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.

Alterations in expression of imprinted genes from the H19/IGF2 loci in a multigenerational model of intrauterine growth restriction (IUGR).

BACKGROUND: The H19/IGF2 imprinted loci have attracted recent attention because of their role in cellular differentiation and proliferation, heritable gene regulation, and in utero or early postnatal growth and development. Expression from the imprinted H19/IGF2 locus involves a complex interplay of 3 means of epigenetic regulation: proper establishment of DNA methylation, promoter occupancy of CTCF, and expression of microRNA-675. We have demonstrated previously in a multigenerational rat model of intrauterine growth restriction the epigenetic heritability of adult metabolic syndrome in a F2 generation. We have further demonstrated abrogation of the F2 adult metabolic syndrome phenotype with essential nutrient supplementation of intermediates along the 1-carbon pathway and shown that alterations in the metabolome precede the adult onset of metabolic syndrome. The upstream molecular and epigenomic mediators underlying these observations, however, have yet to be elucidated fully. OBJECTIVE: In the current study, we sought to characterize the impact of the intrauterine growth-restricted lineage and essential nutrient supplementation on both levels and molecular mediators of H19 and IGF2 gene expression in the F2 generation. STUDY DESIGN: F2 intrauterine growth-restricted and sham lineages were obtained by exposing P1 (grandmaternal) pregnant dams to bilateral uterine artery ligation or sham surgery at gestational day 19.5. F1 pups were allocated to the essential nutrient supplemented or control diet at postnatal day 21, and bred at 6-7 weeks of age. Hepatic tissues from the resultant F2 offspring at birth and at weaning (day 21) were obtained. Bisulfite modification and sequencing was employed for methylation analysis. H19 and IGF2 expression was measured by quantitative polymerase chain reaction. Promoter occupancy was quantified by the use of chromatin immunoprecipitation, or ChIP, against CTCF insulator proteins. RESULTS: Growth-restricted F2 on control diet demonstrated significant down-regulation in H19 expression compared with sham lineage (0.7831 vs 1.287; P < .05); however, essential nutrient supplementation diet abrogates this difference (4.995 vs 5.100; P > .05). Conversely, Igf2 was up-regulated by essential nutrient supplemented diet on the sham lineage (2.0 fold, P = .01), an effect that was not observed in the growth restricted offspring. A significant differential methylation was observed in the promoter region of region H19 among the intrauterine growth-restricted lineage (18% vs 25%; P < .05) on a control diet, whereas the essential nutrient supplemented diet was alternately associated with hypermethylation in both lineages (sham: 50%; intrauterine growth restriction: 84%, P < .05). Consistent with essential nutrient supplementation impacting the epigenome, a decrease of CTCF promoter occupancy was observed in CTCF4 of the growth restricted lineage (2.45% vs 0.56%; P < .05) on the control diet, an effect that was repressed with essential nutrient supplementation. CONCLUSION: Heritable growth restriction is associated with changes in H19 gene expression; these changes are reversible with diet supplementation to favorably impact adult metabolic syndrome.

Keeping the rhythm: light/dark cycles during postharvest storage preserve the tissue integrity and nutritional content of leafy plants.

BACKGROUND: The modular body structure of plants enables detached plant organs, such as postharvest fruits and vegetables, to maintain active responsiveness to environmental stimuli, including daily cycles of light and darkness. Twenty-four hour light/darkness cycles entrain plant circadian clock rhythms, which provide advantage to plants. Here, we tested whether green leafy vegetables gain longevity advantage by being stored under light/dark cycles designed to maintain biological rhythms. RESULTS: Light/dark cycles during postharvest storage improved several aspects of plant tissue performance comparable to that provided by refrigeration. Tissue integrity, green coloration, and chlorophyll content were generally enhanced by cycling of light and darkness compared to constant light or darkness during storage. In addition, the levels of the phytonutrient glucosinolates in kale and cabbage remained at higher levels over time when the leaf tissue was stored under light/dark cycles. CONCLUSIONS: Maintenance of the daily cycling of light and dark periods during postharvest storage may slow the decline of plant tissues, such as green leafy vegetables, improving not only appearance but also the health value of the crops through the maintenance of chlorophyll and phytochemical content after harvest.

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.

Essential nutrient supplementation prevents heritable metabolic disease in multigenerational intrauterine growth-restricted rats.

