Studies of the effect of experimental inflammation on rat liver nucleotide sugar pools

Camp. Biochem. Physiol. Vol. 17A, No. 2, pp. 207-212, Printed in Great Britain

STUDIES

1984 0

OF THE EFFECT OF EXPERIMENTAL ON RAT LIVER NUCLEOTIDE SUGAR

0300-9629/84 $3.00 + 0.00 1984 Pergamon Press Ltd

INFLAMMATION POOLS

H. A. KAPLAN, B. M. R. N. J. WOLOSKI and J. C. JAMIESON Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 (Received 6 May 1983) Abstract- 1. The effect of experimental inflammation on levels of nucleotide sugars was studied in tat liver. 2. There was an increase of about 2-fold in the levels of UDP-N-acetylhexosamines and GDP-Man at 8 and 4 hr after inflammation, respectively. At 48 hr after inflammation GDP-Man had returned close to control values, but UDP-N-acetylhexosamines were still about 50% above controls. 3. There was a 30% reduction in CMP-NeuAc and UDP-Gal at 8-12 hr after inflammation before increasing to slightly above controls at 16-48 hr after inflammation. 4. Inflammation resulted in an increase in activities of glucosamine-6-phosphate synthase and UDPGlcNAcZ-epimerase to about twice control activities at 24 and 8 hr after inflammation, respectively, before declining; CMP-NeuAc synthase activities did not show large changes following inflammation.

INTRODUCTION Inflammation in mammals caused by a variety of conditions including neoplastic disease, rheumatoid arthritis and exposure to chemical inflammatory agents results in changes in a variety of processes affecting lipid, carbohydrate, amino acid and mineral metabolism (Biesel, 1980). Recently, we have reported that inflammation causes an increase in amino acid pools in liver (Woloski et al., 1983). This was attributed to an increase in the flux of amino acids from muscle to plasma and then to liver for utilization by liver for elevated protein synthesis. The major group of liver synthesized proteins that are elevated in inflammation are the acute phase serum glycoproteins, such as rat CQacid glycoprotein which has been extensively studied in our laboratory (e.g. Jamieson et al., 1975, 1982; Shutler et al., 1977; Langstaff et al., 1980). Acute phase glycoproteins, like q-acid glycoprotein, contain carbohydrate chains of the complex type composed of Man, Gal, GlcNAc and NeuAc (Jamieson, 1983). The activated forms of these sugars that are utilized for oligosaccharide chain synthesis are the appropriate nucleotide sugars. In view of the effect of inflammation on hepatic synthesis of acute phase glycoproteins, as well as the earlier reported changes in hepatic amino acid pools found following inflammation, we have now measured the pools of nucleotide sugars in liver following inflammation and compared these results with those obtained from control animals. The results are discussed in terms of the possible relationship to elevated glycoprotein biosynthesis found following Abbreviations: GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine ; Gal, galactose; Man, mannose ; Glc, glucose; NeuAc, N-acetylneuraminic acid ; uridine diphosphate-N-acetylglucosUDP-GlcNAc, amine ; UDP-GalNAc, uridine diphosphate Nacetylgalactosamine ; GDP-man, guanosine diphosphate mannose ; CMP-NeuAc, cytidine monophosphate-Nacetylneuraminic acid. 207

inflammation and should prove to be useful in understanding the complex metabolic changes occurring in liver during injury and stress.

MATERIALS AND

METHODS

Sugars and nucleotide sugars were from Sigma Chemical Corp., St. Louis, MO; UDP-[‘4C] GlcNAc (300 nCi/nmol); GDP-[“VZ]Man (166 nCi/nmol) and CMP-[14C] NeuAc (197 nCi/nmol) were from New England Nuclear Corp., Lachine, Quebec; ACS cocktail was from Amersham Corp., Oakville, Ontario; analytical grade Dowex l-x8 100-200 mesh, Cl- form was from Bio-Rad Laboratories, Richmond, CA; picric acid was from BDH, Poole, U.K. Preparation

