Metabolic Responses to Prolonged Fasting in a Naturally Obese Marine Mammal

Many animals undergo fasting in order to survive during periods of severe weather, food scarcity, migration, or reproduction. While most animals decrease their metabolism while fasting, northern elephant seals fast for several months while undergoing energy-intensive activities such as molting and reproduction. Seals fuel their energy needs with large fat stores that they accumulate while foraging at sea, resembling human patients with diabetes and obesity (insulin sensitivity, high levels of glucose and fat) while fasting. While some of the hormone signals involved in fasting have been identified, the molecular mechanisms that regulate healthy metabolic adaptations to fasting in seals are not fully understood. We used proteome sequencing to examine changes in protein abundance in blood plasma and the main energy-utilizing (skeletal muscle) and energy-storing (blubber) tissues of adult female elephant seals over their five-week molting fast. We found that while blubber and muscle proteomes were remarkably stable over fasting, over 50 proteins changed in abundance in plasma, including those associated with fat storage, metabolism, and transport. Apolipoproteins, which are key components of cholesterol and fat-transporting particles (such as HDL and LDL), dominated proteome responses to fasting. Apolipoproteins associated with fat storage decreased, while those associated with fat burning and HDL function increased over fasting. Our findings suggest that changes in apolipoprotein composition may mediate the metabolic transitions between feeding and fasting and underlie metabolic health in elephant seals. Many of these proteins have not been previously studied in this species and provide intriguing hypotheses about metabolic regulation during prolonged fasting in mammals

Comprehensive spatiotemporal analysis of early chick neural crest network genes

Specification of neural crest progenitors begins during gastrulation at the neural plate border, long before migration or differentiation. Neural crest cell fate is acquired by progressive activation of discrete groups of transcription factors that appear to be highly conserved in vertebrates; however, comprehensive analysis of their expression has been lacking in chick, an important model system for neural crest development. To address this, we analyzed expression of 10 transcription factors that are known specifiers of neural plate border and neural crest fate and compared them across developmental stages from gastrulation to neural crest migration. Surprisingly, we find that most neural crest specifiers are expressed during gastrulation in chick, concomitant with and in similar domains as neural plate border specifiers. This finding suggests that interactions between these molecules may occur much earlier than previously thought, an important consideration for interpretation of functional studies

SOX2 Functions to Maintain Neural Progenitor Identity

Neural progenitors of the vertebrate CNS are defined by generic cellular characteristics, including their pseudoepithelial morphology and their ability to divide and differentiate. SOXB1 transcription factors, including the three closely related genes Sox1, Sox2, and Sox3, universally mark neural progenitor and stem cells throughout the vertebrate CNS. We show here that constitutive expression of SOX2 inhibits neuronal differentiation and results in the maintenance of progenitor characteristics. Conversely, inhibition of SOX2 signaling results in the delamination of neural progenitor cells from the ventricular zone and exit from cell cycle, which is associated with a loss of progenitor markers and the onset of early neuronal differentiation markers. The phenotype elicited by inhibition of SOX2 signaling can be rescued by coexpression of SOX1, providing evidence for redundant SOXB1 function in CNS progenitors. Taken together, these data indicate that SOXB1 signaling is both necessary and sufficient to maintain panneural properties of neural progenitor cells

Transcriptome analysis of northern elephant seal (Mirounga angustirostris) muscle tissue provides a novel molecular resource and physiological insights

BackgroundThe northern elephant seal, Mirounga angustirostris, is a valuable animal model of fasting adaptation and hypoxic stress tolerance. However, no reference sequence is currently available for this and many other marine mammal study systems, hindering molecular understanding of marine adaptations and unique physiology.ResultsWe sequenced a transcriptome of M. angustirostris derived from muscle sampled during an acute stress challenge experiment to identify species-specific markers of stress axis activation and recovery. De novo assembly generated 164,966 contigs and a total of 522,699 transcripts, of which 68.70% were annotated using mouse, human, and domestic dog reference protein sequences. To reduce transcript redundancy, we removed highly similar isoforms in large gene families and produced a filtered assembly containing 336,657 transcripts. We found that a large number of annotated genes are associated with metabolic signaling, immune and stress responses, and muscle function. Preliminary differential expression analysis suggests a limited transcriptional response to acute stress involving alterations in metabolic and immune pathways and muscle tissue maintenance, potentially driven by early response transcription factors such as Cebpd.ConclusionsWe present the first reference sequence for Mirounga angustirostris produced by RNA sequencing of muscle tissue and cloud-based de novo transcriptome assembly. We annotated 395,102 transcripts, some of which may be novel isoforms, and have identified thousands of genes involved in key physiological processes. This resource provides elephant seal-specific gene sequences, complementing existing metabolite and protein expression studies and enabling future work on molecular pathways regulating adaptations such as fasting, hypoxia, and environmental stress responses in marine mammals

Application of phenotypic microarrays to environmental microbiology

Environmental organisms are extremely diverse and only a small fraction has been successfully cultured in the laboratory. Culture in micro wells provides a method for rapid screening of a wide variety of growth conditions and commercially available plates contain a large number of substrates, nutrient sources, and inhibitors, which can provide an assessment of the phenotype of an organism. This review describes applications of phenotype arrays to anaerobic and thermophilic microorganisms, use of the plates in stress response studies, in development of culture media for newly discovered strains, and for assessment of phenotype of environmental communities. Also discussed are considerations and challenges in data interpretation and visualization, including data normalization, statistics, and curve fitting

