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Hepatic polyribosomes and protein synthesis: Seasonal changes in a hibernator
HEPATIC ~~L~~~US~~ES
AND PROTEfN S~ES~S
:
SEASONAL CHANGES IN A HIBERNATOR B. K. WHITI’EN,
1;. 85, SCHRADER,*
R. L. HUSTON,
AND G. R. HONOLD?
U.S. Army Institute of Environm~tal Medicine, Natick, wwtu
or76o, USA., and U.S. Army Medical Research and Nutrition Laboratory, Denver, Cokado 80240, U.S.A.
3. The datasuggw that the ‘ nutritionai state ‘ofthcaxtixi&tllaybeaninlportantfactor in these observed aktxtiom.
THE, relationship between ribosome aggregates (polyribosomcs) and protein synthesis has received considerable attention since several kutigazors demonstrated that these aggregates were the active partides in protein synthesis (Marks, Burka, and SchIessinga, ~$2 ; Warner, Rich, and Hall, 1962 ; Gir, 1963 ; Wetstein, %,a&&, and No& 1963)‘ Recently, Munro (1968) showed that hepatic ribosomal aggregation and protein synthetic capacity were related to the mztritional state of the animal with pixrtieular rcrfcrrncc to amino-acid supply and/or imbalance. Thus %uctuations in nutritive state would be expected to inffnence hepatic ribosomaI aggregation and protein synthetic capacity.’ Hibernators have a seasonal pattern -of winter Iethargia, characterized by prolonged periods of deep hypothermia and fasting during which time they may lose weigh& and summer activity, characterized by rcfading, growth, and rapid weight+in (Pcngc.llcy, x967). This SeasoIlitl p&tern appeared to make the hibanator an ~cellent model * Prucut 3ddrcu: aepartmwat of A$pxlomy, Unidty of wiswMiny Madison, wii3cansin 33106s U.S.A. *+seqt address: Gena-al Mills Research tabotstoraa, MinncapoEs, M;innaota 55427, U.SJL
to study seasonal changes in protein synthesis and to conrelate the nutritive and metabolic condition of the animal with hepatic ribosome aggregation and protein synthetic qacity. Tk+cn”ti grou3xd squirret (C&a&@ ti-) were obtained in the spring, pked ininditiduaIcagqqndfedadietofPur&taLab Chow, carrots, and water ad &i&m. A&m& were randomly assigned to three groups m follows: (a) summer normothcnnic animals w&h were maintaind at 22' C. and sacri%ccd in August; (e) titer noamothcrxnic animala which were maintained at 22'C. and sacrificed in January (at the time of sacrifice these at&n& were acdvc and not iethar& or hyp&xsnic);
!i$.mmcrand winter normotbcrmic anima&wencfedadietofPurinaLabCbaw, carrota and watcrrtdlibihrm. Hibunating animala were without food but had water available ad
sacrifice).
libitlhnl. IAers fhm
4 animrlr in each group WffF pooled and polyribcaomu and cell sap enzytnca a&acted using the following modifications of the mcthodsofWunner,Ekll,andMunro (rg66).The cx&zusion meditun contained 25omM sl+tcmq 5omMH EPES buff& 25mM KU, 25m.M KOH, 5m.M M&Z&, and 3mM glutathicxtc. acpE3 ~6 and the P+iba5onl~ WeE -
197% ID 40%~
SRASONAL
CHANGES
IN PROTEIN
post-mitochondrial supemataut (PMS) was treated with d&late (I ‘67 per cent). A polyxibosome pellet was obtained by layeriog the d-t-treated PMS over a discontinuous gradiew (~5 and wo M sucrose b&x) and cent&zging at 32x.ooog for 1.75 houn. Ceil a&acted at pH 6.4 and saPenzYm@JWe= centrifuged as described for the polyribosomes, except that only 0’5 M aucroae buffer was used for the gradient. An aliquot of the cell sap supernatant was passed through a Sephadex G-IO
407
SYNTHEW
subtracted from complete assay values. All samples were run in triplicate and the experiment was repeated. RESULTS
AND DISCUSSION
polyribosomes isolated from summer normothermic animals were characterized by a large number of high molecular weight aggregates (Fig. IA) and a rapid Hepatic