ofliver extracts

Liver extracts were prepared from control and experimentally inflammed rats as described earlier for the measurement of liver amino acid pools (Woloski et al., 1983) except that [“‘Cl labelled nucleotide sugars were added (typically 0.1 nmol, 30 nCi) prior to deproteinization by homogenization with picric acid; this was necessary to test for recoveries following chromatography on Dowex columns. Samples of 8 ml supernatants were applied to Dowex columns as before (Woloski et al., 1983) and nucleotide sugars were eluted with 40 ml 0.25 N HCI after amino acids, sugars and amino sugars had been washed off the columns with 40 ml 0.04 N HCl (Woloski et al., 1983). Based on recoveries of radioactivity not less than 90% of the nucleotide sugars in the liver homogenates were eluted in the 0.25 N HCI fraction. The 0.25 N HC1 fractions were taken to dryness by rotary evaporation and used for analyses of UDP-N-acetylhexosamines, UDPGal and GDP-Man. For analyses of CMP-NeuAc, cell sap fractions were prepared from 4 g samples of liver by a procedure based on that described by Bley et al. (1973). Livers were perfused with ice-cold 0.154. M KCl-1 mM EDTA adiusted to DH 7.5 with NaOH and homogenized in perfusion buffer using 10 up and down strokes of> Potter-Elvehjem homogenizer at 2000 rev/min. A large granule fraction was sedimented at 20.000 pfor 10 min in the Sorvall RC-2B with the SS-34 rotor and tlhe supernatant centrifuged for 1 hr at 105,OOOg in a Beckman L5-50 fitted with a Ti 50 rotor. The supematant was used for determination of CMP-NeuAc (see

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H. A. KAPLAN et ul

belowl and for assay of CMP-NeuAc synthase and UDPGicNAc-2’qimerasc (see below). For assay of glucosamineh-phosphase synthase activities (see below) homogenates were fractionated as above, but 12 mM gluc{~se-6-phosphate was added prior to homogenization to prevent conversion of fructose-h-phosphate to glucose&phosphate by phosphoglucose isomerase(Miyagi and Tsiuki. 1971 ; Bley et nl., 1973).

The&‘75 HCl effluents from the Dowex coiumns(see above) weredissolved in 2 ml water: 1.5ml were removed for analysis of UDP-N-acetylhexosamines and the remainder was used for analysis of GDP-Man and UDP-Gal. For analysis of UDP-GlcNAc and UDP-CalNAc the sample was adjusted to 2 N HCI by addition of 1.5 ml 4 N HCI and hydrolysed at 100 C for 3 hr under vacuum to release free amino sugars (Molnar e’I al., 1964); samples were taken to dryness in a desiccator over NaOH. Samples were dissolved in 1ml water and appropriate volumes analyzed in an NC-2P Technicon Amino Acid Analyzer System. To test for recovery after hydrolysis. samples of 100 nmol UDP-GlcNAc, UDPGafNAc. glucosamine-NC1 and galactosamine-HCI, either alone or as mixtures, were analyzed as above. Recoveries were not less than 90” For analyses of GDP-Man and UDP-Gal, 0.5 ml 0.02 N HCI and 100 nmol myo-inositol were added to the solution that remained after removal of a sample for hexosamine analyses (see above). Nucleotide sugars were hydrolyzed at 100 C for 20 min to release free sugars. The samples were applied directly to columns of Dowex-1 x 8 as described previously (Woloski et al., 1983) and neutral sugars eluted with 40 ml 0.04 N HCI: the eWuent was taken almost to dryness by rotary evaporation, transferred to l ml Reactivials (Pierce Chemical Co., Rockford, IL) dried under N, at 65 C and used for analyses of sugars by gas-liquid chromatography. Gas-liquid chromatography was performed on alditol acetate derivatives of sugars prepared by the method of Torello er ul. (1980). The equipment used was a PerkinElmer Sigma 2B gas chromatograph fitted with a flame ionization detector. Glass columns 6 ft long and 2 mm i.d. were packed with 3”;; SP-2330 on lm/l20 Supelcoport (Supelco Inc.. Bellefonte, PA). The temperature program was set for 6 min at 1RO’C followed by 2 min at 220°C at a rate of 1 Cjmin and finally for 15 min at 250°C at a rate of S”C/min. Injection port temperature was 22O’C and the detector temperature was 300 C. The carrier gas was N, at a rate of 20 ml:min. Peak areas were determined with a Perkin-Elmer M2 integrator. Analyses for CMP-NeuAc was performed on cell fractions using a method based on that described by van den Eijnden and van Dijk (1972). Samples of 38 nCi CMP-[‘4C]-NeuAc (I 97 nCi,/nmol) were added to 1 ml vol ofcell sap in ice and the entire solution applied to a 40 cm origin on Whatman 3 MM paper (46 x 57 cm) and descending chromatography performed at 4’ C for 24 hr using 95”L ethanol : 0.6 M NH,OH (7:3) as solvent. The band containing CMP-NeuAc was detected under u.v. light or by counting small sections of paper cut from the edges of the chromatogram. Under the conditions used CMP-NeuAc had an R, value of about 0.3 and was well separated from NeuAc which had an R(value of about 0.5. The CMP-NeuAc was eluted from the chromatogram with water and the eluate freeze-dried. The CMPNeuAc typicaily contained 90:: of the radioactivity applied to the chromatogram. The freezedried maferia1 was dissolved in 1 ml water and suitable volumes were removed for counting and for determination of NeuAc using the thiobarbituric acid method of Warren (1959); the method includes a hydrolysis step which releases NeuAc from CMP-NeuAc in siru. The CMP-NeuAc content of cell sap was calculated from the results of quantitative analysis for NeuAc and the radioactivity recovered, after correciing for NeuAc added as CMP-[‘QNeuAc. In all the experiments involving recovery of sugars, results

