Regulation of hepatic protein inflammation and sepsis
synthesis
in chronic
THOMAS C. VARY AND SCOT R. KIMBALL Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Vary, Thomas C., and Scot R. Kimball Regulation of hepatic protein synthesis in chronic inflammation and sepsis. Am. J. Physiol. 262 (Cell Physiol. 31): C445C452, 1992.-The regulation of protein synthesis was determined in livers from control, sterile inflammatory, and septic animals. Total liver protein was increased in both sterile inflammation and sepsis. The rate of protein synthesis in vivo was measured by the incorporation of [“Hlphenylalanine into liver proteins in a chronic (5 day) intra-abdominal abscess model. Both sterile inflammation and sepsis increased total hepatic protein synthesis approximately twofold. Perfused liver studies demonstrated that the increased protein synthesis rate in vivo resulted from a stimulation in the synthesis of both secreted and nonsecreted proteins. The total hepatic RNA content was increased 40% only in sterile inflammation, whereas the translational efficiency was increased twofold only in sepsis. The increase in translational efficiency was accompanied by decreases in the amount of free 40s and 60s ribosomal subunits in sepsis. Rates of peptide-chain elongation in vivo were increased 40% in both sterile inflammation and sepsis. These results demonstrate that sepsis induces changes in the regulation of hepatic protein synthesis that are independent of the general inflammatory response. In sterile inflammation, the increase in protein synthesis occurs by a combination of increased capacity and translational efficiency, while in sepsis, the mechanism responsible for accelerated protein synthesis is an increased translational efficiency. translational efficiency; ribosomal tide-chain elongation; ribonucleic
subunits; acid
perfused liver; pep-
AND SEPSIS induce an acute inflammatory reaction. This response is characterized by leukocytosis, fever, increased oxygen consumption, increased cardiac output, and changes in protein and amino acid metabolism. Distinct and coordinated changes in the concentration of individual plasma proteins, called acute-phase proteins, occur within 24 h following injury (for review seeRef. 11). The acute-phase proteins maximize immune responsiveness to the foreign body and repair to damaged tissues. Because the liver is responsible for the synthesis of these acute-phase proteins, hepatic protein synthesis is accelerated by a wide variety of acute inflammatory insults (7, 9, 18, 22, 23). Despite the accelerated rate of hepatic protein synthesis during inflammation and sepsis, the mechanisms responsible for this stimulation have not been fully elucidated. The general pathway of protein synthesis is composed of many steps, each involving the interaction of several components. The pathway may be broadly divided into transcription and translation. There is ample evidence for regulation of protein synthesis by each of these processes under a variety of conditions. The synthesis of specific proteins is probably regulated transcriptionally during inflammation. For example, mRNA concentrations for the acute-phase proteins are elevated, BOTH TRAUMA
0363-6143/92
$2.00 Copyright
(19, 20, 24) whereas albumin mRNA is decreased (13). These observations suggest that selective changes in the rate of synthesis of specific hepatic proteins are the result of changes in the availability of mRNA. However, increased mRNA concentrations may not wholly account for the overall increased rates of protein synthesis. Instead, translation may also be increased. The translational phase of protein synthesis involves peptide-chain initiation, elongation, and termination. Each of these processes involves a complex series of reactions involving a multitude of enzymes, protein factors, and cofactors. Accelerated rates of translation result from either an increased capacity to synthesize proteins or increased efficiency of the translation process. Increased capacity for protein synthesis refers to an increased amount of ribosomes potentially available for protein synthesis, whereas the efficiency of translation refers to how well the system can transcribe mRNA. Very few studies have examined the regulation of translation during inflammation or sepsis. Total liver RNA concentrations are increased within 24 h following either endotoxin administration (9) or a laparotomy (18), suggesting increased capacity may be important in regulating hepatic protein synthesis. In contrast, burn injury is associated with increased aggregation of polysomes, suggesting that changes in the efficiency of translation may be important (7). All the former studies regarding the effects inflammation or infection on hepatic protein synthesis were performed within 48 h of the injury. However, studies in humans demonstrate that the rise in acute-phase proteins is maintained for days in trauma patients and for weeks in patients with sepsis (28). There is no information available concerning the long-term adaptations of hepatic protein synthesis to inflammation or sepsis. Studies described here examine the consequences of a chronic sterile inflammatory or septic nidus (5 day) on hepatic protein synthesis. Furthermore, the studies investigated possible mechanisms responsible for the increased protein synthesis in response to intra-abdominal abscessformation. METHODS Male Sprague-Dawley rats (Charles River Breeding, Wilmington, MA) weighing -200-300 g were maintained on a 12h light/l2-h dark cycle with rat chow and water supplied ad libitum. Sepsis was induced by the creation of a stable intraabdominal abscess following the implantation of a sterilized fecal-agar pellet (1.5 ml) containing Escherichia coli (serotype 018 AC) (10” CFU) plus Bacteriodes frugilis (ATCC No. 23745) (10’ CFU) (30-33). The sterile inflammation abscess was generated by replacing the bacterial inoculum with an equal volume of sterile saline. Animals were fasted for 24 h and anesthetized
0 1992 the American
Physiological
Society
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C446
PROTEIN
SYNTHESIS
with a combination of ketamine (110 mg/kg body wt) and acepromazine (1 mg/kg body w-t), and the fecal-agar pellet was introduced into the lower abdominal cavity. Control animals were fasted for 24 h. No laparotomy was performed in control rats because hepatic protein synthesis is not changed 3 days following a laparotomy compared with nonoperated rats (9). After recovery from the surgical procedures, the intra-abdominal abscess was allowed to develop for 5-7 days. Previous studies (31) have shown that control, sterile inflammatory, and septic rats consume equal amounts of rat chow per day on days 2 through 7 after implantation of the fecal-agar pellet. Measurement
of
Protein
Synthesis
In uivo. Rates of protein synthesis in vivo were estimated using the flooding dose method described by Scornik (27) as modified by Garlick et al. (5). Five to seven days following the implantation of the fecal-agar pellet, animals from each group were anesthetized with pentobarbital sodium (Nembutal) (50 mg/kg body wt) and placed on a surgical board. As septic animals did not show the same weight gain as control animals following the surgical procedures (33), control and sterile inflammatory animals were weight matched to the septic animals. The weights of the animals in each group at the time of tissue sampling are shown in Table 1. An incision was made in the neck and polyethylene catheters (PE-50 tubing) were inserted into the carotid artery and jugular vein. After catheterization of the carotid artery, a l-ml sample of arterial blood was withdrawn into heparinized tubes for baseline measurements of plasma phenylalanine concentrations. After the removal of the blood sample, a bolus infusion of L- [3H] -phenylalanine (150 mM, 30 &i/ml; 1 ml/l00 gm body wt) was delivered over a loto 15-s interval via the catheter in the jugular vein (5, 9, 33). After injection of the L-[3H]-phenylalanine, the abdomen was opened. Fifteen minutes after injection of the radioisotope, blood (3 ml) was withdrawn from the carotid artery catheter into heparinized tubes for determination of the plasma phenylalanine concentration and radioactivity. A portion of the liver was frozen in situ using clamps precooled to the temperature of liquid nitrogen. The frozen tissue was powdered under liquid nitrogen. A portion of the frozen powdered tissue was homogenized in 5 vol of 10% (wt/vol) trichloroacetic acid (TCA) and centrifuged. The supernatant was used for the measurement of the total tissue free phenylalanine concentration and radioactivity. The concentration of phenylalanine in the plasma samples was measured using supernatants from TCA extracts. Phenylalanine concentrations were measured by high-pressure liquid chromatography method following derivatization with 4dimethylaminoazobenzene-4’-sulfonyl chloride (34). Fractions corresponding to the phenylalanine peak were collected for determination of radioactivity. The intracellular concentration and radioactivity were calculated as the difference between the phenylalanine specific radioactivity in the total tissue and in
Table
1. Effect of sterile inflammation and sepsis on body weight, liver weight, and hepatic protein content Condition
Control Sterile abscess Septic abscess
Animal
Wt, g
259t18 241kll 284k14
Liver
Wt, g
9.1 MO.32 11.28t0.35* 11.35kO.43”
mg Protein/ gwt
207k9 191k7 205&12
g Protein/Liver
1.71&0.06* 2.05tO. 13* 2.27&0.23*
Values shown are means k SE for an n of 9-13 rats in each condition. Liver weights are sum of frozen liver weight and weight of remaining liver tissue excised following sampling. g protein/liver was calculated by multiplying mg protein/g wt times total liver weight. * P < 0.05 vs. control.