FIG. I.-Hepatic polyribosome pro&s made on isokinetic sucrose damity gradients. A, Polyribounnca from summer normothermic ground squirrds. B, PolyriW tPm winter normothermic ground squinels. C, Polyribowmes from winter bibcmatiog ground squirrels. column to remove fm amino-acids. Polyribosome profiles were obtained on isokinetic sucrose gradients as dcacribed by Nell (1967). Protein synthesis was assayed at 37” C. in a cell-free system using polyribosomcs, cell sap, a complete aminoacid mix, 14-carh taggal amino-aci4 and an energy system. The akuay medium (0.7ml. per assay tube) contaioed the fdlowing, in jlmok!s: sucrose 175; H.EP= buffer 35; KCl 17’5; KOH 17’5; MgCl, 3.5; ATP I; GTP WI; creatine phosphate 20; mixture of 19 L-amino-acids 0*x9. Each assay tube also contained 20 pg. aeatine phosphokinase, 0’2 pc. ach of 14-carbon uniformly labdled ~&wine, L-lysine, L-phenylalanine, and L-valine. An aliquot of freshly prqxed L-cylkne (0.01 *ok per assay) was added and the pH was adjusted to 7’3 with HCl at 37” C. Tke assay mixture was pre-incubated at 37’ C. for 2 minutes. Immediately thereafter ~2mLofcellsapando~rmLofpolyribosomc3 (4-5 a3%d0’1 mg. protein respectively) were added and the reaction allowed to proceed for 2 rnin~tea. *Cold _IO per cent TCA containing iLrrmK)sQcL was used to stop the reaction ZLpitate protein. Proteio was isolated and washed uctekvely before detpzmi&g radie activity aaordiog to standard methods. Reactions were run in triplicate and cell sap blank values (incubations without polyribosomcs) were
rate of protein synthesis ( lobi I). Polyribosomes from winter normothermic animals were characterized by very little high molecular weight material, 4 or 5 distinct peaks of lower molecular weight aggregates (Fig. I B), and a lower rate of protein synthesis ( IkHel) . Winter hibernating animals had few polyribosomcs, a majority of monomer or single ribosomal units (Fig. I C), and a very low rate of protein synthesis (lob& I). These results demonstrate that hepatic protein synthetic capacity in hibernators is influenced by season and is related to ribosome aggregation. Summer normothermic animals were active and well fed. In contrast, winter normothermic animals ate less, were lethargic, and lost approximately x5-20 per cent of their body-weight between August and January. Pengelley (1967) reported a similar seasonal decrease in food intake and associated weight-loss in golden-mantled ground squirrels maintained at 22’ C. and suggested that these parameters were influenced by a circannian (seasonal) rhythm.
WHIlTEN ET AL.
408
‘Ikwfke the observed &aggregation of polyrihosomes and decreased protein synthetic capacity in winter normotherfnic animals compared to summer normothermic animals appear to be due, in part, to a circannian rhythm of food inta+ke not associated with cold temperatures or deep To&la I.-In
Vi&zAg
p_s
Sm
wiiter
hibcmating
TheauthorawiahtocxpresstheirthankstoDr. H. Sauberlicfi and Dr. J. Heon of the U.S. Army Medicsl R~~Nu~d~~~, and -Dr. R. Bu&ngton, Dr. M. Landowne, a& Dr. W. Evans of the U.S. Army Ruawch Institute of Environmcr~talMedicine, for their support and beIp&i commmu during this study.
w
* Gromdspuirrsltian suulmer llormotimmic Winter normothermic
ACICNOWLEDGEMWTS
Fro&inSW* 341925*25&r 6224 & I 222 754f 547
*Values
cxpruscd as z&SD of triplicate determinationsfor two replicates. Values rcprc-
sent DPM x4-carbon labelled amino-acids korporated per 2 &utes per rag. pdyxiU prwcin.
hypothermia. Neariy complete disaggregation of polyribosomes, decreased protein synthetic capacity, and a marked weight-loss (30-45 per cent of prehibanation weight) during winter hibernation may be a fiuxction of the deep hypothermia of these animals, prolonged f&sting, or a combination of these factors. These data suppart previous results which showed that a crude hepatic microsomal preparation from winter hibernating &irteen-lined ground squirr& bad a deaeased protein synthetic capacity at 37” C. when compared to a similar preparation from summer normothermic animals at the same incubation temperature (Whitten and Klain, MW Alterations in plasma and liver amino-acid patterns brought about by fasting and aminoacid imbalancea are known to cause disaggregation of polyribosomes and a decrease in protein synthetic capacity (Munro, rg68). The dccmsed food intake ofwinter noRnot&&xi& pW prolo* fasting amino-aad levels (Klain and whitbcrr, x968; Kristoff&sson and Broberg, 1968) in hibernating animals suggut that the ‘ nutritive state’ may be an important factw in the observed d@gregation of hepatic polyribosomes and deueased hcpatic prbtdn synthetic capacity in these animals.
