Hannah V. Carey

Animals in a bacterial world, a new imperative for the life sciences

In the last two decades, the widespread application of genetic and genomic approaches has revealed a bacterial world astonishing in its ubiquity and diversity. This review examines how a growing knowledge of the vast range of animal–bacterial interactions, whether in shared ecosystems or intimate symbioses, is fundamentally altering our understanding of animal biology. Specifically, we highlight recent technological and intellectual advances that have changed our thinking about five questions: how have bacteria facilitated the origin and evolution of animals; how do animals and bacteria affect each other’s genomes; how does normal animal development depend on bacterial partners; how is homeostasis maintained between animals and their symbionts; and how can ecological approaches deepen our understanding of the multiple levels of animal–bacterial interaction. As answers to these fundamental questions emerge, all biologists will be challenged to broaden their appreciation of these interactions and to include investigations of the relationships between and among bacteria and their animal partners as we seek a better understanding of the natural world.

Hibernation: the immune system at rest?

Mammalian hibernation consists of torpor phases when metabolism is severely depressed, and Tb can reach as low as approximately –2°C, interrupted by euthermic arousal phases. Hibernation affects the function of the innate and the adaptive immune systems. Torpor drastically reduces numbers of all types of circulating leukocytes. In addition, other changes have been noted, such as lower complement levels, diminished response to LPS, phagocytotic capacity, cytokine production, lymphocyte proliferation, and antibody production. Hibernation may therefore increase infection risk, as illustrated by the currently emerging WNS in hibernating bats. Unraveling the pathways that result in reduced immune function during hibernation will enhance our understanding of immunologic responses during extreme physiological changes in mammals.

Hibernation induces oxidative stress and activation of NF-κB in ground squirrel intestine

Abstract Dramatic changes in blood flow occur during torpor-arousal cycles in mammalian hibernators that could increase the risk of oxidative stress to sensitive tissues. We used 13-lined ground squirrels (Spermophilus tridecemlineatus) to determine the effect of hibernation on lipid peroxidation and expression of stress-activated signaling pathways in the intestine, a tissue highly susceptible to ischemia-reperfusion injury. Compared with summer-active squirrels, levels of the mitochondrial stress protein GRP75 were consistently higher in intestinal mucosa of hibernators in each of five hibernation states (entrance, short-bout torpid, long-bout torpid, arousal and interbout euthermia). The redox-sensitive transcription factor, nuclear factor-κB (NF-κB), was strongly activated in each hibernation state compared with summer squirrels except for squirrels during an arousal from torpor. In contrast, NF-κB activation in brown adipose tissue (BAT) was low in active and hibernating squirrels regardless of season. Levels of conjugated dienes (products of lipid peroxidation) were higher in intestine of hibernators entering torpor and early in a torpor bout compared with summer squirrels. Conjugated diene levels were also higher in short-bout torpid vs arousing squirrels. The results suggest that the intestinal mucosa is vulnerable to oxidative stress during the hibernation season and in response may activate cellular defense pathways that help minimize severe oxidative damage induced by torpor-arousal cycles.

Reactive nitrogen species inhibit alveolar epithelial fluid transport after hemorrhagic shock in rats.

Our recent experimental work demonstrated that a neutrophil-dependent inflammatory response in the lung prevented the normal up-regulation of alveolar fluid clearance by catecholamines following hemorrhagic shock. In this study, we tested the hypothesis that the release of NO within the airspaces of the lung was responsible for the shock-mediated failure of the alveolar epithelium to respond to catecholamines in rats. Hemorrhagic shock was associated with an inducible NO synthase (iNOS)-dependent increase in the lung production of NO and a failure of the alveolar epithelium to up-regulate vectorial fluid transport in response to β-adrenergic agonists. Inhibition of iNOS restored the normal catecholamine-mediated up-regulation of alveolar liquid clearance. Airspace instillation of dibutyryl cAMP, a stable analog of cAMP, restored the normal fluid transport capacity of the alveolar epithelium after prolonged hemorrhagic shock, whereas direct stimulation of adenyl cyclase by forskolin had no effect. Pretreatment with pyrrolidine dithiocarbamate or sulfasalazine attenuated the iNOS-dependent production of NO in the lung and restored the normal up-regulation of alveolar fluid clearance by catecholamines after prolonged hemorrhagic shock. Based on in vitro studies with an alveolar epithelial cell line, A549 cells, the effect of sulfasalazine appeared to be mediated in part by inhibition of NF-κB activation, and the protective effect was mediated by the inhibition of IκBα protein degradation. In summary, these results provide the first in vivo evidence that NO, released within the airspaces of the lung probably secondary to the NF-κB-dependent activation of iNOS, is a major proximal inflammatory mediator that limits the rate of alveolar epithelial transport after prolonged hemorrhagic shock by directly impairing the function of membrane proteins involved in the β-adrenergic receptor-cAMP signaling pathway in alveolar epithelium.