IN
SEPSIS
the extracellular space, as previously described (32, 33). The specific radioactivity of phenylalanine measured in the plasma was assumed to accurately reflect that in the extracellular space. Total tissue water was determined by weighing a portion of powdered tissue before and after drying in an oven. Another portion of the frozen powdered tissue was used to estimate the rate of incorporation of radioactive phenylalanine into protein. Approximately 0.5 g of powdered tissue was homogenized in 2 ml of ice-cold 3.6% (wt/vol) perchloric acid (HClO,) and centrifuged. The supernatant was decanted while the pellet was washed a minimum of five times with 3.6% (wt/ vol) HClO, to remove any acid-soluble radioactivity. The pellet was washed with acetone, followed by a mixture of chloroform:acetone (l:l, vol:vol) and then water. The pellet was then dissolved in 0.1 M NaOH. A portion of this sample was taken for measurement of protein concentration by the Biuret method, using crystalline bovine serum albumin as a standard (12). Another portion of the sample was used for the measurement of radioactivity in protein by liquid scintillation spectrometry using the proper corrections for quenching [disintegrations per minute (dpm)]. Count rates varied depending upon the size of tissue but were typically 15,000-25,000 dpm for plasma and 2,000-4,000 dpm for liver protein. Rates of protein synthesis were calculated as described previously (33) using the specific radioactivity of the intracellular phenylalanine as the precursor pool. The assumption in using this technique to estimate the rate of protein synthesis in vivo is that the reutilization of nonradioactive phenylalanine from protein stores via degradation is negligible when the plasma, and hence tissue phenylalanine concentration, is elevated to high concentrations. This assumption was verified by measuring the plasma and intracellular phenylalanine concentration following the bolus infusion of the phenylalanine. The plasma phenylalanine concentrations were increased from -70 nmol/ ml (average of all three conditions) to 1,736 t 106,1,559 t 156, and 1,599 t 98 nmol/ml 15 min after the injection of phenylalanine into control, sterile inflammatory, and septic animals. Also, the liver intracellular free phenylalanine concentration was increased in control animals from 47 t 5 to 1,599 & 85 nmol/ml intracellular water, in sterile inflammatory animals from 55 t 7 to 1,778 t 148 nmol/ml intracellular water, and in septic animals from 62 t 10 to 1,407 t 82 nmol/ml intracellular water. Under the condition of elevated plasma phenylalanine concentrations, the specific radioactivity of the intracellular phenylalanine is assumed to be equal to the specific radioactivity of the tRNA-bound phenylalanine. These assumptions have been verified in isolated perfused systems (3, 10). In vitro. Livers were perfused in situ as described previously (l-3). The perfusate consisted of a modified Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 10 mM glucose, 3% (wt/ vol) bovine serum albumin (Pentex bovine albumin, fraction V, Miles), sufficient washed bovine erythrocytes giving a hematocrit -25% (vol/vol), and amino acids at five times the concentrations found in rat arterial plasma, with the exception of leucine, which was 5 mM. The perfusate was maintained at 37°C and was gassed with humidified 95% Oa-5% CO,. The first 50 ml of the perfusate flowing through the liver were discarded. The remaining 100 ml of medium were recirculated through the isolated liver preparation at a flow rate of 1.25 ml/g liver. After perfusion for 30 min to stabilize the liver, [“Hlleucine (250 &i) was added to the remaining 100 ml of perfusion medium. Samples from the perfusate were collected during the course of 120 min of perfusion, centrifuged to remove erythrocytes and frozen for subsequent analysis. At the end of the perfusion, livers were rapidly excised from the carcass, blotted, weighed, and frozen using Wollenberger clamps precooled to the temperature of liquid nitrogen. Perfusate and liver samples were analyzed for incorporation of [3H]leucine into
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PROTEIN
SYNTHESIS
proteins by TCA precipitation (l-3). Rates of protein synthesis were calculated by dividing the incorporation of [3H]leucine into the appropriate protein fraction by the perfusate specific radioactivity of leucine. It has previously been shown that perfusion of livers with 5* mM leucine results in rapid equilibration of the specific radioactivity of aminoacyl-tRNA to a value equal to that of the amino acid in the intracellular and extracellular compartments (10). This approach allows the use of the specific radioactivity of the perfusate [“Hlleucine for accurate determination of rates of protein synthesis (1, 3). Based on a leucine content of 9.1% in total liver proteins, the rate of protein synthesis is expressed as milligrams of protein synthesized per hour per gram liver (25) . Isolation
of Ribosomal
Subunits
Another portion of frozen powdered tissue was homogenized in a Dounce homogenizer in 8 vol of ice-cold buffer A, containing 25 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (pH 7.5), 250 mM potassium chloride, 1 mM magnesium acetate, 1 mM dithiothreitol, 250 mM sucrose, and 0.1 mM EDTA (2). The homogenate was centrifuged at 10,000 g for 10 min, the supernatant was carefully aspirated, and the volume was measured. To the supernatant, 0.1 vol of a solution containing 10% (wt/vol) Triton X-100 and 10% (wt/vol) deoxycholate was added, and the samples were vortexed. Supernatant samples (0.7 ml) were layered onto 0.44-2.0 M exponential sucrose gradients made up in buffer B, which contained as final concentrations 25 mM tris(hydroxymethyl)aminomethane (Tris) (pH 7.7), 12.5 mM potassium chloride, 2.5 mM magnesium acetate, and 2.5 mM ,&mercaptoethanol. The samples were centrifuged at 35,000 rpm in a SW41 rotor (Beckman Instruments) for 20 h at 4°C to resolve the 40s and 60s ribosomal subunits. The absorbance at 254 nm of the gradients was monitored and fractions (0.8 ml) were collected using a density gradient fractionator (Instrument Specialties). RNA contents of the homogenates and sucrose gradient fractions containing the 40s and 60s ribosomal subunits were determined by alkaline hydrolysis. Aliquots (0.5 ml) were treated with 2 ml of 10% (wt/vol) TCA, vortexed, and incubated for 10 min on ice. The samples were centrifuged at 8,800 g for 10 min at 4°C. The supernatant was discarded, and the pellets were washed two times with 2.5 ml of ice-cold 6% (wt/vol) HClO,. The pellets were dissolved in 1.5 ml of 0.3 N KOH and incubated at 37°C for 1 h, after which 0.5 ml of 4 N HClO, was added. The samples were centrifuged at 8,800 g for 10 min at 4°C and the supernatant was carefully aspirated. RNA content was determined spectrophotometrically as the absorbance at by the absorbance 260 nm (A32.6 pg/ml = 1 at 260 nm) corrected at 232 nm (4). The recovery of RNA from sucrose gradients was -31% of total liver RNA and from supernatants prior to addition of the Triton X-lOO/deoxycholate, -52%. RNA content of each sucrose gradient fraction was expressed as micrograms RNA per milligram of homogenate RNA. Measurement
of Relative Rate of Peptide-Chain
Elongation
