Effect of Tumor Necross Factor on AcetylCoenzyme A Carboxylase Gene Expresson and Preadpocyte Dfferentaton Mchael E. Pape and KHan Km Department of Bochemstry Purdue Unversty West Lafayette, Indana 47907 Tumor necross factor () s secreted by macrophages n response to varous stmul and blocks lpd accumulaton durng the converson of preadpocytes to adpocytes n culture. In the present report, we nvestgate the effect of recombnant on the expresson of acetylcoenzymea (CoA) carboxylase, the ratelmtng enzyme for longchan fatty acd bosynthess. We used a preadpocyte cell lne, 30A5, derved from 10T12 mouse fbroblasts after treatment wth 5azacytdne. Treatment of the preadpocyte cell lne wth dexamethasone and nsuln trggers the converson of these cells to mature adpocytes as evdenced by the accumulaton of lpd. The mrna and enzyme levels of acetylcoa carboxylase as well as the enzyme actvty ncrease markedly durng the converson process. prevents the converson of preadpocytes to adpocytes wth a concomtant nhbton n the accumulaton of acetylcoa carboxylase mrna and decrease n enzyme actvty. Ths observed reducton n acetylcoa carboxylase mrna levels s reversble upon removal of. AcetylCoA carboxylase mrna levels and enzyme actvty also decrease when fully dfferentated adpocytes are exposed to but to a much lesser extent. These results suggest that affects de novo lpd synthess n part by alterng the mrna levels of acetylcoa carboxylase. (Molecular Endocrnology 2: 395403, 1988) INTRODUCTION Invasve stmul n the form of vruses, bactera, or parastes elct a varety of host responses desgned to neutralze and destroy the nfectous agent. Retculoendothelal cells and lymphocytes medate such responses, n part, by secretng cytoknes. The acton of these cytoknes appears to affect not only the functons of the mmune system but also of nonlymphatc tssues. 088888098803950403$02.000 Molecular Endocrnology Copyrght 1988 by The Endocrne Socety For nstance, tumor necross factor () whch s secreted by macrophages n response to endotoxn (lpopolysaccharde) challenge (1, 2), affects cellular functons of adpocytes. blocks the ablty of adpocytes to synthesze and assmlate lpds and also promotes the moblzaton of trglycerde stores (35). It has been suggested that ths moblzaton of energy stores serves to fuel the host mmune system n ts fght to destroy the nfectous agent (24). In fact, has been mplcated n cachexa, a condton characterzed by weght loss due to the chronc catabolc state durng development of certan nfectons and malgnances (24, 6). Although the gross physologcal effects of are well documented (3 and references theren), the mechansm of acton has only recently begun to be explaned n bochemcal terms. Of partcular nterest toward explanng acton has been the effect of on lpogeness. has been shown to decrease the actvty and synthetc rate of lpoproten lpase n both n vtro and n vvo studes (710) and also to decrease the mrna levels of glycerophosphate dehydrogenase (4, 11), ap2 (12), adpsn (12), and other developmentally regulated genes of unknown dentty n adpocytes (4). Although the expresson of these genes appears to be assocated wth the mature adpocyte (13), and thus provdes an excellent model system for analyzng dfferentaton mechansms, none of these protens has been shown to be requred for de novo bosynthess of longchan fatty acds; one of the prmary steps n lpogeness. AcetylcoenzymeA (CoA) carboxylase s requred for the de novo synthess of the fatty acds that comprse all lpds; the carboxylase s the ratelmtng enzyme for longchan fatty acd bosynthess and catalyzes the carboxylaton of acetylcoa to malonyl CoA (14, 15). Subsequent aldol condensaton reactons nvolvng malonylcoa form the fatty acds. Snce acetylcoa carboxylase s the ratelmtng enzyme for the only pathway of de novo bosynthess of fatty acds, any agent or dfferentaton process that affects fatty acd bosynthess must alter the expresson of acetylcoa carboxylase. Thus, acetylcoa carboxylase expresson s an Downloaded from https:academc.oup.commendartcleabstract253952713520 by guest on 04 September 2018 395
