Liver proteome response to torpor in a basoendothermic mammal, Tenrec ecaudatus, provides insights into the evolution of homeothermy.

Many mammals use adaptive heterothermy (e.g., torpor, hibernation) to reduce metabolic demands of maintaining high body temperature (Tb). Torpor is typically characterized by coordinated declines in Tb and metabolic rate (MR) followed by active rewarming. Most hibernators experience periods of euthermy between bouts of torpor during which homeostatic processes are restored. In contrast, the common tenrec, a basoendothermic Afrotherian mammal, hibernates without interbout arousals and displays extreme flexibility in Tb and MR. We investigated the molecular basis of this plasticity in tenrecs by profiling the liver proteome of animals that were active or torpid with high and more stable Tb (∼32°C) or lower Tb (∼14°C). We identified 768 tenrec liver proteins, of which 50.9% were differentially abundant between torpid and active animals. Protein abundance was significantly more variable in active cold and torpid compared with active warm animals, suggesting poor control of proteostasis. Our data suggest that torpor in tenrecs may lead to mismatches in protein pools due to poor coordination of anabolic and catabolic processes. We propose that the evolution of endothermy leading to a more realized homeothermy of boreoeutherians likely led to greater coordination of homeostatic processes and reduced mismatches in thermal sensitivities of metabolic pathways.

Antioxidant properties of UCP1 are evolutionarily conserved in mammals and buffer mitochondrial reactive oxygen species.

Mitochondrial uncoupling reduces reactive oxygen species (ROS) production and appears to be important for cellular signaling/protection, making it a focus for the treatment of metabolic and age-related diseases. Whereas the physiological role of uncoupling protein 1 (UCP1) of brown adipose tissue is established for thermogenesis, the function of UCP1 in the reduction of ROS in cold-exposed animals is currently under debate. Here, we investigated the role of UCP1 in mitochondrial ROS handling in the Lesser hedgehog tenrec (Echinops telfairi), a unique protoendothermic Malagasy mammal with recently identified brown adipose tissue (BAT). We show that the reduction of ROS by UCP1 activity also occurs in BAT mitochondria of the tenrec, suggesting that the antioxidative role of UCP1 is an ancient mammalian trait. Our analysis shows that the quantity of UCP1 displays strong control over mitochondrial hydrogen peroxide release, whereas other factors, such as mild cold, nonshivering thermogenesis, oxidative capacity, and mitochondrial respiration, do not correlate. Furthermore, hydrogen peroxide release from recoupled BAT mitochondria was positively associated with mitochondrial membrane potential. These findings led to a model of UCP1 controlling mitochondrial ROS release and, presumably, being controlled by high membrane potential, as proposed in the canonical model of "mild uncoupling". Our study further promotes a conserved role for UCP1 in the prevention of oxidative stress, which was presumably established during evolution before UCP1 was physiologically integrated into nonshivering thermogenesis.

Intestinal perfusion indicates high reliance on paracellular nutrient absorption in an insectivorous bat Tadarida brasiliensis.

Flying vertebrates have been hypothesized to have a high capacity for paracellular absorption of nutrients. This could be due to high permeability of the intestines to nutrient-sized molecules (i.e., in the size range of amino acids and glucose, MW 75-180 Da). We performed intestinal luminal perfusions of an insectivorous bat, Tadarida brasiliensis. Using radio-labeled molecules, we measured the uptake of two nutrients absorbed by paracellular and transporter-mediated mechanisms (L-proline, MW 115 Da, and D-glucose, MW 180 Da) and two carbohydrates that have no mediated transport (L-arabinose, MW 150 Da, and lactulose, MW 342 Da). Absorption of lactulose (0.61±0.06 nmol min(-1) cm(-1)) was significantly lower than that of the smaller arabinose (1.09±0.04 nmol min(-1) cm(-1)). Glucose absorption was significantly lower than that of proline at both nutrient concentrations (10mM and 75 mM). Using the absorption of arabinose to estimate the portion of proline absorption that is paracellular, we calculated that 25.1±3.0% to 66.2±7.8% of proline absorption is not transporter-mediated (varying proline from 1 mM to 75 mM). These results confirm our predictions that 1) paracellular absorption is molecule size selective, 2) absorption of proline would be greater than glucose absorption in an insectivore, and 3) paracellular absorption represents a large fraction of total nutrient absorption in bats.