Upregulation of AMPK during cold exposure occurs via distinct mechanisms in brown and white adipose tissue of the mouse

AMPK (adenosine monophosphate‐activated protein kinase), a key regulator of cellular energy metabolism and whole‐body energy balance, is present in brown adipose tissue but its role in regulating the acute metabolic state and chronic thermogenic potential of this metabolically unique tissue is unknown. To address this, the AMPK signalling system in brown and white adipose tissue was studied in C57Bl/6 mice under control conditions, during acute and chronic cold exposure, and during chronic adrenergic stimulation. In control mice AMPK activity in brown adipose tissue was higher than in any tissue yet reported (3‐fold the level in liver) secondary to a high level of expression of the α1 isoform. During the first day of cold, a time of intense non‐shivering thermogenesis, AMPK activity remained at basal levels. However, chronic (>7 days) cold caused a progressive increase in brown adipose tissue AMPK activity secondary to increased expression of the α1 isoform. To investigate the signalling pathway involved, noradrenaline (norepinephrine) and the β3‐adrenergic‐specific agonist CL 316, 243 were given for 14 days. This increased uncoupling protein‐1 content in brown adipose tissue, but not AMPK activity. In white adipose tissue 15 days of cold increased α1 AMPK activity 98 ± 20%, an effect reproduced by chronic noradrenaline or CL 316 243. We conclude that chronic cold not only increases AMPK activity in brown and white adipose tissue, but that it does so via distinct signalling pathways. Our data are consistent with AMPK acting primarily as a regulator of chronic thermogenic potential in brown adipose tissue, and not in the acute activation of non‐shivering thermogenesis.

Seasonal restructuring of the ground squirrel gut microbiota over the annual hibernation cycle

Many hibernating mammals suspend food intake during winter, relying solely on stored lipids to fuel metabolism. Winter fasting in these species eliminates a major source of degradable substrates to support growth of gut microbes, which may affect microbial community structure and host-microbial interactions. We explored the effect of the annual hibernation cycle on gut microbiotas using deep sequencing of 16S rRNA genes from ground squirrel cecal contents. Squirrel microbiotas were dominated by members of the phyla Bacteroidetes, Firmicutes, and Verrucomicrobia. UniFrac analysis showed that microbiotas clustered strongly by season, and maternal influences, diet history, host age, and host body temperature had minimal effects. Phylogenetic diversity and numbers of operational taxonomic units were lowest in late winter and highest in the spring after a 2-wk period of refeeding. Hibernation increased relative abundance of Bacteroidetes and Verrucomicrobia, phyla that contain species capable of surviving on host-derived substrates such as mucins, and reduced relative abundance of Firmicutes, many of which prefer dietary polysaccharides. Hibernation reduced cecal short-chain fatty acid and ammonia concentrations, and increased and decreased concentrations of acetate and butyrate, respectively. These results indicate that the ground squirrel microbiota is restructured each year in a manner that reflects differences in microbial preferences for dietary vs. host-derived substrates, and thus the competitive abilities of different taxa to survive in the altered environment in the hibernator gut.

Oral IGF-I enhances nutrient and electrolyte absorption in neonatal piglet intestine

The effect of orally administered insulin-like growth factor-I (IGF-I) on small intestinal structure and function was studied in 5-day-old colostrum-deprived piglets. Human recombinant IGF-I (3.5 mg ⋅ kg-1 ⋅ day-1) or control vehicle was given orogastrically for 4 days. Body weights, jejunal and ileal mucosa wet and dry weights, and serum IGF-I levels were similar in the two groups. Small intestinal villus height and crypt depth and jejunal enterocyte microvillar dimensions were also similar between groups. Oral IGF-I produced higher rates of jejunal ion transport because of increased basal Na+ absorption. Short-circuit current responses to mucosal addition ofd-glucose andl-alanine and net transepithelial absorption of 3- O-methylglucose were increased by IGF-I. Carrier-mediated uptake ofd-glucose per milligram in everted jejunal sleeves was greater in IGF-I-treated piglets because of a significantly greater maximal rate of uptake. We conclude that rates of net Na+ and Na+-dependent nutrient absorption are enhanced in piglets treated with oral IGF-I, and this effect is independent of changes in mucosal mass or surface area.The effect of orally administered insulin-like growth factor-I (IGF-I) on small intestinal structure and function was studied in 5-day-old colostrum-deprived piglets. Human recombinant IGF-I (3.5 mg. kg(-1). day(-1)) or control vehicle was given orogastrically for 4 days. Body weights, jejunal and ileal mucosa wet and dry weights, and serum IGF-I levels were similar in the two groups. Small intestinal villus height and crypt depth and jejunal enterocyte microvillar dimensions were also similar between groups. Oral IGF-I produced higher rates of jejunal ion transport because of increased basal Na+ absorption. Short-circuit current responses to mucosal addition of D-glucose and L-alanine and net transepithelial absorption of 3-O-methylglucose were increased by IGF-I. Carrier-mediated uptake of D-glucose per milligram in everted jejunal sleeves was greater in IGF-I-treated piglets because of a significantly greater maximal rate of uptake. We conclude that rates of net Na+ and Na+-dependent nutrient absorption are enhanced in piglets treated with oral IGF-I, and this effect is independent of changes in mucosal mass or surface area.