MOL ENDO1988 396 Vol 2 No. 5 other excellent marker for lpogeness n varous developmental or hormonal states. The mechansm of shortterm carboxylase enzyme control has been extensvely studed (1417). Our laboratory has recently solated cdna (18) for the carboxylase thus, allowng us to nvestgate the role of carboxylase mrna metabolsm n the expresson of acetylcoa carboxylase. Here we report that 1) an ncrease n acetylcoa carboxylase actvty durng 30A5 preadpocyte dfferentaton s due to an ncrease n the amount of acetylcoa carboxylase and ts mrna and 2) dfferentaton of 30A5 preadpocytes and accumulaton of acetylcoa carboxylase mrna s completely and reversbly nhbted by. RESULTS Relatonshp between AcetylCoA Carboxylase Actvty, Amount, and mrna Level The 30A5 preadpocyte cell lne was used to nvestgate the expresson of acetylcoa carboxylase, durng adpocyte converson. The undfferentated 10T12 fbroblast lne was used as the control cell lne. Treatment of confluent 30A5 cells wth dexamethasone and nsuln trggers the converson of these cells to adpocytes as evdenced by the precocous accumulaton of lpd droplets n these cells after several days of treatment (see Fg. 1). Eght days after reachng confluency 85 90% of the cells exhbt lpd accumulaton (Fg. 1 and Table 1). In ths regard the cells are smlar to 3T3L1 cells and ther dervatves (19) and TA1 cells (13), a preadpocyte lne also derved from 10T12 cells. The amount of cytosolc proten ncreases 2fold durng converson of the 30A5 cells but remans relatvely constant for 10T12 cells (Table 1). A smlar ncrease n proten amount has also been observed n the converson of 3T3L1 preadpocytes to adpocytes (20). AcetylCoA carboxylase actvty ncreases markedly durng the converson process (Table 1) wth an ncrease of approxmately 12fold from day 1 to day 8 on a per cell bass. However, the ncrease s 5.5fold when the actvty s expressed per mllgram of soluble proten. We have observed that there s a slght ncrease n cell number from day 1 to day 8 (1.4fold) and therefore data expressed on a per cell bass provdes a more accurate representaton. The ncrease n carboxylase actvty parallels the percent of cells exhbtng lpd accumulaton (Fg. 2A). AcetylCoA carboxylase actvty remans relatvely constant n 10T12 cells that are exposed to the same dfferentaton scheme as 30A5 cells (Table 1 and Fg. 2A). In order to determne whether ths ncrease n carboxylase actvty s due to an ncrease n the level of carboxylase enzyme, an mmunottraton of carboxylase actvty was performed usng acetylcoa carboxylase specfc antbodes (Fg. 3). In ths experment a fxed amount of cell extract s ncubated wth ncreasng amounts of antserum to acetylcoa carboxylase and then carboxylase actvty s assayed. The decrease n enzyme actvty n the presence of antserum s proportonal to the amount of carboxylase and allows determnaton of the equvalence pont,.e. the amount of antserum requred to neutralze carboxylase actvty. A 12fold hgher enzyme actvty n the sample from day 8 requred about 8 tmes more antbodes to neutralze actvty compared to the day1 sample. The ncrease n carboxylase amount correlates well wth the percent of cells exhbtng lpd accumulaton (Fg. 2B). The relatonshp between carboxylase enzyme content and mrna levels was examned by usng labeled acetylcoa carboxylase cdna to measure carboxylase mrna amount n a dotblot assay. Durng