Lipids in mammalian hibernation and artificial hypobiosis

Membrane lipids—phospholipids, fatty acids, and cholesterol—participate in thermal adaptation of ectotherms (bacteria, amphibians, reptiles, fishes) mainly via changes in membrane viscosity caused by the degree of fatty acids unsaturation, cholesterol/phospholipids ratio, and phospholipid composition. Studies of thermal adaptation of endotherms (mammals and birds) revealed the regulatory role of lipids in hibernation. Cholesterol and fatty acids participate in regulation of the parameters of torpor, gene expression, and activity of enzymes of lipid metabolism. Some changes in lipid metabolism during artificial and natural hypobiosis, namely, increased concentration of cholesterol and fatty acids in blood and decreased cholesterol concentration in neocortex, are analogous to those observed under stress conditions and coincide with mammalian nonspecific reactions to environmental agents. It is shown that the effects of artificial and natural hypobiosis on lipid composition of mammalian cell membranes are different. Changes in lipid composition cause changes in membrane morphology during mammalian hibernation. The effect of hypobiosis on lipid composition of membranes and cell organelles is specific and seems to be defined by the role of lipids in signaling systems. Comparative study of lipid metabolism in membranes and organelles during natural and artificial hypobiosis is promising for elucidation of adaptation of mammals to low ambient temperatures.     

The role of homeoviscous adaptation in mammalian hibernation

The bulk membrane fluidity of brain synaptosomes and kidney cortex microsomes of hibernating and active mammals have been compared using the steady-state fluorescence polarization technique. No consistent differences were observed indicating that homeoviscous adaptation may not be an important strategy during hibernation.     

Skeletal muscle hexokinase: regulation in mammalian hibernation

The perfused rat liver responds in several ways to NAD⁺ infusion (20–100 μM). Increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption and gluconeogenesis are some of the effects that were observed. Extracellular NAD⁺ is also extensively transformed in the liver. The purpose of the present work was to determine the main products of extracellular NAD⁺ transformation under various conditions and to investigate the possible contribution of these products for the metabolic effects of the parent compound. The experiments were done with the isolated perfused rat liver. The NAD⁺ transformation was monitored by HPLC. Confirming previous findings, the single-pass transformation of 100 μM NAD⁺ ranged between 75% at 1.5 min after starting infusion to 95% at 8 min. The most important products of single-pass NAD⁺ transformation appearing in the outflowing perfusate were nicotinamide, ADP-ribose, uric acid, and inosine. The relative proportions of these products presented some variations with the time after initiation of NAD⁺ infusion and the perfusion conditions, but ADP-ribose was always more abundant than uric acid and inosine. Cyclic ADP-ribose (cADP-ribose) as well as adenosine were not detected in the outflowing perfusate. The metabolic effects of ADP-ribose were essentially those already described for NAD⁺. These effects were sensitive to suramin (P2_XY purinergic receptor antagonist) and insensitive to 3,7-dimethyl-1-(2-propargyl)-xanthine (A2 purinergic receptor antagonist). Inosine, a known purinergic A3 agonist, was also active on metabolism, but uric acid and nicotinamide were inactive. It was concluded that the metabolic and hemodynamic effects of extracellular NAD⁺ are caused mainly by interactions with purinergic receptors with a highly significant participation of its main transformation product ADP-ribose.     

Suppression of MAPKAPK2 during mammalian hibernation

Metabolic signaling coordinates the transition by hibernating mammals from euthermia into profound torpor. Organ-specific responses by activated p38 mitogen activated protein kinase (MAPK) are known to contribute to this transition. Therefore, we hypothesized that the MAPK-activated protein kinase-2 (MAPKAPK2), a downstream target of p38 MAPK, would also be active in establishing the torpid state. Kinetic parameters of MAPKAPK2 from skeletal muscle of Richardson’s ground squirrels, Spermophilus richardsonii, were analyzed using a fluorescence assay. MAPKAPK2 activity was 27.4 ± 1.27 pmol/min/mg in muscle from euthermic squirrels and decreased by ∼63% during cold torpor, while total protein levels were unchanged (as assessed by immunoblotting). In vitro treatment of MAPKAPK2 via stimulation of endogenous phosphatases and addition of commercial alkaline phosphatase decreased enzyme activity to only ∼3–5% of its original value in muscle extracts from both euthermic and hibernating squirrels suggesting that posttranslational modification suppresses MAPKAPK2 during the transition from euthermic to torpid states. Enzyme S_0.5 and n_H values for ATP and peptide substrates changed significantly between euthermia and torpor, and also between assays at 22 versus 10 °C but, kinetic parameters were actually closely conserved when values for the euthermic enzyme at 22 °C were directly compared with the hibernator enzyme at 10 °C. Arrhenius plots showed significantly different activation energies of 40.8 ± 0.7 and 54.3 ± 2.7 kJ/mol for the muscle enzyme from euthermic versus torpid animals, respectively but MAPKAPK2 from the two physiological states showed no difference in sensitivity to urea denaturation. Overall, the results show that total activity of MAPKAPK2 is in fact reduced, despite previous findings of p38 MAPK activation, and kinetic parameters are altered when ground squirrels enter torpor but protein stability is not apparently changed. The data suggest that MAPKAPK2 suppression may have a significant role in the differential regulation of muscle target proteins when ground squirrels enter torpor.     