In a separate group of animals, an adaptation of the method described by Palmiter (15) was used to measure the rate of peptide-chain elongation in sterile inflammatory or septic rats relative to control rats in vivo. The relative rate of elongation was deduced from the difference in the average incorporation of radioactive methionine into proteins in the soluble fraction and into nascent polypeptide chains of the polysome fraction following ultracentrifugation of a liver homogenate in a sucrose density gradient. Briefly, control, sterile inflammatory, and septic animals were anesthetized with pentobarbital sodium
IN
SEPSIS
c447
(Nembutal) (50 mg/kg body wt) and placed on a surgical board. An incision was made in the neck and polyethylene catheters (PE-50 tubing) inserted in the carotid artery. After catheterization of the carotid artery, 1 mCi of either [3H] - or [35S]methionine was injected via the carotid artery. Five minutes following injection of the radioisotope, the liver was frozen in situ with clamps precooled to the temperature of liquid nitrogen. The frozen tissue was powdered in liquid nitrogen. Approximately equal weights (1 g) of 3H- and 35S-radiolabeled frozen powdered liver were combined, homogenized, and centrifuged (10,000 g) as described above for determination of ribosomal subunits. To the supernatant, 0.1 vol of a solution containing 10% (wt/vol) Triton X-100 and 10% (wt/vol) deoxycholate was added and the samples were vortexed. Supernatant samples (0.7 ml) were layered onto 0.60-2.0 M linear sucrose gradients made up in buffer B. The samples were centrifuged at 41,000 rpm in a SW41 rotor (Beckman Instruments) for 3 h at 4°C. The absorbance at 254 nm of the gradients was monitored. Fractions (1.2 ml) were collected using a density gradient fractionator (Instrument Specialties). To verify that the fractions collected corresponded to the soluble protein fraction and the polysome fraction, individual fractions from two gradients were combined and layered onto a Pharmacia PD-10 column. The fractions were eluted with buffer A diluted 1:l with water to remove sucrose. The washed fractions were layered onto individual exponential sucrose gradients (0.44-2.0 M) and centrifuged at 41,000 rpm in a SW-41 rotor for 16 h at 4°C. The absorbance at 254 nm was estimated for each gradient, and the original fractions corresponding to the 4OS, 60s and 80s ribosomal subunits were determined. Fractions preceding these ribosomal subunit profiles were considered the soluble fraction, whereas fractions after these subunits were considered to be polysomal ribosomes. To 1 ml of each fraction, 0.5 ml of 0.2% (wt/vol) bovine serum albumin and 4 ml of 10% (wt/vol) TCA was added. The samples were vortexed, incubated at 0°C for 10 min, and centrifuged at 10,000 g for 11 min at 4°C. The supernatant was discarded, the pellet was redissolved in 1 ml of 0.25 M NaOH, and 2 ml of 10% TCA were added. The samples were vortexed, placed in a water bath (9OOC) for 10 min, and centrifuged as before. The supernatant was discarded and the pellet was treated as before, excluding heating. After centrifugation, the pellet was redissolved in 0.5 ml of 0.25 N NaOH and 0.4 ml were taken for determination of radioactivity. Radioactivity was measured using an LKB Excel scintillation counter using appropriate corrections for quenching (dpm) and 3H and 35S separations. The relative rate of elongation was estimated as the average ratio S of 3H to 35S in the fractions corresponding to the soluble proteins divided by the average ratio P of 3H to 35S in the fractions corresponding to polysomes (15). A deviation of the S/P ratio from unity indicates that elongation is increased in the one condition relative to the other. Statistical
Analysis
The experimental data for each condition are summarized as means + SE for an n of 5-17 animals in each group. Statistical evaluation of the data was performed using the Student’s t test for unpaired comparisons. Differences among means were considered significant when P < 0.05. RESULTS
Previous reports showed that an increased rate of protein synthesis following an acute (within 24 h) inflammatory or bacterial insult was associated with an increased liver size and protein content (9, 18). To deter-
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C448
PROTEIN
SYNTHESIS
mine whether similar changes occurred in a chronic model of inflammation and inflammation compounded by bacterial infection, the liver weights and concentration of protein in livers were measured 5 days following the implantation of the fecal-agar pellet. The total liver weight was significantly (P < 0.05) increased 24% in both sterile inflammation and sepsis compared with control (Table 1). In contrast, there were no significant differences in the amount of protein in livers from the different groups. Because the wet weight to dry weight ratio is increased in sterile inflammation and sepsis (32), the protein content of the liver was also calculated per gram dry weight. As was observed for the wet weight measurements, there were no significant differences in the protein content among the different groups (data not shown). However, the total amount of protein per liver was significantly (P < 0.05) increased by 20% in sterile inflammation and by 33% in sepsis. Hence, part of the increase in liver weight in sepsis and inflammation results from an increased total liver protein content. The effect of chronic (5 day) inflammation and sepsis on hepatic protein synthesis is presented in Fig. 1A. The rate of incorporation of radioactive phenylalanine into protein in livers from control animals was 534 f 91 nmol. h-l. g liver-‘. This corresponds to a fractional rate of synthesis of 45 & 7%/day. Both the rate of incorporation of radioactive phenylalanine into proteins and the fractional rate of synthesis (89 rfi 14%/day) were increased approximately twofold in livers from rats with a sterile abscess (P < 0.05) compared with control animals. As observed in sterile inflammation, sepsis significantly stimulated both the incorporation of radioactive phenylalanine into proteins (P < 0.05) and the fractional rate of synthesis (96 + 17%/day, P < 0.02) twofold compared with control. Hence, both chronic sterile inflammation and sepsis enhanced rates of total hepatic protein synthesis. Furthermore, the magnitude of the stimulation of protein synthesis was similar in both conditions. Although the results from the in vivo rates of protein synthesis indicate an increase in hepatic protein synthesis, the flooding-dose method used preferentially estimates the synthesis of proteins rapidly being synthesized (5, 18). Furthermore, the short time between injection of radioactive phenylalanine and time of tissue sampling (15 min) estimates the synthesis of both secretory and nonsecretory proteins, as newly synthesized proteins remain in the liver for at least this period. Therefore, alterations in the rate of hepatic protein synthesis were further investigated in experiments using the isolated perfused liver where secreted and nonsecreted protein synthesis could be measured. In these studies, samples of the perfusion medium and of the liver were taken to determine the rates of production of secretory and nonsecretory proteins, respectively. After a lag period of -30 min, which represented the time for synthesis, processing, and secretion of protein into the perfusate, incorporation of radiolabeled leucine into total secreted protein increased linearly over the duration of the perfusion (Fig. 2). The characteristic lag period of 30 min between the addition of radiolabeled leucine into the perfusate and appearance of radiolabeled proteins was not altered by sterile inflammation or sep-
IN
SEPSIS
i -+
--tI I ::.:.I ..:: :.:.
.:
: :; ~.