the dfferentaton process, the amount of carboxylase mrna ncreases about 10fold from day 1 to day 8 (Fg. 4). Ths ncrease s not due to a general elevaton of all mrnas as actn mrna levels follow the opposte trend. The decrease n actn mrna levels durng the converson process occurs n other preadpocyte cell lnes as well (21). The ncrease n carboxylase mrna levels correlates well wth the percent of cells dsplayng lpd accumulaton and also wth the ncreases n enzyme actvty and amount (Fg. 2, C and D). Effect of on AcetylCoA Carboxylase mrna Levels It has been prevously reported that a partally purfed preparaton of cachectn [ s closely related to or dentcal wth cachectn (1)] obtaned from mouse macrophages that had been exposed to Eschercha col lpopolysaccharde nhbted the synthess of acetylcoa carboxylase n 3T3L1 cells (22). However, t has also been observed that recombnant had lttle effect on acetylcoa carboxylase actvty n fully dfferentated 3T3L1 adpocytes (8). Such an observaton led to the speculaton that per se, mght have a mnmal effect on the acetylcoa carboxylase catalyzed pathway (8). In the present studes, we have examned the effect of recombnant human on the amount of acetylcoa carboxylase mrna. When preadpocytes were exposed to dfferent doses of for 8 days, almost completely prevented the accumulaton of acetylcoa carboxylase mrna at a concentraton of 200 Uml (Fg. 5). The effect of on acetylcoa carboxylase mrna s specfc snce 3actn mrna levels were not affected (Fg. 5). A denstometrc scannng of the Northern blot (Fg. 5) showed that at a concentraton of 200 U ml (74 ngml) nhbted carboxylase mrna accumulaton by approxmately 80%, after adjustng the level of carboxylase mrna to the level of the actn mrna. The 10 klobase (kb) carboxylase mrna and the smaller speces (<28 S) were ncluded n the analyss because the smaller speces are degradaton products of the larger carboxylase mrna. 1 Whether the smaller RNA speces were ncluded n the analyss or not, the same degree of effect was observed. We have prevously reported that a 3 kb mrna n rat lver specfcally hybrdzed to a carboxylase cdna probe (18). 1 Pape, M. E., and K.H. Km, unpublshed observaton. Downloaded from https:academc.oup.commendartcleabstract253952713520 by guest on 04 September 2018
Regulaton of AcetylCoA Carboxylase mrna 397 Fg. 1. Converson of 30A5 Preadpocytes to Adpocytes A, 10T12 mouse fbroblasts at confluency; BE, 30A5 cells at varous days after reachng confluency. B, Day 1; C, day 5; D, day 7; E, day 8. Each panel s a 100x magnfcaton of the cell monolayer by phase contrast. Downloaded from https:academc.oup.commendartcleabstract253952713520 by guest on 04 September 2018 In addton to alterng the accumulaton of carboxylase mrna, also decreased acetylcoa carboxylase actvty by about 67% on a per mllgram of proten bass when preadpocytes were exposed to for 8 days (Fg. 6). Under these condtons, the converson of the preadpocytes to adpocytes was completely nhbted (Fg. 7); an effect that could not be overcome by nsuln at concentratons of up to 200 u.gml. Effect of Wthdrawal on AcetylCoA Carboxylase mrna In order to determne f the nhbtory effects of on acetylcoa carboxylase mrna accumulaton could be reversed, we examned the effect of removal from the culture meda of 30A5 cells. Confluent preadpocytes were treated wth dexamethasone and nsuln for