were corrected for losses based on the recovertes of radioactivity obtained following the isolation procedures or, rn the case ofgas-liquid chromatography. by usingmyo-inos~t~~l as an internal standard. Enzyme assays Glucosamine-6-phosphate synthase activities were measured according to Kornfeld (1967). Assay mixtures contained 12 mM glutamine, 1 mM EDTA, 40 mM sodium phosphate buffer, pH 7.5. and up to 0.1 ml 105,OOOg supematant containing 12 mM glucose-6-phosphate (set above) and 6 mM fructose-&phosphate in a total volume 01 0.5 ml. Incubations were for 60 min at 37 ‘C and the reaction was stopped by heating at 1OO’C for 2 min. Protein was removed by centrifugation at 5000 gfor 10 min in a bench centrifuge and 0.2 ml samples of supernatants were analyzed for glucosamine-6-phosphate by a modification of the Margan-Elson method as described by Ghosh et ui. (1960) scaled down to a final volume of 3.6 ml. One unit of glucosamine-&phosphate synthase activity is defined as I nmol glucosamine-6-phosphate formed in 60 min. UDP-GlcNAc-2’-epimerase was assayed according IO Sommar and Ellis (1972). Assay mixtures contained 0.5 pmol UDP-GlcNAc, 12.5 pmol MgSO,, 50 pmol TrisHCl buffer, pH 7.5, and up to 0.1 ml 105.000 gsupernatant in a total volume of0.25 ml. Samples were incubated for 20 min at 37 C and the reaction was stopped by heating at loo’C for 2 min and N-acetyl-b-mannosamine, the reaction product, was determined as described by Spivak and Roseman (1966). One unit of UDP-GlcNAc-2’-epimerase activity is defined as 1 nmol N-acetyl-o-mannosamine formed in 20 min. For assay of CMP-NeuAc synthase the procedure of Kean and Roseman (1966) was used. Each incubation mixture contained 90 pmol Tris-HCI, pH 9.0, 2.5 pmol NeuAc, 2.5 pmol MgCI, and 0.1 ml of 105,000 gsupernatant in a total volume of 0.5 ml. Incubations were for up to 20 min at 37’C and the tubes were cooled in ice. Free NeuAc was destroyed with 0.075 ml 2.5 M NaBH, for I5 min at room temperature and CMP-NeuAc remaining was assayed as above (Warren, 1959). One unit of CMP-NeuAc synthase activity is dehned as 1nmof CMP-NeuAc formed in 20 min. In all enzyme assays linearity was established between enzyme activities and amount of enzyme protein; reactions were also linear with time under the assay conditions used. Protein was assayed as described by Lowry et ul. (1951). but with modified reagents and volumes described by Miller ( 1959). RESLiLTS

The effect of experimental inflammation on hepatic levels of UDP-N-acetylhexosamines is shown in Fig. I, Control rat liver contained 141 nmol of UDP-Nacetylhexosamines/g wet wt liver composed of 95 nmol UDP-GlcNAc and 46 nmol of UDP-GalNAc. In~ammation caused a rapid rise in UDP-Nacetylhexosamine pools reaching a maximum at 8 hr after inflammation where the pool sizes were about twice those found in controls. This was followed by a rapid decline reaching a value of about 50% above controls at 12 hr after inflammation ; a second minor peak was observed at I6 hr after inflammation before a steady state was established where UDP-IVacetylhexosamine pools were about 507; above control values (Fig. 1). Figure I also shows that the response of UDP-GlcNAc to inflammation parallels that of UDP-GalNAc although the pool of UDPGlcNAc was about twice that of UDP-GalNAc. This observation was not surprizing since it is known that UDP-GlcNAc-~-epimerase which forms UDPGalNAc from UDP-GlcNAc maintains steady state