Suppression of MAPKAPK2 during mammalian hibernation

Metabolic signaling coordinates the transition by hibernating mammals from euthermia into profound torpor. Organ-specific responses by activated p38 mitogen activated protein kinase (MAPK) are known to contribute to this transition. Therefore, we hypothesized that the MAPK-activated protein kinase-2 (MAPKAPK2), a downstream target of p38 MAPK, would also be active in establishing the torpid state. Kinetic parameters of MAPKAPK2 from skeletal muscle of Richardson’s ground squirrels, Spermophilus richardsonii, were analyzed using a fluorescence assay. MAPKAPK2 activity was 27.4 ± 1.27 pmol/min/mg in muscle from euthermic squirrels and decreased by ∼63% during cold torpor, while total protein levels were unchanged (as assessed by immunoblotting). In vitro treatment of MAPKAPK2 via stimulation of endogenous phosphatases and addition of commercial alkaline phosphatase decreased enzyme activity to only ∼3–5% of its original value in muscle extracts from both euthermic and hibernating squirrels suggesting that posttranslational modification suppresses MAPKAPK2 during the transition from euthermic to torpid states. Enzyme S_0.5 and n_H values for ATP and peptide substrates changed significantly between euthermia and torpor, and also between assays at 22 versus 10 °C but, kinetic parameters were actually closely conserved when values for the euthermic enzyme at 22 °C were directly compared with the hibernator enzyme at 10 °C. Arrhenius plots showed significantly different activation energies of 40.8 ± 0.7 and 54.3 ± 2.7 kJ/mol for the muscle enzyme from euthermic versus torpid animals, respectively but MAPKAPK2 from the two physiological states showed no difference in sensitivity to urea denaturation. Overall, the results show that total activity of MAPKAPK2 is in fact reduced, despite previous findings of p38 MAPK activation, and kinetic parameters are altered when ground squirrels enter torpor but protein stability is not apparently changed. The data suggest that MAPKAPK2 suppression may have a significant role in the differential regulation of muscle target proteins when ground squirrels enter torpor.     

The role of membrane fatty acids in mammalian hibernation

During mammalian hibernation, cellular membranes continue to function at temperatures approaching 0 C. The molecular mechanisms that confer this capacity to the membranes are unknown but may be related to the fluidity of the membrane and to the level of unsaturated fatty acids. The basic tenets of membrane fluidity and the contribution of cholesterol, polar head groups, and fatty acids toward maintaining a fluid membrane in a liquid-crystalline state are examined in this review. It is shown that although unsaturated fatty acids can enhance membrane fluidity at low temperatures, there does not appear to be a consistent trend toward increased levels of unsatruated fatty acids during hibernation in all tissues of hibernators. Consequently, there may be some other role for the alterations in the composition of membrane fatty acids found during the hibernating cycle other than increasing membrane fluidity to permit continued activity at reduced temperatures.     

A simple molecular mathematical model of mammalian hibernation

A simple model of the dynamics of the body temperature of a hibernating mammal is presented. Our model provides a good match to experimental data, showing the interruption of low-temperature torpor bouts with periodic interbout arousals (IBAs). In this paper we present a mathematical model of the molecules that participate in the initiation, regulation, and maintenance of the hibernating state. This model can be used to describe the role of regulatory molecules, signal transducers, downstream target enzymes, structural proteins, or metabolites. Because many of the biochemical mechanisms are unknown, this is a preliminary and largely phenomenological model that we hope will inspire further investigation.     

Identification of novel blood proteins specific for mammalian hibernation.

Mammalian hibernation is a unique physiological adaptation that allows the sustainment of life under extremely low body temperatures. In the chipmunk, we found four proteins related specifically to hibernation. These proteins started to diminish in concentration in the blood before and disappeared during hibernation. These proteins reappeared in the blood as hibernation ceased and remained during nonhibernation. The complete or partial amino acid sequences of the four proteins showed that three (27-, 25-, and 20-kDa) were previously unknown, whereas another (55-kDa) is highly homologous with alpha 1-antitrypsin. The three novel proteins are homologous, indicating that they are a family. In the NH2-terminal regions of these proteins, a collagen-like amino acid sequence is present, whereas in their COOH-terminal regions, two sequences, Ser-Ala-Phe-Ala-Val-Lys and Val-Trp-Leu-Glu, are conserved. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and gel permeation chromatography under denaturating conditions revealed that the four proteins form a 140-kDa complex in the plasma fraction. The novel proteins were detected in blood of another hibernator, the ground squirrel, but not in rodent nonhibernators, namely tree squirrels and rats. The present finding is the first identification of a hibernation-specific protein. The presence of specific proteins in hibernators suggests the involvement of genetic factors in the control of hibernation. These proteins provide valuable tools for understanding molecular mechanisms of mammalian hibernation.     