MOL ENDO1988 398 Vol 2 No. 5 Table 1. Changes n 30A5 Cells durng Adpocyte Converson Days after Confluence Proten" 1 2 3 4 5 6 7 8 10 % Cells Exhbtng Lpd Accumulaton 30A5 10T12 <1 <1 510 <1 1520 <1 30 <1 45 <1 6570 <1 8590 <1 8590 <1 Cytosolc Proten (mg10 6 Cells) 30A5 10T12 0.18 0.29 0.30 0.19 0.38 0.23 ACC Actvty (MU10 6 Cells) 30A5 10T12 21 46 112 25 243 33 30A5 117 363 635 ACC Actvty (MUmg) a Proten amounts and acetyl CoA carboxylase actvtes are presented as the average of two determnatons on duplcate cell monolayers for each tme pont. (7) J25O u 2 200 () I5O H IOO > I < 50 ACC IO CJ e \ <8 *o \ 6 o < 4 > u 2 or A 1 A J*^ 2 DAYS B 1? A A A, 4 AFTER A ^ A r r 1 1 9^ V A V A A 6 8 10 CONFLUENCE ' f ~ _ IOC) 80 60 40 100 80 60 40 20 O 1 LVUN FER LL. Q () 20 _l _l CJ Q \ REN' u. I DIF U) o 2 4 6 8 10 DAYS AFTER CONFLUENCE 2 4 6 8 10 5 2 4 6 8 10 s < DAYS AFTER CONFLUENCE DAYS AFTER CONFLUENCE Fg. 2. Increases n AcetylCoA Carboxylase Actvty, Enzyme, and mrna Levels durng Adpocyte Converson The data from Table 1 and Fg. 3 and 4 s presented together n graphcal form here. The percentage of 30A5 (O) and 10T12 (A) cells dfferentated s presented n AC. A, AcetylCoA carboxylase actvty (30A5, ; 10T12, A); B, acetylcoa carboxylase enzyme level ( ); C, acetylcoa carboxylase mrna amount (30A5,3; 10T12, A); D, actvty, amount, and mrna levels together. Error bars for actvty measurements represent the range of two determnatons on duplcate cell monolayers. 10T12 159 127 144 250 Downloaded from https:academc.oup.commendartcleabstract253952713520 by guest on 04 September 2018
Regulaton of AcetylCoA Carboxylase mrna 399 1 2 3 2 4 6 8 ACC Ab (xl x I0" 2 ) Fg. 3. Increases n AcetylCoA Carboxylase Enzyme Level durng Adpocyte Converson AcetylCoA carboxylase actvty was mmunottrated as descrbed n Materals and Methods. The equvalence pont was determned by extrapolaton as demonstrated n the graph ( ). Error bars represent the range of two determnatons on duplcate cell monolayers. ACC ACTIN DAYS AFTER CONFLUENCE 1 3 4 5 6 8 10» # * # # Fg. 4. Increases n AcetylCoA Carboxylase mrna Levels durng Adpocyte Converson The dfferentaton scheme as descrbed n Materals and Methods was ntated after 30A5 cells had reached confluency (day 0). Total RNA was solated at varous tmes as ndcated and analyzed wth the acetylcoa carboxylase (ACC) or 3actn ncktranslated cdna. The ACC flter contans dots wth 10 fg and 5 ^g RNA. 18S ACTIN * * Fg. 5. Effect of on AcetylCoA Carboxylase mrna Levels durng Preadpocyte to Adpocyte Converson 30A5 cells were grown n the presence of from day 0 to day 8. Cells were harvested at day 8. Total RNA was solated and analyzed wth a ncktranslated cdna probe for acetylcoa carboxylase or?actn. Lane 1, No ; lane 2, 100 Uml ; lane 3, 200 Uml. Each lane contans 10 ng RNA. AcetylCoA carboxylase mrna s 10 kb n length (18). 3 days and then the meda was changed to nclude nsuln and ; ths contnued untl day 7 at whch pont was removed and fresh meda contanng nsuln was added to the cultures. Carboxylase mrna levels were notceably lower at day 7 n the treated cells. However, 3 days after removal of carboxylase mrna levels ncreased to almost the same level as the control wthout (Fg. 8A). A smlar experment n whch cells were exposed to at an earler tme (day 4 to day 0) before followng the standard dfferentaton scheme, showed a slght lag n the accumulaton of carboxylase mrna n comparson to control cells (Fg. 8B). In addton, the percentage of treated cells that dsplayed lpd durng the converson process was smaller than untreated control cells (data not shown). dd not notceably nhbt the prolferaton of these cells as both cultures reached confluence on the same day. These data demonstrate that the nhbtory effect of on acetylcoa carboxylase mrna accumulaton s reversble. To examne whether could affect the amount of acetylcoa carboxylase mrna n fully dfferentated Downloaded from https:academc.oup.commendartcleabstract253952713520 by guest on 04 September 2018
