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This study used 3H‐L‐alanyl‐L‐alanine to demonstrate dipeptide uptake using in vitro gut sacs prepared from the hindgut of the Pacific hagfish Eptatretus stoutii. Concentration‐dependent kinetic analysis resulted in a sigmoidal distribution with a maximal (± SE) uptake rate (Jmax‐like) of 70 ± 3 nmol cm−2 h−1 and an affinity constant (Km‐like) of 1072 ± 81 μM. Addition of high alanine concentrations to transport assays did not change dipeptide transport rates, indicating that hydrolysis of the dipeptide in mucosal solutions and subsequent uptake via apical amino acid transporters was not occurring, which was further supported by a Km distinct from that of amino acid transport. Transport occurred independent of mucosal pH, but uptake was reduced by 42% in low mucosal sodium. This may implicate cooperation between peptide transporters and sodium‐proton exchangers, previously demonstrated in several mammalian and teleost species. Finally, apical L‐alanyl‐L‐alanine uptake rates (i.e., mucosal disappearance) were significantly increased following a meal, demonstrating regulation of uptake. Overall, this examination of dipeptide acquisition in the earliest extant Agnathan suggests evolutionarily conserved mechanisms of transport between hagfish and later‐diverging vertebrates such as teleosts and mammals. [ABSTRACT FROM AUTHOR]

Hagfish are capable of tolerating extreme hypercapnia (> 30 Torr) by mounting substantial plasma [HCO3−] (hypercarbia) to compensate for CO2-mediated acidosis. The goal of this study was to characterize the mechanistic hypercarbia-recovery strategies in the highly CO2 tolerant hagfish. We exposed hagfish to hypercapnia (30 Torr) for 48 h and allowed a 24 h recovery period in normocapnic seawater. Within 8 h of the recovery period, the compensatory plasma [HCO3−] load (~ 70 mmol L−1) was rapidly offloaded. While increases in both whole-animal HCO3− excretion and glomerular filtration were observed throughout recovery (2-8 h), neither can fully account for the observed rates of whole-animal HCO3− loss, which peaked at ~ 3.5 mmol kg−1 h−1. Inhibition of carbonic anhydrase via acetazolamide revealed that the restoration of plasma [HCO3−] from hypercapnia-induced hypercarbia is likely facilitated in a dualistic manner, initially relying on both carbonic anhydrase mediated CO2 offloading and Cl−/HCO3− exchange processes, both of which are likely either upregulated or further activated as recovery progresses. [ABSTRACT FROM AUTHOR]

Hagfishes are unique to the vertebrate lineage in that they acquire dissolved nutrients across multiple epithelia including the intestine, gill, and skin. This feat has been attributed to their immersive feeding behavior that likely simultaneously provides benefits (nutrient rich) and potentially adverse (hypercapnia, hypoxia, high environmental ammonia) physiological effects. Examinations have been conducted of the ex vivo transport capabilities of specific nutrients as well as in vivo effects of the hypothesized feeding environments, yet the physiological effects of feeding itself have never been elucidated. We examined the post-prandial physiology of Pacific hagfish ( Eptatretus stoutii), identifying changes in oxygen consumption, acid-base balance, ammonia waste excretion, and intestinal morphology following feeding in captivity. Following voluntary feeding, post-prandial oxygen consumption was significantly elevated (1868 ± 272 µmol kg h) 8 h following feeding when compared to control resting metabolic oxygen consumption (642 ± 51 µmol kg h) and resulted in a factorial metabolic scope of 2.92. Changes in acid-base status were not observed following feeding in either the excreted components or the caudal blood samples; however, a significant alkalosis was observed 8 h post-feeding in the major intestinal blood vein. Significant increases (16-fold) in ammonia excretion were recorded in 36 h post-fed hagfish. Finally, significant post-prandial increases in intestinal mucosal thickness and microvilli length were observed, with mucosal thickness remaining significantly increased throughout 36 h and the microvilli length returning to fasted lengths by 36 h. These results demonstrate the post-feeding physiology of the earliest diverging extant craniate and identify correlations between physiology and hindgut morphology 8 h following feeding. [ABSTRACT FROM AUTHOR]

Chondrichthyans play an important role in nutrient cycling of many marine ecosystems, yet little is known about their nutritional physiology particularly relating to nutrient acquisition in the spiral valve intestine. This unique organ poses challenges for examining nutrient transport physiology using traditional reductionist methods owing to its scroll-like morphology. Thus, we established a method for the characterization of nutrient uptake rates in two representative chondrichthyans, the Pacific spiny dogfish ( Squalus suckleyi ) and the Pacific spotted ratfish ( Hydrolagus colliei ). We validated a dual-cannulation method wherein perfusate was circulated through the vasculature of the spiral valve via the anterior and posterior intestinal arteries, and [ 14 C]glucose or [ 3 H]oleic acid was accumulated from the static spiral valve lumen into the anterior and posterior intestinal veins. Radiotracer accumulated at a stable rate in the venous effluent in comparison with measures of mucosal disappearance. Interestingly, similar anterior and posterior glucose uptake was observed in dogfish, yet significantly more oleic acid was accumulated in the posterior veins of ratfish. Further validation of the preparation in dogfish demonstrated sodium dependence of glucose transport as well as an effect of bovine insulin administration to the arterial circulation. Each of these manipulations resulted in significant differences in glucose handling between the anterior and posterior veins, suggestive of heretofore unknown heterogenous functions along the intestine. This preparation demonstrates a new and reliable method for the measurement of nutrient acquisition and regulation thereof in a unique digestive organ. Furthermore, it presents avenues for investigation of differential functional along the spiral valve. NEW & NOTEWORTHY We describe a novel dual cannulation method for investigating radiolabeled nutrient uptake from a unique organ, the spiral valve. Furthermore, we identify functional differences in nutrient transport along the length of the spiral valve which consists of a homogenous gross morphology. Finally, this method reveals a useful way in which to manipulate the arterial supply to better understand postprandial physiology as it varies with metabolites and endocrine factors.