Hepatic gluconeogenesis and mitochondrial function during hibernation

1. The aim of these studies was to investigate a mitochondrial basis for changes in gluconeogenesis during hibernation. 2. State 3 respiration rates in liver mitochondria from hibernating ground squirrels were reduced by 62-66%. The limiting reaction appeared to be electron transport, particularly in respiratory complex III. 3. The mitochondrial ATP + ADP + AMP content was reduced by 29% during hibernation; cellular adenine nucleotide content was unchanged. 4. Pyruvate carboxylation in intact mitochondria was decreased 75% during hibernation, although total pyruvate carboxylase activity was not lower. 5. Rates of gluconeogenesis in intact hepatocytes isolated from hibernators were lower than in cells from non-hibernators.     

The physiological link between metabolic rate depression and tau phosphorylation in mammalian hibernation

Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD). Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with conformational changes of highly phosphorylated tau protein that are typically related to neuropathological alterations. The particular hibernation characteristics of black bears with a continuous torpor period and an only slightly decreased body temperature, therefore, potentially reflects the limitations of this adaptive reaction pattern and, thus, might indicate a transitional state of a physiological process.     

Connexins in mammalian heart function

In heart, the propagation of electrical activity is mediated by intercellular channels, referred to as junctional channels, aggregated into gap junctions and localised between myocytes. These channels consist of structurally related transmembrane proteins, the connexins, three of which (CX43, CX40 and CX45) have been shown to be associated with the myocytes of mammalian heart; a fourth, CX37, was detected exclusively in endothelial cells. In this paper, we review the recent data dealing with the topographical heterogeneity of expression of these connexins in the different cardiac tissues and the unique conductance properties of the channels they form, and attempt to assess the role played by each connexin and the consequences of their multiplicity in the propagation of action potentials.     

Translational initiation is uncoupled from elongation at 18 degrees C during mammalian hibernation

Cellular and organismal homeostasis must be maintained across a body temperature (Tb) range of 0 to 37 degrees C during mammalian hibernation. Hibernators depress biosynthetic activities including protein synthesis, concordant with limited energy availability and temperature effects on reaction rates. We used polysome analysis to show that initiation of protein synthesis ceases during entrance into torpor in golden-mantled ground squirrels (Spermophilus lateralis) when Tb reaches 18 degrees C. Elongation of preinitiated polypeptides continues slowly throughout the torpor bout. As Tb begins to rise, initiation resumes even at temperatures below 18 degrees C, although the euthermic polysome pattern is not reestablished. At precisely 18 degrees C, there is a large increase in initiation events and a complete restoration of euthermic polysome distribution patterns. These data indicate a role for both passive and active depression of translation during torpor and are consistent with a requirement for new protein biosynthesis during each interbout arousal.     

Evaluation of the role of AMP-activated protein kinase and its downstream targets in mammalian hibernation

Mammalian hibernation requires an extensive reorganization of metabolism that typically includes a greater than 95% reduction in metabolic rate, selective inhibition of many ATP-consuming metabolic activities and a change in fuel use to a primary dependence on the oxidation of lipid reserves. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in this reorganization. AMPK activity and the phosphorylation state of multiple downstream targets were assessed in five organs of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) comparing euthermic animals with squirrels in deep torpor. AMPK activity was increased 3-fold in white adipose tissue from hibernating ground squirrels compared with euthermic controls, but activation was not seen in liver, skeletal muscle, brown adipose tissue or brain. Immunoblotting with phospho-specific antibodies revealed an increase in phosphorylation of eukaryotic elongation factor-2 at the inactivating Thr56 site in white adipose tissue, liver and brain of hibernators, but not in other tissues. Acetyl-CoA carboxylase phosphorylation at the inactivating Ser79 site was markedly increased in brown adipose tissue from hibernators, but no change was seen in white adipose tissue. No change was seen in the level of phosphorylation of the Ser565 AMPK site of hormone-sensitive lipase in adipose tissues of hibernating animals. In conclusion, AMPK does not appear to participate in the metabolic re-organization and/or the metabolic rate depression that occurs during ground squirrel hibernation.     

Membrane dynamics in mammalian embryogenesis: Implication in signal regulation

Eukaryotes have evolved an array of membrane compartments constituting secretory and endocytic pathways that allow the flow of materials. Both pathways perform important regulatory roles. The secretory pathway is essential for the production of extracellular, secreted signal molecules, but its function is not restricted to a mere route connecting intra‐ and extracellular compartments. Post‐translational modifications also play an integral function in the secretory pathway and are implicated in developmental regulation. The endocytic pathway serves as a platform for relaying signals from the extracellular stimuli to intracellular mediators, and then ultimately inducing signal termination. Here, we discuss recent studies showing that dysfunction in membrane dynamics causes patterning defects in embryogenesis and tissue morphogenesis in mammals. Birth Defects Research (Part C) 108:33–44, 2016. © 2016 Wiley Periodicals, Inc.