:
: 1:
: ,. 7, I, ,/ ,, I , , ,, / /I, ,, L CONTROL
STERILE
ABSCESS
SEPTIC
ABSCESS
Fig. 1. Rate of protein synthesis (A), RNA concentration (B), and translational efficiency (C) in livers of control, sterile inflammatory, and septic rats. Livers were taken from animals 5 days following intraperitoneal introduction of a sterile fecal-agar pellet with and without a bacterial inoculum. Rates of protein synthesis were measured in vivo following intravenous injection with saline containing [3H]phenylalanine (150 pmol/lOO g body wt). Determination of [3H]phenylalanine incorporation into liver protein and calculation of rates of protein synthesis were performed as described in METHODS. Total RNA was measured in homogenates of liver samples. Synthesis rate/ RNA values were obtained by dividing protein synthesis rate by tissue RNA content for each condition. Values shown are means + SE for 510 animals in each group. A: *P < 0.05 vs. control; B: *P < 0.05 vs. control or septic abscess; C: *P < 0.05 vs. control or sterile abscess.
sis. However, in livers from both sterile inflammatory and septic rats, the rate of production of total secreted proteins was increased markedly compared with values obtained from livers of control rats. These differences, as well as data relating to nonexported proteins in perfused livers, are shown in Table 2. Livers from sterile inflammatory and septic rats produced total secretory proteins at a rate 174% (P < 0.001) and 142% (P < 0.001) of the control rate, respectively. Furthermore, the proportion of total liver protein synthesized destined for export to the plasma was significantly (P < 0.05) in-
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PROTEIN I
I
A
I
I
I
I
SYNTHESIS
I
Control
-.A -a-
-
B
Sterile Septic
20
0
40
60
80
100 120
TIME OF PERFUSION (min) Fig. 2. Production of total secreted protein by perfused livers from control, sterile inflammatory, and septic rats. At zero time, [“Hlleucine was added to a final concentration of 5.0 &i/ml of perfusate. Samples of nerfusate were collected at 2O-min intervals for determination of radioactivity present in total secretory proteins as described in METHODS. Values presented for each time point are means t SE of 5-12 determinations in each condition. Rates of total secretory protein production (given in Table 2) were derived from a least-squares linear regression analysis of values presented here.
Table 2. Effect of sterile inflammation and sepsis on rates of protein synthesis in perfused livers Measurement
Total
secreted
Control
protein
. S~~t~~~~~~~~~x~~~~~~
. . Sypnrphteesi~ onfltifii l:LL protein, mg* g-’ 9h-’ % of total liver protein
secreted
Sterile Abscess
Septic Abscess
1.04kO.07
1.81&0.17*
1.48t0.07*
1.6OkO.08
2.14&0.14?
1.95&0.10*
2.56k0.13
3.94&0.29*
3.37&0.10*
39&2
47+2$
47+1$
Values represent means k SE for determinations derived from 5-12 * P C 0.001 vs. control; t P < 0.005 vs. animals in each condition. control; $ P < 0.05 vs. control. Synthesis of total liver protein is a summation of individual values for total secretory protein production and synthesis of nonexported protein for each liver. Ratio of total secreted protein production to total liver protein synthesis x 100.
creased in both sterile inflammation and sepsiscompared with control. Synthesi .sof nonexported protein s by livers from sterile inflammatory and septic rats was also significantly increased 34% (P < 0.005) and 22% (P < 0.05) compared with control. Likewise, the rate of total liver protein (i.e., secreted plus nonexported) was also significantly increased in sterile inflammation (P < 0.001) and sepsis (P < 0.001). Increased rates of protein synthesis may be due to an increased capacity for protein synthesis or an increased efficiency of the translation process. Ribosomal RNA, which represents the capacity for protein synthesis, accounts for as much as 80% of the total RNA content. Therefore, changes in the total amount of RNA presumably reflects changes in ribosomal RNA. The total RNA concentration in liver is shown in Fig. 1B. The concentration of RNA in livers from control animals was 5.2 t 0.1 mg RNA/g wet wt, which corresponds to 26 t 0.8 pg RNA/mg protein. Sterile inflammation was associated
IN
c449
SEPSIS
with an -30% increase in total hepatic RNA (P c 0.01). This increase in RNA in livers from sterile inflammatory animals was significant whether the data were expressed as per gram wet weight (P < 0.05) (Fig. 1B) or per milligram protein basis (P < 0.01) (data not shown). However, in contrast to sterile inflammation, sepsisdid not induce an increase in total liver RNA content. The RNA concentration in livers from septic animals was not significantly different from control animals but was significantly lower than the values obtained from sterile inflammatory animals (P C 0.05). Because the stimulation of protein synthesis in sepsis was not correlated with increased capacity for protein synthesis, the efficiency of translation, expressed as protein synthesis per RNA, was calculated. These results are shown in Fig. 1C. The translational efficiency was increased, although not significantly in livers from sterile inflammatory animals compared with control. However, in contrast to sterile inflammation, sepsiswas associated with an over twofold (P c 0.001) enhancement of the translational efficiency compared with control. Hence, these data indicate that increased efficiency of translation is an important mechanism for enhanced protein synthesis in sepsis. The effect of sterile inflammation and sepsis on the translational component of protein synthesis was also evaluated by isolating the 40s and 60s ribosomal subunits from liver using sucrose density gradient fractionation. The results of these comparisons are shown in Fig. 3. The content of ribosomal subunits in livers from sterile abscess rats was not significantly different from control in sterile inflammation. In contrast to sterile inflammation, the amount of the 40s and 60s ribosomal subunits was both significantly decreased relative to control in livers of septic rats. To further examine the regulation of protein synthesis, the rate of peptide-chain elongation was measured in I
40
s
L Sterile Abscess
Septic Abscess
CONDITION Fig. 3. Effect of sterile inflammation and sepsis on levels of ribosomal subunits in liver. Samples were taken from animals as described in Fig. 1. 40s and 60s ribosomal subunits were isolated on sucrose gradients and analyzed for RNA as described in METHODS. Values shown are means & SE for 7-8 livers in each condition. *P < 0.01 vs. control; **P < 0.05 vs. control
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c450
PROTEIN
SYNTHESIS
vivo. In this method, one rat is pulse-labeled with [3H]methionine and another animal is pulse-labeled with [35S]methionine. The relative rate of elongation between the two animals is deduced from the difference in the average ratio of isotopes in the polysomal fractions and the average ratio of isotopes in the soluble protein fraction (15). To verify this procedure, it was necessary to demonstrate that the ratio of 3H/35S in the soluble protein fraction (S) divided by the ratio of 3H/35S in the nascent-chain polysome fraction (P), that is the S/P ratio, was unity in control animals. The S/P ratio in livers from control animals was 1.05 t 0.04 in five separate experiments, as expected. This result indicates that the relative rate of peptide-chain elongation was nearly identical in the two control animals. To further validate the procedure, modulation of the rate of peptide-chain elongation should result in the appropriate changes in radioactivity in the fractions corresponding to the soluble proteins and polysomes. Cycloheximide is known to inhibit protein synthesis by blocking elongation at the site of peptide-bond formation on the 80s ribosome. Therefore, the effect of cycloheximide on the rate of elongation was determined in two groups of control rats. One group received an intraperitoneal injection of cycloheximide (2.5 pg/kg body wt) and the other received an equal volume of saline. This amount of cycloheximide was calculated to give an in vivo concentration close to the inhibition constant (Ki) of peptide-bond formation in perfused mouse livers (G. E. Mortimore, personal communication). Forty-five minutes following the injection of either saline or cycloheximide, the appropriate isotope was infused. The S/P ratio was 0.156 in cycloheximide- vs. saline-treated animals. This result indicates that the rate of elongation measured by this procedure was inhibited by 85% in rats injected with cycloheximide (Table 3). With these preliminary studies verifying the procedure, the rate of elongation in sterile inflammatory and septic animals relative to control was measured. In livers from sterile inflammatory animals, the rate of elongation was significantly (P < 0.001) increased 48% relative to control (Table 3). A similar significant (P c 0.005) increase in the rate of elongation relative to control was observed in livers from septic animals relative to control. Hence, the relative rate of elongation was increased to the same extent in both sterile inflammation and sepsis Table 3. Effect of sterile inflammation and sepsis on the relative rate of peptide-chain elongation in vivo Condition
Control Control + cycloheximide Sterile abscess Septic abscess
Rate
of Elongation (% control)
105k4 16k9*
149t8* 140t6t
Data are presented as means t SE for 4-5 animals in each group. 0.001 vs. control; t P < 0.005 vs. control. Rats were injected with 1 mCi of either [3H]- or [35S]methionine and liver samples were frozen in situ 5 min later. Radioactivity in soluble protein and polysome fractions was measured as described in METHODS. The ratio of 3H to “‘S in soluble protein fraction (S) and polysome fraction (P) was calculated. Values shown represent S/P ratio of experimental group to control X 100. *