Aggrecan-based extracellular matrix shows unique cortical features and conserved subcortical principles of mammalian brain organization in the Madagascan lesser hedgehog tenrec (Echinops telfairi Martin, 1838).

The Madagascan tenrecs (Afrotheria), an ancient mammalian clade, are characterized by unique brain anatomy. Striking features are an expanded paleocortex but a small and poorly differentiated neocortex devoid of a distinct granular layer IV. To investigate the organization of cortical areas we analyzed extracellular matrix components in perineuronal nets (PNs) using antibodies to aggrecan, lectin staining and hyaluronan-binding protein. Selected subcortical regions were studied to correlate the cortical patterns with features in evolutionary conserved systems. In the neocortex, paleocortex and hippocampus PNs were associated with nonpyramidal neurons. Quantitative analysis in the cerebral cortex revealed area-specific proportions and laminar distribution patterns of neurons ensheathed by PNs. Cortical PNs showed divergent structural phenotypes. Diffuse PNs forming a cotton wool-like perisomatic rim were characteristic of the paleocortex. These PNs were associated with a dense pericellular plexus of calretinin-immunoreactive fibres. Clearly contoured PNs were devoid of a calretinin-positive plexus and predominated in the neocortex and hippocampus. The organization of the extracellular matrix in subcortical nuclei followed the widely distributed mammalian type. We conclude that molecular properties of the aggrecan-based extracellular matrix are conserved during evolution of mammals; however, the matrix scaffold is adapted to specific wiring patterns of cortical and subcortical neuronal networks.

Transposition and Intermingling of Galphai2 and Galphao afferences into single vomeronasal glomeruli in the Madagascan lesser Tenrec Echinops telfairi.

The vomeronasal system (VNS) mediates pheromonal communication in mammals. From the vomeronasal organ, two populations of sensory neurons, expressing either Galphai2 or Galphao proteins, send projections that end in glomeruli distributed either at the rostral or caudal half of the accessory olfactory bulb (AOB), respectively. Neurons at the AOB contact glomeruli of a single subpopulation. The dichotomic segregation of AOB glomeruli has been described in opossums, rodents and rabbits, while Primates and Laurasiatheres present the Galphai2-pathway only, or none at all (such as apes, some bats and aquatic species). We studied the AOB of the Madagascan lesser tenrec Echinops telfairi (Afrotheria: Afrosoricida) and found that Galphai2 and Galphao proteins are expressed in rostral and caudal glomeruli, respectively. However, the segregation of vomeronasal glomeruli at the AOB is not exclusive, as both pathways contained some glomeruli transposed into the adjoining subdomain. Moreover, some glomeruli seem to contain intermingled afferences from both pathways. Both the transposition and heterogeneity of vomeronasal afferences are features, to our knowledge, never reported before. The organization of AOB glomeruli suggests that synaptic integration might occur at the glomerular layer. Whether intrinsic AOB neurons may make synaptic contact with axon terminals of both subpopulations is an interesting possibility that would expand our understanding about the integration of vomeronasal pathways.

Glycosylation at the fetomaternal interface in hemomonochorial placentae from five widely separated species of mammal: is there evidence for convergent evolution?