Inflammation

and nucleotide

1 I -i 100

90

1 so

Fig. 1. Effect of inflammation on UDP-GlcNAc, -a--; and UDP-GalNAc, ---A---; in liver. Results are means from 3 to 6 experiments ; the bars represent expressed as standard

the range of values obtained errors of the mean.

levels of these nucleotide sugars such that UDPGlcNAc levels are about twice those of UDP-GalNAc (Molnar et al., 1964). Figure 2 shows that the control level of GDP-Man was 4.8 nmol/g wet wt liver. Inflammation resulted in a rapid increase to about 70% above controls within 4 hr after inflammation ; there was then a rapid decline to about half control values at 12 hr followed by a second peak at 24 hr before returning close to control values at 48 hr after inflammation. Although the levels of GDPMan were much lower than those found for UDP-Nacetylhexosamines (Fig. 1) the response of GDP-Man to inflammation did bear a resemblance to that found with UDP-N-acetylhexosamines. For example, all three nucleotide sugars increased rapidly to about twice control values at short times after inflammation (i.e. at 4 hr for GDP-Man and 8 hr for UDP-Nacetylhexosamines ; Figs 1 and 2), declined at 12 hr after inflammation and then increased again at longer times after inflammation (see Figs 1 and 2). The effect of inflammation on hepatic levels of CMP-NeuAc and UDP-Gal are shown in Fig. 3. The

Fig. 2. Effect of inflammation on GDP-Man in liver. Results are expressed as for Fig. 1.

sugars

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control level of CMP-NeuAc was 49 nmol/g wet wt liver, but this was reduced by about 30% at 8-12 hr after inflammation; there was then a rapid increase to give values slightly above control values at 2448 hr after inflammation. The response of UDP-Gal to inflammation seemed to parallel that of CMP-NeuAc (Fig. 3). The control level of UDP-Gal was 31 nmol/g wet wt liver. Like CMP-NeuAc the level of UDP-Gal declined at short times after inflammation before increasing rapidly to just above control values at 1648 hr after inflammation (Fig. 3). In view of the alterations described above in the hepatic levels of UDP-N-acetylhexosamines and CMP-NeuAc found during inflammation, it was of interest to measure the changes in the activities of the regulatory enzymes involved in the synthesis of these two nucleotide sugars. Glucosamine-6-phosphate synthase which converts fructose-6-phosphate to glucosamine-6-phosphate with glutamine as amide donor is the regulatory enzyme for UDP-GlcNAc synthesis ; the enzyme is subject to feedback inhibition by UDP-GlcNAc (Kornfeld et al., 1964; Schachter, 1978). For synthesis of CMP-NeuAc the regulatory enzyme is UDP-GlcNAc-2’-epimerase which catalyzes the formation of N-acetylmannosamine from UDPGlcNAc; this enzyme is subject to feedback inhibition by CMP-NeuAc (Kornfeld et al., 1964; Schachter, 1978). CMP-NeuAc synthase was also measured as an example of a non regulatory enzyme. The effect of inflammation on these three enzyme activities is given in Table 1. Both regulatory enzymes showed increases in specific activities following inflammation. There was a small increase in glucosamine-6-phosphate synthase activity at 8 hr after inflammation, but the major response occurred at 24 hr after inflammation when enzyme activities were about twice those of controls, Although this was followed by a decline in activities, values were still substantially above control values at 48 hr after inflammation. Activities of UDP-GlcNAc2’-epimerase declined to about 60% of controls at 4 hr after inflammation (Table 1); however, this was followed by a rapid rise to over twice control values at 8 hr after inflammation after which activities returned to control values (Table 1). In contrast to the two regulatory enzymes, activities of CMP-NeuAc synthase did not change significantly following inflammation (Table 1).

Fig. 3. Effect of inflammation on CMP-NeuAc, -0~; and UDP-Gal, -A-; in liver. Results are expressed as for Fig. I.

H. A.

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KAPLAN CI ul.

‘Table I. EfTect of inllammation on actlvltiesofglucosamine-6.phosphate GlcNAc-2’-epimerase and CMP-NeuAc aynthase* rime artrr intlammatmn (hrl Control 4 x I2 16 24 4x

Glucosamine-6-P synthase 35.0 38.5 42.5 37.0 59.5 78.0 60.0

k k k f f * f

2.5 2.9 1.5 0.8 5.8 4.4 2.1

UDP-GlcNAc-2’. epimerase 58.6 41.0 127.7 96.7 70.2 60.9 56.8

i_ & * k i_ k +

1.9 2.3 2.0 I9 2.6 1.2 I.2

synth;lxc. IJDP-

CMP-NcuAc synthase 90.0 86.5 x5.0 75.0 71.0 101.0 95.0

c 46 * 4.5 t I4 * 3.4 _ir 3.5 * 77 * 35

*Expressed as specific enzyme activities (units of enzyme activity/mg cell sap protein. see Materials and Methods section). Values are means from six experiments on separate animals i SEM.