MOL ENDO1988 400 Vol 2 No. 5 DAY 0 > DAY 8 DAY 8 ^DAY 11 Fg. 6. Effect of on AcetylCoA Carboxylase Actvty 30A5 cells were grown from day 0 to day 8 wth or fully dfferentated 30A5 cells at day 8 were exposed to for 72 h. Cell extracts were prepared and acetylcoa carboxylase actvty determned. Error bars ndcate the range of two determnatons on duplcate cell monolayers. 30A5, mature adpocytes were treated wth 200 Uml. AcetylCoA carboxylase mrna levels were reduced by about 40% after 72 h of treatment n comparson to control cells (Fg. 9). The effects of on mature adpocytes are not due to changes n cell number and vablty snce cell number and vablty were dentcal for treated and control cells after 96 h of treatment. Durng the 72h treatment, acetylcoa carboxylase actvty decreased only slghtly (<15%) n comparson to control cells (Fg. 6). Cells not treated wth but mantaned n nsuln for 72 h also dsplay the same degree of decreased enzyme actvty n comparson to fully dfferentated cells at day 8 (0 h). These data suggest that acts prmarly by controllng acetylcoa carboxylase mrna levels and any effects of on enzyme actvty may be the result of acton at the mrna level. DISCUSSION Adpocyte dfferentaton s a complex process requrng the orchestraton of varous sgnals n order to trgger the converson (23, 24). Although lttle s known about the sgnals needed to trgger the converson process, varous markers have been employed to assess the dfferentaton state of the cell. The phenotypc markers used are generally ncreases n lpd droplets and nsuln receptors (25) whle an ncrease n glycerophosphate dehydrogenase, malc enzyme, and ATPctrate lyase (26) actvtes have been used as bochemcal markers. Although these enzymatc markers sgnfy a lpogenc Fg. 7. Effect of on Converson of Preadpocytes to Adpocytes Both panels are 30A5 cells 8 days after confluence at 10Ox magnfcaton. A, No ; B, present throughout the 8 days. state, they do not dstngush between de novo synthess of fatty acds and uptake of fatty acds from the meda to effect adpocyte converson. The present studes are of partcular nterest because they nvolve acetyl CoA carboxylase, the ratelmtng enzyme of longchan fatty acd bosynthess thus, provdng a drect marker to assess the cellular state of de novo fatty acd bosynthess. The 30A5 preadpocyte cell lne derved from 10T1 2 mouse fbroblast cells used n ths paper s commtted to adpocyte dfferentaton as are other preadpocyte cell lnes (13, 19). After dfferentaton, whch can be elaborated by treatment wth glucocortcod and nsuln, all of these cell lnes dsplay lpd accumulaton and possess many other characterstcs of the mature adpocyte. One characterstc that should be common to these cell lnes s an ncrease n acetylcoa carboxylase actvty durng the converson of the preadpocyte to adpocyte. Ths s the case for 3T3L1 cells whch show ncreases n carboxylase actvty and amount durng the converson process (20). The underlyng mechansm for such ncreases n carboxylase amount has not been known. Downloaded from https:academc.oup.commendartcleabstract253952713520 by guest on 04 September 2018
Regulaton of AcetylCoA Carboxylase mrna 401 ACC ACTIN ACC ACTIN CONTROL D7 D1O CONTROL D8 24 h D7 48h (D3D7) B CONTROL (D4 DO) D1O D13 D1 D5 08 D1 05 D8 ACC ACTIN Fg. 8. Effect of Wthdrawng on the Accumulaton of AcetylCoA Carboxylase mrna A, Cells were exposed to 200 Uml of from day (D) 3 to day 7. Cells were harvested at days 7, 10, and 13. Total RNA was dotted onto ntrocellulose and probed wth antsense RNA probes derved from acetylcoa carboxylase cdna or actn cdna. The carboxylase flter (ACC) contans 1.7 ng RNA dot and the actn flter 0.85 ng RNAdot. B, Cells were exposed to 200 Uml from day 4 to day 0. Cells were harvested at days 1, 5, and 8. RNA was analyzed as descrbed above. The ACC flter contans 4 *g