Hemomonochorial placentation occurs in diverse species. We have examined placental glycosylation in five widely separated mammals with this type of placentation--lesser hedgehog tenrec (Echinops telfairi), spotted hyena (Crocuta crocuta), nine-banded armadillo (Dasypus novemcinctus), human (Homo sapiens) and guinea pig (Cavia porcellus)--in order to assess whether evolutionary convergence to the hemomonochorial state is accompanied by a similar convergence of glycan expression. Placentae from 2 E. telfairi, 3 C. crocuta, 1 D. novemcinctus, 4 womenand 1 C. porcellus were fixed and processed into epoxy resin. Binding of twenty-three lectins was assessed using a semiquantitative ranking system. The trophoblast apical/microvillous membrane of all five species showed marked similarities in glycosylation. In the N-linked series, there were abundant bi/tri-antennary complex chains, while the non-bisected variants were much scarcer. All species had plentiful N-acetyl lactosamine sequences; at chain termini, binding to Galbeta1,4GlcNAc and Galbeta1,3GalNAc sequences was greatly enhanced after neuraminidase treatment. In all species, terminal NeuNAcalpha2,3 residues were detected. The tenrec had unusually abundant terminal N-acetyl galactosamine. The basal plasma membrane/basal lamina showed glycosylation patterns distinct from the microvillous membrane in each case, indicating chemical diversity of the two opposite faces of trophoblast. Similar classes of glycan at the hemochorial interface suggest conservation of function. The observed lectin binding patterns suggest broad similarities of glycosylation that may have arisen by convergent evolution.

The house musk shrew (Suncus murinus): a unique animal with extremely low level of expression of mRNAs for CYP3A and flavin-containing monooxygenase.

Expression of drug-metabolizing enzymes including cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) in various tissues of Suncus murinus (Suncus) were examined. Northern blot analysis showed that mRNAs hybridizable with cDNAs for rat CYP1A2, human CYP2A6, rat CYP2B1, human CYP2C8, human CYP2D6, rat CYP2E1, human CYP3A4 and rat CYP4A1 were expressed in various tissues from Suncus. The mRNA level of CYP2A in the Suncus lung was very high. Furthermore, it was found that the level of CYP2A mRNA in the Suncus lung was higher compared to the Suncus liver. The expression level of mRNA hybridizable with cDNA for human CYP3A4 was very low. The presence of CYP3A gene in Suncus was proven by the induction of the CYP with dexamethasone. Very low expression levels of mRNAs hybridizable with cDNAs for rat FMO1, rat FMO2, rat FMO3 and rat FMO5 were also seen in Suncus liver. No apparent hybridization band appeared when human FMO4 cDNA was used as a probe. The hepatic expression of mRNAs hybridizable with cDNAs for UDP-glucuronosyltransferase 1*6, aryl sulfotransferase, glutathione S-transferase 1, carboxyesterase and microsomal epoxide hydrolase in the Suncus were observed. These results indicate that the Suncus is a unique animal species in that mRNAs for CYP3A and FMO are expressed at very low levels.

Identification of chicken GnRH II in brains of metatherian and early-evolved eutherian species of mammals.

Two molecular forms of GnRH (chicken GnRH II and a second variant) are present in the brains of species from all the major vertebrate groups. In mammals, two forms are present in metatherian species and early-evolved eutherian species, but chicken GnRH II has not been identified in more advanced eutherian species. We investigated the nature of GnRH molecular forms in several early-evolved mammalian species, using high performance liquid chromatography and radioimmunoassay with specific GnRH antisera. These chromatographic and immunological data indicate that in the brains of a metatherian species (possum, Trichosurus vulpecula) and in two early-evolved eutherian species (order Insectivora: musk shrew, Suncus murinus and mole, Chrysochloris asiatica), both mammalian and chicken II GnRHs are present, while in another relatively early-evolved eutherian species (order Chiroptera: bat, Miniopterus schreibersii) only mammalian GnRH is present. In the adult possum and mole brains the proportion of chicken GnRH II was lower than that of mammalian GnRH, while in the musk shrew brain chicken GnRH II predominated. A peptide likely to be mammalian proGnRH was detected in the brains of the three eutherian species (musk shrew, mole, and bat). These findings suggest that metatherian and primitive eutherian species of mammals continue to express chicken GnRH II as in the vast majority of nonmammalian vertebrates, while the peptide is apparently not expressed in modern placental mammalian species. The functional significance of chicken GnRH II is not yet clear, but there are indications that it has a neurotransmitter or neuromodulator role in addition to that of regulating pituitary hormone release in certain vertebrate species.

Effect of starving and refeeding on lipid metabolism in suncus.