P
synthesis
in chronic
THOMAS C. VARY AND SCOT R. KIMBALL Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Vary, Thomas C., and Scot R. Kimball Regulation of hepatic protein synthesis in chronic inflammation and sepsis. Am. J. Physiol. 262 (Cell Physiol. 31): C445C452, 1992.-The regulation of protein synthesis was determined in livers from control, sterile inflammatory, and septic animals. Total liver protein was increased in both sterile inflammation and sepsis. The rate of protein synthesis in vivo was measured by the incorporation of [“Hlphenylalanine into liver proteins in a chronic (5 day) intra-abdominal abscess model. Both sterile inflammation and sepsis increased total hepatic protein synthesis approximately twofold. Perfused liver studies demonstrated that the increased protein synthesis rate in vivo resulted from a stimulation in the synthesis of both secreted and nonsecreted proteins. The total hepatic RNA content was increased 40% only in sterile inflammation, whereas the translational efficiency was increased twofold only in sepsis. The increase in translational efficiency was accompanied by decreases in the amount of free 40s and 60s ribosomal subunits in sepsis. Rates of peptide-chain elongation in vivo were increased 40% in both sterile inflammation and sepsis. These results demonstrate that sepsis induces changes in the regulation of hepatic protein synthesis that are independent of the general inflammatory response. In sterile inflammation, the increase in protein synthesis occurs by a combination of increased capacity and translational efficiency, while in sepsis, the mechanism responsible for accelerated protein synthesis is an increased translational efficiency. translational efficiency; ribosomal tide-chain elongation; ribonucleic
subunits; acid
perfused liver; pep-
AND SEPSIS induce an acute inflammatory reaction. This response is characterized by leukocytosis, fever, increased oxygen consumption, increased cardiac output, and changes in protein and amino acid metabolism. Distinct and coordinated changes in the concentration of individual plasma proteins, called acute-phase proteins, occur within 24 h following injury (for review seeRef. 11). The acute-phase proteins maximize immune responsiveness to the foreign body and repair to damaged tissues. Because the liver is responsible for the synthesis of these acute-phase proteins, hepatic protein synthesis is accelerated by a wide variety of acute inflammatory insults (7, 9, 18, 22, 23). Despite the accelerated rate of hepatic protein synthesis during inflammation and sepsis, the mechanisms responsible for this stimulation have not been fully elucidated. The general pathway of protein synthesis is composed of many steps, each involving the interaction of several components. The pathway may be broadly divided into transcription and translation. There is ample evidence for regulation of protein synthesis by each of these processes under a variety of conditions. The synthesis of specific proteins is probably regulated transcriptionally during inflammation. For example, mRNA concentrations for the acute-phase proteins are elevated, BOTH TRAUMA
0363-6143/92
$2.00 Copyright
(19, 20, 24) whereas albumin mRNA is decreased (13). These observations suggest that selective changes in the rate of synthesis of specific hepatic proteins are the result of changes in the availability of mRNA. However, increased mRNA concentrations may not wholly account for the overall increased rates of protein synthesis. Instead, translation may also be increased. The translational phase of protein synthesis involves peptide-chain initiation, elongation, and termination. Each of these processes involves a complex series of reactions involving a multitude of enzymes, protein factors, and cofactors. Accelerated rates of translation result from either an increased capacity to synthesize proteins or increased efficiency of the translation process. Increased capacity for protein synthesis refers to an increased amount of ribosomes potentially available for protein synthesis, whereas the efficiency of translation refers to how well the system can transcribe mRNA. Very few studies have examined the regulation of translation during inflammation or sepsis. Total liver RNA concentrations are increased within 24 h following either endotoxin administration (9) or a laparotomy (18), suggesting increased capacity may be important in regulating hepatic protein synthesis. In contrast, burn injury is associated with increased aggregation of polysomes, suggesting that changes in the efficiency of translation may be important (7). All the former studies regarding the effects inflammation or infection on hepatic protein synthesis were performed within 48 h of the injury. However, studies in humans demonstrate that the rise in acute-phase proteins is maintained for days in trauma patients and for weeks in patients with sepsis (28). There is no information available concerning the long-term adaptations of hepatic protein synthesis to inflammation or sepsis. Studies described here examine the consequences of a chronic sterile inflammatory or septic nidus (5 day) on hepatic protein synthesis. Furthermore, the studies investigated possible mechanisms responsible for the increased protein synthesis in response to intra-abdominal abscessformation. METHODS Male Sprague-Dawley rats (Charles River Breeding, Wilmington, MA) weighing -200-300 g were maintained on a 12h light/l2-h dark cycle with rat chow and water supplied ad libitum. Sepsis was induced by the creation of a stable intraabdominal abscess following the implantation of a sterilized fecal-agar pellet (1.5 ml) containing Escherichia coli (serotype 018 AC) (10” CFU) plus Bacteriodes frugilis (ATCC No. 23745) (10’ CFU) (30-33). The sterile inflammation abscess was generated by replacing the bacterial inoculum with an equal volume of sterile saline. Animals were fasted for 24 h and anesthetized
0 1992 the American
Physiological
Society
c445
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C446
PROTEIN
SYNTHESIS
with a combination of ketamine (110 mg/kg body wt) and acepromazine (1 mg/kg body w-t), and the fecal-agar pellet was introduced into the lower abdominal cavity. Control animals were fasted for 24 h. No laparotomy was performed in control rats because hepatic protein synthesis is not changed 3 days following a laparotomy compared with nonoperated rats (9). After recovery from the surgical procedures, the intra-abdominal abscess was allowed to develop for 5-7 days. Previous studies (31) have shown that control, sterile inflammatory, and septic rats consume equal amounts of rat chow per day on days 2 through 7 after implantation of the fecal-agar pellet. Measurement
of
Protein
Synthesis
In uivo. Rates of protein synthesis in vivo were estimated using the flooding dose method described by Scornik (27) as modified by Garlick et al. (5). Five to seven days following the implantation of the fecal-agar pellet, animals from each group were anesthetized with pentobarbital sodium (Nembutal) (50 mg/kg body wt) and placed on a surgical board. As septic animals did not show the same weight gain as control animals following the surgical procedures (33), control and sterile inflammatory animals were weight matched to the septic animals. The weights of the animals in each group at the time of tissue sampling are shown in Table 1. An incision was made in the neck and polyethylene catheters (PE-50 tubing) were inserted into the carotid artery and jugular vein. After catheterization of the carotid artery, a l-ml sample of arterial blood was withdrawn into heparinized tubes for baseline measurements of plasma phenylalanine concentrations. After the removal of the blood sample, a bolus infusion of L- [3H] -phenylalanine (150 mM, 30 &i/ml; 1 ml/l00 gm body wt) was delivered over a loto 15-s interval via the catheter in the jugular vein (5, 9, 33). After injection of the L-[3H]-phenylalanine, the abdomen was opened. Fifteen minutes after injection of the radioisotope, blood (3 ml) was withdrawn from the carotid artery catheter into heparinized tubes for determination of the plasma phenylalanine concentration and radioactivity. A portion of the liver was frozen in situ using clamps precooled to the temperature of liquid nitrogen. The frozen tissue was powdered under liquid nitrogen. A portion of the frozen powdered tissue was homogenized in 5 vol of 10% (wt/vol) trichloroacetic acid (TCA) and centrifuged. The supernatant was used for the measurement of the total tissue free phenylalanine concentration and radioactivity. The concentration of phenylalanine in the plasma samples was measured using supernatants from TCA extracts. Phenylalanine concentrations were measured by high-pressure liquid chromatography method following derivatization with 4dimethylaminoazobenzene-4’-sulfonyl chloride (34). Fractions corresponding to the phenylalanine peak were collected for determination of radioactivity. The intracellular concentration and radioactivity were calculated as the difference between the phenylalanine specific radioactivity in the total tissue and in
Table
1. Effect of sterile inflammation and sepsis on body weight, liver weight, and hepatic protein content Condition
Control Sterile abscess Septic abscess
Animal
Wt, g
259t18 241kll 284k14
Liver
Wt, g
9.1 MO.32 11.28t0.35* 11.35kO.43”
mg Protein/ gwt
207k9 191k7 205&12
g Protein/Liver
1.71&0.06* 2.05tO. 13* 2.27&0.23*
Values shown are means k SE for an n of 9-13 rats in each condition. Liver weights are sum of frozen liver weight and weight of remaining liver tissue excised following sampling. g protein/liver was calculated by multiplying mg protein/g wt times total liver weight. * P < 0.05 vs. control.