RNAdot and the actn flter 2 ng RNAdot. In ths report we nvestgated the relatonshp between acetylcoa carboxylase amount and mrna levels by usng antbodes that are specfc to the carboxylase and also cdna that s specfc for the carboxylase mrna. We have establshed that durng the converson of preadpocytes to adpocytes the ncrease n the level of acetylcoa carboxylase actvty s manly due to an elevaton n the level of the carboxylase mrna and enzyme. AcetylCoA carboxylase actvty ncreases about 12fold from day 1 to day 8 whereas the levels of enzyme and carboxylase mrna ncrease approxmately, 8fold and 10fold, respectvely. The nhbtory effect of on lpoproten lpase s well documented (710). Snce nhbton of ths enzyme prevents the uptake of fatty acds from serumcontanng meda t has been dffcult to assess whether ths s the prmary effect of acton n preventng adpocyte converson or whether also has a major effect on 72h CONTROL D11 Fg. 9. Effect of on AcetylCoA Carboxylase mrna Levels n Fully Dfferentated 30A5 Cells Fully dfferentated 30A5 cells (day 8) were treated wth 200 Uml for varous tmes as ndcated. RNA was analyzed usng antsense RNA probes. The ACC flter contans 1.2 ^g RNAdot and the actn flter 0.6 ng RNAdot. The experment was performed twce wth dentcal results. de novo synthess of fatty acds. The results presented here ndcate that the ablty of to block the converson of preadpocytes to adpocytes s n part due to ts ablty to nhbt de novo bosynthess of fatty acds as evdenced by ts nhbtory effect on the accumulaton of acetylcoa carboxylase mrna and the subsequent producton of carboxylase enzyme. Although t s not possble to assess from these data whether ths s a prmary or secondary effect, the data are consstent wth the noton that de novo fatty acd synthess occurs durng preadpocyte to adpocyte converson and that acetylcoa carboxylase must be present and actve f dfferentaton s to proceed. The nablty of recombnant to cause a marked change n acetylcoa carboxylase actvty n fully dfferentated adpocytes has been prevously reported (8). Our data also ndcate that there s lttle change n carboxylase enzyme actvty when fully dfferentated adpocytes are treated wth. However, we do observe that causes carboxylase mrna levels to decrease, albet to a lesser extent than that observed when s added to preadpocytes. The absence of nsuln from the meda, except that contaned n the serum, dd not alter the effect on carboxylase actvty (see Footnote 1). These observatons suggest that acts prmarly at the level of controllng the mrna content and not drectly at the enzyme level. The extremely slow rate at whch decreases carboxylase mrna levels n fully dfferentated adpocytes may suggest a relatvely long halflfe for the message. Ths appears to be the case for adpsn, a dfferentaton specfc serne protease homologue, whose mrna has an estmated halflfe of greater than 30 h and decreases at a relatvely slow rate upon treatment of adpocytes (12, 21). Based on the knetcs of acetylcoa carboxylase mrna nducton durng dfferentaton, the halflfe of the carboxylase message s Downloaded from https:academc.oup.commendartcleabstract253952713520 by guest on 04 September 2018