Changes in serum adipokines during natural extended fasts in female northern elephant seals

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Rzucidlo, C. L., Sperou, E. S., Holser, R. R., Khudyakov, J., Costa, D. P., & Crocker, D. E. Changes in serum adipokines during natural extended fasts in female northern elephant seals. General and Comparative Endocrinology, 308, (2021): 113760, https://doi.org/10.1016/j.ygcen.2021.113760.Adipose tissue is essential to endotherms for thermoregulation and energy storage as well as functioning as an endocrine organ. Adipose derived hormones, or adipokines, regulate metabolism, energy expenditure, reproduction, and immune function in model systems but are less well studied in wildlife. Female northern elephant seals (NES) achieve high adiposity during foraging and then undergo natural fasts up to five weeks long during haul-outs associated with reproduction and molting, resulting in large changes in adipose reserves. We measured circulating levels of four adipokines: leptin, resistin, adiponectin, and kisspeptin-54, in 196 serum samples from female NES at the beginning and end of their breeding and molting fasts. We examined the relationships between these adipokines and life-history stage, adiposity, mass, cortisol, and an immune cytokine involved in the innate immune response interleukin 6 (IL-6). All four adipokines varied with life-history stage. Leptin concentrations were highest at the beginning of the breeding haul-out. Resistin concentrations were higher throughout the breeding haul-out compared to the molt haul-out. Adiponectin concentrations were highest at the beginning of both haul-outs. Kisspeptin-54 concentrations were highest at the end of the breeding haul-out. Leptin, resistin, and adiponectin were associated with measures of body condition, either adiposity, mass, or both. Resistin, adiponectin, and kisspeptin-54 were associated with circulating cortisol concentrations. Resistin was strongly associated with circulating IL-6, a multifunctional cytokine. Adiponectin was associated with glucose concentrations, suggesting a potential role in tissue-specific insulin sensitivity during life-history stages categorized by high adiposity. Increased cortisol concentrations late in lactation were associated with increased kisspeptin-54, suggesting a link to ovulation initiation in NES. This study suggests dramatic changes in circulating adipokines with life-history and body condition that may exert important regulatory roles in NES. The positive relationship between adiponectin and adiposity as well as the lack of a relationship between leptin and kisspeptin-54 differed from model systems. These differences from biomedical model systems suggest the potential for modifications of expression and function of adipose-derived hormones in species that undergo natural changes in adiposity as part of their life-history.This project was supported by a grant from the Office of Naval Research (#N00014-18-1-2822) to DPC and DEC and the Marine Life Joint Industry Program of the IAGOP. We thank the Año Nuevo State Reserve rangers for logistical support

Terrestrial birth and body size tune UCP1 functionality in seals

The molecular evolution of the mammalian heater protein UCP1 is a powerful biomarker to understand thermoregulatory strategies during species radiation into extreme climates, such as aquatic life with high thermal conductivity. While fully aquatic mammals lost UCP1, most semiaquatic seals display intact UCP1 genes, apart from large elephant seals. Here, we show that UCP1 thermogenic activity of the small-bodied harbor seal is equally potent compared to terrestrial orthologs, emphasizing its importance for neonatal survival on land. In contrast, elephant seal UCP1 does not display thermogenic activity, not even when translating a repaired or a recently highlighted truncated version. Thus, the thermogenic benefits for neonatal survival during terrestrial birth in semiaquatic pinnipeds maintained evolutionary selection pressure on UCP1 function and were only outweighed by extreme body sizes among elephant seals, fully eliminating UCP1-dependent thermogenesis

Complete genome sequence of “Enterobacter lignolyticus” SCF1

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated “Enterobacter lignolyticus”SCF1 on minimal media with alkali lignin as the sole source of carbon. This organism was isolated anaerobically from tropical forest soils collected from the Short Cloud Forest site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are net methane producers. Because of its ability to grow on lignin anaerobically, we sequenced the genome. The genome of “E. lignolyticus” SCF1 is 4.81 Mbp with no detected plasmids, and includes a relatively small arsenal of lignocellulolytic carbohydrate active enzymes. Lignin degradation was observed in culture, and the genome revealed two putative laccases, a putative peroxidase, and a complete 4-hydroxyphenylacetate degradation pathway encoded in a single gene cluster

Complete genome sequence of “Enterobacter lignolyticus” SCF1

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated “Enterobacter lignolyticus”SCF1 on minimal media with alkali lignin as the sole source of carbon. This organism was isolated anaerobically from tropical forest soils collected from the Short Cloud Forest site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are net methane producers. Because of its ability to grow on lignin anaerobically, we sequenced the genome. The genome of “E. lignolyticus” SCF1 is 4.81 Mbp with no detected plasmids, and includes a relatively small arsenal of lignocellulolytic carbohydrate active enzymes. Lignin degradation was observed in culture, and the genome revealed two putative laccases, a putative peroxidase, and a complete 4-hydroxyphenylacetate degradation pathway encoded in a single gene cluster