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IN VITRO APOLIPOPROTEIN B mRNA EDITING ACTNITY CAN BE MODULATED FASTING AND REFEEDING RATS WITH A HIGH CARBOHYDRATE DIET

899-903

BY

Stanley G. Harris and Harold C. Smith* Department of Pathology and the Cancer Center University of Rochester, 601 Elmwood Avenue, Rochester, New York Received

January

15,

1992

Apolipoprotein B mRNA editing in vivo is subject to tissue specific, developmental and metabolic regulation. We demonstratefor the first time that the metabolic modulation of apo B mRNA editing activity can be assayedin vitro using rat liver extracts. The editing activity in extracts from 48h-fasted rats was suppressedrelative to that of normal chow-fed rats. Refeeding with a high-sucrosefat-free chow for 48h stimulatedliver in vitro editing activity to approximately three times that of control liver extracts. The physical properties of editosomesassembledin extracts from fasted/refedrats differed from thoseassembledin control or fasted rat liver extracts. Polypeptide analysisrevealed quantitative alterations of severalproteins in each treatment group suggestinga complex regulatory process. The data corroborate those from in vivo studies and suggestthe potential of the in vitro systemin studying factors responsiblefor metabolic regulation of apo B mRNA editing. 0 1992Academic Press.Inc.

Editing of apolipoprotein B (apo B) mRNA involves the conversion of cytidine to uridine at nucleotide 6666 through a deamidation reaction (1). The conversion of a glutamine codon EAA)

to a translation stop codon (JJAA) enablessynthesisof the high and low molecular weight

isoforms of apo B (apo BB and apo BL respectively) from unedited andedited n-RNA respectively (2-4). Developmental (5), tissue-specific (6) and metabolic (7,8) regulation of apo B mRNA editing have beendescribedin vivo. Most relevant to this study are data which show that hepatic editing in vivo can be decreasedapproximately 1.8-fold by 48 h of fasting (7). At this time, apoB mRNA abundanceincreased by 80% of that found in livers from rats fed with normal chow (control) while the synthesisof both isoforms of apo B increasedslightly. Refeedingrats for 48 h with a high-sucrose,fat-free diet increasedhepatic apo B mRNA editing to 1.3-fold of control or 2.5-fold of the activity in the fasted state. Hepatic apo B mRNA abundanceand apo BL synthesis

korresponding author. Abbreviations: Apo B, apolipoprotein B; LDL, low density lipoprotein; P.A.G.E., polyacrylamide gel electrophoresis;PMSF, phenylmethylsulfonyl fluoride; SDS sodium dodecyl sulfate; VLDL, very low density lipoprotein.

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returned to control levels while apo BH synthesis could not be detected. These data corroborated earlier studies showing alterations in cultured rat hepatocyte triglyceride content and secretion rates following

a similar fasting and refeeding regimen (9,lO).

In the current study, we have taken

advantage of in vitro methods to evaluate whether the editing activity of hepatic extracts will change in parallel with that seen for in vivo regulation.

The data show that factors which were

responsible for metabolic regulation in vivo are extractable and active in the in vitro editing system.

METHODS Male Sprague-Dawley rats weighing 250-300 g were obtained from Charles River and housed under controlled University vivarium conditions until they had grown to 450-500 gm. Aging of rats was performed because fasting was less stressful for iarger rats and improved consistency in the data. In vitro editing activity was identical in hepatic extracts from 250-500 gm control rats. Control rats were maintained on normal rat chow (Purina rat chow; Ralston-Purina, St Louis, MO) while the fasted rats had food withdrawn for 48 h and were provided with drinking water only. Following 48 h of fasting, rats were either sacrificed or refed with high-sucrose, fat-free rat chow (ICN Nutritional Biochemicals, Catalog # 901683, Cleveland, OH) for 48 h and then sacrificed. Unfasted rats were fed this special diet for 48 h prior to sacrifice as a control for sucrose feeding. . IC Extract Preoarath. Rats were sacrificed by cervical dislocation and their livers nerfused in situ with 30 ml of ice cold homogenization buffer (0.33 M sucrose, 50 mM Tris PH= ‘s.0, 5 mM M Clz) containing 0.5 pg eachaprotinin and leipeptin per ml and 1 mM P$lSF. Livers were su%sequently homogenized in an equal volume (w/v> of homogenization buffer with a teflon-to-glass homogenizer. Nuclei were cleared from the cytosol ( 3,500 X g, 10 min) and the S 100 fraction of the cytosol was obtained by ultracenuifugation (100,000 X g, 60 min). In vitro Edi0 uantification. Fifty ~1 editing reactions containing 60 pg of extract protein and 20 fmols of RNA editing substrate (a 498-mer containing the native editing site at nucleotide 6666 within apo B sequences 6413-6860, plus 48 and 2 nucleotides of 5’ and 3’ polylinker sequence respectively) were carried out for 3 h at 30 oC as described previously (11). RNA was purified from each editing reaction and subject to primer extensionanalysisfor the edited nucleotide as describedpreviously (11). In the presenceof high concentrations of ddGTP, two primer extension products are observed subsequent to denaturing gel electrophoresis and autoradiography which correspond to the amount of unedited (CAA) and edited (TAA) RNAs. The primer for thesereactionswas a J2Pend-labeled35-mer (5’ end at nucleotide 6708 of the apo B sequence). The percent editing was determinedby scintillation counting of excised gel bands and calculated asthe dpmsin the excised TAA gel banddivided by the sum of dpms in the CAA andTAA excised gel bands, times 100. Miscellaneous Procedu es Editosomes were resolved on native gels as described previously (11). Protein concentratiois were determinedwith the BioRad assaysystem(BioRad Laboratories, CA). Proteins were resolved by 5-18% gradient, SDS P.A.G.E. In vitro transcription of apo B mRNA substratesand end-labeling of the primer employed reagentsand protocols of Promega (PromegaCo., WI) and United StatesBiochemical (USB, OH) respectively.

