Suppression of protein synthesis in brain during hibernation involves inhibition of protein initiation and elongation

Abstract

Protein synthesis (PS) has been considered essential to sustain mammalian life, yet was found to be virtually arrested for weeks in brain and other organs of the hibernating ground squirrel, Spermophilus tridecemlineatus. PS, in vivo, was below the limit of autoradiographic detection in brain sections and, in brain extracts, was determined to be 0.04% of the average rate from active squirrels. Further, it was reduced 3-fold in cell-free extracts from hibernating brain at 37 degreesC, eliminating hypothermia as the only cause for protein synthesis inhibition (active, 0.47 +/- 0.08 pmol/mg protein per min; hibernator, 0.16 +/- 0.05 pmol/mg protein per min, P < 0.001). PS suppression involved blocks of initiation and elongation, and its onset coincided with the early transition phase into hibernation. An increased monosome peak with moderate ribosomal disaggregation in polysome profiles and the greatly increased phosphorylation of eIF2alpha are both consistent with an initiation block in hibernators. The elongation block was demonstrated by a 3-fold increase in ribosomal mean transit times in cell-free extracts from hibernators (active, 2.4 +/- 0.7 min; hibernator, 7.1 +/- 1.4 min, P < 0.001). No abnormalities of ribosomal function or mRNA levels were detected. These findings implicate suppression of PS as a component of the regulated shutdown of cellular function that permits hibernating ground squirrels to tolerate "trickle" blood flow and reduced substrate and oxygen availability. Further study of the factors that control these phenomena may lead to identification of the molecular mechanisms that regulate this state.

Figure 1

Color-coded transforms of representative autoradiograms, showing rates of cerebral PS in an active (A) and a hibernating (B) ground squirrel at the level of the arcuate nucleus. During hibernation, no autoradiographic evidence of leucine incorporation was detected.

Figure 2

SDS/PAGE separation of proteins in brain, liver, and heart from a hibernating and an active squirrel. A shows the Coomassie-stained gel and B shows the autoradiogram produced by l-[³⁵S]methionine incorporation. During hibernation, incorporation was below the detection limit of the PhosphorImager (1, brain/active; 2, brain/hibernating; 3, liver/active; 4, liver/hibernating; 5, heart/active; 6, heart/hibernating).

Figure 3

In vitro translation in PMS at 37°C. After introduction of l-[3,4,5-³H(N)]leucine into the medium, the reaction was allowed to proceed for 30 min. Incorporation of leucine into TCA-precipitable material was several times higher in PMS from active (Left) compared with PMS from hibernating (Right) brain (−CHX). Incorporation was blocked by 5 mM cycloheximide (+CHX).

Figure 4

Analyses of representative ribosomal profiles in PMS from hibernating and active brains. PMS was analyzed by means of a linear 15–55% sucrose gradient. Note the moderate disaggregation of the polysomal fraction (P) and increase of the monosomal peak (M) in PMS from hibernating brain.

Figure 5

In vitro translational capability of ribosomes isolated from hibernating and active brains. Ribosomes from hibernating (n = 3) and active (n = 3) brains were incubated in ribosome-depleted PMS from active brain. The ribosomes from the two sources showed comparable levels of leucine incorporation, both in the presence and absence of 1 mM 7-methyl-GTP. RNA content was adjusted to 1 OD₂₆₀unit/assay. Bars represent the incorporation of [³H]leucine into protein (mean ± SD).

Figure 6

Immunoblots of total (Upper) and phosphorylated (Lower) eIF2α in brain homogenates from active (lanes 1–3) and hibernating (lanes 4–6) squirrels.

Figure 7

Mean transit time determinations in PMS from active and hibernating squirrel brain. Incorporation of radioactivity in total (solid circles) and soluble (open circles) protein from a representative experiment is plotted against time (Upper). The difference between the time axis intercepts of the linear regression lines corresponds to a one-half transit time (HTT). (Lower) Ribosome transit times (mean ± SD) measured in extracts from five active and five hibernating squirrel brains were significantly different by Student’s t test.