Hepatic fatty acid oxidation of yolk lipoproteins provides the main energy source for chick embryos. Post-hatching these yolk lipids are rapidly exhausted and metabolism switches to a carbohydrate-based energy source. We recently demonstrated that many microRNAs (miRNAs) are key regulators of hepatic metabolic pathways during this metabolic switching. MiRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression in most eukaryotes. To further elucidate the roles of miRNAs in the metabolic switch, we used delayed feeding for 48 h to impede the hepatic metabolic switch. We found that hepatic expression of several miRNAs including miR-33, miR-20b, miR-34a, and miR-454 was affected by delaying feed consumption for 48 h. For example, we found that delayed feeding resulted in increased miR-20b expression and conversely reduced expression of its target FADS1, an enzyme involved in fatty acid synthesis. Interestingly, the expression of a previously identified miR-20b regulator FOXO3 was also higher in delayed fed chicks. FOXO3 also functions in protection of cells from oxidative stress. Delayed fed chicks also had much higher levels of plasma ketone bodies than their normal fed counterparts. This suggests that delayed fed chicks rely almost exclusively on lipid oxidation for energy production and are likely under higher oxidative stress. Thus, it is possible that FOXO3 may function to both limit lipogenesis as well as to help protect against oxidative stress in peri-hatch chicks until the initiation of feed consumption. This is further supported by evidence that the FOXO3-regulated histone deacetylase (HDAC2) was found to recognize the FASN (involved in fatty acid synthesis) chicken promoter in a yeast one-hybrid assay. Expression of FASN mRNA was lower in delayed fed chicks until feed consumption. The present study demonstrated that many transcriptional and post-transcriptional mechanisms, including miRNA, form a complex interconnected regulatory network that is involved in controlling lipid and glucose molecular pathways during the metabolic transition in peri-hatch chicks.

Post-transcriptional gene regulation by small RNAs is now established as an important branch of the gene regulatory system. Many different classes of small RNAs have been discovered; among these are short interfering RNAs (siRNAs) and microRNA (miRNAs). Though differences in the processing and function of small RNAs exist between plants and animals, both groups utilize small RNA-mediated gene regulation in response to pathogens. Host encoded miRNAs and siRNAs are generated from viral RNA function in host defense and pathogenic resistance in plants. In animals, miRNAs are key regulators in both immune system development and in immune function. Pathogens, in particular viruses, have evolved mechanisms to usurp the host’s small RNA-mediated regulatory system. Overall, small RNAs are a major component of host defense and immunity in eukaryotes. The goal of this review is to summarize our current knowledge of the involvement of eukaryotic small RNA pathways in host defense and viral pathogenesis.

Marek’s Disease is a lymphoproliferative disease of chickens caused by Marek’s Disease Virus. Similar to other herpesviruses, Marek’s Disease Virus (MDV) encodes its own small non-coding regulatory RNAs termed microRNAs (miRNAs). We previously found that the expression profile of these viral miRNAs is affected by vaccination with Herpesvirus of Turkeys (HVT). To further characterize miRNA-mediated gene regulation in MDV infections, in the current study we examine the impact of HVT vaccination on cellular miRNA expression in MDV-infected specific-pathogen-free (SPF) chickens. We used small RNA-seq to identify 24 cellular miRNAs that exhibited altered splenic expression in MDV infected chickens (42 dpi) compared to age-matched uninfected birds. We then used Real Time-quantitative PCR (RT-qPCR) to develop expression profiles of a select group of these host miRNAs in chickens receiving the HVT vaccine and in vaccinated chickens subsequently infected with MDV. As was seen with viral miRNA, host miRNAs had unique splenic expression profiles between chickens infected with HVT, MDV, or co-infected birds. We also discovered a group of transcription factors, using a yeast one-hybrid screen, which regulates immune responses and cell growth pathways and also likely regulates the expression of these cellular miRNAs. Overall, this study suggests cellular miRNAs are likely a critical component of both protection from and progression of Marek’s Disease.

To investigate the role of peroxisome proliferator-activated receptor alpha (PPARα) in carnitine status and intestinal fatty acid oxidation in neonates, a total of 72 suckled newborn piglets were assigned into 8 dietary treatments following a 2 (±0.35% clofibrate) × 4 (diets with: succinate+glycerol (Succ), tri-valerate (TC5), tri-hexanoate (TC6), or tri-2-methylpentanoate (TMPA)) factorial design. All pigs received experimental milk diets with isocaloric energy for 5 days. Carnitine statuses were evaluated, and fatty acid oxidation was measured in vitro using [1-¹⁴C]-palmitic acid (1 mM) as a substrate in absence or presence of L659699 (1.6 µM), iodoacetamide (50 µM), and carnitine (1 mM). Clofibrate increased concentrations of free (41%) and/or acyl-carnitine (44% and 15%) in liver and plasma but had no effects in the intestine. The effects on carnitine status were associated with the expression of genes involved in carnitine biosynthesis, absorption, and transportation. TC5 and TMPA stimulated the increased fatty acid oxidation rate induced by clofibrate, while TC6 had no effect on the increased fatty acid oxidation induced by clofibrate (p > 0.05). These results suggest that dietary clofibrate improved carnitine status and increased fatty acid oxidation. Propionyl-CoA, generated from TC5 and TMPA, could stimulate the increased fatty acid oxidation rate induced by clofibrate as anaplerotic carbon sources.

As the chick transitions from embryonic to post-hatching life, its metabolism must quickly undergo a dramatic switch in its major energy source. The chick embryo derives most of its energy from the yolk, a lipid-rich/carbohydrate-poor source. Upon hatching, the chick’s metabolism must then be able to utilize a lipid-poor/carbohydrate-rich source (feed) as its main form of energy. We recently found that a number of hepatically-expressed microRNAs (miRNAs) help facilitate this shift in metabolic processes in the chick liver, the main site of lipogenesis. While adipose tissue was initially thought to mainly serve as a lipid storage site, it is now known to carry many metabolic, endocrine, and immunological functions. Therefore, it would be expected that adipose tissue is also an important factor in the metabolic switch. To that end, we used next generation sequencing (NGS) and real-time quantitative PCR (RT-qPCR) to generate miRNome and transcriptome signatures of the adipose tissue during the transition from late embryonic to early post-hatch development. As adipose tissue is well known to produce inflammatory and other immune factors, we used SPF white leghorns to generate the initial miRNome and transcriptome signatures to minimize complications from external factors (e.g., pathogenic infections) and ensure the identification of bona fide switch-associated miRNAs and transcripts. We then examined their expression signatures in the adipose tissue of broilers (Ross 708). Using E18 embryos as representative of pre-switching metabolism and D3 chicks as a representative of post-switching metabolism, we identified a group of miRNAs which work concordantly to regulate a diverse but interconnected group of developmental, immune and metabolic processes in the adipose tissue during the metabolic switch. Network mapping suggests that during the first days post-hatch, despite the consumption of feed, the chick is still heavily reliant upon adipose tissue lipid stores for energy production, and is not yet efficiently using their new energy source for de novo lipid storage. A number of core master regulatory pathways including, circadian rhythm transcriptional regulation and growth hormone (GH) signaling, likely work in concert with miRNAs to maintain an essential balance between adipogenic, lipolytic, developmental, and immunological processes in the adipose tissue during the metabolic switch.