IN
SEPSIS
the extracellular space, as previously described (32, 33). The specific radioactivity of phenylalanine measured in the plasma was assumed to accurately reflect that in the extracellular space. Total tissue water was determined by weighing a portion of powdered tissue before and after drying in an oven. Another portion of the frozen powdered tissue was used to estimate the rate of incorporation of radioactive phenylalanine into protein. Approximately 0.5 g of powdered tissue was homogenized in 2 ml of ice-cold 3.6% (wt/vol) perchloric acid (HClO,) and centrifuged. The supernatant was decanted while the pellet was washed a minimum of five times with 3.6% (wt/ vol) HClO, to remove any acid-soluble radioactivity. The pellet was washed with acetone, followed by a mixture of chloroform:acetone (l:l, vol:vol) and then water. The pellet was then dissolved in 0.1 M NaOH. A portion of this sample was taken for measurement of protein concentration by the Biuret method, using crystalline bovine serum albumin as a standard (12). Another portion of the sample was used for the measurement of radioactivity in protein by liquid scintillation spectrometry using the proper corrections for quenching [disintegrations per minute (dpm)]. Count rates varied depending upon the size of tissue but were typically 15,000-25,000 dpm for plasma and 2,000-4,000 dpm for liver protein. Rates of protein synthesis were calculated as described previously (33) using the specific radioactivity of the intracellular phenylalanine as the precursor pool. The assumption in using this technique to estimate the rate of protein synthesis in vivo is that the reutilization of nonradioactive phenylalanine from protein stores via degradation is negligible when the plasma, and hence tissue phenylalanine concentration, is elevated to high concentrations. This assumption was verified by measuring the plasma and intracellular phenylalanine concentration following the bolus infusion of the phenylalanine. The plasma phenylalanine concentrations were increased from -70 nmol/ ml (average of all three conditions) to 1,736 t 106,1,559 t 156, and 1,599 t 98 nmol/ml 15 min after the injection of phenylalanine into control, sterile inflammatory, and septic animals. Also, the liver intracellular free phenylalanine concentration was increased in control animals from 47 t 5 to 1,599 & 85 nmol/ml intracellular water, in sterile inflammatory animals from 55 t 7 to 1,778 t 148 nmol/ml intracellular water, and in septic animals from 62 t 10 to 1,407 t 82 nmol/ml intracellular water. Under the condition of elevated plasma phenylalanine concentrations, the specific radioactivity of the intracellular phenylalanine is assumed to be equal to the specific radioactivity of the tRNA-bound phenylalanine. These assumptions have been verified in isolated perfused systems (3, 10). In vitro. Livers were perfused in situ as described previously (l-3). The perfusate consisted of a modified Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 10 mM glucose, 3% (wt/ vol) bovine serum albumin (Pentex bovine albumin, fraction V, Miles), sufficient washed bovine erythrocytes giving a hematocrit -25% (vol/vol), and amino acids at five times the concentrations found in rat arterial plasma, with the exception of leucine, which was 5 mM. The perfusate was maintained at 37°C and was gassed with humidified 95% Oa-5% CO,. The first 50 ml of the perfusate flowing through the liver were discarded. The remaining 100 ml of medium were recirculated through the isolated liver preparation at a flow rate of 1.25 ml/g liver. After perfusion for 30 min to stabilize the liver, [“Hlleucine (250 &i) was added to the remaining 100 ml of perfusion medium. Samples from the perfusate were collected during the course of 120 min of perfusion, centrifuged to remove erythrocytes and frozen for subsequent analysis. At the end of the perfusion, livers were rapidly excised from the carcass, blotted, weighed, and frozen using Wollenberger clamps precooled to the temperature of liquid nitrogen. Perfusate and liver samples were analyzed for incorporation of [3H]leucine into
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PROTEIN
SYNTHESIS
proteins by TCA precipitation (l-3). Rates of protein synthesis were calculated by dividing the incorporation of [3H]leucine into the appropriate protein fraction by the perfusate specific radioactivity of leucine. It has previously been shown that perfusion of livers with 5* mM leucine results in rapid equilibration of the specific radioactivity of aminoacyl-tRNA to a value equal to that of the amino acid in the intracellular and extracellular compartments (10). This approach allows the use of the specific radioactivity of the perfusate [“Hlleucine for accurate determination of rates of protein synthesis (1, 3). Based on a leucine content of 9.1% in total liver proteins, the rate of protein synthesis is expressed as milligrams of protein synthesized per hour per gram liver (25) . Isolation
of Ribosomal
Subunits
Another portion of frozen powdered tissue was homogenized in a Dounce homogenizer in 8 vol of ice-cold buffer A, containing 25 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (pH 7.5), 250 mM potassium chloride, 1 mM magnesium acetate, 1 mM dithiothreitol, 250 mM sucrose, and 0.1 mM EDTA (2). The homogenate was centrifuged at 10,000 g for 10 min, the supernatant was carefully aspirated, and the volume was measured. To the supernatant, 0.1 vol of a solution containing 10% (wt/vol) Triton X-100 and 10% (wt/vol) deoxycholate was added, and the samples were vortexed. Supernatant samples (0.7 ml) were layered onto 0.44-2.0 M exponential sucrose gradients made up in buffer B, which contained as final concentrations 25 mM tris(hydroxymethyl)aminomethane (Tris) (pH 7.7), 12.5 mM potassium chloride, 2.5 mM magnesium acetate, and 2.5 mM ,&mercaptoethanol. The samples were centrifuged at 35,000 rpm in a SW41 rotor (Beckman Instruments) for 20 h at 4°C to resolve the 40s and 60s ribosomal subunits. The absorbance at 254 nm of the gradients was monitored and fractions (0.8 ml) were collected using a density gradient fractionator (Instrument Specialties). RNA contents of the homogenates and sucrose gradient fractions containing the 40s and 60s ribosomal subunits were determined by alkaline hydrolysis. Aliquots (0.5 ml) were treated with 2 ml of 10% (wt/vol) TCA, vortexed, and incubated for 10 min on ice. The samples were centrifuged at 8,800 g for 10 min at 4°C. The supernatant was discarded, and the pellets were washed two times with 2.5 ml of ice-cold 6% (wt/vol) HClO,. The pellets were dissolved in 1.5 ml of 0.3 N KOH and incubated at 37°C for 1 h, after which 0.5 ml of 4 N HClO, was added. The samples were centrifuged at 8,800 g for 10 min at 4°C and the supernatant was carefully aspirated. RNA content was determined spectrophotometrically as the absorbance at by the absorbance 260 nm (A32.6 pg/ml = 1 at 260 nm) corrected at 232 nm (4). The recovery of RNA from sucrose gradients was -31% of total liver RNA and from supernatants prior to addition of the Triton X-lOO/deoxycholate, -52%. RNA content of each sucrose gradient fraction was expressed as micrograms RNA per milligram of homogenate RNA. Measurement
of Relative Rate of Peptide-Chain
Elongation
In a separate group of animals, an adaptation of the method described by Palmiter (15) was used to measure the rate of peptide-chain elongation in sterile inflammatory or septic rats relative to control rats in vivo. The relative rate of elongation was deduced from the difference in the average incorporation of radioactive methionine into proteins in the soluble fraction and into nascent polypeptide chains of the polysome fraction following ultracentrifugation of a liver homogenate in a sucrose density gradient. Briefly, control, sterile inflammatory, and septic animals were anesthetized with pentobarbital sodium
IN
SEPSIS
c447