The liver is an essential energy storage organ in vertebrates. In teleosts and elasmobranchs, previous studies examining hepatic energy balance have used isolated hepatocytes. Although these studies have been informative, the high-fat content in the elasmobranch liver limits isolation of hepatocytes and therefore the utility of this method to understand hepatic metabolic processes. In the present study, we developed an in situ liver perfusion in the North Pacific spiny dogfish Squalus suckleyi . Perfusions were conducted by cannulating the hepatic portal vein (inflowing cannulation) and the sinus venosus through the heart (outflowing cannulation). Changes in major elasmobranch metabolites (glucose and 3-hydroxybutarate [3-HB]) were determined by the arterial (inflow)-venous (outflow) difference in metabolite concentration. Liver preparations were considered viable due to consistent oxygen consumption over 3 h and the maintenance of predictable vasoconstriction following administration of homologous 10 -7 M angiotensin II (ANG II). Removal and reintroduction from the perfusate of metabolites showed endogenous 3-HB production in the isolated perfused livers but did not affect glucose balance. However, the arterial-venous difference of both metabolites did not change following perfusion with heterologous insulin and homologous glucagon, which may be due to the glucose intolerant nature of elasmobranchs. Ultimately, we show the viability of this perfusion for the investigation of hepatic energy mobilization in sharks. NEW & NOTEWORTHY We describe a viable liver perfusion in a shark species for the first time as determined by oxygen consumption and hormone-mediated changes in hemodynamics (angiotensin II, ANG II). In addition, removal of major energy metabolites confirms hepatic ketone [3-hydroxybutyrate (3-HB)] production by an elasmobranch liver. Perfusion with heterologous insulin and homologous glucagon did not cause changes in glucose balance, however, possibly demonstrating differences in glucose metabolism in this taxon as compared with more derived vertebrates.

Hagfish are an excellent model species in which to draw inferences on the evolution of transport systems in early vertebrates owing to their basal position in vertebrate phylogeny. Glucose is a ubiquitous cellular energy source that is transported into cells via two classes of carrier proteins: sodium-glucose-linked transporters (Sglt; Slc5a) and glucose transporters (Glut; Slc2a). Although previous pharmacological evidence has suggested the presence of both sodium-dependent and -independent transport mechanisms in the hagfish, the molecular identities were heretofore unconfirmed. We have identified and phylogenetically characterized both a Slc5a1-like and Slc2a-like gene in the Pacific hagfish ( Eptatretus stoutii ), the latter sharing common ancestry with other glucose-transporting isoforms of the Slc2a family. To assess the potential postprandial regulation of these glucose transporters, we examined the abundance and localization of these transporters with qPCR and immunohistochemistry alongside functional studies using radiolabeled d-[ 14 C]glucose. The effects of glucose or insulin injection on glucose transport rate and transporter expression were also examined to determine their potential role(s) in the regulation of intestinal glucose carrier proteins. Feeding prompted an increase in glucose uptake across the hindgut at both 0.5 mM (∼84%) and 1 mM (∼183%) concentrations. Concomitant increases were observed in hindgut Slc5a1 protein expression. These effects were not observed following either of glucose or insulin injection, indicating these postprandial factors are not the driving force for transporter regulation over this timeframe. We conclude that Pacific hagfish utilize evolutionarily conserved mechanisms of glucose uptake and so represent a useful model to understand early-vertebrate evolution of glucose uptake and regulation.

Hagfish consume carrion, potentially exposing them to hypoxia, hypercapnia, and high environmental ammonia (HEA). We investigated branchial and cutaneous ammonia handling strategies by which Pacific hagfish ( Eptatretus stoutii ) tolerate and recover from high ammonia loading. Hagfish were exposed to HEA (20 mmol/l) for 48 h to elevate plasma total ammonia (T Amm ) levels before placement into divided chambers for a 4-h recovery period in ammonia-free seawater where ammonia excretion ( J Amm ) was measured independently in the anterior and posterior compartments. Localized HEA exposures were also conducted by subjecting hagfish to HEA in either the anterior or posterior compartments. During recovery, HEA-exposed animals increased J Amm in both compartments, with the posterior compartment comprising ~20% of the total J Amm compared with ~11% in non-HEA-exposed fish. Plasma T Amm increased substantially when whole hagfish and the posterior regions were exposed to HEA. Alternatively, plasma T Amm did not elevate after anterior localized HEA exposure. J Amm was concentration dependent (0.05-5 mmol/l) across excised skin patches at up to eightfold greater rates than in skin sections that were excised from HEA-exposed hagfish. Skin excised from more posterior regions displayed greater J Amm than those from more anterior regions. Immunohistochemistry with hagfish-specific anti-rhesus glycoprotein type c (α-hRhcg; ammonia transporter) antibody was characterized by staining on the basal aspect of hagfish epidermis while Western blotting demonstrated greater expression of Rhcg in more posterior skin sections. We conclude that cutaneous Rhcg proteins are involved in cutaneous ammonia excretion by Pacific hagfish and that this mechanism could be particularly important during feeding., (Copyright © 2017 the American Physiological Society.)