RESULTS AND DWXJSSION .

let Affects In v&o EdiaP

. . Actlvltv,

A representative autoradiograph of primer

extension products from RNAs isolated from editing reactions containing hepatic SlOO extracts from control and metabolically conditionedrats is shownin Figure 1. Control S100extracts edited 3-5% of the input RNA substrate. Fasting reduced the in vitro editing activity to background

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Liver extractswerepreparedfrommetabolicallyconditionedratsandassayed for in vitro apoB RNA editing activity asdescribedin Methods. The metabolictreatmentgroupsarelisted abovethe corresponding autoradiographed lanesandthe positionof the radiolabeledprimerand first stop(CAA, uneditedRNA) andsecondstop(TAA, editedRNA) primerextensionproducts areshownto the right. Figure2 Ten ul from editingreactionscontainingextractsfrom control(lanes1 and4), 48hfasted (lanes 2 and 5) and fasted/48hrefed (lanes3 and 6) were resolved on native gels and autoradiographed asdescribedin Methods. Reactionsin lanes4-6 were broughtto 1 mg/ml heparinimmediatelyprior to electrophoresis. B andA indicatethemigrationof controleditosomes andunassembled RNA respectively;- and+ indicate samples which wereloadedwithout and with heparin. Fm

levels. In vitro editing activity wasre-establishedin refed rat liver extracts; reaching2- to 3-fold the level seenin control extracts. Feeding the specialdiet to unfastedrats did not elevate editing activity and actually induced a slight decrease(7% 51.5 SEM, n= 3) in activity.

These data

suggestthat fasting potentiates the refeeding responseof an increasein editing activity. The data clearly show that the editing efficiency in tissueextracts can vary with the metabolic state of the animal and importantly, that factors responsiblefor this modulation of activity are extractable and functional in vitro.

J%ditosome Gel Mob i’lltv IS ’ Altered.

Native gel analysis of editosomes assembly

revealed a small but reproduciblequantitative decreasein editosomes(B complexes)assembledby fasting rat liver extracts and an increasedmobility of thoseassembledin extracts from fasted/refed rats (Fig. 2, lanes 2 and 3 respectively). Faster migrating B complexes were not observed when heparin (1 mg/ml) was added to the samplesprior to electrophoresis(lanes4-6). Moreover, the apparentabundanceof the editosomesassembledby extracts from fasted/refed rats was increased upon addition of heparin (lane 6). These findings are analogousto thoserecently describedin comparing editosomesassembledby liver and enterocyte extracts (12) which suggestedthat the physical properties of editosomeswere different in more active extracts and that heparin could have a normalizing effect. Taken together the data also suggestthat the mechanism(s)for activating liver editing activity may be similar to that responsiblefor elevated editing efficiency in enterocytes. 901

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F&U.& Sixty pg of total S 100 liver extract protein from each metabolic condition were resolved by 518% gradient SDS P.A.G.E. and stained with Coomassie blue. Lanes l-4 correspond to liver SlOO proteins from control rats; 48 h fasted rats; 48 h fasted and high-sucrose, fat-free chow 48 h refed rats and unfasted, high-sucrose, fat-free chow 48 h fed rats. Molecular weight standard proteins were resolved in lane 5 and their Mr are indicated to the right.

Proteins

in SlOO Extracts.