(Nembutal) (50 mg/kg body wt) and placed on a surgical board. An incision was made in the neck and polyethylene catheters (PE-50 tubing) inserted in the carotid artery. After catheterization of the carotid artery, 1 mCi of either [3H] - or [35S]methionine was injected via the carotid artery. Five minutes following injection of the radioisotope, the liver was frozen in situ with clamps precooled to the temperature of liquid nitrogen. The frozen tissue was powdered in liquid nitrogen. Approximately equal weights (1 g) of 3H- and 35S-radiolabeled frozen powdered liver were combined, homogenized, and centrifuged (10,000 g) as described above for determination of ribosomal subunits. To the supernatant, 0.1 vol of a solution containing 10% (wt/vol) Triton X-100 and 10% (wt/vol) deoxycholate was added and the samples were vortexed. Supernatant samples (0.7 ml) were layered onto 0.60-2.0 M linear sucrose gradients made up in buffer B. The samples were centrifuged at 41,000 rpm in a SW41 rotor (Beckman Instruments) for 3 h at 4°C. The absorbance at 254 nm of the gradients was monitored. Fractions (1.2 ml) were collected using a density gradient fractionator (Instrument Specialties). To verify that the fractions collected corresponded to the soluble protein fraction and the polysome fraction, individual fractions from two gradients were combined and layered onto a Pharmacia PD-10 column. The fractions were eluted with buffer A diluted 1:l with water to remove sucrose. The washed fractions were layered onto individual exponential sucrose gradients (0.44-2.0 M) and centrifuged at 41,000 rpm in a SW-41 rotor for 16 h at 4°C. The absorbance at 254 nm was estimated for each gradient, and the original fractions corresponding to the 4OS, 60s and 80s ribosomal subunits were determined. Fractions preceding these ribosomal subunit profiles were considered the soluble fraction, whereas fractions after these subunits were considered to be polysomal ribosomes. To 1 ml of each fraction, 0.5 ml of 0.2% (wt/vol) bovine serum albumin and 4 ml of 10% (wt/vol) TCA was added. The samples were vortexed, incubated at 0°C for 10 min, and centrifuged at 10,000 g for 11 min at 4°C. The supernatant was discarded, the pellet was redissolved in 1 ml of 0.25 M NaOH, and 2 ml of 10% TCA were added. The samples were vortexed, placed in a water bath (9OOC) for 10 min, and centrifuged as before. The supernatant was discarded and the pellet was treated as before, excluding heating. After centrifugation, the pellet was redissolved in 0.5 ml of 0.25 N NaOH and 0.4 ml were taken for determination of radioactivity. Radioactivity was measured using an LKB Excel scintillation counter using appropriate corrections for quenching (dpm) and 3H and 35S separations. The relative rate of elongation was estimated as the average ratio S of 3H to 35S in the fractions corresponding to the soluble proteins divided by the average ratio P of 3H to 35S in the fractions corresponding to polysomes (15). A deviation of the S/P ratio from unity indicates that elongation is increased in the one condition relative to the other. Statistical
Analysis
The experimental data for each condition are summarized as means + SE for an n of 5-17 animals in each group. Statistical evaluation of the data was performed using the Student’s t test for unpaired comparisons. Differences among means were considered significant when P < 0.05. RESULTS
Previous reports showed that an increased rate of protein synthesis following an acute (within 24 h) inflammatory or bacterial insult was associated with an increased liver size and protein content (9, 18). To deter-
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C448
PROTEIN
SYNTHESIS
mine whether similar changes occurred in a chronic model of inflammation and inflammation compounded by bacterial infection, the liver weights and concentration of protein in livers were measured 5 days following the implantation of the fecal-agar pellet. The total liver weight was significantly (P < 0.05) increased 24% in both sterile inflammation and sepsis compared with control (Table 1). In contrast, there were no significant differences in the amount of protein in livers from the different groups. Because the wet weight to dry weight ratio is increased in sterile inflammation and sepsis (32), the protein content of the liver was also calculated per gram dry weight. As was observed for the wet weight measurements, there were no significant differences in the protein content among the different groups (data not shown). However, the total amount of protein per liver was significantly (P < 0.05) increased by 20% in sterile inflammation and by 33% in sepsis. Hence, part of the increase in liver weight in sepsis and inflammation results from an increased total liver protein content. The effect of chronic (5 day) inflammation and sepsis on hepatic protein synthesis is presented in Fig. 1A. The rate of incorporation of radioactive phenylalanine into protein in livers from control animals was 534 f 91 nmol. h-l. g liver-‘. This corresponds to a fractional rate of synthesis of 45 & 7%/day. Both the rate of incorporation of radioactive phenylalanine into proteins and the fractional rate of synthesis (89 rfi 14%/day) were increased approximately twofold in livers from rats with a sterile abscess (P < 0.05) compared with control animals. As observed in sterile inflammation, sepsis significantly stimulated both the incorporation of radioactive phenylalanine into proteins (P < 0.05) and the fractional rate of synthesis (96 + 17%/day, P < 0.02) twofold compared with control. Hence, both chronic sterile inflammation and sepsis enhanced rates of total hepatic protein synthesis. Furthermore, the magnitude of the stimulation of protein synthesis was similar in both conditions. Although the results from the in vivo rates of protein synthesis indicate an increase in hepatic protein synthesis, the flooding-dose method used preferentially estimates the synthesis of proteins rapidly being synthesized (5, 18). Furthermore, the short time between injection of radioactive phenylalanine and time of tissue sampling (15 min) estimates the synthesis of both secretory and nonsecretory proteins, as newly synthesized proteins remain in the liver for at least this period. Therefore, alterations in the rate of hepatic protein synthesis were further investigated in experiments using the isolated perfused liver where secreted and nonsecreted protein synthesis could be measured. In these studies, samples of the perfusion medium and of the liver were taken to determine the rates of production of secretory and nonsecretory proteins, respectively. After a lag period of -30 min, which represented the time for synthesis, processing, and secretion of protein into the perfusate, incorporation of radiolabeled leucine into total secreted protein increased linearly over the duration of the perfusion (Fig. 2). The characteristic lag period of 30 min between the addition of radiolabeled leucine into the perfusate and appearance of radiolabeled proteins was not altered by sterile inflammation or sep-
IN
SEPSIS
i -+
--tI I ::.:.I ..:: :.:.
.:
: :; ~.
:
: 1:
: ,. 7, I, ,/ ,, I , , ,, / /I, ,, L CONTROL
STERILE
ABSCESS
SEPTIC
ABSCESS
Fig. 1. Rate of protein synthesis (A), RNA concentration (B), and translational efficiency (C) in livers of control, sterile inflammatory, and septic rats. Livers were taken from animals 5 days following intraperitoneal introduction of a sterile fecal-agar pellet with and without a bacterial inoculum. Rates of protein synthesis were measured in vivo following intravenous injection with saline containing [3H]phenylalanine (150 pmol/lOO g body wt). Determination of [3H]phenylalanine incorporation into liver protein and calculation of rates of protein synthesis were performed as described in METHODS. Total RNA was measured in homogenates of liver samples. Synthesis rate/ RNA values were obtained by dividing protein synthesis rate by tissue RNA content for each condition. Values shown are means + SE for 510 animals in each group. A: *P < 0.05 vs. control; B: *P < 0.05 vs. control or septic abscess; C: *P < 0.05 vs. control or sterile abscess.
sis. However, in livers from both sterile inflammatory and septic rats, the rate of production of total secreted proteins was increased markedly compared with values obtained from livers of control rats. These differences, as well as data relating to nonexported proteins in perfused livers, are shown in Table 2. Livers from sterile inflammatory and septic rats produced total secretory proteins at a rate 174% (P < 0.001) and 142% (P < 0.001) of the control rate, respectively. Furthermore, the proportion of total liver protein synthesized destined for export to the plasma was significantly (P < 0.05) in-
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PROTEIN I
I
A
I
I
I
I
SYNTHESIS
I
Control
-.A -a-
-
B
Sterile Septic
20
0
40
60
80
100 120
TIME OF PERFUSION (min) Fig. 2. Production of total secreted protein by perfused livers from control, sterile inflammatory, and septic rats. At zero time, [“Hlleucine was added to a final concentration of 5.0 &i/ml of perfusate. Samples of nerfusate were collected at 2O-min intervals for determination of radioactivity present in total secretory proteins as described in METHODS. Values presented for each time point are means t SE of 5-12 determinations in each condition. Rates of total secretory protein production (given in Table 2) were derived from a least-squares linear regression analysis of values presented here.