Intrauterine growth restriction (IUGR) confers heritable alterations in DNA methylation, rendering risk of adult metabolic syndrome (MetS). Because CpG methylation is coupled to intake of essential nutrients along the one-carbon pathway, we reasoned that essential nutrient supplementation (ENS) may abrogate IUGR-conferred multigenerational MetS. Pregnant Sprague-Dawley rats underwent bilateral uterine artery ligation causing IUGR in F1. Among the F2 generation, IUGR lineage rats were underweight at birth (6.7 vs. 8.0 g, P < 0.0001) and obese by adulthood (p160: 613 vs. 510 g; P < 0.0001). Dual energy X-ray absorptiometry studies revealed increased central fat mass (Δ+40 g), accompanied by dyslipidemic (>30% elevated, P < 0.05) serum triglycerides (139 mg/dl), very-LDLs (27.8 mg/dl), and fatty acids (632 µM). Hyperglycemic-euglycemic clamp studies and glucose tolerance testing revealed insulin resistance. Conversely, IUGR lineage ENS-fed rats did not manifest MetS, with significantly lower body weight (p160: 410 g), >5-fold less central fat mass, normal hepatic glucose efflux, and >70% reduced circulating triglycerides and very-LDLs compared with IUGR control-fed F2 offspring (P < 0.01). Moreover, increased methylation of the IGF-1 P2 transcriptional start site among IUGR lineage F2 offspring was reversed in ENS (P < 0.04). This is an initial demonstration that supplementation along the one-carbon pathway abrogates adult morbidity and associated epigenomic modifications of IGF-1 in a rodent model of multigenerational MetS.

Postharvest circadian entrainment enhances crop pest resistance and phytochemical cycling.

The modular design of plants enables individual plant organs to manifest autonomous functions and continue aspects of metabolism, such as respiration, even after separation from the parent plant. Therefore, we hypothesized that harvested vegetables and fruits may retain capacity to perceive and respond to external stimuli. For example, the fitness advantage of plant circadian clock function is recognized; however, whether the clock continues to influence postharvest physiology is unclear. Here we demonstrate that the circadian clock of postharvest cabbage (Brassica oleracea) is entrainable by light-dark cycles and results in enhanced herbivore resistance. In addition, entrainment of Arabidopsis plants and postharvest cabbage causes cyclical accumulation of metabolites that function in plant defense; in edible crops, these metabolites also have potent anticancer properties. Finally, we show that the phenomena of postharvest entrainment and enhanced herbivore resistance are widespread among diverse crops. Therefore, sustained clock entrainment of postharvest crops may be a simple mechanism to promote pest resistance and nutritional value of plant-derived food.

Circadian control of jasmonates and salicylates: the clock role in plant defense.

Plants have evolved robust mechanisms to perceive and respond to diverse environmental stimuli.  The plant phytohormones jasmonates and salicylates play key roles in activating biotic stress response pathways. Recent findings demonstrate that basal levels of both jasmonates and salicylates in Arabidopsis are under the control of the circadian clock and that clock-controlled jasmonate accumulation may underlie clock- and jasmonate-dependent enhanced resistance of Arabidopsis to Trichoplusia ni (cabbage looper), a generalist herbivore. Here we summarize these findings and provide further evidence that a functional plant circadian clock is required for optimal herbivore defense in Arabidopsis.  When given a choice to feed on wild-type plants or arrhythmic transgenics, T. ni prefer plants lacking robust circadian rhythms. Altogether these data provide strong evidence for circadian clock enabling anticipation of herbivore attack and thus contributing to overall plant fitness.

Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior.

Diverse life forms have evolved internal clocks enabling them to monitor time and thereby anticipate the daily environmental changes caused by Earth's rotation. The plant circadian clock regulates expression of about one-third of the Arabidopsis genome, yet the physiological relevance of this regulation is not fully understood. Here we show that the circadian clock, acting with hormone signals, provides selective advantage to plants through anticipation of and enhanced defense against herbivory. We found that cabbage loopers (Trichoplusia ni) display rhythmic feeding behavior that is sustained under constant conditions, and plants entrained in light/dark cycles coincident with the entrainment of the T. ni suffer only moderate tissue loss due to herbivory. In contrast, plants entrained out-of-phase relative to the insects are significantly more susceptible to attack. The in-phase entrainment advantage is lost in plants with arrhythmic clocks or deficient in jasmonate hormone; thus, both the circadian clock and jasmonates are required. Circadian jasmonate accumulation occurs in a phase pattern consistent with preparation for the onset of peak circadian insect feeding behavior, providing evidence for the underlying mechanism of clock-enhanced herbivory resistance. Furthermore, we find that salicylate, a hormone involved in biotrophic defense that often acts antagonistically to jasmonates, accumulates in opposite phase to jasmonates. Our results demonstrate that the plant circadian clock provides a strong physiological advantage by performing a critical role in Arabidopsis defense.