We have previously reported that fatty liver is easily induced in a novel experimental animal, Suncus murinus (suncus) by withholding food, and that apolipoprotein B (apo B) is not actively synthesized in the liver. In the present paper we describe the effect of starving and refeeding on lipid and lipoprotein metabolism in suncus, in order to explore the mechanisms of induction of fatty liver by starving and of its improvement by refeeding. Starvation induced increase in triglyceride content and decrease in glycogen content of the liver. Although the glycogen content returned to the level before starvation at 12 h after refeeding, the triglyceride content decreased gradually but did not reach the prestarvation level even at 24 h after refeeding in suncus. Plasma lipids, glucose, and insulin levels were decreased by starvation and returned to the levels before starvation between 8 and 24 h after refeeding. On the other hand, the plasma levels of free fatty acid and ketone bodies were elevated significantly by starvation and decreased rapidly by refeeding. These responses to starvation and refeeding, except for the change in hepatic triglyceride, are in common with other experimental animals, suggesting that there are no abnormalities in glucose metabolism or in fatty acid metabolism in suncus. In conclusion, the fatty liver induced by starvation in suncus may be caused by impaired triglyceride transport out of the liver, for which apolipoprotein B is mostly responsible, as reported previously.

Water metabolism in the Namib Desert golden mole, Eremitalpa granti namibensis (Chrysochloridae).

1. Laboratory and field studies of energy and water metabolism employing isotopic dilution methods examined the ability of Namib Desert moles to survive on an insect diet without drinking water. 2. Water independence is achieved through efficient renal function while low rates of energy usage and torpor are further effective in reducing overall water requirements.

The testes of moles (Talpa europaea) retain a considerable microsomal capacity for androgen synthesis during seasonal regression.

Seasonal changes in testicular histology and steroidogenesis were investigated in the mole (Talpa europaea). Androgen synthesis was examined by incubating [4-14C]pregnenolone (P) and [4-14C]dehydroepiandrosterone (DHA) with testicular minces in a static incubation system. The metabolites formed were characterized by thin-layer chromatography. Morphological changes were studied by routine histological methods. During sexual quiescence spermatogenesis was arrested. The regressive seminiferous tubules consisted predominantly of Sertoli cells and spermatogonia. On the other hand, histological quantification suggested that season has no significant effects on the number or the nuclear dimensions of Leydig cells in this species. The capacity of the regressive testes (per unit weight) to metabolize P and DHA to testosterone (T) was somewhat greater in regressive (48.5%, 49.4%) than in active (33.2%, 41.6%) testes. The results also suggest that the greater in vitro T production encountered during reproductive quiescence is due possibly to an increase in the activity of 17 beta-hydroxysteroid dehydrogenase (per unit weight). Our data on Leydig cell numbers indicate, however, that the capacity of the individual Leydig cells to produce T is decreased during sexual regression. T. europaea appears to be quite exceptional among seasonally breeding small mammals exhibiting pronounced annual changes in spermatogenesis in that the testes retain a considerable enzymatic capacity to produce testosterone from pregnanes during sexual quiescence. The results suggest that pituitary as well as paracrine regulation of the annual testicular cycle in this species differs from that generally encountered in seasonal breeders.

Alpha, mu and pi glutathione S-transferases: species (Talpa europaea) differences in their expression.

1. Tissue cytosols from Talpa europaea were examined for their glutathione S-transferase isoenzyme content by chromatofocusing, inhibition and immunological techniques and the results compared with data from adult human tissue cytosols. 2. Two sets of glutathione S-transferase isoenzymes were found in liver cytosol of Talpa europaea, they demonstrated similar properties to human alpha and mu isoforms. 3. There was no evidence of expression of the pi isoenzyme set in any of the tissues studied and in this respect Talpa europaea differs from other mammalian species studied so far.

Alanine:glyoxylate aminotransferase activities in liver of Suncus murinus (insectivora).