REFERENcEs ‘Function of aggregated r&Z&CYt~ IibO8oXICSin Dr0tCh.l SWltbiS’. 3. mok i3&d., 4 143-157. * . . KLAIN, G. J., and Wxumm, B. K. (19681, ’ Pimma f&c amino acids in hibernation and G=mt,
A.
(~$31,
arousal‘, Gm&. Biachem. Phpsiol.,q, 617-619. R., and ~OBERC3, s. (x968), ‘F~C~O~~~~~Ofh~~~ in deep hypothcmia and after spontaneous arousals*, E,xj&m& 49 14ar50. M,mxs, P., BURU, E., and f%XiUWNGkR, D. -FFERSSON,
(11~62)~ ‘ protein synthd in aythroid cells, I. Retxulocyte riactive in stimulating amino acid incorporation ‘, Roe. &?I. Acod. sci. U.S.A., &,2x63-2170. Mmo, H. N. (I g68), ’ Roie of amino acid supply in regulating r&oeome fimction’, F& Pm. ikh hL hC.58%-j?. ?iO&, m, I2_3t-1237.
Nom~~~(1$7),
Vtion of oza~octmstantv&city acdimcntation‘, Jv+, w, US, 360-363. ‘PENOELLEY, E. T. (x967), ‘ The relation of aternol conditionsto the onsetand temiuadon of hibmmion pad ceivation ‘, in Mattnmh Hiith (ail. Fmimt, K. C., DAM, A. R., LYXAN,. C. P., ~C~~ONBAUM, E., and Somx, F.Es,:ut~), vol. III, pp. x-129. New York:
wAltNz& j., RICH, A., and Hiu& C. E. (x962), ‘Eltztron lniw studica of liho6ome ~~~~~~‘,~,~~, x38,13gg+403. -
Wm-nw~~, F. O., some ‘, titi,
T., and NOLL, F.
STAEXEUN,
Land, =9f, 430-435.
-
Wxrrrsx, B. IL, and ICLAPI, G. J. (x$8), ‘Protein metabolism in h-tic tique of memating and amuski8 ground my, Am. 3. P&d., ny x3&1362.
Wwsst,
W. H., Bxu, J., and Mumto, H. N.
~1066),‘TIud%ctaffadingwit$atrypte amino nuxturc on nt-llvcr polyxomcs aid ribosomal rihoouclcic acid’, &;&
3ti.
add
J., xos, ht pea.
.
-
AND PROTEfN S~ES~S
:
SEASONAL CHANGES IN A HIBERNATOR B. K. WHITI’EN,
1;. 85, SCHRADER,*
R. L. HUSTON,
AND G. R. HONOLD?
U.S. Army Institute of Environm~tal Medicine, Natick, wwtu
or76o, USA., and U.S. Army Medical Research and Nutrition Laboratory, Denver, Cokado 80240, U.S.A.
3. The datasuggw that the ‘ nutritionai state ‘ofthcaxtixi&tllaybeaninlportantfactor in these observed aktxtiom.
THE, relationship between ribosome aggregates (polyribosomcs) and protein synthesis has received considerable attention since several kutigazors demonstrated that these aggregates were the active partides in protein synthesis (Marks, Burka, and SchIessinga, ~$2 ; Warner, Rich, and Hall, 1962 ; Gir, 1963 ; Wetstein, %,a&&, and No& 1963)‘ Recently, Munro (1968) showed that hepatic ribosomal aggregation and protein synthetic capacity were related to the mztritional state of the animal with pixrtieular rcrfcrrncc to amino-acid supply and/or imbalance. Thus %uctuations in nutritive state would be expected to inffnence hepatic ribosomaI aggregation and protein synthetic capacity.’ Hibernators have a seasonal pattern -of winter Iethargia, characterized by prolonged periods of deep hypothermia and fasting during which time they may lose weigh& and summer activity, characterized by rcfading, growth, and rapid weight+in (Pcngc.llcy, x967). This SeasoIlitl p&tern appeared to make the hibanator an ~cellent model * Prucut 3ddrcu: aepartmwat of A$pxlomy, Unidty of wiswMiny Madison, wii3cansin 33106s U.S.A. *+seqt address: Gena-al Mills Research tabotstoraa, MinncapoEs, M;innaota 55427, U.SJL
to study seasonal changes in protein synthesis and to conrelate the nutritive and metabolic condition of the animal with hepatic ribosome aggregation and protein synthetic qacity. Tk+cn”ti grou3xd squirret (C&a&@ ti-) were obtained in the spring, pked ininditiduaIcagqqndfedadietofPur&taLab Chow, carrots, and water ad &i&m. A&m& were randomly assigned to three groups m follows: (a) summer normothcnnic animals w&h were maintaind at 22' C. and sacri%ccd in August; (e) titer noamothcrxnic animala which were maintained at 22'C. and sacrificed in January (at the time of sacrifice these at&n& were acdvc and not iethar& or hyp&xsnic);
!i$.mmcrand winter normotbcrmic anima&wencfedadietofPurinaLabCbaw, carrota and watcrrtdlibihrm. Hibunating animala were without food but had water available ad
sacrifice).
