Extracts from the brains of hibernating and alert ground squirrels: effects on cells in culture.

Abstract

Aqueous extracts were prepared from pulverized, acetone-dehydrated brains of hibernating and alert ground squirrels. Addition ofthese extracts to Chinese hamster ovary cells in suspension culture resulted in a decrease in the amount of [3H]thymidine incorporated into acid-precipitable material without affecting the transport or phosphorylation of the nucleoside. The inhibition was time-and dose-dependent and full recovery occurred about 2 hr after exposure ofthe cells to the active extract.The active factor is readily oxidized during storage at -700C but full activity can be restored by treatment with 2-mercaptoethanol. The peptide nature of the active material is indicated by its susceptibility to proteases and by loss of activity after alkylation. Fasted or cold-acclimated rats also develop increased levels of active substance in their brains; however, brains of hibernating squirrels contain 10-to 50-fold more of the active substance than brains from either alert squirrels or rats. A time-dependent increase in activity ofextracts from hibernating brain incubated with a homogenate from alert brain suggests that the peptide is activated or generated in the mixture.Attempts have been made to isolate and identify factors that induce or regulate the hibernating state. Dawe and co-workers (1-6) have obtained a fraction from the blood of hibernating woodchucks and ground squirrels ("trigger") that, when injected into alert ground squirrels, induces hibernation even in the summer. Other researchers have failed to obtain a significant induction of hibernation in squirrels (7,8) or hamsters (9) using a similar protocol. Swan et al (10-13) have prepared extracts of brains obtained from hibernating or aestivating animals that have been reported to cause a decline in both body temperature and oxygen consumption when injected intravenously into rats. The large quantities of brain extract required to elicit a response in rats precluded the use ofthis whole animal assay in isolating the factor responsible for lowering metabolic rates. In the present study we have developed a sensitive cell culture bioassay capable ofdistinguishing between extracts prepared from brains of hibernating and alert ground squirrels. Although the levels ofthe active substance in the extract correlate with entrance into hibernation, a similar activity can be detected in brain extracts ofhomeotherms where the levels ofthe active agent respond to altered environmental conditions. The nature of the active substance and the cellular response to it have been investigated. Brains, obtained from animals sacrificed by decapitation, were removed immediately and frozen in either liquid nitrogen or acetone and dry ice. Cold-acclimated squirrels were maintained at 80C and had been in hibernation previously but were not in hibernation on the day ofsacrifice. Hibernating squirrels were also maintained at 80C and had been in hibernation on the observation day previous to the day of sacrifice (2-3 days earlier). Squirrels that were entering hibernation were...

Circannual Control of Hibernation by HP Complex in the Brain

“…Although attempts have been made for over half a century to identify substances responsible for hibernation in the blood and organs of hibernating animals (Amorese et al, 1982;Wang, 1988), there has been little success. In this study, the HP20 complex, HP20c, is identified as a promising candidate hormone controlling hibernation.…”

Potential for Discovery of Neuroprotective Factors in Serum and Tissue from Hibernating Species

“…Attenuation of cellular proliferation in various tissues during torpor occurs in the G1 phase [84], the premitotic phase [85], or in other phases of the cell cycle as reviewed by Lyman [86]. Brain extracts from hibernating TLS decrease cell division in chinese hamster ovary cells (CHO) [87]. If identified, the factors that regulate entrance into and exit from the cell cycle in hibernating animals may prove to be useful in treating cancer or in avoiding neurodegenerative disease states such as Alzheimer disease which show cell cycle dysregulation [88].…”

Sustained torpidity following multi‐dose administration of 3‐iodothyronamine in mice

“…Regarding internal mechanisms underlying hibernation, plasma or brain extracts from hibernating squirrels were shown to effectively induce summer hibernation in squirrels and reduce metabolic rate and T b in non‐hibernator rodents (Dawe et al, 1970; Swan and Schatte, 1977; Amorese et al, 1982). Recent studies have further demonstrated that a one‐time exposure to agents called “hibernation induction triggers” (HIT) was sufficient to induce hibernation‐like state for several hours in laboratory mice (Su, 2000; Scanlan et al, 2004; Blackstone et al, 2005; Lee, 2008).…”