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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.


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Composition of the transcriptome of hypothalamic POMC neurons by total RNA (a), ncRNA (b), lncRNA (c), short ncRNA (d), antisense RNA (e), and sense RNA (complementary to antisense RNA) (f). Only RNAs expressed at levels > 1 CPM were considered for analyses
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Fig5: Composition of the transcriptome of hypothalamic POMC neurons by total RNA (a), ncRNA (b), lncRNA (c), short ncRNA (d), antisense RNA (e), and sense RNA (complementary to antisense RNA) (f). Only RNAs expressed at levels > 1 CPM were considered for analyses

Mentions: Each library yielded on average 42.9 million read-pairs passing Illumina’s quality control filters (PNE: 46.3 million; control: 38.8 million). On average, 30 million or 70.5 % read-pairs per library were aligned to unique sites of the reference genome (PNE: 32.7 million or 71.3 %; Control: 26.8 million or 69.4 %) (Table 2). Read-pairs mapped to 16,014 genes with average expression levels > 1 CPM (Additional file 1: Table S1). Of these, 13,539 (84.5 %) were protein-coding genes, 1708 (10.7 %) were ncRNA genes, and 767 (4.8 %) were pseudogenes (Fig. 5a). NcRNA genes comprised 1124 (65.8 %) lncRNA genes, 265 (15.5 %) short ncRNA genes, and 319 (18.7 %) processed transcripts (Fig. 5b). Mapped lncRNA genes comprised 509 (45.3 %) lincRNA genes, 551 (49 %) antisense RNA genes, 59 (5.2 %) sense-intronic RNA genes, four (0.4 %) sense-overlapping RNA genes, and one (0.1 %) 3′-overlapping ncRNA gene (Fig. 5c). Mapped short ncRNA genes were composed of 116 (43.8 %) microRNA (miRNA) genes, 81 (30.6 %) small nucleolar RNA (snoRNAs) genes, 20 (7.5 %) small nuclear RNA (snRNA) genes, ten (3.8 %) rRNA genes, and 38 (14.3 %) miscellaneous RNA (miscRNA) genes (Fig. 5d). The fraction of expressed short ncRNA genes is likely underestimated because the RNA isolation procedure excluded RNAs smaller than 100 nucleotides. Antisense RNA genes expressed at levels > 1 CPM were almost exclusively non-coding (551 or 99.8 %) and overlapped 529 (87.4 %) protein-coding genes, 61 (10.1 %) ncRNA genes, and 15 (2.5 %) pseudogenes (Fig. 5f).Table 2


Epigenomic and metabolic responses of hypothalamic POMC neurons to gestational nicotine exposure in adult offspring
Composition of the transcriptome of hypothalamic POMC neurons by total RNA (a), ncRNA (b), lncRNA (c), short ncRNA (d), antisense RNA (e), and sense RNA (complementary to antisense RNA) (f). Only RNAs expressed at levels > 1 CPM were considered for analyses
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5015242&req=5

Fig5: Composition of the transcriptome of hypothalamic POMC neurons by total RNA (a), ncRNA (b), lncRNA (c), short ncRNA (d), antisense RNA (e), and sense RNA (complementary to antisense RNA) (f). Only RNAs expressed at levels > 1 CPM were considered for analyses
Mentions: Each library yielded on average 42.9 million read-pairs passing Illumina’s quality control filters (PNE: 46.3 million; control: 38.8 million). On average, 30 million or 70.5 % read-pairs per library were aligned to unique sites of the reference genome (PNE: 32.7 million or 71.3 %; Control: 26.8 million or 69.4 %) (Table 2). Read-pairs mapped to 16,014 genes with average expression levels > 1 CPM (Additional file 1: Table S1). Of these, 13,539 (84.5 %) were protein-coding genes, 1708 (10.7 %) were ncRNA genes, and 767 (4.8 %) were pseudogenes (Fig. 5a). NcRNA genes comprised 1124 (65.8 %) lncRNA genes, 265 (15.5 %) short ncRNA genes, and 319 (18.7 %) processed transcripts (Fig. 5b). Mapped lncRNA genes comprised 509 (45.3 %) lincRNA genes, 551 (49 %) antisense RNA genes, 59 (5.2 %) sense-intronic RNA genes, four (0.4 %) sense-overlapping RNA genes, and one (0.1 %) 3′-overlapping ncRNA gene (Fig. 5c). Mapped short ncRNA genes were composed of 116 (43.8 %) microRNA (miRNA) genes, 81 (30.6 %) small nucleolar RNA (snoRNAs) genes, 20 (7.5 %) small nuclear RNA (snRNA) genes, ten (3.8 %) rRNA genes, and 38 (14.3 %) miscellaneous RNA (miscRNA) genes (Fig. 5d). The fraction of expressed short ncRNA genes is likely underestimated because the RNA isolation procedure excluded RNAs smaller than 100 nucleotides. Antisense RNA genes expressed at levels > 1 CPM were almost exclusively non-coding (551 or 99.8 %) and overlapped 529 (87.4 %) protein-coding genes, 61 (10.1 %) ncRNA genes, and 15 (2.5 %) pseudogenes (Fig. 5f).Table 2

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.


Related in: MedlinePlus