Conditional postnatal deletion of the neonatal murine hepatic circadian gene, Npas2, alters the gut microbiome following restricted feeding.

BACKGROUND: We have recently shown in both non-human primates and in rodents that fetal and neonatal hepatic expression of the circadian transcription factor, Npas2, is modulated by a high fat maternal diet and plays a critical role in establishing life-long metabolic homeostasis. Similarly, we and others have also established the importance of the maternal and early postnatal diet on establishment of the early gut microbiome. OBJECTIVE: We hypothesized that altered circadian gene expression solely in the neonatal liver would result in gut microbiome dysbiosis, especially with diet-induced metabolic stress (ie, restricted feeding). Using a murine model in which we conditionally knock out Npas2 in the neonatal liver, we aimed to determine the role of the circadian machinery in gut dysbiosis with restricted feeding. STUDY DESIGN: We collected fecal samples from liver Npas2 conditional knockout (n = 11) and wild-type (n = 13) reproductive-aged mice before (study day 0) and after the restricted feeding study (study day 17). Extracted DNA was sequenced using the MiSeq Illumina platform using primers specific for the V4 region of the 16S ribosomal DNA gene. The resulting sequences were quality filtered, aligned, and assigned taxonomy. Principal coordinate analysis was performed on unweighted and weighted UniFrac distances between samples with a permutation analysis of variance to assess clustering significance between groups. Microbial taxa that significantly differ between groups of interest was determined using linear discriminate analysis effect size and randomForrest. RESULTS: Principal coordinate analysis performed on weighted UniFrac distances between male conditional knockout and wild-type cohorts revealed that the gut microbiome of the mice did not differ by genotype at the start of the restricted feeding study but did differ by virtue of genotype at the end of the study (P = .001). Moreover, these differences could be at least partially attributed to restricted feeding-associated alterations in relative abundance of the Bacteroides genus, which has been implicated as crucial to establishing a healthy gut microbiome early in development. CONCLUSION: Here we have provided an initial key insight into the interplay between neonatal establishment of the peripheral circadian clock in the liver and the ability of the gut microbiome to respond to dietary and metabolic stress. Because Npas2 expression in the liver is a target of maternal high-fat diet-induced metabolic perturbations during fetal development, we speculate that these findings have potential implications in the long-term metabolic health of their offspring.

Heritable IUGR and adult metabolic syndrome are reversible and associated with alterations in the metabolome following dietary supplementation of 1-carbon intermediates.

Metabolic syndrome (MetS), following intrauterine growth restriction (IUGR), is epigenetically heritable. Recently, we abrogated the F2 adult phenotype with essential nutrient supplementation (ENS) of intermediates along the 1-carbon pathway. With the use of the same grandparental uterine artery ligation model, we profiled the F2 serum metabolome at weaning [postnatal day (d)21; n = 76] and adulthood (d160; n = 12) to test if MetS is preceded by alterations in the metabolome. Indicative of developmentally programmed MetS, adult F2, formerly IUGR rats, were obese (621 vs. 461 g; P < 0.0001), dyslipidemic (133 vs. 67 mg/dl; P < 0.001), and glucose intolerant (26 vs. 15 mg/kg/min; P < 0.01). Unbiased gas chromatography-mass spectrometry (GC-MS) profiling revealed 34 peaks corresponding to 12 nonredundant metabolites and 9 unknowns to be changing at weaning [false discovery rate (FDR) < 0.05]. Markers of later-in-life MetS included citric acid, glucosamine, myoinositol, and proline (P < 0.03). Hierarchical clustering revealed grouping by IUGR lineage and supplementation at d21 and d160. Weanlings grouped distinctly for ENS and IUGR by partial least-squares discriminate analysis (PLS-DA; P < 0.01), whereas paternal and maternal IUGR (IUGR(pat)/IUGR(mat), respectively) control-fed rats, destined for MetS, had a distinct metabolome at weaning (randomForest analysis; class error < 0.1) and adulthood (PLS-DA; P < 0.05). In sum, we have found that alterations in the metabolome accompany heritable IUGR, precede adult-onset MetS, and are partially amenable to dietary intervention.

Fetal hypoxia increases vulnerability of hypoxic-ischemic brain injury in neonatal rats: role of glucocorticoid receptors.

Gestational hypoxia is a common stress to the fetal development and increases the risk of neonatal morbidity. The present study tested the hypothesis that fetal hypoxia results in heightened brain vulnerability to hypoxic-ischemic (HI) injury in neonatal rats via down-regulation of glucocorticoid receptor (GR) in the developing brain. Time-dated pregnant rats were exposed to hypoxia (10.5% O2) from days 15 to 21 of gestation. Brain HI injury was determined in day 10 pups. Maternal hypoxia resulted in asymmetric intrauterine growth restriction in the fetus. The brain HI injury was significantly increased in maternal hypoxia-treated pups as compared with the normoxia control in both males and females. Activation of brain GR by dexamethasone injection into the right lateral ventricle produced a concentration-dependent reduction of HI-induced brain injury in control pups. Maternal hypoxia significantly decreased GR mRNA and protein abundance in the fetal brain and neonatal hippocampus and abolished the dexamethasone-mediated neuroprotective effect in pup brains. This decreased GR expression was resulted from increased DNA methylation, decreased binding of transcription factors Egr-1 and Sp1 to GR gene exon 17 and 111 promoters, and reduced expression of GR exon 17 and 111 mRNA variants. The results demonstrate that gestational hypoxia causes epigenetic repression of GR gene expression in the developing brain resulting in the heightened brain vulnerability to HI injury in neonatal rats.

Dexamethasone protects neonatal hypoxic-ischemic brain injury via L-PGDS-dependent PGD2-DP1-pERK signaling pathway.

BACKGROUND AND PURPOSE: Glucocorticoids pretreatment confers protection against neonatal hypoxic-ischemic (HI) brain injury. However, the molecular mechanism remains poorly elucidated. We tested the hypothesis that glucocorticoids protect against HI brain injury in neonatal rat by stimulation of lipocalin-type prostaglandin D synthase (L-PGDS)-induced prostaglandin D2 (PGD2)-DP1-pERK mediated signaling pathway. METHODS: Dexamethasone and inhibitors were administered via intracerebroventricular (i.c.v) injections into 10-day-old rat brains. Levels of L-PGD2, D prostanoid (DP1) receptor, pERK1/2 and PGD2 were determined by Western immunoblotting and ELISA, respectively. Brain injury was evaluated 48 hours after conduction of HI in 10-day-old rat pups. RESULTS: Dexamethasone pretreatment significantly upregulated L-PGDS expression and the biosynthesis of PGD2. Dexamethasone also selectively increased isoform pERK-44 level in the neonatal rat brains. Inhibitors of L-PGDS (SeCl4), DP1 (MK-0524) and MAPK (PD98059) abrogated dexamethasone-induced increases in pERK-44 level, respectively. Of importance, these inhibitors also blocked dexamethasone-mediated neuroprotective effects against HI brain injury in neonatal rat brains. CONCLUSION: Interaction of glucocorticoids-GR signaling and L-PGDS-PGD2-DP1-pERK mediated pathway underlies the neuroprotective effects of dexamethasone pretreatment in neonatal HI brain injury.