicotine-exposed offspring. RNA-seq further suggested 82 cis-regulatory lncRNA/protein-coding gene interactions, 19 of which involved coding genes regulating neural development and/or function, and revealed expression of several previously unidentified metabolic, neuroendocrine, and neurodevelopment pathways in POMC neurons.

Conclusions: PNE does not result in obesity and type 2 diabetes but instead enhances leptin-melanocortinergic feeding and body weight regulation via POMC neurons in adult offspring. PNE leads to selective upregulation of Gm15851, a lncRNA, in adult offspring POMC neurons. POMC neurons express several lncRNAs and pathways possibly regulating POMC neuronal development and/or function.

Electronic supplementary material: The online version of this article (doi:10.1186/s13073-016-0348-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Ad libitum nicotine intake led to high plasma cotinine levels, moderately reduced fluid intake, and did not alter body weight or food consumption of dams. a Twenty-four-hour fluid intake was determined in group-housed females (n = 4 cages of 3 females/group) before pregnancy and in individually housed females (n = 10/group) during gestation. b Nicotine intake before (n = 4) and during pregnancy (n = 10) was calculated based on 24-h fluid intake. c Plasma cotinine levels were determined by enzyme-linked immunosorbent assay (ELISA) after 4 weeks of nicotine ingestion. Plasma cotinine was detected in all nicotine-exposed females (n = 6) and was undetectable in non-exposed females (n = 6). d Twenty-four-hour food intake was determined in group-housed females (n = 4 cages of 3 females/group) before pregnancy. e Body weight was determined weekly before pregnancy (n = 12/group). Data were analyzed by two-way RM ANOVA followed by Bonferroni post-test (a) or by Mann–Whitney test (c) (**, p < 0.01). All data are expressed as mean ± SEM
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Fig1: Ad libitum nicotine intake led to high plasma cotinine levels, moderately reduced fluid intake, and did not alter body weight or food consumption of dams. a Twenty-four-hour fluid intake was determined in group-housed females (n = 4 cages of 3 females/group) before pregnancy and in individually housed females (n = 10/group) during gestation. b Nicotine intake before (n = 4) and during pregnancy (n = 10) was calculated based on 24-h fluid intake. c Plasma cotinine levels were determined by enzyme-linked immunosorbent assay (ELISA) after 4 weeks of nicotine ingestion. Plasma cotinine was detected in all nicotine-exposed females (n = 6) and was undetectable in non-exposed females (n = 6). d Twenty-four-hour food intake was determined in group-housed females (n = 4 cages of 3 females/group) before pregnancy. e Body weight was determined weekly before pregnancy (n = 12/group). Data were analyzed by two-way RM ANOVA followed by Bonferroni post-test (a) or by Mann–Whitney test (c) (**, p < 0.01). All data are expressed as mean ± SEM