DlSCUSSlON

The 141 value of nmol of UDP-Nacetylhexosamines found for control liver compares favourably with some literature values, but is slightly lower than others. For example, Okubo et al. (1976) reported values ranging from 127 to 534 nmol/g liver from fed rats; with starved rats, Bley et al. (1973), reported 207 nmol/g liver and Bates et al. (1966) reported 270 nmol/g liver. The variability in UDP-Nacetylhexosamine levels could either reflect the use of different strains of rat, or result from variations in the nutritional status of the animals ; UDP-GlcNAc pools have recently been shown to be affected by diet (Tepperman et u[., 1981). Increases in UDP-Nacetylhexosamine pools following trauma have been reported, but the earliest times that were examined were at 18 hr. (Bley et al., 1973 ; Okubo et al., 1976). At times after trauma of 18 hr or longer it was observed that UDP-N-acetylhexosamine pools increased by about 2@30:/, which compares favourably with the 50”/;;increase found in the present work at longer times after inflammation (Fig. 1). The earlier workers, however, would not have detected the sudden rise in UDPN-acetylhexosamine pools to over twice control values that occurred at 8 hr after inflammation, or the subsequent dip in pool levels found at 12 hr after inflammation (Fig. 1). These results show that the response of UDP-N-acetylhexosamine pools to inflammation is much more rapid than was previously believed. Compared to the information available on UDP-Nacetylhexosamine pools in liver, the literature contains much less information on liver pools of the other nucleotide sugars examined in the present studies and how the pools respond to trauma. The value of 49 nmol/g liver found for CMP-NeuAc is slightly higher than the 37 nmol/g liver reported by Carey and Hirschberg (1979), but the response of this nucleotide sugar to trauma has not been reported. It was found that CMP-NeuAc pools were reduced by about 30% at 8-12 hr after inflammation before returning to control levels at about 16 hr after inflammation. A similar response was observed for UDP-Gal pools. The control level of UDP-Gal was 31 nmol/g liver which is substantially lower than the value of 92 nmol/g liver reported by Bauer et al. (1976), using an enzyme assay system, rather than analysis by gas chromatography, to measure the nucleotide sugar. Although the reason for this discrepancy is unclear, Bauer et al. (1976) did

report that UDP-Gal pools fell by 2&307, at short times after trauma induced by sham operation or partial hepatectomy and that this was followed by an increase to control levels at 24-48 hr after operation. The behaviour of UDP-Gal pools in response to trauma reported by Bauer et al. (1976), is thus consistent with the data presented in the present work. The control level of GDP-Man was 4.8 nmol/g liver which is considerably lower than the pool sizes of the other nucleotide sugars studied. Although the literature does not appear to contain information on liver GDP-Man levels, low levels of this nucleotide sugar have been found in other tissues. For example, Mendicino and Rao (1975) reported a value of 12 nmol/g tissue for lymph node. As mentioned in Results the behaviour of the GDP-Man pools after inflammation more closely resembled the changes observed in UDP-Nacetylhexosamine pools, rather than those found for CMP-NeuAc and UDP-Gal. Although the magnitude of the changes were different, pools of UDP-Nacetylhexosamines and GDP-Man both increased rapidly after onset of inflammation, then declined and finally increased again (Figs 1 and 2). The similarities found for the effect of inflammation on the behaviour of the CMP-NeuAc and UDP-Gal pools on the one hand and of the UDP-Nacetylhexosamine and GDP-Man pools on the other, could be explained if we examine the role played by these nucleotide sugars as donors of the glycosyl units for the synthesis of the oligosaccharide chains of liver glycoproteins. Residues of NeuAc and Gal are known to occupy terminal and penultimate positions, respectively, on oligosaccharide chains of liver synthesized glycoproteins, in addition, it is well known that these two sugars are transferred directly from their nucleotide sugars by the appropriate glycosyl transferases to growing oligosaccharide chains (e.g. Schachter. 1978 ; Beyer et al., 1981). Thus, although CMP-NeuAc synthesis is under regulatory control (see Results ; also, see below), the pool levels of CMP-NeuAc and UDPGal might be expected to change in a similar way in response to inflammation ; moreover, the changes are likely to be related to changes in glycoprotein biosynthesis found after inflammation. Therefore, it is interesting to speculate that the decline in CMPNeuAc and UDP-Gal pools at 4-16 hr after inflammation might reflect increased demands for NeuAc and Gal for liver glycoprotein biosynthesis which is substantially increased at 8-24 hr inflammation