MOL ENDO1988 402 Vol 2 No. 5 estmated to be greater than 24 h. It s dffcult to make nferences based on the adpsn studes snce a dfferent cell lne and culture condtons were used. However, these observatons are consstent wth the noton that shuts off transcrpton (4, 23) and the rate at whch specfc mrnas dsappear s lnked to ther normal turnover rates, as suggested by Mn and Spegelman (12). Current work s focused on determnng the effect of on the transcrpton rate of the carboxylase gene and the halflfe of carboxylase mrna. MATERIALS AND METHODS Materals Commercal products were obtaned as follows: Basal Medum Eagle's (M.A. Boproducts); fetal bovne serum (Gbco, Grand Island, NY); dexamethasone and nsuln (Collaboratve Research); 14 Csodum bcarbonate (Research Products Intl., Mt. Prospect, IL; 55.5 mcmmol); guandne sothocyanate (Fluka). AcetylCoA carboxylase was purfed (27) and antserum was prepared as prevously reported (28). Recombnant human tumor necross factoralpha (2.7 x 10 6 Umg) was generously provded by Dr. Tatsuro Nshhara of the Suntory Insttute, Osaka, Japan. Cell Culture A preadpocyte cell lne, 30A5, was derved from 10T12 mouse fbroblasts after treatment of the fbroblast lne wth 5 azacytdne. The cell lne was kndly provded by Dr. Steve Koneczny, Purdue Unversty. Further detals of the solaton and characterzaton of ths cell lne wll be publshed elsewhere. Culture plates were ntally seeded wth approxmately 1.2 x 10 s cells and reached confluency 3 days later. Cells were grown n Basal Medum Eagle's supplemented wth 10% heatnactvated fetal bovne serum. At confluence fresh meda contanng 10~ 6 M dexamethasone and 5 Mgml nsuln were added. After 3 days, the meda were changed to nclude only nsuln and cells were mantaned n ths meda for the remander of the dfferentaton. Ths dfferentaton scheme s the same as that used for TA1 cells (13). The culture meda were changed every 3 days wth fresh meda supplemented wth the approprate hormones. To determne the percent of dfferentated cells at least 200 cells were counted n seven to eght randomly selected felds for each day. Ths was performed for three plates for each tme pont. Those cells dsplayng lpd accumulaton at 200x magnfcaton were scored as adpocytes. AcetylCoA Carboxylase Assays Cells from a 10cm dsh were scraped nto 0.5 ml 5 ITIM sodum phosphate, 10 mm sodum ctrate, 0.5 ITIM EGTA, ph 7.0, and brefly homogenzed (5 sec) n a Polytron homogenzer at low speed (23 settng). Homogenates were centrfuged at 10,000 x o; for 15 mn and the floatng fatpad was asprated off. The supernatant was adjusted to 80 mm phosphate, ph 7.0, and to a fnal volume of 0.85 ml. Onehundred thrtyfve n\ of ths were used to assay acetylcoa carboxylase actvty n a fnal volume of 600 f\ contanng 50 mm sodum phosphate, ph 7.0, 10 mm ctrate, 8 mm magnesum acetate, 1 mm dthothretol, 10 mgml BSA, 2.25 mm ATP, 0.5 mm acetylcoa, and 5 mm 14 Csodum bcarbonate (55.5 mcmmol) at 37 C for up to 12 mn. A reacton mxture wthout ATP, acetylcoa, and sodum bcarbonate was prencubated for 30 mn at 37 C, and the reacton was started by addng these reagents. Under these condtons the reacton was lnear up to 12 mn, and an alquot was taken at 2mn ntervals. The amount of CO 2 fxed n malonylcoa was determned as descrbed (29). One unt of acetylcoa carboxylase actvty was defned as the mcromoles of malonylcoa formed per mnute at 37 C. Determnaton of AcetylCoA Carboxylase Amount The same cell extracts used to determne carboxylase actvty were also used to determne enzyme amount by mmunottraton of carboxylase actvty wth antserum to the carboxylase. The same reagent concentratons were used as descrbed above except all volumes were decreased by onethrd. The enzyme extract was ncubated wth antserum to acetylcoa carboxylase for 30 mn n an cewater bath n the prencubaton soluton descrbed above. The mxture was then transferred to 37 C and ncubated for an addtonal 30 mn before startng the assay. The reactons were stopped after 12 mn. Antserum to acetylcoa carboxylase was serally dluted n rabbt control serum. The control reactons contaned control serum. Determnaton of Proten Amount Proten was measured usng the bcnchonnc acd method (30). The BCA Proten Assay Reagent Kt from Perce Chemcal Co. (Rockford, IL) was used. BSA was used