Mentions: C57BL/6J females were exposed to nicotine ad libitum through drinking water containing nicotine hydrogen tartrate salt at a concentration of 200 μg/mL. Controls received drinking water containing the equivalent amount of pH-matched tartaric acid. Nicotine exposure started at the age of 6 weeks, continued throughout mating at the age of 10 weeks and gestation, and ended at parturition. Since nicotine causes taste aversion [36, 37], the drinking water of both, nicotine-exposed and non-exposed dams was sweetened with 2 % (w/v) saccharin [35]. Nicotine moderately reduced weekly fluid volume intake before and during gestation (Pre-Gestation: F1,12 = 5.41, p = 0.06; Gestation: F1,36 = 23.4, p = 0.0001; RM ANOVA) (Fig. 1a). The mean daily water intake of nicotine-exposed females (≈2 mL/10 g body weight/24 h at 6–8 weeks of age) remained moderately above the mean values reported for wild-type mice of unspecified sex, strain, and age (1.5 mL/10 g body weight/24 h) and those reported for C57BL/6 females aged 7–9 weeks (1.64 mL/10 g body weight/24 h, Mouse Phenome Data Base, The Jackson Laboratory). Mean daily nicotine ingestion calculated based on fluid intake was 0.77 ± 0.03 mg/day (n = 4) before pregnancy and 0.92 ± 0.03 mg/day (n = 10) during pregnancy (Fig. 1b). Moreover, the plasma levels of cotinine, a metabolite of nicotine, and indicator of tobacco smoke exposure in humans [47] determined after 4 weeks of ad libitum nicotine intake were in the range of 137.4–385.1 ng/mL (mean value: 245 ± 0.03 ng/mL; n = 6) (Fig. 1c). These plasma cotinine values are within the range reported in humans who smoke 15–24 cigarettes/day and > 25 cigarettes/day and exhibit serum cotinine concentrations of 230–280 ng/mL and 260–300 ng/mL, respectively [48]. Plasma cotinine was undetectable in age-matched non-exposed dams. Ad libitum nicotine ingestion had no significant impact on maternal food intake (F1,12 = 3.04, p = 0.13; RM ANOVA) (Fig. 1d) or maternal body weight (F1,44 = 3.4, p = 0.08; RM ANOVA) (Fig. 1e) as reported for the C57BL/6 mouse strain [35]. Dams were allowed only one pregnancy. Nicotine-exposed and non-exposed litters displayed no significant difference in mean litter size at postnatal day (PD)1 (PNE: 6.7 ± 0.37; control: 6.1 ± 0.3; n = 10 litters/group; t18 = 1.24, p = 0.23; t test). Postnatal survival of PNE offspring tended to be more compromised than that of control offspring (62.7 % versus 73.8 %) by PD21 but contingency analysis of alive and dead PD21 offspring revealed no significant differences between groups (p = 0.19 by two-sided Fisher’s exact test) and litter sizes were the same in both groups by PD21 (PNE: 4.2 ± 1.02; control: 4.5 ± 0.8; n = 10 litters/group; t18 = 0.22, p = 0.82; t test). However, PNE led to moderate decreases in mean body weight (PNE: 6.55 ± 0.23 g; control: 7.16 ± 0.14 g; n = 42–45/group; p = 0.027; Mann–Whitney test) and mean crown-rump length (PNE: 5.77 ± 0.12 cm; control: 6.15 ± 0.45 cm; n = 42–45/group; p = 0.037; Mann–Whitney test) of PD21 offspring. Therefore, to exclude differences in maternal care as a confounding factor in the assessment of the long-term metabolic consequences of PNE, we selected for the metabolic studies six nicotine-exposed litters and seven non-exposed litters showing similar postnatal survival rates (PNE: 85.7 %; control: 88.4 %), and without differences at PD21 in mean litter size (PNE: 5.7 ± 0.7; control: 5.4 ± 0.5; t11 = 0.28, p = 0.78; t test), mean body weight (PNE: 7.19 ± 0.12 g; control: 7.15 ± 0.15 g; n = 34–38/group; p = 0.47; Mann–Whitney test), and mean crown-rump length (PNE: 6.12 ± 0.06 cm; control: 6.16 ± 0.08 cm; n = 34-38/group; p = 0.41; Mann–Whitney test).Fig. 1


Epigenomic and metabolic responses of hypothalamic POMC neurons to gestational nicotine exposure in adult offspring
Ad libitum nicotine intake led to high plasma cotinine levels, moderately reduced fluid intake, and did not alter body weight or food consumption of dams. a Twenty-four-hour fluid intake was determined in group-housed females (n = 4 cages of 3 females/group) before pregnancy and in individually housed females (n = 10/group) during gestation. b Nicotine intake before (n = 4) and during pregnancy (n = 10) was calculated based on 24-h fluid intake. c Plasma cotinine levels were determined by enzyme-linked immunosorbent assay (ELISA) after 4 weeks of nicotine ingestion. Plasma cotinine was detected in all nicotine-exposed females (n = 6) and was undetectable in non-exposed females (n = 6). d Twenty-four-hour food intake was determined in group-housed females (n = 4 cages of 3 females/group) before pregnancy. e Body weight was determined weekly before pregnancy (n = 12/group). Data were analyzed by two-way RM ANOVA followed by Bonferroni post-test (a) or by Mann–Whitney test (c) (**, p < 0.01). All data are expressed as mean ± SEM
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5015242&req=5