Inflammation and nucleotide sugars (Jamieson et al., 1975). Incorporation of GlcNAc into oligosaccharide chains of glycoproteins occurs in two distinct ways; the first way involves direct transfer from UDP-GlcNAc to a growing oligosaccharide chain in the same way that NeuAc and Gal are added. The second way is quite different and involves the formation of a lipid-oligosaccharide complex which acts as the glycosyl donor for the sugars that form the inner core regions of the carbohydrate chains of serum glycoproteins (Sharon and Lis, 1981; Jamieson, 1983). The lipid-oligosaccharide also contains all of the Man residues found in the oligosaccharide chains of serum glycoproteins. Thus, because of the complex way in which Man and GlcNAc are incorporated into glycoproteins it is harder to relate changes in GDP-Man and UDP-GlcNAc pools in inflammation directly to changes in glycoprotein biosynthesis. However, since transfer of Man and GlcNAc to glycoproteins involves formation of intermediate lipid-oligosaccharide complexes it is not surprising that pools of GDP-Man and UDP-GlcNAc show similar changes following inflammation. Glucosamine-6-phosphate synthase, the regulatory enzyme for UDP-GlcNAc formation has been studied extensively in past years and it has been found to be regulated in a complicated way (e.g. Kornfeld et al., 1964; Bates et al., 1966; Kornfeld, 1967; Miyagi and Tsiuki, 1971). Briefly, the enzyme is subject to feedback inhibition by UDP-GlcNAc, but the degree of inhibition is dependent on secondary factors that can alter the binding constant of UDP-GlcNAc to the enzyme; these factors include UTP, AMP and glucose6-phosphate. Also, it appears that the enzyme is normally about 90% inhibited which leaves substantial latitude for regulatory control. The specific activity of the enzyme in control livers was 35 units/mg protein which is slightly lower than the 40 units/mg protein reported by Bley et al. (1973) and the 35.546 units/mg protein reported by Okubo et al. (1976). In the present studies glucose-6-phosphate was added to the assay system to prevent the conversion of fructose-6phosphate to glucose-6-phosphate by phosphoglucose isomerase which is present in high levels in liver (Miyagi and Tsiuki, 1971). However, as mentioned above, glucose-6-phosphate can enhance the feedback effect of UDP-GlcNAc on the enzyme and so this probably explains the slightly lower activity of glucosamine-6-phosphate synthase found in the present work compared with the data presented by Bley et al. (1973), and Okubo et al. (1976). Inflammation resulted in a small increase in enzyme activity at 8 hr followed by a decline to control values at 12 hr after which there was a rapid rise to about twice that of control activities at 24 hr after inflammation (Table 1). Although there was a decline at 48 hr after inflammation values were still substantially above controls. These results are consistent with the findings of Bley et al. (1973) and Okubo et al. (1976) who reported large increases in glucosamine-6-phosphate synthase activities following laparotomy. The times studied by these workers were 18 hr to 8 days after injury when it was found that the glucosamine-6-phosphate synthase response could be blocked by actinomycin D or cycloheximide suggesting that the elevated activities of glucosamine-6-phosphate synthase found after 18 hr of injury are mainly due to the synthesis of new enzyme