Beneficial Effects of Insulin-like Growth Factor I on Epithelial Structure and Function in Parenterally Fed Rat Jejunum

BACKGROUND & AIMS The functional significance of intestinal hyperplasia stimulated by insulin-like growth factor (IGF)-I is unclear and has not been studied in a model of mucosal atrophy induced by total parenteral nutrition (TPN). The aim of this study was to determine how IGF-I affects intestinal structure and epithelial function in the absence of luminal nutrition caused by TPN. METHODS Rats were maintained with TPN with or without IGF-I (800 micrograms/day), and jejunal histology and epithelial ion transport were measured after 5 days. In a third TPN group without IGF-I, a short-term dose of IGF-I was added during in vitro flux chamber experiments. RESULTS Rats given TPN with IGF-I had greater jejunal mucosal weight, greater protein and DNA content, and increased villus height and crypt depth compared with rats given TPN only. TPN increased ionic permeability and ion transport responses to secretory and absorptive agents. IGF-I in vivo reversed most of these changes; IGF-I in vitro enhanced sodium-dependent glucose absorption but had no other effects. CONCLUSIONS Coinfusion of recombinant human IGF-I with TPN solution stimulates intestinal hyperplasia and attenuates transport changes induced by TPN. The latter effect seems to be primarily associated with the growth state of the epithelium.

Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation

Hibernators are unique among mammals in their ability to survive extended periods of time with core body temperatures near freezing and with dramatically reduced heart, respiratory, and metabolic rates in a state known as torpor. To gain insight into the molecular events underlying this remarkable physiological phenotype, we applied a proteomic screening approach to identify liver proteins that differ between the summer active (SA) and the entrance (Ent) phase of winter hibernation in 13-lined ground squirrels. The relative abundance of 1,600 protein spots separated on two-dimensional gels was quantitatively determined using fluorescence difference gel electrophoresis, and 74 unique proteins exhibiting significant differences between the two states were identified using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Proteins elevated in Ent hibernators included liver fatty acid-binding protein, fatty acid transporter, and 3-hydroxy-3-methylglutaryl-CoA synthase, which support the known metabolic fuel switch to lipid and ketone body utilization in winter. Several proteins involved in protein stability and protein folding were also elevated in the Ent phase, consistent with previous findings. In contrast to transcript screening results, there was a surprising increase in the abundance of proteins involved in protein synthesis during Ent hibernation, including several initiation and elongation factors. This finding, coupled with decreased abundance of numerous proteins involved in amino acid and nitrogen metabolism, supports the intriguing hypothesis that the mechanism of protein preservation and resynthesis is used by hibernating ground squirrels to help avoid nitrogen toxicity and ensure preservation of essential amino acids throughout the long winter fast.

Induction of torpor: Mimicking natural metabolic suppression for biomedical applications†

Mammalian hibernation consists of periods of depressed metabolism and reduced body temperature called “torpor” that are interspersed by normothermic arousal periods. Numerous cellular processes are halted during torpor, including transcription, translation, and ion homeostasis. Hibernators are able to survive long periods of low blood flow and body temperature followed by rewarming and reperfusion without overt signs of organ injury, which makes these animals excellent models for application of natural protective mechanisms to human medicine. This review examines efforts to induce torpor‐like states in non‐hibernating species using pharmacological compounds. Elucidating the underlying mechanisms of natural and pharmacologically induced torpor will speed the development of new clinical approaches to treat a variety of trauma and stress states in humans. J. Cell. Physiol. 227: 1285–1290, 2012.