Fig1: Ad libitum nicotine intake led to high plasma cotinine levels, moderately reduced fluid intake, and did not alter body weight or food consumption of dams. a Twenty-four-hour fluid intake was determined in group-housed females (n = 4 cages of 3 females/group) before pregnancy and in individually housed females (n = 10/group) during gestation. b Nicotine intake before (n = 4) and during pregnancy (n = 10) was calculated based on 24-h fluid intake. c Plasma cotinine levels were determined by enzyme-linked immunosorbent assay (ELISA) after 4 weeks of nicotine ingestion. Plasma cotinine was detected in all nicotine-exposed females (n = 6) and was undetectable in non-exposed females (n = 6). d Twenty-four-hour food intake was determined in group-housed females (n = 4 cages of 3 females/group) before pregnancy. e Body weight was determined weekly before pregnancy (n = 12/group). Data were analyzed by two-way RM ANOVA followed by Bonferroni post-test (a) or by Mann–Whitney test (c) (**, p < 0.01). All data are expressed as mean ± SEM
Mentions: C57BL/6J females were exposed to nicotine ad libitum through drinking water containing nicotine hydrogen tartrate salt at a concentration of 200 μg/mL. Controls received drinking water containing the equivalent amount of pH-matched tartaric acid. Nicotine exposure started at the age of 6 weeks, continued throughout mating at the age of 10 weeks and gestation, and ended at parturition. Since nicotine causes taste aversion [36, 37], the drinking water of both, nicotine-exposed and non-exposed dams was sweetened with 2 % (w/v) saccharin [35]. Nicotine moderately reduced weekly fluid volume intake before and during gestation (Pre-Gestation: F1,12 = 5.41, p = 0.06; Gestation: F1,36 = 23.4, p = 0.0001; RM ANOVA) (Fig. 1a). The mean daily water intake of nicotine-exposed females (≈2 mL/10 g body weight/24 h at 6–8 weeks of age) remained moderately above the mean values reported for wild-type mice of unspecified sex, strain, and age (1.5 mL/10 g body weight/24 h) and those reported for C57BL/6 females aged 7–9 weeks (1.64 mL/10 g body weight/24 h, Mouse Phenome Data Base, The Jackson Laboratory). Mean daily nicotine ingestion calculated based on fluid intake was 0.77 ± 0.03 mg/day (n = 4) before pregnancy and 0.92 ± 0.03 mg/day (n = 10) during pregnancy (Fig. 1b). Moreover, the plasma levels of cotinine, a metabolite of nicotine, and indicator of tobacco smoke exposure in humans [47] determined after 4 weeks of ad libitum nicotine intake were in the range of 137.4–385.1 ng/mL (mean value: 245 ± 0.03 ng/mL; n = 6) (Fig. 1c). These plasma cotinine values are within the range reported in humans who smoke 15–24 cigarettes/day and > 25 cigarettes/day and exhibit serum cotinine concentrations of 230–280 ng/mL and 260–300 ng/mL, respectively [48]. Plasma cotinine was undetectable in age-matched non-exposed dams. Ad libitum nicotine ingestion had no significant impact on maternal food intake (F1,12 = 3.04, p = 0.13; RM ANOVA) (Fig. 1d) or maternal body weight (F1,44 = 3.4, p = 0.08; RM ANOVA) (Fig. 1e) as reported for the C57BL/6 mouse strain [35]. Dams were allowed only one pregnancy. Nicotine-exposed and non-exposed litters displayed no significant difference in mean litter size at postnatal day (PD)1 (PNE: 6.7 ± 0.37; control: 6.1 ± 0.3; n = 10 litters/group; t18 = 1.24, p = 0.23; t test). Postnatal survival of PNE offspring tended to be more compromised than that of control offspring (62.7 % versus 73.8 %) by PD21 but contingency analysis of alive and dead PD21 offspring revealed no significant differences between groups (p = 0.19 by two-sided Fisher’s exact test) and litter sizes were the same in both groups by PD21 (PNE: 4.2 ± 1.02; control: 4.5 ± 0.8; n = 10 litters/group; t18 = 0.22, p = 0.82; t test). However, PNE led to moderate decreases in mean body weight (PNE: 6.55 ± 0.23 g; control: 7.16 ± 0.14 g; n = 42–45/group; p = 0.027; Mann–Whitney test) and mean crown-rump length (PNE: 5.77 ± 0.12 cm; control: 6.15 ± 0.45 cm; n = 42–45/group; p = 0.037; Mann–Whitney test) of PD21 offspring. Therefore, to exclude differences in maternal care as a confounding factor in the assessment of the long-term metabolic consequences of PNE, we selected for the metabolic studies six nicotine-exposed litters and seven non-exposed litters showing similar postnatal survival rates (PNE: 85.7 %; control: 88.4 %), and without differences at PD21 in mean litter size (PNE: 5.7 ± 0.7; control: 5.4 ± 0.5; t11 = 0.28, p = 0.78; t test), mean body weight (PNE: 7.19 ± 0.12 g; control: 7.15 ± 0.15 g; n = 34–38/group; p = 0.47; Mann–Whitney test), and mean crown-rump length (PNE: 6.12 ± 0.06 cm; control: 6.16 ± 0.08 cm; n = 34-38/group; p = 0.41; Mann–Whitney test).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epidemiological and animal studies have reported that prenatal nicotine exposure (PNE) leads to obesity and type-2 diabetes in offspring. Central leptin-melanocortin signaling via hypothalamic arcuate proopiomelanocortin (POMC) neurons is crucial for the regulation of energy and glucose balance. Furthermore, hypothalamic POMC neurons were recently found to mediate the anorectic effects of nicotine through activation of acetylcholine receptors. Here, we hypothesized that PNE impairs leptin-melanocortinergic regulation of energy balance in first-generation offspring by altering expression of long non-coding RNAs (lncRNAs) putatively regulating development and/or function of hypothalamic POMC neurons.