211

protein, rather than by regulatory control. However, it is difficult to reconcile the behaviour of the enzyme at early times after inflammation (i.e. up to 12 hr after turpentine injection) with the 2-fold increase in the pool size of UDP-GlcNAc found at 8 hr after inflammation (Fig. 1). Although there is an elevated level of glucosamine-6-phosphate synthase activity at 8 hr after inflammation the change is not large and it would be unlikely to account entirely for the increased pool of UDP-GlcNAc found at this time after inflammation. Clearly, the feedback control of glucosamine-6phosphate synthase by UDP-GlcNAc during inflammation is a complex process; moreover, the process cannot be considered in isolation since UDP-GlcNAc also acts as the substrate for UDP-GlcNAc-2’epimerase, the first enzyme in the pathway leading to CMP-NeuAc formation. This reaction is not detectably reversible (Sommar and Ellis, 1972) and provides a means to reduce the GlcNAc pool and channel the UDP-GlcNAc into the biosynthetic pathway leading to CMP-NeuAc. The control activity of UDP-GlcNAc-2’-epimerase was 58 units/mg protein which is comparable to the value of 44.7 units/mg protein calculated from the data reported by Kikuchi et al. (1971) (Kikuchi et al. reported 134 units/mg protein of activity defining a unit as 1 nmol product formed/hr; in the present work a unit is 1 nmol product formed/20 min). Inflammation resulted in a rapid reduction in the activity of UDP-GlcNAc-2’epimerase to about 60% of the control value at 4 hr after inflammation. Although it is not clear what the explanation is for this response, one outcome would be a decreased utilization of UDP-GlcNAc which could partially explain the rapid rise in the UDP-GlcNAc pool after inflammation (Fig. 1). At 8 hr after inflammation epimerase activity increased to twice control values. This change might be due to the feedback inhibitor for the enzyme, CMP-NeuAc, which is present in substantially reduced levels at 8-12 hr after inflammation. The acute phase response of glycoproteins has been studied for many years, but so far there has been no satisfactory explanation for the phenomenon. The present studies have focused on one aspect of the process, namely the behaviour of nucleotide sugar pools during inflammation, concentrating on those nucleotide sugars that are the precursors of the sugars found in the oligosaccharide chains of acute phase glycoproteins. In addition, the effect of inflammation on some enzymes of nucleotide sugar metabolism was also studied. Large changes were observed in the activities of glucosamine-6-phosphate synthase and UDP-GlcNAc-2’-epimerase, the two regulatory enzymes studied; but the non-regulatory enzyme, CMPNeuAc synthase, did not change substantially. Although it is difficult to interpret information from in vitro assays of the enzymes in crude preparations, especially when they are under feedback control, it is of interest to note that both regulatory enzymes showed elevated activities during the time when liver glycoprotein biosynthesis is increasing rapidly (i.e. at S-24 hr after inflammation, Jamieson et al., 1975). In addition, it is clear that the response of the nucleotide sugar pools to inflammation is very rapid with substantial increases in GDP-man and UDP-Nacetylhexosamine pools being evident as early as 4 hr

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H. A. KAPLAN et crl

inflammation. Glycoprotein biosynthesis is just starting to show some stimulation at 4 hr after inflammation so changes in hepatic pools of GDPMan and UDP-N-acetylhexosamines appear to precede elevated protein synthesis. The results described in this paper add to our knowledge on changes previously reported (Jamieson et al.. 1975 ; Shutler et al., 1977 ; Langstaff et al., 1980; Woloski et al., 1983) that accompany the acute phase response to inflammation. In addition, the results emphasize the importance of recognizing early biochemical changes in liver and suggest that short time course studies may be crucial in achieving a full understanding of the acute phase response of glycoproteins to inflammation. after

Acknowledyements This work was Natural Sciences and Engineering Canada (grant No. A5394). Two B.M.R.N.J.W.) are indebted to the Studies. University of Manitoba for

supported by the Research Council of of us (H.A.K. and Faculty of Graduate research fellowships.

REFERENCES Bates C. J., Adams W. R. and Hanschumacher R. E. (1966) Control of the formation of uridine diphosphate-hiacetylhexosamine and glycoprotein synthesis in rat liver. J. hiol. Chem. 241, 1705-I 7 12. Bauer C. H., Hassels B. F. and Reutter W. G. (1976) Galactose metabolism in regenerating rat liver. Biochem. J. 154, 141&147. Beyer T. A., Sadler J. E., Rearick J. I., Paulson J. C. and Hill R. L. (1981) Glycosyltransferases and their use in assessing oligosaccharide structure and structure-function relationships. In Adtances in Enzymology (Edited by Meister A.), Vo. 52. pp. 23-174. John Wiley. New York. Beisel W. R. (1980 Effects of infection on nutritional status and immunity. Proc. Fed. Am. Sot. exp. Biol. 39,3 105-3 108. Bley R. L., Okubo H. and Chandler A. M. (1973) Regulation of glucosamine synthesis in injury and partial hepatectomy. Proc. Sot. cup. Biol. Med. 144, 134-140. Carey D. J. and Hirschberg C. B. (1979) Metabolism of Nacetylneuraminic acid in mammals : isolation and characterization of CMP-N-acetylneuraminic acid. Biochemistry 18, 2086-2092. Ghosh S.. Blumenthal H. J., Davidson E. and Roseman S. (1960) Glucosamine metabolism V enzymatic synthesis of glucosamine 6-phosphate. J. biol. Gem. 235, 1265-1273. Jamieson J. C. (I 983) Glycosylation and migration of membrane and secretory proteins. In Plasma Protein Secretion by rhr Lirrr (Edited by Glaumann H., Redman C. and Peters T.). Academic Press, New York (in press). Jamieson J. C., Morrison K. E., Molasky D. and Turchen B. (1975) Studies on acute phase proteins of rat serum. V. Effect of induced inflammation on the synthesis of albumin and r,-acid glycoprotein by rat liver slices. Can. J. Biochem. 53,401-414. Kean E. L. and Roseman S. (1966) The sialic acids. X. Purification and properties of cytidine S’-monophosphosialic acid synthetase. J. hiol. Gem. 241, 5643-5650. Kikuchi K.. Kikuchi H. and Tsuiki S. (1971) Activities of sialic acid synthesizing enzymes in rat liver and rat and mouse tumors. Biochim. hiophys. Acta. 252, 357-368.