1. The distribution of L-alanine:glyoxylate aminotransferase (AGT) activities were found in Suncus liver, 55% in particulate fraction and 45% in supernatant. 2. 65% of AGT activities in particulate were dependent on AGT isoenzyme 2 (AGT 2) having molecular weight 210,000, the remainder (35%) of AGT activities were dependent on AGT isoenzyme 1 (AGT 1) which have aminotransferase activity for serine. AGT activities in supernatant were dependent on AGT 1, AGT 2 and alanine:2-oxoglutarate aminotransferase (GPT), and their activity ratios were 10, 15 and 75%, respectively. 3. Km values for alanine were 0.52 mM; AGT 1, 3.3 mM; AGT 2, 0.88 mM; GPT measuring with AGT activity. AGT activity of GPT was inhibited by addition of glutamate and its Ki value was 1.8 mM. 4. Some other properties of AGT 1, AGT 2 and GPT are described.

[Cerebrosides and sulfocerebrosides in the brain of insectivorous mammals]. / Tserebrotsidy i sul'fotserebrozidy golovnogo mozga nasekomoiadnykh mlekopitaiushchikh.

Brain cerebrosides (C) and sulfocerebrosides (S) of Insectivora, which represent the most ancient and primitive order of placentary mammals, were first studied. The content of C and S is higher in hedgehogs (Erinaceus europaeus, E. auritus) and mole (Talpa europaea) brains as compared to shrew (Sorex araneus) brain. Hydroxy fatty acids predominate over normal fatty acids in C of all studied insectivora brains. The fatty acid content of C and S of the Insectivora brains is similar in being rich in palmitic, stearic and behenic normal acids and hydroxybehenic and hydroxylignocerinic acids. Hydroxy fatty acids of C and S are more saturated and they have a longer chain (sum C24-26 acids) than the normal acids. C and S of insectivora and primate brains are compared. The data may be of importance for the understanding of the biochemical evolution of the nervous system of mammals.

The pineal gland of the mole (Talpa europaea L.). VII. Activity of hydroxyindole-O-methyltransferase (HIOMT) in the formation of 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3-acetic acid, 5-methoxytryptophol and melantonin in the eyes and the pineal gland.

Using a method in which no substrate is added to the incubation medium, the capacity of HIOMT to synthesize 5-methoxytryptophan, 5-methoxytryptamine, 5-methoxyindole-3-acetic acid, 5-methoxytryptophol and melatonin has been determined in the pineal and the eyes of the mole, a mammal having an atrophied visual system. The results demonstrate that the indoleamine metabolism in the retina is similar to the indoleamine metabolism in the pineal. Moreover, in all animals studied both eyes together synthesize 2 to 30 times more of 5-methoxyindoles than the pineal, a result which proves again that the pineal is not the only and not always the most important source of 5-methoxyindoles. With the exception of 5-methoxyindole-3-acetic acid, the synthesis of which is practically neglectable, the production of all other 5-methoxyindoles in the pineal as well as in the retinae is always larger than that of melatonin. In the pineal, 5-methoxytryptophan, for example, is synthesized in a quantity which is 60 to 170 times larger than that of melatonin, while in the retinae the synthesized amount of 5-methoxytryptophan is even 60 to 1000 times larger than that of melatonin.

Preliminary investigations of melatonin and 5-methoxy-tryptophol synthesis in the pineal, retina, and harderian gland of the mole rat and in the pineal of the mouse "eyeless".

Hydroxyindole-O-methyltransferase (HIOMT) activity for the synthesis of melatonin and 5-methoxytryptophol, both 5-methoxyindoles, was measured in the pineal, the Harderian gland and the retina of the mole rat and in the pineal of the mouse "eyeless". In the pineal and the Harderian gland of the mole rat a larger amount of 5-methoxytryptophol than of the melatonin is synthesized. 5-Methoxyindole synthesis is extremely high in the Harderian gland, whereas in the retina HIOMT activity is low and variable. In the pineal of the mouse "eyeless", a low 5-methoxyindole synthesis showing no circadian rhythm is demonstrated. It is concluded that, besides the generally accepted regulation of the indole metabolism by light, in species with atrophied eyes having Harderian glands (mole rat) and in species without eyes other factors than light might be responsible for the indole metabolism in the pineal gland.