libitlhnl. IAers fhm
4 animrlr in each group WffF pooled and polyribcaomu and cell sap enzytnca a&acted using the following modifications of the mcthodsofWunner,Ekll,andMunro (rg66).The cx&zusion meditun contained 25omM sl+tcmq 5omMH EPES buff& 25mM KU, 25m.M KOH, 5m.M M&Z&, and 3mM glutathicxtc. acpE3 ~6 and the P+iba5onl~ WeE -
197% ID 40%~
SRASONAL
CHANGES
IN PROTEIN
post-mitochondrial supemataut (PMS) was treated with d&late (I ‘67 per cent). A polyxibosome pellet was obtained by layeriog the d-t-treated PMS over a discontinuous gradiew (~5 and wo M sucrose b&x) and cent&zging at 32x.ooog for 1.75 houn. Ceil a&acted at pH 6.4 and saPenzYm@JWe= centrifuged as described for the polyribosomes, except that only 0’5 M aucroae buffer was used for the gradient. An aliquot of the cell sap supernatant was passed through a Sephadex G-IO
407
SYNTHEW
subtracted from complete assay values. All samples were run in triplicate and the experiment was repeated. RESULTS
AND DISCUSSION
polyribosomes isolated from summer normothermic animals were characterized by a large number of high molecular weight aggregates (Fig. IA) and a rapid Hepatic
FIG. I.-Hepatic polyribosome pro&s made on isokinetic sucrose damity gradients. A, Polyribounnca from summer normothermic ground squirrds. B, PolyriW tPm winter normothermic ground squinels. C, Polyribowmes from winter bibcmatiog ground squirrels. column to remove fm amino-acids. Polyribosome profiles were obtained on isokinetic sucrose gradients as dcacribed by Nell (1967). Protein synthesis was assayed at 37” C. in a cell-free system using polyribosomcs, cell sap, a complete aminoacid mix, 14-carh taggal amino-aci4 and an energy system. The akuay medium (0.7ml. per assay tube) contaioed the fdlowing, in jlmok!s: sucrose 175; H.EP= buffer 35; KCl 17’5; KOH 17’5; MgCl, 3.5; ATP I; GTP WI; creatine phosphate 20; mixture of 19 L-amino-acids 0*x9. Each assay tube also contained 20 pg. aeatine phosphokinase, 0’2 pc. ach of 14-carbon uniformly labdled ~&wine, L-lysine, L-phenylalanine, and L-valine. An aliquot of freshly prqxed L-cylkne (0.01 *ok per assay) was added and the pH was adjusted to 7’3 with HCl at 37” C. Tke assay mixture was pre-incubated at 37’ C. for 2 minutes. Immediately thereafter ~2mLofcellsapando~rmLofpolyribosomc3 (4-5 a3%d0’1 mg. protein respectively) were added and the reaction allowed to proceed for 2 rnin~tea. *Cold _IO per cent TCA containing iLrrmK)sQcL was used to stop the reaction ZLpitate protein. Proteio was isolated and washed uctekvely before detpzmi&g radie activity aaordiog to standard methods. Reactions were run in triplicate and cell sap blank values (incubations without polyribosomcs) were
rate of protein synthesis ( lobi I). Polyribosomes from winter normothermic animals were characterized by very little high molecular weight material, 4 or 5 distinct peaks of lower molecular weight aggregates (Fig. I B), and a lower rate of protein synthesis ( IkHel) . Winter hibernating animals had few polyribosomcs, a majority of monomer or single ribosomal units (Fig. I C), and a very low rate of protein synthesis (lob& I). These results demonstrate that hepatic protein synthetic capacity in hibernators is influenced by season and is related to ribosome aggregation. Summer normothermic animals were active and well fed. In contrast, winter normothermic animals ate less, were lethargic, and lost approximately x5-20 per cent of their body-weight between August and January. Pengelley (1967) reported a similar seasonal decrease in food intake and associated weight-loss in golden-mantled ground squirrels maintained at 22’ C. and suggested that these parameters were influenced by a circannian (seasonal) rhythm.
WHIlTEN ET AL.