Kornfeld R. (1967) Studies on L-glutamlne I,-fructose hphosphate amidotransferasc: feedback Inhibition by uridine diphosphate-N-acetylglucosaminc. .I. hiol. Chem. 242, 3135-3141. Kornfeld S., Kornfeld R., Neufeld t. b. and O’Brien P. J. (1964) The feedback control of sugar nucleotide blosynthesis in liver. Proc. mrtn. Acad. %i. u.S..4. 53, 371 -379. LangstaB J. M., Burton D. N. and Jamieson J. C. (1980) Studies on the mechamsm of the effects of experimental inflammation on adaptive synthesis of rat liver fatty acid synthctase. Archs Biochrm. Biophys. 204, 294-301. Lowry 0. H., Rosebrough N. J.. Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. .I. hiol. Chem. 193, 265-275. Mendicino J. and Rao A. K. (1975) Regulation ofthe synthesis of nucleoside diphosphate sugars in reticula-endothelial tissues. Eur. J. Biochem. 51, 547-556. Miller G. L. (1959) Protein determination for large mmbers of samples. Aria!,,,,, Chem. 31, 964. Miyagi T. and Tsiuki S. (1971) Effect of phosphoglucose and glucose h-phosphate on UDP-Nisomerase acetylglucosamine inhibition of L-glutamine: D-fructose 6-phosphate aminotransferase. Biochim. hiophys. Acti1 250, 51-62. Molnar J., Robinson G. B. and Winzler R. J. (1964) The biosynthesis of glycoproteins: 111 glucosamine intermediates in plasma glycoprotein synthesis in livers of puromycin-treated rats. J. hiol. Chem. 239, 3 I57 -3 162. Okubo H. and Chandler A. M. (1976) Endocrine status and the response of glucosamine-6-phosphate synthetase to trauma. Expl Molec. Path. 24, 91-104. Schachter H. (1978)Glycoprotein bioaynthesia. In Tlrr G‘!rc,oconjugates(Edited by Horowitz M. 1. and Pigman W.), Vol. 2, pp. 87-181. Academic Press. New York. Sharon N. and Lis H. (198 I) Glycoproteins : research booming on long-ignored, ubiquitous compounds. Ckem. Enq. News 30, 2 144. Shutler G. G., Langstaff J. M., Jamieson J. C. and Burton D. N. (1977) The effects of experimental inflammation on adaptive synthesis of rat liver fatty acid synthetase. Archs Biochem. Biophys. 183, 7 10-717. Sommar K. M. and Ellis D. B. (1972) Uridine diphosphate Nacetyl D-glucosamine-2’-epimerase from rat liver I. Chtalytic and regulatory properties. Biochim. hiophrs. Acto 268, 581-589. Spivak C. T. and Roseman S. (1966) UDP-N-Acetyl-Dglucosamine-2’-epimerase. In Methods rn Enzymoloyy (Edited by Colowick S. P. and Kaplan N. 0.). Vol. 9, pp. 612-615. Academic Press, New York. Tepperman H. M., Silver R., Dewitt J. and Tepperman J. (1981) Effects of high glucose and high lard diets on the activities of rat liver glycosyltransferases. J. Nutr. Ill, 17341741. Torello L. A., Yates A. J. and Thompson D. K. (1980) Critical study of the alditol acetate Method for quantitating small quantities of hexoses and hexosamines in gangliosides. J. Chromat. 202, 195-209. van den Eijnden H. and van Dijk (1972) Convenient method for the preparation of cytidine monophospho-Nacetylneuraminic acid. Hoppr-&>&r ‘r 2. ph~~iol. Chem 353, 1817-1820. Warren L. (1959) The thiobarbituric acid assay of sialic acids. J. hiol. Chem. 234, 1971--1975. Woloski B. M. R. N. J., Kaplan H. A. and Jamieson J. C. (1983) Studies on the efiects of experimental inflammation on rat serum and hepatic amino acid pools. Comp. Biochrm. Physiol. 74A, 8 13-8 16.