Table 2. Effect of sterile inflammation and sepsis on rates of protein synthesis in perfused livers Measurement
Total
secreted
Control
protein
. S~~t~~~~~~~~~x~~~~~~
. . Sypnrphteesi~ onfltifii l:LL protein, mg* g-’ 9h-’ % of total liver protein
secreted
Sterile Abscess
Septic Abscess
1.04kO.07
1.81&0.17*
1.48t0.07*
1.6OkO.08
2.14&0.14?
1.95&0.10*
2.56k0.13
3.94&0.29*
3.37&0.10*
39&2
47+2$
47+1$
Values represent means k SE for determinations derived from 5-12 * P C 0.001 vs. control; t P < 0.005 vs. animals in each condition. control; $ P < 0.05 vs. control. Synthesis of total liver protein is a summation of individual values for total secretory protein production and synthesis of nonexported protein for each liver. Ratio of total secreted protein production to total liver protein synthesis x 100.
creased in both sterile inflammation and sepsiscompared with control. Synthesi .sof nonexported protein s by livers from sterile inflammatory and septic rats was also significantly increased 34% (P < 0.005) and 22% (P < 0.05) compared with control. Likewise, the rate of total liver protein (i.e., secreted plus nonexported) was also significantly increased in sterile inflammation (P < 0.001) and sepsis (P < 0.001). Increased rates of protein synthesis may be due to an increased capacity for protein synthesis or an increased efficiency of the translation process. Ribosomal RNA, which represents the capacity for protein synthesis, accounts for as much as 80% of the total RNA content. Therefore, changes in the total amount of RNA presumably reflects changes in ribosomal RNA. The total RNA concentration in liver is shown in Fig. 1B. The concentration of RNA in livers from control animals was 5.2 t 0.1 mg RNA/g wet wt, which corresponds to 26 t 0.8 pg RNA/mg protein. Sterile inflammation was associated
IN
c449
SEPSIS
with an -30% increase in total hepatic RNA (P c 0.01). This increase in RNA in livers from sterile inflammatory animals was significant whether the data were expressed as per gram wet weight (P < 0.05) (Fig. 1B) or per milligram protein basis (P < 0.01) (data not shown). However, in contrast to sterile inflammation, sepsisdid not induce an increase in total liver RNA content. The RNA concentration in livers from septic animals was not significantly different from control animals but was significantly lower than the values obtained from sterile inflammatory animals (P C 0.05). Because the stimulation of protein synthesis in sepsis was not correlated with increased capacity for protein synthesis, the efficiency of translation, expressed as protein synthesis per RNA, was calculated. These results are shown in Fig. 1C. The translational efficiency was increased, although not significantly in livers from sterile inflammatory animals compared with control. However, in contrast to sterile inflammation, sepsiswas associated with an over twofold (P c 0.001) enhancement of the translational efficiency compared with control. Hence, these data indicate that increased efficiency of translation is an important mechanism for enhanced protein synthesis in sepsis. The effect of sterile inflammation and sepsis on the translational component of protein synthesis was also evaluated by isolating the 40s and 60s ribosomal subunits from liver using sucrose density gradient fractionation. The results of these comparisons are shown in Fig. 3. The content of ribosomal subunits in livers from sterile abscess rats was not significantly different from control in sterile inflammation. In contrast to sterile inflammation, the amount of the 40s and 60s ribosomal subunits was both significantly decreased relative to control in livers of septic rats. To further examine the regulation of protein synthesis, the rate of peptide-chain elongation was measured in I
40
s
L Sterile Abscess
Septic Abscess
CONDITION Fig. 3. Effect of sterile inflammation and sepsis on levels of ribosomal subunits in liver. Samples were taken from animals as described in Fig. 1. 40s and 60s ribosomal subunits were isolated on sucrose gradients and analyzed for RNA as described in METHODS. Values shown are means & SE for 7-8 livers in each condition. *P < 0.01 vs. control; **P < 0.05 vs. control
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c450
PROTEIN
SYNTHESIS
vivo. In this method, one rat is pulse-labeled with [3H]methionine and another animal is pulse-labeled with [35S]methionine. The relative rate of elongation between the two animals is deduced from the difference in the average ratio of isotopes in the polysomal fractions and the average ratio of isotopes in the soluble protein fraction (15). To verify this procedure, it was necessary to demonstrate that the ratio of 3H/35S in the soluble protein fraction (S) divided by the ratio of 3H/35S in the nascent-chain polysome fraction (P), that is the S/P ratio, was unity in control animals. The S/P ratio in livers from control animals was 1.05 t 0.04 in five separate experiments, as expected. This result indicates that the relative rate of peptide-chain elongation was nearly identical in the two control animals. To further validate the procedure, modulation of the rate of peptide-chain elongation should result in the appropriate changes in radioactivity in the fractions corresponding to the soluble proteins and polysomes. Cycloheximide is known to inhibit protein synthesis by blocking elongation at the site of peptide-bond formation on the 80s ribosome. Therefore, the effect of cycloheximide on the rate of elongation was determined in two groups of control rats. One group received an intraperitoneal injection of cycloheximide (2.5 pg/kg body wt) and the other received an equal volume of saline. This amount of cycloheximide was calculated to give an in vivo concentration close to the inhibition constant (Ki) of peptide-bond formation in perfused mouse livers (G. E. Mortimore, personal communication). Forty-five minutes following the injection of either saline or cycloheximide, the appropriate isotope was infused. The S/P ratio was 0.156 in cycloheximide- vs. saline-treated animals. This result indicates that the rate of elongation measured by this procedure was inhibited by 85% in rats injected with cycloheximide (Table 3). With these preliminary studies verifying the procedure, the rate of elongation in sterile inflammatory and septic animals relative to control was measured. In livers from sterile inflammatory animals, the rate of elongation was significantly (P < 0.001) increased 48% relative to control (Table 3). A similar significant (P c 0.005) increase in the rate of elongation relative to control was observed in livers from septic animals relative to control. Hence, the relative rate of elongation was increased to the same extent in both sterile inflammation and sepsis Table 3. Effect of sterile inflammation and sepsis on the relative rate of peptide-chain elongation in vivo Condition
Control Control + cycloheximide Sterile abscess Septic abscess
Rate
of Elongation (% control)
105k4 16k9*
149t8* 140t6t
Data are presented as means t SE for 4-5 animals in each group. 0.001 vs. control; t P < 0.005 vs. control. Rats were injected with 1 mCi of either [3H]- or [35S]methionine and liver samples were frozen in situ 5 min later. Radioactivity in soluble protein and polysome fractions was measured as described in METHODS. The ratio of 3H to “‘S in soluble protein fraction (S) and polysome fraction (P) was calculated. Values shown represent S/P ratio of experimental group to control X 100. *
P