The metabolic regulation

from a reduction in a specific structural or functional protein(s)

described above might arise during fasting followed

by an

increase in this factor(s) upon refeeding. Alternatively, the fasting and refeeding effect might arise from the modulation of a protein(s) which allostericalIy reguIates editing activity. One dimensional 5-18% gradient P.A.G.E. of SlOO extract proteins from the four metabolic conditions revealed that this subset of total cytoplasmic perturbation.

proteins was largely refractory

to this particular

metabolic

A few quantitative alterations specific for each treatment group were observed

(Figure 3). Proteins,

with Mr of 150, 37 and 15 were the only ones to show a trend of an

increased abundance in the fasted state (compare lanes 1 and 2) and a decrease to control level after refeeding (lane 3). The abundance of proteins with Mr of 195 and 160 was markedly reduced in the fasting state but failed to recover upon refeeding.

The abundance of several other proteins

appeared to be sensitive to feeding of the special diet as these alterations occurred regardless of the prefeeding

state of the animal (lanes 3 and 4). Proteins with Mr of 210, 116, 99, 79 and 65

increased in abundance and proteins with Mr of 195, 160, 120, and 103 decreased in abundance whenever the special diet was fed. It is tempting to speculate that decreased editing in the fasted liver results from the increased abundance of inhibitor proteins (Mr 150, 37 and 15) and that down-regulation proteins by refeeding up-regulates editing. Alternatively, 902

of these

a combination of alterations in different

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subsets of proteins during fasting and refeeding may be responsible for the observed alteration. The possibility

also exists however,

that none of the alterations in abundant extract proteins are

relevant to the editing process and that low-abundance extract proteins whose alterations can not be detected by stained P.A.G.E. are responsible.

Moreover, whether protein abundance is regulated

by alterations in synthetic rate, turnover or both remains to be evaluated. In summary, the data shown here are the first to demonstrate that metabolic regulation of apo B mRNA

editing is preserved in extracts used to study editing in vitro..

The physical

properties of editosomes change in the metabolic model. In the subset of total cytoplasmic proteins represented by the S 100 extract, only alterations in the abundance of proteins with Mr 150,37 and 15 appeared to correlate with the metabolic perturbation

Future studies will focus on the potential

structural and functional roles of extract proteins in apo B mRNA editing and its regulation.

,4CKNOWLEDGMENTS This work was supported in part by an NM grant (DK 43739-Ol), a Biomedical Research Support Grant (S7RR05403-29) and an Office of Naval Research Grant (NOO014-89-J1915) awarded to HCS. The authors are grateful to Drs C.E. and J.D. Sparks for helpful discussions and J.M.L. Smith for the preparations of figures.

REFERENCES 1.

Bostrom, K., Garcia, Z., Poksay, K.S., Johnson, D.F., Lusis, A.J., and Innerarity, T.L. (1991) J. Biol. Chem. 265, 22446-22452. 2. Chen, S.H., Habib, G., Yang, C.Y. Gu, Z.W., Lee, B.R. Weng, S.A., Silberman, S.R., Cai, S.J., Deslypere, J.P., Rosseneu, M., Gotto, A.M., Li, W.H., and Chan, L. (1987) Science 238,363-366. Powell, L.M., Wallis, S.C., Pease, R.J., Edwards, Y.H., Knott, T.J., and Scott, J. :: Higuchi, K., Hospattankar, A.V., Law, S.W., Meglin, N., Comight, J., and Brewer, H.B. (1988) Proc. Natl. Acad. Sci. USA 85. 1772-1776. 5. Wu, J.H., Semenkovich, C.F., Chen, S.H., ‘Li, W.H., and Chan, L. (1990) J. Biol. Chem. 265. 12312-12316. 6. Backus, J.W., Eagleton, M-I., Harris, S.G., Sparks, C.E., Sparks, J.D., and Smith, H.C. (1990) B&hem. Biophys. Res. Commun. 170,5 13-518. 7. Baum, CL., Teng, B.B., and Davidson, N.O. (1990) J. Biol. Chem. 265, 19263-19270. 8. Davidson, N.O., Powell, L.M., Wallis, S.C., and Scott, J. (1988) J. Biol. Chem. 263, 13482-13485. 9. Marsh, J.B., and Sparks, C.E. (1982) Proc. Sot. Exp. Biol. Med. 170, 178-181. 10. Davis, R.S., Boogaerts, J.R., Borchardt, R.A., Malone-McNeal, M., and Archambault-Schexnayder, J. (1985) J. Biol. Chem. 260, 14137-14144. 11. Smith, H.C., Kuo, S.K., Backus, J.W., Harris, S.G., Sparks, C.E., and Sparks, J.D. (1990) Proc. Natl. Acad. Sci. USA 88, 1489-1493. 12. Backus, J.W. and Smith, H.C. (1991 Nucleic Acids Res. 19,6781-6786.

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In vitro apolipoprotein B mRNA editing activity can be modulated by fasting and refeeding rats with a high carbohydrate diet.

Apolipoprotein B mRNA editing in vivo is subject to tissue specific, developmental and metabolic regulation. We demonstrate for the first time that th...
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