Methods: C57BL/6J females were exposed ad libitum to nicotine through drinking water and crossed with C57BL/6J males. Nicotine exposure was sustained during pregnancy and discontinued at parturition. Offspring development was monitored from birth into adulthood. From the age of 8&nbsp;weeks, central leptin-melanocortin signaling, diabetes, and obesity susceptibility were assessed in male offspring fed a low-fat or high-fat diet for 16&nbsp;weeks. Nicotine-exposed and non-exposed C57BL/6J females were also crossed with C57BL/6J males expressing the enhanced green fluorescent protein specifically in POMC neurons. Transgenic male offspring were subjected to laser microdissections and RNA sequencing (RNA-seq) analysis of POMC neurons for determination of nicotine-induced gene expression changes and regulatory lncRNA/protein-coding gene interactions.

Results: Contrary to expectation based on previous studies, PNE did not impair but rather enhanced leptin-melanocortinergic regulation of energy and glucose balance via POMC neurons in offspring. RNA-seq of laser microdissected POMC neurons revealed only one consistent change, upregulation of Gm15851, a lncRNA of yet unidentified function, in nicotine-exposed offspring. RNA-seq further suggested 82 cis-regulatory lncRNA/protein-coding gene interactions, 19 of which involved coding genes regulating neural development and/or function, and revealed expression of several previously unidentified metabolic, neuroendocrine, and neurodevelopment pathways in POMC neurons.

Conclusions: PNE does not result in obesity and type 2 diabetes but instead enhances leptin-melanocortinergic feeding and body weight regulation via POMC neurons in adult offspring. PNE leads to selective upregulation of Gm15851, a lncRNA, in adult offspring POMC neurons. POMC neurons express several lncRNAs and pathways possibly regulating POMC neuronal development and/or function.

Electronic supplementary material: The online version of this article (doi:10.1186/s13073-016-0348-2) contains supplementary material, which is available to authorized users.

No MeSH data available.