408
‘Ikwfke the observed &aggregation of polyrihosomes and decreased protein synthetic capacity in winter normotherfnic animals compared to summer normothermic animals appear to be due, in part, to a circannian rhythm of food inta+ke not associated with cold temperatures or deep To&la I.-In
Vi&zAg
p_s
Sm
wiiter
hibcmating
TheauthorawiahtocxpresstheirthankstoDr. H. Sauberlicfi and Dr. J. Heon of the U.S. Army Medicsl R~~Nu~d~~~, and -Dr. R. Bu&ngton, Dr. M. Landowne, a& Dr. W. Evans of the U.S. Army Ruawch Institute of Environmcr~talMedicine, for their support and beIp&i commmu during this study.
w
* Gromdspuirrsltian suulmer llormotimmic Winter normothermic
ACICNOWLEDGEMWTS
Fro&inSW* 341925*25&r 6224 & I 222 754f 547
*Values
cxpruscd as z&SD of triplicate determinationsfor two replicates. Values rcprc-
sent DPM x4-carbon labelled amino-acids korporated per 2 &utes per rag. pdyxiU prwcin.
hypothermia. Neariy complete disaggregation of polyribosomes, decreased protein synthetic capacity, and a marked weight-loss (30-45 per cent of prehibanation weight) during winter hibernation may be a fiuxction of the deep hypothermia of these animals, prolonged f&sting, or a combination of these factors. These data suppart previous results which showed that a crude hepatic microsomal preparation from winter hibernating &irteen-lined ground squirr& bad a deaeased protein synthetic capacity at 37” C. when compared to a similar preparation from summer normothermic animals at the same incubation temperature (Whitten and Klain, MW Alterations in plasma and liver amino-acid patterns brought about by fasting and aminoacid imbalancea are known to cause disaggregation of polyribosomes and a decrease in protein synthetic capacity (Munro, rg68). The dccmsed food intake ofwinter noRnot&&xi& pW prolo* fasting amino-aad levels (Klain and whitbcrr, x968; Kristoff&sson and Broberg, 1968) in hibernating animals suggut that the ‘ nutritive state’ may be an important factw in the observed d@gregation of hepatic polyribosomes and deueased hcpatic prbtdn synthetic capacity in these animals.
REFERENcEs ‘Function of aggregated r&Z&CYt~ IibO8oXICSin Dr0tCh.l SWltbiS’. 3. mok i3&d., 4 143-157. * . . KLAIN, G. J., and Wxumm, B. K. (19681, ’ Pimma f&c amino acids in hibernation and G=mt,
A.
(~$31,
arousal‘, Gm&. Biachem. Phpsiol.,q, 617-619. R., and ~OBERC3, s. (x968), ‘F~C~O~~~~~Ofh~~~ in deep hypothcmia and after spontaneous arousals*, E,xj&m& 49 14ar50. M,mxs, P., BURU, E., and f%XiUWNGkR, D. -FFERSSON,
(11~62)~ ‘ protein synthd in aythroid cells, I. Retxulocyte riactive in stimulating amino acid incorporation ‘, Roe. &?I. Acod. sci. U.S.A., &,2x63-2170. Mmo, H. N. (I g68), ’ Roie of amino acid supply in regulating r&oeome fimction’, F& Pm. ikh hL hC.58%-j?. ?iO&, m, I2_3t-1237.
Nom~~~(1$7),
Vtion of oza~octmstantv&city acdimcntation‘, Jv+, w, US, 360-363. ‘PENOELLEY, E. T. (x967), ‘ The relation of aternol conditionsto the onsetand temiuadon of hibmmion pad ceivation ‘, in Mattnmh Hiith (ail. Fmimt, K. C., DAM, A. R., LYXAN,. C. P., ~C~~ONBAUM, E., and Somx, F.Es,:ut~), vol. III, pp. x-129. New York:
wAltNz& j., RICH, A., and Hiu& C. E. (x962), ‘Eltztron lniw studica of liho6ome ~~~~~~‘,~,~~, x38,13gg+403. -
Wm-nw~~, F. O., some ‘, titi,
T., and NOLL, F.
STAEXEUN,
Land, =9f, 430-435.
-
Wxrrrsx, B. IL, and ICLAPI, G. J. (x$8), ‘Protein metabolism in h-tic tique of memating and amuski8 ground my, Am. 3. P&d., ny x3&1362.
Wwsst,
W. H., Bxu, J., and Mumto, H. N.
~1066),‘TIud%ctaffadingwit$atrypte amino nuxturc on nt-llvcr polyxomcs aid ribosomal rihoouclcic acid’, &;&
3ti.
add
J., xos, ht pea.
.
-