as the standard. Isolaton of RNA Total RNA was solated essentally as descrbed (31) n a modfcaton of the method of Cathala et al. (32). Brefly, cell pellets were homogenzed n buffer contanng 5 M guandne sothocyanate, 50 mm TrsHCI, ph 7.5,10 mm EDTA, 0.14% dethylpyrocarbonate (DEPC), and 8% Smercaptoethanol n a Polytron homogenzer for 5 sec at low speed. The soluton was made 1.7 M n lthum chlorde and RNA was precptated by placng the samples at 4 C overnght. After centrfugaton n a Sorvall SS34 rotor at 5000 rpm for 2 h at 4 C, the pellet was washed n 3 M lthum chlorde and centrfuged agan. The pellet was then resuspended n 50 mm TrsHCI, ph 7.5, 10 mm EDTA, 1% sodum dodecyl sulfate (SDS), 0.14% dethylpyrocarbonate and 8% 3mercaptoethanol, extracted wth phenolchloroform, and precptated wth ethanol. Analyss of RNA For the Northern analyss, RNA was separated by electrophoress under denaturng condtons n agarose gel contanng formaldehyde (33). RNA was then transferred to Zetabnd membrane (AMFCuno, Houston, TX) by electrophoress usng a Transphor apparatus (BoRad, Rchmond, CA) at 30V for 18 h n 10 mm TrsHCI, 5 mm sodum acetate, and 0.5 mm EDTA, ph 7.8. All detectable levels of RNA were removed as evdenced by stanng the gel wth ethdum bromde after transfer. For dotblots, total RNA was dotted onto ntrocellulose (34) usng ether a dot or slotblot apparatus (Schlecher & Schuell, Keene, NH). The ncktranslated cdna nsert of XKH18 (18) and a chcken 0actn cdna clone were used to probe flters under hybrdzaton condtons prevously descrbed (18). For some analyses we also used a 32 Plabeled RNA probe. An antsense RNA probe was generated from a pgem3 vector (Promega, Madson, Wl) that contaned a 1.3 kb par SamHISamHI fragment that les wthn the codng regon of acetylcoa carboxylase. A 0actn antsense RNA probe was also syntheszed n a pgem vector. After the hybrdzaton, flters were washed four tmes for 5 mn n 2x SSC contanng 0.1% SDS at room temperature followed by 2 tmes for 15 mn n 0.1 x SSC and 0.1% SDS at 60 C. Flters were exposed to XAR5 flm at 70 C wth ntensfyng screens. Identcal hybrdzaton and washng condtons were used wth ncktranslated cdna or RNA probes. A RNAse dgeston step was ncluded after the fnal wash of flters probed wth RNA. Quanttaton of Downloaded from https:academc.oup.commendartcleabstract253952713520 by guest on 04 September 2018
Regulaton of AcetylCoA Carboxylase mrna 403 autoradographs was performed usng an LKB laser denstometer and LKB GSXL software. Acknowledgments Receved November 25,1987. Accepted January 21,1988 Address requests for reprnts to: 1 KHan Km, Department of Bochemstry, Purdue Unversty, West Lafayette, Indana 47907. Ths work was supported by a grant from NIH, DK12865. M. E. Pape s a tranee of NASA Tranng Grant NGT50196. Ths s Journal paper number 11341 from the Purdue Unversty Agrcultural Expermental Staton. REFERENCES 1. Beutler B, Greenwald D, Hulmes JD, Chang M, Pan Y CE, Mathson J, Ulevtch R, Ceram A 1985 Identty of tumour necross factor and the macrophagesecreted factor cachectn. Nature 316:552554 2. Beutler B, Mahoney J, Letrang N, Pekala P, and Ceram A 1985 Purfcaton of cachectn, a lpoproten lpasesuppressng hormone secreted by endotoxnnduced RAW 264.7 cells. J Exp Med 161:984995 3. Beutler B, Ceram A 1986 Cachectn and tumor necross factor as two sdes of the same bologcal con. 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Berent JSL, Mahmond M, Torczynsk RM, Bragg PFW, Bollon AP 1985 Comparson of olgonucleotde and long DNA fragments as probes n DNA and RNA dot, southern, northern, colony, and plaque hybrdzatons. Botechnques 3:208220 Downloaded from https:academc.oup.commendartcleabstract253952713520 by guest on 04 September 2018